management of purple blotch of onion, garlic and shallot using antagonists and systemic acquired...

Management of Purple Blotch of Onion,

Garlic and Shallot Using Antagonists

and Systemic Acquired Resistance

Project Leader & Study Leader:

Rizaldo G. Bayot

IMPLEMENTING AGENCY:

National Crop Protection Center,

University of the Philippines Los Baños

PROJECT FUNDING:

Department of Agriculture – BAR #88-521-21

BACKGROUND OF RESEARCH PROJECT

Project was envisioned because of the following

circumstances:

Vegetable RDE Network identified purple blotch

caused by Alternaria porri as a priority research area

in 2000.

Considered as a major constraint in bulb Allium crops

production

Farmers rely heavily on fungicides

“environmental protection should be non-negotiable-

disease management should be done with as little impact

on the environment as possible”

Microbial antagonists and yield increasing bacteria (YIB)

are already used extensively in China

Several biological control agents have been registered in

the USA against plant diseases

These are plant-associated microorganisms that work on

the principle of the “exclusionary” system of plant defense.

Findings of researchers abroad that certain plants can be

induced to become resistant against a broad spectrum of

leaf and root pathogens using biological and chemical

inducers.

If plants can be “immunized” or induced to produce natural

products to combat infection, the use of fungicides can be

reduced; ensuring public safety and environmental

integrity

During project implementation:

Purple blotch was no longer a problem.

Leaf anthracnose caused by Colletotrichum sp.

became very serious in onion and shallot

Project evaluators were informed.

Emphasis of the project shifted to anthracnose.

Objectives:

General:

To develop a biological control- and induced resistance-based

disease management in onion, garlic and shallot in order to

reduce the farmers' dependence on fungicides

Specific:

1) To isolate, characterize and evaluate the efficacy of

indigenous microbial antagonists against Alternaria porri

and Colletotrichum sp. of garlic, onion and shallot.

2) To screen commercially available varieties of garlic, onion

and shallot for systemic acquired resistance (SAR) traits

using SAR-inducing compounds against A. porri and

Colletotrichum sp.

Methodology and Results

1. Biological Control of Purple Blotch Using Microbial

Antagonists

a. Survey of Allium farms and specimen

collection

Onion and shallot farms in Bongabon, Gabaldon, Muñoz and

San Jose City, Nueva Ecija

Garlic farms in Batac, Ilocos Norte

Purple blotch did not develop in the areas surveyed due to

dry weather condition that prevailed

Few plants in Bongabon and Gabaldon developed purple

blotch symptoms late in the season.

b. Isolation of pathogens and pathogenicity

Isolation methods: tissue planting, streaking of spore

suspension, cutting hyphal tip of germinating conidia

Culture media: water agar, potato dextrose agar (PDA),

acidified PDA, PDA amended with onion leaf extract, PDA

amended with 100 ppm streptomycin sulfate, onion leaf extract

amended with 1% glucose.

Spore suspension was prepared and sprayed to onion

seedlings

Inoculated seedlings were incubated in a moist chamber for 48

hours and observed for symptom development in greenhouse.

Alternaria sp. was isolated but did not develop typical

symptoms of purple blotch.

Delayed the other activities of the project

Anthracnose caused by Colletotrichum sp. was severe in the

areas surveyed

The isolated pathogen was highly virulent

Used in subsequent tests

c. Isolation of microbial antagonists

Roots and leaves of garlic, onion and shallot collected

from various locations were used for isolation.

Cut into small pieces 5 gms placed in test tubes or

flasks with sterile water incubated for 30 minutes

with intermittent shaking in vortex mixer suspension

streaked on King’s B medium (KB), nutrient yeast agar

(NYA) and potato dextrose peptone agar (PDPA).

Representative colonies were transferred to PDPA

slants, purified and stored in mineral oil at room

temperature.

80 probable antagonists were isolated from various

sources and maintained in the laboratory using mineral

oil

d. In vitro screening of isolates for antagonism

Dual culture test was used

Garlic Isolates1 Degree of Inhibition2

Against Alternaria sp. Against Colletotrichum sp.

GLC1 - NT

GLC2 ++ NT

GL1W2 + +

GLM1 - -

GLM3 - NT

GLM3a - -

GLM3b + -

GLM4b - -

GLNE1 + +

GLNE2 + -

GRC1a - -

GRC1b - -

GRC2a + +

GRC2b ++ NT

Table 1. In vitro test of probable antagonists for mycelial growth inhibition

of Alternaria sp. and Colletotrichum sp. of Allium using the dual

culture method.



