° MeI)YHapo"HM CMMn03MjYM aKTyenHMM TpeH"oBMMa y 3aWTMTM 6Mlba 2S· 28. cenTeM6ap 2012 6eorpaA, Cp6Mja 360PHMK PAAOBA "International Symposium on Current Trends in Plant Protection" 2S - 28111 September, 2012 Belgrade, Serbia PROCEEDINGS MHCTMyYT 3a 3awTMTY 6Mlba M)KMBOTHY ｃｐｾｍｈｙ＠ M3 6eorpa,.a Institute for Plant Protection and Environment, Belgrade Ro itza Rodeva. Ilija Karov, Zornitsa Stoyanova •... International Symposium: Current Trends in Plant Protection Proceedings 257 UDK: 633.842-24(497.7) PHOMOPSIS CAPSICIAND COLLErOrHICHl/M COCCOIPES INFECTING PEPPER IN MACEDONIA 1 2 2 ROSSITZA RODEVA , ILilA KAROV • ZORNITSA STOYANOVA I, BllJANA KOVACEVIK , V ASILISSA MANOV A', RALITSA GEORGIEV A' phaseolina. O. Gerais. , J., Simpson. J. (phaseolus vilgaris drought stress. lA. (2007): of pathegenicity. Diseases and resistance in 76.646-649. with chlorate Ilnstitute of Plant Physiology and Genetics, 1113 Sofia. Bulgaria 2Goce Delcev University-Stip. Macedonia Phomopsis capsici and Colletotrichum coccodes were found on pepper fruits during a joint expedition carried out in Macedonia. The lesions caused by P. capsici often occurred together and resembled slightly those incited by C. coccodes. Phomopsis lesions could be differentiated on the basis of pliable leathery condition of the affected tissue and of pycnidium presence while C. coccodes produced lesions with regular round shape and abundant acervuli. setae and microsclerotia in coloni1-ed fruit tissue. On some fruits P. capsici caused single infection but mixed infections of Phomopsis and Colletotrichum were observed. as well. C. coccodes is a soil-borne pathogen that produces long-lasting structures (microsclerotia) in the plant debris. The development of this pathogen on pepper might contribute to the building up of inoculum in the soil which could serve as reservoir for other Solanaceae. To our knowledge, this is the first report of P. capsici and C. coccodes on pepper in Macedonia. Variations of hem Iran. Australian Key words: Capsicum annuum, Colletotrichum coccodes, pepper anthracnose, Phomopsis capsici, fruit decay prijatnimi uslovijami INTRODUCTION 12 (I). University of Last years, Phomopsis capsici (Magnaghi) Sacc and several Colletotrichum 'pp. (Colletotrichum gloeosporioides (Penz.) Penz. & Saccardo in Penz., C. acutatum Simmonds ex Simmonds and C. coccodes (WaUr.) SJ. Hughes) occurred on pepper in Bulgaria with increasing frequency (Rodeva et aI., 2009a; 2009b; 2009c), In August 2011 a joint expedition was carried out in Macedonia related to the implementation of ERA 226 project. Two new pepper fungal pathogens were found, isolated, described and characterized. The results are presented in this paper. ,peratures and mineral rophomina phaseolina Biological Control of X'ugoslavia. September s of mineral nutrition. (in Serbian). lnicijevic. M. (2011): eetroot in 2010. Plant ;ugar beet root rot in 2, Brussels. Belgium, MATERIAL AND METHODS Initial isolations from diseased pepper fruits on potato dextrose agar (PDA) revealed the presence of P. capsici and C. coccodes. Four Macedonian (MK26.1, MK26.2, MK7.1, MK7.2) and one Bulgarian (B8.1) isolates of C. coccodes were selected for the investigations. Identification of Colletotrichum spp. was performed on the basis of morphological and cultural characteristics (conidial size and morphology, colony morphology and growth 258 Phomopsis capsici and Colletotrichum coccoides infecting pepper in Macedonia rate, presence or absence of: teleomorph, setae, microsclerotia) (Sutton, 1992; Freeman et aI., 1998; Tozze Jr. et aI., 2006) and pathogenicity tests. Growth rate and colony appearance were studied on three nutrient media: PDA, 0.2% malt extract agar (MEA) and oatmeal agar (OA), which were inoculated with mycelial discs taken from the edge of growing colonies. For the pathogenicity tests the isolates were grown on PDA. Pin pricked