A severe fruit rot of durzan fruits in Bentong and Raub in Pahang; Dengkil in Selangor; Muar

and Kluang in Johore was found to be caused by a Phytophthora sp. Besides fruits, isolates of the

pathogen were also capable of infecting roots, stem and leaves of durian. The isolates were identified

as Phytophthora palmivora, cross-inducing (heterothallic) and of the A 1 mating type, producing

spores when paired with a standard A2 mating type of P. palmivora and another A2 isolate from

cocoa but not with an A 1 mating type of P.parasitica

Chan, L.G

Lim, T.K

Universiti Putra Malaysia Institutional Repository

Pertanika 9(3), 269 - 276 (1986)Fruit Rot of Durian Caused by Phytophthora palmivoraT.K. LIM and L.G. CHANDepartment of Plant Protection,Faculty of Agriculture,Universiti Pertanian Malaysia,43400 Serdang, Selangor, Malaysia.Key words: Durian; fruit rot; Phytophthora palmivoraABSTRAKSuatu reput buah yang teruk pada durian telah dijumpai di Bentong dan Raub di Pahang;Dengkil di Selangor; Muar dan Kluang diJohor disebabkan oleh Phytophthora sp. Selain daripadabuah durian, asingan-asingan patogen juga berupaya menjangkiti akar, batang dan daun pokokdurian. Asingan-asingan dikenalpasti sebagai P. palmivora, 'cross-inducing' (heterotalz'k) dan jenisperpasangan AI, mengeluarkan oospora apabila dzpasang denganjenis perpasangan A2 P. palmivorayang piawai dan suatu lagi perpasangan A2 darzpada koko dan bukan dengan jenzs perpasangan A 1P. parasitica.ABSTRACTA severe fruit rot of durzan fruits in Bentong and Raub in Pahang; Dengkil in Selangor; Muarand Kluang in Johore was found to be caused by a Phytophthora sp. Besides fruits, zsolates of thepathogen were also capable of infecting roots, stem and leaves of durian. The zsolates were identifiedas Phytophthora palmivora, cross-inducing (heterothallic) and of the A 1 mating type, producingoospores when paired with a standard A2 mating type of P. palmivora and another A2 zsolate fromcocoa but not with an A 1 mating type ofP. parasitica.INTRODUCTIONLike most fruit crops, the durian, Duriozibethinus Murr. is susceptible to attacks by pestsand disease-causing microorganisms at all stagesof the crop phenology. Among the latter group isthe fungus Phytophthora palmivora which cancause devastating losses at both the seedling andadult tree stages. The fungus infects the roots,causing root rot (Navaratnam, 1966; Tai, 1970);the trunk, causing patch canker (Thompson,1934; Navaratnam, 1966; Tai, 1970); leaves andshoots, causing leaf blight, defoliation and die-back (Tai, 1970). Recently, durian fruits werealso found tobe infected by a Phytophthora sp.Severe fruit rots were found in several fruitorchards in Bentong and Raub in Pahang;Dengkil in Selangor, and Muar and Kluang inJohore. Fruits on high branches were also attack-ed in several orchards. Affected fruits lost theirmarketability completely.In cognizance of the seriousness of thedisease, investigations were undertaken i) toidentify and characterize the causal fungusthrough cultural studies; ii) to establish patho-genicity on fruits and other parts of the durianplants; and iii) to study the symptomatology ofthe fruit rot.MATERIALS AND METHODSIso.lationsDiseased fruit tissues (5 X 3 X 1 mm) fromadvancing margins were obtained with a steri-T.K. LIM AND L.G. CHANlised scalpel after peeling off the epidermis. Fourpieces of such tissues were embedded in 2 %water agar or Pimaricin Vancomycin PCNBmedia (Ocana and Tsao, 1966). Singlesporangial cultures of two representative isolatesdesignated PDFD (isolate from Dengkil), andPDFK (isolate from Kluang) were obtained andmaintained on Vegetable Juice (V8) Agar (VJA)(Miller, 1955) slants at 24°C and used for thestudies.Cultural Stud(esSix mm mycelial discs taken from theadvancing edge of five-day old VJA cultures ofthe two isolates were separately placed in thecentres of carrot agar (CA), Difco cornmeal agar(CMA), Difco potato dextrose agar (PDA) andVJA plates in two sets of four replicates. Oneset was incubated in continuous darkness at28 ± 1.5°C and the colony