Scientific Papers Series B Horticulture

Scientific Papers. Series B, Horticulture. Vol. LVII, 2013Print ISSN 2285-5653, CD-ROM ISSN 2285-5661, Online ISSN 2286-1580, ISSN-L 2285-5653NEW HOST PLANT FOR VIRUS VECTORNEMATODE XIPHINEMAITALIAE MEYL, 1953(NEMATODA: LONGIDORIDAE) IN ROMANIAMariana BONTA (GROZA) 1 , Ioan ROCA 1 , Claudia COSTACHE 21 University of Agronomic Sciences and Veterinary Medicine of Bucharest,59 Marasti Blvd., 011464, Bucharest, Romania2 Central Phytosanitary Laboratory, 11 Voluntari Blvd., 077190, Bucharest, RomaniaAbstractCorresponding author email: mariana_bonta@yahoo.comExcept direct damage to root system, Xiphinema italiae Meyl, 1953 has been reported to be a vector of Grapevinefanleaf virus (GFLV) (Cohn et al., 1958). Soil samples were collected at a depth of 20-40 cm from orchards andvineyards. Xiphinema italiae was identified in rhisosphere of peach orchard. A polymerase chain reaction protocol andthe morphological and morphometrical characters has been used for the reliable identification of X. italiae.Morphometrics and illustrations of females are provided. Prunus persica L.is a new host plant for Xiphinema italiae forRomania.Key words: Longidoridae, morphology, PCR Multiplex.INTRODUCTIONXiphinema italiae Meyl, 1953 is widespreadmigratory plant parasitic nematode, speciesoccurring in southern and central Europe:Bulgaria (Peneva and Choleva, 1992,Peneva,1997) France (Wang et al., 2003)Greece (Avgelis & Tzortzakakis, 1997,Tzortzakakis et al. 2006) Hungary (Nagy,1999), Italy (Martelli et al.,1966), Moldavia(Polinovskij, 1979), Serbia (Barsi & Lamberti,2003), Slovakia (Liškova et al., 1993) andSpain (Teliz et al., 2007) (Gutiérrez-Gutiérrezet al., 2011).. Outside Europe it was found inCuba (Dias-Silveira & Herrera, 1995), Egypt(Lamberti et al., 1996), Libya (Siddiqui et al.,1987), Nigeria (Khan et al., 1993) and SouthAfrica (Knoetze et al., 2000). Xiphinema italiaehas been reported to be a vector of GFLVaccording to Cohn et al., 1970). In Romania,Romacu, 1971 found X. italiae in associationwith grapevines from sandy soil in Platonetiand Saveni (Ialomia county).MATERIALS AND METHODSFor this study, soil samples were collected fromthe rhizosphere of peach trees at a depth of 20-40 cm from Valul lui Traian (Constanacounty).159Nematodes were extracted from 200cm 3 soil bya sieving and decanting technique, Nematodeswere heat killed at 60ºC for two minutes andfixed in a 4% formaldehyde solution. Thespecimens were processed to mounted onpermanent microscopic glass slides (Seinhorst,1959).The morphological and morphometricalobservations were made using Leica DMLBmicroscope fitted with Leica FDC 295 camera.Multiplex PCR. DNA isolation was carried outby placing 4 nematodes in 10 μL of lysis buffer(1X Platinum Taq DNA polymerase /Invitrogenand 60 μg of proteinase K/mL) between twoglass slides and crushed gently. Thehomogenate was taken up carefully with apipette, transferred to 0,2 mL Eppendorf tubesand frozen at-80°C for 15 min. After the tubeswere incubated at 60°C for 1 h and 95°C for 15min.Amplification was carried out in a 25-μlreaction mixture containing the 2,5 μl lysisbuffer (nematode lysate as PCR template), 1xPlatinum Taq DNA polymerase buffer(Invitrogen), 1.5 mM MgCl 2 (Invitrogen), 0.2mM each of dATP, dCTP, dGTP, and dTTP(Sigma 10mM), 0.8 pmol each primer, and0.5 units of Platinum Taq DNA polymerase