Molecular identification of fungi isolated from diseased grapevine wood in Croatia

Bolesti drva vinove loze, koje uzrokuju fitopatogene gljive sve više dobivaju na važnosti zbog značajnih šteta koje nanose vinovoj lozi. U Hrvatskoj postoji mali broj istraživanja bolesti drva vinove loze. Tijekom 2013., 2014. i 2015. godine sakupljeni su uzorci drva vinove loze iz 5 vinograda na lokalitetima koji su obuhvaćali Dalmaciju, Središnju Hrvatsku i Slavoniju a koji su pokazivali simptome koji se povezuju sa gljivičnim bolestima drva vinove loze. Iz uzoraka su dobivene čiste kulture gljiva te su grupirane prema svojim kulturalnim i morfološkim karakteristikama. Iz svake od navedenih grupa izolata, odabrani su reprezentativni izolati koji su podvrgnuti analizi molekularnim metodama s ciljem njihove identifikacije do razine vrste. Navedenim reprezentativnim izolatima izolirana je ukupna genomska DNA, te su metodom MSP-PCR, uporabom početnice M13 i korištenjem horizontalne gel elektroforeze, dobiveni DNA fingerprint profili. Na temelju analize navedenih DNA fingerprint profila, dio izolata bilo je moguće identificirati do razine vrste temeljem usporedbe njihovih DNA fingerprint profila s odgovarajućim profilima otprije identificiranih vrsta poznatih patogena drva vinove loze. Također, navedena analiza omogućila je provjeru pouzdanosti grupiranja izolata prema morfološkim karakteristikama, a unutar navedenih grupa, prema podudarnosti DNA fingerprint profila, bilo je moguće dodatno razlučiti podgrupe unutar kojih su odabrani reprezentativni izolati za daljnju identifikaciju na temelju molekularnih markera ITS i EF1-. Identifikacija izolata na temelju navedenih molekularnih markera provedena je metodom PCR korištenjem parova početnica ITS5/ITS4, odnosno EF728/EF986. Produkti PCR reakcije su sekvencirani, a dobivene DNA sekvence uspoređene su sa odgovarajućim sekvencama referentnih izolata u međunarodnoj banci gena GenBank korištenjem metode BLAST a po potrebi provedena i njihova filogenetska analiza. Na temelju dokaza dobivenih kombinacijom navedenih metoda, izolati su identificirani, a utvrđene su sljedeće vrste: Diplodia mutila, Botryosphaeria dothidea, Diplodia seriata, Phomopsis ampelina, Diaporthe eres, Sordaria fimicola, Valsa leucostoma, Schizophyllum commune, Stereum hirsutum, Kalmusia variispora.Grapevine trunk diseases caused by fungi are gaining importenance due to significant damage which thay inflict on grapevines. There is small number of researches on grapevine trunk diseases in Croatia. During 2013., 2014. and 2015. samples from five regions, encompassing Dalmatia, Central Croatia and Slavonia, whych exhibited simptoms associated with fungal grapevine trunk diseases, have been collected. From the samples, clean cultures of fungi have been obtained and grouped based on their cultural and morfological characteristics. From each of those groups, representative isolates have been chosen for analisis with molecular methods, and to be identified up to species level. From those representative isolates, complete genomic DNA has been isolated and with method MSP-PCR and primer M13, thrue visualisation by horizontal gel electrophoresis, DNA fingerprint profiles have been obtained. Based on analisis of those DNA fingerprint profiles, for part of isolates, identification up to the level of species was possible due to comparison of their DNA fingerprint profiles with adequate profiles already identified species of known grapevine trunk pathogens. Furthermore, mentioned analisis provided reliability check for grouping based on cultural nad morfological characteristics, and inside those groups, per compatibility of DNA fingerprint profiles, further distinction of subgroups was possible and inside whych representative isolates have been chosen for further identification based on molecular markers ITS and EF1-. Identification of isolates based on molecular markers has been conducted with method PCR by using pairs of primers ITS5/ITS4, or EF728/EF986. Products of PCR reaction have been sequenced and obtained DNA sequences have been compared to corresponding sequences of reference isolates in international gene bank GenBank with method BLAST and if neaded phylogenetic analisis. Based on evidence obtained in combination of mentioned method, isolates have been identified, those species were: Diplodia mutila, Botryosphaeria dothidea, Diplodia seriate, Phomopsis ampelina, Diaporthe eres, Sordaria fimicola, Valsa leucostoma, Schizophyllum commune, Stereum hirsutum, Kalmusia variispora