Indian Phytopath. 65 (4) : 0-0 (2012)

Mango anthracnose caused by Colletotr ichumgloeosporioides Penz. originates from quiescent infectionsin the immature fruit. Resistance of unripe mango fruit to C.gloeosporioides has recently been shown to be due to acombined effect of preformed antifungal substances, 5-subsitituted resorcinols, gallotannins in the peel and chitinasein the latex (Karunanayake et al., 2011). Decline of resorcinolsand gallotannins (Adikaram et al., 2010) during ripeningmakes the r ipe fruits susceptible to anthracnose(Karunanayake et al., 2011). The variation in resorcinols(Hassan et al., 2007), gallotannin and chitinase (Adikaramet al., 2010) concentration explains the differential resistanceof mango cultivars to C. gloeosporioides (Karunanayake,2008). Quiescence of A. alternata in unripe mango wasimplicated to 5-substituted resorcinols in the fruit peel (Drobyet al. 1986).

Although the preexisting barriers such as the cuticle,cell wall and preformed antifungal compounds are sufficientto block infection by most pathogens, the inhibition ofpathogens that penetrate these barriers requires the timelyrecognition and activation of induced defenses. A plant thatcan recognize the pathogen and mount a rapid defenseresponse will prevent pathogen proliferation (a resistantplant), whereas a plant that is incapable of recognizing thepathogen and does not induce a defense response, does soonly slowly, and will be unsuccessful in preventing pathogenspread (a susceptible plant). When constitutive defences arebreached, plants induce new defence responses at structuralor biochemical level locally or systemically. Oxidative burst

is such an early defence response which occurs just afterpathogen recognition and signal transduction. Oxidative burstis characterized by rapid and transient generation of ReactiveOxygen Species (ROS), such as superoxide (O

2-), hydrogen

peroxide (H2O

2) or hydroxyl radicals (OH.) (Wojtaszek, 1997).

ROS are directly toxic to fungal pathogens and also activateother defenses such as HR (Lamb and Dixon, 1997).

HR is the collapse and death of cells immediatelysurrounding the site of infection. This self-sacrifice by theplant holds the pathogen and prevents the infection fromspreading throughout the plant (Morel and Dangle, 1997;Park, 2005). Accumulation of phytoalexins and synthesis ofpathogenisis-related proteins (PR proteins) and cell wallstrengthening proteins are also important induced defencemechanisms employed by plants against pathogens.

In mango, certain pre- and postharvest treatments havefrom time to time been repor ted to enhance defenseresponses. Bion®, Salicylic acid, phosphonates (Zainuri etal., 2001) or BTH treatment (Zhu et al., 2007) increasedalkylresorcinols, chitinase, β-1, 3-glucanase,polyphenoloxidase and PAL activity. Inoculation of the fleshof unripe mango fruits with A. alternata induced antifungalresorcinols (Droby et al., 1987). However, the exact natureof induced defense responses in mango fruit against fungalpathogens is not fully understood and remains unexplored.This paper reports for the first time firm evidence for induceddefense responses in unripe mango fruit when challengedwith C. gloeosporioides.

Differential defence responses expressed in mango (Mangiferaindica) cultivars resistant and susceptible to Colletotrichumgloeosporioides

G.D. SINNIAH, N.K.B. ADIKARAM* and C.L. ABAYASEKARADepartment of Botany, Faculty of Science, University of Peradeniya, Peradeniya 20400, Sri Lanka

ABSTRACT: This paper reports evidence that unripe mango fruit responds to Colletotrichum gloeosporioides, the anthracnosepathogen, by inducing several defence mechanisms. Localized generation of superoxide (O2

-) and hydrogen peroxide (H2O2) wasobserved within hours in the pathogen-challenged epidermal cells, as early defence responses. O2

- production was greater in thecultivar ‘Karutha Colomban’ more resistant to anthracnose than the susceptible ‘Willard’. O2

