Quantitative trait loci from the host genetic background modulate the durability of a resistance gene: a rational basis for sustainable resistance breeding in plants.

The combination of major resistance genes with quantitative resistance factors is hypothesized as a promising breeding strategy to preserve the durability of resistant cultivar, as recently observed in different pathosystems. Using the pepper (Capsicum annuum)/Potato virus Y (PVY, genus Potyvirus) pathosystem, we aimed at identifying plant genetic factors directly affecting the frequency of virus adaptation to the major resistance gene pvr2(3) and at comparing them with genetic factors affecting quantitative resistance. The resistance breakdown frequency was a highly heritable trait (h(2)=0.87). Four loci including additive quantitative trait loci (QTLs) and epistatic interactions explained together 70% of the variance of pvr2(3) breakdown frequency. Three of the four QTLs controlling pvr2(3) breakdown frequency were also involved in quantitative resistance, strongly suggesting that QTLs controlling quantitative resistance have a pleiotropic effect on the durability of the major resistance gene. With the first mapping of QTLs directly affecting resistance durability, this study provides a rationale for sustainable resistance breeding. Surprisingly, a genetic trade-off was observed between the durability of PVY resistance controlled by pvr2(3) and the spectrum of the resistance against different potyviruses. This trade-off seemed to have been resolved by the combination of minor-effect durability QTLs under long-term farmer selection.

Evolutionary Pathways to Break Down the Resistance of Allelic Versions of the PVY Resistance Gene va.

Emergence of viral genotypes can make control strategies based on resistance genes ineffective. A few years after the deployment of tobacco genotypes carrying alleles of the Potato virus Y (PVY) recessive resistance gene va, virulent PVY isolates have been reported, suggesting the low durability of va. To have a broader view of the evolutionary processes involved in PVY adaptation to va, we studied mutational pathways leading to the emergence of PVY resistance-breaking populations. The viral genome-linked protein (VPg) has been described to be potentially involved in va adaptation. Analyses of the VPg sequence of PVY isolates sampled from susceptible and resistant tobacco allowed us to identify mutations in the central part of the VPg. Analysis of the virulence of wild-type isolates with known VPg sequences and of mutated versions of PVY infectious clones allowed us to (i) validate VPg as the PVY virulence factor corresponding to va, (ii) highlight the fact that virulence gain in PVY occurs rapidly and preferentially by substitution at position AA105 in the VPg, and (iii) show that the 101G substitution in the VPg of a PVYC isolate is responsible for cross-virulence toward two resistance sources. Moreover, it appears that the evolutionary pathway of PVY adaptation to va depends on both virus and host genetic backgrounds.

Molecular evolution of the genomic RNA of Apple stem grooving capillovirus.

The complete genome of the German isolate AC of Apple stem grooving virus (ASGV) was sequenced. It encodes two overlapping open reading frames (ORFs), similarly to previously described ASGV isolates. Two regions of high variability were detected between the ASGV isolates, variable region 1 (V1, from amino acids (aa) 532 to 570), and variable region 2 (V2, from aa 1,583 to 1,868). The phylogenetic analysis of the V1 and V2 regions suggested that the ASGV diversity was structured by host plant species rather than geographical origin. The dN/dS ratio between nonsynonymous and synonymous nucleotide substitution rates varied greatly along the ASGV genome. Most of ORF1 showed predominant negative selection except for the two regions V1 and V2. V1 showed an elevated dN and an average dS when compared to the ORF1 background but no significant positive selection was detected. The V2 region of ORF1 showed an elevated dN and a low dS when compared to the ORF1 background with an average dN/dS ≈ 3.0 indicative of positive selection. However, the V2 area includes overlapping ORFs, making the dN/dS estimate biased. Joint estimates of the selection intensity in the different ORFs by a recent method indicated that this region of ORF1 was in fact evolving close to neutrality. This was convergent with previous results showing that introduction of stop codons in this region of ORF1 did not impair plant infection. These data suggest that the elimination of a stop codon caused the overprinting of a novel coding region over the ancestral ORF.

Virus adaptation to quantitative plant resistance: erosion or breakdown?

Adaptation of populations to new environments is frequently costly due to trade-offs between life history traits, and consequently, parasites are expected to be locally adapted to sympatric hosts. Also, during adaptation to the host, an increase in parasite fitness could have direct consequences on its aggressiveness (i.e. the quantity of damages caused to the host by the virus). These two phenomena have been observed in the context of pathogen adaptation to host's qualitative and monogenic resistances. However, the ability of pathogens to adapt to quantitative polygenic plant resistances and the consequences of these potential adaptations on other pathogen life history traits remain to be evaluated. Potato virus Y and two pepper genotypes (one susceptible and one with quantitative resistance) were used, and experimental evolutions showed that adaptation to a quantitative resistance was possible and resulted in resistance breakdown. This adaptation was associated to a fitness cost on the susceptible cultivar, but had no consequence either in terms of aggressiveness, which could be explained by a high tolerance level, or in terms of aphid transmission efficiency. We concluded that quantitative resistances are not necessarily durable but management strategies mixing susceptible and resistant cultivars in space and/or in time should be useful to preserve their efficiency.

