Characterisation of a novel ilarvirus causing grapevine angular mosaic disease

Unusual symptoms were observed on ‘Baresana’ x ‘Baresana’ Vitis vinifera hybrid vines in the Grapevine Variety Collection of the Grapevine Institute, Athens. The affected vines showed sharp angular mosaic on leaves, along the veins and in vein angles, malformations, abortive flowers or very few berries with smaller, wrinkled and non-germinating seeds, as well as gradual decline, severe stunting and death of the vine. Serological tests on diseased vines for the presence of 13 known grapevine viruses gave negative results. An infectious agent was transmitted mechanically to several herbaceous indicator plants. Koch’s Postulates were fulfilled, and the agent, proven to be a virus, was named Grapevine angular mosaic virus (GAMV). Serological tests have been developed for the virus. The most conserved polymerase region showed significant similarity of GAMV with members of subgroup 1 of the Ilarvirus genus; however ML phylogenetic analysis could not support its clustering within this subgroup. GAMV differs serologically and in particle morphology from Grapevine line pattern virus (GLPV) a putative member of the Ilarvirus genus that infects grapevine. It is proposed that GAMV is a novel member of the Ilarvirus genus.     

Genetic variability and population structure of <Emphasis Type="Italic">Grapevine virus A</Emphasis> coat protein gene from naturally infected Italian vines

Grapevine virus A (GVA) is considered one of the viruses associated with rugose wood (RW), one of the most economically important diseases of grapevine. Thirty-seven GVA isolates collected from grapevine cultivars from Marche (central-eastern Italy), Apulia and Campania (southern Italy), were subjected to molecular characterization. The genetic and population diversity was studied in the coat protein (CP) gene by RT-PCR-RFLP analysis with three restriction enzymes (MseI, AluI, and AciI), and nucleotide sequencing. A new primer pair (CP1F/R) allowing amplification of the whole CP gene (621 bp) was developed. RFLP with AciI yielded the highest number of variants in GVA isolates, showing seven different ‘simple’ profiles (A, B, C, D, E, F, and G). ‘Complex’ profiles were also found, and the most common variant combination was A + B in 39% of isolates. The analysis of GVA sequences confirmed the presence of plants infected with more than one GVA variant and suggested that RT-PCR-RFLP is suitable for evaluating population diversity of GVA enabling a screening of different haplotypes. The distribution of RFLP profiles and the phylogenetic analysis were not correlated with the location of infected plants, showing the presence of a GVA population with genetic diversity in the average with those of RNA viruses.     

High prevalence of viruses in table grape from Spain detected by real-time RT-PCR

Table grapes from one of the most important growing area in Spain (Vinalopó, Alicante) protected by the Designation of Origin “Vinalopó bagged table grape”, were surveyed and analysed to determine the prevalence of the five viruses included in the Spanish certification program: Arabis mosaic virus (ArMV), Grapevine fanleaf virus (GFLV), Grapevine fleck virus (GFkV), Grapevine leafroll associated virus-1 (GLRaV-1) and Grapevine leafroll associated virus-3 (GLRaV-3). Ninety five sampling points were selected and the position of grapevine plants georeferenced. Samples were collected in two different vegetative periods and analyses were performed by ELISA and real-time RT-PCR. Purified RNA and immobilized viral targets from plant extracts on nylon membranes were used in parallel assays as templates for PCR assays. In order to analyse these five viral species by real-time RT-PCR, new specific primers and TaqMan probes were designed for detection of ArMV and GFkV. Real time RT-PCR from purified RNA was more sensitive than spot version and ELISA tests. The most prevalent virus was GFLV (95.8%) followed by GLRaV-3 (94.7%), GLRaV-1 (66.3%) and GFkV (65.3%). ArMV was not detected in any sample. The high level of viral infections and the presence of mixed infections suggest that initial infected plant material and uncontrolled traffic of propagation material have played an important role in the spread of viruses.     

Pathogenicity,colony morphology and diversity of isolates of <Emphasis Type="Italic">Guignardia citricarpa</Emphasis> and <Emphasis Type="Italic">G. mangiferae</Emphasis> isolated from <Emphasis Type="Italic">Citrus</Emphasis> spp.

