TransgenicNicotiana Benthamiana and grapevine plants transformed with grapevine virus a (GVA) sequences

As part of a project to construct virus-resistant transgenic grapevines, grapevine virus A (GVA) coat protein (CP) sequences were introduced into grapevine and the model plantNicotiana benthamiana. The engineered constructs were tested for their ability to engender resistance inN. benthamiana. A number of GVA-CP-transformedN. benthamiana lines have become resistant to GVA. Preliminary biological and molecular analyses strongly suggest that in most cases the obtained resistance is RNA-mediated. Several lines of transgenic grapevine were obtained.     

Identification of divergent variants of Grapevine virus A

Eight isolates of Grapevine virus A (GVA), which induced different symptoms in leaves of Nicotiana benthamiana, were recovered from various grapevines. The dsRNA patterns of two isolates, which consistently induced mild vein clearing (referred here as mild isolates of GVA) were similar, but different from those of other isolates of GVA. Analysis based on overall nucleotide (nt) sequence identity in the 3 terminal part of the GVA genome, comprising part of ORF3 (putative movement protein, MP), entire ORF4 (capsid protein, CP), entire ORF5 and part of 3 UTR, revealed that GVA isolates separate into three groups (I, II, III), sharing 91.0–99.8% nt sequence identity within groups and 78.0–89.3% nt sequence identity between groups. Mild isolates of the virus were group III and shared only 78.0–79.6% nt sequence identity with the other isolates. The comparison of predicted amino acid sequences for MP and CP revealed many amino acid alterations, revealing distinct local net charges of these proteins for mild isolates of the virus. Based on both conserved and divergent nt regions in the CP and ORF5, oligonucleotide primers were designed for the simultaneous RT-PCR detection of all GVA isolates and for the specific detection of the most divergent virus variants represented here by mild isolates of the virus.     

Biological control of grapevine crown gall by nonpathogenic <Emphasis Type="Italic">Agrobacterium vitis</Emphasis> strain VAR03-1

A nonpathogenic strain of Agrobacterium vitis VAR03-1 was tested as a biological control agent against crown gall of grapevine (Vitis vinifera L.). A mixture of the nonpathogenic strain VAR03-1 and a tumorigenic strain G-Ag-27 of A. vitis at cell ratios of 1 : 1, 3 : 1, 9 : 1, and 99 : 1 significantly inhibited gall formation and size on stems of tomato (Lycopersicon esculentum Mill.). Strain VAR03-1 also inhibited gall formation on stems of both tomato and grapevine at a 1 : 1 cell ratio with several tumorigenic A. vitis strains isolated from different fields of grapevine in Japan. In biological control tests, when roots of grapevine and tomato seedlings were soaked in a cell suspension of strain VAR03-1 for 24 h before a 1-h soaking in a cell suspension of the pathogen and subsequent planting in pots of infested soil, strain VAR03-1 significantly reduced the incidence of gall formation on both plants.     

Population structure,races, and host range of <Emphasis Type="Italic">Aphanomyces euteiches</Emphasis> from alfalfa production fields in the central USA

Aphanomyces euteiches (races 1 and 2) causes root rot of alfalfa; however, its population biology and distribution are poorly understood where alfalfa is a major crop. The objectives of this study were to (1) characterise the distribution and frequency of races of A. euteiches in Illinois alfalfa fields, (2) determine host range of A. euteiches on cultivated and native legumes, and (iii) to describe genetic diversity and population genetic structure of A. euteiches in alfalfa fields. To accomplish this, soil samples (n = 103) were collected from 30 alfalfa fields in 18 Illinois counties. Using the susceptible cv. ‘Saranac’, 148 isolates of A. euteiches were baited from the soil. The virulence phenotype of isolates representing all 18 counties was tested, and 54% were R1 and 46% were R2. Both races were detected in 61% of the counties, whereas only R1 was detected in 22% and R2 in 17%. Thirteen legume hosts for isolates from alfalfa fields were identified based on symptoms and/or production of oospores in roots. In addition to six previously known hosts, seven species were susceptible to infection: kura clover, purple prairie clover, white prairie clover, ladino clover, hairy vetch, Canadian milk vetch, and Illinois tick trefoil. AFLP analysis revealed high levels of genetic diversity among the isolates from different fields and counties and a lack of genetic structuring of populations based on race or geographical origin. The results suggest that populations of A. euteiches in alfalfa fields are diverse, often composed of races 1 and 2, and create risk for alfalfa and to multiple cultivated and native legume species.     