GRC3 - NT

GRC4 + +

GR1W1a + +

GR1W2 - -

GR1W2a ++ NT

GR1W3 - -

GR1W3b + NT

GR1W5 + -

GRM3a + -

GRM3b + -

GRM4a + -

GRM4b - -

GRM5 + -

GRM6a - -

GRM6b - -

GRNE1a - -

GRNE1b - -

GRNE2b ++ +

GRNE3 - -

GRNE3b - NT

GRNE4a + +

GRNE4b + -

GRNE5a - -

Table 1. Cont…



GRNE5b - -

OLNE1 - -

OLNE2 + -

OLNE3 - -

OLNE4 - NT

OLNE4a + +

OLNE4b + -

ORNE1a + NT

ORNE1b + NT

ORNE2a ++ +

ORNE2b + +

ORUBS1 - +

ORUBS2 - -

Leek Isolates1

LL3 + NT

LR1 + NT

LR2 + NT

LR3 + NT

LR4 ++ NT

Shallot Isolates1

SL1 - NT

SL2 - NT

Table 1. Cont…



SL3 - NT

SL4 - NT

SL5 - NT

SL6 - NT

SL7 + NT

SL8 - NT

BC1 NT +++

BC2 NT ++

B2HT3 ++ ++

B3HT NT +++

B2HTRiFR1 NT +++

BRIHT +++ +++

Bp +++ NT

B. subtilis (Biotech) NT +

B. polymixa (Biotech) NT -

Table 1. Cont…



CHRHT +++ NT

CS2HT ++ ++

ESHT1 ++ NT

ISO2HT ++ ++

M3HT ++ ++

M3MTRifR3 NT +++

ANT1 +++ ++

ANT 2 NT ++

1/ Mycelial disks of the pathogen were placed at the center of PDA plates. Cells of 24-48 hr-old

cultures of antagonists were spotted on the medium about 40 mm from the fungal disk in a X

pattern. Growth inhibition was determined after 3-5 days.

Table . Cont…

d. In vitro screening of isolates for antagonism

Out of 80 isolates, 72 were tested against Alternaria sp. in dual culture

test

4 had strong inhibition, 11 had moderate inhibition, 26 had slight

inhibition, the rest had no effect

53 isolates were tested against Colletotrichum sp.

5 had strong inhibition, 7 had moderate inhibition, 12 had slight

inhibition.

Figure 3. Verification of antagonism of biocontrol agents stored in mineral oil for one year using the dual culture method against the anthracnose pathogen.

Inhibitory effects were retained after 1 year of storage in mineral oil

Figure 4. Paper disk assay of supernatants of biocontrol agents grown in nutrient broth for fifteen days against the anthracnose pathogen.

Filter-sterilized culture filtrate of some antagonists inhibited mycelial growth of

anthracnose pathogen

Spore Germination Test• Prepared spore suspension of Alternaria sp.

100 ul placed on clean glass slides

Antagonist growth (24-48 hr old) was mixed with spore

suspension

Observed microscopically

Germinating conidia of Alternaria sp. after six hours of incubation showing normal germ tube formation in untreated slide (control)(1000x).

Germinating conidia of Alternaria sp. showing swollen germ tube six hours after treatment with B2HT3 isolate (1000x).

Germinating conidia of Alternaria sp. showing large swelling germ tube 18 hours after treatment with B2HT3

isolate (1000x).

Table 2. Influence of selected bacterial antagonists on conidial germination

of Alternaria sp.

1About 100 µL spore suspension of A. porri was placed on a clean glass slide. Small

amount of bacterial growth from 24-48 hours old culture was mixed with the spore

suspension of the pathogen. Glass slides were incubated in petri plates with moist

tissue paper and examined microscopically at different time intervals for any abnormality in spore germination.

Isolate Tested1 Reaction

B2HT3 Swollen germ tubes after 5 hours

Bp Normal spore germination after 5 hours

BR1HT Few spores germinated after 5 hours, swollen germ tubes

ChrHT Few spores germinated after 5 hours, swollen germ tubes

CS2HT Some spores have normal germination, some have swollen germ tubes after 5.5 hours

ESHT1 Most spores have swollen germ tubes after 5.5 hours

ISO2HT Few spores germinated after 5 hours, swollen germ tubes

M3HT Normal spore germination after 5 hours

8 isolates were tested for effects on conidial germination of Alternaria

sp.

5 isolates had strong inhibitory effects on conidial germination .

Germinated conidia have swollen germ tubes

Treatment No. of Lesions 4 Days After Inoculation1

I II Mean

M3HTRifR3 0 0 0

ISO2HT 1.6 0.2 0.9

BRIHT 0.6 2.2 1.4

B2HT3RifR1 2.8 0.8 1.8

CHRHT 4.0 0.6 2.3

B3HT 1.6 3.2 2.4

ESHT1 3.8 3.2 3.5

Control 3.4 4.0 3.7

Table 3. Greenhouse evaluation of antagonists against

Colletotrichum sp. using the “Tanduyong” variety of

shallot (Sept. 2002).

