Phytosanitary status of grapevine in Montenegro

During 2006 and 2007, a survey on the incidence and distribution of fourteen grapevine viruses was carried out in the Skadar Lake basin, one of the two main grapevine‐growing areas of Montenegro. In total 165 samples were collected from four red (‘Vranac’, ‘Krato?ija’, ‘Merlot’ and ‘Cardinal’), two white (‘Chardonnay’ and ‘Rkaciteli’) and a few unknown grapevine varieties in the vicinity of Podgorica and Bar. The phytosanitary status of the collected samples was analysed by DAS‐ELISA and the presence of Grapevine fanleaf virus (GFLV), Grapevine leafroll‐associated virus 1 (GLRaV‐1), Grapevine leafroll‐associated virus 2 (GLRaV‐2) and Grapevine leafroll‐associated virus 3 (GLRaV‐3) was confirmed in some of them. The most frequently found virus in assayed samples was GLRaV‐3 (54.5%), followed by GFLV (23%), GLRaV‐1 (20%) and GLRaV‐2 (0.6%). These serological analyses showed the absence of Grapevine leafroll‐associated virus 6 (GLRaV‐6), Grapevine leafroll‐associated virus 7 (GLRaV‐7), Raspberry bushy dwarf virus (RBDV), Strawberry latent ringspot virus (SLRSV), Tomato ringspot virus (ToRSV), Raspberry ringspot virus (RpRSV), Arabis mosaic virus (ArMV), Tobacco ringspot virus (TRSV), Tomato black ring virus (TBRV) and Cherry leaf roll virus (CLRV) from all tested samples.     

No evidence of transmission of grapevine leafroll-associated viruses by phylloxera (<Emphasis Type="Italic">Daktulosphaira vitifoliae</Emphasis>)

Grapevine leafroll disease is associated with several species of phloem-limited grapevine leafroll-associated viruses (GLRaV), some of which are transmitted by mealybugs and scale insects. The grape phylloxera, Daktulosphaira vitifoliae (Fitch) Biotype A (Hemiptera: Phylloxeridae), is a common vineyard pest that feeds on the phloem of vine roots. There is concern that these insects may transmit one or more GLRaV species, particularly GLRaV-2, a species in the genus Closterovirus. A field survey was performed in vineyards with a high incidence of grapevine leafroll disease and D. vitifoliae was assessed for acquisition of GLRaV. In greenhouse experiments, the ability of D. vitifoliae to transmit GLRaV from infected root sections or vines to co-planted virus-free recipient vines was tested. There were no GLRaV-positive D. vitifoliae in the field survey, nor did D. vitifoliae transmit GLRaV-1, ?2, ?3, or -4LV in greenhouse transmission experiments. Some insects tested positive for GLRaV after feeding on infected source vines in the greenhouse, however there was no evidence of virus transmission to healthy plants. These findings, in combination with the sedentary behaviour of the soil biotype of D. vitifoliae, make it unlikely that D. vitifoliae is a vector of any GLRaV.     

A Natural Population of Recombinant Plum Pox Virus is Viable and Competitive under Field Conditions

The isolate BOR-3, collected in Slovakia in 1996, was recently identified as a natural recombinant between an M and D type of Plum pox virus (PPV). Biological assays demonstrated its capacity to be aphid- and graft-transmitted to various Prunus spp. hosts. A study was carried out to determine the further presence of PPV recombinants in two epidemiologically distinct areas – Slovakia and France. Tools based on PPV-M and D subgroup typing, targeting P3–6K₁, CI and CP regions of the PPV genome were used for recombinant identification. Closely related recombinant variants were detected in different Prunus spp. during a survey conducted in Slovakia in 2001, but not within a set of selected PPV isolates from France collected between 1985 and 2001. Sequence analysis of the (Cter)NIb–(Nter)CP region of 10 recombinant isolates from Slovakia showed their high homology, reaching more than 98%. All the recombinant isolates shared the same recombination breakpoint situated in the C terminus of the NIb gene. Our study demonstrates that the PPV recombinants are viable and competitive with conventional PPV-M and D isolates. The present work indicates that the occurrence of recombinants within PPV isolates might be more common than previously assumed.     

