Sylvie Souche

Mutations in potato virus Y genome-linked protein determine virulence toward recessive resistances in Capsicum annuum and Lycopersicon hirsutum.

The recessive resistance genes pot-1 and pvr2 in Lycopersicon hirsutum and Capsicum annuum, respectively, control Potato virus Y (PVY) accumulation in the inoculated leaves. Infectious cDNA molecules from two PVY isolates differing in their virulence toward these resistances were obtained using two different strategies. Chimeras constructed with these cDNA clones showed that a single nucleotide change corresponding to an amino acid substitution (Arg119His) in the central part of the viral protein genome-linked (VPg) was involved in virulence toward the pot-1 resistance. On the other hand, 15 nucleotide changes corresponding to five putative amino acid differences in the same region of the VPg affected virulence toward the pvr2(1) and pvr2(2) resistances. Substitution models identified six and five codons within the central and C terminal parts of the VPg for PVY and for the related potyvirus Potato virus A, respectively, which undergo positive selection. This suggests that the role of the VPg-encoding region is determined by the protein and not by the viral RNA apart from its protein-encoding capacity.

Different mutations in the genome-linked protein VPg of Potato virus Y confer virulence on the pvr23 resistance in pepper

Five different amino acid substitutions in the VPg of Potato virus Y were shown to be independently responsible for virulence toward pvr2(3) resistance gene of pepper. A consequence of these multiple mutations toward virulence involving single nucleotide substitutions is a particularly high frequency of resistance breaking (37% of inoculated plants from the first inoculation) and suggests a potentially low durability of pvr2(3) resistance. These five mutants were observed with significantly different frequencies, one of them being overrepresented. Genetic drift alone could not explain the observed distribution of virulent mutants. More plausible scenarios were obtained by taking into account either the relative substitution rates, the relative fitness of the mutants in pvr2(3) pepper plants, or both.

Transmission by Olpidium brassicae of Mirafiori lettuce virus and Lettuce big-vein virus, and Their Roles in Lettuce Big-Vein Etiology

ABSTRACT Big-vein disease occurs on lettuce worldwide in temperate conditions; the causal agent has been presumed to be Lettuce big-vein virus (LBVV), genus Varicosavirus, vectored by the soilborne fungus Olpidium brassicae. Recently, the role of LBVV in the etiology of big-vein disease has been questioned because a second soilborne virus, Mirafiori lettuce virus (MiLV), genus Ophiovirus, has been found frequently in big-vein-affected lettuce. LBVV and MiLV, detectable and distinguishable by enzyme-linked immunosorbent assay using specific antisera, were tested for their ability to be transmitted from lettuce to lettuce by mechanical inoculation of sap extracts, or by zoospores of O. brassicae, and to cause big-vein disease. Both viruses were mechanically transmissible from lettuce to herbaceous hosts and to lettuce, but very erratically. LBVV was transmitted by O. brassicae but lettuce infected with only this virus never showed symptoms. MiLV was transmitted in the same manner, and lettuce infected with this virus alone consistently developed big-vein symptoms regardless of the presence or absence of LBVV. With repeated mechanical transmission, isolates of both viruses appeared to lose the ability to be vectored, and MiLV appeared to lose the ability to cause big-vein symptoms. The recovery of MiLV (Mendocino isolate, from Cali-fornia) from stored O. brassicae resting spores puts the earliest directly demonstrable existence of MiLV at 1990.

Biological and molecular variability of lettuce mosaic virus isolates.

ABSTRACT Lettuce mosaic potyvirus (LMV) causes severe disease of commercial lettuce crops. LMV isolates show wide biological variability, particularly in their ability to overcome the resistance genes described in Lactuca sativa. For a better understanding of the molecular interaction between lettuce and LMV, biological and molecular characterization of a collection of 10 LMV isolates known to differ in virulence or aggressiveness was performed. The ability of these isolates to overcome the resistance genes was reevaluated under standardized conditions. To study the molecular variability of LMV, an immunocapture-reverse transcription-poly-merase chain reaction technique, coupled with direct sequencing, was used to obtain nucleotide sequence data from three short regions of the LMV genome. Clustering analysis was performed and compared to the biological properties of the 10 isolates. Three groups of LMV isolates were discriminated based on the molecular data. These groups appear to correlate with the geographic origin of the isolates rather than with their pathogenicity. Sequence comparison with California isolates clearly showed that the California isolates are related to the western European isolates, raising the possibility of past exchanges of LMV between western Europe and California.

