interaction between natural enemies and insecticides … · (rosaceae) md touhidur rahman b.sc...

INTERACTION BETWEEN NATURAL ENEMIES AND INSECTICIDES USED FOR

THE MANAGEMENT OF WESTERN FLOWER THRIPS, FRANKLINIELLA

OCCIDENTALIS (PERGANDE) (THYSANOPTERA: THRIPIDAE) IN THREE

CULTIVARS OF STRAWBERRY, FRAGARIA X ANANASSA DUCHESNE

(ROSACEAE)

Md Touhidur Rahman

B.Sc (Zoology), M.Sc (Zoology)

This thesis is presented for the degree of Doctor of Philosophy of The

University of Western Australia

School of Animal Biology

July 2010

ABSTRACT

Interaction between natural enemies and insecticides used for the management of western

flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) in three

cultivars of strawberry, Fragaria x ananassa Duchesne (Rosaceae)

Keywords: Frankliniella occidentalis, Typhlodromips montdorensis, Neoseiulus cucumeris,

Hypoaspis miles, strawberry IPM, host resistance, spinosad, residual toxicity, LT25, resistance

Integrated pest management (IPM) relies on the use of multiple tactics to reduce pest numbers

below an economic threshold. One of the challenges for the implementation of IPM is using

both insecticides and biological control. This is particularly difficult in horticultural crops where

very little damage can be tolerated. Western flower thrips, Frankliniella occidentalis (Pergande)

(Thysanoptera: Thripidae) is a worldwide pest of economic importance associated with

cultivated crops, ornamentals and weeds. It is considered a major pest of strawberry, Fragaria x

ananassa Duchesne (Rosaceae), and can be responsible for substantial yield loss. Insecticides

are the main method of control for F. occidentalis in strawberry and other crops. Due to the

rapid development of insecticide resistance and the limitations of existing biological control in

Australia, there is a need to incorporate insecticides, natural enemies, and resistant host plants to

keep the population below an economic threshold. This project sought to (i) evaluate

commercial strawberry varieties for feeding and oviposition preferences of F. occidentalis, (ii)

assess the compatibility of natural enemies with an insecticide currently used for F. occidentalis

control in IPM programs, (iii) assess the effectiveness of the release of multiple species of

natural enemies, (iv) determine the residual threshold of an insecticide which controls F.

occidentalis effectively whilst having a reduced effect on natural enemies, and (v) assess the

compatibility of natural enemies with an increased rate of an insecticide to manage an

insecticide-resistant strain.

Frankliniella occidentalis showed a distinctive olfactory preference in a choice trial evaluating

the feeding preference of F. occidentalis to strawberry cultivars (Camarosa, Albion and Camino

Real). Frankliniella occidentalis was attracted most to Camarosa for both feeding and

oviposition, followed by Albion and Camino Real. Frankliniella occidentalis also preferred to

feed on fresh leaves to those that had been fed upon by a conspecific. Of the three varieties

tested, Camino Real was the least preferred cultivar for oviposition. The development period of

F. occidentalis (from eggs to adult emergence) was shortest in Camarosa and longest in Camino

Real. Overall, of the three varieties tested, Camarosa appeared the most favourable for F.

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occidentalis feeding, oviposition and population growth, and the other cultivars might be a

better choice for growers seeking to reduce F. occidentalis populations.

Spinosad (Success™; Dow AgroSciences Australia Ltd) is the only insecticide currently

registered in Australia that is effective against F. occidentalis and regarded to be compatible in

an integrated pest management program. A glasshouse study tested the compatibility of three

predatory mite species, Typhlodromips montdorensis (Schicha) (Acari: Phytoseiidae),

Neoseiulus cucumeris (Oudemans) (Acari: Phytoseiidae), Hypoaspis miles (Berlese) (Acari:

Laelapidae) [commercially available in Australia for thrips management] and spinosad. All

three predatory mites appeared to reduce the F. occidentalis population in strawberry plants.

The efficacy of predatory mites further improved when combined with spinosad. Spinosad

posed no detrimental effect to mites when an interval between spinosad application and the

release of predatory mites was maintained. Releases of the two-species combination T.

montdorensis and H. miles, or all three species (T. montdorensis, N. cucumeris and H. miles)

combined with spinosad applications were more effective in reducing F. occidentalis than single

species releases.

In Western Australia, strawberry is grown in low tunnels. Frankliniella occidentalis populations

remain low during winter (June-August) and increase during spring (late September) to early

summer. Therefore, the use of predatory mites prior to the increase in the F. occidentalis

population in spring might be an approach to the management of F. occidentalis populations in

the tunnel environment. A field trial revealed that predatory mites could be used to control F.

occidentalis in low tunnel-grown strawberry plants in spring. Combined releases of ‘T.

montdorensis and H. miles’ or ‘T. montdorensis, N. cucumeris and H. miles’ were most effective

against F. occidentalis. Their beneficial effect was further increased when combined with

spinosad. It was found that predatory mites performed better when released after a spinosad

spray, compared to mites released before a spinosad application.

At the recommended spinosad application rate (80 mL/100 L, 0.096 g a.i./L) to control F.

occidentalis in strawberry, residues were toxic to the predatory mites. Thresholds for the contact

residual toxicity of spinosad LT25 (lethal time for 25% mortality) were estimated as 4.2 days

(101.63h), 3.2 days (77.72) and 5.8 days (138.83 h) for T. montdorensis, N. cucumeris and H.

miles respectively. The residual threshold increased when predatory mites were simultaneously

fed spinosad-intoxicated F. occidentalis and exposed to residues. Residual thresholds then

increased to 5.4 days (129.67), 4 days (95.09), and 6.1 days (146.68 h) for T. montdorensis, N.

cucumeris, and H. miles respectively. Spinosad residues were also repellent to predatory mites.

According to the standards of the International Organisation for Biological Control (IOBC) the

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results of this study determined that spinosad is a short-lived chemical to N. cucumeris, and is

slightly persistent to H. miles. On the other hand, the recommended rate of spinosad is a short-

lived chemical to T. montdorensis, while with twice the recommended rate it was slightly

persistent. The residual toxicity trial revealed that T. montdorensis, N. cucumeris and H. miles

could be incorporated with a higher application rate of spinosad to combat against a spinosad-

resistant strain of F. occidentalis, if the threshold period is maintained. Residual thresholds of

twice the recommended rate of spinosad for T. montdorensis, N. cucumeris and H. miles were

6.1 days (146.76 h), 5.3 days (127.85 h), and 6.8 days (162.45 h) [LT25] respectively. Thus,

predatory mites can be integrated with a higher application rate of spinosad if required, as long

as the above-mentioned interval between application of spinosad and release of predatory mites

is maintained.

This study contributes to an emerging body of research aimed at developing an integrated

management strategy for F. occidentalis in strawberry crops. Collectively, the findings in this

study suggest that existing biological control agents (predatory mites) can be integrated with

spinosad for the management of F. occidentalis in glasshouse- and field (low tunnel)-grown

strawberry. This management strategy can be further improved by selecting resistant cultivars

that are less suitable to F. occidentalis. However, there is scope to further improve the

effectiveness of this strategy. This includes further field trials, testing of additional cultivars,

and testing different release strategies for the biological control agents.

ACKNOWLEDGEMENTS

First and foremost, I would like to extend my sincere gratitude to my supervisors Dr. Helen

Spafford at The University of Western Australia (UWA), and Dr. Sonya Broughton at the

Department of Agriculture and Food WA, for their advice and supervision. You both provided

untiring and unerring guidance, valuable suggestions, and constructive criticism throughout this

PhD. You have always made me think critically about every aspect of my work and I am

immensely grateful for that. Thank you for making this thesis an enjoyable experience. I

specially thank Helen for allowing me to come to Perth to follow my interest in integrated pest

management.

Mr Anthony Yewers, of Berry Sweet, Bullsbrook, was kind enough to give me strawberry

runners (one of the key components of this project) and allow me to conduct an experiment on

his farm. Mr David Cousins, Department of Agriculture and Food WA, helped me to raise and

maintain strawberry plants and provided me with valuable information and seedlings. It is my

privilege to thank Manchil IPM Services, WA, Biological Services, SA and Beneficial Bug

Company, NSW, for providing predatory mites.

I especially thank Kevin Murray, School of Mathematics and Statistics, UWA, for his great help

in completing some of the statistical analyses presented in this thesis. I thank Peter Turner and

Anna Williams for their friendship and advice, especially in my early days at UWA. I would

also like to thank Peter Langland (School of Animal Biology), Sasha Voss (School of Forensic

Entomology), Sayed Iftekhar (School of Agriculture Economics), Sharif-Ar-Raffi (School of

Plant Biology), and many others at UWA. I benefited from them in many ways. I am also

appreciative of my PhD Review Panel Members, Philip Withers (School of Animal Biology,

UWA), and James Ridsdill Smith (CSIRO) for their advice, especially during the developmental

stage of my project.

My research was funded by UWA, through the University Postgraduate Awards (UPA) and

Scholarship for International Research Fees (SIRF). I also acknowledge add hoc scholarship

from the School of Animal Biology, UWA, and an ad hoc scholarship from Helen Spafford.

Without this financial support, it would not have been possible to complete this project.

Last but not least, I would like to thank my family and friends from Bangladesh. I am

immensely grateful to my beloved parents and all family members for their inspiration, wishes

and blessings during the entire period of the study. They never let me doubt my ability to

achieve my goals. I dedicate this thesis to all my family members, but most especially to my late

father, Mr Badiur Rahman.

ORGANISATION AND DECLARATIONS

The research presented in this thesis is an original contribution to the integrated management of

western flower thrips in glasshouse- and low tunnel-grown strawberry.

This thesis is presented as a series of independent research papers (to be published), preceded by

a general introduction chapter, and followed by general discussion. The central chapters of this

thesis are written and presented as separate manuscripts, so some repetition of basic information

does occur.

I, Touhidur Rahman, carried out the design and conducted the experiments after consultation

with my supervisors, Dr Helen Spafford and Dr Sonya Broughton. All statistical analyses

performed in this thesis were produced by myself after discussions with and review by my

supervisors. I benefited from advice and assistance with the design, laboratory, glasshouse and

fieldwork, analyses and writing from my supervisors and others as duly acknowledged.

I, Touhidur Rahman, certify that this thesis does not incorporate, without acknowledgement,

any material previously submitted for a degree or diploma in any institute, and that it does not

contain any material previously published or written by another person, except where due

reference is made in the text.

_______________

Touhidur Rahman

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TABLE OF CONTENTS

ABSTRACT

ACKNOWLEDGEMENTS

ORGANIZATION AND DECLARATION

TABLE OF CONTENTS

LIST OF FIGURES

LIST OF TABLES

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Chapter I: General introduction and literature review

Introduction

Integrated pest management (IPM)

Western flower thrips: origin and distribution

Western flower thrips: host range, pest status and damage

Chemical control of western flower thrips and insecticide resistance

Biological control of western flower thrips

Thrips predators

Predatory mites

Thrips parasitoids

Fungi

Biological control of western flower thrips in Australia

Host-plant resistance

Cultural methods of control of western flower thrips

IPM programs for control of western flower thrips

Outline of this study

Brief organisation and structure of this thesis

Literature cited

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Chapter II: Variation in preference and performance of western flower thrips,

Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) on three strawberry

[Fragaria x ananassa Duchesne (Rosaceae)] cultivars

Keywords

Abstract

Introduction

Materials and methods

Source cultures

Strawberry cultivars

Western flower thrips (WFT)

Experiment 1: Feeding preference of adult WFT

Experiment 2: Oviposition preference and performance of WFT on caged plants

Experiment 3: Oviposition preference and performance of WFT on leaf discs

Egg hatch

Larval mortality, pupal mortality and adult emergence rate

Developmental time

Data analysis

Results

Experiment 1: Feeding preference of adult WFT

Experiment 2: Oviposition preference and performance of WFT on caged plants

Experiment 3: Oviposition preference and performance of WFT on leaf discs

Discussion

Literature cited

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Chapter III: Effect of spinosad and predatory mites (Acari) on western flower thrips,

Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) in three strawberry

cultivars [Fragaria x ananassa Duchesne (Rosaceae)]

Keywords

Abstract

Introduction


Source cultures

Strawberry cultivars


Predatory mites

Glasshouse experiment: effect of cultivars and predatory mites with or without

spinosad on western flower thrips

Data analysis

Results

Western flower thrips

Predatory mites

Discussion

Literature cited

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Chapter IV: Single versus multiple releases of predatory mites (Acari) combined with a

spinosad application for the management of western flower thrips, Frankliniella

occidentalis (Pergande) (Thysanoptera: Thripidae) in strawberry [Fragaria x ananassa

Duchesne (Rosaceae)]

Keywords

Abstract

Introduction


Source cultures

Strawberry cultivar


Predatory mites

Experiment: effect of single versus multiple species releases of mites combined

with spinosad on WFT

Data analysis

Results


Adults

Larvae

Predatory mites

Discussion

Literature cited

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Chapter V: Use of spinosad and predatory mites (Acari) for the management of western

flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) in

strawberry [Fragaria x ananassa Duchesne (Rosaceae)]: a field study

Keywords

Abstract

Introduction


Study site

Predatory mites

Treatments

Pre-treatment sampling

Post-treatment sampling

Data analysis

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Results

Impact of the spray and predatory mite species combinations on WFT adults

Flower

Fruit

Impact of the spray and predatory mite species combinations on WFT larvae

Flower

Fruit

Impact of the spray and mite species combinations on predatory mites

Single species

Two-species combinations

Three-species release

Species interactions

Discussion

Literature cited

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Chapter VI: Compatibility of spinosad with predaceous mites (Acari) used to control

western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae)

Keywords

Abstract

Introduction


Source cultures

Strawberry plants


Predatory mites

Experiment 1: Direct toxicity of spinosad to WFT and predatory mites


Predatory mites

Experiment 2: Residual toxicity of spinosad to WFT and predatory mites

Mortality of WFT and mites to spinosad residues (contact) over time

Indirect exposure of spinosad to predatory mites via consumption of

intoxicated WFT larvae

Toxicity of spinosad to predatory mites via consumption of intoxicated

WFT larvae and direct exposure to spinosad residues of different ages

Experiment 3: Repellency of spinosad to predatory mites (choice test)

Data analysis

Results

Experiment 1: Direct contact toxicity of spinosad to WFT and predatory mites

Experiment 2: Residual toxicity of spinosad to WFT and predatory mites

Residual (contact) toxicity of spinosad to WFT and predatory mites

Indirect exposure of spinosad to predatory mites via consumption of

intoxicated WFT larvae

Residual toxicity to predatory mites via consumption of spinosad-

intoxicated WFT larvae and direct exposure to spinosad residues

Experiment 3: Repellency of spinosad to predatory mites (choice test)

Discussion

Literature cited

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Chapter VII: Spinosad-resistant western flower thrips, Frankliniella occidentalis

(Pergande) (Thysanoptera: Thripidae) can be managed using spinosad and predatory

mites (Acari) Keywords

Abstract

Introduction


Source cultures

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Strawberry plants


Predatory mites

Experiment 1: Direct toxicity of spinosad


Predatory mites

Experiment 2: Bioassay of spinosad residual toxicity to predatory mites

Experiment 3: Efficacy of predatory mites with spinosad against WFT-resistant

strain

Data analysis

Results

Direct toxicity of spinosad to WFT and predatory mites

Bioassay of spinosad residual toxicity to predatory mites

Efficacy of predatory mites with spinosad against WFT-resistant strain

Effect of spinosad and predatory mite releases on WFT adults

Effect of spinosad and predatory mite releases on WFT larvae

Discussion

Literature cited

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Chapter VIII: General discussion and conclusion

General discussion

Findings and recommendations

Strawberry cultivars distinctively influence western flower thrips’ olfactory and

feeding preference and oviposition preference and performance

Biological control: multiple species versus single species

Combining chemical and biological control

IPM of western flower thrips in low tunnel-grown strawberry

Control of spinosad-resistant western flower thrips strain

Conclusions

Literature cited

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Appendices

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LIST OF FIGURES

Figure captions

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Figure 1.1 Distribution map of Frankliniella occidentalis (Pergande) compiled by

CAB International in association with the European and Mediterranean Plant

Protection Organization (EPPO).

Figure 1.2 (A) Western flower thrips adults, and predatory mites, (B) Typhlodromips

montdorensis (Schicha), (C) Neoseiulus cucumeris (Oudemans) and (D) Hypoaspis

miles (Berlese).

Figure 1.3 Damage of WFT on strawberry fruit and flower.

Figure 2.1 Olfactory preference of WFT adults to strawberry cultivars when offered

(A) ungrazed and (B) grazed (B) leaf discs.

Figure 2.2 Simplex plot showing time spent by WFT adults between strawberry

cultivars when exposed to ungrazed leaf discs and discs previously grazed by

conspecific.

Figure 2.3 Time spent by WFT adults on (A) ungrazed and (B) previously grazed

leaf discs.

Figure 2.4 Preference of WFT adults when given a choice between leaf discs

exposed to conspecifics (grazed) or ungrazed of three different strawberry cultivars.

Figure 2.5 Time [A = total time, B = time spent feeding] spent by WFT adult on

ungrazed and grazed leaf discs.

Figure 2.6 Mean numbers of WFT adults per plant on caged strawberry cultivars at

1, 24, and 48 h post-release.

Figure 2.7 Comparison of WFT adult numbers (Y-axis) at different post-release

periods (hours) on caged plants.

Figure 2.8 Mean numbers of WFT (A) larvae hatched and (B) adults emerged per

plant on caged strawberry cultivars.

Figure 2.9 Comparison of numbers of eggs laid, unhatched eggs, larvae hatched,

pupae developed and adults emerged per leaf disc (Y-axis) on three strawberry

cultivars.

Figure 2.10 Comparison of the percentage of unhatched eggs (U/E), larvae hatched

(L/H), larvae killed (L/K), pupae developed (P), pupae killed (P/K) and adult

emerged (A/E) per leaf disc among cultivars.

Figure 2.11 Comparison of survival rate of WFT among strawberry cultivars.

Figure 2.12 Egg incubation period (IP), larval period (LP), prepupation period

(PPP), pupation period (PP) and total developmental period (TDP) (egg to adult) of

WFT in days, among strawberry cultivars.

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Figure 3.1 Plant covered by a modified cage made from thrips-proof mesh

(105µ; Sefar Filter Specialists Pty Ltd., Malaga)

Figure 3.2 Mean numbers of WFT adults per plant treated with spinosad or

water and either no predatory mites or one of three species of predatory mites.

Figure 3.3 Effects of predatory mites on number of WFT adults per plant over

time (7, 14, or 21 days after release of WFT) sprayed with spinosad on

cultivars (A) Camarosa, (B) Camino Real and (C) Albion.

Figure 3.4 Effects of predatory mites on the number of WFT adults per plant

over time (7, 14, or 21 days after release of WFT) sprayed with water on

strawberry cultivar (A) Camarosa, (B) Camino Real and (C) Albion.

Figure 3.5 Mean numbers of WFT larvae per plant treated with spinosad or

water and either no predatory mites or one of three species of predatory mites.

Figure 3.6 Effects of predatory mites on WFT larvae per plant over times (7,

14, or 21 days after WFT release) sprayed with spinosad on strawberry

cultivar (A) Camarosa, (B) Camino Real and (C) Albion.

Figure 3.7 Effects of predatory mites on WFT larvae per plant over times (7,

14, or 21 days after WFT release) sprayed with water on strawberry cultivar

(A) Camarosa, (B) Camino Real and (C) Albion.

Figure 3.8 Mean numbers of T. montdorensis (A and B) and N. cucumeris (C

and D) per plant in relation to strawberry cultivar and spray treatment

Figure 3.9 Comparison of mean numbers of T. montdorensis and N.

cucumeris per plant sprayed with (A) spinosad and (B) water.

Figure 4.1 Comparison of mean number of WFT adults per plant sprayed with

either spinosad or water and in the presence of no mites or different mite

combinations.

Figure 4.2 Effects of predatory mites on mean number of WFT adults per

plant sprayed with (A) spinosad or (B) water.

Figure 4.3 Comparison of mean number of WFT larvae per plant sprayed with

either spinosad or water and in the presence of no mites or different mite

combinations.

Figure 4.4 Effects of predatory mites on mean number of WFT larvae per

plant sprayed with (A) spinosad or (B) water.

Figure 4.5 Comparison of mean number of T. montdorensis and N. cucumeris

per plant applied with (A) single-species releases of T. montdorensis and N.

cucumeris, (B) double-species releases of T. montdorensis and N. cucumeris,

(C) double species releases of T. montdorensis and H. miles and N. cucumeris

and H. miles, and (D) triple-species release.

Figure 4.6 Mean numbers of (A) T. montdorensis and (B) N. cucumeris per

plant in double-species combinations.

Figure 5.1 Maximum, minimum and average daily air temperature (⁰C), and average

daily temperature (⁰C) inside low tunnel (25 September to 20 November 2008).

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Figure 5.2 Effect of spray treatment and predatory mite species releases on the

number of WFT adults/flower in low tunnel strawberry.

Figure 5.3 Influence of predatory mite species combinations on WFT adults per

flower over time (X-axis) in (A) water, (B) ‘spinosad then mites’ and (C) ‘mites then

spinosad’.

Figure 5.4 Effect of spray treatment and predatory mite species combinations (X-

axis) on the number of WFT adults per fruit (Y-axis).

Figure 5.5 Influence of predatory mites on WFT adults per fruit over time (X-axis)

in (A) water, (B) ‘spinosad then mites’ and (C) ‘mites then spinosad’.


axis) on the number of WFT larvae per flower (Y-axis).

Figure 5.7 Influence of predatory mites on WFT larvae per flower over time (X-axis)

in (A) water, (B) ‘spinosad then mites’ and (C) ‘mites then spinosad’.


axis) on the number of WFT larvae per fruit.

Figure 5.9 Influence of predatory mites on WFT larvae per fruit over time (X-axis)

in (A) control (water), (B) ‘spinosad then mites’ and (C) ‘mites then spinosad’.

Figure 5.10 Comparison of mean number of T. montdorensis (Tm) and N. cucumeris

(Nc) per flower or fruit, when applied alone (single species).

Figure 5.11 Comparison of mean numbers T. montdorensis (Tm) and N. cucumeris

(Nc) applied in double-species combination as (A) ‘T. montdorensis and N.

cucumeris’ and (B) T. montdorensis in ‘T. montdorensis and H. miles’ and N.

cucumeris in ‘N. cucumeris and H. miles’.

Figure 5.12 Comparison of mean numbers of T. montdorensis (Tm) and N.

cucumeris (Nc) when applied as a three-species combination (T. montdorensis, N.

cucumeris and H. miles).

Figure 5.13 Comparison of mean number of (A) T. montdorensis and (B) N.

cucumeris when applied in double-species combination (X-axis).

Figure 6.1 Diagrammatic representation of the testing arena used for toxicity test.

Figure 6.2 Probit mortality of (A) T. montdorensis, (B) N. cucumeris, and (C) H.

miles recorded against spinosad residues of different ages (log10 hrs).

Figure 6.3 Probit mortality of (A) T. montdorensis, (B) N. cucumeris, and (C) H.

miles recorded against spinosad residues of different ages (log10 hrs).

Figure 7.1 Toxicity of spinosad residues to predatory mites after 96 h post-release

exposure period.

Figure 7.2 Relationship of Log10 (hrs) and probit mortality of (A) T. montdorensis,

(B) N. cucumeris and (C) H. miles when exposed to twice the recommended rate of

spinosad residues with different ages.

Figure 7.3 Mean numbers of WFT adults per plant sprayed with spinosad or water

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(control) in the presence or absence of predatory mite.

Figure 7.4 Numbers of WFT adults per plant (Y-axis) sprayed with spinosad (A) or

water (B) with or without predatory mite.

Figure 7.5 Numbers of WFT larvae per plant with spinosad and water (control) in

the presence or absence of predatory mites.

Figure 7.6 Numbers of WFT larvae per plant with spinosad (A) and water (B) in the

presence or absence of predatory mites.

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LIST OF TABLES

Table captions

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Table 1.1 Integration of natural enemies and pesticides registered for glasshouse

vegetables in the Netherlands.

Table 1.2 Insecticides that have been tests for efficacy against WFT.

Table 1.3 Natural enemies evaluated as biocontrol agents of WFT.

Table 1.4 Crops and predatory mites used for WFT control.

Table 5.1 Schedules of treatment applications and sampling.

Table 6.1 Mean (±SE) corrected mortality (%) at different post-release exposure

periods (h) to predatory mites after direct exposure to spinosad (recommended rate,

80 mL/100 L).

Table 6.2 Mean (± SE) corrected mortality (%) of WFT adults and larvae when

exposed to spinosad residues aged 2, 12, 24, 48, 72, 96, and 120 h at different post-

release exposure periods.

Table 6.3 Residual toxicity (contact) of spinosad to predatory mites at 24, 48, and 72

h post release exposure periods. The IOBC classification: 1 = harmless (<25%

mortality), 2 = slightly harmful (25-50% mortality), 3 = moderately harmful (51-

75% mortality) and 4 = harmful (>75% mortality). Persistence class: A = short-lived

(<5 d), B = slightly persistent (5-15 d).

Table 6.4 Probit analysis (Abbott 1925) of the mortality of adult predatory mites

exposed to spinosad residues of different ages.

Table 6.5 Mortality of predatory mites after feeding on spinosad intoxicated WFT

larvae at 24 h, 48 h, and 72 h post-release exposure periods. The IOBC classification:

1 = harmless (<25% mortality), 2 = slightly harmful (25-50% mortality), 3 =

moderately harmful (51- 75% mortality) and 4 = harmful (>75% mortality).

Table 6.6 Residual toxicity of spinosad to predatory mites at 24 h, 48 h, and 72 h

post release exposure periods. Mites were fed spinosad-intoxicated WFT larvae and

simultaneously exposed to residue. The IOBC classification: 1 = harmless (<25%

mortality), 2 = slightly harmful (25-50% mortality), 3 = moderately harmful (51-

75% mortality) and 4 = harmful (>75% mortality). Persistence class: A = short lived

(<5 d), B = slightly persistence (5-15 d).


simultaneously exposed to spinosad via consumption of intoxicated WFT larvae and

contact with spinosad residues of different ages.

Table 6.8 Mean (±SE) numbers of predatory mites on spinosad- and water-treated

strawberry leaf in a choice test (t-test, df = 19).

Table 7.1 Cumulative corrected mortality (%) of spinosad- resistant WFT adults (at

96 h post-release exposure period) and larvae (at 72 h post-release-exposure period)

3

9

13

15

114

150

151

152

153

156

158

159

160

175

xvi

when exposed directly to spinosad spray at different rates.

Table 7.2 Residual toxicity of spinosad (twice the recommended rate) to predatory

mites at 24 h, 48 h, 72 h, and 96 h post-release exposure periods. Mites were fed

spinosad intoxicated WFT larvae and simultaneously exposed to residue. Residual

toxicity were classified: 1 = harmless (<25% mortality), 2 = slightly harmful (25-

50% mortality), 3 = moderately harmful (51-75% mortality), and 4 = harmful (>75%

mortality). Persistence class: A = short lived (<5 d), B = slightly persistent (5-15 d).

Table 7.3 Probit analysis (Abbott 1925) of the mortality of predatory mites exposed

to spinosad residues (by consumption of intoxicated WFT larvae and simultaneous

exposure to residues) of different ages.

177

179

CHAPTER I

General introduction and literature review

1.1 Introduction

There are more than 6.3 billion people worldwide (FAO 2004) and the human population

continues to expand. Thus, there is an increased need for food and this requires large increases

in agricultural production. One of the major threats to human food production is insect pests. It

is estimated current food production worldwide is worth about US$1.3 trillion, with insect pests

causing crop losses estimated at US$500 billion (FAO 2005). Currently there are 10,000 species

of insects considered to be pests of agricultural and horticultural crops (Reuveni 1995). Reuveni

(1995) suggests that the total elimination of pests is not possible, but attempts have been made

to reduce their detrimental effects on plants. In this regard, chemical control of the pest

population has become the principal control tool. Since the first synthetic pesticides were

produced in the 1940s, pesticide usage has increased fiftyfold. Currently 2.5 million tons of

pesticides (US$20 billion) are used each year to combat agricultural pest species worldwide

(Pimentel et al. 1992). Unfortunately, due to a lack of sufficient alternatives and a lack of

grower knowledge, synthetic pesticides are often used prophylactically, too heavily or

inappropriately, and can result in the development of resistant pest populations (Maredia et al.

1992, Eddleston et al. 2002). Currently more than 700 arthropod pest species have been reported

to develop resistance to one or more pesticides (Thacker 2002). Furthermore, pesticides pose

detrimental effects to naturally occurring or inundative biological control agents, which can lead

to secondary pest outbreaks. Therefore, an estimated US$520 million can be attributed for the

additional use of pesticides to control secondary pest populations (Thacker 2002).

In response to growing pest and associated problems and risks with pesticide use, the

agricultural industry has been challenged to develop and implement an integrated approach to

pest management (Stern et al. 1959). One of the other pest management tactics available for

many commodities and pests is biological control. However, the integration of biological

control and pesticide use in a pest management program can be problematic. For example,

spinosad is a biopesticide but it is highly toxic to the predatory mite Neoseiulus fallacies

(Garman) (Villanueva and Walgenbach 2005), and slightly toxic to Amblyseius cucumeris

(Oudemans) (Jones et al. 2005). Thus, before implementation in pest management programs,

compatibility of chemical and biological control agents need to be assessed.


2

This thesis presents the results of a comprehensive study to assess the compatibility of chemical

and biological control for the management of a major economic pest, western flower thrips

(WFT), Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) on strawberry

Fragaria ananassa Duchesne (Rosaceae). When WFT was first detected in Australia in

1993, there were no biological control agents commercially available and control relied solely

on chemicals. Since then, three predatory mite species have become commercially available in

Australia. As with control of WFT in other parts of the world, the challenge is to integrate

chemical and biological control methods in a way that provides an economic reduction in the

pest population. The present project tested the hypothesis that integration of an insecticide

(spinosad) with predatory mite releases [Typhlodromips montdorensis (Schicha) (Phytoseiidae),

Neoseiulus cucumeris (Oudemans) (Phytoseiidae), and Hypoaspis miles (Berlese) (Laelapidae)]

can improve the management of WFT in crops such as strawberry. WFT has developed

resistance to multiple insecticides currently in use. Successful integration of chemical and

biological control would minimise the use of chemicals. The ultimate aim of this approach is to

slow the inevitable evolution of resistance and keep insecticides available to growers. This

study also tested whether multiple releases of predatory mites would provide better WFT

management than single species releases. In addition, the influence of strawberry cultivar on

WFT preference and performance is tested.

1.2 Integrated pest management (IPM)

The term „integrated pest management‟ (IPM) [first appeared in literature in 1967 (Smith and

van den Bosch 1967)] is the use of a range of tactics including biological control, host plant

resistance, cultural control, physical control and minimal use of pesticides (Kogan 1998,

Trumble 1998, Hillocks 2002, Prokopy and Kogan 2003, Feder et al. 2004) to control pest

populations. The United Nations Development Program (UNDP) together with the Food and

Agriculture Organization (FAO) has initiated global programs for the development and

application of IPM in several crops such as rice, cotton, sorghum, millet and vegetable crops

(Peshin et al. 2009). Development of IPM strategies emerged in the USA in the 1950s to

improve pest management in agriculture. In Australia, IPM programs have been developed and

implemented in several crops. Australian examples include the use of classical and inundative

releases of biocontrol agents combined with limited use of selective pesticides for the control of

scale insect and mite pests in citrus (Smith et al. 1997), conservation of native predatory mites

to reduce mite pests in grapes (James and Whitney 1993), predatory mites releases against mite

pests, mating disruption and selective insecticide against moth pests in apple (Thwaite 1997),

and the management of currant lettuce aphid, Nasonovia ribisnigri (Mosley) in lettuce using

Nasonovia-resistant varieties and selective insecticides (McDougall and Creek 2007).


3

IPM adoption has resulted in a decrease in pesticide use from 2.6 billion kg in 2004 (Allan

Woodburn Associates 2005) to 1.7 billion kg in 2007 (Agronova 2008) worldwide. In the USA,

adoption of IPM strategies have saved US$500 million each year (Peshin et al. 2009); no figures

are available for Australia. Despite demonstrated efficacy and cost savings, pesticides remain

and will continue to remain the dominant component in many cropping systems (Dhawan and

Peshin 2009). This is because zero damage is tolerated by some markets (Nothnagl 2006),

alternative control may not be available for some crops and pests, and biological control is not

always effective when pest densities are very high (Malezieux et al. 1992). Moreover, biological

control is expensive compared to chemical control, even for growers of high value crops

(Reuveni 1995). Therefore, one of the biggest challenges of IPM is the use of biological control

agents in conjunction with pesticides.

The first successful integration of biological control agents with pesticides dates back to the

1960s, with the use of dimethirimol (fungicide) integrated with releases of the predatory mite,

Phytoseiulus persimilis Athias-Henriot (Acari: Phytoseiidae), for control of two spotted mite in

cucumber (Reuveni 1995). Since then, the integrated use of pesticides combined with biological

control has been practised in many cropping systems (Table 1.1).

Table 1.1 Integration of natural enemies and pesticides registered for glasshouse vegetables in

the Netherlands after Reuveni (1995).

Pest Beneficials Acaricides/insecticides Remarks

Spider mites

Whiteflies

Leafminers

Thrips Aphids

Noctuids

Predatory mites

Hymenopterous

parasitoid

Hymenopterous

parasitoid Predatory mites,

anthocorid bugs Hymenopterous

parasitoid,

cecidomyiids

lacewings,

coccinellids Egg parasitoid

Fenbutatin oxide,

hexythiazox,

clofentezine Buprofezin

Teflubezuron, Hydrogen

cyanide Oxamyl

- Pirimicarb

Hydrogen cyanide Teflubenzuron

-

Not effective against

Bemisia tabaci and some

strains of Trialeurodes

vaporariorum Harmful to anthocorids Harmful to beneficial

insects Effective against Liriomyza

bryoniae only, selective if

used systemically - Not effective against Aphis

gossypii and some strains

of Myzus persicae As above As above


4

However, pesticide applications are often disruptive to natural enemies and consequently pest

populations may increase to more damaging levels than before treatment occurred (Croft 1990).

Thus, integration of biological control agents with chemical control will not be successful

unless natural enemies survive the pesticides being used in a cropping system (Hoy 1985, Hoy

and Cave 1985). By limiting exposure of a beneficial to a pesticide, using narrow-spectrum

pesticides or biological pesticides, this problem can be reduced (Croft 1990, Greathead 1995).

Tauber et al. (1985) stated, "It is probable that the most dramatic increase in the utilisation of

biological control in agricultural IPM systems could come through the judicious use of selective

pesticides in conjunction with effective natural enemies in specific cropping systems, in specific

geographic regions ….”

In order to develop and implement an effective management strategy, it is necessary to have

information about the pest, the available control strategies and advantages and disadvantages

associated with different control methods. The remainder of this chapter reviews the biology

and management of WFT, and the challenges associated with the management of WFT in

strawberry crops.

1.3 Western flower thrips: origin and distribution

Of more than 5500 species of thrips (Thysanoptera) identified to date (Morse and Hoddle 2006),

a few hundred species, mainly from the family Thripidae, are of economic importance. Of them,

a number of species have become key pests in a wide range of agricultural and horticultural

crops, including WFT. WFT has a cosmopolitan distribution (Figure 1.1) and was first

described in 1895 from specimens collected in California, on apricot, potato, orange and various

weeds (Pergande 1895). Pergande placed WFT in the genus Euthrips, but in 1912, Karny (1912)

moved this species to the newly erected genus Frankliniella. Until 1960, the distribution of the

WFT was supposedly restricted to western North America, Mexico, and Alaska (Bryan and

Smith 1956, Nakahara 1997). However, old records indicate the presence of WFT in New

Zealand in 1934 (Mound and Walker 1982). Since the 1960s, there has been a dramatic increase

in the transport of plant materials throughout the world and WFT has been accidentally moved

(Kiritani 2001). In 1966, it was recorded for the first time on chrysanthemum in a glasshouse in

Pennsylvania [Greenough et al. (1985) cited by Kirk and Terry (2003)]. Subsequently, WFT

was recorded on marijuana in Kansas in 1971, and on African violet in Missouri in 1973, in

North Carolina in 1977 and in Louisiana in 1983 [Greenough et al. (1985) cited by Kirk and

Terry (2003)]. In the early 1980s, WFT was recorded in glasshouses in Canada, where it caused

severe epidemics of Tomato spotted wilt virus (Broadbent et al. 1987).


5

Outside North America, WFT was first recorded in New Zealand in 1934 on lupin (Mound and

Walker 1982, Zur Strassen 1986), but was not considered a pest there during that time. It was

reported from Hawaii in 1955 (Sakimura 1962). In 1983 WFT entered Europe through the

Netherlands (Mantel 1989) and spread quickly across the European continent (Tommasini and

Maini 1995). In the late 1980s, WFT was detected in Eastern Africa (Nakahara 1997),

Colombia, Costa Rica (Baker and Hurd 1968), and South Africa (Gilmore 1989). More recently

it has been recorded from Japan (Hayase and Fukuda 1991), Brazil [1994, (Monterio et al.

2001) cited in Kirk and Terry (2003)], Argentina [1993, (De Santis 1995) cited in Kirk and

Terry (2003)] and Chile [1995, (Gonzalez 1996)]. In Australia, WFT was first recorded in 1993

on glasshouse-grown chrysanthemum in Yangebup, Western Australia (Malipatil et al. 1993).

WFT damage to strawberry was detected in Albany, Western Australia in 1994 (Steiner and

Goodwin 2005)]. This detection was followed by the detection of WFT on strawberry in the

Sydney Basin, New South Wales in 1995, the Yarra Valley, Victoria and the Adelaide Hills,

South Australia in 1998-1999 (Steiner and Goodwin 2005)]. WFT has since spread to all states

except the Northern Territory (Medhurst and Swanson 1999).

Figure 1.1 Distribution map of Frankliniella occidentalis (Pergande) compiled by CAB

International in association with the European and Mediterranean Plant Protection Organization

(EPPO) (CAB International 1999). ● Present: national record, + Present: subnational record.

1.4 Western flower thrips: host range, pest status and damage

WFT is a worldwide pest (Figure 1.1 and 1.2) of economic importance associated with

cultivated crops, ornamental, and wild plants (Lewis 1997b). It is highly polyphagous, adults of

WFT having been reported from at least 240 species from 62 different families of plants (Lewis

1997b). Abundance, biology, and developmental stages of thrips can vary under different

ecological conditions and thus, species compositions of thrips, their occurrence, abundance, and


6

life cycles on plants may differ. Common hosts of WFT include cotton, onion, strawberry,

cabbage, lettuce, capsicum (sweet pepper), tomato, chrysanthemum, gerbera, rose, cucumber,

eggplant, bean, geranium, apple, nectarines, peach, and table grapes [(Elmore 1949, Hightower

and Martin 1956, Allen and Gaede 1963, Oatman and Patner 1969, Zur Strassen 1986, van de

Veire 1987, Brødsgaard 1989b, Buxton and Wardlow 1991) cited in Tommasini (2003)]. In

Australia, WFT utilises a wide range of host plants including ornamentals, fruit crops,

vegetables and weeds (Persley et al. 2009).

Figure 1.2 (A)Western flower thrips adults, and predatory mites, (B) Typhlodromips

montdorensis (Schicha), (C) Neoseiulus cucumeris (Oudemans) and (D) Hypoaspis miles

(Berlese) [Photos: Sonya Broughton and James Altman].

WFT causes substantial crop losses through (i) direct damage (feeding and oviposition), and (ii)

vectoring plant viruses and bacterial diseases (Lewis 1997b). Like all other thrips species, WFT

uses its piercing-sucking stylets (Hunter and Ullman 1989) to penetrate epidermal and sub-

epidermal cells, causing extensive damage to the tissue of leaves, fruits and petals (Kirk 1997).

The degree of feeding damage depends on the plant tissue that is affected, the developmental

stage of the plant, and the susceptibility of the cultivars or the plant species attacked (Childers

and Achor 1995, Childers 1997). Damage includes deformation and growth reduction of the

plant, and silver scars on fruits and leaves (Hunter and Ullman 1989, van Dijken et al. 1994, de

Jager et al. 1995). Scars caused by WFT often induce aesthetic injury to fruits and flowers,


7

making them less valuable or even unmarketable (Parrella and Jones 1987). Frequent symptoms

of WFT damage on ornamentals (e.g. rose, gerbera, chrysanthemum, carnation, geranium,

pansy, marigold etc) are streaking, browning, and distortion of leaves, petals and even buds

(Chisholm and Lewis 1984, Oetting et al. 1993, Harrewijn et al. 1996, Childers 1997). Feeding

on fruits by WFT causes scarring, fruit malformation, and russetting. WFT feeding on immature

cucumber causes silvery scarring or malformation (Rosenheim et al. 1990, Shipp et al. 2000a),

minute scarring on immature nectarine fruits which can develop into serious surface russetting

on mature fruits. Fruits with minor feeding damage may be downgraded at sale, but fruits

displaying serious damage are culled (Pearsall 2000). Feeding on the plant foliage by WFT

negatively affects leaf size and photosynthesis, which can cause significant yield loss (Welter et

al. 1990, Shipp et al. 1998, Shipp et al. 2000a). In addition to feeding damage, WFT can cause

yield loss by oviposition. As in all Terebrantia thrips, the female of WFT features a saw-like

ovipositor with which it drills holes into the parenchymal tissues of leaves, flowers and fruits,

where it deposits kidney-shaped opaque eggs (Brødsgaard 1989b). Oviposition injury by WFT

can result in economic loss in some plants, whilst producing no measurable damage to others

(Brødsgaard 1989b). WFT causes a central russet area surrounded by a white halo on apples due

to oviposition injury (Terry and DeGrandi-Hoffman 1988, Terry 1991). This condition is known

as „pansy spot‟. It has also been reported that oviposition injury of WFT causes surface

deformation in avocado fruit (Fisher and Davenport 1989), black dark scars surrounded by halos

of whitish tissue in grape berries [(Jensen 1973) cited in Childers (1997)], and pale halo

surrounded with puncture marks in tomatoes (Salguero Navas et al. 1991). Plant damage is

aggravated in dry conditions, especially under glasshouse conditions when heavily infested

plants lose moisture rapidly, which can seriously reduce yields and sometimes render crops

uneconomic (Lewis 1997a).

Figure 1.3 Damage of WFT on strawberry fruit and flower [Photo by Sonya Broughton and

Paul Horne].

In addition to direct damage, WFT transmits tospoviruses such as Chrysanthemum stem

necrosis virus, Groundnut ringspot virus, Impatiens necrotic spot virus, Tomato chlorotic spot


8

virus and Tomato spotted wilt virus (Whitfield et al. 2005). Of these, TSWV is considered a

serious disease in several economically important crops worldwide (Cho et al. 1988). TSWV

was first described in 1915 in Australia and has been spread worldwide (McDougall and

Tesoriero 2007). Currently, 1090 plants species in 85 families are hosts of TSWV (Parrella et al.

2003). WFT can also cause secondary infections on plants by transmitting pathogenic fungi or

bacteria (Lewis 1973, Lewis 1997b).

Although accurate information is difficult to obtain, WFT is thought to cause billions of dollars

in yield loss year. For example, in Florida annual crop losses due to WFT and Thrips palmi

exceeded US$10 million (Nuessly and Nagata 1995). In the UK, it is estimated that WFT can

cause crop losses of up to US$76,000 ha-1

each year in glasshouse-grown cucumbers (Nuessly

and Nagata 1995). Annual yield loss attributed to TSWV are US$1 billion alone in the USA

(Goldbach and Peters 1994). As a result, growers spend millions of dollars to manage WFT.

Between 1987 to 1990, the Finland Government spent US$390,000 to eradicate WFT from

glasshouse-grown vegetables and ornamentals (Lewis 1997a)].

In this project, strawberry, Fragaria ananassa Duchesne (Rosaceae) was used as the model host

of WFT. In Australia, strawberry is an intensively managed crop cultivated for its fresh,

aromatic, red berries, with a gross value of approximately AUD$308 million per year

(Anonymous 2009a). WFT is considered a major pest of low tunnel- and greenhouse-grown

strawberries and production is often affected by its direct damage (Ullio 2002). Studies have

shown that flowers may provide WFT with essential resources, either by serving as a mating

site (Rosenheim et al. 1990), or as a source of high-quality food (Trichilo and Leigh 1988).

WFT feeding on fruit typically causes direct puncture damage (Tommasini and Maini 1995).

Medhurst and Steiner (2001) suggested that WFT damage contributes to „seediness‟ of fruit

(Figure 1.3), and that this may be responsible for uneven ripening and yield loss, both of which

reduce grower profits (Houlding and Woods 1995). Feeding by WFT on blossoms may also

cause stigmas and anthers to turn brown and wither prematurely (Zalom et al. 2001). Feeding by

WFT causes significant reduction in flower receptacle size in strawberry (Coll et al. 2007).

However, strawberry is not affected by TSWV (Herron et al. 2007).

1.5 Chemical control of western flower thrips and insecticide resistance

Chemical control has become standard practice for WFT management (Contreras et al. 2001).

Considerable laboratory and field studies have focused on the toxicity of insecticides to WFT

(Table 1.2). In Australia, WFT control currently relies on „older chemistry‟ insecticides

including organophosphates (acephate, chlorpyrifos, dichlorvos, dimethoate, endosulfan,


9

malathion, methamidophos, methidathion, pyrazophos), carbamates (methiocarb, methomyl)

and some newer chemistry insecticides (fipronil, abamectin, spinosad). These are made

available to growers under permits issued by the Agricultural Pesticides and Veterinary

Medicines Authority (APVMA) (Herron and James 2005), or through product registration.

Table 1.2 Insecticides that have been tested for efficacy against WFT.