High genotypic and virulence diversity in Ilyonectria liriodendri isolates associated with black foot disease in New Zealand vineyards

A total of 57 Ilyonectria liriodendri isolates were identified by a combination of species‐specific PCR and DNA sequencing from a collection of 174 Ilyonectria‐like isolates recovered from 101 diseased grapevine samples. These samples were representative of the national vineyard, comprising material contributed by 49 grape growers across seven grape growing areas. This species was predominant, representing 33% of the recovered isolates, and has been reported as a major pathogen of grapevines in other countries. The genetic diversity of the 57 New Zealand isolates was compared to that of isolates from Australia and South Africa using universally primed polymerase chain reaction (UP‐PCR). A total of 66 informative loci distinguished 52 genotypes, of which five contained up to four clonal isolates. Four main clades were identified in a neighbour‐joining (NJ) tree. The international isolates (Australia and South Africa) were placed in a clade that did not include New Zealand isolates. There was a high level of intra‐ and inter‐vineyard genetic variation indicating the free movement of isolates between regions. A subset of nine isolates from different branches of the NJ tree produced two vegetative compatibility groups and hyphal fusion was observed between non‐self pairings. Pathogenicity tests using isolates from different genetic groups inoculated onto either detached roots or 1‐year‐old potted vines showed variability in virulence; however, no correlations were detected.     

A novel type of Potato virus Y recombinant genome,determined for the genetic strain PVYE

Two Potato virus Y (PVY) isolates collected in Brazil, PVY‐AGA and PVY‐MON, were identified as recombinants between two parent genomes, PVY^NTN and PVY‐NE‐11, with a novel type of genomic pattern. The new recombinants had an ordinary PVY^NTN genome structure for approximately 6·7‐kb from the 5′‐end of the genome whereas the 3′‐terminal 3·0‐kb segment had two fragments of NE‐11‐like sequence separated by another small PVY^NTN‐like fragment. PVY strains are defined based on the hypersensitive resistance (HR) response in potato indicators. Both PVY‐AGA and PVY‐MON isolates did not induce the HR in potato cultivars carrying Ny, Nc, or (putative) Nz genes and thus were able to overcome all known resistance genes to PVY. Only one of the two isolates, PVY‐AGA, induced a vein necrosis reaction in tobacco. The biological responses of the potato indicators and tobacco defined PVY‐MON as an isolate of the PVY^E strain. To distinguish PVY‐AGA and PVY‐MON from other PVY^NTN isolates, an RT‐PCR test was developed utilizing new specific primers from the capsid protein gene area and producing a characteristic 955‐bp band. Serological profiling of these PVY isolates with three monoclonal antibodies revealed an unusual reactivity, where one of the two commercial PVY^N‐specific monoclonal antibodies did not recognize PVY‐AGA. The ability of these new PVY recombinants to overcome resistance genes in potato producing mild or no symptoms, combined with the lack of serological reactivity towards at least one PVY^N‐specific antibody may present a significant threat posed by these isolates to seed potato production areas.     

Genetic diversity of Grapevine rupestris stem pitting-associated virus isolates from Tunisian grapevine germplasm