Emerging strains of watermelon mosaic virus in Southeastern France: model-based estimation of the dates and places of introduction.

Where and when alien organisms are successfully introduced are central questions to elucidate biotic and abiotic conditions favorable to the introduction, establishment and spread of invasive species. We propose a modelling framework to analyze multiple introductions by several invasive genotypes or genetic variants, in competition with a resident population, when observations provide knowledge on the relative proportions of each variant at some dates and places. This framework is based on a mechanistic-statistical model coupling a reaction-diffusion model with a probabilistic observation model. We apply it to a spatio-temporal dataset reporting the relative proportions of five genetic variants of watermelon mosaic virus (WMV, genus Potyvirus, family Potyviridae) in infections of commercial cucurbit fields. Despite the parsimonious nature of the model, it succeeds in fitting the data well and provides an estimation of the dates and places of successful introduction of each emerging variant as well as a reconstruction of the dynamics of each variant since its introduction.

First Report of Cucumber mosaic virus in Paeonia lactifera in France.

Chinese peony (Paeonia lactiflora Pall.), a hardy ornamental plant of the family Paeoniaceae cultivated in gardens and for cut flower production, is frequently infected by Tobacco rattle virus (TRV) in the field. The virus usually induces severe mosaic and chlorotic ringspot symptoms in the leaves, decreasing the commercial value of cut flowers. TRV is routinely detected by mechanical inoculation onto Nicotiana tabacum cv Xanthi, where it induces typical necrotic local ringspots in 3 to 7 days, followed by a reverse transcription (RT)-PCR test (2). In 2004, Xanthi test plants inoculated with sap extracts from 4 of 36 P. lactiflora cv. Odile plants grown in a field plot in the region of Hyères (southeast France) showed systemic mosaic symptoms in addition to the TRV-typical response. In each case, Cucumber mosaic virus (CMV) was detected by the reactions of a range of inoculated plants (1), the observation of 30 nm isometric particles in crude leaf extracts with the electron microscope, and by positive reactions in double antibody sandwich (DAS)-ELISAs with specific polyclonal antibodies. In double-immunodiffusion analysis, these isolates were shown to belong to the group II of CMV isolates (3). ELISA of the peony plants confirmed the presence of CMV and revealed two additional infected plants in the spring of 2006. Following isolation from local lesions on Vigna unguiculata and multiplication in Xanthi tobacco plants, one of the isolates was used to inoculate manually or with Myzus persicae aphids 10 CMV-free plants of P. lactiflora cv. Odile obtained from meristem culture. Three months postinoculation, only three of the aphid-inoculated plants were CMV positive by DAS-ELISA. No change was observed at 1 year postinoculation and no symptoms have been observed, even in CMV-infected plants. CMV appears to be latent in P. lactiflora, therefore detection of CMV before vegetative propagation of the plants is advised because of the risks of synergism for symptoms with other viruses such as TRV. To our knowledge this is the first report of CMV in peony. References: (1) L. Cardin et al. Plant Dis. 87:1263, 2003. (2) D. J. Robinson J. Virol. Methods 40:55, 1992. (3) M. J. Roossinck. J. Virol. 76:3382, 2002.

Durability of plant major resistance genes to pathogens depends on the genetic background, experimental evidence and consequences for breeding strategies.

* The breakdown of plant resistance by pathogen populations is a limit to the genetic control of crop disease. Polygenic resistance is postulated as a durable alternative to defeated major resistance genes. Here, we tested this postulate in the pepper-Potato virus Y interaction. * The virus was selected for virulence towards monogenic and polygenic host resistance, using serial inoculations in laboratory and in natural epidemic conditions. The frequency of resistance breakdown and the genetic changes in the virus avirulence gene were analysed. * The monogenic resistance provided by the pvr2(3) gene was defeated at high frequency when introgressed in a susceptible genetic background whereas it was not when combined to partial resistance quantitative trait loci. The suppression of emergence of virulent mutants because of the genetic background resulted both from a differential selection effect and the necessity for the virus to generate multiple mutations. The virus adaptation to the polygenic resistance required a step-by-step selection with a primary selection for virulence towards the major gene, followed by selection for adaptation to the genetic background. * Polygenic resistance proved more durable than monogenic resistance, but breeding strategies giving priority to major resistance factors may jeopardize the progress in durability expected from polygenic resistance.

First Report of Passiflora chlorosis virus in Bituminaria bituminosa in Europe.

