role of constitutive and induced defences in the ... · nimal adikaram, ganga sinniah, chathurika...
TRANSCRIPT
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.