Infected Grapevines Research Articles

Impacts of leafroll‐associated viruses (GLRaV‐1 and ‐3) on the physiology of the Portuguese grapevine cultivar ‘Touriga Nacional’ growing under field conditions

AbstractThe impact of mixed infection of grapevine leafroll‐associated virus 1 and 3 (GLRaV‐1&‐3) on physiological performance of the Portuguese grapevine variety ‘Touriga Nacional’ was evaluated during 3 years with the main purpose of understanding the drastic reduction in yield. Overall, gas exchange was negatively affected in leaves with these leafroll virus infections. Particularly at ripeness stage, the reduction in stomatal conductance (gs) was higher than in net CO2 assimilation rate (A), leading to higher intrinsic water use efficiency (A/gs) in infected leaves. However, the decrease in gs and A were not a consequence of the decrease in bulk water potential, as the water index/normalised difference vegetation index ratio suggested similar magnitude for both treatments. The maximum quantum efficiency of photosystem II was unaffected by GLRaV‐1&‐3, whereas quantum effective efficiency of PSII, apparent electron transport rate and photochemical quenching significantly decreased in infected leaves and these was paralleled by a significant increase of non‐photochemical quenching. Relative to carbon metabolism, the analyses of the net CO2 assimilation rate/photosynthetic photon flux density (A/PPFD) and net CO2 assimilation rate/internal CO2 concentration (A/Ci) curves revealed that virus infection had a negative effect on light saturated rate of CO2 fixation at high irradiances and carboxylation efficiency but, in contrast, apparent quantum yield of CO2 fixation was significantly higher. Meanwhile, the presence of GLRaV‐1&‐3 resulted in a marked decrease in photosynthetic pigments, soluble sugars and soluble proteins contents, while starch and anthocyanins were significantly improved. N, P, Ca, S and Fe leaf concentrations significantly decreased, while K, Mg, B, Cu, Zn and Mn were unaffected by these two leafroll virus species. Infected plants showed a significant decrease in yield, mainly due to a lower cluster weight. These results emphasised the important role of GLRaV‐1&‐3 as a biotic stress for the grapevine physiology and consequently to yield attributes.

Eradication of black rot (Guignardia bidwellii) from grapevines by drastic pruning

A drastic pruning strategy was developed to eradicate the fungal disease black rot (Guignardia bidwellii), which is exotic in Australia, from grapevines, while minimizing the economic cost of returning an affected vineyard to its previous quality and production levels. The protocol involved cutting off vines at the top of the trunk, removing debris from the ground beneath and between vines, mulching the vineyard floor, removing low watershoots during vine regrowth and applying a targeted fungicide programme. The protocol was initially evaluated and consequently modified in Australia using an endemic grapevine disease, black spot or anthracnose (Elsinoe ampelina), as an analogous model system. Then, it was validated in a black‐rot‐infested vineyard in New York, USA. Following two seasons of disease‐conducive weather conditions, no black rot was detected on treated vines, whereas leaf and fruit infections developed on the untreated control vines. These results confirmed the efficacy of the protocol for eradicating black rot from vineyards while allowing vines to return quickly to previous yield and quality levels without replanting. The protocol may have applicability to disease eradication protocols for other perennial crops as well. Evidence is also presented on the efficacy and potential pitfalls of burning infected grapevine material to eradicate E. ampelina.

The Role of Rain in Dispersal of the Primary Inoculum of Plasmopara viticola

Although primary infection of grapevines by Plasmopara viticola requires splash dispersal of inoculum from soil to leaves, little is known about the role of rain in primary inoculum dispersal. Distribution of rain splashes from soil to grapevine canopy was evaluated over 20 rain periods (0.2 to 64.2 mm of rain) with splash samplers placed within the canopy. Samplers at 40, 80, and 140 cm above the soil caught 4.4, 0.03, and 0.003 drops/cm(2) of sampler area, respectively. Drops caught at 40 and 80 cm (1.5 cm in diameter) were larger than drops at 140 cm (1.3 cm). Leaf coverage by splashed drops, total drop number, and drop size increased with an increase in the maximum intensity of rain (mm/h) during any rain period. Any rainfall led to infection in potted grapevines placed outside on leaf litter containing oospores, if the litter contained germinated oospores at the time of rain; infection severity was unrelated to rain amount or intensity. Results from vineyards also indicate that any rain can carry P. viticola inoculum from soil to leaves and should be considered a splash event in disease prediction systems. Sampling for early disease detection should focus on the lower canopy, where the probability of splash impact is greatest.

