Role of constitutive and induceddefences in the resistance of unripe

mangoes to Colletotrichumgloeosporioidesgloeosporioides

Nimal Adikaram, Ganga Sinniah, Chathurika

Karunanayake and Charmalie AbayasekaraDepartment of Botany

University of PeradeniyaSri Lanka

• Anthracnose (Colletotrichum gloeosporioides) and the stem-end rot (Lasiodiplodia theobromae) are two diseases responsible for most of the postharvest losses in ripe mango.

• Immature mangoes are resistant to both C. gloeosporioides and the stem-end rot (SER) pathogen, Lasiodiplodia theobromae.

• However, the resistance declines during • However, the resistance declines during ripening and anthracnose and stem-end rots occur.

Mango fruit defence

• Immature mango fruits have evolved aformidable defence system comprisingconstitutive,- antifungal resorcinols in the latex and- antifungal resorcinols in the latex andperhaps the tissue too.

- antifungal gallotannins in the peel tissue.- chitinase in the latex (distributed in thepeel in a fine net work of minute canals).

Substituted antifungal resorcinols

OH OH

(CH2)

CH

CH

(CH2

OH OH

(CH2)14

CH3

11

)3 5- pentadecyl resorcinolCH3

5- (12- cis-heptadecenyl) resorcinol

Cojocaru et al. (1986)

HPLC showed two additional peeks – one with general features of resorcinols and appears a resorcinol and the other a resorcinol derivative, Sri Lankan cultivars.

Dichloromethane extract

Resorcinol content in unripe mango fruit peel

______________________________________________________________________

Resorcinol level (µg/g FR)• _______________________________________________• Cultivar 5-(12- cis-heptadecenyl) 5-pentadecyl AR19 AR21

• resorcinol resorcinol• ______________________________________________________________________

• ‘Gira’ (R)* 91.5 9.50 15.6 41.23• ‘Karuthacolomban’ (R) 59.9 27.07 532.1 43.16• ‘Rata’ (R) 91.8 26.80 2306.3 79.04• ‘Kohu’ (M) 38.1 8.20 911.4 139.20• ‘Petti’ (S) 20.7 8.60 47.47 15.30• ‘Willard’ (S) 34.4 58.30 14.8 25.70

_____________________________________________________________________

• *Resistant (R) to anthracnose• Moderately susceptible (M) • Susceptible (S)

Resorcinol levels in fruit peel at different stages of ripening in a resistant cultivar (`KC’)

0

20

40

60

80

100

1 3 6 9

Days after harvest

Reso

rcin

ol

am

ou

nt

(m

icro

gra

ms/g

fre

sh

wt.

)

Days after harvest

KC 5-(12 cic heptadecenyl) resorcinol KC 5-pentadecyl resorcinol KC AR21

OR 50 %CLR Break 20 %% decline,

5-(12-cis-heptadecenyl) resorcinol 85%

5-pentadecyl resorcinol 25%

AR 21by approximately 80 %

Resorcinol levels in the peel of fruit of susceptible cultivar

60

80

100

Res

orc

ino

l co

nce

ntr

atio

n

Mic

rog

ram

s/ g

fre

sh w

t.

0

20

40

60

unripe ripe

Cultivar 'Willard '

Res

orc

ino

l co

nce

ntr

atio

n

Mic

rog

ram

s/ g

fre

sh w

t.

5-pentadecyl resorcinol 5-(12 cic heptadecenyl) resorcinol AR21

A mixture of several related Gallotannins

Aqueous methanol extract

Prominent inhibition at Rf 0.00

(Karunanayake et al., 2011)

Cladosporium sp. Colletotrichum gloeosporioides Lasiodiplodia theobromae

MeOH CH2Cl2 MeOH CH2Cl2MeOH CH2Cl2

Gallotannins inhibit C. gloeosporioides and not L. theobromae.

050

100150200250300350400450

Gira KC Rata Kohu Petti Willard

Cultivars

Inh

ibit

ion

are

a o

n T

LC

(mm

2)

Gallotannin activity in unripe peel extracts

Gallotannin activity in ripe peel extracts

54% decline in activity in susceptible cv. ‘Petti’ during ripening.