- production was also greater in C.gloeosporioides-inoculated unripe fruits than in inoculated ripe fruits of both cultivars. Autofluorescence was first observed inchallenged epidermal cells, 12 h after inoculation indicating hypersensitive response. Increased phenylalanine ammonialyase (PAL)activity and accumulation of lignin were observed in the infected peel. There was enhanced chitinase activity in the inoculated peeltissue probably resulting from induced chitinase isoforms with 59.4 kDa and 44.5 kDa molecular weight. In general, the early induceddefence responses were more prominent in the resistant than in the susceptible cultivar. In both cultivars C. gloeosporioidesbecomes quiescent, due to the presence of constitutive antifungal substances in the fruit peel which decline during ripening.Greater resistance induced in the resistant cultivar, `Karutha Colomban’ may be restricting C. gloeosporioides development in theripe fruits, reducing anthracnose disease. Outcome of this study sheds a new insight into the natural disease resistance of mangoagainst C. gloeosporioides.

Key words: Early defence responses, defence gene expression, ROS

RESEARCH ARTICLE

*Corresponding author: n.k.b.adikaram@gmail.com

2 Indian Phytopathology 65 (4) : 2-6 (2012)

MATERIALS AND METHODS

Plant materials

Fruits of mango cultivars, resistant (‘Karutha Colomban’) andsusceptible (‘Willard’) to anthracnose disease, at harvestingmaturity were used for the experiments, unless otherwisestated. The fruits were brought to the laboratory within 24 hafter harvest and blemish-free fruits of uniform size andmaturity were selected and used. The fruits were washedunder running tap water and air-dried at RT (26±2 oC). Fruitswere then wiped with cotton wool soaked in 70% ethanoland allowed to air-dry before they were used for experiments.

Pathogen

C. gloeosporioides isolated from anthracnose lesions in fruitsof cultivar, ‘Karutha Colomban’ and ‘Willard’, was used forinoculation of fruits of respective cultivars. For isolation,diseased tissue segments (5X5 mm2) were cut and surfacesterilized in 1% NaOCl for 3 min and transferred asepticallyto potato dextrose agar (PDA). The plates were incubated atRT and the pathogen was sub-cultured and maintained onPDA at RT.

Fruit inoculation

A suspension of conidia of C. gloeosporioides was preparedby suspending mycelium scraped from 10-14 d old culturesin sterile distilled water (SDW) and filtering through glasswool. The concentration in the filtrate was adjusted to 105

conidia/ml. A set of 15 fruits from each cultivar was inoculatedby applying 20-30 drops (20 µl) of the conidia suspension onthe fruit surface and another set of fruits were treated withdrops (20 µl) of SDW as controls. Inoculated and control fruitswere maintained at 95-100% RH and RT.

Epidermal strips (30 µm thick) beneath the drop ofinoculum were excised using a sharp sterile razor bladeduring a period of 3 - 24 h after inoculation at 3 h intervals,unless otherwise stated. A total of 20 epidermal strips cutfrom four randomly selected replicate fruits were used foreach histochemical staining. In order to observe conidiagermination and associated events, epidermal strips removedwere cleared in boiling ethanol for 2-3 min and rinsed indistilled water. The fungal structures were stained in 10%blue ink (v/v) in 25% acetic acid for 1 min and the excessstain was removed by washing with distil led water(Huckelhoven and Kogel, 1998). The epidermal strips weremounted on glass slides and examined under a lightmicroscope.

