resistance screening and influence of fruit physico ......mango industry is the source of livelihood...

Maria Luz J. Sison*, Cris Q. Cortaga, Ana Kristine S. Barcos, Niño R. Laurel, John Marty C. Mateo, and Fe M. dela Cueva

Institute of Plant Breeding, College of Agriculture and Food Science University of the Philippines Los Banos, College, Laguna 4031 Philippines

Resistance Screening and Influence of Fruit Physico-biochemical Properties of “Carabao”

and Other Mango Varieties Against Oriental Fruit Fly, Bactrocera dorsalis (Diptera: Tephritidae), in the Philippines

Keywords: “Carabao” mango, host plant resistance, oriental fruit fly, physico-biochemical properties

The Philippines is one of the major exporters of fresh and dried mangoes in the world, with “Carabao” (Mangifera indica L.) as its flagship variety. Among the most damaging pests that attack this cultivar is the oriental fruit fly, Bactrocera dorsalis Hendel, which limits the country’s export potential drastically. The control and management of this pest have been developed, however, the search and use of resistant cultivars have not been explored in the Philippines. In this study, a total of 33 mango accessions consisting of “Carabao” and other varieties were evaluated for resistance against B. dorsalis over a period of four years (one trial per year) through ‘choice’ tests. Repeated trials showed that accessions 12-209 (“Carabao” – Tree 203), 12-127 (“Carabao” – Tree 30), 12-103 (“Farrales”), and 16-010 (GES 77 NSIC Carabao variety) exhibited promising resistance ratings (i.e. no damage to moderately susceptible) against oriental fruit fly. Overall correlation analysis of fruit physico-biochemical properties revealed that insect visit is moderately affected by flesh flavonoid content. Meanwhile, adult emergence correlated to peel physico-biochemical properties, especially peel firmness, which considerably affected insect reproduction. Comparison between the most resistant and most susceptible accessions suggests that, apart from high flesh flavonoids and peel firmness, high flesh phenolics and low pH (acidic) levels may also play a defensive role in mango against oriental fruit fly. The accessions characterized can be used as stop-gap varieties or as parental lines for the breeding of resistant “Carabao” mango.

*Corresponding Author: [email protected] [email protected]

INTRODUCTIONThe Philippines is a major exporter of fresh and dried mangoes in the world with USD 91 M in exports and a 4% share of the global market (UNComtrade 2016). The mango industry is the source of livelihood of about 2.5 M Filipino farmers (PCARRD-DOST 2011). Hence, the country’s major strength in the mango global value chain

is its ability to support the cultivation and production of “Carabao” mango, which is reputed as one of the world’s finest, sweetest, and superior quality varieties (Castillo-Israel et al. 2015; Stark et al. 2017). In the last quarter of 2019, the “Carabao” variety contributed the largest share (81.7%) of the total mango production in the Philippines with 22.69 thousand metric tons (PSA 2019). However, the export potential of this variety cannot be maximized due to insect pests and diseases, which are most often difficult to control. Production data in recent years showed that

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Philippine Journal of Science149 (4): 1175-1187, December 2020ISSN 0031 - 7683Date Received: 03 Aug 2020

the country’s mango exports have declined considerably due to failures in complying with the strict sanitary and phytosanitary standards, which is a key requirement in international export markets (Stark et al. 2017).

One of the common and most damaging pests of mango, especially in the “Carabao” cultivar, is the oriental fruit fly, Bactrocera dorsalis Hendel, which limits the country’s export potential and impede international trade due to quarantine restrictions. Recently, the Philippine fruit fly, Bactrocera philippinensis Drew and Hancock, has been synonymized with Bactrocera papayae Drew and Hancock, while B. papayae has been synonymized with B. dorsalis (Boykin et al. 2014; Schutze et al. 2012, 2015). Hence, B. dorsalis is now considered as the senior synonym for all of the prior species mentioned. The oriental fruit fly affects fruit quality through ovipositional punctures and larval damage, which may render the fruits unmarketable with losses ranging from 5–80% (Stonehouse 2001; Choudhary et al. 2018). To control fruit fly infestation, farmers usually rely on the heavy application of chemical pesticides, which are hazardous to health and environment and contribute to increased production costs. Fruit bagging is usually being adapted in the Philippines to avoid application of many pesticides. Apart from this, other cultural practices exist but are often not efficient and may also be very costly. The integration of different pest management strategies has been developed, however, the use or search for possible sources of resistance has not been explored in the Philippines. The use of resistant varieties remains an integral component of any integrated pest management program (Panda and Khush 1995; Smith 2005; Stout 2007). Identification of resistant lines/varieties may serve as a basis for the management of oriental fruit fly and is an important aspect for improvement of the “Carabao” mango through breeding for insect-resistance. Thus, in this study, host plant resistance screening through a “choice” test was carried out for four years using diverse mango accessions consisting of “Carabao” and other varieties to identify resistant accession/s against oriental fruit fly. Alongside, the various physico-biochemical fruit properties of the accessions were analyzed to elucidate mango’s possible mechanism of resistance to this major pest. The information generated in this study can be utilized for the improvement of the “Carabao” mango and the development of resistant varieties.

