Research NoteGrapevine yellows diseased stocks were observed in vineyards of the Alt Emporda area, northern Spain. Phytoplasma was detected and characterized by PCR-RFLP in the region of the 16S rDNA of phytoplasmas or by ELISA. For the first time a FD phytoplasma was detected in Spain

Batlle, A.

Boudon-Padieu, E.

Clair, D.

Kuszala, C.

Larrue, J.

Laviña, A.

English

JKI Open Journal Systems (Julius Kühn-Institut)

Vitis 36 (4), 211-212 (1997) Research Note Detection of flavescence doree phytoplasma in grapevine in northern Spain A. BATLLE 1), A. LAvr"NA1), C. KuszALA2), D. CLArR2), J. LARRUE2l and E. BouooN-PAoiEu2) S u m m a r y : Grapevine yellows diseased stocks were observed in vineyards of the Alt Emporda area, northem Spain. Phytoplasma was detected and characterized by PCR-RFLP in the region of the 16S rDNA of phytoplasmas or by ELISA. For the first time a FD phytoplasma was detected in Spain. K e y wo r d s : phytoplasma, grapevine yellows, detection, tlavescence doree, bois noir. Introduction: Phytoplasma diseases of grapevine, called grapevine yellows (GY), have already been found in many vine growing areas of America, Australia, Europe and in Israel (DArRE et al. 1997 a). In Europe, flavescence dort~e (FD) and bois noir (BN) are most frequent; they cause most serious damage. FD and BN-associated phytoplasmas be-long to the elm yellows (EY) group and the stolbur group, respectively, and are transmitted by different insect vectors. In the last 5 years serial samplings in vineyards of north-eastem Spain, indicated that several diseased plants pre-sented the symptomato1ogy associated with phytoplasmas. The symptoms were: mosaics limited by the leafveins, leaf roll, absence oflignification in autumn, withered flowers and dried berries. Up to now, only the phytoplasma associated to BN had been detected in samples with yellows symptoms (LAvrNA et al. 1995). However, Scaphoideus titanus Ball. the 1eatbopper vector ofFD, is present in all vineyards. Thus the occurrence of FD was kept under a constant watch be-cause the leatbopper species is a highly efficient vector of FD, which in turn is a very dangerous disease because ofits quick spreading when the vector is present. In the fall of 1996 in the Alt Emporda (Catalonia) viticul-ture area near the French border several vineyards were found with a high Ievel of GY -diseased stocks. As FD is causing a severe epidemic on the French side ofthe border, our atten-tion was directed to this new occurrence of a GY in Spain. This paper reports on the first detection ofFD in Spain. Material and methods: Tissue samples were collected in September and October 1996 at different plots ofthe Alt Emporda viticultural area from Grenache gris and Carignan grapevines with typical symptoms ofGY. Control samples of FD or BN-infected grapevines and ofhealthy cuttings were included. Total DNA was extracted from a phytoplasma-enriched fraction prepared from grapevine main leafveins as previ-1) Departament de Patologia Vegetal, IRTA, 08348 Cabrils, Bar-celona, Spain. Fax: +34-3-7533 954. E-mail: batlle@cabrils.irta.es 2 ) Station de Recherehes sur !es Phytoplasmes, INRA, Dijon, France. 211 ously described (DArRE et al. 1992). A nested-PCR proce-dure was used with primers specific for a 16S rDNA region in all known phytoplasma: the first primer pair was P 1 (DENG and HrRUKI 1991) and P7 (SMART et al. 1996) and the second was the pair tU5/rU3 (LORENZ et al. 1995). The amplification mixture contained 0.3 7 5 !JM of each primer, 150 )lM of dNTPs, 1 unit·100 ).!1- 1 of Taq DNA polymerase (Appligene), Taq buffer (Appligene ), overlaid with mineral oil. Amplification was carried out in a total volume of20 )ll containig 5-10 ng DNA, for the first amplification. 2 )ll of the amplification product were used for the second step in a total volume of 40 )ll of amplification mixture. 10 )ll ofthe mixture containing the DNA amplified in the second step w,as directly digested ovemight with Tru 91 restriction enzyme. The RFLP of the fragment allows to distinguish EY and stolbur phytoplasmas (DArRE et al. 1997 a). FD specific primers FD9f/r (DArRE et al. 1997 b) derived from both ends of a cloned 1.3 kb fragment from non-ribosomal FD-DNA, were also used for PCR on the same samples. An indirect ELISA sandwich procedure was applied according to CAUDWELL and KuszALA (1992). Sampies were extracted as described, including the addition of CHAPS (SIGMA) in the grinding buffer. Plates were coated with polyclonal rabbit anti-FD antiborlies and a cocktail of monoclonal anti-FD antiborlies was used as second anti-body, followed by alkaline phosphatase antimouse conju-gate. Each sample was assayed