detached pepper fruits were inoculated with agar plugs containing fungal mycelium. Control fruits were inoculated with sterile PDA discs. Tomato and eggplant fruits were additionally inoculated with C. coccodes for comparison. Fruits were incubated for 7 days at 25°C under 100% relative humidity. Reisolations were made at the end of the experiments. At leasl 100 conidia of each isolate were measured on the images with Carnoy program. Total DNA of investigated Colletotrichum isolates was extracted directly from mycelium by DNeasy Plant mini kit (Qiagen, Hilden, Germany). PCR amplifications were performed with both Colletotrichum-specific primer set CciFlICc2RI and C. coccodesspecific nested primers CclNFlICc2NRl. The gels were visualized by UV transillumination, their electronic images were taken by ImageQuant ISO imager (GE Healthcare) and densitometrically analyzed with ImageQuantTL7 software (GE Healthcare) to determine the approximate length of the resulting PCR products. RossilZa Rodeva, I The C. coccodes colonie rate was recorded on OA lowest - on MEA (39.0±7.6 mm) Macedonian ones on all nutrient ro e nuance mainly in the great number of microsclerotia RESULTS C. coccodes was isolated mainly from fruits, seeds of heavily infected fruits and occasionally from roots although it could infect stems and leaves. The disease symptoms were observed in the area of Kochani (village Dolni Pod log) on variety Kurtov ka kapija and in Strurnica (village Bosilovo) also in the postharvest period. Fruit anthracnose appeared first as small, circular, slightly sunken lesions on the surface of ripening fruits (Fig. la). Majority of infections were observed on ripe or over-ripe fruits. The spots quickly enlarged in concentric circles, coalesced, become deeply sunken with dark brown border and developed a water-soaked appearance directly beneath the skin (epidermis) of the fruit (Fig. I b). At first small rounded acervuli containing rose conidial mass were developed on the surface and beneath the lesion (Fig. Ic,e). Later then the fungus formed small, dark survival structures called sclerotia (Fig. Id). Fig. 2. CoLLetotrichum coccodes: malt extract agar (b) and oatmeal Fig. 1. Colletotrichum coccodes: Symptoms of anthracnose on pepper fruits, early infection (a); coalescent lesions (b); young lesions with sporulating acervuli (c); fully developed Ie ions with rnicrosclerotia (d); sporulating acervuli (e) Rossitza Rodeva, I1ija Karov, Zomitsa Stoyanova, ... 259 The C. coccodes colonies were slowly growing. On the ninth day the high t growth rate was recorded on OA (49.5±5.l mm) and PDA (47.8±4.8 mm) (Fig. 2c,a) and the lowest - on MEA (39.0±7.6 mm) (Fig. 2b). Bulgarian isolate had higher growth rate than Macedonian ones on all nutrient media used in the study. The colony colour w gray with ro e nuance mainly in the periphery, where acervuli with conidia developed. With aging a great number of microsclerotia appeared under mycelium. Fig. 2. Colletotrichum coccodes: Appearance of 10 days old colonies potato dextrose agar (a). malt ex.tract agar (b) and oatmeal agar (c) (left and middle column Macedonian isolates, right column - Bulgarian isolate) 260 Phomopsis capsici and Colletotrichum coccoides infecting pepper in Macedonia Conidia were hyaline, straight, cylindrical, aseptate with two to seven oil globules measuring (19.2) 21.3±1.7 (24.6) x (3.1) 4.l±OA (4.7) 11m (Fig. 3a). Acervuli with elae longer than 100 11m developed (Fig. 3b). Ros iua Rodeva, IIija Karov, PCR amplification with of -450 bp in all isolates (Cullen at aI., 2002) (Fig. SA). single specific PCR band of containing as a template DNA Fig. 3. Colletotrichllm coccodes: Conidia (a) and acervulus with conidiophores, conidia and setae (b) (Scale bars = 10 11m) All investigated C. coccodes isolates were pathogenic for pepper, tomato and eggplant (Fig. 4a,b,c). Water-soaked circular