MATERIALS AND METHODS

Flower Induction and Acquisition of Mango FruitsA total of 33 mango accessions (Mangifera indica L.) located in the provinces of Guimaras, Laguna, Quezon,

and Zambales in the Philippines were used in the resistance screening conducted from 2015–2019 (Table 1). The accessions from Laguna are part of the Mango Field Genebank of the Institute of Plant Breeding (IPB) – University of the Philippines Los Banos (UPLB), while the accessions in Quezon and Zambales are located in private mango orchards. On the other hand, "GES" accessions from Guimaras are official “Carabao” mango varieties registered in the National Seed Industry Council (NSIC) of the Philippines and are maintained at the Bureau of Plant Industry – Guimaras National Crop Research, Development and Production Support Center (BPI-GNCRDPSC). For nearby accessions in Laguna and Quezon, mango trees were induced for flowering through the application of calcium nitrate or Ca(NO3)2 (6 kg/ 200 L water). Upon full bloom, the trees were applied with pesticides – namely, Dithane M-45 Neotec fungicide (562 g/ 200 L water) and Actara 25WG insecticide (12 g/ 200 L water) – to avoid pest infestation and ensure fruit setting. At 50 d after flower induction (DAFI), the fruits were bagged until the harvesting stage to avoid disease and pest attacks. For mango accessions situated in distant places (Guimaras and Zambales), the fruits were acquired from the owners/collaborators through courier services or through actual site visit/harvesting.

Mass Rearing of Oriental Fruit FlyDropped and infested mango fruits were collected from the field and brought to the laboratory for the emergence of oriental fruit flies. The infested fruits were placed in a plastic box containing 2-cm-thick coir dust medium for pupation and then covered with mesh cloth. Adult insects, which emerged at approximately 3–4 wk, were transferred to a rearing cage. The insects were fed with water and an artificial diet (mixture of yeast extract and sugar in a 1:3 ratio). Mating takes place 12–15 d after emergence (varies with the temperature), then a banana fruit was placed inside the rearing cage as an egg-laying host (Jayanthi and Verghese 2002). Gravid females were allowed to lay eggs in a banana for 2 d, then the fruit was placed in a plastic box containing 2 cm thick of coir dust medium for pupation, as described earlier. The second-generation insects and succeeding progenies were maintained and used for resistance screenings. The conditions in the rearing laboratory were 26 °C with 55% relative humidity.

Identity Validation of Reared Fruit FliesThe identity of the reared adult insects was verified as B. dorsalis using the six morphological key characteristics described by Iwahashi (1999). The identity of the reared insects was further validated via DNA sequencing of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene from five randomly sampled insects. DNA was extracted through a modified Dellaporta extraction

Sison et al.: Resistance Screening of Mango Against Oriental Fruit Fly

Philippine Journal of ScienceVol. 149 No. 4, December 2020

1176

method (Dellaporta et al. 1983) and cox1 was amplified via PCR (polymerase chain reaction) using the forward (5’ CGGTCACCCAGGAGCTTTAA 3’) and reverse (5’ ACTGTGAATATGTGATGAGCTCA 3’) primers designed from GenBank accession DQ995281.1. The PCR components (25 μL reaction) include 2.5 μL 10X Thermopol Buffer, 0.75 μL 50 mM MgCl2, 0.5 μL 10

mM dNTP’s, 1.25 μL each of 10 μM forward and reverse primers, 0.1 μL (0.5 U) Taq DNA Polymerase (Invitrogen, USA), 2 μL template DNA, and MilliQ water up to 25 μL. The settings include initial denaturation (2 min, 94 °C); 35 cycles of denaturation (45 s, 94 °C), annealing (30 s, 60 °C), and elongation (90 s, 72 °C); then final extension (10 min, 72 °C) and hold/finish (infinite time,

Table 1. The mango accessions used in this study.

