Sequences of the 3′ Halves of the Genomes of Barley Yellow Dwarf Virus-PAV cpA Isolates that Vary in Symptom Severity

Barley yellow dwarf virus (BYDV)-PAV isolates from USA have been separated into two distinct clusters (Chay et al. (1996) Virology 219: 57–65; Chay et al. (1996) Phytopathology 86: 370–377). Following this finding we have shown that BYDV-PAV is divided into two groups cpA and cpB based on their coat protein gene sequence, and distinct host preferences (Mastari et al. (1998) Phytopathology 88: 818–821). We have sequenced the complete 3 half of the genomes of two lethal and two mild cpA isolates and compared them with those of several known PAV cpA isolates to assess variability and locate potential determinants of severity. Open reading frames (ORFs) 3, 4, 5, 6 and the 3 untranslated regions had different percent homologies between isolates: ORF5 (92–97%), ORF3 (88–98%) 3-translational enhancer (87–100%) ORF4 (85–99%), 3 untranslated region (72–97%) and ORF6 (61–99%). In contrast to the mild isolates, the field-lethal isolates (FHv1 and FHv2) fell into the same cluster, regardless of the genomic region analysed. The isolates FHv1 and FHv2 differed from mild isolates by eight amino acid substitutions in ORFs 3 and 4, and insertions in ORF5. Four amino acid substitutions in the 17-kDa protein encoded by ORF4 caused a change in local net charge in the field-lethal isolates. Two insertions of four amino acids were identified in the C-terminal half of ORF5 of the field-lethal isolates, but were not present systematically in all lethal isolates analysed. The potential relationships of these differences in predicted amino acid sequences to disease severity are discussed.     

Some coat protein constituents from strawberry latent ringspot virus expressed in transgenic tobacco protect plants against systematic invasion following root inoculation by nematode vectors

The coding sequences in RNA2 for the coat proteins (CP) of strawberry latent ringspot virus (SLRSV) were modified and amplified using polymerase chain amplification reactions (PCR) to facilitate their expression inAgrobacterium tumefaciens-transformedNicotiana tabacum Xanthi-nc. The coding sequences for the smaller capsid protein (S, 29kDa) and that for the theoretical precursor of L and S (P, 73kDa) had ATG initiation codon sequences added at the 5-proximal Ser/Gly (S/G) cleavage site in the unmodified sequence. The sequence coding for the larger of the two proteins of mature SLRSV capsids (L, 44kDa) had an ATG codon added at its 5 S/G site and a TAG stop codon sequence added at the 3-proximal S/G site. The P, L and S proteins were expressedin planta to a maximum concentration of 0.01 % of total extractable proteins but did not assemble into virus-like particles. When challenged by mechanical inoculation with virus particles or viral RNA, and compared with control plants, tobacco plants (primary transgenic clones or S1 and S2, kanamycin-resistant seedlings) expressing the virus capsid subunits separately, or their precursor, decreased the accumulation of SLRSV particles in inoculated leaves and fewer plants became invaded systemically. In experiments in which the roots of seedlings were exposed to SLRSV-carrying vector nematodes (Xiphinema diversicaudatum), SLRSV was detected in the roots of non-transformed control tobacco plants (6/20) and in transgenic tobacco expressing the L protein (7/40), but not in any of 25 tobacco plants expressing the S protein or in 35 expressing the P protein. This is the second example of CP-mediated resistance to virus inoculation by nematode vectors.     

Characterisation of a maize-infecting potyvirus from Spain

In order to characterise and classify an unknown maize-infecting potyvirus isolated from fields in northeast Spain, the entire coat protein gene and the C-terminal twothirds of the large nuclear inclusion protein (NIb) gene were cloned and sequenced. Protein sequencing enabled the cleavage site between the two proteins to be deduced and also revealed that on storage the viral coat protein undergoes a specific degradation in which the N-terminal 39 amino acids are removed. Comparison of the nucleotide sequence of the 3 non-coding region of the viral RNA and the predicted amino acid sequence of the coat protein with the equivalent regions of other members of the potyvirus group revealed that the Spanish virus is closely related to maize dwarf mosaic virus strain A.     