lesions appeared three days after in eulalion (dai) that became soft and slightly sunken. Wet, gelatinous conidi.aI mass from fungal fruiting bodies (acervuli) gradually covered the lesions. About IO - 14 dai the central lesion part darkened where abundant microsc1erotia developed. Fig. 4. Colletotrichwn coccodes: Symptoms on artificially inoculated pepper (a), tomato (b) and eggplant (c) fruits - 14 days after inoculation Fig. 5. Molecular identification of with primers CclFlICc2RI ; gel CcINFI/Cc2NRI; Lanes 2-13 (C. MK7.l, MK7.2 and C. sp. isolates water); lanes I: Ro itza Rodeva, l\ija Karov, Zornitsa Stoyanova, ... 261 peR amplification with genus-specific primers (CcIFlICc2RI) gave a single band of -4S0 bp in all isolates analyzed (c. coccodes and C. sp.) as expected from the literature (Cullen at aI., 2002) (Fig. SA). However, with the nested primer set CcINFlICc2NRI, a single specific PCR band of expected size (-3S0bp) was obtained only in those reactions containing as a template DNA from investigated C. coccodes isolates (Fig. S8). and Fig. S. Molecular identification of different Colletotrichllm species: Gel (A): PCR amplification with primers CclFIICc2RI; gel (B): PCR amplification with C. coeeodes-specific primers CcINFl/Cc2NR I; Lanes 2-13 (c. coccodes isolates B8.1, B2.1, B40.la, MK26.1, MK26.2, MK7.1. MK7.2 and C. sp. isolates B27, BI.I, B29,S2, S3); lanes 14: Negative controls (mQ water); lanes 1: DNA marker GeneRuler I kb+ (Fermentas). 262 Phomopsis capsid and Colletotrichwn coccoides infecting pepper in Macedonia P. capsici was found in the village Zubovo, Strumica region, on pepper fruits cv. Zubovska kapija (domestic pepper variety of Kurtovska kapija, which is grown only in this village). Until now P. capsici was not recorded on pepper anywhere else in the country. The symptoms of P. capsici on the fruits appeared as brown rot extending in wavy rings more rapidly longitudinally than laterally in the tissue (Fig. 6a). Infection progress led to fruit decay. The dead tissue became dry and bleached in the centre where black globose to subglobose subepidermal or erumpent pycnidia were noticed. White felt-like mycelium developed inside the damaged fruits. The fungus was i olated not only from pericarp but also from seeds of disea ed pepper fruits. some fruits P. capsid CoLletotrichum were observed To our knowledge, this Macedonia. Recently. C. a\., 2008) . C. coecodes is an structures (microsclerotia) in includes pepper, tomato and The outbreak of this pathogen the soil serving as an important Financial support of acknowledged . Fig. 6. Phomopsis capsid: symptoms (a), colony morphology (b) and alpha and beta conidia (c) P. capsici de eloped fast growing colonies on PDA (Fig. 6b). They were initially white, later developrng light to dark brown patches and reached the Petri dish border' 7 day after inoculatio n. S mall bla k pycnidia (150-250 ｾｭＩ＠ devel ped after 10-12 days. The extruded conidia were vi ible as y lIo wi h globo. e sli me. The reverse s ides of the colonies were grayi h with darker regions coinciding with o nidiomata. The i olates produced abundan t al pha conidia - urUcellular. traight. ovoid to oblo ng-fusoid, hyaline, biguttulate, with averrage dimension 6.8 x 2.9 1ffi1. scarce beta conidia which are unicellular, curved or hamate, e guttulate with averrage dimensions 29. 8 x 1. J..lm and very rarely gamma conidia - unicellular, traight, paddle hape. multiguttulate. with averrage dimensions 11.0 x 2.9 Ilm (Fig. 6c). The first o ne nly are viable and infective. No perithecia were found on the over win ter d di eased pepper fruits or in ullure. Artificial inoculation of detached pepper fru its led to ucce fu l infection ten day after inoculation. DISCUSSION Anthracnose of pepper caused by C. coccodes appeared to be a devastating di ease of ripe fruits cau ing severe damages to both field and post harvest levels in warm and rainy seasons. The infections occurred on green fruits but symptoms were visible after the ripening . During the season the pathogen was spread from infected to healthy fruits with conidia splashed by rain. overhead irrigation, or by picking fruit from wet plants. The lasting structures called sclerotia could survive in soil for up to three years and ｣ ｡