Variety Accession no. Variety – Tree no. Source

“Carabao’” variety

12-012 Carabao – Tree 1 Tiaong, Quezon

12-013 Carabao – Tree 1 IPB-UPLB, Laguna









17-002 Carabao – Tree 2327 Bay, Laguna









12-173 Carabao – Tree R2T10 IPB-UPLB, Laguna





16-008 GES 73 Jordan, Guimaras




Other mango varieties

18-001 Golden Pico Jordan, Guimaras

12-186 Tommy Atkins IPB-UPLB, Laguna

12-103 Farrales Castillejos, Zambales

12-182 Unknown green variety Bay, Laguna

12-070 Unknown red variety IPB-UPLB, Laguna



1177

4 °C). PCR products were resolved in 1.5% agarose gel in 0.5x TAE buffer and viewed using GelDoc™ XR+ gel documentation system (Bio-Rad Laboratories). Amplified cox1 was sequenced for the forward and reverse strands (First BASE Laboratories, Malaysia) and sequences were analyzed using Geneious Prime® (v.2019.0.4) and BLASTn for species confirmation.

Evaluation of Mango Accessions for Resistance Against Oriental Fruit FlyMango fruits were harvested generally at 120 DAFI (or during the harvestable stage depending on the variety) and were brought to the Entomology Laboratory, IPB-UPLB to evaluate their potential resistance to oriental fruit fly through host “choice” test. The fruits were left to ripe for a week prior to resistance screening. The screen cage evaluation technique of Rossetto et al. (2006), with some modifications, was adopted in the screening trials. The experiment was performed with three replications containing three fruit samples per replication. Ripened fruits from different mango accessions were enclosed in a screen cage and each replicate was arranged in random for “choice” test. Gravid female fruit flies were released inside the screen cage (at 1:1 fruit to insect ratio) and allowed to choose and oviposit on their preferred fruits. The number of insect visits was recorded every 0.5, 1, 2, and 4 h. Afterward, each replicate was transferred to a labeled plastic tray containing 2 cm coir dust for pupation, then covered with mesh cloth. The trays were kept until adult offspring emerged. The number of adults emerged was recorded from each replicate and the average was taken per accession. The resistance reaction of the accessions was rated accordingly using a rating scale derived and modified from Rossetto et al. (2006) based on successful adult emergence: no damage (ND; zero adult emergence; highest degree of resistance), least susceptible (LS; 1–10 adults emerged); moderately susceptible (MS; 11–20 adults emerged); and highly susceptible (HS; more than 20 adults emerged). A susceptible commercial “Carabao” variety was used as susceptible control during the trials.

Analysis of Fruit Physico-biochemical PropertiesThe various physico-biochemical properties of mango accessions were analyzed in collaboration with the Postharvest Horticulture Training and Research Center (PHTRC) of UPLB (for peel firmness test) and Analytical Services Laboratory (ASL) of IPB-UPLB (for fruit biochemical analysis). For the peel firmness test, a penetrometer (Aikoh model 2256 digital force gauge penetrometer) was applied on two opposite sides of the fruit with three readings each (upper, middle, and lower parts of the fruit). The peel firmness test was done using three sample fruits per accession, then the average was taken. For the fruit biochemical analysis, the following

parameters were analyzed from the peel and flesh of two fruit samples per accession, then the average was taken: proximate analysis (AOAC 1980) [total ash content, crude fiber, crude fat, total protein content (Nkonge and Balance 1982), and moisture content], fruit pH, total soluble solids (TSS) (AOAC Methods 932.12; 976.20; 983.17), total flavonoids (Ordonez et al. 2006) (with catechin as standard); total phenolics (with gallic acid as standard) (Velioglu et al. 1998); and total antioxidant activity (DPPH scavenging assay) (Choi et al. 2002). All reagents used were analytical grade while all spectrophotometric readings were carried out using Shimadzu UV-mini 1480 (Japan) spectrophotometer.

Data AnalysisAll data obtained were statistically analyzed using one-way analysis of variance (ANOVA) followed by pairwise comparison of means using Fisher’s least significant difference (LSD) test at P < 0.05 significance level. To study the relationship between the fruit physico-biochemical properties and evaluation data, a two-tailed correlation analysis was performed using Pearson’s test at P < 0.05 significance level. Data analyses were performed using Prism version 8.1.2 (GraphPad Software, Inc.).

RESULTS

Evaluation of Mango Accessions for Resistance Against Oriental Fruit FlyThe identity of fruit flies reared and used in the resistance screenings was confirmed as B. dorsalis as shown by 100% alignment of mitochondrial cox1 sequences to this species through BLASTn (top 5 hit: accessions MG689924.1, MG689877.1, MG689876.1, MG689873.1, MG689872.1). For the resistance screening, the mean insect visits are presented in Figure 1, while the mean adult emergence and resistance ratings are presented in Figure 2. In the repeated trials, four accessions (out of 33) consistently showed good resistance ratings, i.e. ND, LS, and MS (Figure 2). This includes accession 12-209 (“Carabao” – Tree 203) with resistance ratings of 1 ND, 1 LS, and 2 MS (Figure 2) and mean insect visits ranging from 1.17–3.25 throughout the trials (Figure 1); accession 12-127 (“Carabao” – Tree 30) with resistance ratings of 2 LS and 1 MS (Figure 2) and mean insect visits ranging from 1–3.5 throughout the trials (Figure 1); and accessions 12-103 (“Farrales”) and 16-010 (‘GES 77’) with 3 and 2 ND resistance ratings, respectively (Figure 2). The mean insect visits for these two accessions throughout the trials ranged from 2.42–3.25 and 2.75–3.5, respectively (Figure 1). On the other hand, two accessions consistently scored as HS for two consecutive trials (Figure 2). These