<Emphasis Type="Italic">Grapevine virus A</Emphasis> transmission by larvae of <Emphasis Type="Italic">Parthenolecanium corni</Emphasis>

Grapevine virus A (GVA, Vitivirus) was transmitted experimentally by first and second instars of the scale insect Parthenolecanium corni from grapevine to grapevine and to the herbaceous host Nicotiana benthamiana. This is the first report of GVA transmission by P. corni. Grapevine leafroll-associated virus-1 (Ampelovirus) was always present in the donor grapevines and, in every case, GVA was transmitted simultaneously with this ampelovirus from grapevine to grapevine, suggesting possible interactions between the two viruses for transmission.     

Characterization of Citrus tristeza virus isolates in northern Iran

The biological and molecular properties of four Citrus tristeza virus (CTV) isolates isolated from infected Satsuma trees imported from Japan, and growing in citrus groves in northern Iran (Mahdasht orchards, Mazandaran Province), were investigated. CTV-infected samples were collected from sweet orange trees and grafted onto Alemow (Citrus macrophylla Wester) seedlings. On indicator plants, these isolates produced various symptoms including vein clearing and stem pitting on Mexican lime, Alemow, and Citrus hystrix, and yellowing and stunting on sour orange and grapefruit seedlings. Citrus samples were also surveyed for CTV using serological tests. The coat protein (CP) gene of these isolates was amplified using specific primers, yielding an amplicon of 672 bp for all isolates. Sequence analysis showed 98%–99% sequence homology of Iranian isolates with the Californian CTV severe stem-pitting isolate SY568 and 97%–98% homology with the Japanese seedling yellows isolate NUagA. The Iranian isolates were compared by restriction fragment length polymorphism (RFLP) analysis of the CP amplicon for further classification.     

葡萄A病毒外壳蛋白原核表达及抗血清制备

 葡萄A病毒（Grapevine virus A,GVA）为线性病毒科（Flexiviridae）葡萄病毒属（Vitivirus）的代表种,是葡萄皱木复合病（rugose wood complex disease）的重要病原之一,可引起葡萄嫁接成活率下降、春季萌芽延迟、生长减弱甚至衰退死亡等危害\[1,2\]。GVA为线状单链RNA病毒,基因组共编码5个开放阅读框（ORF1\|5）,其中ORF4 编码外壳蛋白（coat protein, CP）,是病毒粒子包裹和系统移动所必需的功能蛋白\[3,4\]。GVA自然寄主为葡萄,机械摩擦可侵染本氏烟等草本寄主\[2\],由于嫁接和无性繁殖材料调运等因素造成该病毒远距离传播,目前在世界多个国家和地区均有发生。     

Genetic Structure and Variation in Aggressiveness in European and Australian Populations of the Grapevine Dieback Fungus, Eutypa lata

Eutypa lata is an ascomycete fungus causing a severe dieback in grapevine. The genetic structure of populations of E. lata from seven regions in Australia, France, Italy and Spain was examined using 20 random amplified polymorphic DNA (RAPD) markers. In some regions, populations were subdivided and a total of 14 samples were analysed. A total of 231 RAPD haplotypes were found among the 240 isolates. Vegetative compatibility testing further demonstrated that isolates of the same haplotype were genetically distinct. Gene diversity was the highest in the population from northern Italy and lowest in the Alsace region in France. Linkage disequilibrium between pairs of putative loci was very low and most of the multilocus analyses were consistent with the hypothesis of random association of the loci. This suggests that random mating occurred in every population and that the sexual stage shapes the genetic structure of E. lata populations in the regions sampled. Only 6% of the total variability was attributable to differences between populations. Nevertheless, significant differences in allele frequency appeared with respect to six RAPD markers indicating some genetic differentiation between populations. This differentiation appeared attributable to differences between the Italian and Spanish populations and the other populations. We thus hypothesize that a restriction of gene flow exists within Europe. The population from Australia was genetically closer to the French and Spanish populations than to that from Italy. Genetic diversity is associated with considerable variation in aggressiveness, which was assessed on cuttings in the greenhouse in six populations. All populations included a range of isolates differing in aggressiveness, but the Italian population seemed to have more isolates with low aggressiveness.     