1Each figure is the mean of five leaves.

Greenhouse evaluation of promising antagonists

against anthracnose

First trial used 7 potential antagonists

4 isolates reduced anthracnose lesions in leaves of Tanduyong

variety.

Treatment No. of Lesions 8 Days After Inoculation1

I II III MeanCHRHT 0 0 0 0.01CS2HT 0 0.2 0.2 0.131BC1 0.6 1.0 0.4 0.67 kESHT1 0.2 0.8 1.4 0.8 kBC II 1.0 0.8 1.2 1.0 jB3HT 1.2 1.8 0.5 1.17ijM3HT 1.4 1.2 1.2 1.27 hiB2HT3 1.2 0.4 2.6 1.4 hGR4 1.8 0.6 2.4 1.6 gANT1 1.8 1.2 2.2 1.73 gOLNE3 2.0 2.2 1.8 2.0 fB. poly (Biotech) 2.0 2.8 1.4 2.07 fISO2HT 2.0 4.2 2.0 2.73 eBRIHT 4.0 2.0 2.6 2.87 deANT2 2.2 1.2 5.4 2.93 deSL7 3.0 3.0 2.8 2.93 dB. substilis (Biotech) 3.4 3.4 2.6 3.13 eBp 4.2 3.8 2.2 3.4 bControl (water) 2.0 5.6 1.4 3.0 cdControl (N.B.) 5.4 4.8 7.2 5.8 a

Table 4. Greenhouse evaluation of antagonists against Colletotrichum sp.

using the “Tanduyong” variety of shallot (January, 2003).


Greenhouse evaluation of promising antagonists

against anthracnose

Second trial used 18 antagonists

8 isolates reduced anthracnose lesions on leaves of shallot

(Tanduyong) ranging from 0 to 1.4 lesions per leaf compared to 3

lesions in untreated plants.

Treatment

Disease Severity (%)

Mean1 2 3 4 5

1. Control 50 60 60 60 65 59 ab

2. QST (liquid) 30 40 20 20 20 26 f

3. QST (solid) 50 55 40 45 50 48 cde

4. BC1 (liquid) 50 55 50 40 30 45 de

5. BCI (solid) 50 60 60 60 50 56 abc

6. EPSP (liquid) 30 50 50 45 50 45 de

7. EPSP (solid) 50 60 60 55 40 53 bcd

8. Ant 1 (liquid) 50 60 60 65 65 60 ab

9. Ant 1 (solid) 45 50 45 50 50 48 cde

10. CS2HT (liquid) 50 30 45 45 45 43 e

11. CS2HT (solid) 40 50 55 60 60 53 bcd

12. ESHT l (liquid) 60 65 65 70 65 65 a

13. ESHT l (solid) 60 60 60 70 65 63 a

14. Avirulent

Alternaria

50 60 60 50 40 52 bcde

15. 150 ppm

difenoconazole

5 10 5 10 5 7 g

Table 5. Greenhouse evaluation of bacterial antagonists grown in liquid

and solid media against shallot anthracnose.

Comparison of bacterial antagonists grown

in solid and liquid media

Antagonists grown in liquid medium (PDP broth) were more

effective against anthracnose than those grown in solid

medium (PDPA) except for 1 isolate)

Treatment

Disease severity (%)

Mean1 2 3 4 5

Control 70 75 75 75 80 75 a

QST (liquid) 20 30 30 40 30 30 d

BC1 (liquid) 50 50 50 55 40 49 c

BCl (solid) 55 55 55 70 65 60 b

B2HT3 (liquid) 60 50 55 60 60 57 bc

B2HT3 (solid) 60 70 70 65 60 65 b

150 ppm

(score)

20 40 40 45 45 38 d

Table 6. Greenhouse evaluation of selected promising bacterial

antagonists grown in solid and liquid media against

shallot anthracnose.

M3HT B2HT3 B3HT ChrHT C2H

T

Bp Br1HT B.

poly

B.

sub

ISO2

HT

ESHT1

Motility (-) (-) (-) Slight (-) (-) (-) (-) (-) (-) (-)

Growth in 7%

NaCl

(+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+)

Citrate Utilization (+) (+) (-) (-) (-) (-) (+) (-) (+) (+) (+)

Acid From:

Arabinose

Mannitol

Xylose

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

Table 7. Partial characterization of selected potential antagonists.