In the present study, the pathogenicity of 36 isolates of Guignardia species isolated from asymptomatic ‘Tahiti’ acid lime fruit peels and leaves, ‘Pêra-Rio’ sweet orange leaves and fruit peel lesions, and a banana leaf were characterized. For pathogenicity testing, discs of citrus leaves colonized by Phyllosticta citricarpa under controlled laboratory conditions were kept in contact with the peels of fruit that were in susceptible states. In addition, pathogenicity was related to morphological characteristics of colonies on oatmeal (OA) and potato dextrose agar (PDA). This allowed the morphological differentiation between G. citricarpa and G. mangiferae. Polymerase chain reactions (PCRs) were also used to identify non-pathogenic isolates based on primers specific to G. citricarpa. A total of 14 pathogenic isolates were detected during pathogenicity tests. Five of these were obtained from leaf and fruit tissues of the ‘Tahiti’, which until this time had been considered resistant to the pathogen. Given that the G. citricarpa obtained from this host was pathogenic, it would be more appropriate to use the term insensitive rather than resistant to categorize G. citricarpa. A non-pathogenic isolate was obtained from lesions characteristic of citrus black spot (CBS), indicating that isolation of Guignardia spp. under these conditions does not necessarily imply isolation of pathogenic strains. This also applied to Guignardia spp. isolates from asymptomatic citrus tissues. Using fluorescent amplified fragment length polymorphism (fAFLP) markers, typically pathogenic isolates were shown to be more closely related to one another than to the non-pathogenic forms, indicating that the non-pathogenic isolates display higher levels of genetic diversity.     

Occurrence and incidence of viruses infecting green beans in south-eastern Spain

A study was conducted to determine the identity and prevalence of viruses in 455 greenhouses in the main Spanish green bean growing area. Directed surveys were conducted in 422 crops from 2000–2004 to collect samples from diseased plants displaying symptoms that could be attributed to viruses. The samples were analysed to detect any virus by means of dsRNA extraction, mechanical inoculation to test plants, as well as ELISA and/or RT-PCR tests to detect potyviruses, geminiviruses and viruses previously known to infect beans in Spain. Random surveys were conducted in the years 2002 and 2005 (in 21 and 12 greenhouses, respectively) to study the actual incidence of known viruses in the area. Symptoms were recorded in 23,108 plants from which 664 plants were collected and analysed by ELISA or RT-PCR. The results of the directed surveys showed that all the analyzed crops carried the cryptic virus Phaseolus vulgaris endornavirus (PVuV), whereas phytopathogenic viruses appeared in smaller percentages of the crops: Tomato yellow leaf curl virus (TYLCV) 20.4%, Southern bean mosaic virus (SBMV) 9.0%, Tomato spotted wilt virus (TSWV) 4.0%, and the new species Bean yellow disorder virus (BnYDV) that broke out in 2004 with occurrence values higher than 34.3% that year. From 2000–2004 an important decrease in TYLCV was observed, along with a slight increase in SBMV and a consistently low occurrence of TSWV. The results of the random surveys confirmed the increased occurrence of virus detected during the directed surveys, and furthermore demonstrated the percentage of incidence for each virus.     

Incidence of viruses in <Emphasis Type="Italic">Alstroemeria</Emphasis> plants cultivated in Japan and characterization of <Emphasis Type="Italic">Broad bean wilt virus-2, Cucumber mosaic virus</Emphasis> and <Emphasis Type="Italic">Youcai mosaic virus</Emphasis>

Alstroemeria plants were surveyed for viruses in Japan from 2002 to 2004. Seventy-two Alstroemeria plants were collected from Aichi, Nagano, and Hokkaido prefectures and 54.2% were infected with some species of virus. The predominant virus was Alstroemeria mosaic virus, followed by Tomato spotted wilt virus, Youcai mosaic virus (YoMV), Cucumber mosaic virus (CMV), Alstroemeria virus X and Broad bean wilt virus-2 (BBWV-2). On the basis of nucleotide sequence of the coat protein genes, all four CMV isolates belong to subgroup IA. CMV isolates induced mosaic and/or necrosis on Alstroemeria. YoMV and BBWV-2 were newly identified by traits such as host range, particle morphology, and nucleotide sequence as viruses infecting Alstroemeria. A BBWV-2 isolate also induced mosaic symptoms on Alstroemeria seedlings.     