Influence of water stress on Botryosphaeriaceae disease expression in grapevines.

Several species in Botryosphaeriaceae have been associated with grapevine trunk diseases. To evaluate the effect of water stress on infection of grapevines by Botryosphaeriaceae spp., 1-year-old Shiraz/101-14 Mgt nursery grapevine plants were planted in plastic potting bags and placed outdoors under shade netting. Five weeks after planting, vines were pruned and the pruning wounds inoculated with spore suspensions of Neofusicoccum australe , Neofusicoccum parvum , Lasiodiplodia theobroma e or Diplodia seriata . Control treatments consisted of applications of sterile water or a Trichoderma harzianum spore suspension. Stem inoculations were done by inserting a colonised or uncolonised agar plug into a wound made in each stem. Four different irrigation regimes were introduced 12 weeks after planting to simulate varying degrees of water stress. Measurements of stomatal conductance, photosynthetic rate and leaf spectrometry were made to monitor physiological stress. Eight months after inoculation, vines were uprooted and the root, shoot and plant mass of each vine determined. Lesions observed in the inoculated pruning wounds and stems were also measured. Vines subjected to the lowest irrigation regime were significantly smaller than optimally irrigated vines. Water stressed vines also had significantly lower photosynthetic rates and levels of stomatal conductance compared with vines receiving optimal irrigation, indicating that these plants experienced significantly higher levels of physiological stress. The mean lesion length was significantly longer in the pruning wounds and stems of plants subjected to the lowest irrigation regime, with lesion length declining linearly with increasing irrigation volume. These results clearly indicate that when a grapevine is exposed to water stress, colonisation and disease expression by Botryosphaeriaceae spp. are much more severe.

Effect of Esca disease on the phenolic and sensory attributes of Cabernet Sauvignon grapes, musts and wines

Background and Aims: The impact of Esca, a cryptogamic disease affecting woody tissues of grapevines (trunk, branches and shoots), was investigated on phenolic and sensory qualities of Cabernet Sauvignon grapes, derived musts and wines. Methods and Results: Global phenolic analyses revealed no major difference between healthy and affected grapes whereas high-performance liquid chromatography analyses showed decreases in the skin concentrations of catechin and epicatechin and of anthocyanins for the two vintages assessed. Proanthocyanidin characteristics, and particularly mean degree of polymerisation, were strongly decreased in skin proanthocyanidin polymeric fractions. Chemical analyses of wines made with different percentages of fruit from Esca infected grapevines confirmed the moderate impact of Esca on phenolic composition. Nevertheless, sensory analyses revealed a loss of wine sensory quality perceptible with as little as 5% of affected fruit in wines. Conclusions: Phenolic variations in grapes and in derived wines may be related to changes to grapevine physiology caused by Esca fungi that impact on flavonoid metabolism. The chemical and sensory variations between wines can also be related to the delayed ripening delay of fruit from Esca infected grapevines. Significance of the Study: Esca moderately affected the phenolic composition of grapes and decreased the sensory quality of wines, suggesting a dramatic increase in the economic importance of Esca if no control methods are found.