Grapevine rupestris stem pitting-associated virus (GRSPaV) is one of the most widespread grapevine viruses and is transmitted mainly by grafting. GRSPaV presence was tested in 487 samples representative of the Tunisian grapevine germplasm (including autochthonous, table, wine, wild grape, and rootstock varieties) from different Tunisian regions. GRSPaV infection was detected in 51.3% of samples from different Tunisian regions, among which the table grapevine cultivars were the most commonly infected (68.7%). Genetic variability of GRSPaV isolates from wild and cultivated grapevines was assessed by sequencing the partial capsid protein (CP) gene of 19 Tunisian isolates and 1 Italian GRSPaV isolate from Sicily, and the partial RNA-dependent RNA polymerase (RdRp) gene of 13 Tunisian GRSPaV isolates. According to phylogenetic analysis of CP nucleotide sequences obtained in this study and sequences retrieved from GenBank, Tunisian isolates fell into four phylogenetic groups already described (I, II, III, and IV) and two new phylogenetic groups (VI and VIII). Phylogenetic analysis of the partial RdRp gene revealed that Tunisian isolates of GRSPaV are distributed into four phylogroups. This study highlights the importance of regular monitoring of GRSPaV infections in Tunisia, with special regard to those grapevine accessions employed in conservation and selection programmes. In particular, the presence of new GRSPaV genetic variants and infection of wild grapevines must be taken into account in order to choose a correct control strategy.     

Seasonal dynamics and virus translocation of Grapevine leafroll‐associated virus 3 in grapevine cultivars

Grapevine leafroll‐associated virus 3 (GLRaV‐3) is associated with grapevine leafroll disease, one of the most economically important viral diseases of grapevines. This disease impacts on both vine health and grape quality; reduction in yield, brix and wine colour are among its detrimental effects. Many methods, including serological and molecular procedures, have been developed for the detection of GLRaV‐3; however, there is no PCR‐based assay available to quantify virus populations within plant tissues. A real‐time RT‐PCR assay with TaqMan probe was developed for specific and reliable quantitative detection of GLRaV‐3 in infected tissues. The designed primers and probes target the conserved sequence in the RNA‐dependent RNA polymerase (RdRp) domain of the viral genome to prevent amplification of most subgenomic and defective RNAs. This protocol was used to examine the seasonal dynamics and translocation of GLRaV‐3 in field‐grown grapevines. The results showed that the virus spread quickly from trunks to new growing shoots and leaves early in the growing season, and most samples still harboured detectable virus during late summer and autumn. The seasonal progress of one GLRaV‐3 isolate was compared in four grapevine cultivars (Chardonnay, Cabernet Sauvignon, Italia and Thompson Seedless). Within cultivars there was little variability in the distribution and translocation of GLRaV‐3, except for in Thompson Seedless. This quantitative detection assay will be a valuable tool for GLRaV‐3 diagnosis, disease monitoring and population ecology studies.     

Vegetative compatibility groups in Colletotrichum coccodes from Turkey and their aggressiveness to potato

Black dot, caused by Colletotrichum coccodes, is a common disease of potato in Turkey, affecting tuber quality and yield. The objectives of the current study were to characterize vegetative compatibility groups (VCGs) of C. coccodes isolates from three regions in Turkey, and to assess the correlation between VCGs and aggressiveness of isolates on potato. A total of 147 C. coccodes isolates were recovered from plants showing typical black dot symptoms on stolons, roots and stems. The frequency of nitrate non‐utilizing (nit) nit1/nit3 and NitM phenotypes were 79% and 21%, respectively. Complementation between nit mutants of the isolates and eight European/Israeli EU/I‐VCG tester isolates was used to characterize the VCGs. Amongst the tested isolates, 33.3% were assigned to EU/I‐VCG6, 21.8% to EU/I‐VCG8, 15.7% to EU/I‐VCG4. EU/I‐VCG1, EU/I‐VCG3, EU/I‐VCG5 and EU/I‐VCG7 were classified at 1.4%, 3.4%, 4.8% and 5.4%, frequency, respectively. No isolate was assigned to EU/I‐VCG2 group, while 21 isolates (14.3%) were not assigned to any of the EU/I‐VCGs. The pathogenicity tests indicated significant differences in aggressiveness of the isolates with respect to sclerotia density on potato tissues. The highest densities of sclerotia on roots and crown were obtained with EU/I‐VCG6 isolates and the lowest with EU/I‐VCG1, EU/I‐VCG3 and EU/I‐VCG5 isolates. The results demonstrate that there is significant VCG diversity among C. coccodes isolates from potato plants in Turkey.     