Molecular evidence that a lily-infecting strain of Tulip breaking virus from Japan is a strain of Lily mottle virus

The sequence of the 3-terminal 2074 nucleotides (nts), excluding the 3-poly (A) tail, of RNA of a potyvirus isolated from lily (Lilium Asiatic hybrid cv. Enchantment) in Japan, currently tentatively designated as Tulip breaking virus-li (TBV-li), was determined. The sequence started within a single open reading frame (ORF) that encoded the carboxyl terminus of the large nuclear inclusion protein (NIb) and the complete 275-amino-acid coat protein (CP), followed by a 3-untranslated region (3-UTR) of 204 nts. The CP of TBV-li shared 91% amino acid (aa) sequence identity with that of TBV lily strain Dutch isolate (TBV-lily). The nt sequences of their 3-UTR were 94% identical. However both viruses shared only 60–65% sequence identities with TBV tulip strain Niigata isolate in the corresponding regions. The results suggest that TBV-li is closely related to TBV-lily, and that these two TBV lily strains should be classified into a species different from TBV tulip strains. We therefore support a proposal to rename TBV-lily Lily mottle virus (LMoV), and suggest that TBV-li is another strain of LMoV (LMoV-J).     

A carlavirus-specific PCR primer and partial nucleotide sequence provides further evidence for the recognition of cowpea mild mottle virus as a whitefly-transmitted carlavirus

Cowpea mild mottle virus (CMMV) has physicochemical properties typical of carlaviruses, but has remained unclassified due to a number of unusual properties, including no serological cross-reaction with 18 carlaviruses; production of brush-like inclusion bodiesin vivo; and the ability to be transmitted by whiteflies (Bermisia tabaci). In this paper we report the use of a carlavirus specific PCR primer to identify CMMV as a member of the carlavirus group. This is confirmed by nucleotide sequence (958 nucleotides) from the 3 terminal region of CMMV RNA which contains a partial open reading frame (ORF) having high similarity with the coat proteins of other carlaviruses. The sequence also contains an 11.7K ORF at the 3 terminus, containing a zinc-finger motif which is unique to carlaviruses.     

Inoculation of cucumber roots with zoospores of mycoparasitic and plant pathogenic Pythium species: Differential zoospore accumulation, colonization ability and plant growth response

Hydroponically grown cucumber (Cucumis sativus) seedlings were inoculated with zoospores of 1 mycoparasitic (Pythium oligandrum) and 2 pathogenic (Pythium aphanidermatum and Pythium group F) Pythium spp. During the first 2 days after inoculation, all the Pythium spp. caused reduction in the root length. However, roots treated with Pythium oligandrum quickly reached the length of the control and on the 8th day, and for the rest of the experimental period, stimulation of root elongation was noted. Pythium oligandrum was not pathogenic on cucumber and no differences in the fresh weights of control and Pythium oligandrum inoculated plants were observed in the course of the experiment. Pythium group F and Pythium aphanidermatum were pathogenic on cucumber seedlings, but their pathogenicities differed. Thus, while Pythium group F had a constant, negative influence on root length and plant growth, measured as fresh weight, Pythium aphanidermatum caused generalized necroses of the root system, inhibiting consistently root elongation and plant growth and finally causing plant death. Moreover, the zoospores of 2 mycoparasitic species, Pythium oligandrum and Pythium periplocum, were not attracted to roots of cucumber and accumulated on the roots in very low numbers compared to those of the pathogenic species, Pythium aphanidermatum, which were strongly attracted and accumulated in large numbers. Finally, it was also found that Pythium oligandrum colonized the roots very poorly, while Pythium group F and Pythium aphanidermatum were significantly better root colonizers. The significance of these findings is discussed in relation to the ecology of Pythium species and biocontrol.     