Bituminaria bituminosa (L.) Stirton (pitch trefoil) is a perennial legume endemic to the Mediterranean Basin used as forage in arid areas and for stabilization of degraded soils. Mosaic and chlorotic ringspot symptoms have been observed in leaves of B. bituminosa in the Provence-Alpes-Côte d'Azur and Rhône-Alpes regions (France), Liguria (Italy), and Spain since 1975. In crude leaf extracts from more than 50 samples of diverse geographical origins, flexuous particles 680 to 720 nm long and 12 nm wide and pinwheel-like inclusions have been observed with the electron microscope, suggesting infection with a member of the family Potyviridae. The presence of a virus was confirmed by the use of potyvirus-polyvalent ELISA reagents (Potyvirus group test; Agdia, Elkhart, IN) and by the amplification of a DNA fragment of the expected size (≈1,650 bp) with extracts of isolates from different locations using reverse transcription (RT)-PCR with primers specific to members of the Potyviridae (3) corresponding to the 3' end of the virus genome. The amplified fragment of an isolate from Coaraze (Alpes Maritimes Department, France) was cloned and two cDNA clones corresponding to this amplicon were sequenced (GenBank Accession Nos. EU334546 and EU334547). These two sequences facilitated development of new primers (5'-AAARGCRCCCTATATAGCAG-3' and 5'-TATAAAGGTAACGCTAGGTGG-3') to specifically amplify and sequence the coat protein (CP)-coding region of isolates of the virus from five additional French locations. The amino acid sequences of the CP amplicon were more than 96% identical among the French isolates. Comparison with other virus sequences with the BLASTn program revealed that these isolates belonged to the same species as the potyvirus Passiflora chlorosis virus (2), with 89 to 90% and 95 to 97% identity at the nucleotide and amino acid levels, respectively, for the CP-coding region (1). The host range of the virus was evaluated by manual inoculation with the Coaraze isolate and was found to be very narrow. No symptoms and no infections were obtained in Capsella bursa-pastoris, Capsicum annuum, Claytonia perfoliata, Cucumis melo, Cucumis sativus, Cucurbita pepo, Datura stramonium, Gomphrena globosa, Medicago sativa, Nicotiana benthamiana, N. glutinosa, N. tabacum, Ocimum basilicum, Petunia hybrida, Phaseolus mungo, Physalis peruviana, Pisum sativum, Psoralea glandulosa, Ranunculus sardous, Salvia splendens, Solanum lycopersicum, Trifolium repens, Vicia faba, Vigna unguiculata, or Zinnia elegans. Necrotic local lesions were observed in Chenopodium amaranticolor, C. quinoa, and in all eight cultivars of Phaseolus vulgaris tested. The virus was transmitted either manually or by the green peach aphid (Myzus persicae) to healthy B. bituminosa seedlings. Symptoms appeared in 10 to 15 weeks, and the virus was detected in the symptomatic plants by RT-PCR. To our knowledge, this is the first report of a virus infecting B. bituminosa. References: (1) M. J. Adams et al. Arch. Virol. 150:459, 2005. (2) C. A. Baker and L. Jones. Plant Dis. 91:227, 2007. (3) A. Gibbs and A. M. Mackenzie. J. Virol. Methods 63:9, 1997.

First Report of Cucumber mosaic virus and Turnip vein-clearing virus in Dichondra repens in France, Italy, and China.

During surveys of Dichondra repens (kidneyweed, family Convolvulaceae) turfs in public gardens of the Franco-Italian Riviera from 1993 to 2003, leaf mosaic and yellow ringspot symptoms have been observed in Antibes, Menton, Nice, and Vallauris (France) and San Remo and La Mortola (Italy). Isolates from these six locations and from two locations in China (Shanghai and Kunming) have revealed the presence of Cucumber mosaic virus (CMV) based on the behavior of a range of manually inoculated plants (1), the observation of 30 nm isometric particles in semipurified extracts of inoculated Nicotiana tabacum 'Xanthi' plants with the electron microscope, and positive reactions in double antibody sandwich (DAS)-ELISAs with specific polyclonal antibodies. All isolates were shown to belong to group II of CMV isolates (3) by double-immunodiffusion analysis. CMV was previously identified in D. repens in California in 1972 (4). Following isolation from local lesions on Vigna unguiculata and multiplication in 'Xanthi' tobacco plants, two of the isolates were used to inoculate seedlings of D. repens manually or by Aphis gossypii aphids. Two months later, all inoculated plants showed symptoms similar to those previously observed and were positive in DAS-ELISA. In 2000, a D. repens sample collected in Antibes showing similar symptoms as above, induced necrotic local lesions in inoculated 'Xanthi' plants in 48 h, followed by systemic mosaic symptoms typical of CMV, therefore revealing the presence of a second virus. That virus was separated from CMV in apical, noninoculated leaves of Chenopodium quinoa and then used to inoculate a range of test plants. It was infectious in most plants of the families Solanaceae (including Cyphomandra betacea) and Brassicaceae, together with in Chenopodium amaranticolor, C. quinoa, Claytonia perfoliata, Convolvulus spp. 'Belle de jour', Digitalis purpurea, Gomphrena globosa, Ocimum basilicum, Plantago lanceolata, and Valerianella olitoria. It induced asymptomatic systemic infections in D. repens. Numerous, rod-shaped, 300 nm long particles were observed in sap extracts of infected plants with the electron microscope, suggesting the presence of a tobamovirus. A set of primers polyvalent for tobamoviruses (2) allowed the amplification of a DNA product of approximately 800 bp through reverse transcription-PCR performed with total RNA extracts from inoculated 'Xanthi' plants. The DNA product was cloned and sequenced (GenBank Accession No. EU927306) revealing that the virus belonged to a tobamovirus lineage including Ribgrass mosaic virus and viruses infecting cruciferous plants (Turnip vein-clearing virus [TVCV] and Youcai mosaic virus) and was closest to TVCV (95% amino acid identity; GenBank Accession No. NC_001873). To our knowledge, this is the first report of TVCV in D. repens. References: (1) L. Cardin et al. Plant Dis. 87:200, 2003. (2) A. Gibbs et al. J. Virol. Methods 74:67, 1998. (3) M. J. Roossinck. J. Virol. 76:3382, 2002. (4) L. G. Weathers and D. J. Gumpf. Plant Dis. Rep. 56:27, 1972.