characteristics werecompared after seven days. The other set wasused to assess growth rates at the same tempe-rature where the colony diameter measurementswere taken at right angles on alternate days for aduration of seven days. Data were subjected tostatistical analysis (ANOVA).The procedures were repeated for tempe-rature-growth relations using CMA as themedium and temperature regimes of 16, 20,24,28, 32 and 36°C.Spore Characteristic StudiesMeasurements of sporangium andchlamydospore dimensions; apical thickening;pedicel length and width of exit pore of spo-rangia were based on 100 spores of the testisolates in separate experiments.To study sporangium dimensions and depthof apical thickening, the test isolates were grownon CMA plates under flourescent light for sevendays at 28 ± 1.5°C and harvested for zoo-sporangia as follows: a standardised amount ofsterile distilled water was added to the plates anda bent glass rod was used to dislodge the zoo-sporangia. Drops of sporangium suspension weremounted on glass slides in half-strength lacto-phenol blue to facilitate measurements ofsporangium length, breadth and depth of apicalthickening.The procedures were repeated for measure-ment of chlamydospores from another batch ofCMA plates after fourteen days incubation.To study sporangium caducity, a modifiedmethod of AI-Hedaithy and Tsao (1979) wasused. Mycelial mat colonies of the test isolateswere established in 90 mm petri dishes contain-ing 20 ml of vegetable juice made up of 10%Campbell's V-8 juice and 0.2 %ca1cium carbo-nate and incubated in the light at 28 - 15. °C.After five days incubation, the mats were washedfive times and transferred to petri dishes contain-ing 25 ml of sterile distilled water. After 48 hoursof incubation, the plates were observed fornaturally and mechanically detached sporangiaand their pedicel lengths were measured.To measure the sporangium exit pore, aseparate sporangium suspension was subjected tochilling treatment at 6°C for 25 min. (Lee andVarghese, 1974), and then returned to roomtemperature to facilitate release of zoospores.Sporangia and zoospores were studied using half-strength lactophenol blue and the width of theexit pores of the sporangia were measured.Compatibility Type and Oospore ProductionBoth test isolates were paired among them-selves, between each other, separately betweenstandard Al mating type of P. parasitica (IMI268688), A2 mating type of P. palmivora fromcocoa and an A2 mating type of P. _palmivora(IMI 203533) on VJA medium and incubated inthe dark at 28 ± 1.5°C. All compatibility testingswere made in four replicates and examined foroospore production after two weeks. A hundredoospores were measured per plate.In addition, induction of oospore produc-tion of the test isolates was determined using theTrichoderma method of Brasier (1974). Five-dayold VJA cultures of the isolates were invertedover five-day old PDA culture of T. koningiiand sealed together with paraffin and cello-270 PERTANIKA VOL. 9 NO.3, 1986FRUIT ROT OF DURIAN CAUSED BY PHYTOPHTHORA PALMIVORAphane tape. The combined plates were likewiseincubated and examined for oospore productionafter two weeks.Pathogenicity StudiesArtificial inoculations of the roots, stern,leaves and fruits were carried out. For the firstthree plant parts, six two-month old polybagseedlings were used per test isolate and checktreatment and three mature, large fruits werelikewise used for fruit inoculation. Inoculation ofthe test isolates was repeated on cocoa seedlingsand fruits.Zoospore suspensions prepared as describedfor the sporangium caducity studies were usedfor the root, leaf and fruit inoculations. Theconcentration was adjusted to 5000 spores per mlwith the help of the Neubauer haemocytometer.Root inoculation was done by drenching theloosened soil of the polybag with 200 ml of zoo-spore suspension. Check plants were drenchedwith 200 ml of sterile distilled water. The plantswere covered with polyethylene bags for 48 hoursand were kept water-logged for three days byplacing them on clay saucers filled with water.The plants were watered daily and after threemonths, the plants were removed and the rootswashed and evaluated for .the degree of root·infection. Leaf inoculation was accomplished byspraying the seedlings with 50 ml of the zoosporesuspension using the Desaga spray gun. Checkplants were sprayed with sterile distilled water.The plants were again kept for 48 hours in moistpolybags. Leaves were examined for diseasedevelopment after five days. For fruit inocula-tion, three shallow circular plasticine wells (10mm diameter. 