Flavescence doree in France and Italy - Occurrence of closely related phytoplasma isolates and their near relationships to Palatinate grapevine yellows and an alder yellows phytoplasma

Grapevine yellows (GY) are diseases of Vitis vinifera caused by phytoplasmas. On the basis of DNA analysis, it is possible to distinguish different groups and subgroups among grapevine phytoplasmas. Flavescence doree (FD), the most serious problem in European vineyards, is caused by a phytoplasma which belongs to the elm yellows group (EY or 16SrV), Differences between 7 phytoplasma isolates in this group, found until now in French, Italian and German grapevines, and 4 phytoplasmas in the same group, isolated mainly from elm and alder in Europe and America, were investigated. These 11 EY-group isolates plus 3 non-EY phytoplasmas, were compared by PCR-RFLP analyses of two different DNA fragments using 8 restriction enzymes. Two French and two Italian grapevine isolates, classified as FD, appeared to be closely related and were also closer to an Italian alder phytoplasma. One French and one Italian FD isolate always showed the same restriction pattern. On the opposite, the three German grapevine isolates related to alder phytoplasmas in Germany appeared to be closer to the two elm phytoplasmas from America and Europe.

Diversity of grapevine yellows in Germany

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Acquisition efficiency of Flavescence dorée phytoplasma by Scaphoideus titanus Ball from infected tolerant or susceptible grapevine cultivars or experimental host plants

The rate of Flavescence dorée phytoplasma (FDP) acquisition by the leafhopper vector Scaphoideus titanus Ball was tested under field and glass house conditions confining healthy reared nymphs on canes of FDP-infected grapevines or on FDP-infected cuttings collected in the field during the dormant season. Acquisition tests were performed using FD-tolerant (Merlot) or highly susceptible (Pinot blanc) grapevine cultivars, or alternatively using experimentally infected broadbean plants. Frequency of FDP acquisition by leafhoppers was evaluated using a polymerase chain reaction (PCR) assay. Different batches of insects were confined on the same infected source plants in the vineyard for acquisition access periods (AAP) of 7 d at a time at intervals of 15-20 d during spring and summer. When diseased Pinot blanc grapevines were used as source plants, acquisition by leafhoppers and transmission to healthy grapevines increased over summer, while almost no acquisition or transmission was observed when diseased Merlot grapevines were used as source plants. Tests conducted under controlled conditions confirmed that Merlot is a poorer source of FDP than Pinot blanc; the optimum FDP source for S. titanus was broadbean although this plant is not a natural host of the leafhopper. It is assumed that grapevine cultivars play an important role in influencing the proportion of FDP-infected leafhoppers in the vineyards and therefore influencing the rate of disease progress.

Diversity among mycoplasma-like organisms inducing grapevine yellows in France

As antibodies and molecular probes which were previously obtained for diagnosis of grapevine flavescence doree (FD; a yellows disease induced by a MLO), showed to be highly specific, a survey of grapevine samples collected in different viticultural areas in France was undertaken, using a PCR method with primers allowing amplification of a part of the 16S rRNA gene of most MLOs, and restriction analyses of the amplified products (AHRENS and SEEMÜLLER 1992). The presence of MLO was established in all the different grapevine samples, and their diversity was demonstrated. The typcial pattern yielded by FD sensu stricto-MLO was found in samples from southern vineyards, including a symptomless rootstock. Two different patterns were found in samples affected by bois noir disease of northern French vineyards, one of these patterns being previously undescribed. The present survey was non exhaustive and should be followed in the frame of a large collaboration between viticultural countries. It showed the diversity in causal agents of diseases which converge in symptomatology, and emphasizes on the need of specific diagnosis tools, for identification of each of the vector species, for epidemiological studies, and availability of planting material

A comparison of the phytoplasma associated with Australian grapevine yellows to other phytoplasmas in grapevine

The phytoplasma associated with Australian grapevine yellows (AGY) was compared to other phytoplasma diseases of grapevine using the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. Comparison of eight different Australian isolates suggests that only one type of phytoplasma is associated with this disease. Based on RFLP analysis of the 16S rRNA gene, it was shown that AGY is different from the tomato big bud and sweet potato little leaf phytoplasma strains which are widespread in Australia and that it represents the only other phytoplasma strain recorded in Australia to date. Restriction profiles of grapevine phytoplasmas using Mse I suggest that AGY is unique but most closely resembles those phytoplasmas associated with grapevine diseases in the stolbur group. Sequence analysis of the 16S rRNA gene and adjacent spacer region supports this association. The uniqueness of AGY was confirmed by PCR assays using non-ribosomal primers; the primer pair STOL11f/r2 specific for stolbur phytoplasmas did not result in amplification products in grapevines affected with AGY; the primer pair fMLOl/rMLOl which amplifies a region of the tuf gene from phytoplasmas in the aster yellows cluster, amplified AGY DNA confirming its association within this phylogenetic group. RFLP analysis of the tufPCR product again highlighted a distinction between AGY and other stolbur phytoplasmas occurring in grapevine. The only other phytoplasma in Australia which is in the stolbur group is associated with dieback in papaya, and it has the same RFLP profile of the tuf PCR product as AGY