First Report of Potato virus Y in Nicotiana mutabilis in France.

Nicotiana mutabilis Stehmann & Semir is a recently described perennial plant species from southern Brazil that produces long floral stems with white to deep pink flowers and is used for its ornamental quality. In 2003, leaf mosaic symptoms were observed in all 30 N. mutabilis plants in a nursery in the south of France. Observation of crude sap preparations with the electron microscope revealed numerous flexuous particles, 700 to 730 nm long and approximately 11 nm wide, associated with "pinwheel"-like cytoplasmic inclusions, typical of the family Potyviridae. A range of plant species inoculated with extracts from five of the symptomatic plants showed reactions typical of Potato virus Y (PVY) (2), and the presence of the virus was confirmed by positive reactions in double-antibody sandwich (DAS)-ELISA with polyclonal antibodies raised against PVY. To test if PVY was responsible for the symptoms observed in N. mutabilis, an isolate was multiplied in N. tabacum cv. Xanthi plants after isolation from local lesions on Chenopodium amaranticolor and was then mechanically inoculated to 12 seedlings of N. mutabilis cv. Marshmallow. After 3 weeks, the 12 inoculated plants showed systemic vein clearing symptoms and PVY was detected by DAS-ELISA. Reverse transcription (RT)-PCR tests using PVY-polyvalent primers (5'-GATGGTTGCCTTGGATGATG and 5'-TAAAAGTAGTACAGGAAAAGCCA) covering the coat protein (CP) coding region amplified a single DNA fragment of the expected 900 bp from total RNA extracts from Xanthi plants inoculated with the five isolates. One of these DNA products was directly sequenced (GenBank Accession No. EU252529) and several accepted methods of phylogenetic analysis compared this sequence to 80 available PVY CP coding sequences and showed that the N. mutabilis PVY belonged to the C1 group (1). Similar to the other PVY strains in the C group, the N. mutabilis isolate was able to induce hypersensitive local lesions in leaves of potato genotypes carrying the Nc gene. However, contrary to the other characterized C1 isolates (1), it was unable to infect systemically cv. Yolo Wonder pepper plants. That peculiar behavior makes the N. mutabilis isolate a tool to identify the viral determinants controlling the host range of PVY. References: (1) B. Blanco-Urgoiti et al. J. Gen. Virol. 79:2037, 1998. (2) C. Kerlan. No. 414 in: Descriptions of Plant Viruses. CMI/AAB, Kew, Surrey, UK, 2006.

First Report of Cucumber mosaic virus in Viola hederacea in France and Italy.

Viola hederacea Labill. (Australian violet or trailing violet), family Violaceae, is native to eastern Australia and used for its ornamental quality in humid conditions, especially in terrariums. Mosaic and chlorotic ringspots associated with a mild crinkling on leaves of V. hederacea were observed in gardens in southeast France and La Mortola, Italy in 2001 and 2003, respectively. These symptoms were different from the small chlorotic spots reported in the same species in Queensland, Australia, from which a rhabdovirus was isolated (1). In samples collected from both locations, presence of Cucumber mosaic virus (CMV) was suspected on the basis of the symptoms exhibited by a range of inoculated plants (2) and the observation of isometric particles of approximately 30 nm with the electron microscope in sap preparations. This was confirmed by positive reactions with CMV polyclonal antiserum (3) in double-antibody sandwich-ELISA. Because of the viscosity of the V. hederacea extracts, 0.01% (w/v) of pectolyase in phosphate buffer was used to grind the samples and the homogenate was kept at room temperature for 2 h. In double-immunodiffusion analysis, each isolate was shown to belong to the group II of CMV. Tomato strains D and To were used as positive controls for CMV groups I and II, respectively. The virus was transmitted by aphids (Myzus persicae) in a nonpersistent manner from infected Nicotiana tabacum cv. Xanthi-nc plants to healthy Xanthi-nc plants. To determine if CMV was responsible for the symptoms observed, the French and Italian isolates were multiplied in Xanthi-nc tobacco plants after isolation from local lesions on Vigna unguiculata and mechanically inoculated to 10 plants of V. hederacea. A systemic vein clearing developed on leaves 10 days after inoculation, followed by mosaic and crinkling. CMV was detected in each plant 3 weeks after inoculation. To our knowledge, this is the first report of CMV in V. hederacea in France and Italy. References: (1) D. H. Gowanlock and R. G. Dietzgen. Australas. Plant Pathol. 24:215, 1995. (2) L. Cardin et al. Plant Dis. 87:200, 2003. (3) J.-C. Devergne et al. Ann. Phytopathol. 10:233, 1978.