Insecticide IPM

compatibility* Class Reference

Endosulfan Chlorpyrifos Diclorvos Etrimfos Malathion Methamidophos Monocrotophos Omethoate Acephate Naled Sulfotep Formetanate Furathiocarb Methiocarb Methomyl Bendiocarb Fenoxycarb Deltamethrin Λ-cyhalothrin Bifenthrin Cyfluthrin Fluvalinate Fenpropathrin Resemethrin Abamectin* Azadiractin (neem) Nicotine* Beauveria bassiana* Spinosad*

Yes Yes Yes Yes Yes

Organophosphate Organophosphate Organophosphate Organophosphate Organophosphate Organophosphate Organophosphate Organophosphate Organophosphate Organophosphate Organophosphate Carbamate Carbamate Carbamate Carbamate Carbamate Carbamate Pyrethroid Pyrethroid Pyrethroid Pyrethroid Pyrethroid Pyrethroid Pyrethroid Macrocyclic lactone Botanical Botanical Microbial Spinosyn

1, 5, 7, 12, 16 1, 4, 7, 10, 12, 13, 15 4, 5, 6, 7, 8, 18 3 8, 12 1, 2, 4, 5, 7, 14, 16 1, 13 2, 7, 14 20 20 20 4, 7, 9, 10, 14 3 4, 10, 11, 12, 13, 14, 17 1, 5, 7, 19 20 20 2, 8 5, 10, 14 20 20 20 20 20 5, 13, 14, 15 20 20 20 20

[1.Hamrick (1987), 2. Bohmer and Eilenbach (1987), 3. Freuler and Benz (1988), 4. Paiter

(1990), 5. Bournier (1990), 6. Ramakers (1990), 7. Heungens and Butaye (1990), Heungens et

al. (1989), 8. Ribes (1990), 9. Jover et al. (1990), 10. Ferrer et al. (1990), 11. Puiggros et al.

(1990), 12. Devesa and Iberica (1990), 13. Gokkes (1991), 14. Grasselly et al. (1991), 15.

Nasruddin and Smitley (1991), 16. Bohmer et al. (1992), 17. Pasini et al. (1993), 18. Staay and

Uffelen (1988), 19. Heungens (1994)] cited in Tommasini (2003), 20. McDonough et al.

(2009)].*IPM compatibility (Biobest 2009)

Although chemical control has been the primary tool for WFT management (Contreras et al.

2001), because of the small size, secretive habit, high reproductive potential of WFT,

haplodiploidy and improper use of insecticides, WFT has developed insecticide resistance to

several major classes of insecticides (Broadbent and Pree 1997, Jensen 1998, Jensen 2000c,


10

Bielza et al. 2007b, Bielza et al. 2008). It has developed resistance to one or more insecticides in

different part of the world (Helyer and Brobyn 1992, Brødsgaard 1994) including Australia

(Herron and Gullick 1998, Herron and James 2005). In 1961, the first control failure was

reported in Mexico to control WFT on cotton with toxaphene (organochlorine) [Race (1961)

cited in Jensen (2000b)]. However, it was not until 30 years after the first report that Robb

(1989) carried out an experiment to demonstrate insecticide resistance in WFT. Since then, there

have been numerous reports published on the reduction in efficacy of insecticides to control

WFT, indicating the presence of insecticide resistance [for example (Brødsgaard 1991, 1994,

Robb et al. 1995, Zhao et al. 1995c, Zhao et al. 1995b)]. Strains of WFT have developed

resistance to organochlorines, organophosphates, carbamates and pyrethroids and some newer

reduced-risk insecticides (Schreiber et al. 1990, Immaraju et al. 1992, Brødsgaard 1994, Robb et

al. 1995, Broadbent and Pree 1997, Herron and Gullick 1998, Jensen 1998, Jensen 2000c,

Herron and Gullick 2001, Espinosa et al. 2002a, b, Espinosa et al. 2005, Herron and James

2005, Loughner et al. 2005, Ralf Nauen 2005, Maymo et al. 2006, Bielza et al. 2007b, Bielza et

al. 2007a, Bielza et al. 2008, Zhang et al. 2008). Immaraju et al. (1992) reported high levels of

resistance to pyrethroids (permethrin, bifenthrin, abamectin) and moderate-to-high levels of

resistance to methomyl in some populations of WFT. In Europe and Africa, WFT populations

have shown resistance to acephate and endosulfan (organophosphates), and methiocarb

(carbamate) (Brødsgaard 1994). North American WFT populations showed resistance to

diazinon and methomyl (organophosphates), bendiocarb (carbamate) and cypermethrin

(pyrethroid) (Zhao et al. 1995a). In Australia, WFT has developed resistance to almost all

insecticides currently available under permits issued by the APVMA (Herron and James 2005),

except methiocarb and pyrazophos (Herron and James 2005). In Australia, a high level of

resistance to pyrethroids (cypermethrin, bifenthin, deltamethrin and fluvalinate) was detected

and pyrethroids are no longer recommended for use against WFT (Herron and Gullick 2001).

WFT has also been reported to develop resistance to abamectin (Immaraju et al. 1992,

Kontsedalov et al. 1998), endosulfan (Brødsgaard 1994), DDT, imidacloprid and amitraz (Zhao

et al. 1994, Zhao et al. 1995c, Zhao et al. 1995a, b). Pesticide resistance has become more

problematic since cross-resistance has been observed, for example resistance to methiocarb

exists in populations of WFT that have never been exposed to methiocarb (Brødsgaard 1994,

Jensen 2000a). In addition, some of these insecticides have either direct (e.g. mortality) or

indirect (oviposition, longevity and predation) adverse effects on beneficials resulting in

pest resurgence (Blümel et al. 1999).

Increasing problems with resistance, the availability of insecticides and high cost of chemicals

usage, environmental and health risk and adverse effects on natural enemies have challenged us

to develop ways of using insecticides or new insecticide that can be effective against WFT with


11

no or minimal effect on natural enemies (Zhang and Sanderson 1990, Jensen 2000c, Shipp et al.

2000b, James 2002, Jones et al. 2005), For example, Chapman et al. (2009) experimented with

the release of Trichogramma osttiniae and the use of bio-rational insecticides (spinosad,

indoxacarb and methoxyfenozide). They found that these two tactics provide an

environmentally sound approach to managing Ostrinia nubilalis (Hübner) (Lepidoptera:

Crambidae) in bell peppers, due, in part, to the conservation of generalist predators. Kraiss and

Cullen (2008) evaluated the efficiency of pyrethrin, insecticidal soap and mineral oil (known as

reduced-risk insecticides) and the predatory coccinellid, Harmonia axyridis (Pallas)

(Coleoptera: Coccinellidae), for the management of soybean aphid, Aphis glycine Matsumura

(Hemiptera: Aphididae) on North American soybean (Glycine max (L.)). They found that these

chemicals could be used for the management of A. glycine in the presence of H. axyridis.

The novel insecticide spinosad (Dow AgroSciences USA) is a macro-cyclic lactone bio-

insecticide (Elzen et al. 1998b, a, Ludwig and Oetting 2001). Due to its effective use rate,

safety to the environment, mammals and beneficials, the Environmental Protection Agency

(USA) classified spinosad as an environmentally and toxicologically reduced-risk chemical

(Saunders and Bret 1997). Spinosad is efficacious against several thrips species (Cloyd and

Sadof 2001) including WFT (Eger Jr. et al. 1998, Funderburk et al. 2000), and marketing has

focused on its favourable environmental profile, emphasising its potential for use in IPM

systems (Thompson and Hutchins 1999, Thompson et al. 2000). Because of its effectiveness

and safety, spinosad was also awarded the „Presidential Green Chemistry Challenge Award” in

1999 (Anonymous 2009b). Copping (2001) in his “Biopesticide manual” reported that spinosad

shows no effects on predatory insects such as ladybirds, lacewings, big-eyed bugs or minute

pirate bugs. According to the International Organisation for Biological Control (IOBC), the

recommended rate of spinosad is harmless to most predatory arthropods and has been reported

to be compatible with predatory mites, which are extensively used biocontrol agents against

WFT and spider mites (Bret et al. 1997, Williams 2001, Miles et al. 2003, Williams et al. 2003,

Anh et al. 2004, Kim et al. 2005, van Driesche et al. 2006a). Similarly, Bret et al. (1997)

reported that spinosad is non-toxic to natural enemies including Orius spp., Chrysopa spp.,

coccinellids, and predaceous mites. Holt et al. (2006) tested the compatibility of spinosad and

the predatory mite Phytoseiulus persimilis Athias-Henriot (Phytoseiidae) for the control of two-

spotted spider mites on ivy geraniums and found that it can be used in conjunction with

spinosad, causing no obvious detrimental effects to this predator. However, Cote et al. (2004)

reported that toxicity of spinosad against natural enemies as variable. van Driesche et al.

(2006a) report that direct toxicity testing using fresh residues (2h) of the recommended rate of

spinosad for WFT on greenhouse flower crops had no toxic effect on the mite Neoseiulus

cucumeris (Oudemans), but lowered the survival rate of Iphiseius degenerans (Berlese).


12

Unfortunately, WFT strains have developed resistance to spinosad in some parts of the world

(Loughner et al. 2005, Bielza et al. 2007b, Bielza et al. 2007a, Zhang et al. 2008) including

Australia (Herron and James 2005). In August 2008, Dow AgroSciences voluntarily suspended

the use and sale of multiple spinosyn insecticides from two counties of Florida for 12 months as

WFT has developed resistance (Anonymous 2008). Although spinosad resistance in WFT has

been detected in some parts of the world, it still appears to be a promising insecticide,

particularly since it can be combined with biological control (Jensen 2000c).

1.6 Biological control of western flower thrips

Use of biological control agents for the management of arthropod pests dates back to the 12th

century when farmers in the Oriental region used ants to protect fruit trees from pests (Thacker

2002). Natural enemies have been incorporated into IPM strategies of commercial glasshouses

for WFT management with varying degrees of success (Chambers and Sites 1989, Gillespie

1989, Gilkeson 1990, Brødsgaard 2004b, Shipp and Ramakers 2004). Several species of natural

enemy have been reported to attack WFT (Table 1.3) (van Driesche et al. 1998), including

predators (Riudavets 1995, Sabelis and Van Rijn 1997), parasitoids (Loomans et al. 1997),

entomopathogens, particularly fungi (Butt and Brownbridge 1997) and nematodes (Loomans et

al. 1997).

1.6.1 Thrips predators

Many arthropods are known to be predators of thrips and belong to several families under orders

Hemiptera, Diptera, Neuroptera, Coleoptera, Araneida, and some species of Thysanoptera

(Lewis 1973, Ananthakrishnan 1979). However, the most widely employed predators are

anthocorid bugs of the genus Orius (Anthocoridae) and phytoseiid mites (Acari) (Riudavets

1995, Sabelis and Van Rijn 1997). The genera Orius Wolf, Anthocoris Fallen, Montandoniola

Poppius, Xylocoris Dufour and Scoloposcelis Fieber are predators of several thrips species

(Ananthakrishnan and Sureshkumar 1985) in a wide range of field crops, tree crops and

ornamentals (Herring 1966) in temperate and Mediterranean areas of North America and

Europe. Many Orius species can be effective predators of WFT (Sabelis and Van Rijn 1997)

and have been used in greenhouse-grown capsicum, cucumber, and eggplant (Gilkeson 1990,

van den Meiracker and Ramakers 1991, Chambers et al. 1993, Bolckmans and Tetteroo 2002) in

Europe and the USA. Among Orius spp., substantial studies have been made on O. tristicolor

(White), O. insidiosus (Say), O. majusculus (Reuter), O. niger (Wolff), O. laevigatus (Fieber)

and O. albidipennis (Reuter) (Gilkeson et al. 1990, van den Meiracker and Ramakers 1991,

Dissevelt et al. 1995, Rubin et al. 1996, Brown et al. 1999, Funderburk et al. 2000,


13

Ramachandran et al. 2001, Deligeorgidis 2002, Ludwig 2002, Shipp and Wang 2003,

Tommasini 2003, Brødsgaard and Enkegaard 2005, Chow et al. 2008). Orius spp can be found

on several cultivated and non-cultivated plant species and appear to prey on all different thrips

stages (Ramakers 1990, Sabelis and Van Rijn 1997). However, the Orius population and its

Table 1.3 Natural enemies evaluated as biocontrol agents of WFT (van Driesche et al. 1998).

Natural enemies Generalist/

Specialist Cropping

system Mass production

Predators Heteroptera

Orius spp. Anthocoris nemorum Geocorus spp. Nabis spp. Dicyphus spp. Macrolophus spp.

Thysanoptera (predatory thrips) Aeolothrips spp.

Acari (predatory mites) Amblyseius / Neoseiulus spp. Hypoaspis spp. Typhlodromips montdorensis Iphiseius degenerans

Various predators Neuroptera, Diptera

Parasitoids Ceranisus spp.

Pathogens (nematodes, fungi) Steinernema spp. Heterohabditis spp. Thripinema spp. Verticillium lecanii Beauveria bassiana Metarhizium anisopliae Paecilomyces fumosoroseus

Generalist Generalist Generalist Generalist Generalist Generalist Generalist Generalist Generalist Generalist Generalist Specialist Generalist Generalist Unknown Generalist Generalist Generalist Generalist Generalist

GH/Field GH Field Field GH/Field GH Field GH GH/Field GH GH/Field GH/Field GH/Field GH GH GH/Field GH GH GH/Field GH

Some species

available expensive Possible, expensive Not yet developed Not yet developed Not yet developed Possible, reasonable Not yet developed Some available Possible, cheap Possible, reasonable Available Available Some available Difficult Possible, cheap Possible, cheap Not yet developed Possible, cheap Possible, cheap Not yet developed Not yet developed

*GH = Glasshouse

effectiveness are strongly influenced by prey type, density and environmental factors, especially

temperature (Riudavets 1995). Moreover, some species of Orius are injurious to plants since

they probe plant tissue for moisture, or oviposit on growing tips (Riudavets 1995). In addition,

Orius spp. often leave the crop or glasshouse (Riudavets 1995).

Geocoris pallens (Stal) and G. atricolor Montandon (Anthocoridae) have been described as

predators of WFT (Benedict and Cothran 1980, Riudavets 1995). However, Geocoris spp. are

omnivorous and frequently consume plant juices (Benedict and Cothran 1980, Yokoyama


14

1980). Adults and larvae of Nabis alternatus (Parshley) and N. americoferus (Carayon)

(Nabidae) appear to predate on WFT in alfalfa and bean (Benedict and Cothran 1980). In the

Miridae, Deraecoris tamaninii Wagner and Macrolophus caliginosus Wagner are considered to

be WFT predators (Riudavets et al. 1993b, a, Riudavets 1995). Some thrips species in the genus

Aeolothrips (Thysanoptera), A. fasiatus L. and A. intermedius Bagnall (Lacasa 1980, Baker

1988) cited in Tommasini (2003)] predate on WFT.

1.6.2 Predatory mites

Worldwide, considerable research has focused on the effectiveness of predatory mites (Acari)

against insect and mite pests (Chant 1985, Sengonca and Bendiek 1988, McMurtry and Croft

1997, Sabelis and Van Rijn 1997, Brown et al. 1999, Williams 2001, Berndt 2003, Weintraub et

al. 2003, Amano et al. 2004, Berndt et al. 2004a, Berndt et al. 2004b, Colfer et al. 2004,

Wiethoff et al. 2004, Jones et al. 2005, Cakmak et al. 2006, Ebssa et al. 2006, Holt et al. 2006,

Khan and Morse 2006, Kongchuensin and Takafuji 2006, Rhodes and Liburd 2006, van

Driesche et al. 2006b, Fitzgerald et al. 2007, Gerson and Weintraub 2007, Ochiai et al. 2007).

Biological control of thrips started with observations of predatory mites preying on Thrips

tabaci (Lindeman) in greenhouse crops [Woets (1973) cited in Messelink et al. (2006).

However, the first deliberate attempt to control a thrips population was with releases of

Neoseiulus barkeri (Hughes) (= Amblyseius mckenziei) (Ramakers 1980). However, the

introduction of another indigenous (North European) species, Neoseiulus cucumeris

(Oudemans), was more successful (de Klerk and Ramakers 1986). Thereafter, several predatory

mite species have been used in WFT management programs in field and greenhouse crops

(Table 1.4).

Successful control of WFT with predatory mites varies (Chambers and Sites 1989, Gillespie

1989, Gilkeson 1990, Brødsgaard 2004b, Shipp and Ramakers 2004). Several reasons seem to

attribute to the varying degree of success. Firstly, unlike Orius or other thrips predators,

predatory mites either prey on larval (e.g. N. cucumeris) or pupal (e.g. H. miles) stages.

Secondly, the effectiveness of predatory mites is host-plant-dependent. Brown et al. (1999)

reported that the efficacy of N. cucumeris and I. degenerans varied on host plants [(plants:

Justicia adhatoda, Saurauia nepaulensis DC., Clivia miniata Lindl., Crambe strigosa L‟Her.,

Greyia radkloferi Szyszyl, Dietes bicolour Steud., Crotalaria capensis Jacq., Tephrosia

grandiflora Pers., Peumus boldus Molina, Limonium spectabile (Svent.), Photinia mussia L.,

Capsicum annuum L., Dombeya acutangula Cav.]. Riudavets (1995) stated that control of WFT

by N. cucumeris was lower on cucumber and eggplant compared to capsicum (sweet pepper).

Thirdly, some pesticides applied to control other pests are often harmful to predatory mites


15

(Hassan et al. 1987, Hassan et al. 1988, Kim and Paik 1996, Kim and Seo 2001, Mochizuki

2003, Amano et al. 2004). Fourthly, environmental factors such as high or low temperature,

humidity and short photoperiods attribute to the effectiveness of predatory mites (Malezieux et

al. 1992, Shipp and van Houten 1997, Kiers et al. 2000, Shipp et al. 2000a, Jacobson et al.

2001). Apart from these factors, WFT control by predatory mites might also fail if WFT

densities are very high. Therefore, pesticide applications may be necessary to reduce thrips

populations and these may be detrimental to predatory mites (Malezieux et al. 1992).

Table 1.4 Crops and predatory mites used for WFT control (Gerson and Weintraub 2007).

Crop Predator Reference

Beans Chrysanthemum

Cucumber

Cyclamen Gerbera Impatiens Pepper

Rose Strawberry Tomatoes

Hypoaspis (Geolaelaps) aculeifer Neoseiulus cucumeris Oudemans Stratiolaelaps scimitus (Womersley) H. aculeifer N. cucumeris Amblyseius swirskii Athias-Henriot S. scimitus H. aculeifer N. cucumeris Typhlodromips montdorensis Schicha N. cucumeris N. cucumeris A. swirskii Iphiseius degenerans Berlese A. andersoni Chant N. cucumeris T. montdorensis N. cucumeris

Berndt et al.(2004a) Skirvin et al. (2006) Bennison et al. (2002a) Messelink et al. (2005) Wiethoff et al. (2004) De Courcy Williams (2001) Steiner and Goodwin (2002) van Driesche et al. (2006b) van Houten et al. (2005)

Vänninen and Linnamäki

(2002) Steiner (2002) Shipp and Wang (2003)

1.6.3 Thrips parasitoids

Very little is known about WFT parasitoids. Three chalcid genera Ceranius, Goetheama,

Thripobius (Hymentoptera: Chalcidoidea) and a eulophid Entedonastichus (Eulophidae:

Entodontinae) are known to parasitise certain thrips species (Loomans 2003). Only two species

of Ceranisus: C. menes (Walker) and C. americensis (Girault) are known to attack and develop

on WFT (Loomans et al. 1993, Loomans and van Lenteren 1996, Loomans 2003). However, C.

menes and C. americensis showed very limited searching efficiency and low parasitisation rates

when released against WFT on roses in greenhouses in the Netherlands (Loomans et al. 1995).

Entomopathogenic nematodes (EPNs) in the families Steinernematidae and Heterorhabditidae

are potential biological control agents of thrips (Chyzik et al. 1996, Ebssa et al. 2001, Ebssa et

al. 2004a, Ebssa et al. 2004b, Belay et al. 2005, Ebssa et al. 2006). The genus Thripinema (=


16

Howwardula) (Tylenchida: Allantonematidae) has been reported to parasitise several species of

thrips (Sharga 1932, Nickle and Wood 1964, Greene and Parrella 1993, Tipping et al. 1998). T.

nicklewoodi (Siddiqi) is the most abundant pupal parasite of WFT used in commercial

greenhouses and is able to maintain WFT populations below economic thresholds (Greene and

Parrella 1993, Arthurs and Heinz 2002, Mason and Heinz 2002, Smith et al. 2005). Thripinema

nicklewoodi is abundant in greenhouse-grown ornamentals and appears to attack WFT in

confined areas (Greene and Parrella 1993, Mason and Heinz 2002). Despite its effectiveness, a

commercial product of T. nicklewoodi is not presently available (Arthurs et al. 2003).

Temperature also has a significant effect on the success of T. nicklewoodi, and it is most

successful between 10 and 20⁰C (Arthurs et al. 2003). Another nematode Steinernema feltiae

Filipjev (commercially available in USA) has variable success with control of WFT (Chyzik et

al. 1996, Arthurs and Heinz 2006).

1.6.4 Fungi

The fungal species, Zoophthora radicans (Brefeld), Neozygites parvispora (Macleod and Carl)

(Keller and West 1983); Entomophthora thripidum Samson (Samson et al. 1979,

Ananthakrishnan 1993); Beauveria bassiana Bals. (Dyadechko 1964, Lipa 1985,

Ananthakrishnan 1993), Verticillium lecanii (Zimm.) (Nedstam 1991), Metarhizium anisopliae

(Metchnikoff), Paecilomyces fumosoroseus (Wize) have all been reported as pathogens of WFT.

Mycotal, a product containing V. lecanii is now available in Europe for the control of WFT

(Parker 2006). Metarhizium anisopliae, B. bassiana, Lecanicillium muscarium, and

Trichoderma viride can provide control of WFT in greenhouse vegetables and ornamental crops

(Brownbridge 1995, Butt and Brownbridge 1997, Bradley et al. 1998, Shipp et al. 2002, Ugine

et al. 2005, 2007, Gouli et al. 2008). However, a significant drawback of using pathogenic fungi

is that they are effective only at narrow temperate regimes (Inglis et al. 1997, Inglis et al. 1999).

1.6.5 Biological control of western flower thrips in Australia

Several Orius species have been found to predate on WFT (Cook 2000). Orius armatus Gross

appeared to have a significant impact on WFT in the field (Steiner and Goodwin 2000) and was

available in late 2009 for commercial use. Phytoseiid mites have shown some potential for

controlling WFT in Australia (Steiner and Goodwin 2000). This includes the native

Typhlodromips montdorensis (Schicha) (Figure 1.2B) and Typhlodromus occidentalis (Nesbitt)

(Phytoseiidae) (Steiner and Goodwin 2000), both of which have been trialled in greenhouses. T.

montdorensis occurs on a wide range of plants including Ageratum sp., cucumber, Datura sp.,

Eucalyptus sp., strawberry, tomato, Mucuna sp., Oxalis sp., purple bean, green bean, Sechium


17

edule (Choko) and Sida acuta (Schicha 1979)Steiner et al. (2003). Typhlodromips montdorensis

is widely distributed in unforested coastal areas of Queensland and in irrigated and higher

rainfall areas including Biloela and the Atherton Tablelands. It has been reported from New

South Wales, South Australia and the Northern Territory (Goodwin and Steiner 1996), and is

currently used for WFT and spider mite management (Steiner and Goodwin 1998). Another

indigenous species Typhlodromus lailae (Schicha), also appears to be promising candidate for

development as a commercial biocontrol agent for WFT (Steiner et al. 2003). Worldwide the

most widely employed predatory mite, N. cucumeris (Figure 1.2C) was recently confirmed as

occurring in Australia (Steiner and Goodwin 2000), and is being reared commercially in South

Australia and Western Australia. Hypoaspis (= Stratiolaelaps ) miles (Berlese) (Laelapidae)

(Figure 1.2D) has been available on the Australian market for some time. Although H. miles is

primarily used for fungus gnat control, it is effective against WFT (L. Chilman, Manchil IPM

Service, Pers. Comm.). Little is known about the parasitoids or pathogens present in Australia

that would be suitable for the control of WFT (Steiner and Goodwin 2000).

1.7 Host-plant resistance

Host-plant resistance plays an important role in IPM programs [Smith (1990) cited in Parrella

and Lewis (1997)] and is considered a key method for pest control on crops with low economic

thresholds (Nothnagl 2006). The quality of host plants and their availability plays an important

role in the life cycle of phytophagous insects (White 1969, Wellington 1977, White 1993).

Plants vary in their suitability as WFT hosts between species as well as within species (i.e.

cultivars) and, as a result, larger infestations occur on some plants than on others (Krik 1997). In

addition, development of plant resistance to tospoviruses indirectly reduced direct damage

caused by WFT (Ullman 1996). Studies indicate that varietal differences play an important role

in WFT management (de Jager et al. 1993, van Dijken et al. 1994, de Jager et al. 1995, de Kogel

et al. 1997a, de Kogel et al. 1998, Ohta 2002). Variation in varietal resistance to WFT has been

described in several crops including cotton (Trichilo and Leigh 1988), Chrysanthemum (de

Jager et al. 1993, van Dijken et al. 1994, Ohta 2002), rose (Gaum et al. 1994), pepper (Maris et

al. 2003, Maris et al. 2004), tomato (Kumar et al. 1995), cucumber (Mollema et al. 1995, Soria

and Mollema 1995), strawberry (Toshio 2004), cabbage (Stoner and Shelton 1986) and

groundnut (Robb and Parella 1989). Ananthakrishnan and Gopichandran (1993) reported that

leaf plant morphology such as trichome length and width, density of glandular hairs, the

thickness of the leaf cuticle and waxes can have a profound effect on plant acceptability. Brown

and Simmonds (2006) mentioned that leaf surface undoubtedly plays a role in the selection and

preference of host plants by herbivores. Highly pubescent foliage has been observed to lower

the infestation levels of thrips on cultivars of cotton (Rummel and Quisenberry 1979). The


18

presence of trichomes on leaf surfaces can provide complex mechanisms enabling the plant to

evade thrips infestation (Brown and Simmonds 2006). Trichomes can bioactivate secondary

products that may be used by the plant as a chemical method of defence against herbivores

(Wagner 1991, Bisio et al. 1999, Roda et al. 2003).

1.8 Cultural methods of control of western flower thrips

Several cultural practices may reduce injury by WFT. Mechanical barriers such as mesh,

netting, and plastic sheets (mulch) can contribute to WFT control (Broadbent 1969, Cohen and

Marco 1979, Harpaz 1982). Yudin et al. (1991) studied the effect of aluminium polyvinyl

netting on WFT numbers in a field of lettuce crop. Berlinger et al. (1993) found that commercial

woven screens can reduce the entrance of WFT into greenhouses. Sticky traps can also be used

for mass trapping of thrips (Murai 1988, Brødsgaard 1989a, 1993a, b). The use of plastic

mulches is a standard cultural practice in many parts of the world. Mulch provides several

benefits, including improved retention of irrigation water and soil moisture, conservation

of soil applied fertilisers, modulation of soil temperatures, and weed suppression (Terry

1997). Ultra violet (UV) reflective silver mulches have been shown to reduce thrips

colonization onto tomato (Scott et al. 1989, Greenough et al. 1990, Brown and J. E. Brown

1992, Kring and Schuster 1992) and pepper (Vos et al. 1995, Reitz et al. 2003). Other

cultural practices include the push-pull strategy (Bennison et al. 2002b, Cook et al. 2007),

trap/companion crops and intercropping (Bennison et al. 2002b, Matsuura et al. 2005,

Kasina et al. 2006, Buitenhuis et al. 2007), habitat manipulation (Nicholls et al. 2000,

Groves et al. 2001), irrigation and fertiliser manipulation (Schuch et al. 1998, Chau et al.

2005, Davies Jr. et al. 2005, Chau and Heinz 2006). All can significantly affect the

population dynamics of WFT and associated diseases. However, while some of these

methods have been readily adopted by growers overseas, this is not the case in Australia.

1.9 IPM programs for control of western flower thrips

Increasing problems with resistance, availability and the high cost of chemicals usage,

environmental and health risks, adverse effects on natural enemies and the ineffectiveness of

other control strategies have challenged growers to adopt new management strategies (Jensen

2000c, James 2002). As an IPM approach, a combination of cultural, biological, and chemical

control appears to be effective for the management of WFT in several crops. For example,

Momol et al. (2004) demonstrated that the combined use of spinosad, actigard and high UV-

reflective aluminium mulch effectively reduced TSWV transmitted by WFT as much as 76%,

compared to untreated black plastic mulch. Since 1997, Florida pepper growers have been using


19

reduced-risk insecticides, O. insidiosus and UV-reflective mulch for the management of WFT

(Funderburk et al. 2000, Ramachandran et al. 2001, Reitz et al. 2003). Laboratory and

greenhouse experiments of Thoeming and Poehling (2006) illustrated that an integrated

approach of soil application of azadirachtin (botanical, neem product) integrated with predatory

mites, A. cucumeris and H. aculeifer, is consistently effective against WFT on bean (Phaseolus

vulgaris L.) and resulted in efficacies up to 99% in controlling WFT. Soil drenching of neem

formulation systemically affects the plant sucking life stages, directly affects the soil-dwelling

stages of WFT, while N. cucumeris predate on the larval stage and H. aucleifer predate on the

prepupal and pupal stage.

Where multiple biological control agents may be used, it is important to not only

evaluate the impact of insecticides on them, but also how they interact with each other.

Studies have shown that the timely application of predatory mites and localised application of

anthocorid bugs have been very effective against WFT in glasshouse-grown sweet pepper and

cucumber (Jacobson 1997, Shipp and Wang 2003, Skirvin et al. 2006). In greenhouse-grown

chrysanthemum, a combination of A. cucumeris, Orius sp and V. lecanii has proven to be

effective against WFT (Jacobson 1997). Research has shown that a formulation based on B.

bassiana, M. anisopliae and L. muscarium can reduce WFT significantly in greenhouse-grown

vegetables and floral crops (Brownbridge 1995, Butt and Brownbridge 1997, Bradley et al.

1998, Shipp et al. 2002, Ugine et al. 2005, 2007, Gouli et al. 2008). Premachandra et al. (2003)

evaluated the combined application of biocontrol agents and found that the combined use of

entomopathogenic nematodes, Heterorhabditis bacteriophora Poinar (Rhabditida:

Heterorhabditidae), Steinernema feltiae Filipez (Rhabditida: Steinernematidae) and predatory

mite, H. aucleifer can be effective for WFT management.

The use of multiple natural enemies species in pest management programs has been a

controversial issue for a long time because of interspecific competition (DeBach and Rosen

1991, Bellows and Hassell 1999). As an integrated approach, there is a growing trend to use two

or more species of natural enemies to suppress insect pest populations (Premachandra et al.

2003, Avilla et al. 2004, Blümel 2004, Brødsgaard 2004a, Chau and Heinz 2004, Chow and

Heinz 2004, Hoddle 2004, Shipp and Ramakers 2004, Thoeming and Poehling 2006, Chow et

al. 2008). Anthocorid bugs of the genus Orius and phytoseiid mites of the genus Amblyseius are

commonly used for the control of WFT in greenhouse-grown crops in Europe and North

America (Brødsgaard 2004a, Shipp and Ramakers 2004), though resulting benefits for pest

suppression were not quantitatively validated (Blockmans and Tetteroo 2002, Skirvin et al.

2006). Some studies support the premise of natural enemy compatibility (Gillespie and Quiring

1992, Wittmann and Leather 1997, Brødsgaard and Enkegaard 2005), whilst others are opposed


20

(Magalhăes et al. 2004, Sanderson et al. 2005). Schausberger and Walzer (2001) demonstrated

that interspecific competition may occur when different species of predatory mites are combined

together and prey specificity affects the quality and intensity of predator-predator interactions.

Others such as Schausberger and Walzer (2001), report that in perennial greenhouse-grown

crops, releases of P. persimilis and N. californicus may have complementary effects, and could

enhance the biological control of arthropod pests.

In Australia, insecticide applications and the augmentative release of predatory mites are used

for WFT management in horticultural crops. However, this approach is not always effective in

managing WFT. Moreover, insecticide resistance to WFT is a growing problem in Australia

(Herron and James 2005). In order to effectively manage WFT and slow down (if not eliminate)

resistance, there is a need to explore strategies that involve the harmonious integration of

chemical, biological and other control strategies.

1.10 Outline of this study

The aim of this project was to develop an integrated pest management program for the control

of WFT using a range of tactics including resistant varieties, biological control and insecticide.

Strawberry [Fragaria ananassa L. (Rosaceae)] was used as the model host of WFT. In

Australia, strawberry is an intensively managed crop cultivated for its fresh, aromatic, red

berries, with a gross value of approximately AUD$308 million (Anonymous 2009a). WFT is a

major pest of low tunnel-, greenhouse- and field-grown strawberries and production is often

affected by direct feeding damage (Ullio 2002). Studies have shown that flowers may provide

WFT with essential resources, either by serving as a mating site (Rosenheim et al. 1990), or as a

source of high-quality food (Trichilo and Leigh 1988). Feeding by WFT on blossoms may cause

stigmas and anthers to turn brown and wither prematurely (Zalom et al. 2001), or a significant

reduction in flower receptacle size (Coll et al. 2007). WFT feeding on fruit typically causes

direct puncture damage (Tommasini and Maini 1995). Medhurst and Steiner (2001) suggest that

WFT damage contributes to the „seediness‟ of fruit.

The first section of the present study examined the influence of strawberry cultivars on WFT.

The literature suggested that the host-plant resistance plays an important part in the effective

management of insect pest populations, especially in crops where low damage is required (de

Kogel et al. 1998, Schoonhoven et al. 1998). Studies have indicated that varietal differences

plays an important role in WFT management (de Jager et al. 1993, van Dijken et al. 1994, de

Jager et al. 1995, de Kogel et al. 1997b, de Kogel et al. 1998, Ohta 2002). In Australia, more

than 20 strawberry cultivars are grown. The aim was to explore if strawberry cultivars influence


21

feeding and oviposition of WFT. In the present study, it was not possible to evaluate all

strawberry cultivars for varietal susceptibility to WFT. Instead, three commonly grown

cultivars, Camarosa, Camino Real (Short-day), and Albion (day-neutral) were tested. These

cultivars were selected because they are grown throughout Australia.

The remainder of the study explores the possibilities of integrating an IPM-compatible

insecticide, spinosad, and three predatory mites, Typhlodromips montdorensis (Schicha)

(Phytoseiidae), Neoseiulus cucumeris (Oudemans) (Phytoseiidae) and Hypoaspis miles

(Berlese) (Laelapidae) [available in the Australian market for WFT control] as well as effective

combinations of these predatory mites. Spinosad is a novel pesticide derived from fermentation

of actinomycete Saccharopolyspora spinosa Mertz and Yao. It is a bacterial organism first

isolated from a Caribbean soil sample (Sparks et al. 1998), is classified as an environmentally

and toxicologically reduced-risk chemical (Cleveland et al. 2002, Thompson et al. 2002) and

considered as IPM compatible (Thompson and Hutchins 1999, Thompson et al. 2000).

Laboratory and field evaluation of the selectivity of spinosad indicates that it is less toxic to

natural enemies including predatory mites than to their prey (Bret et al. 1997, Miles and Dutton

2000, Thompson et al. 2000, Medina et al. 2001, Holt et al. 2006, Kim et al. 2006, Arthurs et al.

2007). However, Cote et al. (2004) reported toxicity results of spinosad against natural enemies

as variable. For example, Villanueva and Walgenbach (2005) found it is highly toxic to

Neoseiulus fallacies (Garman). Moreover, WFT has developed resistance to spinosad in some

locations (Herron and James 2005, Bielza et al. 2007b). Typhlodromips montdorensis, N.

cucumeris and H. miles appear to be effective against WFT. However, these mites may not be

very effective alone. Moreover, T. montdorensis predate on first and second instar and most

effective at average temperatures over 20ºC and require high humidity (Steiner and Goodwin

2002, Hatherly et al. 2004). While N. cucumeris predate only the first instar of WFT and their

predation rate is often influenced by alternative food like pollen, which hamper its predation

efficiency. Hypoaspis miles is a soil-dwelling predatory mite that predate on the prepupal and

pupal stages only. Despite resistance to spinosad and spinosad toxicity on some predatory mites,

the literature suggested that the integration of spinosad and predatory mites could be a

promising management tactic for WFT in horticulture crops. The aim was to elucidate the

compatibility of spinosad and T. montdorensis, N. cucumeris and H. miles and measure the

effectiveness against WFT, which in turn might slow down the development of insecticide

resistance.

This study assessed more effective methods to release biological control agents in conjunction

with insecticide applications to improve the control of WFT. The use of different predatory

species singly and in combination against WFT was evaluated. Natural enemies currently


22

available for the biological control of WFT forage either on upper plant parts (e.g. N.

cucumeris) or in the soil (e.g. H. miles) (Berndt et al. 2004a) and it was hypothesised that

combinations of predatory mite species would improve the management of WFT. In the course

of this study, differential population growth of WFT on different strawberry varieties was

observed. Therefore, the application of pesticides and predators was evaluated on different

strawberry varieties.

Thus, this thesis has the following specific objectives:

(i) To determine feeding and oviposition preference and performance of WFT on strawberry

cultivars.

(ii) To evaluate the effectiveness of a combination of cultivars, spinosad and predatory mite

for the management of WFT in glasshouse and low tunnel-grown strawberry.

(iii) To investigate the effectiveness of the combined application of predatory mite and

spinosad for management of spinosad resistant WFT.

(iv) To evaluate the toxicity of spinosad against predatory mite.

1.11 Brief organisation and structure of this thesis

In chapter II, the feeding and oviposition preferences of WFT on strawberry cultivars are

evaluated using a choice-test method. Oviposition rate, survival, incubation period, larval period

and pupal period associated with different strawberry cultivars are also determined.

In chapter III, an integrated management strategy against WFT in greenhouse-grown

strawberry is investigated. This approach included the use of different strawberry cultivars,

insecticide (spinosad) and predatory mite releases (T. montdorensis, N. cucumeris and H. miles).

In chapter IV, impact of single versus multiple releases of predatory mites against WFT in

greenhouse-grown strawberries is determined. This approach includes the use of insecticide

(spinosad) and single or multiple releases of predatory mites (T. montdorensis, N. cucumeris

and H. miles).

In chapter V, a field study that explores the compatibility of the strategy used in glasshouse

(Chapter IV) for the management of WFT is investigated.

In chapter VI, the toxicity of spinosad to predatory mites (T. montdorensis, N. cucumeris and

H. miles) is tested via a series of bioassays.


23

In chapter VII, the effectiveness of an IPM management protocol that combines the use of

spinosad (bioinsecticide) with predatory mite releases (T. montdorensis, N. cucumeris and H.

miles) is assessed against a spinosad-resistant WFT population.

Chapter VIII, discusses this body of work and how it can be used to manage WFT in

strawberry grown in glasshouses and low tunnels.

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CHAPTER II

Variation in preference and performance of western flower thrips, Frankliniella

Occidentalis (Pergande) (Thysanoptera: Thripidae) on three strawberry [Fragaria x

ananassa Duchesne (Rosaceae)] cultivars

Keywords: Frankliniella occidentalis, feeding, oviposition, preference, performance,

developmental period, survival rate, strawberry

Abstract

Western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) is

considered a major pest of strawberry, Fragaria x ananassa Duchesne (Rosaceae), causing

substantial yield loss through direct feeding on flowers and fruit. Frankliniella occidentalis

damage can be influenced by host-plant resistance or tolerance. This study determined whether

three commercial strawberry cultivars (Albion, Camarosa, and Camino Real) differed in their

suitability to F. occidentalis. Determination of resistance of strawberry cultivars to F.

occidentalis was based on the level of olfactory response, feeding damage, ovipositional

preference and host suitability for reproduction. Frankliniella occidentalis adults preferred to

feed on Camarosa compared to Albion and Camino Real. However, if leaves had previously

been fed on by conspecifics, there was no difference in feeding preference. Camarosa was the

most preferred cultivar for oviposition. More eggs were laid by WFT on Camarosa than either

on Albion or Camino Real. More larvae hatched and adults emerged from Camarosa than either

Albion or Camino Real. The percentage of unhatched eggs, larvae and pupae that died was

highest on Camino Real. The survival rate was highest and lowest on Camarosa and Camino

Real respectively. The egg incubation, prepupation, pupation and total developmental periods

(egg to adult) was shorter on Camarosa than on Albion or Camino Real. The larval period was

longer on Camarosa (4.43 ± 0.12 days) than Albion (4.26 ± 0.16 days) and Camino Real (4.06 ±

0.14 days). Overall, the data indicate that Camarosa was most favourable for WFT population

growth. The other cultivars may be a better choice for WFT management.

2.1 Introduction

Western flower thrips (WFT), Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae),

is a serious pest of agricultural and horticultural crops worldwide. Although, it was first

discovered in 1895 in California, USA (Pergande 1895), during 1970s, WFT has spread

worldwide, causing substantial damage to a wide range of crops . (Lewis 1973, Shipp et al.

Chapter II: Preference and performance

44

1991, Robb and Parrella 1995, Tommasini and Maini 1995, Lewis 1997a, b, 1998, Kindt et al.

2003, Kirk and Terry 2003). In Australia, WFT was first recorded in 1993 on chrysanthemum in

a glasshouse in Yangebup, Western Australia (Malipatil et al. 1993). Since then it has spread to

all states except the Northern Territory (Medhurst and Swanson 1999). In Australia, WFT is

considered an economic pest of ornamentals, vegetables and fruits (Persley et al. 2009).

Strawberry, Fragaria ananassa Duchesne (Rosaceae) is an intensively managed crop cultivated

for its fresh, aromatic, red berries. In Australia, the strawberry industry has grown steadily over

the last few years, with a gross value of approximately AUD$308 million per annum

(Anonymous 2009). However, strawberry production is affected by direct damage caused by

WFT (Cook 2000, Ullio 2002, Steiner and Goodwin 2005) and WFT is considered a major pest

in greenhouse-, low tunnel- and open field-grown strawberries (Coll et al. 2007). WFT feeding

on strawberry flowers and fruits causes fruit deformation, uneven ripening, early withering of

stigmas and anthers, and reduction in flower receptacle size leading to yield loss (Buxton and

Easterbrook 1988, Houlding and Woods 1995, Medhurst and Steiner 2001). Chemical control of

WFT is considered the principal control strategy (Broughton and Herron 2009). However,

efficient chemical control of WFT is difficult because of their cryptic feeding behaviour, high

mobility, soil-dwelling life stages, and short generation time combined with high fertility

(Jensen 2000b). In addition, WFT has developed resistance to all major insecticide classes in

different parts of the world (Helyer and Brobyn 1992, Immaraju et al. 1992, Brødsgaard 1994,

Zhao et al. 1994, Zhao et al. 1995, Jensen 2000b, a, c, Bielza et al. 2007), including Australia

(Herron and Gullick 1998, Herron and James 2005). In Australia, resistance to newer

insecticides such as spinosad (Success™), three years after it was first introduced is disturbing,

given that it is widely use as an IPM-compatible insecticide (Herron and James 2005). Spinosad

resistance has been detected in populations in NSW, Queensland (Herron and James 2005) and

Western Australia (S. Broughton, Pers. Comm.) and other parts of the world (Bielza et al.

2007). Alternative approaches for the management of WFT are clearly necessary.

Host-plant resistance is considered a key method for pest control in many crops with low

economic thresholds (Schoonhoven et al. 1998). Differences in varietal resistance to thrips have

been described in several crops including cotton (Trichilo and Leigh 1988), chrysanthemum (de

Jager et al. 1993, Ohta 2002), rose (Gaum et al. 1994), pepper (Maris et al. 2003, Maris et al.

2004), tomato (Kumar et al. 1995a), cucumber (Soria and Mollema 1995), strawberry (Toshio

2004), cabbage (Stoner and Shelton 1986) and groundnut (Robb and Parella 1989). Some

cultivars can compensate for thrips feeding and influence oviposition, and therefore thrips

development (Kumar et al. 1995a). For example, de Kogel et al. (1998) found that feeding,

reproduction and adult survival of WFT were reduced on resistant chrysanthemum cultivars


45

compared to susceptible cultivars. The development of plant resistance to tospoviruses can also

indirectly reduce direct thrips damage (Ullman 1996).

In Australia, growers have access to several strawberry cultivars (e.g. Albion, Camarosa,

Camino Real, Ventana, Gaviota, Festival, Kiewa). Evaluation of cultivars tends to be based on

yield, flavour, sweetness and shelf-life (Phillips and Reid 2008), rather than varietal

susceptibility to WFT. My preliminary observations of WFT on strawberry cultivars suggest

that leaf feeding damage varied with cultivar. If strawberry cultivars can influence WFT

feeding, oviposition and development, then it could be used by industry as part of an IPM

program. Only three cultivars were evaluated [Camarosa (short-day), Camino Real (short-day)

and Albion (day-neutral)], since runners of these cultivars were available at the beginning of the

project. In this study, I seek to assess the: (i) variation in feeding preference and performance of

WFT on strawberry cultivars, (ii) effect of fresh (not previously exposed to conspecifics) and

damaged (previously exposed to conspecifics) leaves on preference and performance of WFT,

(iii) oviposition preference on strawberry cultivars, and (iv) developmental time and survival.

2.2 Material and methods

Experiments were conducted in a controlled temperature (CT) room (25±1⁰C temperature, 50-

60% RH and 16:8 h L:D regime) at the University of Western Australia (UWA), Crawley. The

above conditions were considered most suitable for WFT oviposition (Marullo and Tremblay

1993). At 16.6-36.6ºC the total development period of WFT is 7-13 days (Robb 1989).

Experiments were conducted from August to September 2006.

2.2.1 Source cultures

2.2.1.1 Strawberry cultivars

Three commercial strawberry cultivars [Fragaria x ananassa Duchesne (Rosaceae)]

(Albion, Camarosa and Camino Real) were used in this study. Strawberry runners were

propagated in pots (32.5l x 32.5w x 40.5h cm) containing potting mix (Baileys

Fertilisers, Rockingham, WA) in glasshouses at the Department of Agriculture and Food

WA (DAFWA) and UWA. All pots were fitted with sprinklers with automatic timers.