A multiplex nested-PCR assay for sensitive and simultaneous detection and direct identification of phytoplasma in the Elm yellows group and Stolbur group and its use in survey of grapevine yellows in France

Flavescence dorée and Bois noir (or Vergilbungskrankheit), are two main yellows diseases of grapevines in Europe. The two diseases cannot be distinguished on the basis of symptoms but they are associated with two different phytoplasmas which belong to the Elm yellows (16SrV) group and Stolbur (16SrXII) group, respectively. Their spreading areas are overlapping in France, Italy and Spain but they have different vector insects. Flavescence dorée is an epidemic disease and a quarantine organism. National surveys conducted annually in France require straightforward and sensitive assays to detect phytoplasma that sometimes occur in grapevine with a low titre and to characterize them readily. A bi-specific multiplex nested-PCR procedure was developed, to amplify simultaneously two non-ribosomal DNA fragments, 1150 bp and 720 bp in length, specific for Elm yellows-group and Stolbur-group phytoplasmas, respectively. They were identified using agarose gel electrophoresis of amplification products. The procedure is quick, sensitive and reliable. It was used on 2,525 grapevine samples from the field, in the frame of the French survey in 2002. Mixed samples containing both phytoplasmas displayed a mixed profile in the gel. It was confirmed that the nested-PCR amplimer obtained in the FD9 DNA region with Elm yellows-group phytoplasmas, though shorter than the initial FD9 fragment, nevertheless contained the restriction sites that permit the RFLP identification of geographic phytoplasma isolates already characterized in former studies.

Detección y profilaxis del fitoplasma del amarilleamiento del olmo en Francia

Surveys conducted since 1998, showed that symptoms typical of Elm Yellows (EY) were quite frequent in the elm conservatories of Nogent-sur-Vernisson and Guémené- Penfao, France, and on forest trees in several locations in western Europe. Phytoplasmas were detected in symptomatic trees and plants using Polymerase Chain Reaction amplification followed by Restriction Fragment Length Polymorphism analyses of conserved regions of 16S rDNA of phytoplasmas, or of the EY group-specific non ribosomal DNA fragment FD9. Potential vectors were searched among leafhoppers and planthoppers trapped in conservatories and tested for the presence of phytoplasma in their body. Sanitation of multiplication material was experimented using soaking of dormant cuttings and branches in hot water, according to the method devised for destruction of phytoplasma in grapevine multiplication material. Phytoplasmas detected in a number of the elm clones held in the conservatories and in several forest or hedgerow elm trees, showed some diversity, according to restriction analyses of the FD9 fragment. Both American and European types were characterized. Four leaf- and planthopper species were found to carry an EY phytoplasma. Plants grown from cuttings taken from EY-infected clones and soaked in hot water (50 °C, 45 mn) never developed symptoms and tested phytoplasma negative. Most of untreated control plants were all symptomatic and positive. In the future, EY phytoplasma should be carefully checked, especially in sensitive cultivars. More studies of EY phytoplasma variability and potential vecteurs and the assessment of routine use of hot water therapy in multiplication of material should be conducted.Las observaciones realizadas desde 1998 han mostrado que los síntomas típicos del amarilleamiento del olmo (EY) son muy frecuentes en los bancos de olmo de Nogent- sur-Vernisson y Guémené-Penfao, Francia, y en árboles en campo de varias localidades de Europa occidental. La presencia de fitoplasmas se detectó, mediante el uso de la reacción en cadena de la polimerasa seguida de análisis de polimorfismo de longitud de los fragmentos de restricción en regiones conservadas de rADN de16S de fitoplasmas, o del fragmento FD9 de ADN no ribosómico, específico del fitoplasma del EY, en árboles y plantas sintomáticos. Se buscó vectores potenciales entre homópteros cicadélidos y fulgóridos capturados en los invernáculos y estudiados con el fin de detectar la presencia de fitoplasmas en su cuerpo. Como tratamiento profiláctico del material reproductivo, se experimentó sumergiendo las estaquillas y ramas durmientes en agua caliente, de acuerdo con el método desarrollado para la destrucción de fitoplasmas en material de reproducción de la vid. Según el análisis de restricción del fragmento FD9, los fitoplasmas que fueron detectados en algunos clones de olmo conservados en los bancos clonales, así como en varios olmos de campo, mostraron una cierta diversidad genética. Tanto el tipo americano como el europeo fueron caracterizados. Se encontró que cuatro de las especies de cicadélidos y fulgóridos eran portadoras del fitoplasma del EY. Las plantas que se desarrollaron a partir de estaquillas de clones infectados por el EY y que habían sido sumergidas en agua caliente (50 ºC, 45 minutos) nunca presentaron síntomas. Igualmente, las pruebas para la detección del fitoplasma fueron negativas. Por el contrario, todas las plantas control que no habían sido tratadas presentaron síntomas, y los resultados de las pruebas fueron positivos. En el futuro, la presencia del fitoplasma del EY deberías ser comprobada sistemáticamente, especialmente en cultivares sensibles. Deberían desarrollarse más estudios sobre la variabilidad del fitoplasma del EY y vectores potenciales, y sobre la evaluación del uso continuo del tratamiento con agua caliente en la multiplicación del material