Detection of H2O

2 and O

2-

To detect H2O2, the epidermal strips were immersed in 2 mg/ml 3, 3’-diaminobenzidine (DAB, Sigma) in 50 mM MESbuffer, pH 6.5 for 2 h prior to sampling (Thordal-Christensenet al., 1997; Huckelhoven et al., 1999). For detection of O2

-,the epidermal strips were immersed in 0.1% nitrobluetetrazolium (NBT, Sigma) in 50 mM MES buffer pH 6.5 for 30min (Heath, 1998). Ascorbic acid (10 mM) and 900Usuperoxide dismutase (SOD, Sigma) were used as the

negative controls for H2O

2 and O

2-, respectively. The

epidermal strips were cleared in 95% boiling ethanol andobserved under the light microscope (Olympus CX31 withDigital Camera DP20 and application software DP2-BSWbuild 2738, Olympus Corporation, 2006). The percentageepidermal cells stained with NBT were counted out of 100appressoria-epidermal cell contact sites in 3 microscopicfields from an epidermal strip. Average value of 3 epidermalstrips represented the frequency of interaction sites with NBTstaining for each fruit. This experiment was carried out withunripe and ripe fruits of mango cultivars ‘Karutha Colomban’and ‘Willard’.

Detection of autofluorescence

Epidermal strips were placed in a fixing solution (10%formaldehyde, 5% acetic acid, 45% ethanol, and H

2O) for 15

min and soaked in 50% ethanol for 20 min followed by 95%ethanol overnight (Bowling et al., 1997). The strips weremounted in 70% glycerol and observed under a fluorescencemicroscope [Nikon Ophtiphot, Japan, with EPI-FL filter blockB-2A (BA 520, DM 510, Ex 450-490)] and photographed.

Detection of lignin

Epidermal strips and thin transverse sections cut throughthe fruit peel 12, 24, 36, 48 and 72 h after inoculation wereplaced separately in phloroglucinol-HCl reagent for 1-5 min,rinsed and mounted in distilled water and immediatelyobserved under light microscope for red staining in thelignified cells (Siegel, 1959).

Antifungal activity in the inoculated peel

Fruits at harvesting maturity were inoculated with conidia ofC. gloeosporioides and another set was treated with dropsof SDW as a control. Peels (250 µm thick) beneath theinoculum/water drop were removed 48 h after inoculation ortreatment with SDW. The peel tissues were placed inmethanol: dichloromethane (50:50 v/v) in conical flasks (250ml) with side arms connected to a vacuum pump andextracted by vacuum infiltration with continuous stirring(Karunanayake et al., 2011; Adikaram and RatnayakeBandara, 1998). The extracts were concentrated andsubjected to Thin Layer Chromatography bioassay usingCladospor ium cladosporioides as the test fungus(Karunanayake et al., 2011). The area of inhibition zoneswas measured and their Rf values were recorded.

Chitinase activity in the inoculated fruit peel

Fruits at harvesting maturity were inoculated with conidia ofC. gloeosporioides and another set was treated with dropsof SDW as a control. Chitinase was extracted byhomogenizing peel tissue (1.5 g, 250 µm thick) cut from theinoculated sites at 3 h intervals over a 24 h period afterinoculation in 3 ml 60 mM phosphate buffer (pH 7) containing0.1g polyvinylpolypyrrolidone (PVPP) and 30 µlphenylmethylsulfonyl fluoride (PMSF). Extracts werecentrifuged at 9000 g (Sigma 3K30, DJB Labcare,Buckinghamshire, UK) for 5 min and the supernatant wasused as the enzyme source. Chitinase activity was

Indian Phytopathology 65 (4) : 2-6 (2012) 3

determined by gel diffusion assay using glycol chitin as asubstrate (Zou et al., 2002). Aliquots (10 ml) of the extractsand the enzyme standards (Chitinase EC3.2.1.14, fromSerratia marcescens, Sigma) were pipetted into individualwells cut out in the gel. The plates were incubated at RT for48 h and the gel was stained with 10 ml of 0.1% (w/v)calcofluor white (Fluorescent brightener 28, Sigma) in 500mM Tris-HCl (pH 8.9) for 10 min, washed in SDW with orbitalshaking for 2-3 h and viewed under UV light (365 nm). Thediameter of the dark lytic zones, was recorded. The enzymeactivity was calculated using a regression equation derivedusing the lytic zones of known enzyme standards.