1178

Figure 1. Mean number of insect visits in mango accessions used for resistance screening against oriental fruit fly for four years (Y1–Y4). Red rectangles denote accessions with consistent resistance reaction (refer to Figure 2). ANOVA: F = 6.332; df = 12, 39; P < 0.0001 (Y1); F = 9.511; df = 15, 48; P < 0.0001 (Y2); F = 4.551; df = 18, 57; P < 0.0001 (Y3); F = 3.644; df = 12, 39; P = 0.0011 (Y4). Different letters indicate significant differences between accessions at α = 5% level of Fisher’s LSD test. Error bars represent standard errors of the mean.



1179

accessions were 12-013 (“Carabao” – Tree 1) and 12-175 (“Carabao” – Tree 2) with mean insect visits ranging from 2.67–3.5 and 5.5–6, respectively (Figure 1).

Correlation of Fruit Physico-biochemical Properties and Evaluation DataUpon correlation of the overall evaluation data to fruit physico-biochemical properties, flesh flavonoids showed moderate and significant negative correlation to insect visit (r = –0.40; P < 0.05) (Table 2). Meanwhile, significant correlations to adult emergence were observed in the physico-biochemical properties of the peel: weak to moderate correlations were observed in peel moisture (r = –0.33; P < 0.05), peel protein (r = 0.30; P < 0.05), peel fiber (r = –0.38; P < 0.05), peel ash (r = 0.47; P < 0.01), and peel phenolics (r = 0.41; P < 0.01), while the highest correlation was recorded in peel firmness (r = –0.54; P < 0.01) (Table 2).

Comparison of the Most Resistant and Most Susceptible AccessionsThe resistant accessions generally showed firmer peel (ranging from 0.63–2.33 kg-force) than the susceptible accessions (ranging from 0.32–0.52 kg-force) although the resistant accessions 12-127 and 12-209 (“Carabao” – Tree 30 and 203, respectively) were only slightly firmer and not significantly different to the susceptible accessions (Table 3). Higher flesh flavonoids were generally observed in resistant accessions (ranging from 108.05–658.7 mg catechin equivalent/ 100 g dry weight) than on susceptible accessions (ranging from 59.25–73.6 mg catechin equivalent/ 100 g dry weight) (Table 3). Also, higher flesh phenolics were observed in resistant accessions (ranging from 1,958.28–2,615.02 mg gallic acid equivalent/ 100 g dry weight) than on susceptible accessions (ranging from 1,598.55–1,781.2 mg gallic acid equivalent/ 100 g dry weight) although the resistant accession 12-103 (“Farrales”) appeared to be only slightly higher and not significantly different

Figure 2. Mean number of adult emergences in mango accessions used for resistance screening against oriental fruit fly for four years (Y1–Y4). Red rectangles denote accessions with consistent resistance reaction. Accession 12-209 (“Carabao” – Tree 203) showed resistance ratings of 2 MS, 1 ND, and 1 LS; 12-127 (“Carabao” – Tree 30) showed 2 LS and 1 MS, while 12-103 (Farrales) and 16-010 (GES 77) showed 3 and 2 ND ratings, respectively. ANOVA: F = 7.716; df = 12, 26; P < 0.0001 (Y1); F = 3.258; df = 15, 32; P = 0.0025 (Y2); F = 2.626; df = 18, 38; P = 0.0061 (Y3); F = 17.71; df = 12, 26; P < 0.0001 (Y4). Different letters indicate significant differences between accessions at α = 5% level of Fisher’s LSD test. Error bars represent standard errors of the mean. ND – no damage, LS – least susceptible, MS – moderately susceptible, HS – highly susceptible.



1180

Tabl

e 2.

Cor

rela

tion

coef

ficie

nt (r

) bet

wee

n fru

it ph

ysic

o-bi

oche

mic

al p

rope

rties

and

eva

luat

ion

data

at α

= 5

% le

vel o

f Pea

rson

’s te

st.

IVA

EPF

irm

FMoi

stPM

oist

FFat

PFat

FPro

tPP

rot

FFib

PFib

FAsh

PAsh

FNFE

PNFE

pHT

SSFF

lav

PFla

vFP

hen

PPhe

nFR

SA

AE

0.05

PFir

m0.

04-0

.54

FMoi

st0.

21-0

.12

0.29

PMoi

st0.

