Field Reaction to Anthracnose Caused by Colletotrichum spp. on Pepper Fruits

Field Reaction to Anthracnose Caused by Colletotrichum spp. on Pepper Fruits R. Rodeva, Z. Stoyanova and R. Pandeva Institute of Genetics Bulgarian Academy of Sciences 1113 Sofia Bulgaria N. Petrov Plant Protection Institute National Service for Plant Protection Kostinbrod Bulgaria Keywords: Capsicum, Colletotrichum acutatum, Colletotrichum gloeosporioides, Colletotrichum coccodes, fruit disease, host resistance Abstract Anthracnose caused by Colletotrichum spp. was increasingly observed on pepper fruits in the experimental field of Institute of Genetics, Sofia, during the last several years. In our investigation it was established that the population of Colletotrichum is heterogeneous and at least 3 species (C. acutatum, C. coccodes and C. gloeosporioides) could be associated with pepper anthracnose in Bulgaria C. capsici was not found. The symptoms first appeared as small, watersoaked lesions that expanded rapidly. Fully expanded lesions were usually round and sunken and ranged in color from dark red to tan to black. Often multiple lesions formed on individual fruit. The disease could also be introduced into a crop on infested seed. During warm and wet periods, conidia were splashed by rain or irrigation water from diseased to healthy fruit. Diseased fruit acted as a source of inoculum, allowing the disease to spread from plant to plant within the field. A part of the existing at the Institute of Genetics pepper collection of Capsicum annuum cultivars and lines, C. baccatum var. pendulum accessions and interspecific hybrid progenies was evaluated for natural attack by Colletotrichum spp. All studied C. baccatum var. pendulum accessions and most of C. annuum C. frutescens hybrids showed high field resistance. The most susceptible were the cultivars Pazardjishki edar, ačvanka, C. annuum C. chinense hybrids 85/8, 94/2 and 35/2 6 2. INTRODUCTION In 2005 the project B-1515 financed by the Bulgarian National Science Fund started. In realization of this project directed to Botrytis cinerea and Sclerotinia sclerotiorum on pepper field inspections were made to survey for the fungal diseases caused by them. During the years anthracnose on pepper fruit was increasingly observed in the experimental field of Institute of Genetics, Sofia. The disease became a problem in seasons that were wet and warm. In 2007, the weather was highly conducive to the disease and pepper fruits were severely infected. Colletotrichum gloeosporioides (Penz.) Penz. & Saccardo in Penz., C. capsici (Syd.) E.J. Butler & Bisby, C. acutatum Simmonds ex Simmonds, C. coccodes (Wallr.) S.J. Hughes and C. dematium (Pers.) Grove are reported as causal agents of pepper anthracnose (anandhar et al., 1995; Roy et al., 1997; Lewis Ivey et al., 2004; Costa et al., 2006; Tozze Jr., 2007) and C. coccodes, C. dematium, and C. acutatum of tomato anthracnose (Dal Bello, 2000; Sanogo et al., 2003; Costa et al., 2006). In Bulgaria, C. capsici and C. phomoides (Sacc.) Chester (a synonym of C. coccodes) have been considered as the inciters of pepper and tomato anthracnose, respectively (Elenkov and Christova, 1978; Bahariev et al., 1992; Stancheva, 2000). Lately, C. acutatum has been reported on both vegetable crops (Jelev et al., 2008). The purpose of the investigation was to evaluate a part of the existing at the Institute of Genetics pepper collection for anthracnose appearance and severity under field conditions in order to find differences in susceptibility and to reveal the possible occurrence of more than one Colletotrichum species associated with disease symptoms. Proc. IV th Balkan Symp. on Vegetables and Potatoes Eds.: L. Krasteva and N. Panayotov Acta Hort. 830, ISHS 2009 557