Development of a Pathotype Specific SCAR Marker in Plasmodiophora brassicae

Plasmodiophora brassicae is an obligate biotroph that causes clubroot, one of the most damaging diseases of crucifers. Breeding of clubroot-resistant plants has been hampered by the large variation of pathogenicity in P. brassicae and by the lack of an efficient means for detecting specific isolates. To improve the practicality of P. brassicae pathotype-identification, a molecular approach was developed. RAPD profiles of 37 single-spore-derived isolates belonging to seven different pathotypes were compared. A RAPD marker, OPL14₁₂₀₀, was found in the molecular pattern of all the isolates belonging to one particular pathotype (P1), pathogenic on all differential hosts tested. The DNA band corresponding to this marker was cloned and sequenced. No significant homology to previously characterised nucleotide sequences was found. Primers were designed to specifically amplify the OPL14₁₂₀₀ band. The SCAR marker was observed in all isolates belonging to pathotype P1 and was absent in isolates belonging to other pathotypes and in the different plant hosts analysed. The SCAR marker was also generated from direct amplification of DNA from clubs (mixture of host and pathogen DNA) developed after infection by P1 isolates. This molecular marker may be a valuable tool for rapid and reliable identification of P. brassicae P1 isolates in areas where resistant varieties are cultivated.     

A yellows disease system with differing principal host plants for the obligatory pathogen and its vector

A stolbur‐type phytoplasma is the putative pathogen of grapevine yellows disease that causes economic damage to vineyards in most growing areas around the world. The pathogen is known to be transmitted to vines by two planthoppers, Hyalesthes obsoletus and Reptalus panzer; the latter is found in Europe but has not yet been observed in Israel. The establishment of a vector–pathogen–plant relationship requires that the pathogen and the vector meet on a shared host plant. This does not happen in the ecosystem examined here, where two different principal host plants for the obligate pathogen and its vector exist: the pathogen is established on vines, while its vector, H. obsoletus, develops on Vitex agnus‐castus. The present study verified that: (i) the vector cannot complete its life cycle on vines; (ii) V. agnus‐castus does not grow in the immediate vicinity of vines, and does not harbour the pathogen; and (iii) the pathogen is not vertically transmitted from mother to offspring. Moreover, in a thorough search of plants in vine growing areas, no other plants were found that host both the vector and the pathogen. However, it was found that the planthopper can acquire the phytoplasma from infected vines. Nonetheless, this does not prove the ability of the planthopper to further transmit the pathogen to vines and does not explain the presence of the vector on the non‐preferred vines. Thus, the enigma of the pathogen–vector–host triangle in this system remains unresolved.     

Maize dwarf mosaic virus diversity in the Johnsongrass native reservoir and in maize: evidence of geographical,host and temporal differentiation

The genetic diversity and population structure of Maize dwarf mosaic virus (MDMV) was examined by analysis of the full coat protein gene of 539 isolates collected from maize and Johnsongrass (Sorghum halepense) from eight different maize‐growing areas in Spain. Restriction fragment length polymorphism analysis revealed that the MDMV population consisted of 49 genetic variants, with the three most frequent accounting for 44% of the isolates. This population was spatially structured according to the establishment of maize crops in the area. The highest nucleotide diversity values were observed in the old maize‐growing areas in the northeast of Spain (>0·211) and the lowest in the new maize‐growing areas in the west (<0·019). Moreover, the major genetic variants differed between the old and new maize‐growing areas. Evidence of host‐associated selection was found in the endemic area of the virus (Lleida), and aphid‐transmission studies suggested vector selection pressure. Assessment of the temporal evolution of the MDMV population in northeastern areas indicated that time was only significant in Lleida, where it explained 4·8% of the total variation over nine consecutive years.     