6 mm deep) were constructed onthe fruit surface which had been sterilised withalcohol. The wells were filled with 0.5 ml of thezoospore suspension. The wells on the checkfruits were filled with sterile distilled water. Thefruits were kept in moist chambers and observedfor lesion development up to a week.Stern inoculation was carried out bypricking the tender green stern of the plant to astand.ard_dept~ ?f 2 mm with the tip of a needlecarrying mycelial fragments taken from a seven-day old VJ broth culture of the test isolate. Thewound was covered with moist cotton wool andbounded with transparent polyethylene strips.Check plants were wounded with a clean sterileneedle. The plants were kept moist as describedabove. Length of canker lesions were regularlymeasured for a period of two weeks.Reisolations were made on PVP mediumfrom all positive inoculations.RESULTSIsolations consistently yielded the samePhytopthora sp. Representative isolates, PDFDand PDFK, both produced stellate and striatecolonies with sparse aerial mycelia and welldefined margins on CA, VJA and CMA; whilston PDA, colonies were more fluffy and marginswere slightly irregular. Growth on VJA was thefastest, while PDA registered the slowest growthrate (Table 1). Both isolates grew at tempe-ratures of 16°C to 32°C, with optima at 28°Cand both failed to grow at 36°C. Isolate PDFDexhibited higher growth than isolate PDFK (Fig.1.)Both isolates produced papillate, caducous,ovoid to ellipsoid, rounded-based sporangia witha mean of 53.68 X 35.28 J.l.m (Fig. 2). IsolatePDFK produced larger sporangia than PDFD onCMA under light incubation (Table 2). Thelength: breadth ratio of PDFD was 1.48 and thatof PDFK was 1.78. The sporangia possessedshort, hyaline pedicel, occluded by a plug; themean pedicel length observed was 2.61 ± 0.10pm and 2.56 ± 0.09 pm for PDFD and PDFKrespectively. The mean apical depth recordedfor PDFD was 2.76±0.12 pm and for PDFKwas 3.59 ± 0.14 pm; whilst the mean width ofthe exit pore measured was 5.72 ± 0.14 /Jill and6.12 + 0.14 /Jm for PDFD and PDFK respec-tively.Both isolates produced large, spherical,chlamydospores in abundance after two weeks. ofincubation. Chl'amydospores of PDFD rangedfrom 31.72-43.92 pm (mean 36.37±0.21) indiameter while those produced by PDFK weresmaller and ranged from 30.50 - 39.04 /.lm(mean 34.39 ± 0.18 .ptn).PERTANIKA VOL. 9 NO.3. 1986 271T.K. LIM AND L.G. CHANTABLE 1Growth rate of Phytophthora palmivora isolates from durian fruits on selected media at 28 ± 1.5°CAgar mediumIsolatePDFD PDFKMean growth rate (mm/day)Carrot agarCornmeal agarVegetable juice agarPotato dextrose agar11.95* a + +11.63 a12.11 a7.20 a12.33 a11. 58 a12.62 a6.93 b*Each value is an average of 4 replicate plates.+ +Meari value in column followed by similar letters denote no significant differences at Pby New Duncan Multiple Range Test.0.05 as determinedFig. 1: Effect of temperature on mycelial lineargrowth of Phytophthora palmivora isolatesfrom durian fruit on cornmeal agar.and a standard A2 isolate of P. palmivora.Diameters of oospores produced ranged from20.74 ± 30.50 .J1. m (Table 3). No oospores wereformed by the Trichoderma induction method.However, abundant chlamydospores andmorphogenic changes such as the thickening ofthe colony margin of the isolates, vacuolation ofthe cell contents and hyphallysis were observed.Fig. 2: A typical papillate, caducous, ellzpsoidsporangium of Phytophthora palmivorafrom durian (isolate PDFK) with a shortpedicel.3224Temperature °c20/'10 ./://~:/70 ///10 ,IIi50 