First Report of Cucumber mosaic virus in Echium candicans in France.

Echium candicans (Linn.) Herb. Banks (Pride of Madeira or Viper's Bugloss), family Boraginaceae, is a perennial shrub used in gardens for the ornamental quality of its deep blue inflorescences, especially in coastal areas near the Mediterranean Sea. Mosaic symptoms were observed in leaves of E. candicans in the Alpes Maritimes Department of southeastern France, St Jean Cap Ferrat in 1994, Menton in 2002, and Antibes in 2005. Symptoms exhibited in a range of inoculated plants including Nicotiana tabacum cvs. Xanthi and Samsun, Chenopodium quinoa, C. amaranticolor, Vigna unguiculata cv. Black, and Cucumis sativus cv. Poinsett were typical of Cucumber mosaic virus (CMV). Occurrence of CMV in one sample from each of the three localities was confirmed by the observation of isometric particles (approximately 30 nm) with the electron microscope in crude sap preparations from the infected plants, positive reactions in double-antibody sandwich (DAS)-ELISA to polyclonal antibodies raised against CMV (1), and the nonpersistent transmission of the virus from infected Xanthi to virus-free Xanthi plants by Myzus persicae. In double-immunodiffusion analysis, the three isolates were shown to belong to the CMV subgroup II (1,2). To determine if CMV was responsible for the symptoms observed, the isolate from Antibes was multiplied in Xanthi plants after isolation from local lesions on V. unguiculata and mechanically inoculated to 3-year-old plants of E. candicans tested to be free from CMV before the mechanical inoculation. One month after inoculation, mild mosaic symptoms were observed in young leaves and CMV was detected by DAS-ELISA in 10 of 10 inoculated plants. To our knowledge, this is the first report of CMV in E. candicans. References: (1) J.-C. Devergne and L. Cardin. Ann. Phytopathol. 7:225, 1975. (2) M. J. Roossinck. J. Virol. 76:3382, 2002.

First Report of Tobacco rattle virus and Cucumber mosaic virus in Phlox paniculata in France.

Phlox paniculata L., a perennial plant from the family Polemoniaceae, is cultivated as an ornamental in gardens and for cut-flower production. In spring 2003, two types of symptoms were observed in P. paniculata plants grown for cut flowers on a farm in the Var department, France. Some plants showed a mild leaf mosaic while others showed leaf browning and delayed growth. In plants showing mild mosaic, Cucumber mosaic virus (CMV) was detected on the basis of the symptoms exhibited by a range of inoculated plants, the observation of isometric particles (approximately 30 nm) with the electron microscope in crude sap preparations from the infected plants, and the positive reaction in double-antibody sandwich (DAS)-ELISA to polyclonal antibodies raised against CMV (1). In double-immunodiffusion analysis, the five tested isolates were shown to belong to group II of CMV strains. To determine if CMV was responsible for the symptoms observed, one isolate was multiplied in Nicotiana tabacum cv. Xanthi-nc plants after isolation from local lesions on Vigna unguiculata and mechanically inoculated to 12 1-year-old P. paniculata plants. At 3 months post inoculation (mpi), all plants showed mild mosaic and CMV was detected by DAS-ELISA. In sap preparations from P. paniculata plants showing leaf browning symptoms, rod-shaped particles with two distinct sizes of 190 to 210 and 70 to 90 nm long, typical of those associated with tobraviruses, were revealed using electron microscopy. Local lesions typical of Tobacco rattle virus (TRV) were observed after inoculation of N. tabacum cv. Xanthi-nc, Chenopodium amaranticolor, and C. quinoa. Total nucleic acid preparations were prepared from symptomatic plants, and amplicons of the expected size (463 bp) were generated by reverse-transcription (RT)-PCR using primers specific to TRV RNA 1 (4). The nucleotide sequence of one amplicon was 93.6% identical to the sequence of a reference TRV isolate (GenBank Accession No. AJ586803). Twelve 1-year-old P. paniculata plants were mechanically inoculated with an extract of infected tissues from one symptomatic P. paniculata plant. TRV was detected 2 to 6 mpi in apical leaves of all inoculated plants by RT-PCR, although the plants did not express symptoms. Since no other pathogens were detected in the source plants, it is plausible that the lack of symptoms in back-inoculated plants is either due to a long incubation period or an interaction with particular environmental factors such as cold conditions. The survey of approximately 200 plants revealed that approximately 7, 10, and 1% were infected by TRV, CMV, or by both viruses, respectively. CMV and TRV were previously detected in P. paniculata in Latvian SSR and in Lithuania (2,3). These results show that sanitary selection of P. paniculata prior to vegetative propagation should include a screening for TRV and CMV infections. References: (1) J.-C. Devergne et al. Ann. Phytopathol. 10:233, 1978. (2) Y. Ignab and A. Putnaergle. Tr. Latv. S.-Kh. Akad. 118:27, 1977. (3) M. Navalinskiene and M. Samuitiene. Biologija 1:52, 1996. (4) D. J. Robinson. J. Virol. Methods 40:57, 1992.

