Methods for Detection of Phytophthora fragariae var. rubi on Raspberry

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 (ITS4 and DC6 for first round, DC1and DC5 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 (ITS4 and DC6 for first round, DC1 and DC5 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.