Plants were watered every third day in summer, and once a week in winter and spring . A

liquid fertilizer (Thrive®

, Yates, Australia; NPK: 12.4: 3: 6.2; rate: 5mL/2 L water) was

applied once a month. All plants were covered with a thrips cage (105µ; 45 x 35 cm)


46

made from mesh (Sefar Filter Specialists Pty Ltd., Malaga, WA) and supported by a steel-rod

stand. Prior to experimentation, plants were moved from the glasshouse into a CT room.

2.2.1.2 Western flower thrips (WFT)

WFT were reared on Calendula, Calendula officinalis L. (Asteraceae), planted in plastic

pots (50 x 100 mm) with potting mix and kept in insect -proof Perspex cages (500 x 420

x 400 mm H x D x W), with 105 µ mesh net fitted on a Nylex tote box (432 x 320 x 127

mm; Blyth Enterprises Pty Ltd, Australia). Plants were kept in a glasshouse or net house at

UWA from July 2006 to November 2008. All pots were fit ted with sprinklers with

automatic timers. The plants were watered every third day in summer and once a week

during winter and spring. WFT adults were released at the base of each caged plant.

Every second week, adults were collected from the caged plants and released onto new

potted plants to ensure continuous availability. To obtain uniformly aged WFT, adults (20

individuals) were collected and released onto fresh plants and allowed to lay eggs for 24 h.

After 24 h, all adults were removed with an aspirator. Plants were checked daily for adult

emergence and adults that emerged on the same day were used in trials.

2.2.2 Experiment 1: Feeding preference of adult WFT

Twenty-four hours prior to the experiment, potted strawberry plants (six plants of each cultivar,

total n = 18) were brought to the CT room. Flowers were removed from the plants with a sharp

blade, because WFT shows a preference for flowers (Bailey 1933). Plants of each cultivar were

divided into two groups, and covered with thrips cages as described above. One group of plants

was kept free from WFT (ungrazed), whilst the other group received 50 WFT adults per plant

and allowed to feed for 3-4 hours (grazed). To promote feeding, WFT adults were kept without

food 24 h prior to release. WFT adults were then removed from the plants with an aspirator.

From the above plants, leaf discs were cut from the leaves for use in the experiment. To prepare

leaf discs for trials, leaves were detached with a scalpel. Leaf discs (2.5 cm diam.) were cut

using a cork borer (1450-9, MET-APP Pty Ltd, Victoria, Australia) and placed onto moistened

filter paper in a Petri dish (150 x 15 mm), axial side down. Leaf discs were placed randomly in a

circle by maintaining the same distance between discs. Leaf discs were marked with cultivar

code (AL, CA and CR for Albion, Camarosa and Camino Real respectively), and additionally

coded ‘F’ (ungrazed leaves) or ‘PI’ (grazed leaves). A combination of ungrazed and grazed leaf

discs were placed at the bottom of the Petri dish equidistant apart. Combinations included: (i)

ALF + CRF + CAF, (ii) ALPI + CRPI + CAPI, (iii) ALF + ALPI, (iv) CRF + CRPI and (v) CAF +


47

CAPI. Each combination was replicated 20 times. WFT adults (3 d old) were collected with an

aspirator and kept in Petri dishes (150 x 15 mm) for 24 h to acclimatise them with the

experimental arena. No food was provided; except cotton ball soaked in 10% sugar solution was

placed inside the Petri dish.

After 24 h, individual thrips were transferred to the centre of an experimental Petri dish. The top

of the Petri dish was then covered with mesh (mesh net, 105 µ), and sealed with parafilm

(Parafilm M®, Micro Analytix Pty Ltd). Thrips were observed constantly for 30 mins under a

stereomicroscope (20-x magnification illuminated by 12v/10w bulbs) and their behaviour

recorded. Behavioural events recorded were: (1) olfactory preference, (2) total time spent on a

cultivar, and (3) time spent feeding on a cultivar. The first movement of an individual onto a

leaf disc after release was considered to be an olfactory preference. Feeding preferences were

recorded by measuring times spent feeding (time between probing sylets into the leaf tissue til

withdrawn) on the leaf disc of each cultivar. Of the 30 minutes observation, time spent feeding

as well as total time spent on each leaf disc was recorded. If an individual thrips did not move

from its initial position within two minutes after release, it was discarded and replaced with a

new individual. For each individual, a new Petri dish and leaf disc was used.

2.2.3 Experiment 2: Oviposition preference and performance of WFT on caged plants

The experiment was conducted in a CT room as previously described. Three strawberry plants,

one of each cultivar (4-5 leaves/plant) that had not been previously exposed to WFT, were

placed in a circle on a laboratory bench such that each pot touched. Three-day-old adult WFT

females were collected from the colony and placed in a refrigerator for approximately two

minutes. Cold anaesthetised WFT adults were then transferred to a filter paper in a Petri dish.

For each group of three plants, 20 WFT adults were placed in the centre of the plants and thrips

were allowed to disperse. The plants were then covered with a modified cylindrical thrips cage

(open both ends), ensuring that the cage did not touch either pot or plant parts which was

supported by a cylindrical stand. The cage was 150w x 45h cm in dimension made from mesh

net (105 μ). The bottom of the cage was taped onto the bench with masking tape. The top end

of the cage was folded and closed with a paper clip for easy handling. Plants were checked at

one, 24 and 48 h post-release and adult numbers were recorded on each plant. If previously

released WFT were not found, a hand-held battery-powered magnifying glass (4x bifocal

magnifier) was used. After 48 h, all adults were removed from the plants with an aspirator.

Plants were then checked twice a day (0800 and 1900 h) for one week with a magnifying glass

for larvae. No newly hatched larvae were found after five days. Each day, newly hatched larvae

were removed with a fine brush, counted and transferred to new plants of the same cultivar.


48

Individual plants with WFT larvae were covered with a mesh net cage (45 x 35 cm) as

described above and . These plants were checked twice daily (0800 and 1900 h) for three

weeks, and any emerged adults were counted and removed from the plant. The experiment was

repeated 10 times (replicates).

2.2.4 Experiment 3: Oviposition preference and performance of WFT on leaf discs

The experiment was conducted in a CT room. Three leaf discs, one from each cultivar, were

prepared from fresh strawberry leaves as described in section 2.2.2. They were placed adaxial

side up, two mm apart and equidistant from each other on a moistened filter paper at the centre

of a Petri dish (150 x 15 mm). Prior to the experiment, 20 adult females (5-6 d old, females)

[on average after three days post-emergence, WFT females start laying eggs at 20-25°C

(Marullo and Tremblay 1993)] from the same cohort were collected with an aspirator and kept

in Petri dishes for 24 h to acclimatise with the experimental arena. After 24 h, females were

released at the centre of the Petri dish and allowed to oviposit for 24 h. The Petri dish was

covered with mesh net (105 µ), and the edges sealed with parafilm (15 μm) (de Kogel et al.

1997). After 24 h, WFT adults were removed and each leaf disc was transferred onto moistened

filter paper in separate Petri dishes and covered with parafilm as above. Petri dishes were

checked under a stereomicroscope (20x magnification) twice daily for one week [at 20-25ºC,

eggs hatch within 2-4 days (Pfleger et al. 1995)]. Numbers of hatched larvae were recorded per

leaf disc of each cultivar for seven days, though in this trial, no larva hatched after five days.

After each count, larvae were removed from the leaf disc and transferred to fresh leaf discs of

the respective cultivar. The trial was repeated 20 times (replicates).

2.2.4.1 Egg hatch

After seven days, leaf discs were used to count the numbers of unhatched eggs (if any).

Because WFT adults lay eggs inside the leaf tissue by probing their ovipositor, it is not possible

to count any unhatched eggs directly. In order to count unhatched inside the leaf tissue, leaf

discs of each cultivar were boiled into separate beakers (250 ml) with distilled water into a

microwave oven and heated for three minutes (700 watt) (de Kogel et al. 1997). Leaf discs

were soaked in methyl red, so that eggs were clearly seen under a stereomicroscope (40 x).

Numbers of unhatched in each leaf discs were counted.

2.2.4.2 Larval mortality, pupal mortality and adult emergence rate

To determine the effect of strawberry cultivars on larval mortality, pupal mortality and adult

emergence rate, newly hatched larvae (within 12 h of hatching) were collected each day from


49

leaf discs and transferred to new leaf discs (Ø = 2.5 cm) of the same cultivar. Each disc was

placed in a separate Petri dish on slightly moistened filter paper and covered with clear plastic

film. Fresh leaf discs were added daily. To reduce handling stress, larvae were allowed to move

from the old to the new leaf disc. Once all larvae had moved, the older disk was checked under

a magnifying glass, and then discarded. Leaf discs were checked twice daily under a

binocular stereomicroscope (20x magnification) until all larvae had either pupated or died. Any

dead larvae were removed from the leaf disc. Pupae were checked until adult emergence. If a

WFT adult did not emerge, the pupa was considered dead.

2.2.4.3 Developmental time

To determine the influence of strawberry cultivar on egg, larval, prepupal, and pupal period,

and total development time (egg to adult emergence), the duration of each period was measured

for 40 larvae (replicates) on the three cultivars. Since WFT laid eggs inside leaf tissue, the

exact measurement of the incubation period was not possible. However, as WFT adults were

allowed to lay eggs for 24 h and leaf discs were checked at 12 h intervals, the incubation period

was considered to be the period between half the 24 h period and either the 12 h or 24 h count

(depending on when eggs hatched). Leaf discs were changed every second day.

2.2.5 Data analysis

When each thrips chose a leaf disc to move onto, this was scored as the olfactory preference of

WFT adults between cultivars. The differences in olfactory preference by WFT adults between

cultivars were analysed by a chi-square goodness of fit test. Similarly, olfactory preference

between ungrazed and grazed leaf disc within each cultivar was also scored and analysed with a

chi-square test. Feeding preference of WFT between cultivars was determined by the amount of

time spent feeding on leaf disc of each cultivar. In terms of the amount of time spent feeding,

adults exhibited several feeding behaviours such as approaching to leaf disc, stand-still, feeding,

resting, walking. However, only the total time spent feeding and the total time that WFT

remained on leaf discs was analysed. The time spent by adults among cultivars was expressed as

a proportion. To determine adult preference, the total time and the time spent feeding was

subjected to Compositional Data Analysis separately for each cultivar combination (Aitchison

1986, Aebischer and Robertson 1993). Least square mean difference with 5% significance level

was used to separate means.

In the caged trial, the effect of cultivar on adult numbers per plant at one, 24 and 48 h post

release were analysed by separate one-way ANOVAs [independent variable: cultivar, response


50

variable: WFT adult numbers per plant of each cultivar). The difference in adult numbers

between post-release periods for each cultivar was analysed by repeated measures ANOVA.

The influence of cultivars on mean numbers of larvae hatched and adults emerged was analysed

by two separate one-way ANOVAs. Similarly, the effect of strawberry cultivars on numbers of

eggs laid, unhatched eggs, larvae hatched, larvae killed, pupae developed, pupae killed and

adults emerged were compared with a series of one-way ANOVAs. If ANOVA results were

significant, means were separated by least square means difference (alpha = 0.05).

In the leaf disc trial, the survival of the immature stages and the overall survival rate were

calculated as follows:

Larval hatching rate (%) = 100eggsofNo

hatchedlarvaeofNo

Pupation rate (%) = 100.

testedlarvaethripsofNo

prepupaethripsofNo

Emergence rate (%) = 100.

.

prepupaethripsofNo

adultsthripsofNo

Survival rates (%) = 100.

eggsthripsofNo

adultsthripsofNo

The influence of strawberry cultivars on larval hatching rate, pupation rate, adult emergence rate

and total survival rate (eggs to adult emergence) was analysed with separate one-way ANOVA.

Cultivars effect on eggs, larval and pupal mortality was analysed by separate ANOVAs. If

ANOVAs were significant, means were separated by least square mean difference (α = 0.05).

Data subjected to ANOVAs were transformed for homogeneity using √(x+0.5) (Zar 1999). Data

were reversed transformed for presentation. Statistical analyses were conducted using SAS

software 9.1 (SAS 2002-2003).

2.3 Results

2.3.1 Experiment 1: Feeding preference of adult WFT

WFT adults showed an olfactory preference for cultivar (χ2 2

= 6.34, P = 0.04) leaves not

previously exposed to conspecifics. Camarosa was the most preferred cultivar, followed by

Albion (χ2

1 = 3.66, P = 0.03) and Camino Real (χ

2 1

= 5.76, P = 0.01) (Figure 2.1A). However,

there was no significant difference in olfactory preference of WFT adults between Camino Real

and Albion (χ2

1 = 0.14, P = 0.71) (Figure 2.1A). When offered leaves that had previously been


51

grazed on by conspecifics, adult preference did not differ (χ2

2 = 1.03, P = 0.60) among cultivars

(Figure 2.1B).

When offered fresh leaf discs, the total time (Wilks λ = 0.47, F2, 17 = 9.54, P = 0.0017) and the

time spent feeding (Wilks λ = 0.38, F2, 17 = 13.99, P = 0.0003) differed between cultivars

(Figure 2.2). Total time spent and time spent feeding onto each cultivar was longest on

Camarosa and shortest on Camino Real (Figure 2.3). The proportion of time spent feeding did

not differ between Albion and Camino Real. When offered grazed leaf discs, the total time (λ =

0.51, F2, 17 = 8.28, P = 0.0031) and time spent feeding (Wilks λ = 0.60, F2, 17 = 5.66, P =

0.0131) differed with cultivar (Figure 2.2). In both cases, adults spent the most time and the

highest proportion of their time feeding on Camarosa and lowest on Camino Real (Figure 2.3).

No differences were detected between Camarosa and Albion.

0

20

40

60

NS

*

**

Camarosa Albion Camino Real0

20

40

60

NS

NS

NS

A

B

Per

centa

ge

of

WF

T a

dult

s (%

)

Figure 2.1 Olfactory preference of WFT adults to strawberry cultivars when offered (A)

ungrazed and (B) grazed leaf discs. **Significant at P = 0.01, *P = 0.05, NS

Not-significant at P

= 0.05.


52

Figure 2.2 Simplex plot showing time spent by WFT adults between strawberry cultivars when

exposed to ungrazed leaf discs (A = total time, B = time spent feeding) and discs previously

grazed by conspecifics (C = total time, D = time spent feeding). AL = Albion, CA = Camarosa,

CR = Camino Real. In each figure, each dot represents individual WFT adult.

0

20

40

60

80

100

Albion Camarosa

a

b

b

c

aa

Camino Real

Total time spent Time spent feeding0

20

40

60

80

100

b b

a

b

aa

A

B

Tim

e sp

ent by

WF

T a

du

lt (

%)

(Mea

n

SE

)

Figure 2.3 Time spent by WFT adults on (A) ungrazed and (B) previously grazed leaf discs.

Within group, means with different letters differed significantly (LS means, α =0.05).


53

WFT showed olfactory preference for ungrazed leaf discs over grazed leaf discs for all three

cultivars and difference was significant [Figure 2.4; Albion (χ2

1 = 6.24, P = 0.013), Camarosa

(χ2

1 = 9.75, P = 0.0018) and Camino Real (χ2

1 = 3.50, P = 0.04)].

When offered a choice between leaf discs from ungrazed and grazed leaves, WFT spent the

most time on ungrazed discs compared to grazed discs for all cultivars (Figure 2.5; Albion: λ =

0.09, F1, 8 = 85.43, P < 0.0001; Camarosa: λ = 0.10, F1, 8 = 69.40, P < 0.0001 ; Camino Real: λ

= 0.12, F1, 8= 65.23, P < 0.0001). WFT adults also spent more time feeding on ungrazed leaf

discs compared to grazed leaf discs, regardless of cultivar (Figure 2.5).

Albion Camarosa Camino Real0

20

40

60

80

Ungrazed

**

Grazed

**

Per

cen

tag

e o

f W

FT

ad

ult

s (%

)

Figure 2.4 Preference of WFT adults when given a choice between leaf discs exposed to

conspecifics (grazed) or ungrazed of three different strawberry cultivars. **Significant at P =

0.01, *Significant at P = 0.05.


54

0

20

40

60

80

100

Ungrazed Grazed


20

40

60

80

100

A

B

Tim

e s

pen

t (%

) (M

ean

SE

)

Figure 2.5 Time [A = total time, B = time spent feeding] spent by WFT adult on ungrazed and

grazed leaf discs. For each cultivar, means were significantly different (α = 0.05). In each

cultivar, WFT adults spent longer times on ungrazed leaf discs compared to grazed leaf discs.

2.3.2 Experiment 2: Oviposition preference and performance of WFT on caged plants

More adults were found on Camarosa and the least on Camino Real when counted at one hour

(F2, 27 = 9.21, P = 0.0009), 24 h (F2, 27 = 23.01, P < 0.0001) and 48 h (F2, 27 = 42.01, P <

0.0001) post release (Figure 2.6). Differences in preferences were not observed between Albion

and Camino Real until 24 hours post-release, with more adults on Albion than Camino Real

(Figure 2.6).

In each cultivar, WFT adult numbers changed over times from its initial numbers (Figure 2.7).

For Albion, WFT adult numbers per plant gradually decreased from initial numbers over time,

though the difference (Figure 2.7; F2, 27 = 0.90, P = 0.4201) was not significant. In Camarosa,

WFT adult numbers gradually increased from initial numbers at one hour post-release and was

highest at 48 h post-release, the difference was significant (Figure 2.7; F2, 27 = 4.89, P = 0.015).

WFT adult numbers per plant in Camino Real gradually decreased from its initial numbers; the

difference was significant (Figure 2.7; F2, 27 = 3.76, P = 0.0363).


55

The mean number of larvae hatched (F2, 27 = 45.83, P < 0.0001) and adults emerged (F2, 27 =

77.91,P < 0.0001) were highest on Camarosa (larvae hatched = 39.80 ± 2.11, adults emerged =

20.27 ± 1.49) and lowest on Camino Real (larvae hatched = 15.00 ± 1.31, adults emerged = 4.40

± 0.89) (Figure 2.8). Moreover, significantly more larvae hatched and adults emerged on Albion

compared to Camino Real (Figure 2.8).

1h 24h 48h0

6

12

18Albion Camarosa

a

a

b

a

b

c

a

b

c

Camino Real

Nu

mb

er o

f W

FT

ad

ults

(Mea

n

SE

)

Figure 2.6 Mean numbers of WFT adults per plant on caged strawberry cultivars at 1, 24, and

48 h post-release. Means with different letters differed significantly (LS mean, α = 0.05).


5

10

15

201h 24h

a

aa

aa

b

ab

c

48h

Nu

mber

s o

f W

FT

ad

ult

s (M

ean

SE

)

Figure 2.7 Comparison of WFT adult numbers (Y-axis) at different post-release periods (hours)

on caged plants. Means within cultivar with different letters differed significantly (LS means, α

= 0.05).


56

0

10

20

30

40

50

a

b

c


5

10

15

20

25

a

b

c

A

B

Num

ber

of

WF

T (

Mea

n

SE

)

Figure 2.8 Mean numbers of WFT (A) larvae hatched and (B) adults emerged per plant on

caged strawberry cultivars. Means with different letters differed significantly (LS means, α =

0.05).

2.3.3 Experiment 3: Oviposition preference and performance of WFT on leaf discs

The mean numbers of eggs laid (F2, 57 = 8.74, P = 0.0005), unhatched eggs (F2, 57 = 17.93, P <

0.0001), larvae hatched (F2, 57 = 38.044, P < 0.0001), pupae developed (F2, 57 = 51.89, P <

0.0001) and adults emerged (F2, 57 = 45.03, P < 0.0001) differed significantly between cultivars

(Figure 2.9). The greatest numbers of eggs were laid on Camarosa and the lowest on Camino

Real. There were no statistically significant differences in egg lay between Albion and Camino

Real. The least unhatched eggs were recorded on Camarosa, and more on Camino Real.

Numbers of larvae hatched, pupae developed and adults emerged were highest and lowest on

Camarosa and Camino Real respectively (Figure 2.9). The number of pupae that developed and

adults that emerged did not differ between Albion and Albion (p >0.05).


57

Total eggs Unhatch eggs Larvae Pupae Adults0

5

10

15

20

25Albion Camarosa

a

b

a

a

bb a

b

c

aa

b

aa

b

Camino Real

Nu

mb

er o

f W

FT

(M

ean

SE

)

Figure 2.9 Comparison of numbers of eggs laid, unhatched eggs, larvae hatched, pupae

developed and adults emerged per leaf disc (Y-axis) on three strawberry cultivars. Within each

group (X-axis), means with different letters differed significantly (LS means, α = 0.05).

The development rate of WFT was different between cultivars (Figure 2.10 and 2.11). Mean

percentage of larvae hatched (F2, 57 = 106.70, P < 0.0001), pupae developed (F2, 57 = 7.01, P <

0.0001), adults emerged (F2, 57 = 3.78, P = 0.0284) and survival rate (eggs to adults) (F2, 57 =

24.45, P < 0.0001) was highest on Camarosa and lowest on Camino Real. There were no

significant differences between Albion and Camino Real. The mean percentage of unhatched

eggs (F2, 57 = 104.48, P < 0.0001), larvae (F2, 57 = 7.04, P = 0.0019) and pupae (F2, 57 = 11.13,P

< 0.0001) that died was lowest on Camarosa and highest on Camino Real. There were no

differences between Albion and Camino Real.

U/E L/H L/K P P/K A/E0

20

40

60

80

100Albion Camarosa

a

b

c

a

b

c

a

b b a a

b

a

b b

a

b

Camino Real

a

Per

cen

tag

e o

f W

FT

(%

) (M

ean

SE

)

Figure 2.10 Comparison of the percentage of unhatched eggs (U/E), larvae hatched (L/H),

larvae died (L/K), pupae developed (P), pupae died (P/K) and adult emerged (A/E) per leaf disc

among cultivars. Means within groups with different letters differed significantly (LS means, α

= 0.05).


58

Albion Camarosa Camino0

20

40

60

a

a

bS

urv

ival

rat

e of

WF

T (

Mea

n

SE

)

Figure 2.11 Comparison of survival rate of WFT among strawberry cultivars. Means with

different letters differed significantly (LS means, α = 0.05).

The mean egg incubation period (F (2, 117) = 4.45, P = 0.0137), larval (F (2, 117) = 3.65, P =

0.0290), prepupal (F (2, 117) = 3.19, P = 0.0447), pupal (F (2, 117) = 14.76, P < 0.0001) and total

developmental (F (2, 117) = 4.61, P = 0.0119) periods differed between cultivars (Figure 2.12).

Mean egg incubation, prepupation, pupation and total developmental periods were lowest on

Camarosa and highest on Camino Real. There were no differences between Albion and

Camarosa. Mean larval duration was also highest and lowest on Camarosa and Camino Real

respectively, but did not differ between Camarosa and Albion.

IP LP PPP PP TDP0

5

10

15Albion Camarosa

aa baab b

aa b

ab b

a a

Camino Real

b

Dev

elopm

enta

l per

iod (

Mea

n

SE

) in

day

s

Figure 2.12 Egg incubation period (IP), larval period (LP), prepupation period (PPP), pupation

period (PP) and total developmental period (TDP) (egg to adult) of WFT in days, among

strawberry cultivars. Means within group with different letters differed significantly (LS means,

α = 0.05).


59

2.4 Discussion

Host-plant resistance in the present study is defined as a reduction in insect performance. The

results confirm that WFT shows a preference for particular strawberry cultivars. When given a

choice between three cultivars, Camarosa was the most preferred and Camino Real the least

preferred cultivar. This result was confirmed in leaf disc and whole plant choice tests. In leaf

disc trials, adults showed an olfactory and feeding preference for Camarosa. Adults spent the

most time and the highest proportion of their time feeding on Camarosa and the lowest on

Camino Real. In choice tests with whole plants, adults actively dispersed from the less preferred

cultivars. Whilst no published information is available on strawberry, Maris et al. (2004) found

that WFT adults dispersed from one capsicum cultivar to another cultivar in laboratory trials.

This suggests that given a choice, WFT adults can move from one host to another host that may

be more suitable for feeding or oviposition. Leaf condition (i.e. leaves grazed by conspecifics vs

ungrazed leaves) also strongly influenced adult preference. Adults preferred ungrazed leaves

over leaves that had been previously grazed by conspecifics. When offered previously grazed

leaves, no cultivar preference was detected. This result agrees with Delphia et al. (2007) who

reported that higher numbers of WFT were found on unwounded (fresh) tobacco plants

(Nicotiana tabacum L) than plants wounded by conspecific WFT. Delphia et al. (2007) found

that wounded tobacco plants released higher amounts of volatile chemicals than unwounded

plants. Whether a similar mechanism is operating in strawberry is not known.

The quality of specific cultivars may influence thrips performance (Brødsgaard 1987, Brodbeck

et al. 2002). van Lenteren and Noldus (1990) suggest that a higher fecundity rate, faster

developmental rate and higher survival rate indicate better host-plant suitability. On resistant

cucumber and chrysanthemum cultivars for example, reproduction of WFT is reduced (de Kogel

et al. 1997, de Kogel et al. 1998). Soria and Mollema (1995) similarly found that unsuitable

cucumber varieties severely reduced the population growth of WFT. To determine whether

WFT females chose a strawberry cultivar that offered a possible reproductive advantage,

ovipositional preferences were measured in plant (cage) and leaf disc trials. Ovipositional

preference was determined by counting the number of larvae that hatched and the number of

unhatched eggs. In the cage more larvae developed and adults emerged on Camarosa (larvae

hatched = 39.80 ± 2.11, adult emerged = 20.27 ± 1.49) compared to Camino Real (larvae

hatched = 15.00 ± 1.31, adult emerged = 4.40 ± 0.89). In leaf disc trials, 50.72% of eggs

successfully developed to adult on Camarosa and 15.46% of eggs developed to adult on Camino

Real. The egg incubation, prepupation, pupation and total developmental periods (egg to adult)

were also determined. If adults fail to emerge, the population of the next generation is affected,

which could delay WFT from reaching damaging levels. Whilst all cultivars supported the


60

development of eggs to adult, the percentage of adults that emerged and the total development

period varied. On Camarosa, 50.72% of eggs developed to adult and the total developmental

period was 11.19 ± 0.22 days. In Albion, 21.31% of eggs developed to adult and the total

developmental period was 11.20 ± 0.26 days, while in Camino Real, 15.46% eggs successfully

developed to adults and the total developmental period was 12.03 ± 0.23 days.

The mechanism or mechanisms that may be involved in the selection by WFT of Camarosa over

the two other cultivars is not known, and was beyond the scope of this study. Possible

mechanisms include the release of plant volatiles (Bernays and Chapman 1994, Dobson 1994),

the presence of toxic metabolites, the absence of, or suboptimal amounts of essential nutrients

that are required for insect growth, or the presence of enzymes that inhibit food digestion thus

reducing nutrient utilisation (Saxena 1985). For example, Yang et al. (1993) and Snook et al.

(1994) report that wild peanut species (Arachis sp) sustained less damage from Frankliniella

fusca (Hinds) than cultivated peanut (Arachis hypogea L.). They suggest that this may be due to

differences in culticular lipids, especially phenolic acid and acetates (general digestibility

inhibitors).

Physical characteristics such as leaf morphology may be also important (Ananthakrishnan and

Gopichandran 1993, Kumar et al. 1993, Kumar et al. 1995a, Kumar et al. 1995b). Epidermal

appendages such as trichomes can provide a physical barrier against insects. Bioactive

secondary products produced by glandular trichomes may also be used as a chemical method of

defence (Wagner 1991, Bisio et al. 1999, Roda et al. 2003). Two types of trichomes, simple

trichomes and uniseriate glandular trichomes occur in strawberry (Steinite and Levinsh 2003).

However the affect of trichomes on WFT on strawberry cultivars is not known. However,

Rummel and Quisenberry (1979) reported that infestation of thrips on cotton cultivars varies

because of trichome density in different cotton cultivars. The presence of wax on the leaf

surface may also play an important role in the selection of hosts by herbivorous insects

(Eigenbrode 1996). More than 200 reports on resistance to arthropod pests in vegetables have

shown that tolerance was involved in about 10% of the cases, whereas the remaining cases were

equally attributed to either antixenosis (inability of a plant to serve as a host) or antibiosis (kill

insects or reduce plant digestibility) (Schoonhoven et al. 1998).

In conclusion, feeding and oviposition preference and performance of WFT were affected by

strawberry cultivars. Before implementing the present findings in the field, further research

should be carried out to determine the seasonal variation in preference and performance on

strawberry cultivars. The leaf morphology and nutritional contents of each cultivar should also

be assessed to determine the basis for thrips preference, which could then be incorporated into a


61

screening program for strawberry cultivars. Furthermore, whether WFT injury could cause

economic damage on least preferred cultivar needs to be assessed.


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CHAPTER III

Effect of spinosad and predatory mites (Acari) on western flower thrips, Frankliniella

occidentalis (Pergande) (Thysanoptera: Thripidae) in three strawberry cultivars [Fragaria

x ananassa Duchesne (Rosaceae)]


Hypoaspis miles, spinosad, strawberry cultivars

Abstract

Western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) is a

major pest of strawberry, Fragaria ananassa Duchesne (Rosaceae) in Australia. Spinosad

(Success™, Dow AgroSciences Australia Ltd) is the only insecticide currently registered in

Australia that is efficacious against F. occidentalis, and regarded to be compatible in an

integrated pest management program. This study sought to determine whether three predatory

mite species, Typhlodromips montdorensis (Schicha) (Phytoseiidae), Neoseiulus cucumeris

(Oudemans) (Phytoseiidae) and Hypoaspis miles (Berlese) (Laelapidae) could be used with

spinosad and least-preferred cultivars for the management of F. occidentalis. Typhlodromips

montdorensis and N. cucumeris attack first instar thrips whilst H. miles feeds on thrips pupae. In

the glasshouse, three strawberry cultivars (Camarosa, Camino Real, and Albion) were sprayed

once with spinosad (80 mL/100 L rate, 0.096 g a.i./L) or water (control). Thrips adults were

released onto plants 24 h after spraying and predatory mites released six days later. Spinosad

significantly reduced thrips numbers compared to water. Typhlodromips montdorensis reduced

thrips numbers as did N. cucumeris and H. miles. Spinosad had no effect on predatory mites.

The number of H. miles could not be counted directly, but the numbers of thrips in treatments

with H. miles were lower than those in treatments without the mite. Thrips numbers were lowest

on Camino Real followed by Albion and Camarosa. These results suggest that the use of

Camino Real with spinosad applications can be used to reduce initial F. occidentalis thrips

numbers, followed by releases of predatory mites.

3. 1 Introduction

Western flower thrips (WFT), Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae)

is regarded one of the most important economic pests worldwide (Brødsgaard and Albajes 1999,

Jones et al. 2002, Kirk and Terry 2003), causing extensive crop losses of agricultural and

horticultural crops (Lewis 1998). With its piercing-sucking mandibles, WFT penetrates

Chapter III: Effect of spinosad, cultivar and predatory mites

66

epidermal and sub-epidermal cells, causing extensive damage; this includes deformation and

growth reduction of the plant, and silver scars on fruits and leaves (Hunter and Ullman 1989,

Ullman et al. 1989, van Dijken et al. 1994, de Jager et al. 1995). In addition to direct damage,

WFT cause indirect damage by transmitting tospoviruses (German et al. 1992, Wijkamp et al.

1996, Ullman et al. 1997). Since its detection in 1993 in Western Australia (Malipatil et al.

1993), WFT has spread to each state and territory, except the Northern Territory and has

become a major pest in several crops including strawberry, Fragaria x ananassa Duchesne. In

Australia, strawberry production has grown steadily in the last few years; however, its

production is often affected by direct damage by WFT. This includes fruit deformation, uneven

ripening, early withering of stigmas and anthers, and reduction in flower receptacle size leading

to yield loss (Medhurst and Steiner 2001, Zalom et al. 2001, Coll et al. 2007). Similar damage

occurs in low tunnel-, glasshouse- and field-grown strawberries (Ullio 2002).

Whilst insecticides are the main control method (Herron and Cook 2002), because of its small

size, secretive habit, high reproductive potential, and ability to develop resistance to insecticide,

WFT can be difficult to control (Jensen 2000). It has developed resistance to several major

classes of chemicals (Brødsgaard 1994, Broadbent and Pree 1997, Jensen 1998, Jensen 2000) in

different parts of the world. Spinosad™

(Dow AgroSciences, USA) is a mixture of tetracyclic-

macrolide compounds and is classified as a reduced-risk bioinsecticide (Sparks et al. 1998). It

has been used in over 180 different crops to control a wide range of pests, and is effective

against WFT (Funderburk et al. 2000). Spinosad is relied on heavily by growers in Australia,

but WFT has already begun to develop resistance to this chemical (Herron and James 2005).

WFT resistance to insecticides appears to be a serious threat to its effective management;

therefore, additional management strategies are clearly necessary.

Overseas, predatory mites (Acari) are used to manage WFT in field and glasshouse crops (Chant

1985, van Lenteren and Woets 1988, McMurtry and Croft 1997). Until recently, Australian

growers had no effective biological control options for WFT due to quarantine restrictions

prohibiting their importation (Steiner and Goodwin 2001). Recently four predatory mite species

have become available to Australian growers. Of them, two species are natives: Typhlodromips

montdorensis (Schicha) and Typhlodromus occidentalis (Schicha) (Phytoseiidae) (Steiner and

Goodwin 2000). Neoseiulus cucumeris (Oudemans) (Phytoseiidae) and Hypoaspis miles

(Berlese) (Laelapidae) are native to New Zealand and were recently confirmed as occurring in

Australia. Of the four mite species, only T. montdorensis, N. cucumeris and H. miles have been

used for WFT management (Anonymous 2006). However, augmentative releases of predatory

mites are not always sufficient to manage WFT in crops with a low economic threshold such as

strawberry (Gillespie and Ramey 1988, Bakker and Sabelis 1989, Gillespie 1989).


67

It is possible to integrate biological and chemical control when mites are used as natural

enemies (Reuveni 1995, Kongchuensin and Takafuji 2006). However, there are concerns about

the detrimental effects of insecticides on predatory mites. The negative impact of pesticides

have been reported on several predatory mite species including Phytoseiulus persimilis Athias-

Henriot, Typhlodromus pyri (Scheuten), Amblyseius finlandicus (Oudemans), A. potentillae

(Garman), Stratiolaelaps scimitus (Womersley) and Neoseiulus womersleyi (Schicha) (Hassan

et al. 1987, Hassan et al. 1988, Kim and Paik 1996, Bowie et al. 2001, Kim and Seo 2001,

Amano et al. 2004, Cabrera et al. 2004, Li et al. 2006, van Driesche et al. 2006). Pesticides can

affect behaviour, survival, reproduction, attack-rate, and prey handling time (Li et al. 2006, van

Driesche et al. 2006).

Applications of insecticide followed by releases of natural enemies are one proposed solution to

this problem. Bentz and Neal (1995) reported that an initial application of a natural insecticide

[derived from Nicotiana gossei] followed by the release of the hymenopteran parasitoid,

Encarsia formosa Gahan (Aphelinidae) is effective for the management of whitefly in

glasshouse-grown tomato. An initial application of Acramite® (miticide) followed by the release

of Neoseiulus californicus (McGregor) (Phytoseiidae) was reported by Rhodes and Liburd

(2006) as an effective strategy to control spider mite in glasshouse- and field-grown strawberry.

Spraying of the selective pesticide pyriproxyfen, an insect growth regulator, followed by the

release of the predatory bug, Orius sauteri (Poppius) (Anthocoridae), is common practice for

the management of spider mite in eggplant in Japan (Nagai 1990).

Spinosad can provide effective control of many insect pests, while generally having reduced

toxicity to natural enemies (Brunner et al. 2001, Elzen 2001, Villanueva and Walgenbach

2005b). However, selectivity of spinosad on predators is under review (Pietrantonio and

Benedict 1999, Williams et al. 2003). It is regarded to have low to moderate toxicity to

predatory mites (Pietrantonio and Benedict 1999, Ludwig and Oetting 2001) and the

toxicity varies from species to species (Williams et al. 2003, Cote et al. 2004, Jones et

al. 2005). van Driesche et al. (2006) reported that fresh residues of the recommended rate of

spinosad for WFT on glasshouse flower crops had no toxic effect on N. cucumeris, while it

lowered the survival of Iphiseius degenerans (Berlese). Holt et al. (2006) reported that an

application of spinosad for the control of two-spotted spider mite had no effect on the predatory

mite P. persimilis, one day before or after its release. Spinosad is highly toxic to Neoseiulus

fallacis (Garman), the most abundant predaceous mite in North Carolina apple orchards that

prey on the mite pests Panonychus ulmi (Koch) and Tetranychus urticae Koch (Tetranychidae)

(Villanueva and Walgenbach 2005a). Despite its detrimental effect on some species, spinosad

can be integrated with biological control for WFT management (Funderburk et al. 2000, Ludwig


68

and Oetting 2001) if a period of time between spray and release is maintained (Jones et al. 2005,

Khan and Morse 2006, Kongchuensin and Takafuji 2006).

The integration of spinosad and commercially available predatory mites in Australia for the

management of WFT has not been evaluated. Given that cultivar has an effect on thrips survival

(Chapter 2), the objective of this study was to investigate the effectiveness of commercially

available predatory mites [T. montdorensis, N. cucumeris, and H. miles] and the influence

of strawberry cultivars (Camarosa, Camino Real and Albion), with or without a

spinosad application on WFT.

3.2 Materials and Methods

The trial was conducted in a glasshouse (25±2⁰C, 60-70% RH, 16:8 L: D regimes) at The

University of Western Australia (UWA) from September to November 2007.


3.2.1.1 Strawberry cultivars

Strawberry [Fragaria x ananassa Duchesne (Rosaceae)] cv Camarosa and Camino Real

(short-day), and Albion (day-neutral) were used in experiments. Strawberry runners

were obtained from a commercial grower in June 2006, and propagated in pots (32.5l x

32.5w x 40.5h cm) with potting mix (Baileys Fertilisers, Rockingham, WA) in

Figure 3.1 Plant covered by a modified cage made from thrips-proof mesh (105µ; Sefar

Filter Specialists Pty Ltd., Malaga, W. Australia).


69

glasshouses at the Department of Agriculture and Food Western Australia (DAFWA) and

UWA. All potted plants were covered with thrips-proof cages (45 x 35 cm) made from mesh

net (105µ; Sefar Filter Specialists Pty Ltd., Malaga, WA; Figure 3.1) and supported by steel-

rod stands. At the beginning, all runners propagated were checked daily for any insects

including WFT and any disease symptoms. Plants with any insect or with disease symptoms

were removed from the glasshouses. This was done to ensure healthy plants used in experiments

in later stages. All pots were fitted with a sprinkler operated with automatic timers.

During summer, plants were watered every third day, while in winter and spring

watering was once a week. A liquid fertiliser (Thrive®

, Yates, Australia; NPK: 12.4: 3:

6.2; rate: 5mL/2 L water) was applied once a month.


WFT was reared on calendula, Calendula officinalis L. (Asteraceae), planted in plastic

pots (50x100 mm) with potting mix (Baileys Fertilisers, Rockingham, WA) kept in an

insect-proof Perspex cage (500 mm high, 420 mm deep and 400 mm wide) with 105 µ

mesh net fitted on a Nylex tote box (320mm x 420mm; Blyth Enterprises Pty Ltd, Australia).

Plants with WFT were kept in a glasshouse at UWA from July 2006 to November 2008.

All pots were fitted with sprinklers operated with automatic timers. Plants were watered

every third day during summer and once a week in winter and spring. Every second

week, adults were collected from caged plants and released onto new potted plants at

the base to ensure the continuous availability of WFT.

To obtain uniformly aged WFT for this trial, adults (20 individuals) were collected and released

onto fresh caged plants and allowed to lay eggs for 24 h. After 24 h, all adults (20 individuals)

were removed with a small aspirator. Plants were then checked daily for larvae. The newly

hatched larvae were removed, and released onto a strawberry leaf that was placed on a

moistened filter paper in a Petri dish (150 x 15 mm). The time and date of collection were

recorded. Larvae that hatched on the same day were grouped together and maintained on

strawberry leaves in Petri dishes until pupation. Strawberry leaves were changed as required.

Pupae were maintained on a moistened filter paper in a Petri dish until adult emergence. Adults

that emerged on the same day were used in this trial.

3.2.1.3 Predatory mites

Predatory mites [T. montdorensis, N. cucumeris and H. miles] used in the study were

obtained from commercial suppliers (Biological Services, SA; Chilman IPM Services,


70

WA; and Beneficial Bug Company, NSW). Mites were provided in plastic buckets

containing vermiculite. Trials were conducted immediately upon receipt of mites.

3.2.2 Glasshouse experiment: effect of cultivars and predatory mites with or

without spinosad on western flower thrips

In order to evaluate the effect of cultivar and efficacy of predatory mites with or

without spinosad on WFT, an experiment with a split plot design with factors: cultivar,

spray treatment and predatory mite was carried out in a glasshouse (25 ± 2 ⁰C, 60-70%

RH) at UWA. Eighty potted strawberry plants of each cultivar (Camarosa, Camino Real

and Albion) at the 2-3 leaf stage (2-3 weeks old) were divided into two groups. One

group of plants was sprayed with a spinosad solution (80 mL/100 L rate, 0.096 g a.i./L)

using a hand-held atomiser (Hills Sprayers, BH220063) until run-off. The other group

of plants was sprayed with distilled water (control). All plants were then covered with a

thrips-proof cage as described in 3.2.1.1. The bottom end of the cage was secured with

tape. The top end of the cage was closed with a rubber band.

WFT adults and predatory mites were collected with an aspirator f rom the stock culture,

and kept in separate glass vials. On the appropriate release date, opened vials with WFT

or predatory mites were placed at the base of the plant. Fifteen WFT adults (2 d old)

were released onto each plant one day after spraying. Each group of sprayed plants from

each cultivar with WFT adults was further divided into four groups (treatments), with

10 plants per treatment: (i) No mites, (ii) T. montdorensis, (iii) N. cucumeris, and (iv)

H. miles. Six mites were released on each plant-receiving mites six days after the spray

treatment. Each plant was considered a replicate. During experimental periods,

experimental plants were kept flowerless. Plants were checked daily and any blooms

developed were removed by a pair of sharp scissors.

Twenty-four h after WFT release, WFT adults and larvae on each plant were counted

daily for three weeks. Each day, all leaves of each plant were checked thoroughly with a

hand-held illuminated magnifying glass [50mm (2") diameter 2x power with 4x bifocal

magnifier] and numbers of WFT adults and larvae were recorded. WFT counts were

made between 0600 to 0800 h.

In order to determine the number of predatory mites (T. montdorensis and N. cucumeris)

per plant, plants were cut at the base at the end of the trial and immediately placed in a

container (500 ml) with 80% ethyl alcohol. Later, plant materials were sieved with a


71

double-layer sieve (mesh size 105 µ) and checked under a stereomicroscope, and the

numbers of T. montdorensis and N. cucumeris per plant were recorded. To determine the

numbers of H. miles (soil dwelling), the plants were cut as described above, but the top

soil (2-3 cm) from pots was also collected and placed immediately into a container with

80% ethyl alcohol. Later, the plant materials and the soil were sieved as mentioned

above and were checked under stereomicroscope for H. miles. Unfortunately, no H. miles were

recovered.

All trial pots were fitted with a sprinkler so that water did not reach the leaves and upper

parts of the plant. This was intended to avoid any wash out of WFT or mites. Although

the sprinkler was adjusted so that water could not reach plant leaf or upper parts, as an

extra precaution, the watering time was set for the afternoon (1900 h) and plants were

watered once per week.

3.2.2 Data analysis

To determine the effect of cultivars (Camarosa, Camino Real and Albion) and predatory

mites (no mites, T. montdorensis, N. cucumeris and H. miles) with or without spinosad

on WFT over time (days), adults and larvae were analysed separately with a repeated

measures ANOVA (independent fixed variables: cultivars, spray treatments, predatory

mites, sampling days; random variable: plant number; response variables: adults, larvae)

(Proc Mixed Procedure). The data from days seven, 14, and 21 were used (end data

point of each week). Due to significant interaction of cultivars, spray and predatory

mites over time (days) on WFT adults and larvae, additional ANOVAs (repeated

measures, Proc Mixed Procedure) were conducted for each cultivar separately (Quinn

and Keough 2002). Because there was a significant interaction of spray and predatory

mites over time, further ANOVAs (repeated measures, Proc Mixed Procedure) were

conducted for each spray and each cultivar. Due to the number of post hoc multiple tests, an

adjustment to the significance level was made [α = 0.00833= (0.05/3*2)]. If ANOVAs were

significant, means were separated using least square means (SAS 2002-2003).

The influence of cultivars and spray on predatory mites (T. montdorensis and N. cucumeris) was

determined by two-way ANOVAs (Proc Mixed Procedure) separately for T. montdorensis and

N. cucumeris. The difference in mean numbers of T. montdorensis and N. cucumeris was

assessed using two-sample t-test (Proc ttest Procedure) separately for spray treatments in each

cultivar.


72

Square root was applied to meet the assumption of homogeneity of variances (Zar

1999). Data were reverse transformed for presentation. All statistical analyses were

performed using Statistical Package SAS 9.1(SAS 2002-2003). Figures were constructed

using Graphpad Prism 5.0 (GraphPad 2007).

3.3 Results

3.3.1 Western flower thrips

The numbers of WFT adults per plant were influenced by predatory mites (T.

montdorensis, N. cucumeris, and H. miles) and cultivar (Camarosa, Camino Real and

Albion) in the presence or absence of spinosad, and these effects changed over time

(Interaction: F = 9.2612, 432, P < 0.0001). Consequently, a series of ANOVAs for each

cultivar (Camarosa: F 6, 144 = 9.75, P < 0.0001; Camino Real: F 6, 144 = 4.29, P = 0.0005;

Albion: F 6, 144 = 13.02, P < 0.0001) were performed (Appendix, 3.1). As interactions in

these three-way ANOVAs were also significant, the effects of predatory mites on WFT

adults were determined by further ANOVAs for each spray treatment in each cultivar

(Appendix 3.1).