Evidence for positive selection and recombination hotspots in Deformed wing virus (DWV).

Deformed wing virus (DWV) is considered one of the most damaging pests in honey bees since the spread of its vector, Varroa destructor. In this study, we sequenced the whole genomes of two virus isolates and studied the evolutionary forces that act on DWV genomes. The isolate from a Varroa-tolerant bee colony was characterized by three recombination breakpoints between DWV and the closely related Varroa destructor virus-1 (VDV-1), whereas the variant from the colony using conventional Varroa management was similar to the originally described DWV. From the complete sequence dataset, nine independent DWV-VDV-1 recombination breakpoints were detected, and recombination hotspots were found in the 5' untranslated region (5' UTR) and the conserved region encoding the helicase. Partial sequencing of the 5' UTR and helicase-encoding region in 41 virus isolates suggested that most of the French isolates were recombinants. By applying different methods based on the ratio between non-synonymous (dN) and synonymous (dS) substitution rates, we identified four positions that showed evidence of positive selection. Three of these positions were in the putative leader protein (Lp), and one was in the polymerase. These findings raise the question of the putative role of the Lp in viral evolution.

First Report of Alfalfa mosaic virus on Rhamnus alaternus in France.

Rhamnus alaternus L. (evergreen buckthorn), family Rhamnaceae, is a small, hardy shrub from Mediterranean regions grown for its ornamental persistent green or variegated foliage. Chlorotic oak leaf or ringspot symptoms on R. alaternus leaves have been observed in southern France (Bellegarde, Gard department in 1998; Fanjaux, Aude department in 2000; and Saint-Jean-Cap-Ferrat, Alpes-Maritimes department in 2002). Samples from these three localities revealed the presence of Alfalfa mosaic virus (AMV) due to (i) the symptoms observed in an inoculated diagnostic host range previously described (1), (ii) observation of typical bullet-shaped virion particles of different sizes with transmission electron microscopy, (iii) nonpersistent transmission to Nicotiana tabacum cv. Xanthi-nc by Myzus persicae, and (iv) positive reaction in double-antibody sandwich-enzyme-linked immunosorbent assays (DAS-ELISA) to antibodies raised against AMV (gift of G. Marchoux). In addition, in Fanjaux, Viburnum tinus L. plants located close to the infected R. alaternus plants were also infected by AMV and exhibited typical intense calico mosaics (3). The close species, R. frangula L., was previously identified as a natural host for AMV in Italy (2). Following isolation from local lesions on Vigna unguiculata, the Fanjaux isolate was grown in cv. Xanthi-nc plants, where it induced a severe mosaic and stunting of the plants, and inoculated to 2-year-old virus-free R. alaternus plants either mechanically or with M. persicae (10 plants each). Plants were subsequently kept in an insect-proof greenhouse. At 8 and 12 months postinoculation, only one aphid-inoculated plant showed symptoms on young leaves and was AMV-positive in DAS-ELISA, while no mechanically infected plants were infected. This low infection level together with the rare observation of symptoms in natural conditions suggest that R. alaternus is not frequently infected by AMV. References: (1) L. Cardin and B. Moury. Plant Dis. 84:594, 2000. (2) F. Marani and L. Giunchedi. Acta Hortic. 59:97, 1976. (3) N. Plese and D. Milicic. Phytopathol. Z. 72:219, 1971.

Occurrence of Alternaria dichondrae, Cercospora sp., and Puccinia sp. on Dichondra repens in France and Italy.