Native and sulfated oligoglucuronans as elicitors of defence-related responses inducing protection against Botrytis cinerea of Vitis vinifera

During the past two decades, β-(1,4)-d-polyglucuronic acids (glucuronans) have been intensively described in literature for their physico-chemical and biological properties in different models. In this study, a bacterial glucuronan partially acetylated was degraded by a fungal glucuronan lyase to obtain unsaturated oligogluguronans, finally tested for their potential as elicitors of natural defences in Vitis vinifera. The objective was to correlate biological activities with specific structural features using oligoglucuronans varying in degree of polymerization, acetylation and sulfation. The results demonstrated that different defence reactions elicited by these anionic oligomers were dependent on degree of polymerization and nature of substituents (acetyl or sulfate groups). Acetylated oligoglucuronans with degrees of polymerization of 10 and 14 induced transient production of H2O2 and expression of some defence-related genes (PAL, STS, Chit4c and PGIP). The infection of grapevine leaves by Botrytis cinerea was reduced (23%) after treatment with acetylated oligoglucuronans having degree of polymerization of 14. Sulfated oligoglucuronans were not effective for production of H2O2 but induced strong expression of other defence-related PIN, PGIP genes. This work opens the way to new and valuable strategies to protect V. vinifera and other plants against pathogens.

Vector preference for hosts differing in infection status: sharpshooter movement and Xylella fastidiosa transmission

1. Epidemiological theory predicts that vector preference for hosts differing in infection status (i.e. healthy or infected) affects disease dynamics.2. Numerous studies have documented strong vector preference for or discrimination against infected hosts. However, the significance of these behaviours for pathogen transmission and spread has been poorly described.3. We conducted a series of choice assays to evaluate orientation preference, feeding preference, and movement rates of an important group of vectors, the sharpshooter leafhoppers, based on host infection status for the generalist plant pathogen, Xylella fastidiosa Wells et al.4. Sharpshooters did not discriminate between healthy versus infected‐but‐asymptomatic grapevines, but they oriented preferentially to healthy grapevines more frequently than either symptomatic vines or those artificially coloured to mimic disease symptoms.5. In a field trial three sharpshooter species showed different movement rates and preferences for feeding site, but all species exhibited similar and significant preference for healthy hosts.6. Although there was no significant difference in acquisition efficiency among vector species, those individuals that spent more time on healthy hosts tended to be less likely to acquire the pathogen.7. These results suggest that sharpshooters discriminate against infected grapevines, which are likely to be of poorer quality, with visual cues playing a role in host selection. Preference by these vectors may affect pathogen acquisition, which could affect disease spread in the field.

Changes in Carbohydrate Metabolism in Plasmopara viticola-Infected Grapevine Leaves

The oomycete Plasmopara viticola is responsible for downy mildew, a severe grapevine disease. In infected grapevine leaves, we have observed an abnormal starch accumulation at the end of the dark period, suggesting modifications in starch metabolism. Therefore, several complementary approaches, including transcriptomic analyses, measurements of enzyme activities, and sugar quantification, were performed in order to investigate and to understand the effects of P. viticola infection on leaf starch and-to a larger extent-carbohydrate metabolism. Our results indicate that starch accumulation is associated with an increase in ADP-glucose pyrophosphorylase (AGPase) activity and modifications in the starch degradation pathway, especially an increased α-amylase activity. Together with these alterations in starch metabolism, we have observed an accumulation of hexoses, an increase in invertase activity, and a reduction of photosynthesis, indicating a source-to-sink transition in infected leaf tissue. Additionally, we have measured an accumulation of the disaccharide trehalose correlated to an increased trehalase gene expression and enzyme activity. Altogether, these results highlight a dramatic alteration of carbohydrate metabolism correlated with later stages of P. viticola development in leaves.

