Antigenic characteristics as taxonomic criterion of differentiation of Alternaria spp., pathogenic for carrot and parsley

Abstract

Identification of Alternaria genus species is a very complicated process which demands broadly designed investigations and studying of great number of properties which together can be considered as satisfying taxonomic criteria. The main objective of these investigations was examining the possibilities of applying the antigenic characteristics of Alternaria spp. phytopathogenic fungi as a taxonomic criterion, as well as introducing the serological methods for their identification. Conducting the examination of Alternaria spp., pathogenic for Apiaceae plants in Serbia, several isolates were obtained and identified as Alternaria radicina, A. petroselini, A. dauci and A. alternata, based on the conventional mycological methods and host range, as well as on molecular detection and partial characterization. The investigation included 12 isolates from plant leaves, seeds and soil which were pathogenic mainly to carrot and parsley and were identified as A. radicina, A. petroselini, A. dauci and A. alternate. Investigated isolates were compared with each other, as well as with standard isolates for the mentioned species (a total of 5 isolates, originating from USA and EU). During the investigation of serological characteristics of Alternaria spp. firstly a polyclonal antiserum was prepared against one isolate from Serbia identified as A. dauci. This antiserum was specific to Alternaria genus while there was no reaction with antigens from other phytopathogenic fungi genera (Fusarium, Rhizoctonia and Agaricus). Antiserum titer, determined by slide agglutination test, was 1/32. Antigenic characteristics of Alternaria genus fungi were examined by Electro-Blot-Immunoassay serological method (EBIA, Western blot), i.e. their protein profiles were compared. Investigated Alternaria spp. isolates showed different protein band profiles in gel and on nitrocellulose paper, and the observed differences were in complete correlation with the results of the previous identification. All investigated isolates, both domestic and the standards, were similar to each other, and they could be correctly identified to the species level using EBIA. Besides grouping to the species level, antigenic characteristics indicated similarities and differences among the isolates within the same and different species, showing their complex relationships which properly reflect their diversity in nature. In all the previous investigations of Alternaria genus fungi up to now, there have been no data about their serological characteristics as possible taxonomic criteria. Introduction of this group of characteristics represents an important contribution both to the taxonomy and implementation of fast and accurate methods of phytopathogenic fungi identification.

Identifikacija vrsta u okviru roda Alternaria je veoma složen proces koji zahteva široko postavljena ispitivanja i proučavanje većeg broja osobina koje zajedno predstavljaju zadovoljavajuće taksonomske kriterijume. Osnovni cilj ovog rada je bio da se ispita mogućnost primene antigenih osobina fitopatogenih gljiva iz roda Alternaria kao taksonomskog kriterijuma, kao i uvođenje seroloških metoda za njihovu identifikaciju. Proučavajući gljive iz roda Alternaria koje su patogene za gajene biljke iz fam. Apiaceae u našoj zemlji, dobijeno je više izolata koji su, na osnovu proučavanja konvencionalnim metodama i kruga domaćina, kao i molekularne detekcije i delimične karakterizacije, razvrstani u četiri vrste: Alternaria radicina A. petroselini, A. dauci i A. alternata. U ispitivanja je bilo uključeno 12 izolata poreklom sa lista, semena ili iz zemlje, koji su ispoljavali patogenost prvenstveno prema mrkvi i peršunu i identifikovani da pripadaju vrstama A. radicina, A. petroselini, A. dauci i A. alternata. Ispitivani izolati su međusobno upoređivani kao i sa standardima za navedene vrste (ukupno 5 izolata, poreklom iz SAD i EU). Prilikom ispitivanja seroloških osobina Alternaria spp., prvo je pripremljen poliklonalni antiserum na izolat iz Srbije, koji je identifikovan kao A. dauci. Antiserum je ispoljio specifičnost za rod Alternaria, dok nije reagovao sa antigenima iz drugih rodova fitopatogenih gljiva (Fusarium, Rhizoctonia, Agaricus). Titar antiseruma, ispitivan metodom aglutinacije na pločici, bio je 1/32. Antigene osobine gljiva iz roda Alternaria proučavane su primenom elektroforetsko-adsorpciono-imunoenzimske serološke metode (EBIA, Western blot), odnosno upoređivani su njihovi proteinski profili. Ispitivani izolati Alternaria spp. ispoljili su različite profile proteinskih traka u gelu i na nitroceluloznom papiru, a uočene razlike su, u potpunosti, odgovarale rezultatima prethodne identifikacije. Navedeni izolati, domaći kao i standardi, ispoljili su međusobne sličnosti i mogli su biti pravilno identifikovane do nivoa vrste, primenom EBIA. Pored grupisanja do nivoa vrsta, antigene osobine su ukazale na postojanje sličnosti i razlika između izolata unutar istih, odnosno različitih vrsta, ukazujući na njihove veoma složene međuodnose koji verno oslikavaju diverzitet postojanja ovih gljiva u prirodi. U dosadašnjim proučavanjima gljiva iz roda Alternaria, ne postoje podaci o njihovim serološkim osobinama kao mogućim taksonomskim kriterijumima. Uvođenje ove grupe osobina, predstavlja značajan doprinos kako u taksonomiji, tako i u primeni brzih i tačnih metoda identifikacije fitopatogenih gljiva.