Effects of onion yellow dwarf and leek yellow stripe viruses on symptomatology and yield loss of three french garlic cultivars

This study was conducted to determine the effect of two potyviruses, onion yellow dwarf virus (OYDV) and leek yellow stripe virus (LYSV), on the symptoms, growth, and potential yield loss of garlic (Allium sativum). For 2 consecutive years, the impact on leaf length, pseudostem diameter, and bulb weight was evaluated after mechanical inoculation of cultivars Messidrome, Germidour, and Printanor, the three main garlic cultivars grown in France. The reduction in bulb weight due to OYDV ranged from 39% for Germidour to about 60% for the two other cultivars. For LYSV, the reduction in bulb weight was less on Messidrome (17%) and Germidour (26%) than on Printanor (54%). Coinfection with both viruses further reduced growth and bulb weight. When cloves originating from bulbs infected by each virus alone or a mixture of both viruses were planted, results indicated that such chronic infection induced further yield reduction. An assay designed to evaluate the role of LYSV inoculation date on yield revealed that yield losses were the lowest for late-season infections. However, yield loss was greater than 30% when the inoculation was performed at the end of April, the time when natural contamination generally occurs in southern France. A comparison of the impact of mixed infections of OYDV and LYSV from different origins suggested that the isolates did not differ significantly in their effects on yield loss.

Molecular and Biological Characterization of Lettuce mosaic virus (LMV) Isolates Reveals a Distinct and Widespread Type of Resistance-Breaking Isolate: LMV-Most.

ABSTRACT Lettuce mosaic virus (LMV) causes an economically important seedborne and aphid-transmitted disease of lettuce and ornamental crops worldwide. The genetic diversity among 73 LMV isolates was examined based on a 216-nucleotide sequence at the variable region encoding the NIb-coat protein junction. Three clusters of LMV isolates were distinguished: LMV-Yar, LMV-Greek, and LMV-RoW. In the latter cluster, two subgroups of isolates, LMV-Common and LMV-Most, accounted for a large proportion of the LMV isolates analyzed. These two subgroups included the seedborne isolates, consistent with this property contributing a selective advantage and resulting in widespread distribution. In addition to being seedborne, LMV-Most isolates overcome the two resistance genes commonly used in lettuce, mo1(1) and mo1(2), and thus represent a potential threat to lettuce cultivation. The complete sequence of an LMV-Most isolate (LMV-AF199) was determined, allowing a better definition of the genetic relationships among LMV-Most, LMV-Common, and an additional isolate of the LMV-RoW cluster.

Serological, Molecular, and Pathotype Diversity of Pepper veinal mottle virus and Chili veinal mottle virus

ABSTRACT Variability within the pepper-infecting potyviruses Pepper veinal mottle virus (PVMV) and Chili veinal mottle virus (ChiVMV) in Africa and Asia was investigated. Coat protein (CP) gene sequence diversity revealed three clades that corresponded to three geographic locations and there was no evidence of presence of the ChiVMV/Asian group in western or central Africa. These clades included closely related isolates that potentially belong to two viral species, which is consistent with current nomenclature. These clades could not be unambiguously identified with polyclonal antisera; however, reverse transcription-polymerase chain reactions allowed differentiation of the isolates into two species based on a large indel in the CP gene. PVMV and ChiVMV isolates were classified into three and two pathotypes, respectively, in relation to pepper genotypes carrying different resistance factors. Specificity of resistance only partially corresponded to molecular diversity of the isolates. Only one isolate of PVMV could infect pepper genotypes carrying the two recessive genes pvr6 and pvr2 (2); however, these genotypes were not infected by PVMV in field trials in Senegal, despite a high prevalence of PVMV in the surrounding pepper plants.