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.     

Differentiation of <Emphasis Type="Italic">Erwinia amylovora</Emphasis> strains from Bulgaria by PCR-RFLP analysis

Fifty strains of Erwinia amylovora isolated in Bulgaria from different host plants and locations as well as in different years were analysed by RFLP analysis of the pEA29 PstI amplified fragment with HpaII. All the strains formed three well-resolved fragments (large—from 365 to 440 bp, medium—about 341 bp and small—about 180 bp).The strains were classified into three RFLP groups based on the polymorphism in the length of the largest fragment. This fragment was of intermediate size for 63% of the strains, and it was the longest (from 410 to 440 bp) for 29% of the strains. The variable region was sequenced for five strains. The DNA sequence analysis confirmed the different size of the largest fragment. Ten or more than ten SSRs were found for the strains in the group with the largest size of the largest fragment. Some correlation between the RFLP profiles and the origin of the strains was revealed. The RFLP profiles displayed stability in certain strains isolated from the same trees and orchards, but in different years. The number of SSRs was different in strains isolated from one and the same host plant, orchard and year, and also in strains isolated from the same host plant and orchard, but in different years. This could indicate that under natural conditions the fire blight symptoms might be caused by a mixture of E. amylovora strains with different SSR numbers, and so coexistence of distinguishable strains or a change in the population could be assumed.     

Morphological,pathological and genetic variations among isolates of <Emphasis Type="Italic">Cochliobolus sativus</Emphasis> from Nepal

Spot blotch, caused by Cochliobolus sativus (Ito & Kuribayashi) Drechs. ex Dastur, is one of the important diseases of wheat worldwide. The main objective of this study was to investigate the phenotypic and genotypic variability among C. sativus isolates from the hills and plains in Nepal. A total of 48 monoconidial isolates of C. sativus from the hills (n = 24 isolates) and plains (n = 24 isolates) in Nepal were analyzed for morphology, aggressiveness and genetic structure. C. sativus isolates were grouped into three categories on the basis of their colony texture and mycelia colour. Thirteen isolates from the hills and plains belonging to three morphological groups were randomly selected and evaluated for aggressiveness on eight wheat cultivars (Chirya 1, Chirya 7, Milan/Shanghai 7, SW 89–5422, PBW 343, BL 1473, BL 3036, and RR 21) at the seedling stage. Nonparametric analysis revealed that the isolates from the plains (median disease rating of 5) were significantly (P = 0.0001) more aggressive than the isolates from the hills (median disease rating of 3). A significant (P = 0.0001) isolate by cultivar interaction was demonstrated and the isolates from the same geographic region and morphological group displayed different degrees of aggressiveness on wheat cultivars tested. Combined IS-PCR and rep-PCR analyses revealed moderate gene diversity (H = 0.24 and 0.25 for the hills and plains, respectively). Low linkage disequilibrium (LD) value and non-significant (P = 0.001) population differentiation (G″_ST = 0.05) were detected, indicating that isolates of C. sativus from the hills and plains in Nepal were genetically similar. Analysis of molecular variation (AMOVA) revealed low (7%) levels of genetic variation between the hill and plain populations, whereas >93% of genetic variation was found within populations. Overall, C. sativus isolates from Nepal are pathologically and genetically diverse, and such information will be useful in developing wheat cultivars resistant to C. sativus.     