Before presenting the more detailed results, some general trends were apparent. Overall,

mean numbers of WFT adults per plant were lower on plants treated with spinosad

(19.95 ± 1.73) compared to water (32.96 ± 1.74) across the different mite treatments

(Figure 3.2). The number of WFT on the plants with mites (T. montdorensis: 21.47 ±

1.66, N. cucumeris: 24.33 ± 1.86, H. miles 26.89 ± 2.02) was lower than those without

mites (33.14 ± 1.56). There were fewest WFT on Camino Real (19.89 ± 0.94), followed

by Albion (21.66 ± 1.06); most WFT were found on Camarosa (24.84 ± 1.25).


73

No mites Tm Nc Hm0

10

20

30

40

50Spinosad Water

Num

ber

of

WFT

ad

ult

s p

er p

lan

t (M

ean

SE

)

Figure 3.2 Mean numbers of WFT adults per plant treated with spinosad or water and

either no predatory mites or one of three species of predatory mites. Within each group

(X-axis), means were separated by LS means (α = 0.05). Tm = T. montdorensis, Nc = N.

cucumeris, Hm = H. miles.

At day seven, the number of WFT adults on any of the cultivars in the spinosad -treated

plants did not differ (Figure 3.3). At days 14 and 21 on plants sprayed with spinosad,

predatory mite species reduced the number of WFT adults over time, regardless of

cultivar (Camarosa F6,72 = 28.22, P < 0.0001, Camino Real F6,72 = 39.04, P < 0.0001,


74

0

20

40

60

80

No mites Nc Hm

a

a

a a a

a

bca

b

cd

Tm

0

20

40

60

80

a a a a a ab bc

a bc

d

Num

ber

of

WFT

adults

per

pla

nt (M

ean

S

E)

7D 14D 21D0

20

40

60

80

a a a aa

bb

ca

b

cd

A

B

C

Figure 3.3 Effects of predatory mites on number of WFT adults per plant over time (7,

14, or 21 days after release of WFT) sprayed with spinosad on cultivars (A) Camarosa,

(B) Camino Real and (C) Albion. Means with different letters within each day were

significantly different from others (LS means, α = 0.00835). Tm = T. montdorensis, Nc

= N. cucumeris, Hm = H. miles.

Albion F6, 72 = 19.00, P < 0.0001, Figure 3.3). For all three cultivars, plants with no

mites had the highest numbers of WFT adults and differed significantly from the rest

(Figure 3.3). The number of WFT adults was lowest when plants received T.

montdorensis. However, on day 14, the numbers of WFT adults were not different on

Camarosa (Figure 3.2A) or Camino Real (Figure 3.2B) with T. montdorensis and N.

cucumeris, but were on Albion plants (Figure 3.2C). By day 21 there was a distinct


75

difference between the numbers of WFT adults on all cultivars with each of the different

mite species (Figure 3.3).

The response of WFT adults to the cultivars and predatory mites on those plants sprayed

with water was similar to those on plants sprayed with spinosad. There was no

difference in the mean number of WFT adults among any of the treatments on day seven

(Figure 3.4), and mites reduced the number of thrips adults across all cultivars (Figure

3.3). There was, however, a difference in the effect of predatory mites on the number of

WFT adults across all three cultivars over time: Camarosa (F 6, 72 = 22.90, P < 0.0001),

Camino Real (F 6, 72 = 19.7, P < 0.0001) and Albion (F 6, 72 = 83.33, P < 0.0001)

(Appendix 3.1, Figure 3.4). On days 14 and 21, plants of each cultivar with T.

montdorensis had the lowest numbers of WFT adults. However, on day 14 in Camarosa

and Camino Real, WFT adult numbers were not different between T. montdorensis- and

N. cucumeris-treated plants, but did differ in Albion. Similarly, on day 14, WFT adult

numbers were not different between Camarosa and Camino Real plants that received N.

cucumeris and H. miles, but differed on Albion. By day 21 the mean numbers of WFT

adults were different in all predatory mite treatments with a consistent trend for the

least number of thrips being on the plants with T. montdorensis, followed by N.

cucumeris and then H. miles (Figure 3.4 A, B, and C).

Overall, the number of WFT larvae per plant was lowest on plants sprayed with

spinosad (20.67 ± 1.12) compared with those that received water only (31.38 ± 1.57)

across the different mite treatments (Figure 3.5). The number of WFT larvae on the

plants with mites (20.85 ± 1.14, 22.88 ± 1.19 and 27.59 ± 1.45 for T. montdorensis, N.

cucumeris and H. miles respectively) was lower than those without mites (32.77 ± 1.60).

There were fewest WFT larvae on Camino Real (22.41 ± 1.14), followed by Albion

(26.53 ± 1.32). The highest number of WFT larvae was on Camarosa plants (29.13 ±

1.58). There was a significant interaction between the cultivars, spray, and predatory

mites over time (days) (F 12, 432 = 2.31, P = 0.0073) that influenced WFT larvae

numbers. Consequently, further ANOVAs for each cultivar were performed (Appendix

3.2).


76

0

20

40

60

80No mites Tm Nc

aa aa

a ab

bc

ab

c

d

Hm

0

20

40

60

80

a a aa a

ab

c

d

ab b

c

Num

ber

of

WFT

ad

ult

s p

er p

lan

t (M

ean

S

E)

7D 14D 21D0

20

40

60

80

a

a aa a

b

cd

ab

cd

A

B

C

Figure 3.4 Effects of predatory mites on the number of WFT adults per plant over time

(7, 14, or 21 days after release of WFT) sprayed with water on strawberry cultivar (A)

Camarosa, (B) Camino Real and (C) Albion. Means with different letters within each

day were significantly different from others (LS means, α = 0.00835). Tm = T.

montdorensis, Nc = N. cucumeris, Hm = H. miles.


77

No mites Tm Nc Hm0

10

20

30

40

50Spinosad Water

Nu

mb

er o

f W

FT

lar

vae

per

pla

nt (M

ean

S

E)

Figure 3.5 Mean numbers of WFT larvae per plant treated with spinosad or water and

either no predatory mites or one of three species of predatory mites. Within each group

(X-axis), means were significantly different (LS means, α = 0.05). Tm = T.


An interaction of spray and predatory mites treatment and time was evident in each

cultivar (Camarosa: F 6, 144 = 3.78, P = 0.001; Camino Real: F 6, 144 = 2.5,P = 0.022;

Albion: F 6, 144 = 2.76, P = 0.011); therefore, the impact of mite treatment and time on

the number of WFT larvae was evaluated separately by an ANOVA for each spray in

each cultivar (Appendix 3.2).

In those plants sprayed with spinosad, the number of WFT larvae was reduced in each

cultivar over time (days) when predatory mites were present (F 6, 72 = 8.77Camarosa

7.25Camino Real 5.71Albion, P < 0.0001) (Appendix 3.2, Figure 3.6). In each cultivar, plants

that received T. montdorensis had the lowest numbers of WFT larvae per plant, except

in Albion on day seven, when the N. cucumeris-treated plant had the lowest numbers of

WFT larvae (Figure 3.6C). However, on days seven and 14 in Camarosa (Figure 3.6A),

on day 14 in Camino Real (Figure 3.6B) and on day seven in Albion (Figure 3.6C),

mean numbers of WFT larvae were not different between the plants that received T.

montdorensis and N. cucumeris. On the other hand, in each cultivar, WFT larvae

numbers were highest on plants that received ‘no mites’. However, on day seven, in

Camarosa as well as in Albion, there was no difference in WFT larvae between plants

that received ‘no mites’ and H. miles (Figure 3.6).


78

0

20

40

60No mites Tm Nc

a ab

baa

b

c

ab

cd

Hm

0

20

40

60

d

ab

ca

ab

c

a b

c

d

Nu

mb

er o

f W

FT

lar

vae

per

pla

nt (M

ean

S

E)

7D 14D 21D0

20

40

60

aa

bb a bc

d

ab

c

d

A

B

C

Figure 3.6 Effects of predatory mites on WFT larvae per plant over times (7, 14, or 21

days after WFT release) sprayed with spinosad on strawberry cultivar (A) Camarosa,

(B) Camino Real and (C) Albion. Means with different letters within each day were



Similar to the spinosad treatment, WFT larvae numbers per plant were significantly

influenced by predatory mites (Camarosa: F 6, 72 = 3.99, P = 0.0017; Camino Real: F 6,

72 = 3.87, P = 0.0021; Albion: F 6, 72 = 3.59, P = 0.0036) (Appendix 3.2, Figure 3.7).

Typhlodromips montdorensis-treated plants had the lowest numbers of WFT larvae,

except on day seven in Albion, when the N. cucumeris-treated plants had the lowest

numbers of WFT. However, on day 7 in Camarosa and Albion and on day 14 in Camino


79

Real, there was no difference in WFT larvae between plants that received T.

montdorensis and N. cucumeris. On the other hand, at all times in each cultivar, mean

numbers of WFT larvae were highest on plants that received ‘no mites’.

0

20

40

60No mites Tm Nc

aa

bc

a

bc

d

a

b

cd

Hm

0

20

40

60

ab

bc aa

bc

ab

cd

Nu

mb

er

of

WFT

larv

ae p

er

pla

nt (M

ean

S

E)

7D 14D 21D0

20

40

60

aa bc

ab

cdc

a

a

b

A

B

C

Figure 3.7 Effects of predatory mites on WFT larvae per plant over times (7, 14, or 21

days after WFT release) sprayed with water on strawberry cultivar (A) Camarosa, (B)

Camino Real and (C) Albion. Means with different letters within each day were




80


The numbers of T. montdorensis and N. cucumeris per plant were unaffected by

spinosad (F T. montdorensis = 3.57 1, 54, P = 0.06; F N. cucumeris = 0.052 1, 54, P = 0.116) or

cultivar (F T. montdorensis = 1.11 2, 54, P = 0.336; F N. cucumeris = 0.51 2, 54, P = 0.604) (Figure

3.78). In both spray treatments, T. montdorensis numbers were higher than N. cucumeris

(Figure 3.9), but the difference was not significant in Camarosa ( tspinosad = 0.78 18, P =

0.44; twater = 0.22 18, P = 0.83), Camino Real (tspinosad = 1.64 18, P = 0.12; twater = 1.33

18, P = 0.20) nor in Albion (tspinosad = 1.85 18, P = 0.08; twater = 1.61 18 P = 0.13).

0

5

10

15

20

25

Camarosa Camino Real Albion0

5

10

15

20

25

Spinosad Water

A B

C D

Nu

mber

of

pre

dat

ory

mit

es (

Mea

n

SE

)

Figure 3.8 Mean numbers of T. montdorensis (A and B) and N. cucumeris (C and D) per

plant in relation to strawberry cultivar and spray treatment.


81

0

5

10

15

20

25N. cucumerisT. montdorensis

Camarosa Camino Real Albion0

5

10

15

20

25

A

B

Num

ber

of

pre

dat

ory

mit

es p

er p

lant

(Mea

n

SE

)

Figure 3.9 Comparison of mean numbers of T. montdorensis and N. cucumeris per plant

sprayed with (A) spinosad and (B) water.

3.4 Discussion

This study suggests that it is possible to apply spinosad and subsequently release predatory

mites (T. montdorensis, N. cucumeris and H. miles) with a resultant reduction in WFT adults

and larvae, and little or no negative effect on natural enemies. Control of WFT can be further

improved by the use of resistant cultivars. As was presented in Chapter 2, it appears that

strawberry cultivars had a significant influence on WFT numbers, with Camino Real being least

favourable to WFT and Camarosa being most favourable. As in Chapter 2, this study did not

explore the specific factors or mechanisms that may be responsible for differences in WFT

adults. However, this study does demonstrate that the varieties interact with other tactics for

control of WFT.

All three predatory mites were more successful in reducing WFT numbers in cultivar Camino

Real than Albion or Camarosa, although the actual number of predatory mites (T. montdorensis

and N. cucumeris) did not differ amongst cultivars. Brown et al. (1999) tested the predation

efficacy of predatory mites, N. cucumeris and Iphiseius degenerans (Berlese) (Acari:

Phytoseiidae) against WFT on 12 plant families and found that the WFT predation rate of these


82

mites was different on different plant species. The variation in predation efficacy might be due

to differences in plant architecture (Kareiva and Sahakian 1990), surface texture (Kareiva and

Sahakian 1990) and plant chemistry (Price et al. 1980). The susceptibility of herbivores to

predators is often related to the nutritional quality of plants on which the herbivores are feeding

(Price et al. 1980). WFT numbers were low on the cultivar Camino Real, even in the absence of

mites. The difference in WFT numbers on different cultivar-mite combinations suggests that

cultivars not only influence WFT numbers, but also influence the effectiveness of predatory

mites.

Regardless of cultivar, all three mite species reduced the numbers of WFT adults and larvae, but

T. montdorensis appears to be the most effective species in suppressing WFT, followed by N.

cucumeris and H. miles. The efficiency of natural enemies in a pest management program often

varies from species to species of both predator and prey (Chyzik et al. 1996, Berndt et al. 2004a,

Berndt et al. 2004b), and the reasons are likely be multi-factorial. These may include differences

in prey preference, predation rate, distribution and population development (Chyzik et al. 1996,

Berndt et al. 2004b). Bakker and Sabelis (1989) reported that N. cucumeris is able to predate on

only the smallest thrips, which effectively limits them to attacking first instar WFT larvae.

Similarly, T. montdorensis feeds on first instar WFT larvae. The other mite species tested, H.

miles, inhabits the top soil layer (at about 1.3 cm depth) and preys only on WFT pupae

(Glockemann 1992), although some studies suggest that this mite also preys on late second

instar larvae (Berndt 2002, Berndt 2003). Berndt et al. (2004b) found that the predatory mite,

Hypoaspis aculeifer (Canestrini) (Acari: Laelapidae) is more effective against WFT soil

dwelling stages compared to Stratiolaelaps (=Hypoapsis) miles (Berlese) (Acari: Laelapidae),

mainly because the predation rate of H. aculeifer is higher than S. miles. Similarly, Brødsgaard

(1989) and van Houten et al. (1995) reported that N. cucumeris consumed higher numbers of

WFT larvae compared to Amblyseius barkeri (Swirskii) (Acari: Phytoseiidae). Neoseiulus

cucumeris is reported to feed on an average of six WFT larvae (first instar) per day (Zilahl-

Balogh et al. 2007), whilst T. montdorensis feeds on 7-14 larvae per day (Steiner et al. 2003).

Rhodes and Liburd (2006) also reported variation in performance of predatory mites against

two-spotted spider mites (Tetranychus urticae) in field-grown strawberry. Rhodes and Liburd

(2006) found that Neoseiulus californicus and Phytoseiulus persimilis both significantly reduced

T. urticae, though their performance varied from each other. Phytoseiulus persimilis took longer

(one week) than N. californicus to bring two-spotted spider mites under control (<10 mite per

leaflet, ETL). Wiethoff et al. (2004) also found that the efficacy of predatory mites against WFT

varied. In cucumber, N. cucumeris is more successful in reducing WFT numbers than H.

aculeifer (Wiethoff et al. 2004).


83

The distribution of predatory mites often influences their effectiveness in pest management

programs. Typhlodromips montdorensis is a generalist predator and has the ability to distribute

rapidly on different plant parts (Steiner and Goodwin 1998, 2001) which may give this predator

an advantage in reducing WFT over other predatory mites. However, it has been reported that

the within-plant distribution of N. cucumeris is uneven and it prefers the lower part of the plant,

while WFT prefer to remain on the upper part of the plants (cucumber) (Messelink et al. 2006).

Variation in population development rate among different predatory mites might also account

for the variation in effectiveness (Messelink et al. 2006). The present results indicate that T.

montdorensis numbers were slightly, but not significantly, higher than N. cucumeris. However,

even this small difference might contribute to a difference in effectiveness of WFT suppression.

This study did not explore the specific factors that influence the effectiveness of predatory mites

in suppressing WFT; nonetheless, all three predatory mites provide some control of WFT.

Typhlodromips montdorensis seems to be the most effective. However, to maximise the

efficiency of predatory mites in WFT management, future research is needed to evaluate factors

that influence predatory mite efficiency.

In the present study, the numbers of WFT were lower in treatments with one of the mite species

and when spinosad was applied. Spinosad was not detrimental when applied before mite

releases. This suggests that spinosad had no adverse effects on predatory mites at least seven

days after insecticide application at the recommended rate. Thoeming and Poehling (2006)

reported that an application of the botanical azadirachtin (NeemAzal-U, 17% azadirachtin) with

two predatory mite species reduced the numbers of WFT by up to 99% without causing any

significant harm to predatory mites. The previous findings of Ludwig and Oetting (2001) and

Ludwig (2002) indicated that the combined application of spinosad and the predatory

anthocorid bug, Orius insidiosus Say (Heteroptera: Anthocoridae) significantly reduced WFT

numbers in glasshouse potted chrysanthemums and marigold, compared to the control (without

spinosad or Orius). Ludwing and Oetting (2001), Ludwig (2002), and Funderburk et al. (2000)

demonstrate that spinosad had no or little effect on O. insidiosus. However, laboratory studies

by Elzen et al. (1998) and Pietrantonia and Benedict (1999) suggest that spinosad has low

toxicity to O. insidiosus. Similarly, Kongchuensin and Takafuji (2006) reported that there was a

significant negative effect of spinosad on eggs and the immature stage of the predatory mite N.

longispinosus, if exposed to fresh residues within 48 hrs. However, there was no or very little

negative influence of spinosad on adults, eggs or the immature stage of N. longispinosus, if

exposed seven days after spinosad is applied (Kongchuensin and Takafuji 2006).

In conclusion, the management of WFT can be improved by releasing predatory mites after a

spinosad application without any apparent detrimental effect. Moreover, again, Camino Real


84

appears to be the least preferred cultivar to WFT. Of the three predatory mite species, T.

montdorensis in conjunction with spinosad appears to be the most effective combination for the

management of WFT. However, because of the low economic threshold of WFT in strawberry,

single species releases of mites such as T. montdorensis may not be sufficient to suppress WFT.

Multiple species release of predatory mites (Wiethoff et al. 2004, Premachandra et al. 2005)

combined with a spinosad application could be a better strategy for WFT management. Thus, it

is worthwhile to test the compatibility of T. montdorensis, N. cucumeris, and H. miles in

multiple releases with spinosad. In addition, increasing the numbers of predatory mites

(decreasing the ratio of predatory mites to WFT) and using a shorter period between spray

application and predatory mite release could further enhance WFT control. However, a shorter

time lapse between application and predatory mite release could be toxic to predatory mites.

Thus, a study is required to evaluate the residual toxicity of spinosad to predatory mites. This

study was a small-scale study (plants covered with mesh net) and so the conclusions that can be

drawn at this point are somewhat limited. A large-scale experiment in either the glasshouse, low

tunnel environment or both is needed to confirm the present findings before any

recommendation to commercial growers can be made.





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CHAPTER IV

Single versus multiple releases of predatory mites (Acari) combined with a spinosad

application for the management of western flower thrips, Frankliniella occidentalis

(Pergande) (Thysanoptera: Thripidae) in strawberry, Fragaria x ananassa Duchesne

(Rosaceae)


Hypoaspis miles, spinosad, single species releases, multiple species release, strawberry

Abstract


major pest of strawberry in Australia. Three predatory mites, Typhlodromips montdorensis

(Schicha) (Phytoseiidae), Neoseiulus cucumeris (Oudemans) (Phytoseiidae) and Hypoaspis

miles (Berlese) (Laelapidae) are commercially available in Australia for thrips management.

This study sought to determine the effect of single species versus multiple species release of

these predatory mites in combination with an insecticide on F. occidentalis. Spinosad is

currently registered in Australia and is efficacious against thrips and regarded to be compatible

in an IPM program. In the glasshouse, strawberry plants (cv Camino Real) were sprayed once

with either spinosad at the recommended rate (80 mL/100 L rate, 0.096 g a.i./L) or water

(control). Frankliniella occidentalis adults were released onto plants 24 h after spraying, and

mites were released six days later. Mites were released as single-species, two-species, or three-

species combinations. Spinosad significantly reduced F. occidentalis numbers compared to the

control (water). Typhlodromips montdorensis, N. cucumeris and H. miles significantly reduced

F. occidentalis numbers compared to the control (no mites). Spinosad had no effect on mites, as

their numbers (T. montdorensis and N. cucumeris) were higher at the end of the trial than when

initially released. Numbers of Typhlodromips montdorensis and N. cucumeris did not differ

between spinosad and water-treated plants. As H. miles is a soil-dwelling mite, their numbers

could not be counted. Mites released in combination with spinosad were more effective at

reducing thrips numbers than individual applications of predatory mites alone: ‘T. montdorensis

and H. miles’ was the most effective combination. The effectiveness against F. occidentalis was

not different between releases of ‘T. montdorensis and H. miles’ and ‘T. montdorensis, N.

cucumeris and H. miles’. When T. montdorensis and N. cucumeris were released as single

species separately, there was no significant difference in their numbers, though T. montdorensis

numbers were relatively higher than N. cucumeris. When released as a double-species

combination, there were significantly more T. montdorensis than N. cucumeris. In the triple-

Chapter IV: Effects of cultivar and predatory mites

91

species combination, T. montdorensis and N. cucumeris numbers were not significantly

different. The results suggest that spinosad followed by releases of either ‘T. montdorensis and

H. miles’ or ‘T. montdorensis, N. cucumeris and H. miles’ can reduce thrips number effectively.

4. 1 Introduction

Western flower thrips (WFT), Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae)

is an important economic pest worldwide (Brødsgaard and Albajes 1999, Jones et al. 2002, Kirk

and Terry 2003), causing extensive crop losses (Lewis 1998). With its piercing-sucking

mandibles, WFT penetrates epidermal and subepidermal cells causing extensive damage: this

includes deformation and growth reduction of the plant, and silver scars on fruits and leaves

(van Dijken et al. 1994, de Jager et al. 1995). In addition to direct damage, WFT also causes

indirect damage by transmitting tospoviruses (Wijkamp et al. 1996, Ullman et al. 1997). Since

its detection in 1993 in Western Australia (Malipatil et al. 1993), WFT has spread to each state

and territory, except the Northern Territory and become a major pest in several crops including

strawberry. Strawberry [Fragaria x ananassa Duchesne (Rosaceae)] is an intensively managed

crop cultivated for its fresh, aromatic, red berries. In Australia, the strawberry industry has

grown steadily over the last few years with a gross value of approximately $AUD308 million

per annum (Anonymous 2009). However, strawberry production is often hampered by direct

damage caused by WFT in low tunnel, open fields, and glasshouses (Ullio 2002).

Whilst insecticides are the main control method (Herron and Cook 2002), because of its small

size, secretive habit, high reproductive potential, and ability to develop resistance to insecticide,

WFT can be difficult to control (Jensen 2000). WFT has also developed resistance to several

major classes of chemicals (Brødsgaard 1994, Jensen 1998, Jensen 2000) throughout the world.

As an integrated approach, there is a growing trend to use two or more species of natural

enemies to suppress insect pest populations (Premachandra et al. 2003, Avilla et al. 2004,

Blümel 2004, Brødsgaard 2004, Chau and Heinz 2004, Chow and Heinz 2004, Hoddle 2004,

Shipp and Ramakers 2004, Thoeming and Poehling 2006, Chow et al. 2008). Overseas,

phytoseiid mites are used to manage WFT in field and glasshouse crops (Chant 1985, van

Lenteren and Woets 1988, McMurtry and Croft 1997). For example, anthocorid bugs of the

genus Orius and phytoseiid mites of the genus Amblyseius are commonly used for the control of

WFT in glasshouse-grown crops in Europe and North America (Brødsgaard 2004, Shipp and

Ramakers 2004), but resulting benefits have not quantitatively validated (Blockmans and

Tetteroo 2002, Skirvin et al. 2006). Some of the studies support the premise of biological

control agent compatibility (Gillespie and Quiring 1992, Wittmann and Leather 1997,


92

Brødsgaard and Enkegaard 2005), while, others oppose this view (Magalhăes et al. 2004,

Sanderson et al. 2005). Schausberger and Walzer (2001) demonstrated that interspecific

competition may occur when different species of predatory mites are combined together and

prey specificity can influence the quality and intensity of predator-predator interactions.

Schausberger and Walzer (2001) reported that in perennial, glasshouse-grown crops, the release

of Phytoseiulus persimilis Athias-Henriot and Neoseiulus californicus (McGrgor) (Acari:

Phytoseiidae) released to control carmine spider mite, Tetranychus cinnabarinus

(Tetranychidae), could have complementary effects. However, the augmentative release of

predatory mites in single or multiple releases is not always sufficient to manage WFT in crops

with the low economic threshold (Gillespie and Ramey 1988, Bakker and Sabelis 1989,

Gillespie 1989), particularly when low damage is required.

Until recently, Australian growers had no effective biological control options for WFT due to

quarantine restrictions prohibiting their importation (Steiner and Goodwin 2001). Recently four

predatory mite species have become available to Australian growers, two of which are natives:

Typhlodromips montdorensis (Schicha) and Typhlodromus occidentalis (Schicha)

(Phytoseiidae) (Steiner and Goodwin 2000). Neoseiulus cucumeris (Oudemans) (Phytoseiidae)

and Hypoaspis miles (Berlese) (Laelapidae), native to New Zealand, were recently confirmed as

occurring in Australia, despite no record of deliberate introduction. Typhlodromips

montdorensis, N. cucumeris and H. miles are commercially available and have been used for

WFT management (Anonymous 2006).

The compatibility of these predatory mites has not been tested, nor has their effect in

conjunction with insecticides currently used for WFT management in Australia been evaluated.

Spinosad™

(Dow AgroSciences, USA), a mixture of tetracyclic-macrolide compounds, has been

classified as a reduced-risk bio-insecticide (Sparks et al. 1998) and is the primary insecticide

used in Australia for WFT control. However, impact of spinosad on predators is varied

(Williams et al. 2003, Cote et al. 2004, Jones et al. 2005, Villanueva and Walgenbach

2005, van Driesche et al. 2006). Thus, the objectives of this study were to (i) investigate the

effectiveness of commercially available predatory mites [T. montdorensis, N. cucumeris and H.

miles] with or without spinosad against WFT, and (ii) determine the effectiveness of single

versus combined release of predatory mites for the management of WFT.

4.2 Materials and methods

The experiment was conducted in a glasshouse (25 ± 2⁰C, 60-70% RH, 16: 8 L: D cycle)

at the University of Western Australia (UWA) from November 2007 to January 2008.


93


4.2.1.1 Strawberry cultivar

Strawberry [Fragaria ananassa Duchesne (Rosaceae)] cv Camino Real (short-day length

cultivar) was used in this study. In previous experiments (Chapters 2 and 3), Camino

Real was found to be less preferred to WFT and was used in this experiment. Strawberry

runners were obtained from a commercial grower in June 2006, and propagated in pots

(32.5l x 32.5w x 40.5h cm) containing potting mix (Baileys Fertilisers, Rockingham, WA)

in glasshouses at the Department of Agriculture and Food Western Australia (DAFWA) and

UWA. All potted plants were covered with a modified thrips cage (45 x 35 cm) made from

thrips-proof mesh net (105µ, Sefar Filter Specialists Pty Ltd., Malaga, WA; see Chapter 3) and

supported by quadrate steel-rod stands. All pots were fitted with sprinklers. The plants

were watered every third day. A liquid fertiliser (Thrive®

, Yates, Australia; NPK: 12.4:

3: 6.2; rate: 5mL/2 L water) was applied once a month.


WFT were initially collected from calendula, Calendula officinalis L. (Asteraceae) at

DAFWA, South Perth and reared on calendula in pots (50x100 mm). Pots were kept in

thrips proof Perspex cages (500 x 420 x 400 mm H x D x W), fitted with 105 µ mesh

net. The cage fitted on top of a Nylex tote box (Blyth Enterprises Ptd ltd, Australia; 320

x 420 mm). Cages were kept in a glasshouse and tunnel houses at UWA from July 2006 to

November 2008. Plants were watered as described above. Every second week, adults

were collected from caged plants using an aspirator and released onto new potted

calendula plants to ensure the continuous availability of WFT during study periods.

To obtain uniformly aged WFT, 20 adults were collected from the colony, released onto fresh

caged plants, and allowed to lay eggs for 24 h. After 24 h, the adults were removed with a small

aspirator. The plants were checked daily for larvae emergence. Newly hatched larvae were

removed and released onto a strawberry leaf on a moistened filter paper in a Petri dish (150 x 15

mm). The leaf petiole was covered with cotton, soaked in a 10% sugar solution to extend the life

of the leaf. The top of the Petri dish was covered with thrips-proof mesh (105 µ) and the edges

of the Petri dish and mesh were sealed with paraffin film (Parafilm M®, Micro Analytix Pty

Ltd)] and kept in a controlled temperature room (25±1⁰C, 50-60% RH, 16:8 h L: D regime).

Larvae hatched on the same day were transferred to a new Petri dish as above and allowed to

pupate. Adults that emerged on the same day were used in trials.


94


Predatory mites [T. montdorensis, N. cucumeris and H. miles] used in the study were

sourced from commercial suppliers (Biological Services, SA; Chilman IPM Services,

WA; and Beneficial Bug Company, NSW). Mites were provided in plastic buckets

containing vermiculite. Trials were conducted immediately upon receipt of mites.

4.2.2 Experiment: effect of single versus multiple species releases of mites

combined with spinosad on WFT

To determine the effectiveness of predatory mites with or without spinosad, an

experiment with a split-plot design was conducted in a glasshouse at UWA. One

hundred and sixty potted strawberry plants, 2-3 weeks old with 2-3 leaves (excess

leaves were pruned), were divided into two groups (80 per group), and sprayed with

either spinosad at the recommended rate (80 mL/100 L rate, 0.096 g a.i./L) or water

with a hand-held atomiser (Hills Sprayers, BH220063) until run-off (after van Driesche

et al. (2006)). Plants were then covered with a modified thrips cage (45 x 35 cm, open

both ends) made from thrips-proof mesh (105 µ), supported by quadrate steel-rod

stands. The bottom end of the cage was taped to the pot. The top end of the cage was

closed with a rubber band.

Fifteen previously collected 15 WFT adults (2 d old) were released onto each plant 24 h

after spraying. Each group of 80 plants (spinosad or untreated control) were further

divided into eight treatments (mite release) with 10 plants per treatment. All possible

combinations (single and multiple species) of mite were included:

(i) no mites

(ii) T. montdorensis

(iii) N. cucumeris

(iv) H. miles

(v) T. montdorensis and N. cucumeris

(vi) T. montdorensis and H. miles

(vii) N. cucumeris and H. miles

(viii) T. montdorensis, N. cucumeris and H. miles.

Predatory mites were released six days after spraying (Khan and Morse 2006). The

numbers of predatory mites released per plant were (i) six (ii) three + three and (iii) two


95

+two + two for single, double and triple species combinations respectively. WFT (adults

and larvae) were counted every third day for three weeks (from the release of WFT to

the end of the third week). Thrips were counted between 0600 to 0800 h, when they

were less active. Each plant was checked with a battery-powered magnifying glass

[50mm (2") illuminated round 2x power with 4x bifocal magnifier].

To assess the numbers of predatory mites, at the end of the trial, plants were removed

from pots and preserved in a container with 80% ethyl alcohol. The plant was washed

onto a double-layer sieve (made from 105µ mesh) and checked under a

stereomicroscope, and the numbers of T. montdorensis and N. cucumeris per plant were

recorded. To determine the numbers of H. miles (soil-dwelling), plant parts and top soil

(2-3 cm) from pots [H. miles released pots] were collected and preserved as above. The

plant materials and soils were washed onto a double-layer sieve as above and checked

under a stereomicroscope. However, no H. miles were recovered.

The trial pots were fitted with sprinklers (watering every third day) in such a way that

water did not reach the leaf and upper parts of the plant to avoid washing out WFT or

mites. As an extra precaution, the watering program was set for the afternoon (1900 h) .

4.2.3 Data analysis

The effectiveness of mite treatments (single-, double- or triple species-releases) with or

without spinosad on WFT numbers over time, were analysed with repeated measures

ANOVAs (Proc Mixed Procedure) with split-plot design. WFT adults and larvae were

separately analysed (independent fixed variables: mites treatments, spray treatment and

time; random variable: plant numbers; response variables: adults and larvae). Because

there was a significant three-way interaction between the main effects of spray,

predatory mites and time (days), additional ANOVAs (repeated measures) were

performed for each spray treatment (Quinn and Keough 2002). Due to the number of

multiple tests, an adjustment to the significance level was made [α = 0.025 (0.05/2)] . If

significant differences among means were detected, the means were separated using least square

means with the adjusted significance level (SAS 2002-2003).

The difference in predatory mite numbers for single species releases (T. montdorensis

and N. cucumeris) and treatment (spinosad, water) was analysed with two-way ANOVA

(Proc Mixed Procedure; independent fixed variables: spray, mite species; response

variable: numbers of mites). Similarly, two-way ANOVAs were used to determine the


96

difference in numbers of T. montdorensis and N. cucumeris in double-species (T.

montdorensis and N. cucumeris) and triple-species (T. montdorensis, N. cucumeris and

H. miles) combinations. A two-way ANOVA was used to determine the difference of T.

montdorensis and N. cucumeris when released in double-species combination with H.

miles (‘T. montdorensis and H. miles’ and ‘N. cucumeris and H. miles’). Two separate

two-way ANOVAs were used to determine the difference in numbers of T. montdorensis

in double species-releases (combinations: ‘T. montdorensis and N. cucumeris’ and ‘T.

montdorensis and H. miles). Similarly, two separate two-way ANOVAs were used to

evaluate N. cucumeris numbers in double species releases (combinations: T.

montdorensis and N. cucumeris’ and ‘N. cucumeris and H. miles).

Data were subjected to square root transformations when appropriate, to meet the

assumption of homogeneity of variances (Zar 1999) before the data were subjected to

statistical analysis. Data were reverse transformed for presentation in figures. All

statistical analyses were computed using the SAS 9.1 Statistical Package (SAS 2002-

2003), while figures were constructed using Graphpad Prism 5.0 (GraphPad Software

Inc 2007).

4.3 Results


4.3.1.1 Adults

Across all mite combination treatments, spinosad-treated plants had significantly fewer

WFT adults than water-treated plants (Figure 4.1, F 7,144 = 14.06, P < 0.0001; Appendix

4.1). Since there was a significant interaction (F 35, 720 = 2.52, P < 0.0001) between

spray, predatory mite treatments and time (days), further repeated measures ANOVAs

were carried out separately for each spray treatment (Appendix 4.1).


97

No mites Tm Nc Hm Tm+Nc Tm+HmNc+Hm Tm+Nc+Hm0

10

20

30

40

Spinosad Water

Num

ber

of

WFT

ad

ults

(Mea

n

SE

)

Figure 4.1 Comparison of mean number of WFT adults per plant sprayed with either

spinosad or water and in the presence of no mites or different mite combinations.

Within each group, means were significantly different (α = 0.05). Tm = T.


When plants were sprayed with spinosad, the numbers of WFT on plants with different

combinations of predators varied over time (F 35, 360 = 23.94, P < 0.0001) (Appendix

4.1, Figure 4.2A). Mites appeared to take some time to establish. Six and nine days after

WFT release, the numbers of WFT adults per plant were not different between mite

treatments (Figure 4.2A). From days 12 to 21, the mean numbers of WFT adults were

lowest on plants that received the ‘T. montdorensis and H. miles’ two-species

combination only. From days 15 to 21, the numbers of WFT adults did not differ

between plants with ‘T. montdorensis and H. miles’ and ‘T. montdorensis, N. cucumeris and

H. miles’ combinations. From days 12 to 21, the numbers of WFT adults were highest on

plants that received no mites.

When plants were sprayed with water only (control), the number of WFT adults on

plants with different combinations of predatory mites varied significantly over time (F

35, 360 = 37.21, P < 0.0001) (Appendix 4.1, Figure 4.2B). As with the spinosad

treatment, it took mites a few days to establish on plants. On day six, the mean numbers

of WFT adults were not different among the mite treatments. Differences in numbers of

WFT adults between mite treatments began to appear on day nine. From days 9 to 21,

plants with the two-species combinations of T. montdorensis and H. miles had the

lowest numbers of WFT adults, similar to the spinosad treatment. Plants that did not

receive any mites had the highest numbers of WFT.


98

0

20

40

60No mites Tm Nc Hm Tm+Nc Tm+Hm Nc+Hm Tm+Nc+Hm

6D 9D 12D 15D 18D 21D0

20

40

60

A

B

Nu

mb

er o

f W

FT

ad

ult

s (M

ean

S

E)

Figure 4.2 Effects of predatory mites on mean number of WFT adults per plant sprayed

with (A) spinosad or (B) water. X-axis represents days after initial WFT release. Tm =

T. montdorensis, Nc = N. cucumeris, Hm = H. miles.

No mites Tm Nc Hm Tm+Nc Tm+HmNc+Hm Tm+Nc+Hm0

10

20

30

40 WaterSpinosad

Num

ber

of

WFT

lar

vae

(M

ean

S

E)

Figure 4.3 Comparison of mean number of WFT larvae per plant sprayed with either

spinosad or water and in the presence of no mites or different mite combinations.

Within each group, means were significantly different (α = 0.05). Tm = T.


4.3.1.2 Larvae

Similar to WFT adults, there were fewer WFT larvae (F 7, 144 = 46.62, P < 0.0001) on

plants sprayed with spinosad compared to the water (control) Figure 4.3). There was a


99

significant three-way interaction (F 35, 720 = 2.52, P < 0.0001) between spray treatment,

predatory mite treatment and time (days) that influenced the numbers of WFT larvae.

Further ANOVAS for each spray (spinosad, water) were carried out (Appendix 4.1).

In plants sprayed with spinosad, the number of WFT larvae on plants with different

combinations of predatory mites varied over time (Figure 4.4A; F 35, 360 = 6.64, P <

0.0001). On days six and nine, the least numbers of WFT larvae were found on plants

that received ‘T. montdorensis and N. cucumeris’. From days 12 to 21, the two-species

mite combination of ‘T. montdorensis and H. miles’ had the lowest numbers of WFT

larvae. From days 15 to 21, WFT larvae numbers did not differ between plants treated

with the two-species combination of ‘T. montdorensis and H. miles’ or the three-species

combination. WFT larvae were generally highest on the plants that were not treated with

mites, except on day six, when no difference was found between plants that were not

treated with mites and plants treated with H. miles.

0

10

20

30

40

50

No mites Tm Nc Hm Tm+Nc Tm+Hm Nc+Hm Tm+Nc+Hm

6D 9D 12D 15D 18D 21D0

10

20

30

40

50

A

B

Num

ber

of

WF

T l

arvae

(M

ean

SE

)

Figure 4.4 Effects of predatory mites on the mean number of WFT larvae per plant

sprayed with (A) spinosad or (B) water. X-axis represents days after initial WFT

release. Tm = T. montdorensis, Nc = N. cucumeris, Hm = H. miles.


100

On plants sprayed with water, the number of WFT larvae on plants with different

combinations of predatory mites tended to increase over time (Figure 4.4B; F 35, 360 =

5.21, P <0.0001). Mites, in any combination, reduced the numbers of WFT larvae per

plant (Figure 4.4B). On day six, plants with ‘T. montdorensis, N. cucumeris and H.

miles’ had the lowest numbers of WFT larvae but there was little difference in the

numbers of thrips in any of the other mite treatments. The numbers of WFT larvae were

low on plants with all three predatory mite species throughout the rest of the trial,

though these did not differ between plants treated with ‘T. montdorensis and H. miles’

or ‘T. montdorensis and N. cucumeris’. By 21 days, plants that received ‘T.

montdorensis and H. miles’ had the lowest numbers of WFT larvae. However, the

number of WFT larvae did not differ between plants receiving ‘T. montdorensis and H.

miles’ or ‘T. montdorensis, N. cucumeris and H. miles’.


When T. montdorensis and N. cucumeris were applied singly, there was no significant

interaction between the spray treatment and mite species (F 1, 36 = 0.12, P = 0.73).

Overall (spinosad and water), the mean numbers of T. montdorensis (20.18 ± 0.86) and

N. cucumeris (19.75 ± 0.88) did not differ (F 1, 36 = 3.65, P = 0.06). When released as a

single-species, the overall mean numbers of predatory mites (T. montdorensis and N.

cucumeris) between spinosad (19.55 ± 1.22) and water (20.38 ± 0.88) treatments were

not different (F 1, 36 = 00.85, P = 0.36; Figure 4.5A). Similar to single-species releases,

in double-species releases (T. montdorensis and N. cucumeris), there was no interaction

between spray and mite treatments (F 1, 36 = 0.49, P = 0.49). However, there were

significantly more T. montdorensis (10.50 ± 0.49) than N. cucumeris (8.55 ± 0.55) (F (1,

36) = 15.29, P = 0.0004; Figure 4.5B). Overall, mean numbers of predatory mites (T.

montdorensis and N. cucumeris) per plant between spinosad (9.10 ± 0.55 mites/plant)

and water (9.95 ± 11.35 mites/plant) treatments did not differ (F 1, 36 = 2.95, P = 0.10).

When either T. montdorensis or N. cucumeris were released with H. miles, there was no

significant interaction of spray and mite treatments on mite numbers per plant (F 1, 36 =

0.01, P = 0.10). There was also no significant difference (F 1, 36 = 0.24, P = 0.63 Figure

4.5C) between the mean numbers of T. montdorensis (12.10 ± 0.91) and N. cucumeris

(12.35 ± 0.52). Similarly, overall mean numbers of predatory mites (T. montdorensis

and N. cucumeris) did not differ (F 1, 36 = 0.77, P = 0.39) between spinosad (12.00 ±

11.51 mites/plant) and water (12.45 ± 1.32 mites/plant) treatments. For the three -species

combination (T. montdorensis, N. cucumeris and H. miles), there was no interaction (F 1,

36 = 0.43, P = 0.52) of spray and mite treatments that affected predatory mite numbers


101

per plant. Though more T. montdorensis were found per plant (8.35 ± 1.42) than N.

cucumeris (7.50 ± 1.22), the difference (F 1, 36 = 1.22, P = 0.198) was not significant

(Figure 4.5). Similarly, overall mean numbers of predatory mites did not differ between

spinosad (7.95 ± 0.99) and water (8.95 ± 1.31) treatments.

0

5

10

15

20

25

b a

Tm Nc0

5

10

15

20

25

Tm Nc

Num

ber

of

pre

dat

ory

mit

es p

er p

lant

(Mea

n

SE

) A B

C D

Figure 4.5 Comparison of mean number of T. montdorensis and N. cucumeris per plant

applied with (A) single-species releases of T. montdorensis and N. cucumeris, (B)

double-species releases of T. montdorensis and N. cucumeris, (C) double species

releases of T. montdorensis and H. miles and N. cucumeris and H. miles, and (D) triple-

species release. Means with different letters differed significantly (α = 0.05). Tm = T.

montdorensis, N. cucumeris, Hm = H. miles.

When T. montdorensis were released with N. cucumeris and H. miles in two separate

combinations, spray and mite combination had no significant influence (F 1 , 36 = 0.73, P

= 0.398) on T. montdorensis numbers per plant. It also appeared that spray had no

significant (F 1, 36= 2.92, P = 0.096) influence on the overall numbers of T.

montdorensis per plant (10.90 ± 0.46 and 11.72 ± 0.55 for spinosad and water treatment

respectively). The mean numbers of T. montdorensis per plant were higher when

combined with H. miles than N. cucumeris (F 1, 36= 11.73, P = 0.002; Figure 4.6). When

N. cucumeris was released with T. montdorensis and H. miles in double-species

combinations, spray and mites combination had no significant interaction (F 1, 36 = 0.34,

P = 0.566). The mean numbers of N. cucumeris per plant were higher when combined

with H. miles than T. montdorensis and the difference (F 1, 36 = 49.51, P < 0.0001) was

significant (Figure 4.6). The overall mean numbers of N. cucumeris did not differ (F 1, 36


102

= 0.86, P = 0.361) between spinosad (10.19 ± 0.59) and water (10.70 ± 0.48)

treatments.

Tm+Nc Tm+Hm0

5

10

15

ab

Spinosad Water

Tm+Nc Nc+Hm0

5

10

15

a

b

Spinosad Water

T. montdorensis

N. cucumeris

Num

ber

of

pre

dat

ory

mites

(M

ean

SE

)

Figure 4.6 Mean numbers of (A) T. montdorensis and (B) N. cucumeris per plant in

double-species combinations. Tm = T. montdorensis, Nc = N. cucumeris, Hm = H.

miles.

4.4 Discussion

The results of the present study suggest that mites can reduce the WFT population. However,

single mite species may not be able to keep WFT populations under the economic threshold

level. According to Steiner and Goodwin (2005), 45% of flowers with five or more WFT adults

is the economic threshold in strawberry. In this study the plants were kept flowerless therefore it

is not possible to make a direct assessment of the impact of predatory mites on lowering the

thrips below an economic threshold. Nevertheless, the most effective treatment combination in

terms of lowering the thrips population was an application of spinosad followed by multiple

predatory mite releases (T. montdorensis, N. cucumeris and H. miles). The two-species

combination of T. montdorensis and H. miles, or the three-species combination provided better

management of WFT than single-species releases or other two-species combinations. Predatory

mite releases combined with a spinosad application also provided a higher suppression of WFT

adults and larvae than either spinosad or mites alone. This confirms the findings of the previous

study (Chapter 3), that integration of T. montdorensis, N. cucumeris or H. miles with spinosad is

more successful in reducing WFT in glasshouse-grown strawberry than applying either spinosad

or releasing predatory mites alone.