Dichondra repens (kidneyweed or ponysfoot), family Convolvulaceae, is a perennial plant with persistent leaves and is grown alone or in association with turfgrass in subtropical and Mediterranean regions. Because of its prostrate growth habit, it does not need to be mowed. It is also used as a potted plant for house decoration. During surveys of lawns in public gardens of the Franco-Italian Riviera conducted from 1993 to 2003, we noticed 0.1- to 0.5-cm-diameter, brownish, necrotic spots on leaves of D. repens in Antibes, Cannes, Menton, Nice, and Vallauris (France) and in Arma di Taggia, Diano Marina, Imperia, La Mortola, Ospedaletti, San Remo, and Ventimiglia (Italy). Symptoms were more intense in the spring on young leaves but lesions remained all year on older leaves. Two species of fungal pathogens were frequently isolated from these spots. One fungus produced brown, erect conidiophores with brown, pear-shaped conidia and bifid, subhyaline beaks. Conidia formed singly, were composed of 8 to 10 cells with transverse and longitudinal crosswalls, and had one to four hyaline spurs frequently longer than the conidia. Conidia measured 90 to 260 × 16 to 29 µm. The pathogen, identified as Alternaria dichondrae (1), was previously characterized in Italy, New Zealand, and Argentina. The second fungus species produced clumps of erect, brown conidiophores with hyaline, filiform conidia composed of 10 to 20 cells. These conidia measured 90 to 310 × 3 to 3.5 µm. This fungus was identified as a Cercospora sp. (2), a genus not previously reported on D. repens. For both fungi, necrotic spots similar to those observed in natural infections were obtained after spraying a suspension of mycelium and conidia onto leaves of D. repens seedlings that had two to four expanded leaves that had been pricked with a pin. The plants were maintained under high humidity. Assays of mycelium growth on agar media containing various fungicides showed that 1 ppm of pyremethanil completely inhibited the growth of A. dichondrae, whereas a mixture of 10 ppm of diethofencarb and 10 ppm of carbendazine completely inhibited Cercospora sp. growth. Telia were also observed on the lower surface of D. repens leaves, sometimes in association with disease symptoms of A. dichondrae and Cercospora sp. Disease symptoms of the rust were yellowing and curling of the leaf surface with erect petiole, whereas healthy plants were prostrate with plane leaf surfaces. The two-celled teliospores had smooth cell walls, a single germinative pore per cell, and measured 32 to 34 × 12 to 13 µm with a thin unattached pedicel. This rust fungus was consequently classified in the genus Puccinia (2), also not previously reported as a pathogen of D. repens. It is possible that Poaceae plants such as Poa pratensis grown in association with D. repens were the inoculum source. Whereas A. dichondrae and Cercospora sp. do not induce severe diseases and are not widespread, the prevalence of Puccinia sp. tends to increase over time, requiring appropriate treatments to manage infected turf grasses. References: (1) P. Gambogi et al. Trans. Br. Mycol. Soc. 65:322, 1975. (2) G. Viennot-Bourgin. Les Champignons Parasites des Plantes Cultivées, Masson ed. Paris, 1949.

Occurrence of Mosaic Caused by Cucumber mosaic virus in Lobelia Hybrids in France and Italy.

In 2002, mosaic symptoms associated with yellowish ringspots were observed on leaves of a hybrid of lobelia (Lobelia spp.) grown in a public garden in Alsace (France). In 2003, similar symptoms were observed in Lobelia laxiflora in the Hanbury botanical garden (La Mortola, Italy) and the botanical garden of Nice (France). Cucumber mosaic virus (CMV) was identified in samples collected from the three locations on the basis of the following: symptoms exhibited by a host range of inoculated plants previously described (1); the observation of isometric particles (approximately 30 nm) with an electron microscope in crude sap preparations from inoculated plants and semipurified extracts of Claytonia perfoliata; and the positive reaction in double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) to antibodies raised against CMV (2). In double-immunodiffusion analysis, each isolate was shown to belong to the group II strains of CMV (4). In these experiments, no differences were observed among the isolates collected. To test if CMV was responsible for the symptoms observed in Lobelia spp., an isolate from Alsace was grown in Xanthinc tobacco plants following isolation from local lesions on Vigna unguiculata and then mechanically inoculated to L. × speciosa cv. Compliment mix (10 plants), L. siphilitica (10 plants), L. inflata (Indian tobacco) (10 plants), L. erinus cvs. Crystal and Empereur Guillaume (5 plants), L. erinus pendula cvs. Saphyr and Cascade (5 plants), L. laxiflora (10 plants), and L. × gerardii cv. Vedrariensis (5 plants) and grown in a hydroponic system. Eight weeks postinoculation, all plants except L. laxiflora exhibited systemic mosaic and chlorotic ringspot symptoms on leaves and resulted in strong DAS-ELISA reactions for CMV, whereas mock-inoculated controls remained symptomless and virus free. Symptoms were particularly severe on L. siphilitica and L. × speciosa, but mild on L. inflata and L. × gerardii. Foliar mosaic symptoms appeared only 6 months postinoculation in 7 of 10 inoculated L. laxiflora plants. Only these plants were CMV positive using DAS-ELISA. No symptoms were observed in flowers of any plants infected with CMV. CMV has been previously reported in other species of the family Lobeliaceae including L. cardinalis, L. erinus, L. gracilis, and L. tenuior following natural or experimental infection (3) but Koch's postulates were not completed. This study validates that CMV is responsible for mosaic diseases in Lobelia spp., and shows that hybrids from L. cardinalis such as L. × speciosa and L. × gerardii also are susceptible to CMV. Mosaic symptoms in L. siphilitica and L. × speciosa are particularly damaging to their ornamental quality. Moreover, perennial plants such as L. laxiflora can be sources of CMV contamination by aphid transmission. References: (1) L. Cardin et al. Plant Dis. 87:1263, 2003. (2) J. C. Devergne and L. Cardin. Ann. Phytopathol. 7:225, 1975. (3) L. Douine et al. Ann. Phytopathol. 11:439, 1979. (4) M. J. Roossinck. J. Virol. 76:3382, 2002.

Mosaic Symptoms Induced by Cucumber mosaic virus in Polygala myrtifolia in France and New Zealand.