Compatible GLRaV-3 viral infections affect berry ripening decreasing sugar accumulation and anthocyanin biosynthesis in Vitis vinifera

Virus infections in grapevine cause important economic losses and affect fruit quality worldwide. Although the phenotypic symptoms associated to viral infections have been described, the molecular plant response triggered by virus infection is still poorly understood in Vitis vinifera. As a first step to understand the fruit changes and mechanisms involved in the compatible grapevine-virus interaction, we analyzed the berry transcriptome in two stages of development in the red wine cultivar Cabernet Sauvignon infected with Grapevine leaf-roll-associated virus-3 (GLRaV-3). Analysis of global gene expression patterns indicate incomplete berry maturation in infected berries as compared to uninfected fruit suggesting viral infection interrupts the normal berry maturation process. Genes with altered expression in berries harvested from GLRaV-3-infected vines as compared to uninfected tissue include anthocyanin biosynthesis and sugar metabolism genes. The reduction in transcript accumulation for sugar and anthocyanin metabolism during fruit development is consistent with a dramatic reduction in anthocyanin biosynthesis as well as reduced sugar levels in berries, a hallmark phenotypic change observed in virus infected grapevines. Analysis of key regulatory factors provides a mechanism for the observed gene expression changes. Our results provide insight into commonly observed phenotypic alterations in virus infected vines and the molecular mechanisms associated with the plant response to the virus during berry ripening.

Evaluation of Vineyard Weeds as Potential Hosts of Black-Foot and Petri Disease Pathogens

Weeds were sampled in grapevine rootstock mother fields, open-root field nurseries, and commercial vineyards of Albacete, Alicante, Castellón, Murcia, and Valencia provinces in Spain between June 2009 and June 2010 and evaluated as potential hosts of black-foot and Petri disease pathogens. Isolations were conducted in the root system and internal xylem tissues for black-foot and Petri disease pathogens, respectively. Cylindrocarpon macrodidymum was successfully isolated from the roots of 15 of 19 weed families evaluated and 26 of 52 weed species. Regarding Petri disease pathogens, one isolate of Phaeomoniella chlamydospora was obtained from Convolvulus arvensis, and three isolates of Cadophora luteo-olivacea were obtained from Bidens subalternans, Plantago coronopus, and Sonchus oleraceus. Pathogenicity tests showed that Cylindrocarpon macrodidymum isolates obtained from weeds were able to induce typical black-foot disease symptoms. When inoculated in grapevines, isolates of Cadophora luteo-olivacea and Phaeomoniella chlamydospora were also shown to be pathogenic on grapevine cuttings. Our ability to recover grapevine pathogens from vineyard weeds and to demonstrate pathogenicity of recovered strains on grape suggests that these weeds may serve as a source of inoculum for infection of grapevine.

Biological, molecular, and serological studies of a novel strain of grapevine leafroll-associated virus 2

In California, a novel closterovirus was detected in "Redglobe" grapevine, associated with graft incompatibility and given a trivial name "Grapevine rootstock stem lesion associated virus (GRSLaV)." The biological properties of the putative virus were ascertained when asymptomatic yet infected Redglobe scion buds were graft-inoculated onto test plants of Cabernet Sauvignon propagated on 18 different rootstocks. It proved lethal on test plants growing on rootstocks 1616C, 5BB, 5C, 3309C, and 1103 P, whereas latent infections occurred on the remaining scion-rootstock combinations. In contrast, GLRaV-2 type (type strain) produced only typical leafroll symptoms. In a different experiment, GLRaV-2 type was successfully sap-transmitted to N. benthamiana, whereas sap transmission of GRSLaV was unsuccessful. Double-stranded RNA was extracted from infected Redglobe grapevines, cloned, sequenced, and determined a genome length of 16,527 nucleotides. Computer-assisted analysis of open-reading frames (ORFs) revealed a genome organization typical of monopartite viruses in the genus Closterovirus with nine ORFs (range 71-79% identity) with GLRaV-2 type, the closest similar virus species within the family Closteroviridae. Also the 3'-UTR of GRSLaV consisted of 223 nucleotides with an extended oligo(A) tract similar to that of GLRaV-2 type, Beet yellow stunt virus, and Beet yellows virus. Recombinant GRSLaV coat protein was expressed in E. coli, purified, and immunized a rabbit to produce polyclonal antiserum. Serological data matched the molecular data, whereby exposed plant tissue extracts of grapevines infected by both viruses (GRSLaV and GLRaV-2) reacted positively with homologous and heterologous viral antisera but not with healthy grapevine extracts in ELISA and Western blot tests. Based on the comparative sequence data and shared antigens, GRSLaV is now considered a strain of GLRaV-2 and redesignated as Grapevine leafroll associated virus-2 Redglobe (GLRaV-2RG). Primers specific for GLRaV-2RG were developed, which did not amplify GLRaV-2 type strain. When both sets of specific primers were used in assays of different grapevine collections, the incidence of the respective viruses varied considerably, e.g., 1.7 and 13.5%, respectively, for GLRaV-2RG and GLRaV-2 type.