Population genetic structure of <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> on canola in Iran

The genetic structure of 276 Sclerotinia sclerotiorum isolates representing 37 field populations from four provinces in northern Iran were analysed with six polymorphic microsatellite loci. In total, 80 haplotypes were detected with 19 haplotypes (23.7%) shared amongst at least two regional populations. None of the haplotypes were shared among all four regional populations. Of the 80 haplotypes, 32 haplotypes (40%) occurred in low frequencies represented by only one isolate. Moderate levels of gene diversity (H = 0.51 to 0.61) and genotypic diversity (Ĝ = 12.0 to 22.0; clonal fraction = 0.39 to 0.67) for regional populations were observed. Genotypic diversities (Ĝ) did not differ significantly among populations. All regional populations were in linkage equilibrium indicating the occurrence of outcrossing. Low to moderate levels of population subdivision (0.03 to 0.07), were observed among regional populations. Only one large panmictic population was inferred by Structure, indicating no significant population structure. A Mantel test showed no significant isolation by distance (r = −0.43; P = 0.18), indicating anthropogenic movement of inoculum. The results demonstrated that S. sclerotiorum populations in northern Iran, are randomly mating and have a number of shared haplotypes among regional populations; this possibly represents recent founder populations and/or a high occurrence of anthropogenic migration of infected plant material among populations.     

Grapevine crown gall caused by <Emphasis Type="Italic">Rhizobium radiobacter</Emphasis> (Ti) in Japan

In 2005, characteristic symptoms of crown gall on grapevines (Vitis vinifera L. cv. Muscat of Alexandria, and cv. Seto Giants) were observed in a commercial greenhouse-orchard in Okayama Prefecture, Japan. Isolations from diseased tissues consistently yielded bacterial colonies that were white, glistening, and produced abundant polysaccharide on potato semi-synthetic agar (PSA) medium. Ten representative isolates were chosen for further characterization. A multiplex polymerase chain reaction (PCR) assay showed these strains were not Rhizobium vitis but did possess a Ti plasmid. The bacteriological characteristics of the isolates corresponded well with R. radiobacter. The almost complete 16S ribosomal DNA sequences of isolates AT06-1 and AT06-2, selected from 10 grapevine isolates, were determined and corresponded to sequences of R. radiobacter. The pathogenicity of the isolates was tested on young grapevine and tomato (Lycopersicon esculentum Mill.) plants. Gall symptoms developed on both plant species after inoculation, and bacteria with the same colony morphology as those inoculated were reisolated. Based on these results, the isolates were identified as R. radiobacter (Ti). This report is the first of the occurrence of R. radiobacter (Ti) on grapevine in Japan. Phylogenetic analyses using the partial nucleotide sequences of virC operon located on a Ti plasmid showed that the isolate of R. radiobacter (Ti) isolated from grapevine and some strains of R. vitis (Ti) belonged to the same monophyletic group, which differed from the groups of R. radiobacter (Ti) isolated from plants other than grapevine and of the majority of R. vitis (Ti) strains isolated from grapevine. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accessions AB306890, AB306891, and AB465432–AB465459.     

Characterization of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">melongenae</Emphasis> isolates from eggplant in Turkey by pathogenicity,VCG and RAPD analysis

Fusarium wilt is an economically important fungal disease of common eggplant (Solanum melongena) cultivated in the eastern Mediterranean region of Turkey. Seventy-four isolates of Fusarium oxysporum isolated from diseased eggplant displaying typical Fusarium wilt symptoms were screened for pathogenicity on the highly susceptible cv. ‘Pala’. All the isolates tested were pathogenic to eggplant and designated as Fusarium oxysporum f. sp. melongenae (Fomg). Genetic diversity among a core set of 20 Fomg isolates that were selected based upon geographic locations, were characterized by using pathogenicity, vegetative compatibility grouping (VCG), and random amplified polymorphic DNA (RAPD) analysis. The area under the disease progress curve (AUDPC) was calculated for each Fomg isolate until 21 days after inoculation (DAI). The most virulent isolate was identified as Fomg10 based on AUDPC, disease severity and vascular discoloration measurements at 21 DAI. At this date, a good correlation was observed between disease severity and AUDPC values for all isolates (r = 0.73). UPGMA (unweighted pair group method with arithmetic average) cluster analysis of RAPD data using Dice’s coefficient of similarity differentiated all the Fomg isolates tested, and indicated considerable genetic variation among Fomg isolates, but isolates from the same geographic region were grouped together. There was no direct correlation between clustering in the RAPD dendrogram and pathogenicity testing of Fomg isolates. Twenty isolates of Fomg were assigned to VCG 0320.     