Molecular characterization of potato virus YN isolates by PCR-RFLP

Based on the sequence polymorphism in the 5 terminal part of the viral genome, a range of PVY^N isolates were characterized by polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP). Three pairs of primers selected in the 5 non-translated and P1 protein region were tested. Two of them yielded PCR products of about 1Kb from all isolates tested. Restriction analysis of the PCR products gave two distinct electrophoretic patterns, whichever of the three enzymes was used. In this way, the 18 isolates were separated into two easily identifiable subgroups. All tuber necrosing isolates (PVY^NTN) were clustered in the same subgroup.     

Hydrogen peroxide localization and antioxidant status in the recovery of apricot plants from European Stone Fruit Yellows

Hydrogen peroxide (H₂O₂) localization and roles of peroxidases, malondialdehyde and reduced glutathione were compared in leaves of apricot (Prunus armeniaca) plants asymptomatic, European Stone Fruits Yellows (ESFY)-symptomatic and recovered. Nested PCR analysis revealed that Candidatus Phytoplasma prunorum, is present in asymptomatic, symptomatic and recovered apricot trees, confirming previous observations on this species, in which recovery does not seem to be related to the disappearance of phytoplasma from the plant.H₂O₂was detected cytochemically by its reaction with cerium chloride, which produces electron-dense deposits of cerium perhydroxides. H₂O₂was present in the plasmalemma of the phloem cells of recovered apricot plant leaves, but not in the asymptomatic or symptomatic material. Furthermore, by labelling apricot leaf tissues with diaminobenzidine DAB, no differences were found in the localization of peroxidases.Protein content in asymptomatic, symptomatic and recovered leaves was not significantly different from one another. In contrast, guaiacol peroxidase activity had the following trend: symptomatic > recovered > asymptomatic, whereas reduced glutathione content followed the opposite trend: asymptomatic > recovered > symptomatic. Moreover, no differences were observed in malondialdehyde concentrations between asymptomatic, symptomatic and recovered leaves. The overall results suggest that H₂O₂ and related metabolites and enzymes appear to be involved in lessening both pathogen virulence and disease symptom expression in ESFY-infected apricot plants.     

Analysis of gene sequences for the nucleocapsid protein from <Emphasis Type="Italic">Tomato spotted wilt virus</Emphasis> for promoting RNA-mediated cross-protection using the <Emphasis Type="Italic">Potato virus X</Emphasis> vector system

Virus interactions between Tomato spotted wilt virus (TSWV) and Potato virus X (PVX) containing the nucleocapsid protein (N) gene sequences were examined to evaluate the capacity of the N gene sequences from TSWV to promote RNA-mediated cross-protection. Plants simultaneously inoculated with TSWV and PVX containing the 3 96bp of the N gene were highly resistant to TSWV infection, whereas no such resistance was observed in plants inoculated with TSWV and PVX containing the 5 96bp. These results suggest that the 3 portion of the N gene has a higher capacity for promoting RNA-mediated cross-protection of TSWV.     

Molecular Identification of Oat Mosaic Virus as a Bymovirus

A partial sequence of Oat mosaic virus (OMV) has been obtained for four isolates of the virus from four European countries. This represents the first available sequence data for this important disease of winter-sown oats. The longest clone of 1699 nucleotides was obtained from infected English oats using a degenerate primer, designed to members of the Potyviridae family. Alignment of the predicted amino acid sequence with members of the Potyviridae showed closest identity with viruses of the Bymovirus genus. The predicted amino acid sequence has one open reading frame corresponding to part of the NIb and capsid protein, with a 3 untranslated region of 351 nucleotides, followed by a poly(A) tail. PCR primers were designed to the coat protein and NIb gene of members of the Bymovirus genus and used to obtain partial sequences of 1441 nucleotides at the 3 end of infected oats from both Wales and France. A specific primer set designed to the English isolate was used to generate a product of 701 nucleotides from OMV-infected oat leaves from Ireland. All four isolates are highly conserved at the amino acid level.The first two authors contributed equally to the work     