25-0

.33

0.52

0.79

FFat

-0.1

5-0

.30

0.12

-0.3

2-0

.15

PFat

-0.0

70.

20-0

.25

-0.3

3-0

.37

0.43

FPro

t0.

040.

16-0

.10

-0.3

8-0

.28

0.10

-0.0

4

PPro

t0.

100.

30-0

.11

-0.1

5-0

.22

-0.1

5-0

.28

0.66

FFib

-0.1

30.

08-0

.22

-0.4

5-0

.42

0.12

0.17

0.24

0.10

PFib

-0.0

3-0

.38

0.35

0.22

0.15

0.33

-0.1

3-0

.18

0.01

-0.0

4

FAsh

0.03

0.24

-0.1

30.

110.

04-0

.01

0.05

0.08

0.06

-0.1

0-0

.23

PAsh

-0.0

60.

47-0

.56

-0.1

8-0

.43

0.03

0.23

0.34

0.33

0.08

-0.2

30.

43

FNFE

0.05

-0.0

7-0

.28

-0.9

9-0

.75

0.60

0.41

0.60

0.17

0.17

-0.0

7-0

.38

-0.0

4

PNFE

0.07

0.28

-0.4

9-0

.85

-0.9

70.

530.

600.

580.

290.

380.

04-0

.16

0.26

0.83

pH0.

210.

030.

10-0

.03

0.03

0.28

0.46

-0.1

0-0

.03

0.08

0.25

-0.1

5-0

.15

-0.1

70.

11

TSS

-0.3

50.

07-0

.21

-0.7

9-0

.72

0.27

0.16

0.37

0.15

0.38

-0.1

4-0

.12

0.12

0.79

0.76

-0.0

6

FFla

v-0

.40

0.04

-0.3

1-0

.13

-0.3

0-0

.26

-0.2

9-0

.02

-0.0

20.

09-0

.29

0.09

0.19

0.09

0.31

-0.7

30.

29

PFla

v0.

28-0

.03

0.14

0.26

0.24

-0.0

3-0

.57

0.16

0.51

-0.2

40.

280.

120.

09-0

.18

-0.1

4-0

.02

-0.2

2-0

.14

FPhe

n0.

130.

20-0

.14

0.10

0.07

-0.2

4-0

.29

0.12

0.42

-0.0

7-0

.09

0.24

0.19

-0.2

4-0

.25

-0.2

4-0

.07

0.11

0.44

PPhe

n-0

.15

0.41

-0.4

50.

02-0

.18

0.05

0.23

-0.1

6-0

.03

-0.0

5-0

.22

0.28

0.50

-0.4

3-0

.05

-0.0

6-0

.01

0.33

0.12

0.34

FRSA

0.07

0.27

-0.6

30.

03-0

.29

0.19

0.35

0.18

0.17

0.25

-0.1

90.

200.

66-0

.13

0.20

0.06

-0.0

30.

36-0

.07

0.39

0.59

PRSA

0.17

0.34

-0.2

0-0

.15

-0.1

6-0

.15

0.22

-0.1

4-0

.05

-0.1

6-0

.50

0.12

0.35

0.10

-0.2

00.

340.

02-0

.17

0.24

0.30

0.61

0.20

IV –

inse

ct v

isit,

AE

– ad

ult e

mer

genc

e, P

Firm

– p

eel f

irmne

ss, F

/PM

oist

– fl

esh/

peel

moi

stur

e, F

/PFa

t – fl

esh/

peel

fat,

F/PP

rot –

fles

h/pe

el p

rote

in, F

/PFi

b –

flesh

/pee

l fib

er, F

/PA

sh –

fles

h/pe

el a

sh, F

/PN

FE –

fles

h/pe

el n

itrog

en-f

ree

extra

ct, T

SS –

tota

l sol

uble

solid

s, F/

PFla

v –

flesh

/pee

l tot

al fl

avon

oids

, F/P

Phen

– fl

esh/

peel

tota

l phe

nolic

s, F/

PRSA

– fl

esh/

peel

rela

tive

scav

engi

ng a

ctiv

ity

sign

ifica

nt a

t P <

0.0

1 (tw

o-ta

iled)

sign

ifica

nt a

t P <

0.0

5 (tw

o-ta

iled)



1181

Tabl

e 3.

Mea

n (±

SEM

) pee

l firm

ness

, pH

, TSS

, tot

al fl

avon

oid

cont

ent (

flesh

and

pee

l), a

nd to

tal p

heno

lic c

onte

nt (f

lesh

and

pee

l) of

the

mos

t res

istan

t and

mos

t sus

cept

ible

acc

essio

ns.

Acc

essi

on n

o.Va

riet

y –

Tree

no.