ATERIALS AND ETHODS Plant material involved C. annuum cultivars (19 local and 18 foreign). In addition, accessions of C. baccatum var. pendulum (10) were included. Interspecific hybrids at different stages of stabilization were studied. ore important of them originated from the combinations of C. annuum var. annuum with C. chinense (25) and C. frutescens (5). Evaluation for natural attack caused by Colletotrichum spp. on pepper fruits was made under field conditions using a diagrammatic scale (Azevedo et al., 2007). Isolations were made from diseased pepper fruits with typical of anthracnose symptoms. Pieces were cut, surface-sterilized and plated onto petri dishes containing potato-dextrose agar (PDA). For the growth studies cultures were incubated at 25 C with 12 h dark/light periods in enclosed cabinet with fluorescent light. Digital images were recorded with a Canon PowerShot A95 digital camera. easurements were made with Carnoy program. At least 100 conidia of each isolate were measured on images. Identification of Colletotrichum spp. was made on the basis of morphological and cultural characters (conidial morphology, colony color, pigment production, growth rate, appressorium features, presence of: setae, teleomorph, microsclerotia) (Sutton, 1992; Freeman et al., 1998; Tozze Jr., 2006). The pathogenicity tests were performed with seven selected monoconidial cultures (5 obtained from pepper and 2 from tomato). They were grown on PDA and inoculated on green apples, as described by Talhinhas et al. (2008). Wounded and notwounded pepper fruits of highly susceptible accession 85/8 were inoculated with mycelial disc or with 20 μl of the conidial suspension (concentration approximately 1 x 10 5 conidia/ml). Control fruits were inoculated with 20 μl sterile distilled water or with sterile PDA disc. Fruits were incubated for 7 days at 25 C under 100% relative humidity. olecular characterization of the Colletotrichum isolates was made at the Plant Protection Institute, Kostinbrod. The isolates were grown in glucose-casein broth (Sreenivasaprasad et al., 1992). DNA was extracted following the procedure described by Freeman et al. (2000). Universal primers ITS1 and ITS4 and specific for C. acutatum CaInt2 (Sreenivasaprasad et al., 1996) and for C. gloeosporioides CgInt (ills et al., 1992) each in conjunction with the primer ITS4 have been used for identifying Colletotrichum species. RESULTS Fruit rot was the most important symptom although the disease could damage other parts of the plant. Fruit symptoms initially began as water-soaked lesions that became soft and slightly sunken. Lesions progressively increased in number on the fruit, coalesced and quickly covered most of the fruit surface. Fully expanded lesions were usually round and sunken and ranged in color from dark red to tan to black. The lesion surface became covered with the wet, gelatinous conidia from fungal fruiting bodies (acervuli). The seeds of colonized fruits were also infected. Small dark microsclerotia were observed on and inside some of diseased pepper fruits. During warm and wet periods, conidia were splashed by rain or irrigation water from diseased to healthy fruit. Diseased fruit acted as a source of inoculum, allowing the disease to spread from plant to plant within the field. The used diagrammatic scale for field evaluation allowed to divide the accessions into three groups by the diseased area: 1) fruits apparently sound, symptomless (0% diseased area); 2) fruits with 1 to 5 lesions, with diseased area ranging from 1 to 20%, and lesion diameter <0.5 cm; 3) fruits with more than 20% of diseased area, with one or more lesions with >0.5 cm of diameter (Table 1). Anthracnose incidence and intensity differed among the accessions. The most of accessions under study were added to the second group. All C. baccatum var. pendulum accessions and most of C. annuum C. frutescens hybrids showed high field resistance. The most susceptible were the cultivars Pazardjishki edar, ačvanka, C. annuum C. chinense hybrids 85/8, 94/2 and 35/2 6 2. Isolates obtained were determined as C. acutatum, C. gloeosporioides and C. coccodes based on phenotypic characterization (cultural morphology, conidial size and 558