SDS-PAGE: Extracts of peel (10 g) cut from inoculated orcontrol fruits (cultivar ‘Willard’) 15 h after inoculation weresubjected to SDS-PAGE in 12% acrylamide gels containing0.1% (w/v) SDS and 0.01% glycol chitin at 50 V for 2-3 h.The gel was stained with calcoflour white in 500 mm Tris-HCl buffer (pH 8.9) for 10 min. The stain was removed andlytic zones were visualized by placing the gel on atransilluminator (SXT 20M UVitec, France) (Trudel andAsselin, 1989).

Assay for PAL

Fruits at harvesting maturity were inoculated with conidia ofC. gloeosporioides and another set was treated with dropsof SDW as a control. Peel tissues (1.0 g, 250 µm thick) wereremoved from fruits inoculated with C. gloeosporioides andcontrols, at every two-day intervals for 8 days andhomogenized in 10 ml cold acetone (-20 oC) for 2 min at11,000 rpm in a homogenizer (ULTRA TURAX® model T25basic, IKA LABORTECNIK,. Selangor, Malaysia). Thehomogenate was filtered through Whatman No 1 paper andthe powder retained was homogenized twice more in coldacetone and air-dried. PAL was extracted from the powder(100 mg) by stirring for 2 h at 4 °C with 5 ml of 0.1 M sodiumborate buffer (pH 8.8). The extract was centrifuged at 10,000g (Sigma 3K30, DJB Labcare, Buckinghamshire, UK) for 20min at 4 °C and the supernatant was used as the enzymesource. The reaction mixture consisted of 10 mM L-phenylalanine (Sigma), 0.1 M borate buffer (pH 8.8) and 1.5ml of enzyme extract in a final volume of 5 ml. The reactionwas carried out at 37 °C for 60 min and terminated by adding0.1 ml of 5 N HCl. Cinnamic acid produced was extractedinto diethyl ether (7 ml) and quantified by measuring the trans-cinnamic acid formed at 269 nm (Cintra 10e-UV visiblespectrophotometer; GBC Spectral,Victoria, Australia). trans-4-hydroxy-3-methoxycinnamic acid (99%, ALDRICH®) wasused as the standard. The amount of cinnamic acid in thetest samples was calculated from a standard curve preparedusing absorbance values of a series of solutions (10-50 µg/ml) in diethyl ether and expressed as µg cinnamic acid in 1.0g peel (Beno-Moualem and Prusky, 2000).

Statistical analysis

Each experiment was repeated at least twice. One-wayANOVA was performed to find out significant difference inO2

- production between the two mango cultivars and the twodevelopment stages at a particular experimental time point.Chitinase and PAL activity data obtained were analyzed by

two-way ANOVA and the means were separated by Duncan’sMultiple Range test using SAS computer software at 95%significant level (SAS Corporation, Release 6.12).

RESULTS

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

The conidia germinated on the unripe fruit surface of bothresistant and susceptible cultivars 3 h after inoculation. Aseptum was formed in the conidium prior to emergence ofgerm-tube. Within 6 h non-melanized appressoria wereobserved and all appressoria appeared melanized 12 h afterinoculation and the lobes were clearly visible in them. Infectionhyphae that emerged beneath the appressoria were clearlyvisible in the TS taken through the peel, 24 h after inoculation.A considerable number of epidermal cells in contact withinfection hyphae had turned brown (Fig. 1a).

Formation of H2O

2 and O

2-

Production of H2O2 was observed as brownish and diffusibleDAB polymers and O2

- as blue colour, granulated formazan(Figs. 1b & c). First evidence of H

2O

2 was observed 12 h

after inoculation, however, at subsequent time points it wasdifficult to distinguish the DAB stain from induced cellbrowning.