Peel

firm

ness

(kg-

forc

e)pH

% T

SSTo

tal fl

avon

oid

cont

ent

(mg

cate

chin

equ

ival

ent/

100g

dry

wei

ght)

Tota

l phe

nolic

con

tent

(mg

galli

c ac

id e

quiv

alen

t/ 10

0g d

ry w

eigh

t)

Fles

hPe

elFl

esh

Peel

R

12-1

27C

arab

ao –

Tre

e 30

0.75

± 0

.03a

4.70

± 0

b14

.75

± 0.

45ab

189.

24 ±

46.

42b

1,48

8.91

± 1

7.86

c2,

615.

02 ±

113

.7b

18,4

94.6

6 ±

151.

6c

16-0

10G

ES 7

71.

73 ±

0.1

6b6.

35 ±

0.0

5d15

.40

± 0.

4abc

108.

05 ±

3.3

6ab

1,61

0.00

± 0

d2,

500.

22 ±

0b

11,5

00.0

0 ±

0a

12-2

09C

arab

ao –

Tre

e 20

30.

63 ±

0.0

3a4.

20 ±

0a

19.0

0 ±

1d65

8.70

± 3

5c68

0.98

± 2

5.46

b2,

445.

02 ±

226

.4b

22,4

23.9

6 ±

79.2

4d

12-1

03Fa

rral

es2.

33 ±

0.6

8b6.

05 ±

0.0

5c18

.15

± 0.

15cd

182.

10 ±

17.

86b

467.

74 ±

17.

85a

1,95

8.28

± 7

9.24

a11

,900

.25

± 37

9a

S12

-013

Car

abao

– T

ree

10.

32 ±

0.0

3a7.

20 ±

0.1

e17

.00

± 2a

bcd

73.6

0 ±

4.1a

515.

15 ±

4.0

5a1,

781.

20 ±

11.

4a14

,272

.65

± 22

8.4b

12-1

75C

arab

ao –

Tre

e 2

0.52

± 0

.02a

7.05

± 0

.05e

12.5

0 ±

0.5a

59.2

5 ±

2.05

a66

6.40

± 4

.1b

1,59

8.55

± 3

4.25

a18

,383

.20

± 22

8.4c

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to the susceptible accessions (Table 3). In terms of pH, the resistant accessions generally have acidic pH levels (4.20–6.35) while the susceptible accessions were within the neutral pH level (7.05–7.20) (Table 3). Meanwhile, TSS, peel flavonoids, and peel phenolics showed varying results with no trend between the contrasting genotypes (resistant vs. susceptible) (Table 3).

DISCUSSIONOne of the major insect pests attacking mango in many parts of the world is the oriental fruit fly. Although the pest itself has been widely studied and various pest management and control systems have been developed, no sources of resistance for the “Carabao” mango have been identified in the Philippines until this study. In the span of four-year resistance trials of mango against oriental fruit fly, four trials have been performed (one trial per year) through “choice” tests in the screen cage experiment. “Choice” test has the advantage of determining insect preference and host-suitability while the screen cage set-up provided a controlled environment to confirm the resistance reaction of a variety/accession, as demonstrated by Rossetto et al. (2006). Using 33 different mango accessions/varieties, the four-year data on insect visit and adult emergence (Figures 1 and 2, respectively) showed different levels of resistance to oriental fruit fly in the screened accessions, which can be attributed to the broad genetic variability of the collection. However, not all of the accessions listed in this study have been tested for four times or in a yearly basis because of the seasonal availability of fruits which is affected by many factors (e.g. unsuccessful flower induction, the occurrence of tropical typhoons, insufficient daylight length and temperature for flowering, etc.) and the capacity of mango owners/collaborators from different places to provide samples during the conduct of the resistance trials. Hence, the pooled or combined average per accession throughout the resistance trials was not computed. The “choice” tests were instead individually analyzed and rated according to the scoring system derived and modified from Rossetto et al. (2006), based on the number of successful adult emergence. With this, four accessions were observed to exhibit promising resistance ratings in repeated trials ranging from ND to MS and were, therefore, considered as resistant accessions. These were accessions 12-209 (“Carabao” – Tree 203); 12-127 (“Carabao” – Tree 30), 12-103 (“Farrales”), and 16-010 (‘GES 77’) (Figure 2). The insect visits in these accessions, which broadly represents ovipositional preference (Rossetto et al. 2006), were also relatively low compared to other accessions in their batch during the trials (Figure 1). For the accessions 12-103 (“Farrales”) and 16-010 (‘GES 77’), although