Coat protein gene-mediated protection in Lactuca sativa against lettuce mosaic potyvirus strains.

Lettuce mosaic potyvirus (LMV) can be very destructive on lettuce crops worldwide. The LMV strain 0 (LMV-0) coat protein (CP) gene was engineered for expression in plants. It was introduced into three susceptible cultivars of Lactuca sativa using an improved procedure for transformation and regeneration of lettuce, by co-cultivation of leaf explants with Agrobacterium tumefaciens. Several transformants accumulated detectable levels of LMV CP. The R1 progeny of twelve R0 transformants (four plants per cultivar) with T-DNA integration at one single locus, was studied for protection against LMV. The progeny from five R0 transformants showed resistance to LMV-0, with the effectiveness of resistance depending on the development stage of the plants at the time of inoculation. The R1 and R2 progeny from one of these R0 transformants, Cocarde-9a, were more extensively analysed. The homozygous but not the hemizygous R1 plants displayed protection to LMV-0. The R2 progeny from one homozygous R1 plant were shown to be resistant to infection by LMV-0 and other LMV strains. As previously observed in other cases of potyvirus sequence-mediated protection, a phenomenon of recovery was observed in some plants, as well as complete resistance. However, this recovery phenotype was not always maintained, as opposed to the previous described cases, leading to a late progression of viral infection.

Construction of full-length cDNA clones of lettuce mosaic virus (LMV) and the effects of intron-insertion on their viability in Escherichia coli and on their infectivity to plants

SummaryA full length cDNA copy of the genomic RNA of lettuce mosaic virus (LMV) was constructed under the control of an enhanced CaMV 35S promoter and of the NOS terminator. This construct was found infectious when inoculated to lettuce plants. The intron II of the bean nitrite reductase gene was engineered into the LMV FL cDNA in order to relieve possible deleterious effects of viral sequences to Escherichia coli cells and to evaluate the effects of the presence of the intron on the FL cDNA infectivity. The intron-less FL cDNA was found to be as stable as its intron-containing counterpart in E. coli. Sequence analysis of progeny RNA derived from plants inoculated with the intron-containing FL cDNA demonstrated that the inserted intron was perfectly spliced out. The symptoms induced in lettuce by either the intron-less or the intron-containing constructs were identical to those caused by the wild-type virus. However a slight delay in the establishment of infection in lettuce and a more obvious lag in Nicotiana benthamiana were observed with the intron-containing FL cDNA.

Occurrence of Mirafiori lettuce virus and Lettuce big-vein virus in relation to development of big-vein symptoms in lettuce crops

Big-vein disease (BV) of lettuce has been attributed to infection by Lettuce big-vein virus (LBVV), vectored by the soil fungus Olpidium brassicae. The finding of a second soil-borne virus in lettuce, Mirafiori lettuce virus (MiLV), led to a re-investigation of the role of LBVV in big-vein disease, with evidence emerging that both MiLV and LBVV are vectored by O. brassicae, and that MiLV, not LBVV, is the cause of BV (Lot et al. (2002), Phytopathology 92: 288–293). The two viruses have coat proteins of similar size but have different morphologies and are serologically unrelated. We tested individual lettuce plants in BV-prone fields and protected crops in France and Italy for the presence of the two viruses, using DAS-ELISA and antisera specific for each virus. Both MiLV and LBVV were found at high incidence, often together but sometimes separately. Symptoms were frequently found to be associated with MiLV alone or both viruses, but rarely LBVV alone. However, no absolute correlation emerged, because sometimes MiLV was present in the absence of symptoms, and vice versa. To clarify the situation, individual lettuce plants were examined over a period of time in two further surveys. In surveys of protected crops in France, plants with big-vein were always ELISA-positive for MiLV, or else symptomless plants positive for MiLV were later seen to develop big-vein symptoms. Presence or absence of LBVV appeared to have no effect on symptom development. In surveys of open fields in Italy, all combinations were found: presence of both viruses, apparent absence of both viruses, or presence of each one alone, in plants that developed BV. At the end of the observation period, nearly all plants had BV and contained both viruses.