Association between genetic variability and titre of Grapevine Pinot gris virus with disease symptoms

The present work was carried out in order to verify the possible association between a new grapevine disease, characterized by leaf mottling and deformation, and the genetic variability and concentration of Grapevine Pinot gris virus (GPGV), a recently identified virus tentatively associated with the pathology. After vineyard surveys and the establishment of real‐time qPCR assays, characterization of GPGV isolates and evaluation of GPGV titre were assessed in more than 100 samples of grapevine Glera, collected from plants regardless of whether or not they showed the symptomatology. Results showed that there was an important association between the GPGV variants and manifestation of the symptoms, and that grapevines with symptoms harboured significantly higher GPGV titre than symptomless vines. Moreover, an interesting relationship among the phylogenetic clustering of the isolates originating from plants with symptoms and some epidemiological characteristics of the disease was found. The current study confirmed the role of GPGV in the emergent disease characterized by grapevine leaf mottling and deformation.     

Distribution of grapevine leafroll associated virus-3 variants in South African vineyards

Three genetic variants of Grapevine leafroll-associated virus 3 (GLRaV-3) were identified from vineyards of the Western Cape, South Africa. In a previous study, three full genome sequences of isolates representing each of the variant groups were determined. The three variant groups were represented by accessions 621, 623 and PL-20, of variant groups I, II and III respectively. A specific single strand conformation polymorphism (SSCP) profile was assigned to each variant which was used as a quick, reliable detection and differentiation method. In this study we analysed the occurrence of these three GLRaV-3 variants in mother blocks in different cultivars and from different vine growing regions using SSCP. The majority of the plants studied, were infected with the group II variant, similar to isolates 623 and GP18. The distribution of three GLRaV-3 variants within a spatio-temporally recorded cluster of diseased plants was studied by means of SSCP profile analysis of ORF5 amplified PCR products. We showed that different GLRaV-3 variants are transmitted to adjacent plants in an infection cluster. Results showed that, in some leafroll disease clusters, the variant that was present in the original GLRaV-3 infected plant of a cluster was transmitted to adjacent plants in a row and across rows.     

Biological and molecular variation amongst Australian Turnip mosaic virus isolates

Turnip mosaic virus (TuMV) causes crop losses worldwide. Eight Australian TuMV isolates originally obtained from five different species in two plant families were inoculated to 14 plant species belonging to four families to compare their host reactions. They differed considerably in virulence in Brassicaceae crop species and virus indicator hosts belonging to three other families. The isolates infected most Brassica species inoculated, but not Raphanus sativus, usually causing systemic mosaic symptoms, so they resembled TuMV biological host type [B]. Whole genome sequences of seven of the Australian isolates were obtained and had lengths of 9834 nucleotides (nt). When they were compared with 37 non‐recombinant TuMV genomes from other continents and another whole genome from Australia, six of them formed an Australian group within the overall world‐B phylogenetic grouping, while the remaining new genome sequence and the additional whole genome from Australia were part of the basal‐B grouping. When the seven new Australian genomes and the additional whole genome from Australia were subjected to recombination analysis, six different recombination events were found. Six genomes contained one or two recombination events each, but one was non‐recombinant. The non‐recombinant isolate was in the Australian grouping within the overall world‐B group while the remaining recombinant isolates were in the basal‐B and world‐B phylogenetic groups.     

Comparison of foliar and soil formulations of neonicotinoid insecticides for control of potato leafhopper, Empoasca fabae (Homoptera: Cicadellidae), in wine grapes