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insect visits were recorded (Figure 1), no successful adult emergence was noted throughout the trials (Figure 2). The accessions 12-209 (“Carabao” – Tree 203), 12-127 (“Carabao” – Tree 30), and 16-010 ("GES 77") – being “Carabao” mango accessions – are characterized by yellowish pericarp typical to ripened fruits of this variety (Figure 3a). The accession 16-010, namely GES 77, is an official NSIC-registered “Carabao” mango variety from Guimaras – an island province that is considered as the mango capital of the Philippines. Meanwhile, the accession 12-103 (“Farrales”) is a non-“Carabao” mango landrace, which is characterized by pink-blushed pericarp (Figure 3b). Due to its distinctive characteristics apart from “Carabao” and other red/pink-blushed mango varieties, this accession is currently being considered by NSIC for potential release as a new mango variety in the Philippines. On the other hand, the most susceptible accessions were 12-013 (“Carabao” – Tree 1) and 12-175 (“Carabao” – Tree 2), which consistently scored as HS for two consecutive trials (Figure 2).

al. 2015). Flavonoids and isoflavonoids influence the behavior, growth, and development of insect pests, thereby protecting the plant against them (Simmonds 2003). An example of which is the ovipositional deterrence (reduced insect visit) and ovicidal action (reduced egg hatching) of the plant flavonoid quercetin against B. dorsalis, B. cucurbitae, and B. correcta fruit flies (Sharma and Sohal 2013, 2016; Jaleel et al. 2020). On the other hand, significant correlations to adult emergence were observed in the physico-biochemical properties of the peel, which may suggest the role of the fruit peel in the success/failure of insect reproduction (Table 2). Among the parameters in the fruit peel, peel firmness showed the highest negative correlation with adult emergence (r = –0.54; P < 0.01) (Table 2). This result suggests that firm fruit peel may have affected the ovipositional (egg-laying) capacity of fruit flies leading to reduced insect reproduction and adult emergence. Felicitas et al. (2012) observed that the resistant mango variety, which had the firmest peel, showed the lowest pupal recovery of B. invadens (syn. B. dorsalis) while the opposite was observed in susceptible variety with the least firm peel. Peel firmness and not peel thickness was considered in this study since the resistance of mango to fruit fly has been reported to be reliably correlated to the former and not to the latter (Joel 1980; Rattanapun 2009; Rattanapun et al. 2009). Peel firmness is also a major non-preference factor of B. dorsalis to the near-threatened Mangifera camptosperma Pierre (Jayanthi et al. 2020). The weak to moderate significant correlations of peel physico-biochemical properties (i.e. peel moisture, peel fiber, peel ash, and peel phenolics) to adult emergence may pertain to their influence in maintaining (or reducing) peel firmness, as shown also by the significant correlation of these parameters with peel firmness (Table 2). Rossetto et al. (2006) observed that when eggs of Mediterranean fruit fly were inserted in the flesh of a resistant mango variety, a high rate of adult emergence was observed, which suggests that fruit peel could be responsible for fruit resistance. This may not be true to all mango varieties; however, this could explain why only peel physico-biochemical properties were correlated in the overall analysis of adult emergence using diverse mango accessions/varieties.

Verghese et al. (2012) and Choudhary et al. (2018) compared the fruit phenolic content (peel and flesh) between resistant and susceptible accessions and demonstrated that this biochemical component also plays a defensive role in mango against B. dorsalis. In addition, high fruit phenolic content and high fruit acidity are correlated to reduced insect infestation and impaired larval development of Bactrocera species in various fruit-bearing crops (Rattanapun 2009; Rattanapun et al. 2009; Gogi et al. 2010; Felicitas et al. 2012; Haldhar et al. 2013, 2015; Damodaram et al. 2015; Choudhary

Figure 3. Typical “Carabao” mango variety with yellowish pericarp upon ripening (A) and “Farrales” accession with pink-blushed pericarp (B).

The relationship of mango evaluation data to various fruit physico-biochemical properties was inferred through correlation analysis (Table 2) to determine any inhibitory fruit properties against B. dorsalis. Overall correlation analysis suggests that flesh flavonoid content moderately affected insect visit and broadly, ovipositional preference (r = –0.40; P < 0.05) (Table 2). The deterrent and negative effects of fruit flavonoids have been demonstrated in previous studies on mango against B. dorsalis (Damodaram et al. 2015; Choudhary et al. 2018) and on watermelon against B. cucurbitae (Haldhar et