Somatic embryogenesis efficiently eliminates viroid infections from grapevines

Indirect somatic embryogenesis is effective at eliminating the most important viruses affecting grapevines. Accordingly, this technique was tested as a method for eradicating two widespread viroids, Grapevine yellow speckle viroid 1 (GYSVd-1) and Hop stunt viroid (HSVd), from four grapevine cultivars. Both viroids were detected by RT-PCR in grapevine floral explants used for initiating embryogenic cultures, as well as in undifferentiated cells of embryogenic and non-embryogenic calli from anthers and ovaries. In contrast, somatic embryos differentiated from these infected calli were viroid-free, and viroids were not detected in embryo-derived plantlets even 3 years after their transfer to greenhouse conditions. A wider spatial distribution of HSVd than GYSVd-1 within proliferating calli was revealed by in situ hybridization, whereas no hybridization signal was detected in the somatic embryos. In addition, GYSVd-1 and HSVd were localised in the nucleus of infected cells, conclusively showing the nuclear accumulation of representative members of Apscaviroid and Hostuviroid genera, which has been only an assumption so far. Somatic embryogenesis was compared to in vitro thermotherapy, a technique routinely used for virus eradication. After thermotherapy, HSVd and GYSVd-1 were detected in all in vitro plantlets of the cultivar Roussan, and in all lines analysed after 3 years of culture in greenhouse. The high efficiency with which somatic embryogenesis may eliminate viroids and viruses from several infected grapevine cultivars, should allow the availability of virus- and viroid-free material, which would be useful not only for sanitary selection but also for basic research on plant-virus and plant-viroid interactions in grapevine.

Spatial Distribution of Pierce’s Disease Related to Incidence, Vineyard Characteristics, and Surrounding Land Uses

A four-year (2001 to 2004) census was conducted in 220 vineyard blocks in the San Joaquin Valley (Kern County, CA) to characterize the spatial distribution patterns of Pierce’s disease (PD) of grapevine caused by <i>Xylella fastidiosa</i>. Geostatistics and spatial analysis with distance indices were used to investigate relationships of PD incidence (i.e., percentage of vines with PD) and occurrence (i.e., presence or absence of PD) with vineyard characteristics and the surrounding environment using a geographic information system. The census confirmed 52 vineyard blocks with at least one <i>X. fastidiosa</i>-infected grapevine and PD incidence in those vineyard blocks ranged from 0.001 to 29.8%. A total of 36 of the 52 blocks had fewer than five infected vines, so spatial analysis was not valid for these blocks. For the remaining 16 blocks, the distribution of <i>X. fastidiosa</i>-infected vines could be categorized as no structure, random, spatial trend, and aggregation as the incidence increased. Eleven blocks showed PD distributions that were consistent with primary <i>X. fastidiosa</i> spread, while two blocks showed aggregations that suggested secondary or vine-to-vine spread. The distribution in one block indicated a mixture of primary and secondary spread. Two blocks had recurring aggregations of <i>X. fastidiosa</i>-infected vines within rows that could be the result of vector feeding on adjacent plants within rows or bacterial spread on pruning implements. Significantly higher PD occurrence was found in Flame Seedless vineyard blocks, and other vineyard characteristics such as vineyard age, pruning method, and planting density were not related to PD occurrence. Although PD occurrence was not spatially related with surrounding environment, PD incidence was spatially related to citrus. This study provides spatial information concerning the epidemiology and sampling of PD in vineyards.