Superoxide generation was evident in the infected cells,9 h after inoculation, in both cultivars tested. The percentageof infected cells showing superoxides increased with timeand reached a peak at 15-21 h after inoculation anddecreased sharply. About 50% of the infected cells showedsuperoxide generation 15-18 h after inoculation in the unriperesistant cultivar ‘Karutha Colomban’. Only 29-35% of theinfected cells showed superoxide generation 15-18 h afterinoculation in the ripe fruits. In susceptible ‘Willard’, the

Fig. 1. (a) Photomicrographs of epidermal peel of unripe mango fruitof cultivar Karutha Colomban 24 h after inoculation with C.gloeosporioides showing appressoria formed on epidermalcells and infection hyphae (magnification 1000X), (b) brownishcells with diffusible DAB polymers indicating H2O2, (c) bluecolour cells with granulated formazan indicative of O2

-, (d)autoflourescence of invaded and browned cells underfluorescent microscopy (magnification 400X)

4 Indian Phytopathology 65 (4) : 2-6 (2012)

percentage cells that generated superoxides was significantlylower than ‘Karutha Colomban’ and a maximum of 33% cellsproduced superoxides 21 h after inoculation. Superoxidegeneration by the ripe ‘Willard’ cells was very low and thepercentage of infected cells showing NBT staining was lessthan 10% at all the time points tested. The difference inpercentage of interaction sites (epidermal cells in contactwith appressoria) that produced O2

- between cultivars as wellas between unripe and ripe stages was statistically significant(P>0.05) (Fig. 2).

Autofluorescence

In both cultivars, the epidermal cells that were penetrated byinfection hyphae and had turned brown after 24 h, showedautofluorescence as localized bright yellow-greenfluorescence (Fig. 1d). The first evidence of autofluorescencewas observed in the cell walls 12 h after inoculation. Withtime autofluorescence was observed in cells surrounding thepathogen invaded cell.

Histochemical test for lignin

In both cultivars, the infected brown cells and the cellsimmediately surrounding these gave positive results for lignin.Red staining was first observed 36-48 h after inoculation andcontinued at latter time points examined.

Antifungal activity in the fruit peel inoculated with C.gloeosporioides

Several inhibition zones were visible at Rf 0.16-0.18, 0.21-0.22, 0.26-0.28, 0.48-0.50 and 0.53-0.59 in thechromatograms (Fig. 3) with dichloromethane phase of peelextract from inoculated or healthy fruits of both cultivars.However, the area of inhibition produced by the extract ofinoculated fruit tissue was slightly larger and stronger. Thesize of the inhibition zones produced by the compounds atRf 0.48-0.59 were 157±32 mm2 and 305±15 mm2 in healthyand inoculated ‘Karutha Colomban’ fruit peel, respectively.In ‘Willard’ the area of inhibition was 175 ±20 mm2 and 287±30

mm2 respectively, in healthy and inoculated fruit peel. Therewere no additional inhibition areas in the chromatograms withinoculated fruit extract compared to the uninoculated controls(Fig. 3). The size of the inhibition area produced by themethanol fraction of both inoculated and control tissueextracts corresponding to gallotannins was similar in thecultivar ‘Karutha Colamban’. However, a slight increase ingallotannin activity was observed in the inoculated peelextract of the cultivar ‘Willard’ (data not shown).

Fig. 2. Histochemical analysis of O2- generation in epidermal cells of

‘Karutha Colomban’ (KC) and ‘Willard’ fruit after inoculationwith conidia of C. gloeosporioides

Fig. 3. Thin layer chromatogram with extracts of (a) healthy mangofruit peel and (b) inoculated with C. gloeosporioides andbioassayed with C. cladosporioides.

Chitinase activity

Peel chitinase activity fluctuated with time after inoculationin unripe fruits of both cultivars. The peak activity wasobserved 12 h after inoculation in ‘Karutha Colomban’ (Fig.4). However, the difference in chitinase activity observed atdifferent time points was not significant (P>0.05) in the peelof unripe fruit of cultivar ‘Willard’. Four chitinase bandscorresponding to molecular weights 69.7, 65.4, 44.5 and 34.2kDa were visible in peel extract of healthy fruit of cultivar‘Willard’. The inoculated peel extract showed an additionalband corresponding to 59.4 kDa. The band correspondingto 44.5 kDa was more prominent in the inoculated peel extractthan that of the healthy peel extract.