The 3' terminal sequence of RNA1 of wheat spindle streak mosaic virus canadian isolate (WSSMV-C)

The sequence of the 3 terminal 1722 nucleotides (nts) of RNA1 of the type (Canadian) isolate of wheat spindle streak mosaic bymovirus (WSSMV-C) was determined. The sequence started within a single open reading frame (ORF), which was expected to encode the carboxyl terminus of the nuclear inclusion b protein (NIb) and the capsid protein (CP) of 294 amino acids, followed by a 3 untranslated region (UTR) of 237 nucleotides. The NIb and CP of WSSMV-C share 99 and 100% amino acid sequence identity with the corresponding proteins of WSSMV-French isolate (WSSMV-F), but only 89 and 77% with wheat yellow mosaic virus (WYMV-J), respectively. The 3UTR of RNA1 of WSSMV-C shares 94% nucleotide sequence identity with that of WSSMV-F but only 73% with WYMV-J and WYMV-Chinese isolate (WYMV-Chi). The results support the classification of WSSMV-C and WSSMV-F as strains of the same virus species which is distinct from WYMV.     

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.     

Viruses of stone fruits in Albania

Surveys were carried out in the main stone-fruit growing areas of Albania to assess the phytosanitary status of Prunus in conimercial orchards and varietal collections. The presence of virus and virus-like diseases and their identification was ascertained through field observations, sap transmission to herbaceous hosts, graft transmission to woody indicators, ELISA and IEM tests. The mean infection level was 42%. In particular, infections in apricot and almond were 12 and 16%, respectively, i.e. lower than in plum and cherry (47 and 56%, respectively). The following viruses were identified: plum pox potyvirus (PPV). apple chlorotic leaf spot trichovirus (ACLSV). prunus necrotic ringspot (PNRSV) and prune dwarf (PDV) ilarviruses. PPV infection was very severe in plum, and limited in apricot and peach. Apple mosaic ilarvirus (ApMV), and six nepoviruses tested for (SLRV, TBRV, RRV, CLRV, ArMV and ToRSV) were not encountered in Primus.     

‘Plantibodies’: a flexible approach to design resistance against pathogens

Engineering resistance against various diseases and pests is hampered by the lack of suitable genes. To overcome this problem we started a research program aimed at obtaining resistance by transfecting plants with genes encoding monoclonal antibodies against pathogen specific proteins. The idea is that monoclonal antibodies will inhibit the biological activity of molecules that are essential for the pathogenesis. Potato cyst nematodes are chosen as a model and it is thought that monoclonal antibodies are able to block the function of the saliva proteins of this parasite. These proteins are, among others, responsible for the induction of multinucleate transfer cells upon which the nematode feeds. It is well documented that the ability of antibodies to bind molecules is sufficient to inactivate the function of an antigen and in view of the potential of animals to synthesize antibodies to almost any molecular structure, this strategy should be feasible for a wide range of diseases and pests.Antibodies have several desirable features with regard to protein engineering. The antibody (IgG) is a Y-shaped molecule, in which the domains forming the tips of the arms bind to antigen and those forming the stem are responsible for triggering effector functions (Fc fragments) that eliminate the antigen from the animal. Domains carrying the antigen-binding loops (Fv and Fab fragments) can be used separately from the Fc fragments without loss of affinity. The antigen-binding domains can also be endowed with new properties by fusing them to toxins or enzymes. Antibody engineering is also facilitated by the Polymerase Chain Reaction (PCR). A systematic comparison of the nucleotide sequence of more than 100 antibodies revealed that not only the 3-ends, but also the 5-ends of the antibody genes are relatively conserved. We were able to design a small set of primers with restriction sites for forced cloning, which allowed the amplification of genes encoding antibodies specific for the saliva proteins ofGlobodera rostochiensis. Complete heavy and light chain genes as well as single chain Fv fragments (scFv), in which the variable parts of the light (V_L) and heavy chain (V_H) are linked by a peptide, will be transferred to potato plants. A major challenge will be to establish a correct expression of the antibody genes with regard to three dimensional folding, assembly and intracellular location.     

