Methods for Detection of Phytophthora fragariae var. rubi on Raspberry

Abstract

Phytophthora fragariae var. rubi (Wilcox & Duncan), a causal agent of raspberry root rot, is a serious soil-borne pathogen listed by EPPO as an A2 quarantine pest. Root samples were collected from badly diseased raspberry plants showing a variety of characteristic and often dramatic symptoms during surveys carried out in western Serbia in 2002. Identification of the causal agent was performed in collaboration work with the Scottish Crop Research Institute (S.C.R.I.), Dundee, UK. Necrotic roots were plated on selective French bean agar (incorporating ampicilin, ryfamicin, bavistin and hymexasol). Detection of isolates was based on cultural and morphological features compared with referent cultures. DNA was extracted directly from the sampled roots using extraction buffer (200 mM Tris- HCl pH 8.5, 250 mM NaCl, 25 mM EDTA, 0.5% SDS), purified by multi spin separation columns [Thistle Scientific (Axygen)] or in 24:1 mixture of chlorophorm- iso-amyl alcohol and amplified by nested PCR (ITS 4 and DC 6 for first round, DC 1 and DC 5 for second round). Diluted DNA extracts were also amplified by conventional PCR with modified ”universal” Phytophthora primers (ITS 6, ITS 7 and ITS 8, Cooke et al., 2000) and digested with Msp1. Digestion patterns of the universal primers PCR product from infected roots matched those of Scottish strains. P. fragariae var. rubi occured on 8 out of 14 sites. Our results indicate that nested PCR (ITS 4 and DC 6 for first round, DC 1 and DC 5 for second round) or digestion of the ”universal” Phytophthora primers PCR product for detection of P. fragariae var. rubi are more sensitive and less time-consuming and therefore recommended for use.

Phytophthora fragariae var. rubi (Wilcox & Duncan), prouzrokovač truleži korena maline, je veoma značajan zemljišni patogen A2 karantinskog statusa u EPPO. Uzorci korena su sakupljeni sa jako zaraženih biljaka maline koje su ispoljavale čitav niz karakterističnih i često dramatičnih simptoma tokom pregleda koji su vršeni u zapadnoj Srbiji 2002. godine. Identifikacija prouzrokovača je izvršena u saradnji sa Scottish Crop Research Institute (S.C.R.I.), Dundee, UK. Nekrotični korenčići su unošeni u selektivnu French bean podlogu, koja je sadržala ampicilin, rifamicin, bavistin i himeksazol. Detekcija dobijenih izolata je vršena posmatranjem kulture i morfoloških karakteristika i poređenjem sa autentičnim kulturama. DNK je ekstrahovana direktno iz uzorkovanih korenova pomoću ekstrakcionog pufera (200 mM Tris-HCl pH 8,5, 250 mM NaCl, 25 mM EDTA, 0,5% SDS), prečišćena Multi spin separacionim kolonama (Thistle Scientific (Axygen) ili 24:1 mešavinom hloroforma i izoamil alkohola i amplifikovana nested PCR (ITS 4 i DC 6 za prvi krug, DC 1 i DC 5 za drugi krug). Rastvoreni DNK ekstrakti su takođe amplifikovani putem konvencionalnog PCR sa modifikovanim „univerzalnim” Phytophthora prajmerima (ITS 6, ITS 7 i ITS 8, Cooke i sar., 2000) i digestirani sa Msp1. Digestioni paterni proizvoda univerzalnih prajmera iz zaraženih korenova su se poklapali sa škotskim izolatima. P. fragariae var. rubi se javila na 8 od 14 lokaliteta. Naši rezultati ukazuju da su nested PCR (ITS 4 i DC 6 za prvi krug, DC 1 i DC 5 za drugi krug) i digestija produkta univerzalnih Phytophthora prajmera u cilju detekcije P. fragariae var. rubi senzitivnije i zahtevaju manje vremena, pa ih stoga preporučujemo za korišćenje.