Fig. 4. Chitinase activity in the peel of mango cultivar ‘KaruthaColomban’ and ‘Willard’ following inoculation with C.gloeosporioides

Indian Phytopathology 65 (4) : 2-6 (2012) 5

PAL activity

The inoculated peels showed about two fold increase in PALactivity (P<0.05) than the control peels in both ‘KaruthaColomban’ and ‘Willard’ at all the time points tested (Fig.5).PAL activity was higher in the cultivar ‘Karutha Colomban’than that of ‘Willard’ throughout the period. There was anincrease in PAL activity with time from the initial level (Day 0)in the inoculated and control fruit peel of the both cultivars.

a direct antimicrobial effect (Thordal-Christensen et al., 1997)or trigger other defence responses such as papillae formation(Huckelhoven et al., 1999; Thordal-Christensen et al., 1997),protein cross-linking and accumulation of phenoliccompounds associated with plant defence (Borden andHiggins, 2002). Further, ROS production in mango fruit peelseems to be dependent on the physiological stage of thefruit development. In both cultivars, the percentage ofinteraction sites that produce O

2- was higher in the unripe

than ripe fruits. This result suggests that ROS productiondecreases with ripening which probably makes ripe fruitsmore susceptible to pathogen attack.

H2O

2 and O

2- accumulation 12 h after inoculation was

restricted to the sites where host cell browning had occurred.There was indicated intense autoflourescence in the infectedbrown cells and neighbouring host cells. The occurrence ofautoflourescence was due to accumulation of polyphenolsat the site of infection. Towards 36-48 h after infection, theaccumulation of lignin was also evident in the brown cells.Accumulation of autofluorescent compounds is associatedwith hypersensitive response (HR) and browning andaccumulation of phenols are thought to be responses thatfollow cell death (Park, 2005). It appears that the epidermalcells of mango fruit peel undergo HR like cell death inresponse to infection by C. gloeosporioides though visiblenecrotic symptoms appeared 3-4 days after inoculation in‘Willard’ and 6-7 days in ‘Karutha Colomban’ (G.D. Sinniah,unpublished data). Oxidative burst is considered as one ofthe earliest events in the HR (Jabs et al., 1997; Able et al.,2003) and localization of H2O2 and O2

- at the site of cellbrowning and autoflourescence suggests that ROS may havea role in the accumulation of polyphenols and associatedcell death in mango-C. gloeosporioides interaction.

There was a significant increase in PAL activity, a keyenzyme in phenolic biosynthesis, after inoculation in bothcultivars. The increased PAL activity corresponds with theaccumulation of phenolic compounds and lignification at theinoculated sites. These results provide further evidence forenhanced localized production of secondary metabolites thatare needed to inhibit pathogen infection, directly or indirectly.Increased PAL activity and phenolic content have also beenreported in freckle infected banana (Abayasekara et al.,1998), Fusarium infected banana (De Ascensao and Dubery,2000) and C. gloeosporioides infected avocado pericarp(Beno-Moualem and Prusky, 2000).

Accumulation of PR-proteins is an important feature ofplant defence responses upon infection by pathogens(Ferreira et al., 2007). The rapid enhancement of chitinaseactivity in mango peel was observed after infection by C.gloeosporioides. The induction of chitinase activity wascommon to both resistant ‘Karutha Colomban’ andsusceptible ‘Willard’ cultivars. However, the increase wassignificant only in the resistant cultivar. Higher chitinaseactivity observed in the peel of ‘Willard’ is a cultivar effect. Itwas reported that chitinase activity per unit area of peel andin a given volume of latex was higher in ‘Willard’ than that of‘Karutha Colomban’ (Karunanayake, 2008). Preformedchitinases present in the mango latex have been shown tohydrolyze the walls of conidia of C. gloeosporioides in vitro

Fig. 5. PAL activity in unripe ‘Karutha Colomban’ and ‘Willard’ fruitpeel following inoculation with C. gloeosporioides.