103

Present study demonstrates that either of the predatory mites and their combination performed

better against WFT when released after spinosad spray. While, in water treatment, either mite

species was able to reduce WFT population to some extent, which might not be enough to

reduce WFT population effectively. Previous studies have also shown that WFT can be

controlled with an insecticide application followed by the release of beneficials (Ludwig and

Oetting 2001, Ludwig 2002, Thoeming and Poehling 2006). Thoeming and Poehling (2006)

reported that an application of neem (Botanical, 17% azadiractin) combined with the release of

a combination of two predatory mite species increased efficacy to 99%, without causing any

significant harm to predatory mites. The previous findings of Ludwing and Oetting (2001) and

Ludwig (2002) suggest that applications of spinosad and the predatory bug, Orius insidiosus

Say (Hemiptera: Anthocoridae) to glasshouse- potted chrysanthemums and marigold

significantly reduced WFT compared to the control (without spinosad or Orius). Ludwing and

Oetting (2001), Ludwig (2002) and Funderburk et al. (2000) demonstrate that spinosad had no

or little effect on O. insidiosus. However, the laboratory studies of Elzen et al. (1998) and

Pietrantonia and Benedict (1999) showed that spinosad has low toxicity to O. insidiosus.

Similarly, Kongchuensin and Takafuji (2006) reported that fresh spinosad residues (up to 48 h

old) have a significant, negative effect on eggs and the immature stage of the predatory mite

Neoseiulus longispinosus (Evans) (Acari: Phytoseiidae). However, spinosad was not harmful to

N. longispinosus seven days after application. This study and the previous study (Chapter 3)

suggest that spinosad poses no detrimental effect to T. montdorensis, N. cucumeris and H. miles

if these mites are released six days after spraying.

Multiple-species release of predatory mites can be more effective in reducing WFT populations

than single-species releases. However, release of multiple species of predator may result in

intraguild predation (IGP) which can have a significant effect on the prey suppression (Losey

and Denno 1998). Although IGP is common in many predators (Vance-Chalcraft et al. 2007)

including several predatory mites (Schausberger and Walzer 2001, Walzer and Schausberger

2005), it is not known whether any intraguild predation can occur among T. montdorensis, N.

cucumeris and H. miles. Brodeur et al. (2002) recognised that the release of multiple predator

species is an effective strategy that would suppress pest populations in a manner that is more

economically viable than the use of single predator species. Wiethoff et al. (2004) reported that

cucumber plants had fewer WFT when N. cucumeris and H. miles were applied together than

either N. cucumeris or H miles alone. Similarly, combined releases of Phytoseiulus persimilis

(Athias-Henriot) and Neoseiulus californicus (McGregor) (Acari: Phytoseiidae) can provide

long-term suppression of carmine spider mite (Tetranychus cinnabarinus Boisduval

(Tetranychidae)) in potted gerbera plants, compared to individual-species releases

(Schausberger and Walzer 2001). Premachandra et al. (2005) reported that the emergence of


104

WFT adults was significantly lower if entomopathogens were released with the predatory mite,

Hypoaspis aculeifer (Berlese) (Acari: Laelapidae), than applications of either entomopathogens

or H. aculeifer alone. Premachandra et al. (2005) also showed that when foliage-inhabiting (T.

montdorensis) and soil-dwelling (H. miles) mite species were applied together, they provided

the highest suppression of WFT. Synergistic effects can be expected if a plant-dwelling predator

can evoke the escape behaviour of the prey, making the prey available for ground-dwelling

predators (Losey and Denno 1999).

In the present study, two foliage foraging predatory mites combined together provided better

results in suppressing WFT than their single-species release. However, this combination was

less effective than when combined with release of the soil-dwelling H. miles. This can be

explained by the resource competition between two predators, which occurs if two predators

compete for a shared prey (Janssen et al. 1998). As a result, one species might outcompete the

other. When T. montdorensis and N. cucumeris are released together, there were more T.

montdorensis than N. cucumeris, which is not the case when they are combined with H. miles

separately. When all three species were released, no interaction seemed to occur between T.

montdorensis and N. cucumeris. One possibility is that as in the two-species combination, plants

had higher numbers of T. montdorensis and N. cucumeris, which might increase the

interspecific competition. In the triple-species combinations, plants had less T. montdorensis

and N. cucumeris, and perhaps less competition for WFT. Thus for the successful

implementation of multiple releases of predatory mites in a pest management program, further

studies need to be carried out to determine if cannibalism, intra-guild predation and preference

between primary prey (pest population) and secondary prey (predator population) occurs,

especially when two or more predators utilise the same resource. Furthermore, it is also

important to determine the optimal release rate of predatory mites that can effectively reduce the

pest population, without having any negative impact on each other.

In conclusion, the integration of spinosad and multiple-species releases of predatory mites could

be a sustainable management strategy for WFT management in strawberry. When single-species

release is the only option, T. montdorensis in conjunction with spinosad appears to be the best

combination for the management of WFT in strawberry. WFT management could be further

improved by reducing the interval between spray application and predatory mite release.

However, the residual toxicity of spinosad to predatory mites would first need to be determined.


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of synchrony. Ecological Application 9: 378-386.










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Australian strawberry crops: within-plant distribution characteristics and action

thresholds. Australian Journal of Entomology 44: 175-185.









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influence of intraguld predaation on prey suppression and prey release: A meta-analysis.

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475.







Jersey.

CHAPTER V

Use of spinosad and predatory mites (Acari) for the management of western flower thrips,

Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) in strawberry [Fragaria x

ananassa Duchesne (Rosaceae)]: a field study

Key words: Frankliniella occidentalis, Typhlodromips montdorensis, Neoseiulus cucumeris,

Hypoaspis miles, spinosad, strawberry, low tunnel, integration, multiple release

Abstract

The efficacy of single- and multiple- species releases of predatory mites (Acari), Typhlodromips

montdorensis Schicha (Phytoseiidae), Neoseiulus cucumeris Oudemans (Phytoseiidae) and

Hypoaspis miles Berlese (Laelapidae) and their compatibility with spinosad (Success™

, Dow

AgroSciences, Australia) for the control of western flower thrips, Frankliniella occidentalis

(Pergande) (Thysanoptera: Thripidae) was evaluated in commercial strawberry. The trial was

carried out in a commercial strawberry [Fragaria x ananassa Duchesne (Rosaceae)] farm at

Bullsbrook, Western Australia, from September to November 2007 (spring). Naturally

occurring F. occidentalis infestations on low tunnel-grown strawberry were sprayed with water

(control), ‘spinosad (80 mL/100 L rate, 0.096 g a.i./L) then mites’ or ‘mites then spinosad (80

mL/100 L rate, 0.096 g a.i./L)’ applications. Predatory mites (Acari), Typhlodromips

montdorensis Schicha (Phytoseiidae), Neoseiulus cucumeris Oudemans (Phytoseiidae) and

Hypoaspis miles Berlese (Laelapidae) were released as single-, two-, and three- species

combinations. Predatory mites reduced the number of F. occidentalis on strawberry plants

sprayed with either water or spinosad, compared to the no mite treatment. Frankliniella

occidentalis numbers were lower on spinosad-treated plants that received predatory mites than

on the plants sprayed with water and received predatory mites. Spinosad posed no negative

effect to predatory mites, as mite numbers on plants sprayed with spinosad did not differ from

the water treated plants. However, predatory mites were most effective in reducing thrips when

released after spinosad was applied (‘spinosad then mite’ treatment). The three species

combination of predatory mites appeared to perform better in reducing thrips numbers compared

to their individual release. The two species combination of T. montdorensis (foliage inhabiting)

and H. miles (soil dwelling) appeared to be the most effective in suppressing F. occidentalis.

The next most effective combination was a triple-species release (T. montdorensis, N. cucumeris

and H. miles). The double-species combination of T. montdorensis and N. cucumeris was the

least effective, and may interact with each other. Although spinosad and predatory mites were

able to reduce thrips numbers, F. occidentalis numbers increased five weeks after treatment.

Chapter V: Compatibility of predatory mites and spinosad

110

This suggests that a further application of predatory mites, spinosad or both is required. Single-

and multiple-species release of predatory mites combined with spinosad for F. occidentalis

management in low tunnel-grown strawberry are discussed.

5.1 Introduction

Western flower thrips (WFT), Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae),

is considered one of the most devastating pest thrips in a range of horticultural crops, including

strawberry (Lewis 1973, Mound 1997). In Australia, the strawberry [Fragaria ananassa

Duchesne (Rosaceae)] industry is worth AUD$308 million annually (Anonymous 2009), and is

an intensively managed crop cultivated for its fresh, aromatic, red berries. However, strawberry

production is often hampered by direct damage caused by WFT (Ullio 2002). WFT damage

contributes to the ‘seediness’ of strawberry fruit (Medhurst and Steiner 2001), and is

responsible for uneven ripening and yield loss (Houlding and Woods 1995). WFT feeding on

strawberry blossoms may also cause stigmas and anthers to turn brown and wither prematurely

(Zalom et al. 2001), and reduce flower receptacle size (Coll et al. 2007).

Because of its cryptic behaviour, minute size, and high reproductive rate, WFT is difficult to

control using pesticides. Moreover, it has developed resistance against major classes of

insecticides that are currently in use in many parts of the world (Helyer and Brobyn 1992,

Brødsgaard 1994), including Australia (Herron and James 2005). Because of the inadequacy of

chemical control, there is a need to develop integrated control methods. One particular

challenge is to integrate biological and chemical control, as many pesticides have lethal and

sublethal effects on biological control agents. Spinosad is a novel pesticide, derived from the

fermentation of the actinomycete Saccharopolyspora spinosa Mertz and Yao (Sparks et al.

1998), and is classified as an environmentally and toxicologically reduced-risk chemical

(Sparks et al. 1998, Cleveland et al. 2002, Thompson et al. 2002). Since its discovery, spinosad

has been used in over 180 different crops to control a wide range of pests worldwide (Bret et al.

1997, Thompson et al. 1997, Zhao et al. 2002). In Australia, spinosad has been used for the

control of Lepidopteran and Thysanopteran pests (Downard 2001), and is highly effective

against WFT (Funderburk et al. 2000). Spinosad is regarded to have no or reduced toxicity to

natural enemies (Brunner et al. 2001, Elzen 2001, Villanueva and Walgenbach 2005a).

However, the selectivity of spinosad on predatory insects is under review (Pietrantonio and

Benedict 1999, Williams et al. 2003). Spinosad is regarded to have low to moderate toxicity

to predatory mites and the toxicity can vary from species to species (Williams et al.

2003, Cote et al. 2004, Jones et al. 2005). van Driesche et al. (2006) reported that fresh

residues of spinosad applied at the recommended rate to control WFT on glasshouse flower


111

crops, had no toxic effect on the predatory mite Neoseiulus cucumeris (Oudemans) (Acari:

Phytoseiidae), but lowered the survival of Iphiseius degenerans (Berlese) (Acari: Phytoseiidae).

Spinosad is reported to be harmless to Phytoseiulus persimilis Athias-Henriot (Acari:

Phytoseiidae), widely used for the control of two-spotted spider mites (Holt et al. 2006), but is

highly toxic to Neoseiulus fallacis (Garman) (Acari: Phytoseiidae), which is used in North

Carolina apple orchards to control European red mite (Panonychus ulmi Koch) and two-spotted

mite (Tetranychus urticae Koch) (Villanueva and Walgenbach 2005b). Despite its detrimental

effect on some species, spinosad can be integrated with biological control agents for WFT

management (Funderburk et al. 2000), if a period of time is maintained between pesticide

application and release of natural enemies (Jones et al. 2005, Khan and Morse 2006).

Several species of natural enemies are reported to attack above- and below-ground stages of

WFT (van Driesche et al. 1998). Several predatory mite species (Acari) prey on either the larval

or pupal stages, and are currently used to control WFT in protected crops (e.g. glasshouses) with

some success (Riduavets 1995, Sabelis and Van Rijn 1997). Most attention has focused on three

species: Amblyeius barkeri (Hughes), N. cucumeris and I. degenerans (Macgill 1927,

Rodrìguez-Reina et al. 1992). The efficacy of N. cucumeris is limited because the adults only

feed on thrips first instar larvae (Gillespie and Ramey 1988); it is the most commonly used

biocontrol agent for thrips in protected cropping. Neoseiulus cucumeris is particularly

successful in glasshouse capsicum (Ramakers 1988) in the Netherlands, but to my knowledge,

little is known about the use of phytoseiid predators in semi-open or open fields.

Recently, several strawberry growers in Western Australia have begun releasing P. persimilis, a

predatory mite for the control of the two-spotted mite, T. urticae, in the glasshouse and field,

and N. cucumeris for control of WFT in glasshouses. The use of predatory mites in

semiprotected crops such as low tunnels (floating covers) is limited. In Western Australia, field

populations of WFT are low during winter (June-August), and increase during late September as

temperature increases (L. Chilman, pers. comm. 2006). Therefore, the release of predatory mites

before the spring population increase might be a useful approach for managing WFT in low

tunnel-grown strawberry. Four species of predatory mite are commercially available in Australia

(Neoseiulus cucumeris, Typhlodromips montdorensis, Hypoaspis miles and Hypoaspis

aculeifer) (Biological Services 2009). Neoseiulus cucumeris and T. montdorensis are plant-

dwelling species that predate on first instar WFT larva (Steiner and Goodwin 2002). Hypoaspis

miles is a soil-dwelling species that predates on WFT pupae (Glockemann 1992). However,

recent studies suggest that H. miles also predates on second instar WFT larva (Berndt 2002,

Berndt 2003). The difference in their use on different parts of the plant, and predation on

different WFT life stages raises the question of their compatibility in single versus combined


112

releases. The overall objective of the present study was to evaluate the efficacy of T.

montdorensis, N. cucumeris, and H. miles as single-species and multiple-species releases, and

their compatibility with spinosad for WFT control in low tunnel-grown strawberry. Specifically,

this study had three main aims:

i. Determine if spinosad and mites can be used effectively in combination to reduce WFT.

It is expected that the combined application of T. montdorensis, N. cucumeris, and H.

miles with spinosad would provide better suppression of WFT.

ii. Determine if spinosad affects predatory mites.

iii. Determine if a single-species release is more, or less effective than multiple-species

releases in reducing WFT numbers. A combination of plant and soil dwelling mites

could provide better control of WFT than plant dwelling mite alone.


5.2.1 Study site

The trial was carried out on a commercial strawberry farm at Bullsbrook (S 31°39.294’, E

115º58.589), 54 km north of Perth, Western Australia, from 20 September to 26 November

2007. Strawberry (cv. Camarosa) was grown on silver plastic mulch under floating row covers.

The grower made available four tunnels for experimentation. Each tunnel was 1.5 m wide and

50 m long, with a total of 668 (4 x 167) strawberry plants per tunnel. Strawberry runners were

transplanted on 28 April 2007, with 30 cm intervals between runners within and between rows.

After transplanting, the tunnels were covered with clear plastic sheets to keep the tunnel hot

during the winter. Plants received no pesticide sprays three weeks before commencement of the

experiment.

A data logger (Hobo Pro Series, HO8-031-08, Onset, USA) was installed inside each tunnel to

record air temperature. Before installation, the data logger was programmed using Boxcar®

Pro

4.3 software to log ambient air temperature at one-hour intervals during the experimental

period. At the end of the experiment, temperature data was extracted from the data logger and

averaged to obtain daily mean temperature (⁰C). Maximum, minimum and daily average air

temperatures were collected from the Bureau of Meteorology, WA. The closest weather station

was the RAAF Air Base, Pearce, approximately 22 km north-west of Bullsbrook (Figure 5.1).


113

30

-Sep

5-O

ct

10

-Oct

15

-Oct

20

-Oct

25

-Oct

30

-Oct

4-N

ov

9-N

ov

14

-No

v

19

-No

v

0

10

20

30

40

Max air temp Min air temp Ave Temp inside tunnelAve temp

Tem

pera

ture (C

)

Figure 5.1 Maximum, minimum and average daily air temperature (⁰C), and average daily

temperature (⁰C) inside low tunnel (25 September to 20 November 2008). Maximum and

minimum air temperature collected from RAAF, Pearce (22 km north-west of Bullsbrook).

Temperature inside the tunnel was recorded using a data logger.


Predatory mites [Typhlodromips montdorensis, Neoseiulus cucumeris and Hypoaspis

miles] used in the study were sourced from commercial Australian suppliers (Biological

Services, SA; Chilman IPM Services, WA; and the Beneficial Bug Company, NSW).

Mites were provided in plastic buckets or plastic bag containing vermiculite. Trials were

conducted immediately upon receipt of predatory mites.

5.2.3 Treatments

The treatments were applied in a two-factor, split-plot design (three spray treatments x eight

mite treatments). Each of the four tunnels was divided into three plots, 1.2 m wide and 16.5 m

long, with 220 (4 x 55) plants. Within each tunnel, spray treatments were randomly assigned to

a plot and plants were sprayed with either water (control) or spinosad (Success™

, Dow

AgroSciences Australia) (Table 5.1). Spinosad treatments were applied either before the mites

were released (‘spinosad then mites’), or after the mites were released (‘mites then spinosad’).

The lapse of time between a spinosad application and predatory mite release was six days.

Water or spinosad was applied with a Knapsack Sprayer (12 L, Rapid Spray™

; Tank

Management Ltd, Australia) until run-off. Because the experiment was carried out in low tunnel


114

where the environmental conditions somewhat similar to glasshouse, plants were sprayed until

run-off). Spinosad (0.096 g a.i./L) was applied at the recommended rate of 80 mL/100 L.

Each spray plot within each tunnel was further divided into eight ‘mite release’ sub-plots, 1.2 m

wide and 1.9 m long consisting of 24 (4 x 6) plants. Mite release treatments were:

(i) No mites (control)

(ii) T. montdorensis

(iii) N. cucumeris

(iv) H. miles

(v) T. montdorensis and N. cucumeris

(vi) T. montdorensis and H. miles

(vii) N. cucumeris and H. miles

(viii) T. montdorensis, N. cucumeris and H. miles.

All mite treatments within each spray plot were randomly assigned. Within each tunnel, spray

plots were separated by a row of plants. Similarly, a row of plants was kept as a buffer zone

between each sub-plot, which was treated with neither spinosad nor mites. On either side of the

buffer row, a mesh net (105 µ, Sefar Filter Specialists Pty Ltd., Malaga, WA) supported by a

wooden frame created a barrier to prevent movement of WFT and predatory mites between sub-

plots.

Table 5.1 Schedules of treatment applications and sampling.

Treatment Weeks

1-3 3 4 4-8

Water then

mites

Pre-treatment

sampling

Water

sprayed

Mites

released

Post-treatment

sampling

Spinosad then

mites

Spinosad

sprayed

Mites

released

Mites then

spinosad

Mites

released

Spinosad

sprayed

5.2.3.1 Pre-treatment sampling

Before treatment application, plants in each plot (within each tunnel) were sampled for three

weeks at weekly intervals to determine if there were any differences in WFT populations within

or between tunnels. At each sample, three plants were randomly selected from a plot within

each tunnel. Three flowers and three fruits were selected from each plant and removed with a

pair of sharp scissors. Each flower or fruit was placed into a separate glass container (with 80%


115

ethyl alcohol) and labelled. In the laboratory, the numbers of WFT larvae and adults in each

flower and fruit were counted under a binocular stereomicroscope. Across plots, the numbers of

WFT larvae and adults were averaged per flower or fruit.

5.2.3.2 Post-treatment sampling

After the third sample was collected, the spray treatment areas were treated with water or

spinosad, or sprinkled with predatory mites (Table 5.1). After six days and after the fourth

sample had been collected, predatory mites were released onto the water- or spinosad-treated

plants. Plots that had previously received predatory mites (week three) were sprayed with

spinosad. Approximately 300 mites per m-2

were sprinkled over plants in the single-species

treatment (de Courcy Williams 2001). In the two-species combination, the same release rate was

used, but each species made up 50% of the total. Similarly, for the three-species combination,

the above release rate was used, with each species comprising a third of the total.

Plots were sampled for four more weeks at weekly intervals as described above. In the

laboratory, collected samples were checked under a binocular stereomicroscope and the

numbers of WFT larvae and adults, T. montdorensis and N. cucumeris in each flower and fruit

were recorded. Across plants, WFT larvae and adults, T. montdorensis and N. cucumeris

numbers were averaged per flower or fruit. Since the H. miles count was not successful in

previous experiments (chapters 3 and 4), no attempt was made to count H. miles.

5.2.4 Data analysis

To determine if counts of WFT differed within and between tunnels, before mite-release

treatments, the numbers of WFT larvae and adults on the flowers and fruits were analysed with

repeated measures ANOVA, with a split design (Proc Mixed Procedure). Independent fixed

factors were sub-plot (within factor) and time (sampling week, repeated factor); random factor:

plant number, block factor: tunnel (between); response variables: WFT adults and larvae. There

were no significant differences in WFT adult and larval numbers within and between tunnels.

The influence of spray application and predatory mite treatments on WFT adults and larvae over

time from week four to eight (post-treatment) was subjected to repeated measures ANOVA with

split-plot design (Proc Mixed Procedure). Independent fixed factors were spray treatment and

mite treatments, time (repeated factor); random factors were plant numbers; block factor:

tunnel; and response variables were WFT adults and larvae on flower and fruits. However,

because there was a significant interaction of spray treatment, mite treatments and time, a series

of repeated measures ANOVA with a split-plot design (Proc Mixed Procedure) was conducted


116

for each spray treatment (Quinn and Keough 2002). Since multiple comparisons were made, an

adjustment to the significance level was required [α = 0.01667 (0.05/3]. If ANOVAs indicated a

significant difference, means were subjected to pair wise comparison.

Because of the different release rates of predatory mites (T. montdorensis and N. cucumeris) in

different mite combinations, several repeated measures ANOVAs with split-plot design (Proc

Mixed Procedure) were used. Since mites were released in either week four (‘spinosad then

mite’) or week three (‘mites then spinosad’ treatment), data collected from weeks five to eight

were used to analyse differences in mite numbers. When released as single species, the effect of

spray treatment and time were subjected to repeated measures ANOVA with a split-plot design

[Proc Mixed Procedure; Fixed independent variable: spray, mite species, time (repeated factor);

random factor: tunnel; response variables: flower, fruit]. Influence of spray treatment and time

on predatory mites numbers (T. montdorensis and N. cucumeris) for double-species and triple-

species releases were evaluated with a series of repeated measures ANOVA with a split-plot

design [fixed independent variable: spray, mite species/combinations, times (repeated factor);

random factor: tunnel; response variables: flower, fruit]. Additionally, the influence of spray

treatment and species combination on T. montdorensis numbers in releases of T. montdorensis

with the other mite species, and N. cucumeris with the other mite species, were analysed with

repeated measures ANOVA with split-plot design [Proc Mixed Procedure; Fixed independent

variable: spray, mite combinations, times (repeated factor); random factor: tunnel; response

variables: T. montdorensis and N. cucumeris on flower and fruit]. If ANOVAs were significant,

means were separated using least square means difference (α = 0.05).

The data were transformed with square root before analysis, to meet the assumption of

homogeneity of variances (Zar 1999). However, actual means are presented in figures

and tables. All statistical analyses were performed with SAS 9.1 Statistical Package

(SAS 2002-2003). Figures were constructed using GraphPad Prism 5.0 (GraphPad,

2007).

5.3 Results

There were no pre-treatment differences in the mean number of WFT adults and larvae on

flowers or fruit within and between tunnels (Appendix 5.1). An average of 7.37 ± 0.55 WFT

adults were collected from flowers and 4.67 ± 0.67 from fruits. An average of 7.09 ± 0.34 WFT

larvae was collected from flowers and 4.64 ± 0.22 from fruits. The number of WFT adults in

flowers was above the economic threshold established for strawberry, which is 45% of flowers

with five or more adult WFT (Steiner and Goodwin 2005).


117

5.3.1 Impact of the spray and predatory mite species combinations on WFT adults

5.3.1.1 Flower

When applied in different combinations, predatory mites and spray treatments significantly

affected (F14, 63 = 10.63, P < 0.0001) the number of WFT adults (Appendix 5.2, Figure 5.2).

Across mite treatments, there were fewer WFT adults on flowers on ‘spinosad then mites’

treated plants and most on water-treated plants. However, there were significant interactions

between spray treatment, predatory mite species combinations and time (F56, 288 = 18.93, P <

0.0001), and the effect of mite species combinations on WFT adults was evaluated with a series

of ANOVAs by spray treatment (Appendix 5.2).

No mites Tm Nc Hm Tm*Nc Tm*Hm Nc*Hm Tm*Nc*Hm0

2

4

6

8

10

Water Spinosad then mites

b

aa

a

b

c

aa

b

a

c

c

ab

b

a

a

b

a

ab

a

c

a

b

Mites then spinosad

Num

ber

of

WF

T a

dult

per

flo

wer

(M

ean

S

E)

Figure 5.2 Effect of spray treatment and predatory mite species releases on the number of WFT

adults/flower in low tunnel strawberry. Tm = T. montdorensis, Nc = N. cucumeris, Hm = H.

miles. Means with different letters within each group differed significantly (LS means, α =

0.05).*indicates mite species combinations.

The mean number of WFT adults on plants treated with water (F28, 96 = 276.74, P < 0.0001),

‘spinosad then mites’ (F 28, 96 = 86.57, P < 0.0001) or ‘mites then spinosad’ (F 28, 96 = 157.72, P

< 0.0001), were affected by predatory mite species combinations (Figure 5.3, Appendix 5.2).

The lowest number of WFT adults was recorded from plants where T. montdorensis and H.

miles had been released in combination, or where all three mite species had been released

(Figure 5.3). In the water and ‘spinosad then mites’ treatments, there was no significant

difference in the number of WFT adults at week four (Figure 5.3). Across time, the number of

WFT adults per flower was highest on plants that did not receive any mites (Figure 5.3).


118

2

4

6

8

10

12

No mites Tm Nc Hm

Tm*Nc Nc*Hm Tm*Nc*HmTm*Hm

2

4

6

8

10

12

Nu

mb

er o

f W

FT

ad

ult

s p

er

flo

wer

(Mea

n

SE

)

PoT1 PoT2 PoT3 PoT4 PoT52

4

6

8

10

12

A

B

C

Figure 5.3 Influence of predatory mite species combinations on WFT adults per flower over

time (X-axis) in (A) water, (B) ‘spinosad then mites’ and (C) ‘mites then spinosad’. WFT

adults’ counts were commenced at weekly interval. PoT = Post-spinosad spray/mites release.

Within each week, means were separated by LS means (α = 0.017). Tm = T. montdorensis, Nc

= N. cucumeris, Hm = H. miles. *indicates predatory mite species combination.

5.3.1.2 Fruit

Spray treatments and predatory mite species combinations affected the number of WFT adults

per fruit (F14, 63 = 9.63, P < 0.0001; Figure 5.4, Appendix 5.2). WFT adult numbers were


119

highest on water-treated plants and on plants that did not receive any mites (> 4 WFT

adults/fruit), and lowest, on ‘spinosad then mites’ treated plants (<3.75 WFT/fruit; Figure 5.4).

There was a significant interaction between spray treatment, predatory mite species and time, (F

56, 288 = 3.78, P < 0.0001). The influence of mite species combinations on WFT adults/fruit over

time was therefore evaluated by separate ANOVAs for each spray treatment (Appendix 5.2).


2

4

6

8Water Spinosad then mites

a b

c

ab

c

a

b

c

a

a

b

a

b

c

a

bc

aa

b

aa

Mites then spinosad

a

Num

ber

of

WFT

adults

per

fru

it (

Mea

n

SE

)

b

Figure 5.4 Effect of spray treatment and predatory mite species combinations (X-axis) on the

number of WFT adults per fruit (Y-axis). Tm = T. montdorensis, Nc = N. cucumeris, Hm = H.


0.05). *indicates mite species combination.

Predatory mite species combinations and time affected the number of WFT adults per fruit in

control (water) (F 28, 96 = 3.22, P < 0.0001), ‘spinosad then mite’ (F 28, 96 = 32.59, P < 0.0001)

and ‘mites then spinosad’ (F 28, 96 = 47.34, P < 0.0001) treatments (Appendix 5.2, Figure 5.5).

In all spray treatments, the lowest numbers of WFT adults per fruit were on plants where T.

montdorensis and H. miles had been released in combination. However, for the water (control)

treatment, WFT adults were lowest on plants with the triple-species combination, five weeks

after treatment. There was no difference in the number of WFT adults /fruit on plants in water

(control) and ‘mite then spinosad’ treatments on which ‘T. montdorensis and H. miles’ and ‘T.

montdorensis, N. cucumeris and H. miles’ combinations were released. In addition, in the

control (water) and ‘spinosad then mites’ treatments, WFT adults per fruit on week four (post-

treatment sampling, PoT1) among mite treatments were not different. For all spray treatments

across time, WFT adults per fruit were highest on plants with no mites.


120

1

3

5

7

No mites Tm Nc Hm


1

3

5

7

Nu

mb

er o

f W

FT

ad

ult

s p

er f

ru

it (

Mea

n

SE

)


3

5

7

A

B

C

Figure 5.5 Influence of predatory mites on WFT adults per fruit over time (X-axis) in (A)

water, (B) ‘spinosad then mites’ and (C) ‘mites then spinosad’. WFT adults’ counts were

commenced at weekly interval. PoT = Post-spinosad spray/mites release. Tm = T. montdorensis,

Nc = N. cucumeris, Hm = H. miles. Within each week, means were separated by LS means (α =

0.017). *indicates predatory mite species combination.

5.3.2 Impact of the spray and predatory mite species combinations on WFT larvae

5.3.2.1 Flower

Spray treatments and predatory mite release treatments affected the number of WFT larvae per

flower (F14, 63 = 20.77, P < 0.0001; Figure 5.6). The lowest numbers of WFT larvae per flower


121

were on ‘spinosad then mites’-treated plants (3.72 ± 0.34 larvae/flower), and highest on water-

treated plants (6.75 ± 0.27 larvae/flower). However, because there was a significant interaction

of spray, mite release treatment and time (F56, 288 = 37.11, P < 0.0001), the effect of mite

species combinations over time were evaluated with separate ANOVAs for each spray treatment

(Appendix 5.3).


2

4

6

8

10Water Spinosad then mites

a a

b

a

b

c

a

b

c

a

b

c

a

b

c

a

b

c

a

b

c

a

b

c

Mites then spinosad

Nu

mb

er o

f W

FT

la

rvae

per

flo

wer

(M

ean

S

E)


number of WFT larvae per flower (Y-axis). Tm = T. montdorensis, Nc = N. cucumeris, Hm = H.


0.05).*indicates mite species combination.

The highest number of WFT larvae was on plants with no mite releases (Figure 5.7). Predatory

mite species combinations had a significant effect on the number of WFT larvae per flower

(Appendix 5.3, Figure 5.7). For all spray treatments over the five weeks, the lowest numbers of

WFT larvae were recorded from plants where T. montdorensis and H. miles had been released in

combination (Figure 5.7). There were no differences in the number of WFT larvae on plants

treated with either T. montdorensis or H. miles, or all three species, except at week five post-

treatment.


122

4

6

8

10

12

No mites Tm Nc Hm


1

3

5

7

9

Nu

mb

er o

f W

FT

la

rv

ae p

er f

low

er (

Mea

n

SE

)


4

6

8

10

A

B

C

Figure 5.7 Influence of predatory mites on WFT larvae per flower over time (X-axis) in (A)

water, (B) ‘spinosad then mites’ and (C) ‘mites then spinosad’. WFT larvae counts commenced

at weekly interval. PoT = Post-spinosad spray/mites release. Within each week, means were

separated by LS means (α = 0.017). Tm = T. montdorensis, Nc = N. cucumeris, Hm = H. miles. *indicates predatory mite species combination.

5.3.2.2 Fruit

Predatory mite species combinations and spray treatment had a significant effect on the number

of WFT larvae per fruit (F14, 63 = 25.43, P < 0.000; Appendix 5.3, Figure 5.8). The lowest

number of larvae was on plants treated with ‘spinosad then mites’, and the highest on water-

treated plants. There was a significant interaction of spray, predatory mite species combinations


123

and time (week) (F56, 288 = 9.15, P < 0.0001). Separate ANOVAs were used to evaluate the

effect of predatory mite species combinations on spray treatment (Appendix 5.3).


2

4

6

8

Water Spinosad then mites

ab

c

a

b

c

ab

c

aa

b

a

b

c

a

b

c

a

b

c

a

b

c

Mites then spinosad

Nu

mb

er o

f W

FT

la

rv

ae p

er f

ru

it (

Mea

n

SE

)


number of WFT larvae per fruit. Tm = T. montdorensis, Nc = N. cucumeris, Hm = H. miles.

Means with different letters within each group differed significantly (LS means, α = 0.05). *indicates mite species combination.

Any combination of predatory mite species appeared to have a significant effect on the number

of larvae per fruit over the five weeks trial period, regardless of spray treatment (Appendix 5.3,

Figure 5.9). The mean number of WFT larvae per fruit was lowest on plants receiving ‘T.

montdorensis and H. miles’ and highest on plants with no mites. The mean number of WFT

larvae per fruit did not differ across predatory mite treatments one week after treatment. There

was no difference in mean number of larvae on plants that received either T. montdorensis or H.

miles.


124

3

4

5

6

7

No mites Tm Nc Hm

Tm*NcNc*Hm Tm*Nc*HmTm*Hm

1

2

3

4

5

6

Nu

mb

er o

f W

FT

la

rva

e p

er p

lan

t (M

ean

S

E)


2

3

4

5

6

A

B

C

Figure 5.9 Influence of predatory mites on WFT larvae per fruit over time (X-axis) in (A)

control (water), (B) ‘spinosad then mites’ and (C) ‘mites then spinosad’. PoT = Post-spinosad

spray/mites release. Within each week, means were separated by LS means (α = 0.017). Tm = T.

montdorensis, Nc = N. cucumeris, Hm = H. miles. *indicates predatory mite species

combination.

5.3.3 Impact of the spray and mite species combinations on predatory mites

5.3.3.1 Single species

There was no interaction of spray treatment and time (F 6, 63 = 0.97, P = 0.46; Appendix 5.4) on

predatory mite numbers (T. montdorensis and N. cucumeris) per flower. The mean number of T.

montdorensis and N. cucumeris per flower was significantly different (P = 0.26; Appendix 5.4,


125

Figure 5.10). However, the mean number of predatory mites per flower differed significantly (F

2, 6 = 38.63, P = 0.0004) between spray treatments (Appendix 5.4, Figure 5.10). There was no

difference in the number of predatory mites per flower in the control or ‘spinosad then mites’

treatments (Figure 5.10).

When mites were applied as single-species treatments, there was no significant interaction

between spray treatment and time (P = 0.85; Appendix 5.4). There were significantly more T.

montdorensis per fruit than N. cucumeris (F 1, 11 = 29.39, P = 0.0002; Figure 5.10). The overall

mean numbers of predatory mites differed significantly amongst spray treatments (F 2, 6 = 29.97,

P = 0.0008; Appendix 5.4, Figure 5.10). Overall, the highest numbers of predatory mites were

found in the control (water) treatment and lowest on the ‘mites then spinosad’ treatment.

However, there was no difference in the number of predatory mite numbers in the control and

‘spinosad then mites’ treatments.

0

2

4

6

abb

Tm Nc0

2

4

6

ab

Mite species

Water S-M M-S

a

bb

Spray

Flower

Fruit

Nu

mber

of

pre

dat

ory

mit

es (

Mea

n

SE

)

Figure 5.10 Comparison of the mean number of T. montdorensis (Tm) and N. cucumeris (Nc)

per flower or fruit, when applied as single species. Left side of the figure compares mean

numbers of T. montdorensis and N. cucumeris. Right side compares the combined mean

numbers of mites (T. montdorensis and N. cucumeris) between spray treatments. Means with

different letters within group were significantly different (LS means α = 0.05). S-M = ‘Spinosad

then mites’, M-S = ‘Mites then spinosad’.


126

5.3.3.2 Two-species combinations

When T. montdorensis and N. cucumeris were applied in a double-species combination, spray

treatment and time had no significant effect on predatory mite numbers (flower: F 6, 63 = 0.22, P

= 0.9680; fruit: F 6, 63 = 0.07, P = 0.9986; Appendix 5.4). However, there were significantly

more T. montdorensis than N. cucumeris (flower: F 1, 11 = 10.53, P = 0.0092; fruit: F 1, 11 = 9.99,

P = 0.0081; Figure 5.11). The predatory mite numbers per flower (F 2, 6 = 11.95, P = 0.0081) or

fruit (F 2, 6 = 13.95, P = 0.0062) differed between spray treatments (Figure 5.11). In both

flowers and fruits, most predatory mites were found in the control treatment and least in ‘mites

then spinosad’ treatment (Figure 5.11). The mean numbers of predatory mites in the control and

‘spinosad then mites’ treated plants did not differ.

For two-species combinations with H. miles, there was no interaction between spray treatment

and time on predatory mite numbers (flowers: F 6, 63 = 0.50, P =0.81; fruit: F 6, 63 = 0.90, P =

0.50; Appendix 5.4). The mean number of predatory mites differed between spray treatments

(flower: F 2, 6 = 30.70, P =0.0007; fruit: F 2, 6 = 21.41, P = 0.0019; Figure 5.11), and were

highest in the control and lowest in the ‘mites then spinosad’ treatment (Figure 5.11). The

predatory mite numbers on control plants and ‘spinosad then mites’ treatments did not differ

(Figure 5.11).


127

0

1

2

3

4

ab

abb

0

1

2

3

4

ab

ab

b

0

1

2

3

4

a

bb

Tm Nc0

1

2

3

4

Mite species

Water S-M M-S

a

bb

Spray

A

B

Flower

Fruit

Flower

Fruit

Num

ber

of

pre

dat

ory

mit

es (

Mea

n

SE

)

Figure 5.11 Comparison of the mean numbers T. montdorensis (Tm) and N. cucumeris (Nc)

applied in double-species combination as (A) ‘T. montdorensis and N. cucumeris’, and (B) T.

montdorensis in ‘T. montdorensis and H. miles’ and N. cucumeris in ‘N. cucumeris and H.

miles’. Left side of the figure compares mean numbers of T. montdorensis and N. cucumeris.

Right side compares combined mean numbers of predatory mites (T. montdorensis and N.

cucumeris) between spray treatments. Means with different letters within a group were

significantly different (LS means, α = 0.05). S-M = ‘Spinosad then mites’, M-S = ‘Mites then

spinosad’.


128

5.3.3.3 Three-species release

Spray treatment and time had no significant effect on the overall mean numbers predatory mites

(T. montdorensis and N. cucumeris) (flower: F 6, 63 = 0.08, P = 0.9982; fruit: F 6, 63 = 0.09, P =

0.9974; Appendix 5.4). Similarly, the mean number of T. montdorensis and N. cucumeris were

not different on flowers (F 1, 11 = 0.15, P = 0.7070) or fruits (F 1, 11 = 0.15, P =0.7078) (Figure

5.12). However, overall mean numbers of predatory mites differed significantly among spray

treatments (flowers: F 2, 6 = 8.04, P = 0.0201; fruit: F 2, 6 = 8.03, P = 0.0201; Figure 5.12). The

highest and lowest numbers of predatory mites were in the water and ‘mites then spinosad’

treatments respectively (Figure 5.12). However, the mean number of predatory mites per flower

or fruit did not differ between control and ‘spinosad then mites’ treatments.

0

1

2

3

a

bb

Tm Nc0

1

2

3

Mite species

Water S-M M-S

a

bb

Spray

Flower

Fruit

Num

ber

of

pre

dat

ory

mit

es (M

ean

SE

)

Figure 5.12 Comparison of mean numbers of T. montdorensis (Tm) and N. cucumeris (Nc)

when applied in a three-species combination (T. montdorensis, N. cucumeris and H. miles). Left

side of the figure compares mean numbers of T. montdorensis and N. cucumeris per flower/fruit.

Right side compares overall mean numbers of predatory mites between spray treatments. Means

within each group with different letters were significantly different (LS means α = 0.05). S-M =

‘Spinosad then mites’, M-S = ‘Mites then spinosad’.


129

5.3.3.4 Species interactions

When T. montdorensis was applied with either N. cucumeris or H. miles, mite species

combinations and time had no effect on the number of T. montdorensis (flower: F 6, 54 = 0.60, P

= 0.73; fruit: F 6, 54 = 0.08, P = 0.10; Appendix 5.5). More T. montdorensis were found when

applied with H. miles, and least when applied with N. cucumeris on flowers (F 1, 9 = 48.66, P <

0.0001) and fruit (F 1, 9 = 11.46, P = 0.0081) (Figure 5.13). When N. cucumeris was applied as a

double-species combination with T. montdorensis or H. miles, mite species combinations and

time had no significant effect on the mean numbers of N. cucumeris (flowers: F 6, 54 = 0.31, P =

0.93; fruit: F 6, 54 = 0.50, P = 0.81; Appendix 5.5). However, N. cucumeris numbers were

highest on flowers (F 1, 9 = 20.43, P = 0.001) and on fruit (F 1, 9 = 14.42, P = 0.002), when

applied with H. miles and lowest with T. montdorensis (Figure 5.13).

Tm*Nc Tm*Hm0

1

2

3

4

ab

Tm*Nc Tm*Hm

ab

Tm*Nc Nc*Hm0

1

2

3

4

ab

Tm*Nc Nc*Hm

a

b

Flower Fruit

Flower Fruit

A

B

Num

ber

of

pre

dat

ory

mit

es (

Mea

n

SE

)

Figure 5.13. Comparison of mean number of (A) T. montdorensis and (B) N. cucumeris when

applied in double-species combination (X-axis). Tm = T. montdorensis, Nc = N. cucumeris, Hm

= H. miles. Means within each group (flower/fruit) with different letters were significantly

different (LS means, α = 0.05). *indicates mites species combinations.


130

5.4 Discussion

The integration of reduced-risk chemicals and biological control for arthropod pest management

may provide more comprehensive management than either approach alone. Unfortunately,

insecticides are often found to be detrimental to natural enemies (Li et al. 2006, van Driesche et

al. 2006). The negative impact of pesticides has been reported on several phytoseiid species

used for WFT management (Hassan et al. 1987, Hassan et al. 1988, Kim and Paik 1996, Kim

and Seo 2001, Amano et al. 2004, Li et al. 2006, van Driesche et al. 2006). Therefore, before

recommending the integrated used of chemicals and biological control agents for commercial

use, the effectiveness of the strategy needs to be evaluated. However, to my knowledge, there

are limited numbers of studies that test the effectiveness of the combined use of chemical and

biological control agents in Australia. In addition, no research has focused on the integration of

insecticide and predatory mites for the management of WFT. The aim of this study was to

investigate the compatibility of a reduced-risk chemical (spinosad) and predatory mites in

single- versus multiple-species releases, and their effectiveness against WFT in low tunnel-

grown strawberry in spring. As an integrated pest management approach, this study also

evaluated whether predatory mites should be released before (‘mites then spinosad’) or after

(‘spinosad then mites’) spinosad is applied.

The results indicate that T. montdorensis, N. cucumeris and H. miles can be used in low tunnel-

grown strawberry for WFT management in spring. During the study period, the air temperature

varied between 10-29⁰C (17⁰C ± 0.43), however, the temperature inside the tunnel was higher

(range 15 to 35⁰C, 21.18⁰C ± 0.48) since it was heated by the sun. Gillespie and Ramey (1988)

reported that N. cucumeris can survive at a constant temperature of 9⁰C for months, and

oviposited within three days when returned to room temperature (20-22⁰C). Steiner et al. (2003)

found that T. montdorensis did not diapause when reared under 10⁰C under long day conditions,

and was able to survive at 8⁰C (Steiner and Goodwin 2002). This suggests that, based on

temperature, T. montdorensis and N. cucumeris can survive in low tunnels during spring.

However, the lower spring temperature may affect their reproduction, as there was no

significant increase in mite numbers over time. Hypoaspis miles was not counted in this study,

and its ability to survive in low tunnels during spring is not known. However, there were fewer

WFT where plants had been treated with H. miles, suggesting that H. miles was able to survive

and effectively reduce WFT.

The present study suggests that releases of T. montdorensis, N. cucumeris or H. miles combined

with spinosad is more effective in managing WFT in low tunnel-grown strawberry than either

spinosad applications or mite releases alone. Glasshouse studies also indicated that the


131

integration of T. montdorensis, N. cucumeris, or H. miles with spinosad is more successful in

controlling WFT than spinosad or predatory mites alone (Chapters 3 and 4). Thoeming and

Poehling (2006) reported that the application of azadiractin (Botanical, Neem Azal-U, 17%

azadiractin) with N. cucumeris and H. aculeifer increased efficacy to 99% to control WFT.

Ludwig and Oetting (2001) and Ludwig (2002) report that when the predatory bug Orius

insidiosus Say (Heteroptera: Anthocoridae) was released after spinosad was applied to potted

chrysanthemum and marigold, better control of WFT was achieved than with either approach

alone. This combined approach will only be effective if the pesticide has little or no effect on

the natural enemy, and is efficacious against the pest. Spinosad poses no detrimental effects to

O. insidiosus, but is highly efficacious against WFT (Funderburk et al. 2000, Ludwig and

Oetting 2001, Ludwig 2002). Chapman et al. (2009) state that an environmentally sound

approach to managing Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) is possible by

releasing a parasitic wasp (Trichogramma ostriniae Pang et Chen (Hymenoptera:

Trichogrammatidae)) and applying biorational insecticides (spinosad, indoxacarb and

methoxyfenozide) to bell peppers. The combined use of reduced-risk insecticides (pyrethrin,

insecticidal soap and mineral oil) and the ladybird Harmonia axyridis (Pallas) (Coleoptera:

Coccinellidae), provides better management of the soybean aphid, Aphis glycine Matsumura

(Hemiptera: Aphididae) on North American soybean (Kraiss and Cullen 2008). In the present

study, spinosad initially reduced WFT numbers on strawberry plants, but WFT reached the

economic threshold one week after application (>5 WFT adults per flower, (Steiner and

Goodwin 2005) This also occurred when predatory mites were applied alone (without spinosad).

Effective management of WFT in low tunnel-grown strawberry was achieved by integrating

spinosad and predatory mite releases. In this strategy, the WFT population is initially reduced

by spinosad. Mites were released six days after spinosad was applied, and spinosad did not

appear to impact negatively on them. When a lapse of time is maintained between pesticide

application and mite release, spinosad has likely been degraded by photolysis (Viktorov et al.