morphology, growth rate on PDA, microsclerotial production) (Sutton, 1992) and pathogenicity test. C. coccodes was easily separated from C. acutatum and C. gloeosporioides as a distinct species on the basis of abundant microsclerotia and scarce mycelium production. C. capsici forming conidia falcate, fusiform, gradually tapered towards each end, ranging from 18 to 23 μm in length and 3.5 to 4 μm in width was not found in our investigation (Sutton, 1992). C. acutatum and C. gloeosporioides developed morphologically similar gray colonies and it was difficult to differentiate them on the basis of the phenotype only. The presence of setae was observed in one isolate on PDA. None of the isolates under study formed the teleomorph in culture. The isolates producing microsclerotia and determined as C. coccodes (1 from pepper and 1 from tomato) expressed low pathogenicity on green apple fruits. C. acutatum isolates produced large brown lesions with scattered acervuli protruding the peel surface and abundant salmon-colored conidial mass. C. gloeosporioides isolates incited large brown to dark brown lesions slightly sunken and partially covered with light grey mycelium. The pathogenicity test carried out on pepper showed that all isolates under study caused severe infection especially on pin-wounded fruits irrespective of inoculum applied (mycelial disc or suspension). The least aggressive C. coccodes was more commonly found in 2005 and 2006 but primarily at the end of growing season. In warm and wet 2007, C. acutatum was the prevalent species. Reisolations were successfully made from inoculated apple and pepper fruits but not from control one. A single DNA fragment of approximately 590 kb was amplified for 8 isolates using the universal primers ITS1 and ITS4. PCR with C. acutatum-specific primer, CaInt2, confirmed the identity of 5 isolates (4 isolates from pepper and 1 from tomato) (Fig. 1). Two other isolates reacted with C. gloeosporioides-specific primer, CgInt (Figure is not shown). A part of isolates, however, did not react with any of the primers tested. DISCUSSION The anthracnose symptoms caused by Colletotrichum spp. on pepper fruits were similar and microscopic analysis was necessary to identify the species. The lesions from which C. gloeosporioides was isolated were indistinguishable from those caused by C. acutatum. Both of these fungi were found on fruit with C. gloeosporioides causing disease on ripe fruit and C. acutatum causing disease on both immature and ripe fruit (Lewis Ivey et al., 2004). C. coccodes also was found on pepper fruits with similar symptomatology at first stage of lesion development, however, this species had different micromorphology producing abundantly microsclerotia in fruit tissue as well as in culture (Costa et al., 2006; Tozze Jr. et al., 2007). C. capsici forming falcate conidia was not found in our investigation although this species was pointed as the only inciter of pepper anthracnose in Bulgaria before 2008 (Elenkov and Christova, 1978; Bahariev et al., 1992; Stancheva, 2000). C. coccodes and C. gloeosporioides were not reported on pepper in Bulgaria up to now but former was shown as the inciter of tomato anthracnose using an older synonym C. phomoides. C. acutatum has already been reported by other authors in Bulgaria (Jelev et al., 2008) and confirmed in our study. The severe form of anthracnose caused by this species