III40 I/30 I/!20 isolate PDFKisolate PDFD10No oospores were formed when the isolateswere selfed, paire~ between each other or pairedwith the Al mating type of P. parasitica. Abun-dant oospores with amphigynous antheridia wereobserved when the isolates were separatelypaired with a P. palmivora isolate from cocoaBoth isolates were capable of infecting theroots, stem, leaves and fruits of the durian, butwere avirulent on cocoa. They caused a reduc-tion in the amount of rootlets, necrosis of therootlets and tap roots of seedlings. Both isolateswere equally virulent on the durian stem (Table4), causing extensive canker lesions which girdl-272 PERTANIKA VOL 9 NO.:i, 1986"tlt""l~....,>z;:J::><:or-<.0Zo.(jO<.000O'lt'-:--.J;",:TABLE 2Sporangium morphology of Phytophthora palmivora isolates from durian on cornmeal agar after 7 days light inoculation at 28 ± 1.5°CIsolate Range of Mean Range of Mean Length/ Mean Mean Meanlength length* breadth breadth* breadth pedicel apical width ofratio length* depth* exit pore*( tL m ) ( tL m ) ( p.m) ( tL m ) ( tL m ) ( P.m) ( tLm) ( tL m )PDFD 39.04 - 73.20 52.94 ± 1.29 26.84 - 46.36 35.92±0.83 1.48 2.61 ± 01 2.76±0.12 5.73±0.14PDFK 46.36 - 68.32 56.46 ± 0.94 24.40 - 41.48 36.06 ± 0.63 1. 78 2.56 ± 0.09 3.59 ± 0.14 6.12±0.14*Average of 100 spores.>rj~c::i~o,.,o>rjt:lC~:;ZCJ:>cC/lt'I1t:ltxl0<~"'<:..,o~~g::t..~t'"'~~g::t..T.K. LIM AND L.G. CHANTABLE 3Dimension of oospores obtained from pairings between Phytophthora palmivora isolates fro!lldurian (PDFD, PDFK) and A 2 mating types on vegetable juice agarCrossingsPDFD X PCl (P. palmivora A 2 cocoa isolate)PDFK X PCl (P. palmivora A 2 cocoa isolate)PDFD X A 2 P. palmivora (IMI 203533)PDFK X A 2 P. palmivora (IMI 203533)PDFK X AlP. parasitica (IMI 268688)PDFD X AlP. parasitica (IMI 268688)Range (f..l.m) Mean diameter (1J..ifm)*21.96 - 30.50 25.54 ± 0.3920.74-28.06 25.14 ± 0.3020.74 - 26.84 23.91 ± 0.2320.74 - 26.84 23.18 ± 0.230 00 0*Average of 100 oospores count per replicate plate.TABLE 4Pathogenicity of Phytophthora palmivora durian isolates on durian seedlingsIsolatePDFDPDFKBlank agarNumber of days after inoculation3 6 10 14I Mean stem lesion (mm)10.75* a** 110 a 176.5 a dieback8.50 a 102.3 a 186 a dieback0 b 0 b 0 b 0*Average of 4 replicates consisting of 6 durian seedlings per replicate.**Mean values followed by the same letter i~ each column are not significantly different at Pmined by New Duncan Multiple Range Test.0.05 as deter-ed the stems, leading to dieback of the seedlings.When inoculated on the leaves, symptoms werevisible after three days. Leaf lesions were charac-terised by small off-coloured, water-soaked spotswhich darkened and coalesced into largerpatches. Infected leaves drooped and abortedprematurely. On fruits •.symptoms first appearedas hydrotic patches which turned brown andthen dark-brown to black. A whitish bloom ofmycelia and sporangia Jormed on the lesion afterfive days (Fig. 3).The test isolates were reisolated from allpositive inoculations. All check inoculations didnot produce disease symptoms.Fig. 3: Durian fruit naturally. infected byPhytophthora palmivora. Note the whitebloom of mycelia and sporangia of thepathogen on the necrotic lesion (arrowed).274 PERTANIKA VOL. 9 NO.3, 1986FRUIT ROT OF DURIAN CAUSED BY PHYTOPHTHORA PALMIVORADISCUSSIONThe above studies clearly demonstrated thatthe Phytophthora isolates from durian fruitbelonged to the P. paimivora (Butler) Butlergroup. The cultural and morphological charac-teristics of the isolates subscribed to those of P.paimivora as described by Newhook et al.