Raspberry bushy dwarf virus: A grapevine pathogen in Serbia

During a field survey in 2005 in vineyards and grapevine nurseries at localities in central Serbia, a few plants with unusual virus-like symptoms were observed. Leaf samples were analyzed by DAS-ELISA for the presence of nine viruses. Besides other viruses frequently occurring on grapevine, Raspberry bushy dwarf virus (RBDV) was detected in two samples. Results were confirmed by nested-PCR and sequence analysis of the fragment in 5? part of RNA-1. Obtained sequence shared at least 93% of nucleotide identity with the compared RBDV sequence originating from raspberry. The finding of Raspberry bushy dwarf virus on grapevine in Serbia is a second finding of this pathogen on grapevine worldwide. The first natural infection of grapevine with this virus was reported in Slovenia in 2003.

Response of the Vitis vinifera L. cv. ‘Nebbiolo’ proteome to Flavescence dorée phytoplasma infection

Flavescence dorée is a serious phytoplasma disease affecting grapevine in several European countries. We studied the interaction of Flavescence dorée phytoplasma with its natural plant host by monitoring the effects of infection on the protein expression profile. Among the 576 analyzed spots, 33 proteins were differentially regulated in infected grapevines. Grouping into MIPS functional categories showed proteins involved in metabolism (21%), energy processes (9%), protein synthesis (3%), protein fate (18%), cellular transport and transport routes (6%), cell defense and virulence (42%). Among the differentially regulated proteins, we selected six targets (thaumatin I, thaumatin II, osmotin-like protein, plant basic secretory protein, AAA(+) Rubisco activase and proteasome α5 subunit) and we analyzed their expression by quantitative RT-PCR on samples collected in 2008 and 2009 in several vineyards in Piedmont region, Italy. There was a positive correlation between mRNA and protein expression for most of the genes in both the years. We discuss the involvement of these proteins in the specific response to phytoplasma infection. To our knowledge, this work is the first to investigate the response of the grapevine proteome to Flavescence dorée phytoplasma infection, and provides reference protein profiles for future comparative proteomic and genomic studies.

Analysis of genetic diversity and phylogeny of partial coat protein domain in Czech and Italian GFLV isolates

The genetic diversity of Grapevine fanleaf virus (GFLV) was evaluated in 4 isolates sampled from naturally infected grapevines from South Moravia (Czech Republic) and 2 Italian isolates from Bari (Italy). Conserved regions within sequences in databases were found and new primers corresponding to these regions were designed and tested for RT-PCR amplification of the CP codifying region. After sequencing of obtained amplicons the similarity of isolates was analysed via alignments of sequences and by means of dendrograms

Five phylogenetic groups identified in the coat protein gene of grapevine leafroll-associated virus 3 obtained from Portuguese grapevine varieties

The genetic variability and population structure of grapevine leafroll-associated virus 3 (GLRaV-3) variants were updated by examining the diversity within the viral coat protein (CP) gene among 174 isolates belonging to a collection of Vitis vinifera representing most of the Portuguese varieties. Phylogenetic analysis revealed the existence of five well-defined clusters. Three of these correspond to previously defined groups, another corresponds to variants from Chile for which only one sequence has been previously identified, and an additional new group includes only Portuguese variants. A typing tool based on asymmetric PCR-ELISA (APET) was developed within the frame of this population structure. This tool was used to assess the prevalence of each phylogenetic group among the infected grapevine varieties. Although most of the isolates harbour variants from groups 1 and 2, variants from the remaining three groups exist in a number of varieties, reinforcing the notion that they are genuine genomic variants and are not isolated, atypical cases.

