Morphological and molecular characterization of Colletotrichum coccodes isolated from pepper cultivated in Bulgaria and Macedonia

Abstract

Colletotrichum coccodes has been recognized as one of the causal agents of pepper fruit anthracnose in Bulgaria. Recently, this species has been recorded in pepper fruits in Macedonia. In Bulgaria, the fungus has also been isolated from roots of premature senescent pepper plants but in Macedonia it has not been isolated yet. The purpose of the investigation was to make comparative morphological, cultural and molecular characterization of C. coccodes isolates obtained from pepper fruits and roots in Bulgaria and Macedonia. Additionally, a technique was applied to differentiate among the C. coccodes isolates obtained from roots and other microsclerotia-producing fungi. On the host tissue, C. coccodes developed acervular conidiomata with cup-shaped fruiting bodies accompanied with dark-pigmented, unbranched, thick-walled sterile hyphae called setae. A slimy mass of hyaline, straight, unicellular, fusiform conidia appeared on nutrient media. In a short time, numerous small dark globose setose microsclerotia emerged in the colony starting from its centre and distributing proportionally throughout agar plates. Two PCR primer sets were used to sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions. Single products of ~450 bp and ~350 bp were amplified by the genus-specific (Cc1F1/Cc2R1) and the species-specific primers (Cc1NF1/Cc2NR1), respectively. Morphological, cultural and molecular characterization of the isolates from roots and fruits showed that root rot and fruit anthracnose of pepper were caused by one and the same causal agent determined as C. coccodes. The isolates from roots showed rapid mycelial growth, gave rise to numerous minute microsclerotia and produced conidia only on SSPA. SSPA supported significantly more mycelium growth and sporulation than all other media tested and could be recommended for production of large quantity of conidia. No pattern of genetic variation associated with the organ or geographic origin of the isolates was determined.Colletotrichum coccodes se javlja kao jedan od uzrocnika antraknoze u plodovima paprike u Bugarskoj. Nedavno je potvrdjeno njeno prisustvo i na plodovima paprike iz Makedonije. U Bugarskoj je ova gljivica izolovana iz korena prerano sazrelih biljaka ali u Makedoniji nisu zabelezena slicna zapazanja. Cilj ovog rada je da se izvrsi komparativna morfoloska, kulturoloska i molekularna karakterizacija izolata C. coccodes dobijenih iz plodova i korena paprike uzgajane u Bugarskoj i Makedoniji. Dalje, ova tehnika je primenjena za utvrdjivanje razlika izmedju izolata C. coccodes dobijenih iz korena i ostalih gljivica koje mogu da formiraju mikrosklerocije. Na tkivu domacina, C. coccodes je razvila acervularne konidiomate sa plodonosnim telom u obliku solje sa tamno obojenim, nerazgranatim, sterilnim hifama sa debelim zidom koje se zovu sete. Na hranljivoj podlozi, razvile su se prave, jednocelijske, vretenaste konidije sastavljene od sluzaste hijalinske mase. Za kratko vreme pojavio se velik broj sitnih, tamnih, globularnih, cekinjastih mikrosklerocija pocevsi od centra kolonije i proporcionalno se sireci preko agarne ploce. Dva seta PCR prajmera je koriscen za sekvencioniranje ribozomalnih internih (ITS1 i ITS2) regija. Pojedinacni odzivi na ~450 bp i ~350 bp su pojacani koriscenjem rod-specificnih (Cc1F1/Cc2R1) i vrsta-specificnih (Cc1NF1/Cc2NR1) prajmera, respektivno. Morfoloska, kulturoloska i molekularna karakterizacija izolata iz korena i plodova paprike je pokazala da je uzrocnik truleza korena i antraknoze plodova jedan te isti patogen, C. coccodes. Izolati iz korena su pokazali sposobnost brzog micelarnog rasta i stvaranja velikog broja sitnih mikrosklerocija a konidije su proizvodili samo na SSPA. Na SSPA podlozi je dobijen znacajno veci micelarni rast i intenzitet sporulacije u odnosu na ostale ispitivane podloge i moze se preporuciti za proizvodnju vecih kolicina konidija. Nije zapazena nikakva genetska varijacija u izolatima u odnosu na biljni organ iz kojeg su izolovani ili geografsko poreklo biljaka.