(1978); Brasier and Griffin, (1979); and Water-house et ai. (1983). The failure of both isolates togrow at the temperature of around 36°C furtherdistinguish them from P. nicotianae var. nico-tianae and P. nicotianae var. parasitica whichcould grow well at temperatures above 35°C(Newhook et ai., 1978; Waterhouse et ai., 1983;Weste, 1983). The ability of the two Phytoph-thora isolates to produce chlamydospores inabundance in agar culture also distinguish themfrom other Phytophthora species recorded inMalaysia like P. meadii, P. hevea and P.botryosa which do not or rarely producechlamydospores in culture (Waterhouse, 1974).The caducous nature of their sporangiawith short pedicels of < 3 p.m, their stellate andstriate colonies and their inability to formoospores by Trichoderma induction are similarto the traits described for Morphological Form 1(MF1 )in the P. paimivora group (Newhook etai.) 1978; Brasier and Griffin, 1979; Waterhouseet ai., 1983). To avoid taxonomical confusion,Brasier and Griffin (1979), reiterated that theMF1 designation should be discarded and allMF1 isolated be regarded as typical P. palmivoraspecies.The formation of oospores by the isolateonly when crossed with A2 mating type of P.paimivora tester and from cocoa confirmed thatthe isolates are heterothallic or cross-inducing(Ko, 1978), and of the Al compatibility type.The Phytophthora isolates were capable ofinfecting both subterranean and aerial plantparts similar to some aerial-soil parasites like P.cactorum and P. parasitica (Ko, 1980).Although P. paimivora has an extensive hostrange (Chee, 1969), some specificity do existamong isolates from various hosts as areexemplified by the durian isolates which wereavirulent on cocoa.ACKNOWLEDGEMENTSGrateful acknowledgements are due to Dr.Jean Stamps of the Commonwealth MycologicalInstitute for supplying the A1 tester strain of P.parasitica (IMI 268688) and A2 tester strain ofP. paimivora (1MI 203533).REFERENCESAL-HEDIATHY, S.S.A. and P.H. TSAO. (1979):Sporangium pedicel length in Phytophthoraspecies and the consideration of its uniformity indetermining sporangium caducity. Trans. Bn·t.Mycol. Soc. 72(1): 1 -13.BRASIER, C.M. (1975): Stimulation of sex organ for-mation in Phytophthora by antagonistic species ofTn·choderma. 1. The effect invitro. NewPhytologist 74: 183 - 194.BRASIER, C.M. and M.J. GRIFFIN. (1979): Taxonomyof Phytophthora palmivora on cocoa. Trans.Bn·t. Mycol. Soc. 72: 111-143.CHEE. K.H. (1969): Hosts of Phytophthora palmivora.Rev. Appl. Mycol. 48: 337 - 344.Ko, W.H. (1978): Heterothallic Phytophthora: Evi-dence for hormonal regulation of sexual repro-duction.]. Gen. Microbial. 107: IS - 18.Ko, W.H. (1980): Sexuality, evolution and origin ofPhytophthora. Plant. Prot. Bull. Taiwan 22:141-151.LEE, B.S. and G. VARGHESE. (1974): Studies of thegenus Phytophthora in Malaysia. Reproductionand sexuality. Mal. Agri. Res. 3: 137 - 149.MILLER, P.M. (1955): V-8 juice agar as a generalmedium for fungi and bacteria. Phytopathol. 45:461 - 462.NAVARATNAM, S.J. (1966): Patch canker of the duriantree. Mal. Agri.J. 45(3): 291 - 294.NEWHOOK. F.J., G.M. WATERHOUSE and D.]. STAMPS.(1978): Tabular keys to the species of Phyto-phthora de Bary. eMf. Mycol. Paper No. 143,20pp.OCANA, G. and P.H. TSAO. (1966): A selective agarmedium for the direct isolation and enumerationof Phytophthora in soil. A bstr. Phytopathol. 56:893.TAl, L.H. (1970): Studies of Phytophthora palmivora,the causal organism of patch canker disease ofdurian. Cawangan Perkembangan, Jabatan Per·tanian, K. Lumpur. 7 pp.PERTANIKA VOL. 9 NO.3, 1986T.K. LIM AND L.G. CHANTHOMPSON, A. (1934): A disease of the durian tree.Mal. Agric.J. 22: 367 - 371.WATERHOUSE, G.M. (1974): Other Phytophthoraspecies recorded in cacao. In: P.H. Gregory (ed.)Phytophthora disease of cocoa. London:Longman, pp 71 - 79.WATERHOUSE, G.M., FJ. NEWHOOK and OJ. STAMPS.(1983): Present criteria for classification ofPhytophthora. In: D.C. Erwin, S. Barknicki-Gracia', and P.H. Tsao (eds.) Am. Phytopathol.Soc. St. Paul, Minnesota. pp 139 - 147.WESTE, G. (1983): Population dynamics and survivalof Phytophthora. In: D.C. Erwin, S. Barknicki-Gracia, and P.H. Tsao (eds.), Phytophthora -its biology, taxonomy, ecology and pathology.Am. Phytopathol. Soc. St. Paul, Minnesota. pp237-257.(Received 28 May, 1986)276 PERTANIKA VOL. 9 NO.3, 1986

Fruit Rot of Durian Caused by Phytophthora palmivora

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Fruit Rot of Durian Caused by Phytophthora palmivora

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