The testing of seeds using both the blotter and potato dextrose agar showed infection of Colletotrichum

capsici to be well established both within and on the external surfaces of chilli seeds. Microtome

sections showed that mycelia were present in the outer and inner layers of the testa and in the endosperm

region. Formation of the acervulus was initiated below the seed coat and also in the endosperm and

emerged to the surface after disrupting the seed coat. Parenchymatous tissues were also distorted. The

pathogen finally grows on the seed surface

Meon, Sariah

Wan Nik, Wan Zainun

Universiti Putra Malaysia Institutional Repository

Pertanika 11(3), 341-344 (1988)Seed-borne Infection and Development of Colletotrichum capsiciin Naturally Infected Chili SeedSARIAH MEON & W. ZAINUN W. NIKDepartment ofPlant ProtectionFaculty ofAgricultureUniversiti Pertanian Malaysia43400 UPM, Serdang, Selangor, MalaysiaKey words: Colletotrichum capsid; chili seed; site of infection.ABSTRAKPengujian biji benih menggunakan kaedah kertas serap dan agar dekstros menunjukan penjangkitanColletotrichum capsici yang jelas di dalam dan di permukaan luar biji benih cili. Hirisan microtomemenunjukkan miselium didapati dalam lapisan luar dan dalam kulit dan dalam endosperma. Pembentukanaservulus dimulakan di bawah kulit biji bentKdan juga di kawasan endosperma dan keluar dengan meme-cahkan kulit biji. Tisu-tisu parenkima juga bertukar bentuk. Patogen akhirnya hidup di atas permukaanbiji benih.ABSTRACTThe testing of seeds using both the blotter and potato dextrose agar showed infection of Colleto-trichum capsici to be well established both within and on the external surfaces ofchilli seeds. Microtomesections showed that mycelia were present in the outer and inner layers of the testa and in the endospermregion. Formation of the acervulus was initiated below the seed coat and also in the endosperm andemerged to the surface after disrupting the seed coat. Parenchymatous tissues were also distorted. Thepathogen finally grows on the seed surface.INTRODUCTIONAnthracnose or ripe fruit rot of chillies (Capsicumannuum, L) caused by Colletotrichum capsid(Syd.) Butler & Bisby has been found to be aserious disease in the chilli growing areas of Malay-sia. The disease attacked the leaves, stems, flowersand was most damaging on mature ripe fruits. Di-sease symptoms were occasionally observed ongreen fruits as well. The ftrst indication of thedisease is the appearance of small, elliptical oroblong straw-coloured, slightly sunken lesions onthe surface of the mature fruit. This is followedby the development of black acervuli arranged inconcentric rings, giving a target board appearance.Current observations in Peninsular Malaysia showsthat the reduction in yield of marketable fruitsdue to anthracnose generally ranges from 10%-60% depending on certain seasons of the year(Mah, 1987).The pathogen persists in the soil, infectedcrop residues and weeds (Saxena et al. 1982).Several workers reported that Colletotrichumcapsid was seed-borne (Higgins, 1930; Grover andBansod, 1970; Mordue, 1971; Rout and Rath,1972; Holliday, 1980). Hence, the use of anthrac-nose infected seeds can give rise to weak seedlingswhich then become the primary inoculum sourcein a chilli fteld. From the primary inoculumsource, spread of the disease can be through wind-borne spores or through rain splash. Although,control of C. capsid affecting foliage, branchesand fruits of chillies had been well documented,no attention had been paid so far to the determi-nation of the seed-borne nature of C. capsid.Hence this study is initiated to determine thesite of infection and subsequent development inthe infected seed.SARIAH MEONMATERIALS AND METHODSChilli seeds used in this study were collected fromdiseased fruits of the variety MC-4 obtained froman experimental field in Universiti PertanianMalaysia. Four hundred seeds were used for eachmethod of isolation. External seed-borne infectionwas determined by directly plating the seeds onmoist blotter. Internal seed-borne infection wasdetermined by surface - sterilising the seeds with10% chlorox for 3 minutes, followed by repeatedwashings with sterilised water and plating on pota-to dextrose agar. The plates were incubated forseven days at 25 C in alternating cycles of 12hrnear ultraviolet-light (NVY) and darkness, andexamined for fungal infection using a stereo and acompound microscope. The experiment wasarranged in a completely randornised design andthe mean percentage of isolation was based on400 seeds.On the fourth day of incubation, naturally-infected seeds were fIXed in 3% glutaraldehyde in0.025M phosphate buffer and vacuum extractedfor half an hour. They were then dehydrated ineight graded alcohol series (30,40, 50, 60, 70, 80,90 and 100 % ethanol) and embedded in gelatincapsules with JB-4 embedding mixture (polys-ciences, Inc. , Warington, PA 18976). Sectionswere cut on an ordinary rotary microtome andstained with 0.1% toluidine blue in 0.1% sodiumborate (0' Brien et ai, 1964).In addition, scanning electron microscopywas also used as an additional tool in this study.Seed samples were fIXed in 6% buffered glutral-dehyde, washed three times with phosphatebuffer and post-fIXed with 1% osmium tetroxide.The samples were then dehydrated in acetoneseries and dried in a Polaron Critical Point dryingapparatus. The dried specimens were stuck tocopper stubs and shadowed with gold in a PolaronSputter Coater. Micrographs were taken in aJOEL JSM 35C Scanning electron