Methods for Detection of Phytophthora fragariae var. rubi on Raspberry
Metode za detekciju Phytophthora fragariae var. rubi na malini
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Author
Koprivica, Mirjana
Dulic-Markovic, Ivana
Jevtic, Radivoje
Cooke, Dave E.L.
Keywords
Phytophthora fragariae var. rubiRaspberry root rot
PCR methods
trulež korena maline
PCR metode detekcije
Metadata
Show full item recordAbstract
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.