Ineffectiveness of pruning to control citrus huanglongbing caused by <Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter americanus

The huanglongbing (HLB) disease of citrus trees, caused by Candidatus Liberibacter asiaticus and Ca. Liberibacter americanus, was first reported in Brazil in March, 2004. The presence of the disease has caused serious concerns among growers. Pruning experiments were conducted to determine if removal of symptomatic branches or the entire canopy (decapitation) would eliminate infected tissues and save HLB-affected trees. Pruning was done in five blocks on a total of 592 3- to 16 year-old ‘Valência’, ‘Hamlin’ or ‘Pêra’ sweet orange trees showing no symptoms or with two levels of symptom severity. Ten decapitated trees per block were caged and all trees were treated with insecticides to control the psyllid vector, Diaphorina citri. Mottled leaves reappeared on most symptomatic (69.2%) as well on some asymptomatic (7.6%) pruned trees, regardless of age, variety, and pruning procedure. Presence of the pathogen (Ca. Liberibacter americanus) in all symptomatic trees was confirmed by PCR. In general, the greater the symptom severity before pruning the lower the percentage of trees that remained asymptomatic after pruning.     

Molecular and biological characterization of <Emphasis Type="Italic">Chrysanthemum stem necrosis virus</Emphasis> isolates from distinct regions in Japan

Three isolates of Chrysanthemum stem necrosis virus (CSNV) were obtained from chrysanthemum plants in distinct regions of Japan in 2006 and 2007. All the original host plants showed severe necrotic symptoms on the leaves and stems. Amino acid sequence data of the nucleocapsid protein genes of the three isolates (CbCh07A, TcCh07A, and GnCh07S) showed high identities with those of two other CSNV isolates, HiCh06A L1 from Japan and Chry1 from Brazil. Furthermore, for the first time the complete nucleotide sequence of the S RNA was determined for CSNV (isolate HiCh06A). In phylogenetic analysis based on the non-structural protein genes from the genus Tospovirus, HiCh06A L1 was placed in the same genetic group as Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus. Host range examination for isolates HiCh06A L1 and CbCh07A showed that green pepper (cv. ‘Kyoyutaka’, ‘Saitamawase’, ‘Tosakatsura’, ‘L3 sarara’ and ‘L3 miogi’) and tomato (cv. ‘Sekaiichitomato’) were systemically susceptible hosts, whereas TSWV-resistant Solanaceae species, Capsicum chinense, Lycopersicon peruvianum and a TSWV-resistant cultivar of green pepper (cv. TSR miogi), were resistant.     

Characterization of Grapevine Algerian latent virus isolated from nipplefruit (Solanum mammosum) in Japan

Alfalfa mosaic virus (AMV), Cucumber mosaic virus (CMV), Potato virus Y (PVY), Tomato bushy stunt virus nipplefruit strain (TBSV-Nf), and an unknown spherical virus were isolated from nipplefruit (Solanum mammosum) cultivated in Chiba Prefecture, Japan. The spherical virus was identified as Grapevine Algerian latent virus nipplefruit strain (GALV-Nf) from the genus Tombusvirus, based on its physical properties, serological relationships, and analysis of genomic RNA. The genomic RNA of GALV-Nf is 4731 nucleotides long and encodes five open reading frames as well as those of other tombusviruses. Nipplefruit infected with GALV-Nf had severe stunting, leaf deformation, and clear mosaic symptoms. This is the first report of an isolation of GALV in Japan. An erratum to this article is available at .     

A new furovirus infecting barley in France closely related to the Japanese soil-borne wheat mosaic virus