microscope.RESULTS AND DISCUSSIONColleto trichum capsici was isolated most fre-quently both by the blotter and potato dextroseagar, suggesting that the pathogen could be presenton the seed surface as well as inside the seed(Table 1). Similar results were reported by Groverand Bansod (1970) and Rout and Rath (1972) onchilli seeds. In addition, eight genera of fungiwere also isolated (Table 1). Rhizopus stolonifer,Chaetomium globosum and Pestalotiopsis sp. wereisolated from chilli seeds and represent newrecords of chilli seed-borne infection.Transverse sections of seeds naturally infec-ted by Colletotrichum showed a number ofTABLE 1Percentage isolation of fungi from chilli seeds using the blotter method and potato-<1extrose agarColletotrlchum capsici (Syd.) Butler & BisbyFusarium spp.Aspergillus spp.Drechslera rostrata (Drechsl.) Richardson & FraserCurvularia lunata (Waller) & BoedijinColletotrlchum gloesporiodes (penz.) saccChaetomium globosum KzeRhizopus stoloniler (Ehrenb. ex Fr.) LindPestalotiopsis sp.* Percentage isolation *Blotter Potato-Dectrosemethod Agar method37 2925 3117 316 193 110.3 00.4 01.3 00 7342 PERTANIKA VOL. II NO.3, 1988SEED-BORNE INFEcrlON AND DEVELOPMENT OF C. CAPSICI IN NATURALLY INFECTED CHILI SEEDacervuli over the seed surfaces. After infection,acervulus primordia were formed beneath theinner layers of the testa and within the endospermregion (Plate 1). Subsequently, the young acer-vulus enlarged and caused the testa to bulgeoutwards by exerting pressure on the testa (Plate2).Plate 1. Acervulus primordia (a"owed) beneath seedcoat (Sc . seed coat; Ap • Acervulus primordia;p. parenchyma; e . endosperm).Plate 2. Acervulus primordia emerging through seedcoat. (YA . young acervulus; AP . acervulusprimordia)Ultimately, the testa ruptured and the youngacervulus emerged. Each mature acervulus borenumerous setae and masses of conida (Plates 3A,B). On bursting, conidia were liberated in the formof ooze. Mycelia were also detected in the endos-perm region (Plates 4A. B) just below the innertesta. The mycelium could grow directly into theinner layer of the testa surrounding the endos-perm, or could remain dormant until the seedgerminated. Infected parenchymatous tissues ofthe seed appeared distorted.Plate 3. (A) Tranverse section, (B) Whole mount (SEMmicrograph) of mature acervulus showingnumerous setae. (S· setae; MA· matureacervulus)It is evident from these studies that C.capsici was borne intraembryonal in chilli seeds.Disruption of seed tissues could be due to theactivity of cellulolytic and pectionlytic enzymesproduced by C. capsici (Sariah, 1980). Sporesoozing out from acervuli can serve as a primaryinoculum source for the spread of the disease.The fungus can then spread from the seed tothe placenta of the fruit. The pathogen could thenpenetrate the developing ovules, or young seedwith unlignified testa at any point on their surface.Infection of seeds could also occur directly fromPERTANIKA VOL. 11 NO.3, 1988 343SARIAH MEONPlate 4 Transverse section of infected seed showingthe presence of hyphae in the endospermregion (A) light microscopy and (B) SEMmicrograph (H - hyphae).the mother plant: through the pedicel, funiculusor integuments into the developing ovule. Also,conidia of C capsici could also be mechanicallyattached to the surface of the testa, remainingdormant until the seed germinated.With seed-borne inocula, the way in whichthe inoculum was carried within or on the seedsinfluenced the type of control measure used(Neergaard, 1979). Therefore, with internal seed-borne infection as was the case here, systemicfungicides will provide a more practical andeffective control measure. Besides anthracnoseinfected seeds should not be used for planting.REFERENCESGROVER, R.K. and R.D. BANSOD. (1970): Seed bornenature of Colletotrichum capsici in chilli seeds andits ~ontrol by seed dressing fungicides. IndianPhytopathol., 23: 664-668.HIGGINS, B.B. (1930): A pepper fruit rot new to theUnited States Georgia Expert. Stat. Bull., no. 162,10 pp. Rev. Appl. Mycol. 10: 56-57 (Abstract).HOLLIDAY, P. (1980): Fungus Disease of Tropicalcrops. Cambridge University Press. 607 pp.MAH, S.Y. (1987): Anthracnose fruit rot of chilli(Capsicum annuum. L): Some aspects of its etiology,epidemiology and control in Peninsular Malaysia.M. Agric. Sc. Thesis. Universiti Pertanian Malaysia.MORDUE, J.E.M. (1971): Colletotrichum capsici C.M.I.Discription of pathogenic fungi and bacteria No.317, Commonwealth Mycological Institute, Kew.NEERGAARD, P. (1979): Seed Pathology vols. 1 & 2.2nd edition. The Macmillan Press Ltd. London.840 pp & 843 pp. respectively.O'BRIEN, T.P., N. FEDER and M.E. MCCULLY. (1964):Polychromatic staining of plant cell walls bytoludine blue. Protoplasrrla 59: 368-373.ROUT. R.K. and G.C. RATH. (197 2): Note on seed-borne desease of chilli (Capsicum annuum, L.)Indian Phytopathol. 25: 5 97-598.SARIAH, M. (1980). Pectinolytic and cellulolytic enzymeproduction by Colletotrichum capsici. (Syd.) Butler& Bisby. Malay. Appl Bioi. 9: lOS-II O.SAXENA, A.K. I.J. HOSHI and J.B. SAXENA. (1982):Additions to the host range of Colletotrichumcapsici. India Phytopathol. 35: 166.(Received 29 April, 1988)344 PERTANIKA VOL. II NO.3, 1988

Seed-borne Infection and Development of Colletotrichum capsici

in Naturally Infected Chili Seed

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Seed-borne Infection and Development of Colletotrichum capsici in Naturally Infected Chili Seed

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