21% decline in the resistant cv. ‘KC’

‘KC’>‘Gira’>’Rata’>’Kohu’>’Petti’>’Willard’

Gallotannin antifungal activity in different mango cultivars

200 400

Gallotannins content and disease level are negatively correlated (62%)

0

20

40

60

80

100

120

140

160

180

200

1 3 5 7 9

Days

Lesio

n a

rea/

mm

2

0

50

100

150

200

250

300

350

400

Inh

ibit

ion

are

a o

n T

LC

/ m

m2

Lesion area Inhibition area

Mango latex

• When removed from the fruit, mango latex separates in to an oily phase and an aqueous phase. phase.

• The oily phase contains antifungal resorcinols & the aqueous phase chitinase.

Aqueous phase

Oily phase

Chitinase activity in the aqueous phase of mango latex

2 min 180 min

Lasiodiplodia theobromae

Chitinase activity in the aqueous phase of latex in selected mango cultivars

c d ef

a b

0.40.60.8

11.21.41.61.8

2

Act

ivity

in u

nits

/10

mic

ro li

tre

s

g h

(a) ‘Gira’ (b) ‘Karuthacolomban’ (c) ‘Kohu’,(d) ‘Petti’ (e) ‘Rata’ (f) ‘Willard’ (g) Papaya latex

(+positive control) (h) Phosphate buffer (- control)

00.20.4

‘Gira

’‘K

C’

‘Rata

’

‘Koh

u’

‘Pet

ti’

‘Willa

rd’

Act

ivity

in u

nits

/10

mic

ro li

tre

s

‘Rata’ & ‘Kohu’ >> ‘Willard’ & ‘Petti’ >> ‘Gira’ & ‘KC’

Cultivar Resistance/Susc-eptibility

Relative gallotannincontent

Latex chitinaseactivity

(units/l)

Volume of latex

Anthracnose SER

Ambalavi MR S -- 0.083±0.025

Dilpasan S R -- 0.087±0.010

Gira R S 355/207*(42%) 0.094±0.004 3.1

KC R S 295/231 (21.9%) 0.029±0.012 8.36±0.49

Kohu MR R 271/171 (36.9%) 0.123±0.011 3.25Kohu MR R 0.123±0.011 3.25

Malvana R R -- 0.118±0.032

Neelam R R -- 0.139±0.031

Petti S R 321/146 (54.3%) 0.189±0.051 3.4

Rata MR R 318/178 (44%) 0.253±0.040 5.16±0.46

Seylum MR R -- 0.039±0.019

Willard S R 148/100 (33%) 0.089+0.046 4.11+0.39

cultivar Volume, ml

Oil phase % Oil phase Interiediate Aqueous % Aqueousphase phase

Total volume

‘KC’ (R)

1.5±0.15 17.8±1.1 0.53±0.24 6.33±0.33 75.9±3.6 8.36±0.49

‘Rata’ (MR)

1.06±0.14 20.4±0.95 0.4±0.25 3.7±0.1 72.4±4.7 5.16±0.46

‘Willard’(S)

0.43±0.03 10.5±0.17 0.16±0.11 3.5±0.24 85.8±2.1 4.11±0.39

Volumes of latex

(S)

‘Kohu’(MR)

0.4 12.3 0.05 2.8 86.15 3.25

‘Petti’(S)

0.3 8.8 0.1 3.0 88.2 3.4

‘Gira’(R)

0.5 16.13% 0.1 2.5 80.6 3.1

The % of the oil phase is correlated with the amount of resorcinols (Hassan, 2006). The % of the oil phase was greater in more resistant cultivars

Anthracnose development in fruits in which latex was retained

0

50

100

150

200

250

300

350

1 2 3 4 5 6 7 8 9 10

Days after inoculation

Lesio

n a

rea (

mm

2) ‘Willard’ Anthracnose was significantly (P<0.05) less in latex-retained fruits of the susceptible cv.