In April 2001, stunted barley plants bearing mosaic symptoms were observed in a field in France (Marne Department, 51). Rod-shaped and flexuous particles were visualized by electron microscopy and positive serological reactions were detected by ELISA with Barley yellow mosaic virus (BaYMV) and Soil-borne cereal mosaic virus (SBCMV) polyclonal antisera. The tubular virus which was soil transmissible to barley cv. Esterel was separated from BaYMV by serial mechanical inoculations to barley cv. Esterel. This furo-like virus, in contrast to a French isolate of SBCMV, could be transmitted to Hordeum vulgare, Avena sativa, Beta vulgaris and Datura stramonium. RT-PCR was used to amplify the 3′-terminal 1500 nucleotides of RNA1 and the almost complete sequence of RNA2. Nucleotide and amino acid sequence analyses revealed that the French virus infecting barley is closely related to a Japanese isolate of Soil-borne wheat mosaic virus (SBWMV-JT) which was originally isolated from barley. This French isolate was named SBWMV-Mar. The 3′ UTRs of both RNAs can be folded into tRNA-like structures which are preceded by a predicted upstream pseudoknot domain with seven and four pseudoknots for RNA1 and RNA2, respectively. The four pseudoknots strongly conserved in RNAs 1 and 2 of SBWMV-Mar show strong similarities to those described earlier in SBWMV RNA2 and were also found in the 3′ UTR of Oat golden stripe virus RNAs 1 and 2 and Chinese wheat mosaic virus RNA2. Sequence analyses revealed that the RNAs 2 of SBWMV-Mar and -JT are likely to be the product of a recombination event between the 3′ UTRs of the RNAs 2 of SBWMV and SBCMV. This is the first report of the occurrence of an isolate closely related to SBWMV-JT outside of Japan.     

Partial characterisation of a novel <Emphasis Type="Italic">Tobamovirus</Emphasis> infecting <Emphasis Type="Italic">Actinidia chinensis</Emphasis> and <Emphasis Type="Italic">A. deliciosa</Emphasis> (Actinidiaceae) from China

Actinidia chinensis and A. deliciosa plants from China, showing a range of symptoms, including vein clearing, interveinal mottling, mosaics and chlorotic ring spots, were found to contain ~300 nm rod-shaped virus particles. The virus was mechanically transmitted to several herbaceous indicators causing systemic infections in Nicotiana benthamiana, N. clevelandii, and N. occidentalis, and local lesions in Chenopodium quinoa. Systemically- infected leaves reacted with a Tobacco mosaic virus polyclonal antibody in indirect ELISA. PCR using generic and specific Tobamovirus primers produced a 1,526 bp sequence spanning the coat protein (CP), movement protein (MP), and partial RNA replicase genes which showed a maximum nucleotide identity (88%) with Turnip vein clearing virus and Penstemon ringspot virus. However, when the CP sequence alone was considered the highest CP sequence identity (96% nt and 98% aa) was to Ribgrass mosaic virus strain Kons 1105. The morphological, transmission, serological and molecular properties indicate that the virus is a member of subgroup 3 of the genus Tobamovirus.     

Characterisation of a Quantitative Resistance to Vector Transmission of Tomato yellow leaf curl virus in Lycopersicon pimpinellifolium

Two wild genotypes from the same species Lycopersicon pimpinellifolium, WVA106 (susceptible) and INRA-Hirsute (so-called ‘resistant’), were compared with respect to their reaction to Tomato yellow leaf curl virus isolate Réunion (TYLCV-Mld[RE]), using both whitefly-mediated inoculation and graft inoculation. Disease incidence and symptom severity were scored. Presence and quantification of viral DNA were assessed by dot blot hybridisation. Upon insect inoculation, accession INRA-Hirsute showed a moderate resistance against TYLCV that was overcome by a high inoculation pressure obtained by increasing the cumulative number of inoculative whiteflies. Temporal analyses of the disease progress in relation to this criterion exhibited that the protection was quantitative, mainly reducing the TYLCV-Mld[RE] incidence by at maximum 50% at low inoculation pressure. When graft inoculated, the final TYLCV-Mld[RE] disease incidence was 100% in both susceptible and resistant genotypes with severe symptoms, suggesting a reduction of virus transmission by a vector resistance as a possible mechanism. Implications of using such type of resistance in breeding programmes are discussed.     

Differentiation of Bean pod mottle virus isolates based upon host symptoms

Accessions from Glycine, Phaseolus, and Vigna genera were screened for their reactions to different subgroups of isolates of Bean pod mottle virus (BPMV) in order to establish a differential host system. Screening results indicated that the BPMV isolates differed in pathogenic aggressiveness but not in virulence. No major resistance genes were found in soybean (Glycine max) or G. soja since all screened accessions showed mosaic or necrotic symptoms to BPMV inoculation. However, these accessions expressed differences in severity of symptoms when challenged by various BPMV isolates. The inoculation of G. tomentella accessions did not result in mosaic symptoms, and some accessions did not support systemic infection of some of the isolates. Resistance, presented as a hypersensitive reaction, was observed in some of Phaseolus and Vigna genotypes, and resistant response or susceptibility was stable to all the isolates used in the screening. In conclusion, the selected G. soja genotypes PI 407019, PI 464889A, and PI 464928, and ‘Amsoy 71’ soybean may help to separate severe (reassortant) from mild isolates of BPMV based upon their phenotypic reactions.     