Impact of alfalfa mosaic virus subgroup I and II isolates on terpene secondary metabolism of Lavandula vera D.C., Lavandula×alardii and eight cultivars of L. hybrida Rev.

Lavandula vera D.C., Lavandula×alardii and eight different L. hybrida Rev. cultivars cultivated at the Herb Garden of Casola Valsenio (Italy), found naturally infected by Alfalfa mosaic virus (AMV), have been evaluated for the influence of their pathologic status on essential oil biosynthesis and quality. The viral disease, consisting of a “yellow mosaic” on leaves and stems, was identified by means of electron microscopy, RT-PCR-RFLP and PAS-ELISA assays. Molecular characterization of the coat protein gene of four AMV isolates suggested that three of them belonged to subgroup I (from cvs. Ordinario and Grosso, Lavandula×alardii), while the isolate from cv. Sumiens showed molecular features of subgroup II. The infection of Lavandula plants by AMV (of the two subgroups) was found to decrease essential oil secretion. Changes in the relative composition regarded the linalool-linalyl acetate ratio; the overall increase in terpenic esters and in some cases increased sesquiterpenic abundance. Infection caused by AMV subgroup II isolate influenced negatively the quality of the essential oil much more than AMV isolates belonging to subgroup I. The importance of the phytopathological status of essential oil bearing crops is outlined.     

Population structure of the rice sheath blight pathogen <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> AG-1 IA from India

The population structure of Rhizoctonia solani AG-1 IA causing rice sheath blight from India was evaluated for 96 isolates using seven RFLP loci. Nineteen of the isolates did not hybridise to R. solani AG-1 IA RFLP probes and rDNA analyses subsequently confirmed that they were either Ceratobasidium oryzae-sativae isolates or another Rhizoctonia sp. The population structure of the remaining 77 R. solani AG-1 IA Indian isolates was similar to that of a previously characterized Texas population. Clonal dispersal of R. solani AG-1 IA in India was moderate within fields and no clones were shared among field populations. Low levels of population subdivision and small genetic distances among populations were consistent with high levels of gene flow. Frequent sexual reproduction was indicated by the fact that most populations were in Hardy–Weinberg equilibrium (HWE). The two loci (R68 and R111) that deviated significantly from HWE showed an excess of heterozygosity. Although Texas and Indian populations were geographically very distant, they exhibited only moderate population subdivision, with an F_ST value of 0.193.     

Genetic control of partial resistance to ‘collar’ and ‘root’ isolates of <Emphasis Type="Italic">Phoma macdonaldii</Emphasis> in sunflower

Phoma macdonaldii is one of the most important pathogens of sunflower (Heliantus annuus) in France. In order to determine the inheritance of resistance to the disease, five sunflower genotypes with wide genetic variability for resistance to two ‘collar’ and two ‘root’ Phoma isolates were crossed in a diallel programme. Four separate experiments were undertaken under controlled conditions. In each one, the response of parental genotypes and their F1 hybrids were evaluated with one of the four Phoma isolates. Analysis of variance was performed to determine the effects of genotype on disease severity score when inoculated with ‘collar’ or ‘root’ Phoma isolates and showed significant variability among parents and F1 hybrids for disease severity score. Diallel analysis showed that general combining ability (GCA) and specific combining ability (SCA) effects for resistance to ‘collar’ and ‘root’ Phoma isolates were highly significant for each of the four isolates indicating that both kinds of gene effects were important in controlling the resistance. The GCA/SCA ratios were more than one for three out of four isolates showing that additive genetic effects were more important than non-additive effects for resistance to three of the studied Phoma isolates. Hence, conventional breeding methods could be recommended to achieve genetic improvement to such ‘collar’ and ‘root’ Phoma isolates.