2002) by the time that the mites are released. However, apart from direct mortality, spinosad

could affect survival, reproduction or prey handling efficiency (Li et al. 2006, van Driesche et

al. 2006). Laboratory and semi-field studies suggest that the direct application of pesticides and

even aged residues can be harmful to many natural enemies (Li et al. 2006, van Driesche et al.

2006), including predatory mites (Hassan et al. 1987, Hassan et al. 1988, Kim and Seo 2001,

Amano et al. 2004). Although spinosad is considered harmless to predatory mites, direct

application and fresh residues are toxic to some species. Kongchuensin and Takafuji (2006)

demonstrated that fresh spinosad residues up to 48 h old (12% suspensible concentrate)

significantly reduced the number of eggs and the immature produced by Neoseiulus

longispinosus (Evans) (Phytoseiidae). However, there was no effect on N. longispinosus if


132

exposed to residues after seven days. van Driesche et al. (2006) reported that fresh spinosad

residues had no effect on the survival of N. cucumeris or Iphiseius degenerans (Berlese)

(Phytoseiidae), but it lowered their oviposition rate. Khan and Morse (2006) tested the impact of

four pesticides on the predatory mite Euseius tularensis Congdon and found a significant effect

if mites were released five - six days after a spinosad application, but no effect if released after

seven days. Since the toxicity of a given pesticide varies from species to species, a bioassay is

needed to evaluate the toxicity of the recommended rate of spinosad for WFT management on

T. montdorensis, N. cucumeris, and H. miles. This will allow mites to be released to avoid any

toxicity posed by spinosad, while providing more effective control of WFT.

The efficiency of natural enemies in the a pest management program often varies from species

to species (Chyzik et al. 1996, Berndt et al. 2004a, Berndt et al. 2004b, Wiethoff et al. 2004).

The present study demonstrates that T. montdorensis, N. cucumeris, or H. miles were effective

against WFT in low tunnel-grown strawberry. However, the effectiveness of these predatory

mites against WFT appeared to differ. When released as a single species, T. montdorensis

appeared to be the most effective predator, resulting in fewer WFT, followed by N. cucumeris

and H. miles. This validates the previous findings (see Chapters 3 and 4) that T. montdorensis

performed better in reducing WFT over the other two mite species. The success of natural

enemies in a pest management program depends on several factors such as predation on prey

stage, predation rate, within-plant distribution of predators and prey, and availability of

supplemental food sources. Neoseiulus cucumeris is reported to predate on first instar larvae

(Bakker and Sabelis 1989), which is a potentially limiting characteristic. Hypoaspis miles preys

on WFT pupae (Glockemann 1992), though recent studies found that H. miles may also prey

upon second instar larvae (Berndt 2003). The predation rate also varies from species to species.

Berndt et al. (2004b) reported that efficiency of the predatory mites, Hypoaspis aculeifer

Canestrini (Laelapidae) and H. miles against WFT were different, mainly because H. aculeifer

ate more WFT compared to H. miles. Similarly, Brødsgaard (1989) and van Houten et al. (1995)

reported that N. cucumeris consumed more thrips compared to H. aculeifer and Neoseiulus

barkeri (Hughes) (Phytoseiidae). Rhodes and Liburd (2006) report variation in the performance

of predatory mites against two-spotted spider mites in strawberry. In field-grown strawberry,

Phytoseiulus persimilis Athias-Henriot takes a longer time to bring two-spotted spider mites

under control, compared to Neoseiulus californicus McGregor (Rhodes and Liburd 2006). It

was reported that T. montdorensis can prey 7-14 first instar WFT larvae per day (Steiner et al.

2003). While, on an average, N. cucumeris and H miles can prey six and two first instar WFT

larvae per day respectively (Berndt et al. 2004b, Zilahl-Balogh et al. 2007). The distribution of

predatory mites can also influence their effectiveness in pest management programs.

Typhlodromips montdorensis is a generalist predator and has the ability to distribute rapidly on


133

different parts of the plant (Steiner and Goodwin 1998, 2001), which may give it an advantage

over the other two species. Steiner and Goodwin (1998, 2001) also reported that the when

release, T. montdorensis rapidly distribute over the whole plants, whilst the within-plant

distribution of N. cucumeris is uneven i.e. distribute to certain part of the plant (Messelink et al.

2006). Neoseiulus cucumeris prefers the lower part of the of plant, while WFT prefer to remain

on the upper part of the plants (Messelink et al. 2006). Hypoaspis miles is a soil-dwelling

predator, which limits its prey to thrips pupae.

Variation in the reproduction and development of predatory mites often plays an important role

in their success (Messelink et al. 2006). The present field trial indicates that in single-species

release, N. cucumeris numbers were fewer than T. montdorensis. However, it is not known why

there were more T. montdorensis than N. cucumeris, as the same numbers of both species were

released. The efficacy of predatory mites may be influenced by the presence of supplemental

food such as pollen. In chrysanthemum, for example, the presence of pollen as supplemental

food reduced the predation efficiency of N. cucumeris on WFT by up to 55% (Skirvin et al.

2007). Similarly, van Rijn and Tanigoshi (1999) and van Rijn et al. (2002) reported that

Iphiseius degenrans Berlese (Acari) also feeds on pollen, effecting the predation of WFT on

cucumber. The presence of pollen as a supplement food has no influence on T. montdorensis

(Steiner and Goodwin 2002). Foraging efficiency of commercially supplied predators varies

from species to species which may also affect their effectiveness in reducing pest population

(Steiner and BjØrnson 1996).

Although the single-species release of predatory mites in all treatments appears to be effective

for WFT management in low-tunnel grown strawberry, the release of a foliage inhabiting (T.

montdorensis or N. cucumeris) and a soil-dwelling predator (H. miles) was more effective.

There is a growing trend to use two or more species of natural enemies to manage insect

populations effectively (Premachandra et al. 2003, Avilla et al. 2004, Blümel 2004, Brødsgaard

2004, Chow and Heinz 2004, Hoddle 2004, Shipp and Ramakers 2004, Thoeming and Poehling

2006, Chow et al. 2008) might be partly due to the complementary effects on each other.

Brodeur et al. (2002) recognised that the release of multiple-predator species as an effective

strategy that would ideally suppress pest populations in a manner that is more economically

viable than the use of single-predator species. Wiethoff et al. (2004) reported cucumber plants

had WFT when N. cucumeris and H. miles were applied together than either N. cucumeris or H

miles released alone. The combined release of P. persimilis and N. californicus also provided

long-term effective suppression of carmine spider mite Tetranychus cinnabarinus (Boisduval)

(Tetranychidae) in potted gerbera plants, compared to their individual release (Schausberger and

Walzer 2001). Premachandra et al. (2005) reported that the emergence of WFT adults was


134

significantly lower in the combined application of entomopathogenic pathogens and the

predatory mite, H. aculeifer, than applications of either alone. The synergistic effects can be

expected if a plant-dwelling predator evokes escape behaviour of the prey and makes the prey

available for ground-dwelling predators (Losey and Denno 1999). However, the release of T.

montdorensis and N. cucumeris may not be effective since they may compete for the same prey

(Janssen et al. 1998). As a result, one species might outcompete the other. Although the present

study did not address this, it appears that when T. montdorensis and N. cucumeris are released in

combination, T. montdorensis numbers were higher than N. cucumeris. However, when applied

in triple-species combinations, mite performance was only second to the double-species

combination of T. montdorensis and H. miles. When all species were released together there

appeared to be no interaction between T. montdorensis and N. cucumeris. When these two

predatory mites were applied in a double-species combination, 150 mites of each species were

released per m-2

, but only 100 individuals per m-2

of each species in the triple-species

combination. The higher ratio used in the double species released may have increased the

chance of interspecific competition. Therefore, for the successful implementation of multiple

releases of predatory mites in a pest management program, further studies need to be carried out

to determine if intraguild predation occurs and preference between primary prey (pest),

secondary prey (predator) and other interactions. Furthermore, it is important to determine the

optimal release rate of predatory mites that can effectively reduce the pest population, whilst

having no negative impact on each other.

In conclusion, the integration of spinosad and multiple combinations of predatory mites (T.

montdorensis, N. cucumeris, and H. miles) could be a sustainable strategy for WFT management

in low tunnel-grown strawberry during spring. Although both spinosad treatments (‘spinosad

then mites’ or ‘mites then spinosad’) appeared to be effective against WFT, applying spinosad

before releasing the mites seems to be the more effective strategy, which has several

advantages. Firstly, the initial WFT population is reduced by spinosad. It is known that

biological control of arthropod pests by predatory mites can fail if the initial pest population is

very high (Malezieux et al. 1992). Secondly, direct toxicity of spinosad is harmful to T.

montdorensis, N. cucumeris and H. miles. Spraying first eliminates the problem of direct

exposure. Thirdly, this strategy enhances the chance to eliminate resistant populations. If the

lapse of times between spinosad application and mite release could be further shortened, control

could be further improved. Studies based on residual toxicity of spinosad to predatory mites will

allow the precise residual threshold to be determined. In addition, the present study commenced

during spring. The residual toxicity of spinosad to predatory mites may vary from season to

season. van de Veire et al. (2002) reported that abamectin (bioinsecticide) is more persistent in


135

spring than summer. Therefore, seasonal variation also needs to be considered and the

supplementary release of predatory mites after five weeks, may also be needed.






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Control 40: 160-167.

CHAPTER VI

Compatibility of spinosad with predaceous mites (Acari) used to control western flower

thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae)

Keywords: Spinosad, Frankliniella occidentalis, Typhlodromips montdorensis, Neoseiulus

cucumeris, Hypoaspis miles, direct toxicity, residual toxicity, preference, LT25

Abstract

Spinosad™

(Dow AgroSciences, USA) is a biopesticide widely used for control of western

flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae). Spinosad is

reported to be non-toxic to several predatory mite species used for the biological control of

thrips, and is recommended for use in integrated pest management programs for this reason.

Predatory mites (Acari) have recently become available to Australian growers for the control of

thrips. This includes Typhlodromips montdorensis (Schicha), Neoseiulus cucumeris (Oudemans)

and Hypoaspis miles (Berlese), which feed on thrips larvae or pupae. This study investigated the

impact of direct and residual toxicity (contact) of spinosad (recommended rate: 80 mL/100 L,

0.096 g a.i./L) on F. occidentalis and direct and residual (contact, indirect via consumption of

intoxicated thrips larvae and simultaneous exposure via contact and consumption of intoxicated

thrips larvae) toxicity on predatory mites. This study also investigated the repellency of

spinosad residues to mites. Direct contact with spinosad effectively reduced the numbers of F.

occidentalis adults and larvae, causing >96% mortality. Two to 96 h old spinosad residues were

also toxic to F. occidentalis. Direct exposure to spinosad resulted in >90% mortality of all three

mite species. Thresholds for the residual toxicity (contact) of spinosad LT25 (lethal time for 25%

mortality) were estimated as 4.2, 3.2 and 5.8 days for T. montdorensis, N. cucumeris and H.

miles respectively. When mites were simultaneously exposed to spinosad residues and fed

spinosad-intoxicated thrips larvae, toxicity increased. Residual thresholds were re-estimated as

5.4, 3.9 and 6.1 days for T. montdorensis, N. cucumeris, and H. miles respectively. Spinosad

residues were also repellent to mites. Residues aged two to 48 h repelled T. montdorensis and H.

miles, and residues aged two to 24 h repelled N. cucumeris. These data suggest that mites could

be safely released six days after spinosad is applied for the management of F. occidentalis.

6.1 Introduction

Spinosad (Success™

; Dow AgroSciences Australia) is a novel pesticide derived from

fermentation of the actinomycete, Saccharopolyspora spinosa Mertz and Yao (Sparks et al.

Chapter VI: Bioassay

142

1998). It is active against Lepidoptera, Diptera, and Thysanoptera (Cloyd and Sadof 2001).

Spinosad is classified as an environmentally and toxicologically reduced-risk chemical

(Cleveland et al. 2002, Thompson et al. 2002). Spinosad was first registered in Australia in 1998

and the USA in 1997 for use in cotton (Thompson and Hutchins 1999), and is now used in more

than 180 crops worldwide (Zhao et al. 2002). The marketing of spinosad has focused on its

favourable environmental profile, emphasising its potential for use in the integrated pest

management (IPM) systems (Thompson and Hutchins 1999, Thompson et al. 2000). Several

studies suggest that spinosad is less toxic to natural enemies including predatory mites, than

their prey (Miles and Dutton 2000, Thompson et al. 2000, Medina et al. 2001, Holt et al. 2006,

Kim et al. 2006, Arthurs et al. 2007). However, it has been reported that spinosad toxicity to

natural enemies is variable (Cote et al. 2004).

Spinosad is the primary insecticide used to control western flower thrips (WFT), Frankliniella

occidentalis (Pergande) (Thysanoptera: Thripidae) in strawberry in Australia. However, there

are concerns about WFT evolution of resistance to spinosad (Herron and James 2005, Bielza et

al. 2007). Biological control has been successfully incorporated with pesticide use to manage

WFT in commercial glasshouses in North America and Europe (Chambers and Sites 1989,

Gillespie 1989, Gilkeson 1990, Brødsgaard 2004, Shipp and Ramakers 2004). The most widely

employed natural enemies are predatory phytoseiid mites (Acari) that feed on thrips larvae

(Riudavets 1995, Sabelis and Van Rijn 1997). In Australia, predatory mites Typhlodromips

montdorensis (Schicha) (Phytoseiidae), Neoseiulus cucumeris (Oudemans) (Phytoseiidae) and

Hypoaspis miles (Berlese) (Laelapidae), have shown some potential for controlling WFT

(Steiner and Goodwin 2000). Nevertheless, it is not known what effect spinosad has on these

species of predatory mites that could be used in an IPM program in Australian strawberry

production. To determine whether spinosad could be integrated with mites into existing

Australian strawberry pest management, a series of laboratory bioassays were carried out to

evaluate the toxicity of spinosad to the mites and to determine if predator-prey interactions

would be affected. Specifically, the objectives of this study were to evaluate:

(i) Toxicity of direct contact with the recommended rate of spinosad to WFT and predatory

mites.

(ii) Residual toxicity of spinosad to WFT.

(iii) Residual toxicity (contact, indirect via consumption of intoxicated WFT and,

simultaneous exposure to spinosad via direct contact and consumption of treated prey)

and residual thresholds of spinosad for T. montdorensis, N. cucumeris and H. miles.

(iv) Repellency of spinosad residues to mites.


143


Trials were conducted in a controlled temperature (CT) room (25±1⁰C, 50-60% RH, 16:8 h L:D

regime) from June to September 2008 at the University of Western Australia (UWA).

6.2.1 Source of cultures

6.2.1.1 Strawberry plants

Strawberry runners [Fragaria ananassa Duchesne (Rosaceae)] cultivar ‘Camino Real’

were planted into pots (32.5l x 32.5w x 40.5h cm) containing potting mix (Baileys


WA (DAFWA) and UWA. All pots were fitted with sprinklers with automatic timers. The

plants were watered every third day. A liquid fertiliser (Thrive®

, Yates, Australia NPK:

12.4: 3: 6.2; rate: 5mL/2 L water) was applied once a month.


A glasshouse colony of WFT was established from individuals initially collected from

calendula flowers, Calendula officianalis L. (Asterales: Asteraceae) in a glasshouse at

DAFWA. From July 2006, WFT colonies were maintained on potted calendula at UWA.

Calendulas were grown from seeds collected from calendula plants maintained at

DAFWA. Calendula seeds were sown in plastic pots (50 x 100 mm) containing potting

mix, and kept in insect-proof Perspex cages (500 mm high, 420 mm deep and 400 mm

wide). The cages were fitted with thrips proof mesh net (105 µ; Sefar Filter Specialists Pty

Ltd., Malaga, WA), and the entire cage was fitted on top of a Nylex tote box (320 x 420 mm;

Blyth Enterprises Pty Ltd, Australia). When calendula plants were flowering, WFT adults were

released at the base of the plant to maintain the colony.


Predatory mites [Typhlodromips montdorensis, Neoseiulus cucumeris, and Hypoaspis miles]

were sourced from commercial Australian suppliers (Biological Services, SA; Manchil IPM

Services, WA; and Beneficial Bug Company, NSW). Mites were provided in plastic buckets or

plastic bag containing vermiculite. Trials were conducted immediately upon receipt of

predatory mites.


144

6.2.2 Experiment 1: Direct toxicity of spinosad to WFT and predatory mites

6.2.2.1 Western flower thrips

The experiment was conducted in the controlled temperature (CT) room. Twenty cold-

anaesthetised WFT adults (2-3 d old) collected from the colony were placed on a paper towel

and lightly sprayed with either 5mL of diluted spinosad (treatment) or water (control) with a

hand-held atomiser (Hills Sprayers, BH220063) . Spinosad (Success™, 120 g/L emulsifiable

concentrate, Dow AgroSciences Australia Ltd) was applied at the recommended rate of 80

mL/100 L rate (0.096 g a.i./L). After spraying excess spinosad or water (if any) was gently

removed with a soft tissue. Thrips (n = 20) were then transferred to a glass Petri dish (150 x 15

mm) containing an excised strawberry leaf placed adaxial side up on a piece of moistened filter

paper. The filter paper was glued to the bottom of the Petri dish to ensure that thrips could not

hide between the Petri dish and filter paper. The leaf petiole was covered with cotton wool

soaked in 10% sugar solution to extend leaf life and the edge of the strawberry leaf was glued to

the filter paper. The Petri dish was covered with a screen (mesh net 105 µ), and the side of the

Petri dish was sealed with paraffin film (Parafilm M®, Micro Analytix Pty Ltd) to prevent thrips

escaping (Figure 6.1). This procedure was repeated 20 times to produce 20 Petri dishes each

with 20 adult thrips. The procedure was repeated a second time instead using first instar thrips

larvae to produce 20 Petri dishes each with 20 first instar larvae.

Figure 6.1 Diagrammatic representation of the testing arena used for toxicity test.

Petri dishes were then placed (randomly) on a laboratory bench and thrips were monitored for

mortality. Spinosad is slow acting (Bret et al. 1997) and cumulative mortality of a test organism

usually plateaus at 2 days (48 h) to 6 days (144 h) after exposure (Viñuela et al. 2001, Cisneros

et al. 2002). Consequently, Petri dishes with WFT adults were examined at 6, 24, 48, 72, and 96

h post-release exposure periods under a stereomicroscope. The WFT larvae were checked at 6,

24, 48 and 72 h post-release exposure periods (after 72 h post-release exposure periods, all

larvae had pupated). WFT adults or larvae were recorded as dead if they did not respond to

probing with a fine paintbrush.


145


To assess the effect of direct exposure to spinosad on the three species of predatory mites, I used

the same bioassay method as described above except for the following differences. A thin

barrier of Tac-Gel (Stickem™

, The Olive Centre, Australia) was applied to the edge of the leaf

to prevent the escape of predatory mites. In addition, first or second instar WFT larvae were

added to Petri dishes to provide food for the mites. In each Petri dish, there were 200 first instar

thrips larvae for T. montdorensis [average 10 larvae per mite (Steiner et al. 2003)], 100 first

instar larvae for N. cucumeris [average six larvae per mite (Zilahl-Balogh et al. 2007)] and 40

second instar for H. miles [two larvae per mite (Berndt et al. 2004)]. During the trial period,

additional thrips larvae were added to the Petri dishes as required. Mortality of the mites was

monitored at 6, 24, 48 and 72 h post-release exposure periods (as no mite mortality occurred

after 72 h post-release exposure period). Mites were recorded as dead if they did not respond to

probing with a fine paintbrush. There were 20 individuals per Petri dish and 20 Petri dishes for

each species.

6.2.3 Experiment 2: Residual toxicity of spinosad to WFT and predatory mites

6.2.3.1 Mortality of WFT and mites to spinosad residues (contact) over time

In this experiment, WFT and mites were placed on strawberry leaves with different levels of

spinosad residue. Prior to spraying, 3-4 weeks old potted strawberry plants (CA Camino Real)

were moved to the CT room and split into groups (2, 12, 24, 48, 72, 96, 120 and 144 h old

residues and control). Leaves were marked with a permanent marker at the base of the petiole,

to enable leaves with spinosad residues to be tracked. Spinosad (at the recommended rate of

80mL/100 L, 0.096 g a.i./L) or distilled water (control) was applied to both sides of the leaves

with a hand-held atomiser until run-off (method devised after Eger et al. (1998)). In order to

get residues with different ages at the same times, first those plants in the 144 h old residue

treatment were sprayed once, followed by the spray on subsequent group of plants. Strawberry

plants in the control group were sprayed with distilled water 24 h prior to the experiment.

Separate atomisers were used for the spinosad and the water. After spraying, all plants were

covered with a modified thrips-cage (45 x 35 cm) made from mesh (105 µ) supported by a

quadrate steel-rod stand to exclude thrips from the plants. The bottom end of the cage

was taped to the pot with tape. The top end of the cage was closed with a rubber band.

Plants covered with mesh cage were then returned to the glasshouse and kept until use.


146

Spinosad was also applied to the Petri dishes (150 x 15 mm; testing arena) at the same time in a

similar fashion mentioned above in order to obtain strawberry leaves and dishes of the same

exposure age. Petri dishes were held with the open end sidewise on a stand and sprayed the

inner side with spinosad solution using an atomiser until run-off. Both parts of a Petri dish were

sprayed with spinosad. After spraying, once the excess liquid (if any) run-off, the Petri dishes

were kept on a tray and allowed to dry in a glasshouse for one and half hours. Dried Petri dishes

were stored in separate plastic trays according to residue treatments (2, 12, 24, 48, 72, 96, 120,

and 144 h old residues and the control) and kept in the CT room until use.

At the onset of the experiment, strawberry plants sprayed with spinosad or water were brought

to the CT room. Strawberry leaves were removed from the sprayed plants and placed adaxial

side up at the bottom of a glass Petri dish (150 x 15 mm) with the same residue ages. Each leaf

was glued to the bottom of a Petri dish to prevent arthropods from hiding underneath. In

addition, a thin barrier of Tac Gel was applied to the edge of the leaf to prevent escape;

however, WFT adults can fly. A spinosad-treated Petri dish was used in order to keep the test

subject in contact with spinosad residues, if any test individual somehow escaped the treated

leaf surface. The petiole was covered with cotton soaked in a 10% sugar solution to extend leaf

life. Twenty WFT adults (2-3d old), 20 WFT larvae (1d old), or 20 adult mites (each species)

were released onto the leaf surface. A screen cover (mesh net 105 µ) was placed over the Petri

dish as previously described. The strawberry leaf and Petri dish was sprayed with distilled water

to be used as a control. Petri dishes with WFT and predatory mites were then randomly arranged

on a laboratory bench in the CT room. For each of the eight residue times and the control

(water) there were 20 replicates of each mite species and WFT adults and larvae. For the mite

bioassays, WFT larvae were added (at a rate described above) as food each day for 2-3 hrs, and

then removed to ensure that mites were not consuming spinosad-intoxicated larvae.

Mortality was checked under a stereomicroscope. WFT (larvae and adults) and predatory mites

(T. montdorensis, N. cucumeris and H. miles) were recorded as dead if they did not respond to

probing with a fine paintbrush. WFT adults were checked at 6, 24, 48, 72, and 96 h post-release

exposure periods. WFT Larvae were checked at 6, 24, 48, and 72 h post-release exposures (after

72 h all the larvae had pupated). Predatory mite mortality was checked at 24, 48 and 72 h post-

release exposure periods (no mortality occurred after 72 h post-release exposure).

6.2.3.2 Indirect exposure of spinosad to predatory mites via consumption of intoxicated

WFT larvae

Twenty-four hours prior to the trial, newly emerged first instar WFT larvae were collected from

the stock colony. Larvae were transferred to a glass Petri dish (150 x 15 mm) containing a


147

cotton wool ball soaked in a 10% sugar solution, then stored in the CT room for 12 h (to ensure

feeding on spinosad- or water-treated leaf at a later stage). Larvae were then released onto

excised strawberry leaves previously treated with spinosad (2, 12, 24, 48, 72, 96, 120, or 144 h

old residues) or control (water) in a Petri dish and allowed to feed for 12 hours. A thin barrier of

Tac Gel was applied to the edge of the leaf to prevent larvae from escaping the leaf. After 12 h,

WFT larvae were transferred onto a fresh, untreated strawberry leaf in a Petri dish (150 x 15

mm), containing 20 adult mites of one species of mite. The leaf petiole was covered with cotton

soaked in a 10% sugar solution to keep the leaf fresh during the trial period. A thin barrier of

Tac Gel was applied to prevent either WFT larvae or predatory mites escaping the leaf surface.

In each Petri dish, 200, 120 and 40 first or second instar of WFT larvae that had not been

exposed to spinosad were released for T. montdorensis, N. cucumeris and H. miles respectively.

During the trial period, additional thrips larvae were added to the Petri dishes as required. All

Petri dishes with WFT larvae and predatory mites were covered with mesh net (105 µ) and

sealed with paraffin film. All Petri dishes with predatory mites were kept on a laboratory bench

(randomly arranged) and checked under stereomicroscope at 24, 48 and 72 h post-release

exposure periods. Predatory mites were recorded as dead if they did not respond to probing with

a fine paintbrush. Each treatment (residue age) and control (water) was replicated 20 times (20 x

20 = 400 individuals) for each species of predatory mites.

6.2.3.3 Toxicity of spinosad to predatory mites via consumption of intoxicated WFT

larvae and direct exposure to spinosad residues of different ages

This bioassay evaluated mortality of predatory mites (T. montdorensis, N. cucumeris and H.

miles) via two exposure routes: (i) via consumption of intoxicated WFT larvae, and (ii)

simultaneous direct exposure to spinosad residues on strawberry leaves. WFT larvae were

collected and exposed to spinosad as described in 6.2.3.2; strawberry leaves were prepared as

described in 6.2.3.1. Testing arenas were prepared using the same age residue of leaf, Petri dish,

and WFT larvae. A thin layer of Tac Gel was applied at the edge of the leaf to prevent escape of

WFT larvae or predatory mites from the testing arena. Intoxicated first instar WFT larvae were

released onto the leaf surface in a Petri dish at a rate described above. Twenty mite adults of one

species were then released onto the leaf surface and the Petri dish was covered and sealed as

described above. A Petri dish and strawberry leaf were sprayed with distilled water to be used as

a control. All Petri dishes were arranged randomly on the laboratory bench in the CT room and

mortality was checked under a stereomicroscope at 24, 48, and72 h post-release exposure

periods. The above procedure was repeated 20 times (20 x 20 = 400 individuals) with spinosad

residues aged 2, 12, 24, 48, 72, 96, 120 and 144 h for each species of predatory mite.


148

6.2.4 Experiment 3: Repellency of spinosad to predatory mites (choice test)

To evaluate the possible repellency of different aged residues of spinosad (2, 12, 24, 48, 72, and

96 h old) to T. montdorensis, N. cucumeris and H. miles, a choice test was conducted in the CT

room using a method modified from van Driesche et al. (2006). A test arena was constructed by

cutting spinosad-treated (leaves with spinosad treated residues 2, 12, 24 48, 72 and 96 h old) or

water-treated (control) strawberry leaves in half along the mid-vein. Leaf halves (one half

consisting of a spinosad treated leaf, the other the control leaf) were then taped onto filter paper

to form a full leaf, with a 2 mm channel between the halves for mite releases. A thin barrier of

Tac Gel was applied to the edges of the leaf and filter paper. Additionally, at the centre of each

leaf half, a rounded arena was created using Tac-Gel where WFT larvae were released as an

attractant. The Tac-Gel barrier prevented WFT larvae moving from the spinosad treated leaf to

water treated leaf half. In a pilot experiment, it was found that most of the adults of predatory

mite (T. montdorensis, N. cucumeris, or H. miles) did not move from its release point if no WFT

larvae were provided as an attractant. Before predatory mites were released into the test arena,

15 WFT larvae (first instar for T. montdorensis and N. cucumeris and second instar for H. miles)

were released onto each leaf half. Ten adult mites of one species were then placed on the filter

paper channel in each Petri dish. The test arena was observed three times during a 60 min period

at three 20 min intervals (three observations) and the number of predatory mites on the

spinosad- and the water-treated leaf surfaces was counted at each observation. With each

species of predatory mite, the experiment was repeated 20 times (20 x 10 = 200 individuals of

each species) for each residue (aged 2, 12, 24, 48, 72 and 96 h). A new testing arena was used

for each test.

6.2.5 Data analysis

For each experiment in which mortality was assessed, the number of individuals that died at

each observation was counted and expressed as a percentage of the total number of individuals

in the arena and corrected using Abbott’s formula (Abbott 1925):

Abbott’s formula takes into account the proportion of control thrips or mites dying in the trial

that have not been exposed to the spinosad, and amongst those that have been exposed to

spinosad, some may die of natural causes. In these trials, control mortality of either thrips or

mites never exceeded 5%.


149

The cumulative mortality of predatory mites due to direct toxicity of spinosad was analysed

with one-way ANOVA (Proc Mixed Procedure) to differentiate mortality amongst species. In

addition, if mortality differed between post release exposure periods, mortality data of each

species of predatory mite (T. montdorensis, N. cucumeris, and H. miles) was subjected to

separate one-way ANOVAs. To determine if residues of different ages affected the mortality of

WFT, the mortality of adults and larvae was analysed with a one-way ANOVA for each post-

release exposure period. Cumulative mortality of WFT adult and larvae was also analysed with

separate one-way ANOVAs. Similarly, the influence of residual age of spinosad on mortality of

each species of predatory mites was analysed with separate one-way ANOVAs for each post

release exposure period. Least square means significant difference tests at the 5% probability

level were used to test for treatment differences.

Based on the mortality of predatory mites, results were classified into four categories following

the IOBC (International Organization of Biological Control) guidelines and ranked as:

1 = harmless (<25% mortality)

2 = slightly harmful (25-50% mortality)

3 = moderately harmful (51- 75% mortality)

4 = harmful (>75% mortality) (Sterk et al. 1999).

The persistence of spinosad for each species of predatory mites was also classified according to

the time taken to lose toxicity (<30% mortality):

A = short lived (<5 days)

B = slightly persistent (5-15 days) (Hassan et al. 1994, Sterk et al. 1999).

To determine the contact or contact and indirect toxicity (via consumption of intoxicated WFT

larvae and simultaneous exposure to residues), the residual threshold of spinosad for T.

montdorensis, N. cucumeris and H. miles was estimated with Probit analyses (Finney 1971).

The LT25 (lethal time of 25% mortality) was used, which is considered an acceptable level

(Shipp et al. 2000).

The repellency of spinosad residues (2, 12, 24, 48, 72 and 96 h old) to predatory mites was

analysed by paired t -test (Proc ttest Procedure).

Prior to statistical analyses data were transformed using √(x + 0.5) (Healy and Taylor 1962);

however, untransformed means were shown in tables and figures. All statistical analyses were

computed with SAS 9.1 statistical package, Carry, NC, USA (SAS 2002-2003). Figures were

drawn with GraphPad Prism 5.0 software (GraphPad Software Inc 2007).


150

6.3 Results

6.3.1 Experiment 1: Direct contact toxicity of spinosad to WFT and predatory mites

When exposed directly to spinosad, WFT adults experienced 98% mortality (at 96 h post-

release exposure) and larvae experienced 96% mortality (at 72 h post-release exposure).

All species of predatory mites had greater than 90% mortality when exposed directly to

spinosad, but mortality differed significantly amongst mite species (F 2,57 = 8.58, P = 0.0006),

with the highest mortality recorded for H. miles (95.27% ± 2.71) and the lowest for N.

cucumeris (90.25% ± 2.71). However, there was no significant difference (p > 0.05) in

mortality between T. montdorensis (91.31% ± 2.04) and N. cucumeris. Spinosad toxicity

declined over time. Mortality of mites was highest at 24 h post-release exposure period (Table

6.1). Mortality was lowest at 96 h post-release exposure period for T. montdorensis and H. miles

and 72 h post-release exposure period for N. cucumeris. No mortality of N. cucumeris was

recorded at 96 h post-release exposure period.

Table 6.1 Mean (±SE) corrected mortality (%) at different post-release exposure periods (h) to

predatory mites after direct exposure to spinosad (recommended rate, 80 mL/100 L).

Post-release

exposures period(h)

Corrected mortality (%) of predatory mite species (Mean ± SE)

T. montdorensis* N. cucumeris* H. miles*

6

24

48

72

96

6.00 ± 0.78b

60.35 ± 3.19d

15.25 ± 1.72c

6.46 ± 1.56b

3.25 ± 0.91a

6.55 ± 0.93b

62.33 ± 1.95d

14.90 ± 1.05c

6.47 ± 1.59b

0.0†

9.23 ± 1.46c

68.32 ± 2.00d

11.41 ± 0.89c

5.56 ± 1.35b

0.75 ± 0.41a

F

df

P

106.09

4 , 95

<0.0001

204.28

3, 76

<0.0001

256.40

4 , 95

<0.0001

* Within column values followed by the same letter do not differ significantly at α = 0.05. †No

mortality of N. cucumeris at 96 h post-release exposure period was detected, and was therefore

not included in analysis.

6.3.2 Experiment 2: Residual toxicity of spinosad to WFT and predatory mites

6.3.2.1 Residual (contact) toxicity of spinosad to WFT and predatory mites

Spinosad residues aged 2, 12, 24, 48, 72 and 96 h old were toxic to WFT adults (46 to 93%

mortality), causing death until 96 h post-release exposure period (Table 6.2). The highest and


151

lowest mortality occurred when WFT adults were exposed to 2 h and 96 h old spinosad residues

respectively (Table 6.2). Adult mortality was high and not significantly different (p > 0.05)

among 2, 12, and 24 h old spinosad residues (Table 6.2). Similar to WFT adults, spinosad

residues declined in toxicity to WFT larvae over time. The mean percentage of cumulative

mortality to WFT larvae was highest and lowest when exposed to 2 h and 96 h old spinosad

residues respectively (Table 6.2).

Table 6.2 Mean (± SE) corrected mortality (%) of WFT adults and larvae when exposed to

spinosad residues aged 2, 12, 24, 48, 72, 96, and 120 h at different post-release exposure

periods.

Residue

age (h)

Corrected mortality (%) (Mean ± SE)

Post-release exposure periods (h) Cumulative

mortality* 6h* 24h* 48h* 72h* 96h*

Adults

2h

12h

24h

48h

72h

96h†

6.6±0.77c

4.8±0.7bc

2.8±0.75b

1.3±0.5a

0.0±0.0a

0.0±0.0a

-

55.8±2.9c

52.2±2.9c

50.1±2.7c

31.0±1.6b

21.2±2.1a

18.4±1.9a

-

25.8±1.9a

25.9±1.9a

27.5±2.1a

28.7±1.9a

24.4±1.9a

22.2±1.8a

-

3.8±0.95a

4.5±1.14a

5.8±1.11a

7.5±1.18a

7.0±0.92a

5.5±0.88a

-

0.0±0.00a

0.0±0.00a

0.5±0.34a

1.8±0.75b

0.8±0.4ab

0.0±0.00a

-

92.9±1.58c

89.6±1.72c

89.0±1.83c

71.3±3.32b

54.2±3.06a

46.8±2.49a

-

F

df

P

25.17

5 & 114

< 0.0001

47.93

5 & 114

< 0.0001

1.45

5 & 114

0.2144

1.91

5 & 114

0.0987

3.36

5 & 114

0.0072

66.88

5 & 114

< 0.0001

Larvae

2h

12h

24h

48h

72h

96h

120h†

8.1±0.9c

7.1±0.9c

4.1±0.7b

1.5±0.6a

0.5±0.3a

0.0±0.0a

-

63.6±3.0d

59.9±2.6d

36.5±1.9c

22.4±2.2b

11.7±1.1a

8.8±1.1a

-

17.5±1.9b

22.0±2.1bc

29.7±2.1c

27.9±2.1c

15.9±1.9ab

9.8±1.6a

-

4.8±0.9a

4.5±1.1a

5.5±0.9ab

9.8±1.2b

6.5±0.9ab

4.0±0.9a

-

-

-

-

-

-

-

-

94.9±2.7e

94.7±1.1d

79.6±2.6d

62.0±2.8c

35.2±2.3b

22.6±1.8a

-

F

df

P

37.94

5 & 114

< 0.0001

129.82

5 & 114

< 0.0001

16.89

5 & 114

< 0.0001

3.92

5 & 114

0.0026

172.75

5 & 114

< 0.0001

* Within column for each WFT stage, values followed by the same letter do not significantly

differ at α = 0.05. †No mortality of adults and larvae was found for 120 h old spinosad residue.

Spinosad residues aged 2, 12, 24, 48, 72, 96 and 120 h old were toxic to mites, causing death

until 72 h post-release exposure period (Table 6.3). Mortality due to residual contact toxicity of

spinosad differed with residue age. Mortality was the highest and lowest for all three species

when mites were exposed to 2 h and 144 h old residues respectively. There was no significant

difference in mortality when T. montdorensis or N. cucumeris were exposed to 2 h and 12 h old

residues. For H. miles, mortality did not differ between 2 h, 12 h and 24 h old residues.


152

According to the IOBC toxicity classification, contact toxicity of spinosad ranged from

harmless (<25% mortality) to harmful (>75% mortality) (Table 6.3). Spinosad residues aged 2 h

to 96 h were slightly to moderately harmful to T. montdorensis at 72 h post-release exposure

Table 6.3 Residual toxicity (contact) of spinosad to predatory mites at 24, 48, and 72 h post

release exposure periods. The IOBC classification: 1 = harmless (<25% mortality), 2 = slightly

harmful (25-50% mortality), 3 = moderately harmful (51- 75% mortality) and 4 = harmful

(>75% mortality). Persistence class: A = short-lived (<5 d), B = slightly persistent (5-15 d).

Residue

age (h)

Corrected mortality (%) (mean ± SE) at

post release exposure period†

Toxicity class Persistence

class

24h* 48h* 72h* 24h 48h 72h

T. montdorensis

2h

12h

24h

48h

72h

96h

120h

144h

48.5±4.1g

44.7±3.7fg

39.9±2.6ef

33.3±2.2e

21.0±2.4d

13.9±2.2c

4.6±0.5b

0.0±0.0a

57.34.6±5.1f

57.01±4.8ef

56.0±4.5e

49.7±4.6e

38.0±3.8d

31.1±3.3cd

8.4±0.7b

0.0±0.0a

70.6±5.1f

63.01±4.9ef

62.7±4.9e

59.4±5.1e

45.4±3.8d

33.7±3.6c

9.2±0.9b

0.8±0.4a

2

2

2

2

1

1

1

1

3

3

3

2

2

2

1

1

3

3

3

3

2

2

1

1

A

F

df

P

91.62

7, 152

< 0.0001

110.43

7, 152

< 0.0001

105.95

7, 152

< 0.0001

N. cucumeris

2

12

24

48

72

96

120

144

44.4±3.2e

42.9±3.1e

29.8±1.9d

23.5±1.8c

18.2±1.82c

6.6±0.6b

0.8±0.4a

0.0±0.0a

71.7±5.1g

70.2±4.9g

53.1±4.3f

41.1±2.8e

32.1±3.2d

13.5±0.9c

4.6±0.5b

0.5±0.3a

76.0±4.9f

74.5±4.9f

58.2±4.4e

45.7±2.9d

36.2±3.2d

14.8±1.1c

4.6±0.5b

0.5±0.3a

2

2

1

1

1

1

1

1

3

3

3

2

2

1

1

1

4

3

3

2

2

1

1

1

A

F

df

P

174.95

7, 152

< 0.0001

128.75

7, 152

< 0.0001

145.63

7, 152

< 0.0001

H. miles

2

12

24

48

72

96

120

144

55.5±3.6f

54.0±3.6f

52.34±1.9f

46.7±3.9e

37.0±1.7d

17.2±1.7c

9.9±1.5b

2.4±0.7a

78.9±2.5f

75.3±3.0f

69.4±2.2f

63.9±4.4e

55.2±2.4d

42.5±2.5c

17.3±1.6b

8.5±1.3a

82.34±2.5f

80.6±3.3f

76.45±2.6ef

73.49±4.6de

62.9±2.3d

44.6±2.8c

27.4±2.1b

10.5±1.7a

3

3

3

2

2

1

1

1

4

4

3

3

3

2

1

1

4

4

4

3

3

2

2

1

B

F

df

P

183.76

7, 152

< 0.0001

139.47

7, 152

< 0.0001

121.37

7, 152

< 0.0001

†no mortality was recorded after 72 h post-release exposure period. *within column, for each

species of predatory mites, means with different letters differed significantly (α = 0.05).


153

period. Meanwhile, spinosad residues aged 2 h to 72 h were slightly harmful to harmful to N.

cucumeris at 72 h post-release exposure period. In case of H. miles, spinosad residues aged from

2 h to 120 h were slightly harmful to harmful at 72 h post-release exposure period. According to

the IBOC guidelines, spinosad toxicity for T. montdorensis and N. cucumeris was short-lived,

while slightly persistent for H. miles.

The relationships between mite mortality and spinosad residues, with mortality as a function of

residue age (log hrs) are presented in Figure 6.2. The LT25 (25% mortality of non-target

organisms caused by residual toxicity) indicates that the release of each species of predatory

mite after a spinosad application would cause only 25% mortality, allowing 75% survival of the

predatory mites. LT25 of spinosad differed for each species and were estimated as 4.2 days

(101.63 h; Antilog102.01

), 3.2 days (77.72 h; Antilog101.89

) and 5.8 days (138.83 h; Antilog102.143

)

for T. montdorensis, N. cucumeris and H. miles respectively (Table 6.4; Figure 6.2).

Table 6.4 Probit analysis (Abbott 1925) of the mortality of adult predatory mites exposed to

spinosad residues of different ages.

Spp Slope ± SE LT25

(hrs)

95% CL χ

2 df P Lower Upper

T. montdorensis

N. cucumeris

H. miles

-2.09 ± 0.078

-2.11 ± 0.091

-3.18 ± 0.131

101.63

77.72

138.83

91.34

70.27

127.79

114.39

86.719

152.40

16.11

13.53

21.02

6

6

6

0.013

0.035

0.002


154

0.0

0.2

0.4

0.6

0.8

1.0

0.0

0.2

0.4

0.6

0.8

1.0

Pro

bit

mo

rtality

0 1 2 3 40.0

0.2

0.4

0.6

0.8

1.0

Log10 (hrs)

A

B

C

Figure 6.2 Probit mortality of (A) T. montdorensis, (B) N. cucumeris, and (C) H. miles recorded

against spinosad residues of different ages (log10 hrs).

6.3.3.2 Indirect exposure of spinosad to predatory mites via consumption of intoxicated

WFT larvae

This bioassay evaluated mortality of predatory mites (T. montdorensis, N. cucumeris and H.

miles) via consumption of intoxicated WFT larvae. WFT larvae were allowed to feed for 12

hours on strawberry leaves previously treated with spinosad (2, 12, 24, 48, 72, 96, 120 or 144 h

old residues). The consumption of intoxicated WFT larvae killed predatory mites until 72 h

post-release exposure periods as shown in Table 6.5. Mortality varied among residue ages and

mortality was highest when mites fed on intoxicated WFT larvae that had fed on leaves with 2 h

old residue. Meanwhile, mortality was lowest when T. montdorensis and N. cucumeris fed on

intoxicated WFT larvae that had fed on 120 h old residue. No mortality of T. montdorensis or N.


155

cucumeris was recorded when mites fed on WFT larvae that had fed on 144 h old residue. In

case of H. miles, mortality was lowest when mites fed on WFT larvae that had fed on 144 h old

residue.

According to the IOBC classification, spinosad via indirect exposure was harmless to

moderately harmful to predatory mites (Table 6.5). Spinosad residues aged 2 h to 24 h were

slightly to moderately harmful to T. montdorensis at 24 h and 48 h post-release exposure

periods, and moderately harmful at 72 h post-release exposure period. Meanwhile, 72 h to 120 h

old residues of spinosad via indirect exposure were harmless to T. montdorensis. For N.

cucumeris, indirect toxicity of 2 h and 12 h old residues was slightly harmful when examined at

24 h and 48 h post-release exposure periods and moderately harmful at 72 h post-release

exposure period. Residues 24 h and 48 h old (at 72 h post-release exposure period) were only

slightly toxic to N. cucumeris. For H. miles, 2 h, 12 h and 24 h old residues were moderately

harmful, though 12 h and 24 h old residues were classified as slightly harmful when examined

24 h post-release exposure period. Forty-eight hour and 72 h old residues were slightly harmful

for H. miles at 72 h post-release exposure period.


156

Table 6.5 Mortality of predatory mites after feeding on spinosad intoxicated WFT larvae at 24

h, 48 h, and 72 h post-release exposure periods. The IOBC classification: 1 = harmless (<25%

mortality), 2 = slightly harmful (25-50% mortality), 3 = moderately harmful (51- 75%

mortality) and 4 = harmful (>75% mortality).