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et al. 2018). However, in this study, peel phenolics in the “Carabao” mango appear to affect peel firmness as shown by its significant and moderate negative correlation with peel firmness (r = –0.45; P < 0.01) (Table 2). This may be related to the natural increase of peel phenolics associated with a decrease in peel firmness in mature/ripe “Carabao” mango fruits used in the trials (Jha et al. 2013; Yaptenco et al. 2013; Ajila and Prasada Rao 2013; Shamim et al. 2015). In the overall correlation analysis, no clear correlation of evaluation data has been established to fruit phenolics and acidity (e.g. pH), probably due to the lack of direct relationship of these parameters to the evaluation data using diverse mango accessions/varieties with broad variability for oriental fruit fly-resistance. An example is the accession 12-103 (“Farrales”), which exhibited high resistance against oriental fruit fly despite having relatively low flesh phenolics content (Table 3; Figure 2). The other three resistant accessions, on the other hand, exhibited relatively higher flesh phenolic content, which is significantly different from the HS accessions 12-013 (“Carabao” – Tree 1) and 12-175 (“Carabao” – Tree 2) (Table 3). Comparison of the contrasting genotypes, i.e. most resistant and most susceptible accessions showed that the former generally have higher flesh phenolics and higher flesh flavonoids than the latter (Table 3). Conversely, the resistant accessions appeared to have an acidic pH level while the HS accessions were within the neutral pH level (Table 3). The pH/acidity level is known to be largely influenced by organic acids in mango (Liu et al. 2013; Oliveira et al. 2016). These results may mean that aside from the negative effects of flesh flavonoids and peel firmness to the oriental fruit fly, the flesh phenolics and pH/acidity may also play a defensive role in “Carabao” mango, as reported in other mango varieties and other crops upon a comparison between contrasting genotypes (resistant vs. susceptible) (Gogi et al. 2010; Felicitas et al. 2012; Verghese et al. 2012; Haldhar et al. 2013, 2015; Choudhary et al. 2018). Moreover, most of the chemical reactions in the insect gut, which involve digestive enzymes, are known to be influenced by pH and may affect larval survival (Cohen 2004; Chaudhury and Skoda 2009). Aside from the studied fruit biochemical components, Rashmi et al. (2017) also reported that tannins in mango peel, especially during increased fruit maturity stage, were significantly higher in the resistant varieties than in susceptible varieties, which suggests its important role in deterring or preventing fruit fly damage in mango.

The described fruit physico-biochemical characteristics in the resistant accessions may have hampered the ovipositional capacity of the female fruit flies, incapacitated the fruits to instigate oviposition in gravid flies, or caused insect non-preference and impaired development of larvae. On the contrary, the opposite characteristics

observed in 12-013 (“Carabao” – Tree 1) and 12-175 (“Carabao” – Tree 2) may have resulted in their consistent high susceptibility to oriental fruit flies. The role of TSS, as a function of fruit sugars (Shamim et al. 2015), in the susceptibility/resistance of mango is unresolved in the current study, although it has been reported that higher TSS/ fruit sugars may lead to high insect preference and development (Rattanapun 2009; Rattanapun et al. 2009; Choudhary et al. 2018). TSS showed a significant and strong negative correlation to fruit moisture (flesh and peel) and strong positive correlation to fruit nitrogen-free extract (NFE) (flesh and peel), while it showed a weak but significant positive correlation to flesh protein, fiber, and flavonoids (Table 2).

In summary, based on the evaluation trials, four mango accessions were regarded as resistant against oriental fruit fly. Since the “Carabao” mangoes currently available in the local and international markets are generally susceptible to this insect pest, the accessions characterized in this study can be used as stop-gap varieties or as parental for the improvement of “Carabao” mango with resistance to the oriental fruit fly. Moreover, insights regarding the mechanisms of insect-resistance in mango were provided in this study through correlation analysis of fruit physico-biochemical properties.

ACKNOWLEDGMENTSThis study was funded by the Department of Science and Technology – Philippine Council for Agriculture, Aquatic, and Natural Resources Research and Development under the project entitled “Characterization of “Carabao” and other mango varieties with resistance to fruit fly and anthracnose” (Fund Code: N90672). The authors would like to thank the following collaborating farm owners and agencies that were instrumental in the identification and provision of mango accessions: National Seed Foundation, National Plant Genetic Resources Laboratory, and Fruit and Ornamental Crops Section of IPB-UPLB; BPI-GNCRDPSC; Adel Rapadas (Rapadas Farm, Tiaong, Quezon) and Perlita Farrales (Castillejos, Zambales); to Elenita Castillo and Teresita Maligalig of the ASL-IPB-UPLB for the assistance in fruit biochemical analysis and PHTRC-UPLB for the assistance in peel firmness test; to Raquel Javier, Kristine Joy de Castro, Regina Herrera, Imelda Vibal, Rizalina Tiongco, Cris Urriza, and Enrique Ramos for the assistance in insect mass-rearing, harvesting of fruits, crop management, and data gathering; to the mango program teammates: Scientist Lolita Valencia (former Mango Program Leader), Carolyn Alcasid, and Gino Hernandez for the identification and provision of mango accessions, and technical assistance.



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STATEMENT ON CONFLICT OF INTERESTThe authors declare that they have no conflict of interest.

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resistance screening and influence of fruit physico ......mango industry is the source of livelihood...

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