Myrtle-leaf milkwort or sweet pea shrub (Polygala myrtifolia L.), family Polygalaceae, is a shrub from South Africa and is well adapted to Mediterranean-type conditions and used as an ornamental plant in gardens and pots or as cut flowers. During 2002 and 2003, mosaic symptoms and leaf distortion were observed in P. myrtifolia in Menton, Roquebrune-Cap Martin, Golfe Juan, and Antibes (Alpes Maritimes Department, France) in public gardens and potted plants. Occasionally, white streaks were observed in flowers. Cucumber mosaic virus (CMV) was identified in samples collected from the four locations on the basis of transmission to and symptoms exhibited by a range of diagnostic host plants (1), observation of isometric particles (≅30 nm) in crude sap preparations from the infected plants by electron microscopy, and positive reaction using double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) with polyclonal antibodies raised against CMV (2). Each isolate was shown to be a group II CMV strain (3) using double-immunodiffusion analysis. During 2004, CMV was also detected using DAS-ELISA in P. myrtifolia samples collected in New Zealand (Christchurch, Akaroa, and Roturoa). To confirm that CMV was responsible for pathogenicity, the Menton isolate was isolated from local lesions on Vigna unguiculata, amplified in Nicotiana tabacum cv. Xanthi-nc, and then mechanically inoculated into 1-year-old P. myrtifolia, P. myrtifolia cv. Grandiflora, and P. myrtifolia cv. Compacta (synonymous to cv. Nana) plants. The D strain of CMV, a reference tomato strain from subgroup I (2), was used for comparison. All experimental plants were propagated from cuttings, grown hydroponically and all tested negative for CMV using DAS-ELISA prior to inoculation. At 12 weeks postinoculation, systemic symptoms were observed on leaves from all inoculated plants (10 plants per genotype for the Menton isolate and 5 plants per genotype for the D strain), except for two P. myrtifolia plants inoculated with the Menton isolate. CMV was detected in apical, noninoculated leaves using DAS-ELISA in all symptomatic plants. A total recovery from symptoms was observed in P. myrtifolia and P. myrtifolia cv. Grandiflora but not in P. myrtifolia cv. Compacta at 6 months postinoculation (mpi) in 7 of 15, 10 of 15, and 15 of 15 DAS-ELISA positive plants, respectively. At 7 mpi, the plants were pruned and planted in soil and at 8 mpi, CMV was detected using DAS-ELISA in most of the plants, and symptoms developed in a few stems of some of the plants. Tessitori et al. (4) described similar symptoms and have detected CMV in P. myrtifolia from Italy, but they did not reproduce the disease in healthy plants. Our results show that CMV is responsible for the symptoms observed and that both CMV subgroups are infectious in P. myrtifolia. Since P. myrtifolia is generally vegetatively propagated by cuttings, frequent CMV tests on the mother stock plants are recommended because of fluctuations in virus titer and symptom expression in some genotypes. To our knowledge, this is the first report of this CMV host in France and New Zealand. A voucher specimen will be deposited at the Station de Pathologie Végétale at INRA, Montfavet. References: (1) L. Cardin et al. Plant Dis. 87:1263, 2003. (2) J. C. Devergne and L. Cardin. Ann. Phytopathol. 7:225, 1975. (3) M. J. Roossinck. J. Virol. 76:3382, 2002. (4) M. Tessitori et al. Plant Dis. 86:1403, 2002.

Durable virus resistance in plants through conventional approaches: a challenge.

Breeding for virus resistance is often considered the most efficient and simplest way to avoid the losses due to plant virus diseases. Resistance mechanisms are very diverse and interact with various stages of the virus cycle in the host plant. Resistances may also differ in their specificity, stability and durability. Breeding for resistance is a long and costly process, therefore to be cost effective it should provide durable protection. Three pathosystems are discussed to illustrate some of the field and laboratory approaches that can be used to assess resistance durability: Cucumber mosaic virus-specific resistance in melon, Zucchini yellow mosaic virus tolerance in zucchini squash, and extreme resistance to Potato virus X in potato. The possibility of predicting resistance durability is discussed in relation to the nature of the resistance, the genetic changes required for a virus to overcome the resistance and the effects of such changes on virus fitness.

Enzyme-Linked Immunosorbent Assay Testing of Shoots Grown In Vitro and the Use of Immunocapture-Reverse Transcription-Polymerase Chain Reaction Improve the Detection of Prunus necrotic ringspot virus in Rose.

We developed and evaluated two different methods to improve the detection of the most prevalent virus of rose in Europe, Prunus necrotic ring-spot virus (PNRSV). Immunocapture-reverse transcription-polymerase chain reaction was estimated to be about 100 times more sensitive than double-antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) and showed an equivalent specificity. Based on the observation that PNRSV multiplies actively in young growing tissues (axillary shoots and cuttings), an in vitro culture method allowing rapid (about 15 days) and homogeneous development of dormant axillary buds with high virus titers was standardized. ELISA tests of these young shoots showed, in some cases, a 10(4) to 10(5) increase in sensitivity in comparison to adjacent leaf tissues from the rose mother plants. Between 21 and 98% (depending on the season) more samples were identified as positive by using ELISA on samples from shoot tips grown in vitro rather than on leaves collected directly from the PNRSV-infected mother plants. This simple method of growing shoot tips in vitro improved the confidence in the detection of PNRSV and eliminated problems in sampling appropriate tissues.