Days after inoculation

Fruits from w hich latex w as drained Fruits from w hich latex w as not drained

020

4060

80100

120140

160180

1 2 3 4 5 6 7 8 9 10 11

Days after inoculation

Les

ion

are

a (m

m2)

Fruits from w hich latex w as drained Fruits from w hich latex w as not drained

‘Karuthacolomban’ Anthracnose was less in fruits from which latex was not drained, but not significant (resistant cv.)

Cultivar Chitinase activity, units/g FWDay 1 Day 3 Day 6

`Willard’ Latex drained Control

0.32 +0.16 0.26 + 0.03 --0.48 +0.06 0.30 + 0.03 --

`KC’ `KC’ Latex drained Control

0.75 0.62 + 0.5 0.62 + 0.53 0.58 0.58 + 0.5 0.58 + 0.30

Induced defence responses

• Mango peel tissue responds to C. gloeos-porioides by producing Reactive Oxygen Species (O2

-) & H2O2, increased peroxidase and chitinase activity, increased phenols during early hours.during early hours.

• No phytoalexins induced.

Germination and appressoria formation by C. gloeosporioides on unripe mango fruit

GT

NA

MAS

L

PP

a b

BCA

IPA

BC

a b

• Germination 3h after inoculation

• Appressoriaformation 6 h

• Melanization of appressoria 9-12 h

• Infection hyphae24 h

9 h 18 h

48 h

O2- generation in epidermal cells of mango peel

following inoculation with C. gloeosporioides

a b

12 h 24 h

Karuthacolamban’

Epidermal peels stained with 0.1% Nitroblue tetrazolium (NBT)

c dWillard’

H2O2 generation in epidermals cells following inoculation with C. gloeosporioides

a b

First observed 9-12 h after inoculation; peels were stained with diaminobenzidine (DAB)

a

Autofluorescence and cell browning12 h 24 h 48 h

Autofluorescence

Cell browning Accumulation of

phenols Cell death HR??

Chitinase activity in the peel of mango cultivar ‘Karuthacolamban’ and ‘Willard’ following inoculation with

C. gloeosporioides.

0.8

1.2

1.6

2

Ch

itin

ase

acti

vity

(U

)

65.4

71.269.7

59.4

65.4

0

0.4

0.8

0 3 6 9 12 15 18 21 24

Time after inoculation (h)

Ch

itin

ase

acti

vity

(U

)

Karuthacolamban Willard

33.4

45.0

34.2

44.5

Infected Healthy

Induced isozymes 59.4 & 44.5 kDa

20

40

60

80

100

Intr

eact

ion

sit

e (%

)

0

9 12 15 18 21 24

Time after inoculation (h)

Resistant (KC) Susceptible (Willard)

Interaction site (%): Percentage of appressoria showing blue stainingin the target cells/surrounding in a field of vision.

Differentially expressed genes in the interaction between mango fruits and C. gloeosporioides

Expressed bands‘Karutha

colamban’‘Willard’

Differentially Differentially

expressed bands6 1

Up regulated 2 2

Down regulated 3 7

Conclusions

• Resistance of mango is due to a combination of the constitutive defences - antifungal resorcinols, gallotannins and the enzyme chitinase.

• In addition, some defence responses appear to be induced when the fruit is challanged with C.gloeosporioides.C.gloeosporioides.

• Resistant cultivars have higher levels of one or more of the constitutive defences, and also induce higher levels new defences.

• Latex plays a defensive role. Fruit resistance to anthracnose may be enhanced by latex management.

• Treatments that retain/enhance constitutive defences and induce new defences will help disease management.

• Some of these information (e.g. gallotannin • Some of these information (e.g. gallotannin content, chitinase activity) would be useful in selection of resistant cultivars.

Acknowledgement

• Australian Centre for International Agricultural Research (ACIAR)

H-T11A, OPA11

02

4 48

H-T11A, OPB6

0 24 48

H-T11C, OPD6

0 24 48

KU1

KDD4 KD1

KD2

KDD5

Differential display profile of RNA samples isolated at different times followinginoculation with C. gloeosporioides from unripe mango cultivars ‘Karuthacolamban’(K) using the anchor primer HT11M (Where H- AAGC, M can be either G, A, or C). incombination with random primers given at the top. DD- differentially displayed band,U-up regulated bands, D- down regulated bands.