Sanitary status of stone-fruit trees in East Anatolia (Turkey) with particular reference to apricot

Field surveys were carried out in the main stone-fruit-growing areas of East Anatolia (Turkey) to assess the sanitary status of varietal collections, mother blocks and commercial orchards. The presence of virus and virus-like diseases was ascertained by enzyme-linked immunosorbent assay (ELISA), sap transmission to herbaceous hosts, graft transmission to peach cv. GF305 and molecular hybridization tests. A total of 1019 samples was tested by ELISA (859 apricot, 120 cherry, 21 almond and 19 peach). The sanitary status of apricot was extremely satisfactory, as the infection level was less than 0.3%. Cherry and almond, however, showed 21% and 33% infection respectively. The viruses identified were apple chlorotic leaf spot trichovirus (ACLSV), prune dwarf ilarvirus (PDV) and prunus necrotic ringspot ilarvirus (PNRSV). The commonest virus was PDV. Plum pox potyvirus (PPV), apple mosaic ilarvirus (ApMV) and the nepoviruses tomato black ring (TBRV), raspberry ringspot (RpRSV), strawberry latent ringspot (SLRV), cherry leaf roll (CLRV), arabis mosaic (ArMV) and tomato ringspot (ToRSV) were not encountered. Peach latent mosaic viroid (PLMVd) and hop stunt viroid (HSVd) were not detected either.     

Interactions between Plasmopara helianthi, Glomus mosseae and Two Plant Activators in Sunflower Plants

Interactions between Plasmopara helianthi, Glomus mosseae and two plant activators DL--amino-n-butyric acid (BABA) and CGA 245704 (acibenzolar-S-methyl (BTH)) in sunflower plants susceptible to downy mildew were studied in four experiments using different methods of treatment and pathogen inoculation. Both chemicals were applied as soil drenches and foliar sprays, whereas P. helianthi infection was obtained by root and cotyledon inoculations of the seedlings. Soil drenches at the rates of 50 and 100mgkg^–1 soil of BABA and BTH given 1 and 3 days before P. helianthi inoculation, respectively to mycorrhizal plants, provided moderate protection against the pathogen (about 50–55%). Morphological changes and decrease in mycorrhizal colonization in roots of BTH-treated plants and in BTH-treated mycorrhizal plants were also observed. Delay in the emergence and reduction of the root systems were more evident at the highest concentration but decreased with time. These effects were absent with the BABA treatment.Foliar spray treatment of BABA and BTH, applied at 4000 and 200µgml^–1, respectively (1 day post-inoculation) to mycorrhizal plants provided good protection (about 80%) against P. helianthi foliar infections. No effects on mycorrhizal colonization or on root systems were observed. In vitro tests on the effect of the compounds on the mycorrhizal fungus showed that the germination of G. mosseae sporocarps increased with BABA treatment whereas it was greatly inhibited by BTH treatment.     

Relative susceptibilities of eleven potato cultivars and breeders' clones toGlobodera pallida pathotype Pa 3, with a discussion of the interpretation of data from pot experiments