Multiple virus infections of lablab [<Emphasis Type="Italic">Lablab purpureus</Emphasis> (L.) Sweet] in Nigeria

Leaf samples of Lablab purpureus collected from two agroecological zones of Nigeria—the northern guinea savanna zone (NGSZ) and the derived savanna zone (DSZ)—were infected with viruses when serologically indexed against available antisera. Approximately 31.1 and 81.1% of the leaf samples collected from the NGSZ and DSZ, respectively, were infected. Seven viruses were found: Bean common mosaic virus (BCMV), Cowpea aphid-borne mosaic virus (CABMV), Cucumber mosaic virus (CMV), Cowpea mottle virus (CPMoV), Cowpea severe mosaic virus (CPSMV), Southern bean mosaic virus (SBMV) and Tobacco mosaic virus (TMV) were detected from samples collected from NGSZ, while CMV, CPMoV, Cowpea mosaic virus (CPMV) and CPSMV were detected from samples from DSZ.     

First report of a <Emphasis Type="Italic">Grapevine Algerian latent virus</Emphasis> disease on statice plants (<Emphasis Type="Italic">Limonium sinuatum</Emphasis>) in Japan

A viral disease was found in Nagano Prefecture, Japan, on statice (Limonium sinuatum) with chlorotic leaf spot, necrotic stunt, and dwarfing. Spherical virus particles 30 nm in diameter were isolated from infected plants and statice seedlings and caused identical symptoms 4 weeks after mechanical inoculation. Nucleotide and deduced amino acid sequences of the coat protein showed 98% and 98.7% identities with those of Grapevine Algerian latent virus (GALV) nipplefruit strain. This is the first report in Japan of a viral disease on statice caused by GALV. The nucleotide sequence data reported here are available in the DDBJ/EMBL/GenBank databases under accession AB461854.     

A new Badnavirus species detected in Bougainvillea in Brazil

Badnavirus in Bougainvillea spectabilis showing virus-like symptoms was identified by the presence of bacilliform particles, measuring 125–130 × 30–40 nm in leaf-dip preparations and by analysis of its putative open reading frame 3 sequence. The virus, tentatively named Bougainvillea bacilliform virus (BBV), had the highest identities (up to 60%) with Spiraea yellow leaf spot virus, Gooseberry vein banding associated virus, Taro bacilliform virus, and Citrus yellow mosaic virus. In phylogenetic analysis, BBV clustered with Badnavirus putative species. Attempts to transmit the virus to several hosts failed. This is the first report of a new Badnavirus detected in Bougainvillea.     

Olfactory response of the ladybird beetle <Emphasis Type="Italic">Stethorus gilvifrons</Emphasis> to two preys and herbivore-induced plant volatiles

The spider mites Tetranychus urticae Koch and Panonychus ulmi (Koch) (Tetranychidae) cause severe economic losses to vegetable farms and deciduous fruit orchards in Turkey. One of their predators, the ladybird beetle Stethorus gilvifrons (Muls.) (Col., Coccinellidae), aggregates on mite-infested patches of plants. The present study assessed whether there is a role for herbivore-induced plant volatiles (HIPVs) and/or odors emitted directly from these two mite species in the aggregative response of ladybird beetles. The olfactory responses of the predator females to volatiles from T. urticae- and/or P. ulmi-infested sweet pepper (four cultivars, viz. ‘Demre’, ‘Yalova Carliston’, ‘Kandil Dolma’ and ‘Yag Biberi’), kidney bean (cv. ‘Barbunya’) and apple (M9 rootstock) were investigated using a two-choice olfactometer. Our results showed that HIPVs emitted from both T. urticae- and P. ulmi-infested plants significantly attracted S. gilvifrons adults for all plants except the sweet pepper cv. Yag Biberi. In addition, it was found that volatiles from apple plants infested by T. urticae and, especially, P. ulmi are more attractive for S. gilvifrons females than those emitted by other infested plants. The results also suggest that the odors of T. urticae adults and their products might influence the attraction of S. gilvifrons females.