Characterization of a F-box gene up-regulated by phytohormones and upon biotic and abiotic stresses in grapevine

F-box proteins are key components of the ubiquitin (Ub)/26S proteasome pathway that mediates selective degradation of regulatory proteins involved in a wide variety of cellular processes affecting eukaryotic cells. In plants, F-box genes form one of the largest multigene superfamilies and control many important biological functions. Among the F-box genes characterized to date only few have been involved in the regulation of plant defense responses. Moreover, no F-box genes have been studied and characterized in grapevine. Using a differential display approach we isolated a F-box gene (BIG-24.1), which is up-regulated during Botrytis cinerea infection of grapevine leaves. BIG-24.1 encodes a polypeptide of 386 amino acids with a conserved F-box domain in the N-terminus region and a kelch domain. By investigating expression profiles of BIG-24.1, we show that the gene expression is strongly stimulated in B. cinerea infected berries and in grapevine cells challenged by MAMP rhamnolipids, a non-host bacterium and an endophytic rhizobacterium. The gene is also strongly induced by abiotic stresses including UV-C and wounding or by salicylic acid, methyl-jasmonate, ethylene and abscisic acid that are known to be involved in defense signalling pathways. In addition, sequence analysis of the BIG-24.1 promoter revealed the presence of several regulatory elements involved in the activation of plant defense responses.

Genetic variability of the stolbur phytoplasma vmp1 gene in grapevines, bindweeds and vegetables

Evaluation of the genetic variability of stolbur phytoplasma infecting grapevines, bindweeds and vegetables, collected in different central and southern Italian regions. Phytoplasma isolates belonging to stolbur subgroup 16SrXII-A were subjected to molecular characterization by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP), to investigate two different nonribosomal genes: tuf and vmp1. In grapevines, 32% of samples were infected by tuf-a type and 68% by tuf-b type, with different relative incidences in the regions surveyed. All herbaceous samples (bindweeds, tomato, tobacco, pepper, celery) were infected by tuf-b. The gene vmp1 showed higher polymorphism in grapevines (nine profiles) than herbaceous plants (six) by RFLP analysis, in agreement with nucleotide sequences' analysis and virtual digestions. The phylogenetic analysis of vmp1 gene sequences supports the RFLP data and demonstrates the accuracy of RFLP for preliminary assessments of genetic diversity of stolbur phytoplasmas and for screening different vmp types. Stolbur represents a serious phytosanitary problem in the areas under investigation, owing to heavy economic losses in infected grapevines and vegetables. Molecular information about the complex genotyping of the vmp1 gene provides useful data towards a better understanding of stolbur epidemiology. Moreover, this study clarifies some different vmp1 genotype classifications of stolbur, providing molecular data in comparison with previous investigations.

Co-operational PCR Coupled with Dot Blot Hybridization for the Detection of Phaeomoniella chlamydospora on Infected Grapevine Wood

The technique consisting of the co-operational PCR coupled with dot blot hybridization and posterior colorimetric visualization was developed for the detection of Phaeomoniella chlamydospora, one of the major pathogenic fungi involved in the Petri disease of grapevine. A partial region of the fungal rDNA including the internal transcribed spacer (ITS) region was amplified through co-operational PCR for P. chlamydospora and 17 additional grapevine-associated fungi included in the genera Botryosphaeria, Cryptovalsa, Cylindrocarpon, Dematophora, Diplodia, Dothiorella, Eutypa, Fomitiporia, Lasiodiplodia, Neofusicoccum, Phaeoacremonium, Phomopsis and Stereum, by using the primer pairs NSA3/NLC2 (external pair) and NSI1/NLB4 (inner pair). A specific probe (Pch2D) targeting the ITS2 region in the rDNA was developed for the detection of P. chlamydospora. Dot blot hybridizations carried out with the PCR products showed the specificity of the probe. Results indicated that Pch2D only hybridized with DNA amplicons of P. chlamydospora isolates, thus proving the specific detection of this fungus, while the 17 remaining species tested for the Pch2D probe resulted in negative results. Sensitivity of the technique was established below 0.1 pg of genomic DNA. This technique was further validated using artificially inoculated grapevine cuttings with P. chlamydospora. The efficacy of detection was established at 80% after two independent blind assays.