Curves according to the equationPf=M (1-e ^–aPi/M ) fitted well to the relations according to Seinhorst's (1993) modelPf=y _e y _h M(1-e ^–aPi/M ) between initial egg densities of potato cyst nematodes (Globodera rostochiensis, G. pallida)Pi up to 5T _h (T _h =the tolerance limit of haulm weight) andPf at the end of the growing season (a=maximum rate of reproduction,M, M=different theoretical maximum egg densities). Variation of estimates ofM, due to variation of the parameters of the submodelsy _e andy _h for the effect of weight reduction of haulms (and, therefore, of roots) on cyst production and damage to root tissue on egg production, respectively, was small enough to be ignored relative to experimental error. Therefore, ratios of values ofM, determined in simultaneous pot experiments with different potato cultivars, are reliable measures of the relative host status of these cultivars at initial egg densitiesPi of these nematodes up to about 5T _h . Variation between potato cultivars of growth reduction and damage to root tissue by the nematodes reduces the reliability of ratios of rates of relative susceptibility of these cultivars.The ratios between the maximum rates of reproduction ofG. pallida, pathotype Pa 3, on 8 out of 9 cultivars and one breeder's clone of potatoes and this rate on the susceptible cvs Bintje and Irene (relative susceptibilitiesrs _a ) could be considered to be equal to the ratios of maximum population densitiesM on these cultivars (relative susceptibilitiesrs _M ) (relative susceptibilities independent of initial egg density). The latter ratios were 0.59 times the first (relative susceptibilities negatively correlated with initial egg density) in one cultivar and one breeder's clone. Relative susceptibilitiesrs _a andrs _M of the tested cultivars and breeders clones suggest the existence of continuous ranges of both relative susceptibilities between 0.50 and 0.15 with, in a great majority of cases,rs _a =rs _M .     

Development and use of detection methods specific for <Emphasis Type="Italic">Cucumber vein yellowing virus</Emphasis> (CVYV)

Two methods for the detection of Cucumber vein yellowing virus (CVYV) on infected plants were developed, based on the information provided by cDNA clones covering the 3-end of the genome of a Spanish isolate (CVYV-AILM). The sequenced portion of the CVYV-AILM genome showed a 96.6% aminoacid identity with that of a reported sequence of another CVYV isolate from Israel (Lecoq et al., 2000). The first detection method used a RNA specific probe for hybridization with nucleic acids extracted from infected plants. The probe was complementary to a portion of the CVYV genome including the C-terminal part of the NIb and most of the coat protein (CP) coding regions. The second detection method employed polyclonal antisera raised against recombinant viral CP expressed in bacteria. The specific antibodies were used to detect the presence of virus particles in plant extracts. Both procedures resulted in a highly specific detection of CVYV in plants infected with different isolates of the virus. No interference was observed with other cucurbit-infecting viruses. Sensitivities achieved were sufficient for routine diagnosis of the presence of the virus in plants.     

A streak disease of pearl millet caused by a leafhopper-transmitted geminivirus

The cause of a streak disease of pearl millet (Pennisetum glaucum), originating from Nigeria, has been attributed to a geminivirus belonging to the African streak virus cluster. A full-length, infectious clone of the virus was obtained which was transmissible by the vectorCicadulina mbila (Naudé). Analysis of the complete nucleotide sequence of the coat protein gene of this virus shows it to be most closely related to sugarcane streak virus. The possible evolutionary implications of this finding are discussed.     

Morphology and intracellular localization of bacilliform virus particles associated with the clover enation disease

In ultrathin sections of tumorous tissues of white clover plants artificially infected by grafting with the clover enation virus nuclei and nucleoli were very much enlarged. Such nuclei contained massive amounts of tubular particles in crystalline array. In spaces between the nuclear membrane lamellae such particles had an additional coat and usually occurred in irregular accumulations. These bacilliform particles measured about 200 × 80 m. This virus was not found in normal tissues.Two other viruses with elongated particles of 475 and 665 m were also observed in non-neoplastic tissues. They were easily detected in negatively stained chop preparations, rapidly transmitted by sap, and considered to be mere contaminants.Enation virus diseases are heterogeneous in epidemiology and etiology. Several have recently been found associated with dense accumulations of big spherical or polyhedral virus particles. The virus detected here closely resembles several bacilliform viruses described in recent years. These viruses are quite different in symptomatology and epidemiology.