on immature and mature pepper fruits is an emerging disease that may threaten the profitability of pepper crops in areas where it becomes established (Lewis Ivey et al., 2004). The phenotypic and genotypic characteristics in our investigation showed that the population of Colletotrichum is heterogeneous and at least 3 species could be associated with pepper anthracnose in Bulgaria. A number of examples are known where more than one Colletotrichum species is associated with the same host, and a single Colletotrichum species is able to infect a range of hosts (Freeman et al., 1998). The role and relative importance of C. acutatum in pepper fruit anthracnose and co-occurrence with other Colletotrichum species in Bulgaria need to be defined more precisely. Knowledge of the predominant Colletotrichum spp. and their distribution in the country is essential for development of the most effective control strategies. Since no cultivars have shown to be highly resistant those of them demonstrating 559

at least moderate resistance could be chosen. All studied C. baccatum var. pendulum accessions and most of C. annuum C. frutescens hybrids showed high field resistance. The most susceptible were the cultivars Pazardjishki edar, ačvanka, C. annuum C. chinense hybrids 85/8, 94/2 and 35/2 6 2. Reliable level of resistance to anthracnose was reported in some line of C. baccatum, C. chinense and C. frutescens (Black and Wang, 2007; Kim, 2007; Henz et al., 2007). Availability of stable resistant source is an important prerequisite to any disease resistance program. C. baccatum var. pendulum as a group seemed to be the most promising species. ACKNOWLEDGEENTS Financial support by the Bulgarian National Science Fund (B-1515) is gratefully acknowledged. Literature Cited Azevedo, C.P., Henz, G.P. and Filho, A.C. 2007. Anthracnose scale for field evaluations. In: D.G. Oh and K.T. Kim (eds.), Proc. First Intl. Symposium on Chili Anthracnose. Convention Center, Seoul Natl. Univ., Korea. Bahariev, D., Velev, B., Stefanov, S. and Loginova, E. 1992. Diseases, weeds and pests on vegetavle crops. Zemizdat, Sofia (in Bulg.). Black, L.L. and Wang, T.C. 2007. Chili Anthracnose Research at AVRDC 1993-2002. In: D.G. Oh and K.T. Kim (eds.), Proc. First Intl. Symposium on Chili Anthracnose. Convention Center, Seoul Natl. Univ., Korea. Costa,.H.D., Pfenning, L.H. and Pozza, E.A. 2006. Occurrence of Colletotrichum coccodes, a pathogen of Solanaceae in Brazil. Fitopatologia Brasileira 31:315. Dal Bello, G.. 2000. First Report of Colletotrichum dematium on tomato in Argentina. Plant Disease 84:198. Elenkov, E. and Christova, E. 1978. Diseases and pests on vegetable crops. Ch.G. Danov, Plovdiv (in Bulg.). Freeman, S., Katan, T. and Shabi, E. 1998. Characterization of Colletotrichum species responsible for anthracnose diseases of various fruits. Plant Disease 82:596 605. Freeman, S., Shabi, E. and Katan, T. 2000. Characterization of Colletotrichum acutatum causing anthracnose of anemone (Anemone coronaria L.). Applied Environmental icrobiol. 66:5267 5272. Henz, G.P., Reis, A., Filho, A.C.C. and Boiteux, L.S. 2007. Present situation of the anthracnose disease in sweet and hot pepper in Brazil and search for sources of resistance. In: D.G. Oh and K.T. Kim (eds.), Proc. First Intl. Symposium on Chili Anthracnose. Convention Center, Seoul Natl. Univ., Korea. Jelev, Z.J., Bobev, S.G., inz, D., aymon,. and Freeman, S. 2008. First report of anthracnose fruit rot caused by Colletotrichum acutatum on pepper and tomato in Bulgaria. Plant Disease 92:172. Kim, B.C. 2007. Country report of anthracnose research in Korea. In: D.G. Oh and K.T. Kim (eds.), Proc. First Intl. Symposium on Chili Anthracnose. Convention Center, Seoul Natl. Univ., Korea. Lewis Ivey,.L., Nava-Diaz, C. and iller, S.A. 