BACKGROUND: The potential of systemic neonicotinoid insecticides to control potato leafhopper, Empoasca fabae (Harris), a damaging pest of wine grapes in the eastern United States, was investigated. Soil or foliar applications were made to potted or field‐grown vines, and the response of leafhoppers was determined in clip cages over the following month on young or mature leaves. RESULTS: Foliar application of imidacloprid caused immediate and long‐lasting reductions in E. fabae survival on both leaf ages, whereas the activity of soil‐applied imidacloprid was delayed. Clothianidin, imidacloprid and thiamethoxam all provided long‐lasting reduction in leafhopper survival on young and mature foliage when applied through either delivery route. However, the percentage of moribund nymphs was significantly greater on foliar‐treated vines and increased over time in mature and immature leaves compared with soil‐treated vines. Residue analysis of foliar‐applied imidacloprid showed an 89% decline in mature leaves from day 1 to day 27, and a 98% decline in immature leaves over the same time period. Comparison of soil‐applied clothianidin, imidacloprid and thiamethoxam in field‐grown vines showed significant reduction in E. fabae only on mature leaves of vines treated with thiamethoxam. CONCLUSIONS: Neonicotinoids can control E. fabae in small vines, even in rapidly expanding foliage where this pest causes greatest injury. Soil application provides superior long‐term vine protection because declining residues on foliar‐treated vines lead to suboptimal activity within 2–3 weeks. Vineyard managers of susceptible cultivars may take advantage of this approach to E. fabae management by using foliar applications of the three neonicotinoids tested here, or by using soil‐applied thiamethoxam. Copyright © 2011 Society of Chemical Industry     

Occurrence of Grapevine Pinot gris virus in Friuli Venezia Giulia (Italy): field monitoring and virus quantification by real‐time RT‐PCR

Since 2003 the presence of a new syndrome characterized by symptoms of stunting, chlorotic mottling, leaf deformation, reduced yields and quality has been reported in some white berry varieties of Vitis vinifera in Trentino‐Alto Adige and Friuli Venezia Giulia vineyards. The identification of a new virus, provisionally called Grapevine Pinot gris virus (GPGV), in a cv. Pinot gris vine suggested an association between this new syndrome and the virus presence (Giampetruzzi et al., 2012), however the contemporary presence of GPGV in both symptomatic and asymptomatic plants has still to be explained. In this work, a large‐scale monitoring over a 3‐year period (2012–14) of Friuli Venezia Giulia vineyards and nurseries has shown a widespread presence of GPGV in symptomatic plants and also in asymptomatic vines, even if at a slightly lower percentage. Quantitative analyses of the virus titer revealed a great variability in the viral content of both symptomatic and asymptomatic plants but the mean GPGV quantity in symptomatic vines was significantly higher than in asymptomatic plants.     

Virulence and pCM1 plasmid carriage are related to BOX-PCR fingerprint type in strains of Clavibacter michiganensis subsp. michiganensis that cause bacterial wilt and canker of tomato in Argentina

Clavibacter michiganensis subsp. michiganensis (Cmm) causes bacterial wilt and canker in tomato, producing important economic losses worldwide. Its virulence has been related to several putative virulence factors present on a chromosomal pathogenicity island and on plasmids pCM1 and pCM2, in strain NCPPB382. We genotypically characterized a collection of Cmm isolates from the main greenhouse tomato-producing areas of Argentina by BOX-PCR fingerprinting and screened for the presence of genes and plasmids involved in pathogenicity by PCR. In addition, we evaluated in vitro cellulolytic activity and virulence in planta of selected strains. BOX-PCR fingerprinting clustered strains into four groups. Group II was dominant and included the most virulent strains, while Group III was the smallest and had the least virulent strains. All local strains exhibited similar cellulolytic activity. Most of the examined strains carry two plasmids of similar size to those of NCPPB382, although there were strains with one or three plasmids. By PCR amplification of repA, pCM1 was detected only in strains belonging to Group III, which includes local strains closely related to reference strain NCPPB382. All analysed pathogenicity genes were widespread among strains, and so in strains belonging to Groups I and II, celA found on pCM1 in NCPPB382 could be found in the chromosome or in plasmids other than pCM1. This study contributes to a better understanding of the diversity of Cmm genetic profiles and virulence of strains present in Argentina. Such information could be useful for the selection of strains for screening of host resistance and development of resistant tomato varieties.