Residue

age (h)

Corrected mortality (%) (mean ± SE) at post-release

exposure period†

Toxicity class

24h* 48h

* 72h

* 24h 48h 72

T. montdorensis 2h

12h

24h

48h

72h

96h

120h

144h††

43.25±2.06f

40.25±3.05f

32.75±2.40e

22.75±2.09d

11.75±1.27c

6.5±0.97b

0.75±0.41a

-

45.23±2.72e

42.24±3.39de

40.35±3.35de

37.38±2.13d

22.75±2.07c

10.75±1.27b

3.75±0.71a

-

56.32±2.80e

52.32±3.52e

51.23±3.58e

40.36±2.11d

24.75±2.03c

10.75±11.27b

3.75±0.71a

-

2

2

2

1

1

1

1

-

2

2

2

2

1

1

1

-

3

3

3

2

1

1

1

-

F

df

P

115.73

6, 133

< 0.0001

140.98

6, 133

< 0.0001

1156.59

6, 133

< 0.0001

N. cucumeris 2

12

24

48

72

96

120

144††

44.27±2.41f

37.93±3.01f

30.07±1.92e

17.68±1.72d

11.91±1.73c

1.56±0.53b

0.71±0.34a

-

42.36±3.92e

40.36±3.91de

37.89±3.99de

30.20±2.67d

20.85±3.12c

8.16±0.83b

1.11±0.41a

-

51.23±3.64f

50.78.36±3.80f

40.93±3.99e

32.80±2.68d

22.74±3.36c

8.16±0.95b

1.11±0.41a

-

2

2

2

1

1

1

1

-

2

2

2

1

1

1

1

-

3

3

2

2

1

1

1

-

F

df

P

164.90

6, 133

< 0.0001

167.00

6, 133

< 0.0001

183.16

6, 133

< 0.0001

H. miles 2

12

24

48

72

96

120

144

50.14±3.23g

46.56±3.45fg

40.34±2.25ef

35.78±3.89e

20.76±2.60d

11.65±1.39c

5.06±0.62b

0.00±0.00a

53.35±2.44e

50.01±2.93e

44.35±2.74de

37.25±4.42d

22.13±3.37c

20.13±2.05c

9.72±1.79b

0.95±0.39a

62.34±2.11f

59.99±3.28f

55.45±2.91f

40.56±2.40e

34.20±3.67d

23.64±2.05c

9.72±1.79b

0.95±0.39a

3

2

2

2

1

1

1

1

3

3

3

2

1

1

1

1

3

3

3

2

2

1

1

1

F

df

P

218.32

6, 152

< 0.0001

192.998

6, 152

< 0.0001

201.76

6, 152

< 0.0001

†no mortality was recorded after 72 h post-release exposure.

††no mortality of T. montdorensis

and N. cucumeris was recorded for 144 h old spinosad residue. *Within column, for each species

of predatory mites, means with different letters differed significantly (LS means, α = 0.05).


157

6.3.5 Residual toxicity to predatory mites via consumption of spinosad-intoxicated WFT

larvae and direct exposure to spinosad residues

Toxicity of spinosad residues via consumption of intoxicated WFT larvae and simultaneous

exposures to residues is shown in Table 6.6. The mean mortality of T. montdorensis and N.

cucumeris was highest when exposed to 2 h old residue and lowest when exposed to 144 h old

residue. There was no difference in T. montdorensis or N. cucumeris mortality between 2 h and

12 h old residues. The mortality of H. miles was similarly highest when exposed to 2 h old

residue and lowest when exposed to 144 h old residue. Hypoaspis miles mortality was high and

not significantly different among 2 h, 12 h and 24 h old residues.

Indirect and contact toxicity of spinosad residues were classified as harmless to harmful (Table

6.6). Spinosad residues aged 2 h, 12 h and 24 h were harmful to T. montdorensis at 72 h post-

release exposure period. Meanwhile, 48 h to 96 h old residues were slightly harmful to

moderately harmful to T. montdorensis at 72 h post-release exposure period. For N. cucumeris,

2 h and 12 h old residues were harmful at 48 h and 72 h post-release exposure periods.

Meanwhile, 24 h and 48 h old residues appeared to be moderately harmful for N. cucumeris at

72 h post-release exposure period. Spinosad residue aged 72 h was slightly harmful at 48 h and

72 post-release exposure periods only. Spinosad residues aged 96 h, 120 h and 144 h were

classified harmless to N. cucumeris. For H. miles, 2 h, 12 h, and 24 h old residues were

moderately harmful at 24 h post-release exposure period and became harmful when examined at

48 h and 72 h post-release exposure periods. Spinosad residues aged 48 and 72 h were

moderately harmful to harmful for H. miles at 72 h post-release exposure period. Residues 96 h

and 120 h old were classified as slightly harmful to H. miles.

As no harmful effects were found when T. montdorensis and N. cucumeris were exposed to

residues aged 120 h, spinosad was categorised as short-lived (persistence <5 d) (Table 6.6).

However, 120 h old spinosad residue posed some harmful effect to H. miles and was ranked as

slightly persistent.

The relationships between mortality of the predatory mites and spinosad residues were analysed

by probit procedure (Table 6.7; Figure 6.3) and mortalities as a function of residue ages (log

hrs) are presented in Figure 6.3. The residual thresholds (LT25) were estimated as 5.4 d (129.67

h, Antilog102.11285

), 3.9 d (95.09 h, Antilog101.97816

) and 6.1 d (146.68 h, Antilog102.16638

) for T.

montdorensis, N. cucumeris and H. miles respectively.


158

Table 6.6 Residual toxicity of spinosad to predatory mites at 24 h, 48 h, and 72 h post release

exposure periods. Mites were fed spinosad-intoxicated WFT larvae and simultaneously exposed

to residue. The IOBC classification: 1 = harmless (<25% mortality), 2 = slightly harmful (25-

50% mortality), 3 = moderately harmful (51- 75% mortality) and 4 = harmful (>75% mortality).

Persistence class: A = short lived (<5 d), B = slightly persistence (5-15 d).

Residue

age (h)

Corr. mortality (%) (mean ± SE) at post-

treatment periods† Toxicity class

Persist.

class

24h* 48h

* 72h

* 24h 48h 72h

T. montdorensis

2h

12h

24h

48h

72h

96h

120h

144h

51.39±2.04f

50.63±2.47f

42.82±2.93e

35.77±1.59d

31.74±1.81d

15.62±0.89c

5.79±0.90v

0.00±0.00a

81.47±2.61f

81.47±2.66f

73.09±3.77ef

70.05±2.68e

48.98±3.11d

28.68±1.07c

9.13±1.09b

3.27±0.40a

87.56±2.84f

86.55±2.73f

76.90±3.94f

73.86±2.56e

55.33±3.58d

31.73±1.54c

9.14±1.09b

3.27±0.40a

3

3

2

2

2

1

1

1

4

4

3

3

2

2

1

1

4

4

4

3

3

2

1

1

A

F

df

P

240.55

6, 152

< 0.0001

288.40

6, 152

< 0.0001

287.23

6, 152

< 0.0001

N. cucumeris

2

12

24

48

72

96

120

144

53.02±1.80g

45.23±2.91fg

39.70±1.45ef

31.16±1.63d

22.11±1.29c

10.55±0.86b

1.28±0.40a

0.00±0.00a

80.97±2.65f

76.14±3.81ef

67.01±2.69e

48.73±2.44d

43.15±1.86d

19.29±1.47c

5.33±0.66b

1.21±0.43a

84.52±2.81f

80.46±3.70ef

72.08±2.51e

53.30±2.57d

46.45±2.40d

20.56±1.57c

5.33±0.66b

1.21±0.43a

3

2

1

1

1

1

1

1

4

4

3

2

2

1

1

1

4

4

3

3

2

1

1

1

A

F

df

P

325.02

6, 152

< 0.0001

313.48

6, 152

< 0.0001

322.23

6, 152

< 0.0001

H. miles

2

12

24

48

72

96

120

144

71.14±3.23f

70.13±2.11f

65.06±2.20f

50.64±2.70e

38.48±1.53d

17.72±1.15c

10.88±1.13b

4.56±0.41a

87.98±2.44f

86.19±2.55f

83.89±1.97f

70.84±3.19e

57.03±2.61d

43.73±1.44c

32.23±1.85b

11.76±0.90a

94.63±2.36f

91.05±2.84f

89.26±2.12ef

78.26±2.91e

63.68±2.72d

48.59±2.27c

32.99±1.99b

11.76±0.90a

3

3

3

3

2

1

1

1

4

4

4

3

3

2

2

1

4

4

4

4

3

2

2

1

B

F

df

P

257.59

6, 152

< 0.0001

200.28

6, 152

< 0.0001

211.91

6, 152

< 0.0001

†no mortality was recorded after 72 h post-treatment in either of the species.

*within column, for

each species, means with different letters differed significantly (α = 0.05).


159


simultaneously exposed to spinosad via consumption of intoxicated WFT larvae and contact

with spinosad residues of different ages.

Species Slope ± SE LT25

(hrs)

95% CL

χ2 df P

Lower Upper

T. montdorensis

N. cucumeris

H. miles

-1.46±0.12

-1.43±0.10

-1.81±0.15

129.67

95.09

146.68

118.52

87.17

135.91

143.42

104.61

159.89

14.36

12.35

15.68

6

6

6

0.0258

0.0546

0.0155

0.0

0.2

0.4

0.6

0.8

1.0

0.0

0.2

0.4

0.6

0.8

1.0

Pro

bit

mort

ality

0 1 2 3 40.0

0.2

0.4

0.6

0.8

1.0

Log10 (hrs)

A

B

C

Figure 6.3 Probit mortality of (A) T. montdorensis, (B) N. cucumeris, and (C) H. miles recorded

against spinosad residues of different ages (log10 hrs).

6.3.6 Experiment 3: Repellency of spinosad to predatory mites (choice test)

Results showed that T. montdorensis, N. cucumeris and H. miles were repelled by spinosad

residues, though this repellence decreased with residue age (Table 6.8). There were more T.


160

montdorensis and H. miles on the water-treated strawberry leaves than leaves with 2 h, 12 h, 24

h and 48 h old spinosad residues. However, 72 h and 96 h old residues did not repel T.

montdorensis or H. miles (Table 6.8). While, N. cucumeris were repelled by residues aged 2 h,

12 h and 24 h, but not by residues aged 48-96 h.

Table 6.8. Mean (±SE) numbers of predatory mites on spinosad- and water-treated strawberry

leaf in a choice test (t-test, df = 19).

Species Residual

age (h)

Mean ±SE t-value P-value

Control (Water) Spinosad

T. montdorensis

2

12

24

48

72

96

7.17 ± 0.16

7.13 ± 0.14

7.07 ± 0.16

5.97 ± 0.21

4.65 ± 0.09

4.62 ± 0.11

1.87 ± 0.19

1.85 ± 0.16

1.97 ± 0.17

2.58 ± 0.22

4.40 ± 0.11

4.67 ± 0.15

15.66

18.72

16.22

8.83

1.48

-0.23

< 0.0001

< 0.0001

< 0.0001

< 0.0001

0.1556

0.8242

N. cucumeris

2

12

24

48

72

96

6.98 ± 0.23

7.02 ± 0.18

6.45 ± 0.17

4.65 ± 0.09

4.72 ± 0.09

4.83 ± 0.08

1.17 ± 0.16

1.13 ± 0.12

2.22 ± 0.18

4.48 ± 0.12

4.53 ± 0.11

4.75 ± 0.11

15.74

24.67

14.90

1.01

1.21

0.58

< 0.0001

< 0.0001

< 0.0001

0.3248

0.2423

0.5663

H. miles

2

12

24

48

72

96

7.40 ± 0.19

7.47 ± 0.19

7.52 ± 0.19

5.73 ± 0.22

4.82 ± 0.17

4.46 ± 0.14

1.03 ± 0.13

0.95 ± 0.13

1.07 ± 0.15

3.68 ± 0.22

4.48 ± 0.18

4.73 ± 0.16

24.78

25.19

25.17

4.98

1.03

-0.98

< 0.0001

< 0.0001

< 0.0001

< 0.0001

0.3171

0.3394

6.4 Discussion

This laboratory bioassay confirms that spinosad is efficacious against WFT, causing 98%

mortality of adults and 96% mortality of larvae when applied at the recommended rate (80

mL/100 L spinosad). Morishita (2001) similarly found that spinosad (25% WP) killed 100%

WFT larvae in Petri dish bioassays. Apart from direct contact, spinosad residues also killed

WFT adults and larvae, with 2 h old residue causing 100% mortality (Jones et al. 2005, van

Driesche et al. 2006). The present study demonstrated that spinosad residues were highly toxic

for longer: 6-48 hours under laboratory conditions. After 72 h, spinosad was either less or no

longer toxic to WFT. Whilst it is difficult to extrapolate results from the laboratory to the field,

there could also be a residual effect in the field.

Spinosad also affected T. montdorensis, N. cucumeris, and H. miles both directly and indirectly

and choice tests indicate that spinosad residues are repellent to mites. Residues aged 2 h to 48 h


161

repelled T. montdorensis and H. miles, and residues aged 2 h to 24 h repelled N. cucumeris.

Villanueva and Walgenbach (2006) reported that spinosad residues repelled two-spotted spider

mite Tetranychus urticae Koch, but not European red mite, Panonychus ulmi (Koch).

Tetranychus urticae females also congregated in untreated areas where they laid more eggs

(Villanueva and Walgenbach 2006). While, van Driesche et al. (2006) found no significant

difference in the numbers of N. cucumeris and the predatory mite Iphiseius degenerans

(Berlese) on untreated or spinosad treated leaves, although they found relatively more mites on

water-treated leaves.

Spinosad was highly toxic to T. montdorensis, N. cucumeris and H. miles when applied at the

recommended rate (80 ml/100 L), causing >90% mortality. Elzen et al. (1998) using a bean-pod

bioassay, found that spinosad killed >90% adult Orius insidiosus (Say) (Hemiptera:

Anthocoridae). Spinosad residues were also toxic to mites and based on the age of the residue,

the IOBC toxicity rating ranged from harmless to harmful. Spinosad residues aged 2 h to 72 h

old were moderately to slightly harmful to N. cucumeris; residues aged 2 h to 96 h old were

moderately to slightly harmful to T. montdorensis. Residues aged 2 h to 120 h old were harmful

to slightly harmful to H. miles. This agrees with the data of Kongchuensin and Takafuji (2006)

and Villanueva and Walgenbach (2005), who reported that spinosad residues were toxic to

Neoseiulus longispinosus (Evans) and Neoseiulus fallacies (Garmen) (Acari: Phytoseiidae).

Based on IOBC testing, Kopperts reports that spinosad is persistent to N. cucumeris (1-2 weeks)

(Kopperts 2009), while Biobest reports that spinosad is not persistent to N. cucumeris (Biobest

2009). Contrary to the present findings, Jones et al. (2005) and van Driesche et al. (2006)

reported that 2 h old spinosad residues were only slightly toxic to N. cucumeris. However,

differences in results may be due to differences between N. cucumeris populations, such as

previous exposure to spinosad. No spinosad has been applied in the insectaries in which the

mites were reared (pers. comm., Chilman, Manchil IPM Services, WA).

The present bioassay also indicates that toxicity increased when mites simultaneously ate WFT

larvae that had previously fed on spinosad-treated leaves and contacted to spinosad residues.

For example, contact of T. montdorensis with 2 h old spinosad residue killed 70% adults, but

mortality increased to 87% when T. montdorensis was also fed intoxicated WFT larvae.

Villanueva and Walgenbach (2005) reported that adult Neoseiulus fallacies (Garman), a

predator of two-spotted spider mite (T. urticae), died after feeding on T. urticae eggs exposed to

spinosad. Thresholds for the residual (contact) toxicity of spinosad LT25 (lethal time for 25%

mortality) were estimated as 4.2 days (101.63h), 3.2 days (77.72) and 5.8 days (138.83 h) for T.

montdorensis, N. cucumeris and H. miles respectively. When mites fed on intoxicated WFT

larvae and were simultaneously exposed to residues, toxicity further increased. Thresholds were


162

re-estimated as 5.4 d (129.67 h), 4 d (95.09 h), and 6.1 d (146.68 h) for T. montdorensis, N.

cucumeris, and H. miles respectively. This suggests that T. montdorensis, N. cucumeris and H.

miles are likely to survive if released six days after spinosad is applied. This is in general

agreement with studies on other predatory mites. Kongchuensin and Takafuji (2006) stated there

was no or very little negative influence of spinosad on adults, eggs or immature of N.

longispinosus if exposed to spinosad seven days after application. Khan and Morse (2006)

found a significant effect if the predatory mite, Euseius tularensis Congdon (Acari:

Phytoseiidae) was released within five to six days of a spinosad application, but not if E.

tularensis was released seven days after application. Residual thresholds were also higher for all

three mite species when indirect exposure and contact toxicity was taken into account.

Therefore, the interval between pesticide application and predatory mites release should be

based on persistence and harmfulness of indirect and contact toxicity, not contact toxicity alone.

In conclusion, studies of the effects of pesticides on natural enemies often aim to assess the

suitability of pesticides for IPM. To minimise any detrimental effects on non-target organisms,

selectivity tests are performed with the aim of choosing pesticides with a high degree of lethal

toxicity against the target pests and minimal non-target lethal toxicity. Although, this study was

conducted under controlled conditions, it still provides valuable information on the likely trends

of toxicity of spinosad residues. However, the sublethal effects of spinosad on these mites also

need to be examined. Natural enemies subjected to multiple routes of exposure to pesticides

may respond in unexpected ways that would be impossible to predict based on single route,

laboratory toxicity tests (Kunkel et al. 2001). Side effects can include changes in behaviour,

impact on fecundity and immature development (Desneux et al. 2007).


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beneficial arthropods. Annual Review of Entomology 52: 81-106.

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Control 40: 160-167.

CHAPTER VII

Spinosad-resistant western flower thrips, Frankliniella occidentalis (Pergande)

(Thysanoptera: Thripidae) can be managed using spinosad and predatory mites (Acari)


Hypoaspis miles, resistance, spinosad, residual toxicity, LT25

Abstract


serious pest on a wide range of crops and has developed resistance to one or more insecticides.

In Australia, F. occidentalis has developed resistance to the biopesticide, spinosad. To control

spinosad-resistant F. occidentalis, growers could double the recommended application rate (80

mL/100 L to 160 mL/100 L). However, increasing the rate could have a detrimental effect on

predatory mites (Acari) which are used as biological control agents in an integrated pest

management (IPM) approach. This study assessed the effects of applying spinosad (Success™

,

Dow AgroSciences, Australia) at twice the recommended rate to spinosad-resistant F.

occidentalis and to the predatory mites, Typhlodromips montdorensis (Schicha) (Phytoseiidae),

Neoseiulus cucumeris (Oudemans) (Phytoseiidae) and Hypoaspis miles (Berlese) (Laelapidae).

Direct exposure to twice the recommended rate of spinosad killed 100% of all mite species.

Spinosad residues aged two, 24, and 48 h were also highly toxic to all three mite species,

causing 96-100% mortality. The persistence of spinosad was rated as short-lived for N.

cucumeris, and slightly persistent for T. montdorensis and H. miles. Comparative toxicity

indicates that spinosad residues aged 48 to 168 h were less toxic to N. cucumeris followed by T.

montdorensis and H. miles. The residual thresholds (LT25) of twice the recommended rate of

spinosad for T. montdorensis, N. cucumeris, and H. miles were calculated as 6.1, 5.3, and 6.8

days respectively. By maintaining an interval, of 6-7 days between spinosad application at twice

the recommended rate and mite release, F. occidentalis can be effectively controlled.

Typhlodromips montdorensis appears to be the most successful species in reducing thrips

numbers followed by N. cucumeris and H. miles. Information that could be included in

resistance management of F. occidentalis is discussed.

7.1 Introduction

During the last few decades, many arthropod species have developed resistance to a number of

insecticides and insecticide classes (Georghiou 1990). This presents a serious challenge to food

Chapter VII: Resistance management

167

production when chemical control is the primary tactic used to manage pest populations.

Development of a new pesticide takes a significant investment and may not be developed

quickly enough to benefit growers. Thus, strategies must be adopted to prolong the effectiveness

of current insecticides while new technologies are being developed. Western flower thrips

(WFT), Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) is a worldwide pest of

economic importance (Lewis 1998), infesting a wide range of crops including ornamentals,

fruit, and vegetable crops (Tommasini and Maini 1995). Chemical control is the principal

management strategy for WFT in many parts of the world (Contreras et al. 2001), including

Australia (Herron and Gullick 2001, Herron and James 2005, Herron et al. 2007). However,

WFT has developed resistance to several insecticides (Brødsgaard 1994, Broadbent and Pree

1997, Jensen 2000, Espinosa et al. 2002, Herron and James 2005), including newer pesticides

such as imidacloprid and amitraz (Zhao et al. 1994, Zhao et al. 1995). In Australia, WFT control

relies on a limited number of pesticides (abamectin, acephate, chlorpyrifos, dichlorvos,

dimethoate, endosulfan, fipronil, malathion, methamidophos methidathion, methiocarb,

methomyl, pyrazophos and spinosad) (Herron and James 2005). However, high levels of

resistance to pyrethroids (cypermethrin, bifenthin, deltamethrin and fluvalinate) has been

detected and pyrethroids are no longer recommended for use against WFT (Herron and Gullick

2001). WFT also developed resistance to spinosad in Australia (Herron and James 2005) and

other parts of the world (Bielza et al. 2007). Spinosad is considered to be not toxic or less toxic

on beneficials and is often included in pest management programs that incorporate biological

control (Miles et al. 2003, Anh et al. 2004, Kim et al. 2005). Although spinosad resistance has

been detected in Australian populations, it may be possible to continue to use this pesticide in

conjunction with biological control (Jensen 2000).

In Australia, three species of predatory mites, Typhlodromips montdorensis (Schicha)

(Phytoseiidae), Neoseiulus cucumeris (Oudemans) (Phytoseiidae) and Hypoaspis miles

(Berlese) (Laelapidae) are commercially available for the management of WFT. Susceptibility

of these mites to spinosad has not yet been evaluated. However, it is expected that spinosad

application will reduce mite populations (Williams et al. 2003). For example, Kongchuensin

and Takafuji (2006) reported fresh spinosad residues (up to 48 h old) posed significant

detrimental effects on eggs immature stages of the predatory mite Neoseiulus longispinosus

(Evans). van Driesche et al. (2006) reported that fresh residues of spinosad applied at the

recommended rate for WFT control on glasshouse flower crops can lower the survival of

Iphiseius degenerans (Berlese). Spinosad is reported to be highly toxic to Neoseiulus fallacis

(Garman) used in North Carolina apple orchards to control pest mites (Panonychus ulmi Koch

and Tetranychus urticae Koch) (Villanueva and Walgenbach 2005).


168

Given the current high reliance on spinosad for WFT control and the increasing probability that

resistance will spread, it is important to explore ways in which spinosad can be used with

biological control agents effectively. One approach is to use an initial high dose of an

insecticide to reduce the resistant population of the pest, and thereafter release natural enemies

to maintain the population below the economic threshold (Tabashnik and Croft 1982). However,

the use of a high dose of an insecticide is likely to increase the detrimental effect on natural

enemies, particularly if there is any residual activity. Therefore, it is important to evaluate the

threshold period of an insecticide for natural enemies when there is a need to increase the dose.

This study investigated the possibility to integrate chemical control (high dose) and biological

control for the management of a spinosad-resistant WFT population. The objectives of this

study were to evaluate: (i) the residual threshold of high dose spinosad for T. montdorensis, N.

cucumeris, and H. miles, and (ii) the effectiveness of T. montdorensis, N. cucumeris, and H.

miles with a higher rate of spinosad to manage spinosad-resistant WFT.


Trials were conducted in a controlled temperature (CT) room (25±1⁰C, 50-60% RH, 16:8 h L: D

regime) from October 2008 to January 2009 at the University of Western Australia (UWA).


7.2.1.1 Strawberry plants

Strawberry runners [Fragaria x ananassa Duchesne (Rosaceae)] cultivar Camino Real

were planted into pots (32.5 l x 32.5 w x 40.5 h cm) containing potting mix (Baileys


WA (DAFWA), South Perth and UWA, Crawley. All pots were fitted with sprinklers with

automatic timers. During summer, the plants were watered every third day, while in

winter and spring plants were watered once a week. A liquid fertiliser (Thrive®

Yates,

Australia; NPK: 12.4: 3: 6.2; rate: 5mL/2 L water) was applied once a month.


A glasshouse colony of WFT was established from individuals initially collected from

calendula flowers [Calendula officianalis L. (Asterales: Asteraceae)] from glasshouses

at DAFWA. WFT colonies were maintained on potted calendula from July 2006 at

UWA. Potted calendula was grown from seeds collected from calendula plants.


169

Calendula seeds were sown in plastic pots (50x100 mm) with potting mix (Baileys

Fertilisers, Rockingham, WA) and plants were kept in insect proof Perspex cage (500

mm high, 420 mm deep and 400 mm wide), fitted with 105 µ mesh net (Sefar Filter

Specialists Pty Ltd., Malaga, WA) fitted on a Nylex tote box (320 x 420 mm; Blyth

Enterprises Pty Ltd, Australia). To maintain the WFT colony, every fortnight WFT was

collected from caged calendula plants and released onto fresh plants.

Spinosad-resistant WFT used in this study were obtained from NSW Agriculture, which was

initially collected from hydroponic lettuce in the Sydney basin, NSW (S. Broughton, pers.

comm.). The colony was maintained on calendula in insect-proof Perspex cages as described

above from July 2007 at UWA.


Predatory mites [T. montdorensis, N. cucumeris and H. miles] were sourced from commercial

Australian suppliers (Biological Services, SA; Chilman IPM Services, WA; and Beneficial Bug

Company, NSW). Mites were provided in plastic buckets containing vermiculite. Trials were

conducted immediately upon receipt of mites.

7.2.2 Experiment 1: Direct toxicity of spinosad


Toxicity of spinosad (Success™, 120 g/L EC, Dow AgroSciences Australia Ltd) at (i) the

recommended rate (80mL/100 L, 0.096 g a.i./L), (ii) twice the recommended rate and (iii) three

times the recommended rate to spinosad-resistant WFT adults and larvae were evaluated.

Twenty-four hours prior to the trial, WFT adults (5-6 d old) from the resistant strain were

collected from calendula plants with an aspirator, and brought to the CT room. WFT adults were

transferred to a glass Petri dish (150 x 15 mm) containing a strawberry leaf. The top of the Petri

dish was covered with mesh net (105 µ) and sealed with paraffin film (Parafilm M®, Micro

Analytix Pty Ltd)] and kept for 24 h for acclimatisation to the experimental arena. At the onset

of the experiment, 20 cold-anaesthetised WFT adults were placed on a paper towel and lightly

sprayed once with either 5mL of spinosad solution (diluted in distilled water) at one of the

above rates or distilled water only (control) with a hand-held atomiser (Hills Sprayers,

BH220063). After spraying, excess liquid (if any) was gently removed with a soft tissue. WFT

adults (n = 20) were then transferred to an excised strawberry leaf (adaxial side up) on a

moistened filter paper in a single Petri dish (150 x 15 mm). The leaf petiole was covered with


170

cotton was soaked in a 10% sugar solution to extend the leaf life. The leaf edge was glued to the

filter paper so that WFT could not go underneath the leaf. The top of the Petri dish was covered

with a mesh net (105 µ) sealed with a paraffin film to prevent thrips escaping. There were 20

replicates (20 x 20 = 400 individuals) of each treatment and a control. The experiment was

repeated using the same protocol as above but with WFT larvae (first instar) instead of adults.

All Petri dishes were then placed on a laboratory bench in a random block design in the CT

room (25±1⁰C, 50-60% RH and 16:8 h light-dark cycle) and thrips were monitored for

mortality. Spinosad is a slow-acting insecticide (Bret et al. 1997) and cumulative mortality of a

test organism usually plateaus at 2 days (48 h) to 6 days (144 h) after exposure (Viñuela et al.

2001, Cisneros et al. 2002). Consequently, Petri dishes with WFT adults were examined at 6,

24, 48, 72, and 96 h post-release exposure periods under a stereomicroscope. The WFT larvae

were checked at 6, 24, 48 and 72 h post treatments (after 72 h post-release exposure period

those larvae were lived had pupated). WFT adults or larvae were recorded as dead if they did

not respond to probing with a fine paintbrush.


Bioassay methods were the same as for thrips, except that a thin barrier of Tac Gel (Stickem™

,

The Olive Centre, Australia) was applied to the edge of the leaf to keep mites on the leaf

surface. In addition, first or second instar WFT larvae were added to the Petri dishes to provide

food for the mites. In each Petri dish, there were 200 larvae for T. montdorensis [average 10

first instar larvae/ mite (Steiner et al. 2003)], 100 larvae for N. cucumeris [average six fist instar

larvae/ mite (Zilahl-Balogh et al. 2007) and 40 larvae for H. miles [two second instar larvae/

mite (Berndt et al. 2004)]. During the trial period, additional thrips larvae were added to the

Petri dishes as required. Mortality of the mites was monitored at 6 h, 24 h, 48 h, 72 and 96 h

post-release exposure periods. Mites were recorded as dead if they did not respond to probing

with a fine paintbrush. Each treatment (spray) was replicated 20 times (20 x 20 = 400

individuals) for each species of predatory mite.

7.2.3 Experiment 2: Bioassay of spinosad residual toxicity to predatory mites

This bioassay evaluated the mortality of predatory mites (T. montdorensis, N. cucumeris and H.

miles) via consumption of intoxicated WFT larvae and simultaneous exposure (contact) to

spinosad residues. Predatory mites were placed on strawberry leaves that had been treated with

spinosad solution at twice the recommended rate (80 mL/100 L), and were allowed to feed on

intoxicated WFT larvae that had been exposed to the same aged residue. Twenty-four hours


171

prior to the trial, newly emerged first instar WFT larvae were collected from the stock colony.

Larvae were transferred to a glass Petri dish (150 x 15 mm) containing a cotton wool ball

soaked in a 10% sugar solution, and then stored in the CT room for 12 h (to ensure feeding on

spinosad- or water-treated leaf at a later stage). Larvae were then released onto excised

strawberry leaves previously treated with spinosad (2, 24, 48, 72, 96, 120, 144 or 168 h old

residues) or control (water) in a Petri dish and allowed to feed for 12 hours. A thin barrier of

Tac Gel was applied to the edge of the leaf to prevent larvae from escaping the leaf. The dose of

twice the recommended rate of spinosad was chosen for testing residual toxicity on predatory

mites in this experiment, because, in the previous experiment, this dose appeared effective in

controlling 50% or more of the spinosad-resistant WFT population. Prior to spraying, potted

strawberry plants Camino Real (3-4 weeks old) were brought to the CT room and split into

groups (2, 24, 48, 72, 96, 120, 144 and 168 h old residue). To obtain the required residues

available for experimentation at the same time, strawberry plants were sprayed at different times

with spinosad solution until run-off with a hand-held atomiser. Those plants in the 168 h old

residue treatment were sprayed first, followed by spray of the subsequent group of plants. In a

similar manner, a second set of plants was sprayed with spinosad solution by maintaining 12 h

interval from the time when the first set of plants was sprayed for each treatment (residue ages).

Strawberry plants in the control group were sprayed with distilled water 24 h prior to the

experiment. To avoid contamination, separate atomisers were used for the water and spinosad.

Leaves were marked with a permanent marker at the base of the petiole, to enable leaves with

residues to be accurately selected. After spraying, plants were covered with a modified thrips-

cage (45 x 35 cm, open both ends) made from mesh net (105µ) and supported by quadratic

steel-rod stands. The bottom end of the cage was taped to the pot. The top end of the

cage was closed with a rubber band. All treated plants were then returned to a glasshouse for

natural degradation of spinosad and were kept until use.

Spinosad was also applied to the Petri dishes (150 x 15 mm; testing arena) at the same time as it

was sprayed on the plants in order to obtain strawberry leaves and dishes of the same exposure

age. The Petri dishes were held with the open end sidewise and sprayed inner on the side with

spinosad solution using an atomiser until run-off. Both parts of a Petri dish were sprayed with

spinosad. Petri dishes were kept on a tray and allowed to dry in a glasshouse for one and half

hours. Dried Petri dishes were stored in separate plastic trays and kept in the CT room until use.

The Petri dishes were sprayed with distilled water to be used as the untreated control.

At the beginning of the experiment, the first set of spinosad treated plants was brought to the CT

room. A strawberry leaf excised from a treated plant was placed adaxial side top at the bottom

of a glass Petri dish. A thin barrier of Tac Gel was applied to the edge of the leaf to prevent


172

WFT larvae escape from the leaf surface. First instar WFT larvae collected from the colony

were released onto the treated leaf in the Petri dish and allowed to feed for approximately 12

hrs. This was done to get the intoxicated WFT larvae. Twelve hours later, a second set of

spinosad treated strawberry plants [plants sprayed by maintaining a 12 h interval of each

respective residue age group] were brought to the CT room. Testing arenas were prepared using

the same age residue of leaf, Petri dish and WFT larvae as above. Intoxicated first instar WFT

larvae [the number of WFT release per Petri dish was the same as mentioned in section 7.2.2.2)

were released first onto the leaf, followed by the release of predatory mites. During the trial

periods, additional intoxicated WFT larvae with same residue age were added to the Petri dish if

required. Twenty adult mites of one species were placed on the leaf surface in a Petri dish

covered with a mesh net (105µ) and sealed with paraffin film. The leaf petiole was covered

with cotton was soaked in a 10% sugar solution to keep the leaf fresh and extend the life. Before

releasing WFT larvae or predatory mites, a thin barrier of Tac Gel was applied to the edge of the

leaf surface to prevent escape of WFT larvae and predatory mites. There were 20 replicates (20

x 20 individuals) of each treatment (residue ages). The experiment was repeated using the same

protocol above but with two other species of predatory mites. The Petri dish and strawberry leaf

were sprayed with distilled water to be used as the control (20 x 20 individuals). All Petri dishes

with predatory mites were arranged randomly on a laboratory bench and mortality was checked

in the same manner as explained previously for WFT. Spinosad is slow-acting (Bret et al. 1997)

and cumulative mortality of a test organism usually plateaus at two days (48 h) to six days (144

h) after exposure (Viñuela et al. 2001, Cisneros et al. 2002). Predatory mites were recorded as

dead if they did not respond to probing with a paintbrush.

7.2.4 Experiment 3: Efficacy of predatory mites with spinosad against WFT-resistant

strain

This experiment evaluated the efficacy of predatory mites when combined with twice the

recommended rate of spinosad (80ml/100 L) against spinosad-resistant WFT strain. In order to

obtain uniformly aged WFT adults for this trial, ten adults were collected from the resistant

colony, then released onto a fresh (not previously exposed to any insects including WFT)

calendula plant in insect-proof Perspex cages as described in 7.2.1. WFT adults were

allowed to lay eggs for 24 h. After 24 h, all adults were removed from the plants with an

aspirator. Plants were checked daily for larval emergence. Adults that emerged on the same day

were used in this trial.

Prior to the trial, WFT adults (spinosad-resistant) were collected from the stock colony, brought

to the CT room and released onto freshly caged strawberry plants. Strawberry plants with WFT


173

adults were kept in the CT room for 24 h to acclimatise with the experimental environment. At

the onset of the trial, 40 strawberry plants (3-4 leaves stage) cultivar Camino Real, were brought

to the CT room. Plants were randomly divided into two groups and sprayed with twice the

recommended rate of spinosad or distilled water (control) with a hand-held atomiser until run-

off. Plants were then covered with a modified thrips cage (45 x 35 cm) made with mesh net

(105µ) and supported by a quadrate steel-rod stand. The treated plants were allowed to

dry for two hours. Previously acclimatised spinosad-resistant WFT adults of the same age

were then released onto the plants. Fifteen WFT adults per plant were released. Plants with

WFT were further divided into four groups and received:

(i) No mites

(ii) six T. montdorensis

(iii) six N. cucumeris

(iv) six H. miles.

Based on the residual threshold (estimated from the previous experiment), , N. cucumeris, T.

montdorensis or H. miles were released on to plants at six, five or seven days after spinosad

application respectively. Pots were randomly arranged on a laboratory bench in the CT room.

There were five plants (pot = replicate) per treatment (mite species). Twenty-four hours after

WFT release, the plants were checked with a battery-powered, hand-held magnifying glass

[50 mm (2") illuminated round 2x power with 4x bifocal magnifier] for live WFT. Thereafter,

every fifth day for five weeks, plants were checked and the number of live WFT adults and

larvae per plant were recorded. Plants were checked early in the morning (0600 to 0800 h)

as WFT was found to be less active at this time. Plants were watered as required.

7.2.5 Data analysis

For each experiment in which mortality was assessed, the number of individuals that died at

each observation was counted and expressed as a percentage of the total number of individuals

in the arena and corrected using Abbott’s formula (Abbott 1925).

Abbott’s formula takes into account the proportion of control thrips or mites dying in the trial

that have not been exposed to the spinosad, and amongst those that have been exposed to

spinosad, some may die of natural causes. In these trials, control mortality of either thrips or

mites never exceeded 5%.


174

The differences in corrected cumulative mortality (mortality after 96 h post-release exposure

period) of each residue ages among predatory mites were analysed by one-way ANOVA (Proc

Mixed Procedure), except cumulative mortality due to two and 24 h old residues, as these two

residues caused 100% mortality of all three species of predatory mites. To determine the

difference in toxicity amongst residue ages, corrected mortality of T. montdorensis, N.

cucumeris, and H. miles at each period post-release exposure period was subjected to one-way

ANOVA separately. Mortality data were transformed using arcsine transformation (Zar 1999)

(Healy and Taylor 1962) to normalise before analysis, though actual means are reported. If

ANOVA results showed significant differences, mortality means were separated by least square

mean differences at 5% probability.

The residual toxicity of spinosad to T. montdorensis, N. cucumeris, and H. miles at each post-

release exposure period was classified following International Organization of Biological

Control (IOBC) guidelines:

1 = harmless (<25% mortality)

2 = slightly harmful (25-50% mortality)

3 = moderately harmful (51- 75% mortality)

4 = harmful (>75% mortality) (Sterk et al. 1999).

The persistence of spinosad for each species of predatory mites was also classified according to

the time taken to lose toxicity (<30% mortality, IOBC persistence class):

A = short-lived (<5 d)

B = slightly persistent (5-15 d) (Hassan et al. 1994, Sterk et al. 1999).

The residual toxicity threshold of spinosad for T. montdorensis, N. cucumeris and H. miles was

estimated with Probit analyses (Finney 1971) by Proc Probit Procedure. The LT25 (lethal time of

25% mortality) was used, which is considered an acceptable level (Shipp et al. 2000).

The effect of spinosad and predatory mite releases on the numbers of the spinosad-resistant

WFT strain (adults and larvae) were compared by two-way ANOVA (Proc Mixed Procedure)

(independent variables: spray treatment and predatory mite releases; response variables: WFT

adults or larvae) separately at each observation, 10 to 35 DAS (days after spray). Since there

was a significant interaction between spray (spinosad and water) and predatory mite releases (no

mites, T. montdorensis, N. cucumeris and H. miles), additional one-way ANOVAs were

performed (Quinn and Keough 2002). WFT numbers (adults or larvae) at each observation

period were analysed with two separate one-way ANOVAs (one for each spray treatment). Due


175

to multiple comparisons, an adjustment to the significance level was made, α = 0.025 (0.05/2).

Meanwhile, to determine the difference in WFT adults and larvae between groups before mite

release, WFT adults and larval data for spinosad and water treatment of one and five DAS were

analysed by one-way ANOVAs (Proc Mixed Procedure). If ANOVA results were significant,

means were separated with least square mean differences. Data were transformed using

√(x + 0.05) (Healy and Taylor 1962) to normalise before analysis. Data were reversed

transformed for presentation.

All analyses were computed using SAS 9.1, SAS Institute, 2003, Cary, NC, USA (SAS 2002-

2003). Figures were drawn with GraphPad Prism 5.0 software (GraphPad Software Inc 2007).

7.3 Results

7.3.1 Direct toxicity of spinosad to WFT and predatory mites

Bioassays confirmed that the recommended rate of spinosad was not more toxic to adults or

larvae of the spinosad-resistant strain of WFT used in this study, than the distilled water spray

(control) (Table 7.1). Doubling the recommended rate killed >70% of WFT adults and larvae

(Table 7.1). Meanwhile, direct exposure to triple the recommended rate of spinosad killed 100%

of WFT adults and larvae. In the water treatment, 0.5% WFT adults were killed, whereas no

mortality of WFT larvae was recorded in the water treatment.

As it was found that double of the recommended rate of spinosad could be effective against

spinosad-resistant WFT population, toxicity of double the recommended rate of spinosad was

tested against T. montdorensis, N. cucumeris and H. miles. Direct exposure to twice the

recommended rate was very toxic to predatory mites T. montdorensis, N. cucumeris, and H.

miles, resulting in 100% mortality. In this trial, in the water treatment, mite mortality never

exceeded 5% (1.7%, 2.0% and 3.25% of T. montdorensis, N. cucumeris and H. miles were killed

respectively).

Table 7.1 Cumulative corrected mortality (%) of spinosad- resistant WFT adults (at 96 h post-

release exposure period) and larvae (at 72 h post-release-exposure period) when exposed

directly to spinosad spray at different rates.

Spinosad application rate

Corrected mortality (%) of spinosad-resistant

WFT (Mean ± SE)

Adults Larvae

Recommended rate (80 ml/100 L)

Double the recommended rate

Triple the recommended rate

0.50 ± 0.46

87.94 ± 2.36

100

0.00

78.57 ± 3.45

100


176

7.3.2 Bioassay of spinosad residual toxicity to predatory mites

Spinosad residues of all ages of double the recommended rate were toxic to some degree to T.

montdorensis, N. cucumeris and H. miles (Figure 7.1), although toxicity declined as the residual

period increased. Spinosad residues aged 2 h and 24 h were very toxic to T. montdorensis, N.

cucumeris and H. miles and caused 100% mortality within 24 h of exposure. Cumulative

mortality of 48 h old residue at 96 h post-release exposure period was not different (F 2, 57 =

3.01, P = 0.057) among predatory mite species and was close to 100%. However, each mite

species experienced different mortality when exposed to 72-168 h old residues (72 h: F 2, 57 =

35.08, P < 0.0001; 96 h: F 2, 57 = 5.64, P = 0.0058; 120 h: F 2, 57 = 9.61, P 0.0002; 144 h: F 2, 57

= 25.41, P < 0.0001; 168 h: F 2, 57 = 35.43, P < 0.0001) old residues at 96 h post-release

exposure period (Figure 7.1). For each residue age (72 h to 168 h), H. miles mortality was the

highest and N. cucumeris mortality was the lowest (Figure 7.1). However, mortality of T.

montdorensis and N. cucumeris was not different when exposed to 72 h old residue. Similarly,

when exposed to 96 h old residue, mortality of T. montdorensis and H. miles was not different.

In the control treatment, less than 5% (1.5%, 2% and 2.5% mortality of T. montdorensis, N.

cucumeris and H. miles respectively) mortality was found in the predatory mite species.

48h 72h 96h 120h 144h 168h0

20

40

60

80

100

T. montdorensis N. cucumeris

a a a

aa

b

a

bb

ab

c

a

bc

a

bc

H. miles

Corr

ecte

d m

ort

ality (

%)

(Mea

n

SE

)

Figure 7.1 Toxicity of spinosad residues to predatory mites after 96 h post-release exposure

period. Within each residue age, means with different letters differed significantly (α = 0.05).

It also appears that the patterns of toxicity of spinosad of double the recommended rate to T.

montdorensis, N. cucumeris and H. miles differed during the post-release exposure periods

(Table 7.2). For all three mite species there was a significant decline in mortality during each

post-release exposure period as residue time increased. When spinosad was applied at twice the


177

Table 7.2 Residual toxicity of spinosad (twice the recommended rate) to predatory mites at 24

h, 48 h, 72, h and 96 h post-release exposure periods. Mites were fed spinosad intoxicated WFT

larvae and simultaneously exposed to residue. Residual toxicity was classified: 1 = harmless

(<25% mortality), 2 = slightly harmful (25-50% mortality), 3 = moderately harmful (51-75%

mortality), and 4 = harmful (>75% mortality). Persistence class: A = short lived (<5 d), B =

slightly persistent (5-15 d).

Res.

Age

Corrected mortality (%) (mean ± SE) at post-

release periods Toxicity class

Per.

Class 24h* 48h

* 72h

* 96h

* 24

h

48

h

72

h

96

h

T. montdorensis

2h

24h

48h

72h

96h

120h

144h

168h

100e

100e

91.3±2.3e

50.3±1.4d

33.8±1.6c

10.0±0.9b

9.5±0.8b

3.0±0.9a

100f

100f

96.0±1.7f

60.5±1.5e

45.8±1.7d

22.4±1.3c

17.6±1.3b

8.3±0.9a

100f

100f

100f

67.5±1.6e

54.1±1.9d

30.5±1.6

24.6±1.7b

16.5±1.5a

100f

100f

100f

71.1±1.9e

56.2±1.5d

31.0±1.8c

24.6±1.7b

16.8±1.4a

4

4

4

3

2

1

1

1

4

4

4

3

2

1

1

1

4

4

4

3

3

2

1

1

4

4

4

3

3

2

1

1

B

F

df

P

709.26

7 & 152

< 0.0001

669.08

7 & 152

< 0.0001

409.94

7 & 152

< 0.0001

372.11

7 & 152

< 0.0001

N. cucumeris

2h

24h

48h

72h

96h

120h

144h

168h

100g

100g

91.5±2.2f

52.5±1.5e

29.8±1.4d

14.8±1.0c

3.3±1.1b

0.0±0.0a

100g

100g

94.4±1.8f

58.5±1.7e

38.5±1.9d

21.0±1.5c

10.7±1.2b

5.8±0.6a

100g

100g

96.4±1.3f

66.1±1.6e

45.2±2.0d

26.8±1.4c

16.3±1.2b

7.4±0.9a

100f

100f

96.7±1.3f

68.9±1.8e

48.7±2.1d

26.8±1.4c

16.3±1.2b

7.4±0.9a

4

4

4

3

2

1

1

1

4

4

4

3

2

1

1

1

4

4

4

3

2

1

1

1

4

4

4

3

2

2

1

1

A

F

df

P

881.76

7 & 152

< 0.0001

647.50

7 & 152

< 0.0001

636.77

7 & 152

< 0.0001

621.14

7 & 152

< 0.0001

H. miles

2h

24h

48h

72h

96h

120h

144h

168h

100f

100f

95.8±1.8f

79.8±2.1e

40.8±1.5d

27.3±2.4c

10.5±0.6b

3.8±1.0a

100f

100f

100f

86.5±2.4e

49.4±1.4d

35.9±2.2c

23.2±1.3b

11.5±1.2a

100e

100e

100e

86.9±2.4d

51.8±1.5c

37.4±2.0b

32.3±1.8b

22.3±1.8a

100e

100e

100e

89.7±1.9d

57.6±2.5c

37.4±2.0b

32.3±1.8b

22.3±1.8a

4

4

4

4

2

2

1

1

4

4

4

4

3

2

2

1

4

4

4

4

3

2

2

1

4

4

4

4

3

2

2

1

B

F

df

P

537.45

7 & 152

< 0.0001

471.23

7 & 152

< 0.0001

348.82

7 & 152

< 0.0001

373.99

7 & 152

< 0.0001

*For each species, within column, means with different letters differed significantly (α = 0.05).