2004. Identification and management of Colletotrichum acutatum on immature bell peppers. Plant Disease 88:1198-1204. anandhar, J.B., Hartman, G.L. and Wang, T.C. 1995. Semiselective medium for Colletotrichum gloeosporioides and occurrence of three Colletotrichum species on pepper plants. Plant Disease 79:376 379. ills, R.P., Sreenivasaprasad, S. and Brown, A.E. 1992. Detection and differentiation of Colletotrichum gloeosporioides isolates using PCR. FES icrobiol. Lett. 98:137 144. Roy, K.W., Killebrew, J.F. and Ratnayake, S. 1997. First report of Colletotrichum capsici on bell pepper in ississippi. Plant Disease 81:693. Sanogo, S., Stevenson, R.E. and Pennypacker, S.P. 2003. Appressorium formation and tomato fruit infection by Colletotrichum coccodes. Plant Disease 87:336 340. 560

Sreenivasaprasad, S., Brown, A.E. and ills, R.P. 1992. DNA sequence variation and interrelationships among Colletotrichum species causing strawberry anthracnose. Physiol. ol. Plant Pathology 41:265 281. Sreenivasaprasad, S., Sharada, K., Brown, A.E. and ills, R.P. 1996. PCR-based detection of Colletotrichum acutatum on strawberry. Plant Pathol. 45:650 655. Stancheva, J. 2000. Atlas of agricultural crop diseases. I. Vegetable crop diseases. Pentsoft, Sofia-oskow (in Bulg.). Sutton, B.C. 1992. The genus Glomerella and its anamorph Colletotrichum. p.1 26. In: J.A. Bailey and.j. Jeger (eds.), Colletotrichum. Biology, Pathology and Control. CAB Intl., Wallingford, Oxon, UK. Talhinhas, P., uthumeenakshi, S., Neves-artin, J., Oliveira, H. and Sreenivasaprasad, S. 2008. Agrobacterium-mediated transformation and insertional mutagenesis in Colletotrichum acutatum for investigating varied pathogenicity lifestyles. ol. Biotechnol. 39:57 67. Tozze Jr., H.J., ello,.b.a. and assola Jr., N.S. 2006. orphological and physiological characterization of Colletotrichum sp. isolates from solanaceous crops. Summa Phytopathologica 32:71 79. Tozze Jr., H.J., Gioria, R., Suzuki, O., Brunelli, K.R., Braga, R.S. and assola Jr., N.S. 2007. Natural occurrence of Colletototrichum coccodes (Wallr.) Hughes causing anthracnose on pepper (Capsicum annuum L.) in Brazil. Summa Phytopathologica 33:419. Tables Table 1. Field reaction of Capsicum accessions to anthracnose caused by Colletotrichum spp. on pepper fruits. Diseased area 1 (0-8 scale) Class 0 0 1-20% 1-4 ore than 20% 5-8 Accessions 1 Based on a 0 8 scale (Azevedo et al., 2007). C. baccatum var. pendulum accessions (1, 2, 3, 4, 5, 6, 7, 8, 10, 65), C. annuum C. frutescens hybrids (3 1 /4 7 1, 3 1 /4 7 2, 3 1 /4 12 4, 3 2 /7 1 3, Royal purple, Fiesta, inito, Zulu, ulato, Rouge Long Ordinaire, Rino, Clovis, Doux Long des Landes, Hebar, Fitostop, Zlaten medal, Albena, Pazardzishka kapiya, Kurtovska kapiya, Biala kapiya, Sofiiska kapiya, Kalinkov, Vibo, Gorogled, Sivriya, Chorbadziiski, Kozi rog, Palanačka kapija, Duga bela, Župska rana, C. annuum x C. chinense hybrids (4/1, 4/2, 4/2 1, 4/3, 4/3 1, 6/1, 6/2, 6/3, 7/2 2, 85/3 1, 85/4 1, 85/9 1, 85/9 2, 35/2 1, 35/2 1 4, 35/2 6 4, 35/3 6, ХІІ 3, ХІІ 11, ХІІ 24, XII 25, ХІІ 26) Pirin, Buketen, Bulgarian ratund, Fehërëson, Fehërëson synthetic, Striama, C. annuum x C. frutescens hybrid (3 2 /7 5 4 ІІ), Tondo picante calabrese, Yolo Wonder, Pazardjishki edar, ačvanka, C. annuum C. chinense hybrids (85/8, 94/2, 35/2 6 2 ) 561

Figures 1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 6 7 8 9 10 11 12 13 590 bp 490 bp Fig. 1. PCR of the ribosomal region, which includes the two non-coding ITS1 and ITS2, and the 5.8S rrna gene, amplified with primers ITS1, ITS4 and CaInt2. Columns 4, 6, 8, 9 and 11 correspond to Colletotrichum acutatum isolates and 7, 12 and 13 to Colletotrichum spp. All isolates were obtained from pepper except 6 and 7 isolated from tomato. = marker 100 bp-dna ladder. Agarose gel 1.8%. 562