ｵ ｾ＠ infections either directly or by producing secondary spores. The lesions caused by P. capsici often occurred together and resembled slightly those resulting from infection by C. coecodes. Phomopsis lesions could be differentiated on the basis of pliable leathery condition of the affected tissue and of pycnidium presence while C. coeeodes produced lesions with regular round shape and abundant microsclerotia in colonized fruit tissue. On CuUen. D. W .• Lees, A. K.. coccodes from soil and Plant Pathology, 51: 281 Freeman. S.• Katan, T. Shabi. E. for anthracnose diseases Rodeva. R.. Pandeva, R., caused by Phomopsis Rodeva. R., Stoyanova, Z., caused by Colletotrichum 562. Rodeva, R .• Stoyanova, Z., pepper fruits in Bulgaria. Sulton. B. C. (1992) : The Colletotrichum. Biology, CAB lnt!.. Wallingford, Tozze Jr., H. J .• Mello, M. B. A.. characterization of Phytopathologica, 32: 71 TrkuJja. V. , StojCic. 1., Brkljat. G., mogucnosti njegova sUlbijar I Macedon ia Rossitza Rodeva, Ilija Karov, Zornitsa Stoyanova, ... 'er fruits cv. only in this the country. wavy ri ngs Igress led to ( globose to ｾ＠ mycelium ;>ericarp but some fruits P. capsici caused single infection but mixed infections of Phomopsis and Colletotrichum were observed, as well. To our knowledge, this is the first report of C. coccodes and P. capsici on pepper in Macedonia. Recently, C. coccodes has been reported in Bosnia and Herzegovina (Trkulja et aI., 2(08). C. coccodes is an important soil-borne pathogen that produces long-lasting structures (microsclerotia) in the dying plant parts, with host range in Solanaceae that includes pepper, tomato and eggplant, causing anthracnose and potato, causing black-dot. The outbreak of this pathogen on pepper can lead to an enrichment of inoculum density in the soil serving as an important source of inoculum for other solanaceous crops. ｾｲ･＠ ACKNOWLEDGEMENTS Financial support of SEE-ERA.NET PLUS project ERA 226 is gratefully acknowledged. REFERENCES conidia (c) initially borders 7 2 day . The he colonies ; produced biguttulate, " curved or rna conidia 11.0 x 2.9 -und on the pepper I 263 Cullen, D. W., Lees, A. K., Toth, I. K., Duncan, J. M. (2002). Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real-time PCR Plant Pathology, 51: 281-292. Freeman, S., Katan, T. Shabi, E. (1998). Characterization of Colletotrichwn species responsible for anthracnose diseases of various fruits . Plant Disease, 82: 596-605. Rodeva, R, Pandeva, R., Stoyanova, Z. (2009a) . A new fruit disease of pepper in Bulgaria caused by Phomopsis capsici. Acta Horticulturae (ISHS), 830: 551-556. Rodeva, R., Stoyanova, Z., Pandeva, R, Petrov, N. (2009b). Field reaction to anthracnose caused by Colletotrichum spp. on pepper fruits . Acta Horticulturae (ISHS), 830: 557562. Rodeva, R, Stoyanova, Z., Pandeva, R. (2009c). Occurrence of Colletotrichum coccodes on pepper fruits in Bulgaria. Plant Protection (Skopje), 20: 65-69 . Sutton, B. C. (1992): The genus Glomerella and its anamorph Colletotrichum. In: Colletotrichum. Biology, Pathology and Control. J. A. Bailey and M. J. Jeger (eds.), CAB Inti., Wallingford, Oxon, UK, pp. I-26. Tozze Jr., H. J., Mello, M. B. A., Massola Jr., N. S. (2006). Morphological and physiological characterization of Colletotrichum sp. isolates from solanaceous crops. Summa Phytopathologica, 32: 71-79. Trkulja, V., ｓｴｯｪｾｩ｣Ｌ＠ 1., Brkljac, G., Zavisic, N. (2008). Pojava Colletotrichum coccodes u BiH i mogucnosti njegova suzbijanja. Glasnik zastite bilja, 31: 43-53.