178

recommended rate, mortality of T. montdorensis was different among spinosad residues at each

post-release exposure period. Mortality of T. montdorensis was highest and lowest when

exposed to 2 h and 168 h old residues respectively. However, at 24 h or 48 h post-release

exposure periods, there was no significant difference in T. montdorensis’ mortality when

exposed to 2 h and 24 h old residues. In addition, when examined at 72 h or 96 h post-release

exposure periods, T. montdorensis’ mortality did not differ between 2 h, 24 h and 48 h old

residues. Similarly, at each post-release exposure period, mortality of N. cucumeris or H. miles

was highest and lowest when exposed to 2 h and 168 h old residues respectively. However, at

24 h, 48 h or 72 h post-release exposure periods, N. cucumeris mortality did not differ between

2 h and 24 h residues. While at 96 h post-release exposure period, there was no difference in N.

cucumeris’ mortality when exposed to 2 h, 24 h and 48 h old residues. For H. miles, mortality

due to 2 h, 24 h and 48 h old residues was not different. Moreover, mortality of each species of

predatory mites due to spinosad residues was increased as the mites remain for longer periods in

the Petri dish.

Spinosad residues declined in toxicity with age (Table 7.2). According to the IOBC toxicity

classification, residues aged 2 h to 48 h were harmful (>75% mortality) to all three species for

as long as 96 hours after the mites were first exposed. Hypoaspis miles was the most sensitive to

spinosad residues, with 96 h old residue classified as moderately harmful. Based on the IOBC

toxicity ratings, spinosad caused no harmful effect to N. cucumeris five days post-release (rating

1= < 25% mortality) and was considered to be short-lived (persisting for less than five days).

For T. montdorensis and H. miles, spinosad was classified as slightly persistent (persisting for 5-

15 days) with 120 h (5 days) old residue for T. montdorensis and 120 h (5 days) and 144 h (6

days) old residues for H. miles classified as slightly harmful (25-50% mortality).

The relationships between predatory mite mortality and spinosad residues, with mortality as a

function of residue age (log hrs) are presented in Figure 7.2. The LT25 (25% mortality of non-

target organisms caused by residual toxicity) indicates that the release of each species of

predatory mite after a spinosad application would cause only 25% mortality, allowing 75%

survival of predatory mites. The LT25 of spinosad differed for each species and was estimated as

6.1 days (146.76 h; Antilog102.16660

) for T. montdorensis, 5.3 days (127.85 h; Antilog102.10669

) for

N. cucumeris and 6.8 days (162.45 h; Antilog102.21071

) for H. miles (Table 7.3).


179

Table 7.3 Probit analysis (Abbott 1925) of the mortality of predatory mites exposed to spinosad

residues (by consumption of intoxicated WFT larvae and simultaneous exposure to residues) of

different ages.

Species Slope ± SE LT25

(hrs)

95% CL χ

2 df P

Lower Upper

T. montdorensis

N. cucumeris

H. miles

-4.55±0.25

-5.36±0.19

-5.37±0.37

146.76

127.85

162.45

141.51

124.24

156.86

152.82

131.85

169.02

12.64

10.20

16.56

6

6

6

0.0491

0.1167

0.0111

0.0

0.5

1.0

0.0

0.5

1.0

Pro

bit

mo

rtality

1.0 1.5 2.0 2.5 3.00.0

0.5

1.0

Log10 (hrs)

A

B

C

Figure 7.2 Relationship of Log10

(hrs) and probit mortality of (A) T. montdorensis, (B) N.

cucumeris and (C) H. miles when exposed to twice the recommended rate of spinosad residues

with different ages.


180

7.3.3 Efficacy of predatory mites with spinosad against WFT-resistant strain

7.3.3.1 Effect of spinosad and predatory mite releases on WFT adults

Predatory mites (T. montdorensis, N. cucumeris, and H. miles) were more effective at reducing

WFT adult numbers after spinosad is applied (Figure 7.3). Since there was a statistically

significant interaction between spray [spinosad, water (control)] and mite releases (no mites, T.

montdorensis, N. cucumeris, H. miles) (Appendix 7.1), treatments (spinosad, water) were

separately analysed at each observation [DAS = days after spray] (Appendix 7.2).

0

10

20

30

40

50 10 DAS

Spinosad Water

15 DAS

0

10

20

30

40

5020 DAS 25 DAS

No mitesTM NC HM0

10

20

30

40

50 30 DAS

No mitesTM NC HM

35 DASNu

mb

er o

f W

FT

ad

ults

(Mea

n

SE

)

Figure 7.3 Mean numbers of WFT adults per plant sprayed with spinosad or water (control) in

the presence or absence of predatory mite. TM = T. montdorensis, NC = N. cucumeris, HM = H.

miles. DAS = Days after spray. Means with different letters differed significantly (α = 0.025).

There were no treatment differences in WFT adult numbers (plants assigned to mites release),

prior to mite releases at 1 DAS (days after spray) (Fspinosad = 0.583, 16, P = 0.6368; Fwater = 2.573,


181

16, P = 0.0905) and 5 DAS (Fspinosad = 0.463, 16, P = 0.7152; Fwater = 0.923, 16, P = 0.4551)

(Figure 7.4). From 10 to 35 DAS, mean numbers of WFT adults were significantly different

among predatory mite treatments (no mites, T. montdorensis, N. cucumeris, H. miles), except at

10 DAS in the control treatment (water) (Appendix 7.2; Figure 7.4B). Plants treated with ‘no

mites’ had the highest numbers of WFT adults compared to plants that were treated with

spinosad and received predatory mites (Figure 7.4A). Similarly, in the water treatment (control),

plants treated with ‘no mites’ had the highest numbers of WFT adults compared to plants that

received predatory mites (Figure 7.4B). In both spinosad and water (control) treatments, T.

montdorensis was the most effective species at reducing WFT adult numbers, except at 10 and

15 DAS. Hypoaspis miles was most effective at 10 and 15 DAS in both spinosad and control

(water) treatments, but the least effective species from 20 to 35 DAS compared with T.

montdorensis and N. cucumeris.

0

10

20

30

40

No mites TM NC HM

D1* D5* D10 D15 D20 D25 D30 D350

10

20

30

40

A

B

Num

ber

of

adult

s per

pla

nt

(Mea

n

SE

)

Figure 7.4 Numbers of WFT adults per plant (Y-axis) sprayed with spinosad (A) or water (B)

with or without predatory mite. In each observation (D10 to D35, D = days after spray), means

were separated by least square mean difference at α = 0.025. *WFT adults’ numbers per plant

before mite release.

7.3.3.2 Effect of spinosad and predatory mite releases on WFT larvae

Predatory mites (T. montdorensis, N. cucumeris, and H. miles) were more successful at reducing

WFT larvae numbers after spinosad is applied (Figure 7.5). Because of the statistically

significant interaction between spray [spinosad, water (control)] and mite treatment (no mites,

T. montdorensis, N. cucumeris, H. miles), effects of mite treatment on WFT larvae were


182

separately analysed for spinosad and water spray at each observation [DAS = days after spray]

(Appendix 7.1 and 7.2).

0

10

20

30

4010 DAS

a

b

a

b

a

b a

b

Spinosad Water

15 DAS

a

b

ab

a

b

a

b

0

10

20

30

4020 DAS

a

b

a

ba

b ab

25 DAS

a

b

a

b

a

ba

b

No mitesTM NC HM0

10

20

30

40

a

b

a

b

a

b aa

30 DAS

No mitesTM NC HM

35 DAS

aa

a

ba

ba a

Num

ber

of

WF

T l

arvae

per

pla

nt

(Mea

n

SE

)

Figure 7.5 Numbers of WFT larvae per plant with spinosad and water (control) in the presence

or absence of predatory mites. DAS = days after spray. TM = T. montdorensis, NC = N.

cucumeris, HM = H. miles. Means within group with different letters differed significantly (α =

0.025).

When plants were examined at one DAS, no WFT larvae were found. At five DAS prior to

predatory mites release, there was no differences in WFT larvae numbers per plant among

treatments (plants assigned to predatory mites release) in spinosad (F 3, 16 = 2.00, P = 0.1546) or

water (control) (F 3, 16 = 0.80, P = 0.5111) treatments (Figure 7.6). From 10 to 35 DAS, mean

numbers of WFT larvae per plant were significantly different among predatory mite treatments

(no mites, T. montdorensis, N. cucumeris or H. miles) in both spinosad and water (control)

treatments (Appendix 7.2; Figure 7.6). In spinosad treatment, plants treated with ‘no mites’ had

the highest numbers of WFT larvae compared to plants received predatory mites. Similarly, for

the water treatment (control), plants treated with ‘no mites’ had the highest numbers of WFT

larvae compared to plants that received predatory mites. For both spinosad and water (control)


183

treatments, T. montdorensis was the most effective species in reducing WFT larvae, except at 15

DAS in spinosad treatment and 10 DAS in the water (control) treatment. Neoseiulus cucumeris

was the most effective in reducing WFT larvae at 15 DAS and 10 DAS in spinosad and water

(control) treatments respectively. H. miles was the least effective species in reducing WFT

larvae compared with T. montdorensis and N. cucumeris.

0

10

20

30

No mites TM NC HM

D1* D5* D10 D15 D20 D25 D30 D350

10

20

30

A

B

Num

ber

of

WF

T l

arvae

per

pla

nt

(Mea

n

SE

)

Figure 7.6 Numbers of WFT larvae per plant with spinosad (A) and water (B) in the presence

or absence of predatory mites (TM = T. montdorensis, NC = N. cucumeris, HM = H. miles). At

each observation (D = days after spray), means were separated by least square mean difference

at α = 0.025. *WFT nymphs per plant before release of mites.

7.4 Discussion

The present study suggests that to control 50% or more of a spinosad-resistant WFT strain, the

recommended rate of spinosad (80 mL/100L, 0.096 g a.i./L) will need to be doubled (160

mL/100L, 0.192 g.a.i./L). However, by doubling the rate, toxicity to predatory mites (T.

montdorensis, N. cucumeris and H. miles) released for biological control of WFT increased and

caused 100% mortality. The predatory mites used in this study cannot tolerate direct contact

with spinosad at the above rate.

Fresh to relatively fresh residues (2 to 72 h old) of double the recommended rate of spinosad

were also harmful to T. montdorensis, N. cucumeris, and H. miles, causing 100% mortality.

Spinosad is considered to be a short-lived insecticide that rapidly degrades in nature (Thompson

et al. 2000, Williams et al. 2003). Under laboratory conditions; however, spinosad is considered


184

to be highly stable and capable of causing high mortality to a parasitoid, up to 1 month after

being applied to foliage or artificial surfaces (Bernando and Viggiani 2000). Semi-field and

field studies suggest that spinosad residues pose little or no toxicity to natural enemies after

three to 7 days based on species (Boyd and Boethel 1998, Ruberson and Tillman 1999, Crouse

et al. 2001). This study was conducted in the laboratory. However, the effect of spinosad on

predatory mites suggests that the effects of spinosad residues decreases over time, and would

pose little threat to the predator if the initial exposure to the pesticide occurs after a period of

time. Based on residual threshold (LT25) calculations and confirmed with laboratory trials, mites

can be released 5 to 7 days after a spinosad application. Neoseiulus cucumeris could be released

after 5.3 days (127.85 h), T. montdorensis after 6.1 days (146.76 h), and H. miles after 6.8 days

(162.45 h). Despite the increase in the rate of spinosad applied, the results presented here

suggest that predatory mites (T. montdorensis, N. cucumeris and H. miles) can be integrated

with an increased rate of spinosad application for the effective management of spinosad-

resistant WFT.

In the bioassay, spinosad was classified to have short-lived persistence against T. montdorensis

and N. cucumeris, while slightly persistent for H. miles. The variable effect of spinosad residues

is not unexpected. There is variation in the residual effect of insecticides including spinosad on

other species. It has been reported that LC50 values of spinosad exceeded 960 ppm for Podius

nigrispinus (Dallas) (Hemiptera: Pentatomidae) (Torres et al. 1999), while LC50 was only 50

ppm for Podius maculiventris (Say) (Hemiptera: pentatomidae) (Viñuela et al. 1998). Similar to

the present findings, Sáenz-de-Cabezón Irigaray et al. (2007) stated that the miticide, abamectin

was short-lived to the western predatory mite, Galendromus (= Typhlodromus, = Metaseiulus)

occidentalis Nesbitt (Acari: Phytoseiidae), and slightly persistent to Phytoseiulus persimilis

Athias-Henriot (Acari: Phytoseiidae). Khan and Morse (2006) tested the impact of four

pesticides on the predatory mite Euseius tularensis Congdon, and found residual effects if mites

were released five to six days after spinosad was applied. But spinosad had no residual effect on

E. tularensis if released seven days after the spinosad application (Khan and Morse 2006).

As with previous findings (Chapters 3, 4 and 5), the efficacy of the mites in reducing WFT

differed. Typhlodromips montdorensis appears to be the most effective species in suppressing

WFT, followed by N. cucumeris and H. miles. There may be a variety of reasons for such

variation (Brødsgaard 1989, van Houten et al. 1995, Steiner and Goodwin 1998, 2001, Berndt et

al. 2004, Messelink et al. 2006, Skirvin et al. 2007). Nevertheless all three predatory mites

tested in this study provided better control of WFT when released after spinosad was applied.

However, to get maximum suppression of WFT using these predatory mites, a further study

detailing factors that influence their success needs to be carried out.


185

In summary, the challenge for managing resistant pest populations is that management with

insecticides often requires that the dose applied has to be increased in order to be effective

(Broughton and Herron 2007). The consequential effects on natural enemy means that often the

two tactics cannot be used together. However, the results herein suggest that spinosad-resistant

WFT could be managed with an increased rate of spinosad and could be further reduced by

releasing predatory mites. However, caution is required as direct exposure to spinosad or

relatively fresh residues are toxic to T. montdorensis, N. cucumeris, and H. miles. Since

predatory mites predate on either first instar larvae or pupal stages but not the adult stage, the

adult WFT population needs to be reduced before predatory mites are released. As this study

was conducted in a controlled environment, pesticide risk assessments need to be validated with

semi-field and/or field studies.


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Chapter VIII

General discussion and conclusion

8.1 General discussion

In this thesis, I hypothesised that chemical control, biological control, and resistant host-plants

could be effectively combined to reduce pest numbers on a horticultural commodity. I sought to

test this hypothesis with a significant and difficult to manage pest in a crop with high aesthetic

value, and in Australia, where few alternatives to pesticides are available for management. Since

its first detection in 1895 in California, western flower thrips, Frankliniella occidentalis

(Pergande) (Thysanoptera: Thripidae) has spread throughout the world and is considered a

significant pest. Frankliniella occidentalis is hard to control with pesticides and this has led to

increased efforts towards the implementation and use of integrated pest management (IPM). In

Australia, an IPM approach for F. occidentalis that incorporates multiple tactics has been

difficult, because few alternatives to pesticides have been available besides pesticides. This is

now changing now with the commercial availability of predatory mites, a predatory bug,

predatory beetle and nematodes (Australian Biological Control 2009). However, to use

biological and chemical control effectively, the tactics need to be carefully applied.

Frankliniella occidentalis is a pest of many commercial crops and is particularly damaging to

strawberry. Several varieties of strawberry are grown in Australia but none has been explicitly

developed for resistance to F. occidentalis. The aim of this body of work was to evaluate the

compatibility of chemical (spinosad) and biological control (predatory mites), along with host-

plant resistance for the management of F. occidentalis in strawberry.

To test the hypothesis, this project sought first to establish the influence of strawberry cultivars

on feeding preference and oviposition preference and performance of F. occidentalis. Secondly,

this study tested the efficacy of three commercially available predatory mites [Typhlodromips

montdorensis (Schicha) (Phytoseiidae), Neoseiulus cucumeris (Oudemans) (Phytoseiidae), and

Hypoaspis miles (Berlese) (Laelapidae)] with or without spinosad along with three strawberry

cultivars (Camarosa, Camino Real, and Albion) against F. occidentalis. Thirdly, this

project investigated the efficiency of single- versus multiple-species release of

predatory mites with or without spinosad in the management of the F. occidentalis

population. Fourthly, this project sought to test the compatibility of predato ry mites and

the release the protocol for the management of F. occidentalis in low tunnel-grown

strawberry. Fifthly, this study evaluates the residual threshold of spinosad for the

predatory mites. Lastly, this study investigates the possibility of the integration of these

Chapter VIII Summary

190

predatory mites with an increased application rate of spinosad [twice the recommended

rate for F. occidentalis] to control a spinosad-resistant F. occidentalis strain.

8.2 Findings and recommendations

8.2.1 Strawberry cultivars distinctively influence western flower thrips’ olfactory and

feeding preference and oviposition preference and performance

Host-plant resistance to arthropod pests generally plays an important role in pest management

programs (White 1969, Wellington 1977, White 1993, Parrella and Lewis 1997) and is

considered a key method for pest control in many crops with a low economic threshold

(Schoonhoven et al. 1998). To my knowledge, no strawberry cultivars have been tested for their

resistance to F. occidentalis. I tested three strawberry cultivars developed by the University of

California for differences in their suitability as hosts of F. occidentalis. These were Camarosa

and Camino Real, which are short-day varieties, and Albion, an ever-bearing (day-neutral)

variety. These cultivars are grown commercially in Australia and overseas (California

Strawberry Commission 2009).

The experiments conducted in this study suggest that different cultivars influence the feeding

and oviposition behaviour of F. occidentalis. Of the three cultivars tested, there was clear

variation in thrips behaviour and survival. Camarosa was most preferred by F. occidentalis

adults and it appeared to favour F. occidentalis development. The cultivar Camino Real was the

least preferred cultivar and the most unfavourable for F. occidentalis. There may be other

cultivars that are less favourable to F. occidentalis than Camino Real. Growers currently base

their cultivar selections on marketable yield, fruit colour, fruit size, taste and resistance to

diseases such as verticillium wilt and phytophthora crown rot (Phillips and Reid 2008). Further

screening of these cultivars would enable resistance information to be provided to growers, and

allow them to include thrips resistance in their decision-making. This study did not determine

the specific factors or characteristics that influence thrips preference and performance, so future

work should be conducted in this area. In addition, host-plant resistance to herbivores is

influenced by seasonal changes, and this should also be addressed.

8.2.2 Biological control: multiple species versus single species

All three predatory mites tested are effective predators of F. occidentalis. Typhlodromips

montdorensis was the most effective at reducing F. occidentalis, followed by N. cucumeris and

H. miles. Multiple-species release of predatory mites provided better management of F.


191

occidentalis than releases of a single species of predatory mites. As an integrated approach,

there is a growing trend to use two or more species of natural enemies to suppress insect pest

populations (Premachandra et al. 2003, Avilla et al. 2004, Blümel 2004, Brødsgaard 2004, Chau

and Heinz 2004, Chow and Heinz 2004, Hoddle 2004, Shipp and Ramakers 2004, Thoeming

and Poehling 2006, Chow et al. 2008). By maintaining a lapse of time between spinosad

application and predatory mite release, both methods can be incorporated to reduce F.

occidentalis numbers. Management can be further improved when combined with the cultivar

Camino Real. However, multiple-species release of natural enemies in pest a management

program is not always quantitatively validated (Blockmans and Tetteroo 2002, Skirvin et al.

2006). Some of the studies support the premise that species are compatible (Gillespie and

Quiring 1992, Wittmann and Leather 1997, Brødsgaard and Enkegaard 2005), whilst others

suggest otherwise (Magalhăes et al. 2004, Sanderson et al. 2005). This study found that any

combination of multiple-species release of predatory mites (T. montdorensis, N. cucumeris, and

H. miles) performed better in reducing F. occidentalis than respective single species. However,

predatory mite species varied in their compatibility. In this study, when combined together, T.

montdorensis and N. cucumeris were less effective than other combinations. However, they

performed better when applied in triple-species combination (second-best combination). When

predatory mites were released in double-species combinations, their numbers (as a proportion of

total mites released) were higher than in triple-species combination. This may increase the

chance of interspecific competition as the mites share the same resource. Further research

should study the optimal release rate, which might eliminate interspecific competition. One of

the limitations of the predatory mites used in this study is that they are either larval or pupal

predators. This means that adult F. occidentalis escaped predation. Therefore, future study

should explore combining mite releases with other predators such as the anthocorid bug, Orius

armatus, which preys on both larval and adult stage or other natural enemies (Baez et al. 2004).

8.2.3 Combining chemical and biological control

In addition to host-plant resistance, it has been argued that the integration of chemical and

biological control could provide better management of arthropod pests (Funderburk et al. 2000,

Ludwig and Oetting 2001, Ludwig 2002, Thoeming and Poehling 2006, Funderburk 2009) than

either tactic alone. Spinosad is a biopesticide classified as an environmentally and

toxicologically reduced-risk chemical (Cleveland et al. 2002, Thompson et al. 2002). It is

registered as an IPM-compatible insecticide in Australia (Thompson and Hutchins 1999).

Spinosad is regarded to have low to moderate toxicity to predatory mites but the toxicity

varies from species to species (Williams et al. 2003, Cote et al. 2004, Jones et al. 2005).


192

Therefore, for successful integration of chemical and biological control, it is important

to evaluate the residual threshold of a chemical on its respective natural enemies.

I found that a spinosad application followed by the release of commercially available predatory

mites could effectively reduce the F. occidentalis population but only if there was sufficient

time between spraying and release. As expected, the direct toxicity of recommended rate of

spinosad (F. occidentalis management, 80 ml/100 L, 0.096 g. a.i./L) as well some aged

residues were toxic to T. montdorensis, N. cucumeris and H. miles, causing substantial

mortality. Thresholds for residual toxicity of spinosad LT25 (lethal time for 25% mortality)

were estimated as 4.2 days (101.63h), 3.2 days (77.72) and 5.8 days (138.83 h) for T.

montdorensis, N. cucumeris and H. miles respectively. At this time, spinosad posed no

significant negative effect to mites. Based on the bioassays, spinosad was characterised as short-

lived (degraded quickly) for T. montdorensis and N. cucumeris (degrade quickly) and slightly

persistent for H. miles. It is important to note that residual threshold increased when predatory

mites fed on intoxicated F. occidentalis and were simultaneously exposed to spinosad residues.

This was estimated as 5.4 days (129.67), 4 days (95.09), and 6.1 days (146.68 h) for T.

montdorensis, N. cucumeris, and H. miles respectively. Natural enemies subjected to multiple

routes of exposure to pesticides may respond in unexpected ways that would be impossible to

predict based on single-route exposure (Kunkel et al. 2001). Thus, estimates of residual

thresholds should be based on persistence and harmfulness of indirect and contact toxicity, not

contact toxicity alone.

Spinosad can degrade quickly in nature (Thompson et al. 2000, Williams et al. 2003). The

bioassays conducted in this study were in the laboratory under controlled conditions. However,

chemical degradation may be influenced by environmental factors such as temperature,

photoperiod, and seasonal variation (van de Veire et al. 2002, Zilahl-Balogh et al. 2007).

Persistence of a chemical also varies from season to season (van de Veire et al. 2002, Zilahl-

Balogh et al. 2007). Spinosad may thus have more or less of a detrimental effect on the

predatory mites than presented in these experiments.

8.2.4 IPM of western flower thrips in low tunnel-grown strawberry

Control of F. occidentalis and other thrips with natural enemies in glasshouse crops is

successful in many parts of the world (Ramakers 1988). However, little is known about the

efficacy of predatory mites in semi-open or open field conditions. It has been established that

predatory mites are effective in glasshouse conditions where temperatures and humidity are

higher. Can predatory mites be effective in low tunnel-grown crops during spring, when


193

conditions are much cooler? I found that predatory mites reduced F. occidentalis in low tunnel

grown strawberry. The mites T. montdorensis, N. cucumeris and H. miles can be used in

combination in low tunnels for F. occidentalis management even in the relatively cooler

conditions. Gillespie and Ramey (1988) reported that N. cucumeris can survive at a constant

temperature of 9⁰C for months and oviposit within three days when returned to room

temperature (20-22°C). Typhlodromips montdorensis did not diapause when reared under 10⁰C

with long day conditions (Steiner et al. 2003) and can survive at 8⁰C or above (Steiner and

Goodwin 2002). In the low tunnel, the plastic cover keeps the tunnel warmer than the ambient

air, which might provide a suitable environment for predatory mites at least during the time of

year that the experiment was conducted. Longer-term studies on the population dynamics of the

mites in the field environment should be conducted to determine the temperature range at which

they can sustain their populations and remain active.

As an integrated approach, this study tested whether the use of predatory mites before or after

spinosad application would be the better option for F. occidentalis management in low-tunnel

grown strawberry. Spraying of spinosad followed by the release of predatory mites is more

effective as shown in this strategy, spinosad provided effective reduction of F. occidentalis

while having no significant detrimental effects on natural enemies. In the present study, the

numbers of predatory mites were significantly lower in the ‘mites then spinosad’ treatment

compared to the ‘spinosad then mites’ treatment, suggesting that there is a detrimental effect of

spinosad on predatory mites when applied after the mites are released. In a low tunnel

environment, the integration of spinosad and predatory mites was effective at reducing F.

occidentalis population below the economic threshold (5 or more thrips per flower in 45% of

flowers, (Steiner and Goodwin 2005)), but only for a limited period. Thus, there might be a

need for a further application of either spinosad or of predatory mites or both to maintain the F.

occidentalis population below the economic threshold throughout the cropping period.

8.2.5 Control of spinosad-resistant western flower thrips strain

Given the current high reliance on spinosad for F. occidentalis control and the increasing

probability that resistance in Australian populations will spread (Herron et al. 2007), it is

important to explore ways in which spinosad can be used with biological control agents

effectively when resistance is evident. One approach is to use an initial high dose of an

insecticide to reduce the resistant population, and thereafter use natural enemies to maintain the

population at a low level. However, use of the high dose of an insecticide would likely increase

the detrimental effect of a pesticide to natural enemies, particularly if there is any residual

activity of the insecticide. This study found that twice the recommended rate of spinosad


194

(recommended rate for F. occidentalis control, 160 mL/100 L, 0.192 g a.i./L) is very toxic to T.

montdorensis, N. cucumeris and H. miles, causing 100% mortality. Moreover, the residues

(depending on age) were also very toxic to the predatory mites. Despite the increased rate,

spinosad can degrade rapidly by photolysis and is short-lived. It persisted for less than five days

for N. cucumeris, and was classified as slightly persistent to T. montdorensis and H. miles. The

residual threshold (LT25) of twice the recommended rate was found to be 5.3 days (127.85 h) 6.1

days (146.76 h), and 6.8 days (162.45 h) for N. cucumeris, T. montdorensis, and H. miles. By

maintaining a lapse of 6-7 days between spray and predatory mite release, an increased rate of

spinosad could be used for F. occidentalis management when required.

This study only tested the compatibility of predatory mites with spinosad for WFT management.

However, the prey-predator ratio in which optimal management could be achieved was not

determined. Strawberry crops are often infested by other pests such as spider mites, rootworm,

leafrollers, root weevils , crown borers. These pests might also require chemical or biological

control. Therefore, compatibility of these predatory mites with other chemicals, or other

beneficials used for other pest control may also need to be evaluated.

8.3 Conclusions

This thesis evaluated the compatibility of an existing chemical (spinosad) and predatory mites

(T. montdorensis, N. cucumeris and H. miles), combined with cultivar resistance for the

management of F. occidentalis in strawberry. This thesis provides information for F.

occidentalis management in strawberry, which can also be adopted for pest management in

other horticultural crops where F. occidentalis is a problem. Differential performance of F.

occidentalis on strawberry cultivars has been demonstrated in this thesis, suggesting that host-

plant resistance could be used. Cultivar selection for thrips management has not been

considered for the Australian strawberry industry. Likewise, careful integration of biological

and chemical control has not been previously used for F. occidentalis management in Australia.

The findings presented here provide multifaceted support towards an effective, integrated

approach to manage F. occidentalis in field-grown strawberries. Pests continue to develop

resistance to insecticides and this is the case with F. occidentalis and spinosad. I demonstrated

that resistant strains of a pest could be managed by incorporating a pesticide with natural

enemies, thus retaining both pesticides and natural enemies in the pest management toolbox.

Where F. occidentalis has already begun to develop resistance to spinosad in Australia, it is

critical that we find alternatives to its management.


195


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Anthocoridae) on life stages and species of Frankliniella flower thrips (Thysanoptera:

Thripidae) in pepper flowers. Environmental Entomology. 33: 662-670.












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fruiting vegetables. Florida Entomologist 92: 1-6.






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Herron, G., M. Steiner, B. Gollnow, and S. Goodwin. 2007. Western flower thrips (WFT)

insecticide resistance management plan. New South Wales Department of Primary

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Kunkel, B. A., D. W. Held, and D. A. Potter. 2001. Lethal and sublethal effects of bendiocarb,

halofenozide and imidacloprid on Harpalus pennsylvanicus (Coleoptera: Carabidae)

following different modes of exposure in turf grass. Journal of Economic Entomology

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prey. Behavioural Ecology 16: 364-370.

Parrella, M. P., and T. Lewis. 1997. Integrated pest management (IPM) in field crops, pp.

595-638. In T. Lewis [ed.], Thrips as crop pests. CAB International, Wallingford, Oxon,

UK.

Phillips, D., and A. Reid. 2008. New strawberry varieties for WA - trial results in 2005, 2006

and 2007, pp. 9. Department of Agriculture and Food Western Australia, Kensington,

South Perth WA.




Ramakers, P. M. J. 1988. Population dynamics of the thrips predators Amblyseius mckenziei

and Amblyseius cucumeris (Acarina: Phytoseiidae) on sweet pepper. Netherland Journal

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against the predatory bug Orius laevigatus. Phytoparasitica 30: 525-528.

Wellington, W. G. 1977. Returning the insect to insect ecology: some consequences for pest

management. Environmental Entomology 6: 1-8.

White, T. C. R. 1969. An index to measure weather-induced stress of trees associated with

outbreaks of psyllids in Australia. Ecology 50: 905-909.

White, T. C. R. 1993. The inadequate environment: nitrogen and the abundance of animals.

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xvii

APPENDICS

Appendix 3.1 Summary of repeated measures ANOVAs tested the effect of cultivars

and predatory mites with or without spinosad on mean numbers of WFT adults per plant

at days 7, 14, and 21.

Source df (Num & Den) F value P value

All cultivars

Cultivar

Spray

Mites release

Day

Cultivar*spray

Cultivar*mites release

Cultivar*day

Spray*mites release

Spray*day

Mites release*day

Cultivar*spray*mites release

Cultivar*Spray*day

Cultivar*mites release*day

Spray*mites release*day

Cultivar*spray*mites release*day

Camarosa

Spray

Mites release

Day

Spray*mites release

Spray*day

Mites release*day


Camino Real

Spray

Mites release

Day

Spray*mites release

Spray*day

Mites release*day


Albion

Spray

Mites release

Day

Spray*mites release

Spray*day

Mites release*Day


2, 216

1, 216

3, 216

2, 432

2, 216

6, 216

4, 432

3, 216

2, 432

6, 432

6, 216

4, 432

12, 432

6, 432

12, 432

1, 72

3, 72

2, 144

3, 72

2, 144

6, 144

6, 144

1, 72

3, 72

2, 144

3, 72

2, 144

6, 144

6, 144

1, 72

3, 72

2, 144

3, 72

2, 144

6, 144

6, 144

2245.37

4062.00

516.31

4776.66

3.28

5.99

14.14

21.93

256.04

144.53

2.38

14.64

4.88

7.32

9.26

1191.60

102.90

1600.00

6.62

21.43

38.83

9.75

1651.47

2209.65

1241.62

9.77

111.28

45.93

4.29

1259.91

227.12

2107.92

10.61

185.13

79.63

13.02

<0.0001

<0.0001

<0.0001

<0.0001

0.0395

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

0.0303

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

0.0005

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

0.0005

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

xviii


and predatory mites with or without spinosad on mean numbers of WFT larvae per plant

at days 7, 14, and 21.


All cultivars

Cultivar

Spray

Mites release

Day

Cultivar*spray

Cultivar*mites release

Cultivar*day

Spray*mites release

Spray*day

Mites release*day

Cultivar*spray*mites release

Cultivar*Spray*day

Cultivar*mites release*day


Cultivar*spray*mites release*day

Camarosa

Spray

Mites release

Day

Spray*mites release

Spray*day

Mites release*day


Camino Real

Spray

Mites release

Day

Spray*mites release

Spray*day

Mites release*day


Albion

Spray

Mites release

Day

Spray*mites release

Spray*day

Mites release*Day


2, 216

1, 216

3, 216

2, 432

2, 216

6, 216

4, 432

3, 216

2, 432

6, 432

6, 216

4, 432

12, 432

6, 432

12, 432

1, 72

3, 72

2, 144

3, 72

2, 144

6, 144

6, 144

1, 72

3, 72

2, 144

3, 72

2, 144

6, 144

6, 144

1, 72

3, 72

2, 144

3, 72

2, 144

6, 144

6, 144

447.65

143.41

181.43

254.80

241.03

2.81

51.50

3.40

9.17

22.80

4.96

15.43

1.20

2.91

2.31

62.75

74.66

17.62

6.78

9.32

12.45

3.78

556.01

48.08

52.67

4.35

19.52

5.55

2.56

22.40

63.53

264.96

2.60

10.88

7.73

2.76

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

0.0457

<0.0001

0.0186

0.0001

<0.0001

<0.0001

<0.0001

0.2782

0.0087

0.0073

<0.0001

<0.0001

<0.0001

0.0004

0.0002

<0.0001

0.0010

<0.0001

<0.0001

<0.0001

0.0071

<0.0001

<0.0001

0.0217

<0.0001

<0.0001

<0.0001

0.0590

<0.0001

<0.0001

0.0114

xix


and predatory mites (combined application) with or without spinosad on mean numbers

of WFT (adults and larvae) on days 6, 9, 12, 15, 18, and 21.


WFT adults

Spray

Mites release

Day

Spray*mites release

Spray*day

Mites release*day


Spinosad

Mites release

Day

Mites release*day

Water

Mites release

Day

Mites release*day

WFT larvae

Spray

Mites release

Day

Spray*mites release

Spray*day

Mites release*day


Spinosad

Mites release

Day

Mites release*day

Water

Mites release

Day

Mites release*day

1, 144

7, 144

5, 720

7, 144

5, 720

35, 720

35, 720

7, 72

5, 360

35, 360

7, 72

5, 360

35, 360

1, 144

7, 144

5, 720

7, 144

5, 720

35, 720

35, 720

7, 72

5, 360

35, 360

7, 72

5, 360

35, 360

5129.93

585.99

855.30

14.06

10.47

56.53

2.52

193.12

312.30

23.94

469.60

596.93

37.21

2964.87

134.16

46.62

19.86

77.28

9.64

1.75

46.71

14.13

6.64

90.18

88.97

5.21

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

0.0052

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

xx

Appendix 5.1 Summary of ANOVA results for WFT on flower and fruit of strawberry grown in

low tunnels before spray/mite treatments.

Source of variations Num df Den df F value P value

Adults

Flower

Tunnel (between)

Mites treatment (within tunnel)

Times (weeks)

Fruit

Tunnel (between)


Times (weeks)

Nymphs

Flower

Tunnel (between)


Times (weeks)

Fruit

Tunnel (between)


Times (weeks)

3

7

2

3

7

2

3

7

2

3

7

2

21

21

62

21

21

62

21

21

62

21

21

62

2.58

1.17

6.24

1.90

1.21

5.21

1.14

1.12

2.29

2.11

1.53

2.64

0.0815

0.3607

0.0034

0.1606

0.3404

0.0088

0.3559

0.3876

0.0385

0.1291

0.2114

0.0187

xxi

Appendix 5.2 Summary of ANOVA results for WFT adults on flower and fruit (split-plot

repeated measures ANOVA) of strawberry grown in low tunnels.


Flowers

Tunnel

Spray regime

Predatory mites

Times (weeks)

Spray regime*predatory mites

Spray regime*times

Predatory mites*times

Spray regime*predatory mites*times

Water spray

Tunnel

Predatory mites

Times (weeks)

Predatory mites*Times

Spinosad sprayed then mites released

Tunnel

Predatory mites

Times (weeks)


Mites released then spinosad sprayed

Tunnel

Predatory mites

Times (weeks)


Fruits

Tunnel

Spray regime

Predatory mites

Times (weeks)


Spray regime*times



Water spray

Tunnel

Predatory mites

Times (weeks)



Tunnel

Predatory mites

Times (weeks)

Predatory mites*Times


Tunnel

Predatory mites

Times (weeks)


3

2

7

4

14

8

28

56

3

7

4

28

3

7

4

28

3

7

4

28

3

2

7

4

14

8

28

56

3

7

4

28

3

7

4

28

3

7

4

28

6

6

63

288

63

288

288

288

21

21

96

96

21

21

96

96

21

21

96

96

6

6

63

288

63

288

288

288

21

21

96

96

21

21

96

96

21

21

96

96

0.90

361.64

344.31

564.74

10.73

110.72

50.66

18.93

2.13

145.73

331.68

32.71

1.97

765.74

40.44

101.42

1.88

41.57

562.37

7.04

1.72

698.13

130.16

70.61

9.63

118.24

26.03

3.78

2.88

13.62

1.98

3.22

0.67

284.76

74.93

32.59

0.70

85.62

103.29

47.34

0.4942

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

0.1494

<0.0001

<0.0001

<0.0001

0.1640

<0.0001

<0.0001

<0.0001

0.2617

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

0.0602

<0.0001

0.1029

<0.0001

0.5789

<0.0001

<0.0001

<0.0001

0.5607

<0.0001

<0.0001

<0.0001

xxii

Appendix 5.3 Summary of repeated measures ANOVA results for WFT larvae on flower and

fruits of strawberry grown in low tunnels.


Flowers

Tunnel

Spray regime

Predatory mites

Times (weeks)


Spray regime*times



Water spray

Tunnel

Predatory mites

Times (weeks)



Tunnel

Predatory mites

Times (weeks)



Tunnel

Predatory mites

Times (weeks)


Fruits

Tunnel

Spray regime

Predatory mites

Times (weeks)


Spray regime*times



Water spray

Tunnel

Predatory mites

Times (weeks)



Tunnel

Predatory mites

Times (weeks)



Tunnel

Predatory mites

Times (weeks)


3

2

7

4

14

8

28

56

3

7

4

28

3

7

4

28

3

7

4

28

3

2

7

4

14

8

28

56

3

7

4

28

3

7

4

28

3

7

4

28

6

6

63

288

63

288

288

288

21

21

96

96

21

21

96

96

21

21

96

96

6

6

63

288

63

288

288

288

21

21

96

96

21

21

96

96

21

21

96

96

1.80

150.17

430.84

569.23

20.77

24.03

274.06

37.11

2.85

332.62

407.16

276.74

2.53

93.85

154.91

86.57

1.24

219.91

191.86

157.72

0.15

104.20

483.08

303.37

25.43

59.89

53.76

9.15

1.91

175.90

13.55

107.71

1.83

238.96

233.93

31.33

1.19

113.75

103.60

24.23

0.1781

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

0.0619

<0.0001

<0.0001

<0.0001

0.0848

<0.0001

<0.0001

<0.0001

0.3203

<0.0001

<0.0001

<0.0001

0.9279

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

0.1583

<0.0001

<0.0001

<0.0001

0.1726

<0.0001

<0.0001

<0.0001

0.3370

<0.0001

<0.0001

<0.0001

xxiii

Appendix 5.4 ANOVA results for predatory mites, T. montdorensis and N. cucumeris per

flower/fruit when applied in different combinations in different spray treatments.


Sin

gle

sp

rel

ease

Flower

Tunnel

Mite species

Spray

Times (tunnel)

Spray*times

Fruit

Tunnel

Mite species

Spray

Times (tunnel)

Spray*times

3

1

2

3

6

3

1

2

3

6

6

11

6

63

63

6

11

6

63

63

3.06

1.42

38.63

1.55

0.97

1.05

29.39

299.7

2.58

0.43

0.1131

0.2592

0.0004

0.2097

0.4551

0.4358

0.0002

0.0008

0.0615

0.8562

Double

sp r

elea

se (

Tm

*N

c)

Flower

Tunnel

Mite species

Spray

Times (tunnel)

Spray*times

Fruit

Tunnel

Mite species

Spray

Times (tunnel)

Spray*times

3

1

2

3

6

3

1

2

3

6

6

11

6

63

63

6

11

6

63

63

0.58

10.53

11.95

1.89

0.22

1.24

9.99

13.95

1.28

0.07

0.6497

0.0078

0.0081

0.1404

0.9680

0.3753

0.0092

0.0062

0.2882

0.9986

Double

sp r

elea

se (

Tm

*H

m,

Nc*

Hm

)

Flower

Tunnel

Mite species

Spray

Times (tunnel)

Spray*times

Fruit

Tunnel

Mite species

Spray

Times (tunnel)

Spray*times

3

1

2

3

6

3

1

2

3

6

6

11

6

63

63

6

11

6

63

63

0.62

4.58

30.70

1.58

0.50

1.32

4.69

21.41

1.83

0.90

0.6179

0.0556

0.0007

0.2040

0.8089

0.3259

0.0534

0.0019

0.1516

0.4992

Tri

ple

sp

rel

ease

(T

m*

NC

*H

m) Flower

Tunnel

Mite species

Spray

Times (tunnel)

Spray*times

Fruit

Tunnel

Mite species

Spray

Times (tunnel)

Spray*times

3

1

2

3

6

3

1

2

3

6

6

11

6

63

63

6

11

6

63

63

0.57

0.15

8.04

0.18

0.08

1.35

0.15

8.03

0.22

0.09

0.6556

0.7070

0.0201

0.9121

0.9982

0.3454

0.7078

0.0201

0.8801

0.9974 *indicates species combinations. Tm = T. montdorensis, Nc = N. cucumeris, Hm = H. miles.

xxiv

Appendix 5.5 Repeated measures ANOVA results of the effects of spray, mite species

combination (double) and time (weeks) on T. montdorensis (when applied with N. cucumeris

and H. miles) and N. cucumeris (when applied with T. montdorensis and H. miles) in double-

species combinations (Repeated measures ANOVA with split-plot design).


T. montdorensis

Flowers

Tunnel

Spray

Mite combination

Times (tunnel)

Spray*mite combination

Spray*times

Mite combination*Times

Spray*mite combination*times

Fruits

Tunnel

Spray

Mite combination

Times (tunnel)


Spray*times

Mite combination*times


N. cucumeris

Flowers

Tunnel

Spray

Mite combination

Times (tunnel)


Spray*times



Fruits

Tunnel

Spray

Mite combination

Times (tunnel)


Spray*times



3

2

1

3

2

6

3

6

3

2

1

3

2

6

3

6

3

2

1

3

2

6

3

6

3

2

1

3

2

6

3

6

6

6

9

54

9

54

54

54

6

6

9

54

9

54

54

54

6

6

9

54

9

54

54

54

6

6

9

54

9

54

54

54

0.20

15.88

48.66

2.41

2.93

0.43

0.46

0.60

0.09

8.41

11.46

2.12

0.15

0.25

0.04

0.08

1.39

11.76

20.43

2.34

2.73

0.20

0.08

0.31

1.53

11.57

14.42

0.79

1.03

0.59

0.35

0.50

0.8954

0.0040

<0.0001

0.0770

0.1047

0.8557

0.7093

0.7283

0.9613

0.0182

0.0081

0.1088

0.8656

0.9555

0.9903

0.9979

0.3332

0.0084

0.0014

0.0836

0.1183

0.9766

0.9725

0.9300

0.2467

0.0009

0.0018

0.5073

0.3799

0.7368

0.7927

0.8055

xxv

Appendix 7.1 ANOVA results showing the interaction of spray (spinosad, water) and predatory

mite (no mites, T. montdorensis, N. cucumeris, H. miles) on WFT adults and larvae.

Obs. Source F df P

Adults

10 DAS

15 DAS

20 DAS

25 DAS

30 DAS

35 DAS

Larvae

10 DAS

15 DAS

20 DAS

25 DAS

30 DAS

35 DAS

Spray*mites

16.75

6.96

4.75

3.28

8.45

14.50

38.43

7.86

7.89

17.99

26.97

12.16

3, 32

< 0.0001

0.0010

0.0075

0.0333

0.0003

< 0.0001

< 0.0001

0.0005

0.0004

< 0.0001

< 0.0001

< 0.0001

xxvi

Appendix 7.2 ANOVA results of the effects of predatory mites (no mites, T. montdorensis, N.

cucumeris, H. miles) on WFT adults and larvae in spinosad- and water-treated plants.

Obs. Source F df P

Adults

Spinosad

10 DAS

15 DAS

20 DAS

25 DAS

30 DAS

35 DAS

Water

10 DAS

15 DAS

20 DAS

25 DAS

30 DAS

35 DAS

Larvae

Spinosad

10 DAS

15 DAS

20 DAS

25 DAS

30 DAS

35 DAS

Water

10 DAS

15 DAS

20 DAS

25 DAS

30 DAS

35 DAS

Predatory mites

release

21.70

554.34

51.72

30.85

56.80

112.26

2.38

70.70

58.51

48.61

38.88

104.33

137.59

31.69

25.89

213.26

61.49

136.26

25.64

47.78

59.92

69.59

51.21

89.75

3, 16

< 0.0001

< 0.0001

< 0.0001

< 0.0001

< 0.0001

< 0.0001

0.1082

< 0.0001

< 0.0001

< 0.0001

< 0.0001

< 0.0001

< 0.0001

< 0.0001

< 0.0001

< 0.0001

< 0.0001

< 0.0001

< 0.0001

< 0.0001

< 0.0001

< 0.0001

< 0.0001

< 0.0001

interaction between natural enemies and insecticides … · (rosaceae) md touhidur rahman b.sc...

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