in 4 replicates and the OD 405 nm values were recorded after 150 min of incubation with the enzyme substrate. Results and Discussion: In 7 samples out of 9 from diseased grapevines, an EY group phytoplasma was detected in the nested PCR procedure followed by RFLP analysis (Figure ). The same samples were positive when FD9f/r prim-ers were used, and a 1.3 kb amplification product was ob-Tab I e ELISA readings of grapevine samples harvested in 2 localities of Alt Emporda. Infected controls were samples from FD- and BN-diseased grapevines from France. Healthy cuttings were ground in greenhouse. Sampies 2 and 6 were considered negative for FD ELISA readings Sampie Origin Variety OD 405 nm 1 Locality 1 Grenache gris 0.377 2 (Alt Emporda) 0.078 3 0.207 4 Locality2 Carignan 0.664 5 (Alt Emporda) 0.330 6 0.093 7 0.525 8 0.288 FDcontrol Languedoc Carignan 0.283 (France) 0.594 BN control Vallee du Rhöne Chardonnay 0.036 (France) 0.056 Healthy control greenhause Chardonnay 0.027 0.037 212 A. BATLLE, A. LAVrNA, C. KuszALA, D. CLAIR, J. LARRUE and E. BouooN-PADIEU kbp L 1.6 0.5 bp l. 590 270 185 125 80 65 2 3 4 5 6 7 8 9 10 11 12 A 2 3 4 5 6 7 8 9 10 11 12 B Figure: A: agarose gel ( 1.2%) electrophoresis of 16S rDNA nested-PCR amplified fragments (860 bp). B: polyacrylamide gel (10%) electrophoresis ofthe amplified products shown in A after Tru 91 restriction. (I) healthy Carignan, (2) Chardonnay infected with stolbur (BN), (3) Alicante naturally infected with FD from France, ( 4, 5, 6, 12) Grenache gris from Alt Emporda, (7, 8, 9, 10, II) Carignan from Alt Emporda. L. Molecular weight marker: A, 1 Kb DNA (GIBCO BRL); B, pBR 322/Hae JJI (Appligene). tained (data not shown). With the two procedures one of the samples gave only a weak response (lane 6). With the use ofFD antibodies in ELISA, 6 samples out of8 and the FD control gave positive results; healthy and BN infected controls gave negative results (Table ). This shows that a EY -group phytoplasma is also asso-ciated with grapevine yellows in Spain. Further restriction analysis showed that this phytoplasma isolate is similar to the FD isolate detected in grapevines in southem France (DAIRE et al. 1997 b ). The roJe of S. titanus will be checked in the next seasons by transmission trials and direct analysis on wild specimens of the leafhopper species collected in affected vineyards. The occurrence ofthe disease in Spain underlines the importance to start control measures by Iimi-tation ofvector populations and the use of cleaned planting material (CAUDWELL et al. 1990). Careful watch ofpossible new foci ofthe disease by a survey ofGY occurring in neigh-bouring viticulture areas seems to be appropriate. CAUDWELL, A.; KuszALA, C.; 1992: Mise au point d' un test ELISA sur les ti ssus de vignes atteintes de flavescence don!e. Res. Microbiol. 143, 791-806. - -; LARRU E, J.; VAL AT, C.; ÜRENAN, S.; 1990: Les traitements a l'eau chaude des bois de vigne atteints de Ia Flavescence doree. Progr. Agric. Vitic. I 07, 281-286. DArRE, X.; BounoN-PADIEU, E.; BERVILLE, A.; ScHNEIDER, B.; CAUDWELL, A.; 1992: Cloned DNA probes for detection of grapevine Flavescence don!e mycoplasma-like organ ism (MLO). Ann. Appl. Bio!. 121 , 95-103. - -; CtAIR, D.; LARRUE, J.; BouooN-PADIEU, E.; 1997 a: Survey for grape-vine yellows in diverse European countries and Israel. Vitis 36 (I) , 53-54. - -;- -; REINERT, W.; BouooN-PADIEU, E.; 1997 b: Detection and differ-entiation of grapevine yellows phytoplasmas belonging to the elm yellows group and to the stolbur subgroup by PCR amplifica-tion of non-ribosomal DNA. Eur. J. Plant Pathol. 103, 507-514. DENG, S. ; HIRUKI , C. ; 1991: Amplification of 16s rRNAgenes from culturable and nonculturable mollicutes. J. Microbiol. Meth. 14, 53-61. LAVINA, A.; BATTLE, A.; LARRUE, J. ; DAIRE, X.; CLAIR, D.; BOUDON-PADIEU, E.; 1995: First report of grapevine bois noir phytoplasma in Spain. Plant Dis., 79 (10), 1075. LoRENZ, K. H.; ScHNEIDER, B.; AHRENS, U.; SEEMÜLLER, E.; 1995: Detec-tion of the apple proliferation and pear decline phytoplasmas by PCR amplification of ribosomal and non-ribosomal DNA . Phytopathology 85, 771-776 . SMART, C. D.; ScHNEIDER, B.; BLOMQUIST, C. L. ; GuERRA, L. J.; HARRISON, N. A. ; AHRENS, U.; LORENZ, K. H.; SEEMÜLLER, E.; KIRCHPATRICK, B. C.; 1996: Phytoplasma-specific PCR primers based on sequences of the 16S-23S rRNA spacer region. Appl. Environ. Microbiol. 62 (8), 2988-1993. 

Detection of flavescence dorée phytoplasma in grapevine in northern Spain

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Detection of flavescence dorée phytoplasma in grapevine in northern Spain

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