Study 2. Systemic Acquired Resistance in Disease

Management of Onion and Shallot

Preliminary evaluation of compounds for induction

of resistance in shallot. Two varieties of shallot

were evaluated (Tanduyong and Batanes)

Various concentrations of acetylacetate and

K2HPO4 were evaluated

Results are not conclusive although the

Tanduyong and Batanes varieties showed some

degree of induced resistance

Mean No. of Lesions 6 Days After Inoculation1

Treatment 1 Day After Induction 3 Days After

Induction

Tanduyong Batanes Tanduyong Batanes

0.1% Acetylsalicylate 1.0 1.0 1.0 2.2

0.075% Acetylsalicylate 0 0.4 2.4 3.4

0.05% Acetylsalicylate 0.8 2 2.6 2.0

100 mM K2HPO4 1.6 1.6 4.4 2.8

75 mM K2HPO4 3.6 1.0 0.8 1.4

50 mM K2HPO4 1.4 1.8 4.0 3.2

Control (untreated) 1.0 1.0 6.4 4.4

Table 8 Efficacy evaluation of various concentrations of selected

chemicals for induction of resistance of shallot varieties

against Colletotrichum sp (September 2002).


Treatment Disease Severity (%) 8 days after inoculation

I II III Mean

Control 30 42 48 40.0 abc

100 ppm (BTH) 25 26 19 23.3 d

10 mM INA 38 28 36 34.0 abcd

20 mM INA 29 23 35 29.0 bcd

10 mM SA 46 44 33 41.0 ab

20 mM SA 44 40 35 39.7 abc

10 mM Oxal 43 35 44 40.7 abc

20 mM Oxal 33 24 26 27.7 cd

50 mM K2HPO4 44 39 54 46.7 a

75 mM K2HPO4 24 46 36 35.3 abcd

Table 9. Efficacy evaluation of various compounds for induced resistance

of shallot (cv. Tanduyong) against Allium anthracnose.

1Plants were inoculated four days after induction.

Second trial, isonicotinic acid (INA), salicylic acid (SA),

oxalic acid and K2HPO4 were compared with

benzothiodiazole (BTH) or acibenzolar-s-methyl

Only BTH reduced anthracnose severity (23.3%) compared to

40% in untreated plants

Plants sprayed with 20 mM oxalic acid had 27.7% disease

severity

Treatment % Disease Severity 5 Days After Inoculation Mean

1 2 3 4 5

Control (untreated) 50 40 30 40 30 38 de

150 ppm BTH 40 30 30 30 45 35 e

Acacia (fresh) 50 55 60 60 60 57 ab

Acacia (compost) 50 60 60 50 50 54 abc

(fresh) 30 40 40 30 30 34 e

(compost) 60 60 60 70 60 62 a

Chromolaena (fresh) 50 50 40 40 30 42 cde

Chromolaena

(compost)

60 40 55 60 60 55 ab

Narra (fresh) 60 60 30 60 30 48 bcd

Narra (compost) 60 70 30 60 55 55 bcd

Colletotrichum

(mango)

30 30 40 50 40 38 de

Table 10. Greenhouse evaluation of various plant extracts as possible

inducers of shallot resistance against anthracnose.

None of the plant extracts (fresh or compost extract) increased the resistance

of test plants against onion anthracnose

Field evaluation of promising antagonists and chemical inducers

against shallot anthracnose

Set-up of field experiment for evaluation of selected antagonists and

chemical inducers against leaf anthracnose of shallot.

Treatment

Disease Severity (%)1

MeanI II III

Control 50 56 62 56.0 a

150 ppm difenoconazole 36 44 34 38.0 c

BTH (150 ppm) 39 38 36 37.7 c

BC1 54 48 50 50.7 ab

B2HT3 46 40 44 43.3 bc

QST 38 42 44 41.3 c

Oxalic acid (20 mM) 38 32 46 38.7 c

Table 11. Field evaluation of selected bacterial antagonists and

chemical inducers against shallot anthracnose.

1Each figure is the mean of five plant samples.

Field evaluation of promising antagonists and chemical inducers

against shallot anthracnose

Antagonists evaluated were: BC1, B2HT3 and QST (commercial

product)

Chemical inducers evaluated: oxalic acid and BTH

Antagonist B2HT3 was comparable to QST (commercial product)

Chemical inducer oxalic acid (20mM) was comparable to BTH

(commercial product)

The treatments (except BC1) were comparable to the effect of

fungicide difenoconazole (150 ppm)

Conclusion and Recommendation

Purple blotch of bulb Allium crops caused by Alternaria porri was no longer a

serious threat to bulb Allium crops production from 2002 to 2005.

Foliar anthracnose caused by Colletotrichum sp., and later on, twister disease

caused by Colletotrichum sp. and Fusarium sp. became serious fungal diseases of

these crops.

Some indigenous bacterial antagonists may be effective against foliar

anthracnose of shallot

“Tanduyong” variety of shallot may be induced to become resistant to

anthracnose by certain chemical activators

Further studies are necessary to fine-tune the biocontrol-based and SAR-based

disease management of foliar anthracnose in bulb Allium crops..

management of purple blotch of onion, garlic and shallot using antagonists and systemic acquired...

Documents