DISCUSSION

The resistance of unripe mango fruit to C. gloeosporioideshas been implicated to the presence of constitutiveresorcinols (Hassan et al., 2007) and gallotannins in the peeland chitinase in the latex (Karunanayake et al., 2011). Thenature of immature mango fruits to respond to infection byinducing defence responses remained relatively unknown.There were occasional reports of PR-proteins in mango inresponse to defence elicitor treatment (Zainuri et al., 2001).The present study provides clear evidence for induceddefence responses in mango in during C. gloeosporioides.

Light microscopic observation showed no morphologicaland temporal difference in the pattern of germination andappressorium formation by conidia of C. gloeosporioides inthe unripe fruit between ‘Karutha Colomban’ and ‘Willard’.This suggests that the pre-penetration events are similar inboth resistant and susceptible cultivars and that theresistance responses trigger following penetration. Similarly,there were no morphological differences in pre-penetrationevents of C. acutatum between the resistant and susceptibleblueberry cultivars (Wharton and Schilder, 2008).

Accumulation of H2O

2 and O

2- was observed in the

epidermal cells as an early defence response (6-9 h afterinoculation) in mango fruit peel following infection by C.gloeosporioides. O2

- generation was clearly identified by theformation of blue colour formazan when stained with NBT.However, pathogen-induced cell browning interfered withyellow-brown DAB staining of H2O2 and made thequantification difficult. The rapid and higher level of O2

-

generation in the resistant ‘Karutha Colomban’ compared tothe susceptible ‘Willard’ suggests an active role of O

2- in

defence. The highly localized accumulation of ROS may have

6 Indian Phytopathology 65 (4) : 2-6 (2012)

within 2-3 h after exposure (Adikaram et al., 2010). Theinduction of chitinase following infection may enhancehydrolytic degradation of mycelial walls of invading fungi. Themolecular weights of induced chitinase isozymes were shownto be 59.4 kDa and 44.5 kDa. Induction of chitinase enzymeswas also shown in Vicia faba during Botrytis fabae infection(Attia et al., 2007). Two chitinase isozymes with molecularweights, 18 kDa and 12 kDa, were induced in freckle infectedbanana fruits var. ‘Embul’ (Wanigasekara, 2009). In rice cellstreated with an elicitor from Rhizoctonia solani induced achitinase having a molecular weight 35 kDa (Velazhahan etal., 2000). mRNA differential display experiments revealedtranscriptional activation of defence genes in fruits of theresistant (`Karutha Colomban’) and susceptible ‘Willard’cultivars. A higher number of differentially expressed cDNA’swas shown in the inoculated fruit peel of ‘Karutha Colomban’than that of ‘Willard’ (unpublished results). Avocado fruits havealso been shown to respond to C. gloeosporioides infectionby a sophisticated molecular activating structural andbiochemical defence mechanisms (Djamni-Tchatchou et al.,2012).

In conclusion, the unripe mango fruit responded toinoculation with conidia of C. gloeosporioides by inducingseveral early defences. These responses are followed byincreased activity of chitinase, PAL and accumulation ofphenolics and lignification of cell walls. These responses weregenerated more in unripe mango fruits than in ripe fruits inboth mango cultivars tested and the resistant cultivar toanthracnose ‘Karutha Colomban’ showed defense responsesearlier and in greater magnitude than susceptible cultivar‘Willard’. The study revealed that inducible defences also playan important role in the resistance of unripe mango to C.gloeosporioides when the preformed defences are overcomeby the pathogen.

ACKNOWLEDGEMENT

The authors acknowledge the Australian Centre forInternational Agricultural Research (ACIAR) for financialassistance.

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