Occurrence of Different Tospoviruses in Vegetable Crops in Argentina

Significant damage on vegetable crops by tospoviruses had occurred sporadically in Argentina in the past but since 1994, severe outbreaks have been recorded every year. The crops that have been most affected, tomato, lettuce, and pepper, were surveyed in the provinces of Mendoza and Buenos Aires in 1994–95 and 1995–96. A few weeds and miscellaneous crops were also collected. A total of 543 samples showing symptoms typical for tospoviruses were analysed by double-antibody sandwich-enzyme-linked immunosorbent assay with polyclonal antibodies to groundnut ringspot tospovirus (GRSV), impatiens necrotic spot tospovirus (INSV) and tomato spotted wilt tospovirus (TSWV). The 339 samples collected in 1995–96 were also assayed for tomato chlorotic spot tospovirus (TCSV). In addition, the incidence of tospoviruses in tomato crops was assessed in 41 farms representing 310 ha and 10 cultivars. GRSV was identified in 222 samples (40.8%), TSWV in 194 samples (32.7%), TCSV in 50 samples (14.7%), INSV was not detected and 77 samples did not react with the antisera used. TSWV was found to prevail in Buenos Aires and GRSV in Mendoza. Mixed infections were not found in this survey. In tomato crops the mean incidence of tospoviruses was 33%. These results show that the disease formerly assigned to TSWV, is caused by at least three tospoviruses.     

The molecular population genetics of the Tomato spotted wilt virus (TSWV) genome

RNA viruses are characterized by high genetic variability resulting in rapid adaptation to new or resistant hosts. Research for plant RNA virus genetic structure and its variability has been relatively scarce compared to abundant research done for human and animal RNA viruses. Here, we utilized a molecular population genetic framework to characterize the evolution of a highly pathogenic plant RNA virus [Tomato spotted wilt virus (TSWV), Tospovirus, Bunyaviridae]. Data from genes encoding five viral proteins were used for phylogenetic analysis, and for estimation of population parameters, subpopulation differentiation, recombination, divergence between Tospovirus species, and selective constraints on the TSWV genome. Our analysis has defined the geographical structure of TSWV, attributed possibly to founder effects. Also, we identify positive selection favouring divergence between Tospovirus species. At the species level, purifying selection has acted to preserve protein function, although certain amino acids appear to be under positive selection. This analysis provides demonstration of population structuring and species-wide population expansions in a multisegmented plant RNA virus, using sequence-based molecular population genetic analyses. It also identifies specific amino acid sites subject to selection within Bunyaviridae and estimates the level of genetic heterogeneity of a highly pathogenic plant RNA virus. The study of the variability of TSWV populations lays the foundation in the development of strategies for the control of other viral diseases in floral crops.     

Diagnostic assays for two closely related tospovirus species, <Emphasis Type="Italic">Watermelon bud necrosis virus</Emphasis> and <Emphasis Type="Italic">Groundnut bud necrosis virus</Emphasis> and identification of new natural hosts

Watermelon bud necrosis virus (WBNV) and Groundnut bud necrosis virus (GBNV) are two closely related tospovirus species infecting several crops in India. In the present study, specific diagnostic assays for these two most predominant tospoviruses were developed by comparing host reactions and genome sequence information. Bioassay was developed by sap transmission of both the viruses to a set of host plants, cowpea, groundnut and watermelon. WBNV induced only local symptoms including chlorotic spots on cowpea cv. Pusa Komal, typical chlorotic ring spots on cv. C-152, bud necrosis on watermelon and no symptoms on groundnut. Whereas, GBNV induced both local and systemic symptoms including chlorotic/necrotic spots and veinal necrosis on both the cowpea cultivars, systemic yellowing and bud necrosis on groundnut and no symptoms on watermelon. Single and duplex RT-PCR was developed for diagnosis of WBNV and GBNV by designing primers based on differential sequence from Gn/Gc genes located on M RNA and from nucleocapsid protein (NP) gene located on S RNA genome. The duplex RT-PCR using NP gene specific primers was successfully utilised for the diagnosis of natural infection of GBNV in dahlia and muskmelon and mixed infection of GBNV and WBNV in chrysanthemum. The amplified products of duplex RT-PCR were sequenced and phylogenetic analysis confirmed the authenticity of the RT-PCR assay as well as confirmed infection of WBNV and GBNV in the new natural hosts. The bioassay and RT-PCR developed in this study will be useful in detecting single and mixed infection of GBNV and WBNV strains in different crops.     

Complementation between Two Tospoviruses Facilitates the Systemic Movement of a Plant Virus Silencing Suppressor in an Otherwise Restrictive Host

Background

New viruses pathogenic to plants continue to emerge due to mutation, recombination, or reassortment among genomic segments among individual viruses. Tospoviruses cause significant economic damage to a wide range of crops in many parts of the world. The genetic or molecular basis of the continued emergence of new tospoviruses and new hosts is not well understood though it is generally accepted that reassortment and/or genetic complementation among the three genomic segments of individual viruses could be contributing to this variability since plants infected with more than one tospovirus are not uncommon in nature.

Methodology/Principal Findings

Two distinct and economically important tospoviruses, Iris yellow spot virus (IYSV) and Tomato spotted wilt virus (TSWV), were investigated for inter-virus interactions at the molecular level in dually-infected plants. Datura (Datura stramonium) is a permissive host for TSWV, while it restricts the movement of IYSV to inoculated leaves. In plants infected with both viruses, however, TSWV facilitated the selective movement of the viral gene silencing suppressor (NSs) gene of IYSV to the younger, uninoculated leaves. The small RNA expression profiles of IYSV and TSWV in single- and dually-infected datura plants showed that systemic leaves of dually-infected plants had reduced levels of TSWV N gene-specific small interfering RNAs (siRNAs). No TSWV NSs-specific siRNAs were detected either in the inoculated or systemic leaves of dually-infected datura plants indicating a more efficient suppression of host silencing machinery in the presence of NSs from both viruses as compared to the presence of only TSWV NSs.

Conclusion/Significance

Our study identifies a new role for the viral gene silencing suppressor in potentially modulating the biology and host range of viruses and underscores the importance of virally-coded suppressors of gene silencing in virus infection of plants. This is the first experimental evidence of functional complementation between two distinct tospoviruses in the Bunyaviridae family.     

Summer weeds as hosts for Frankliniella occidentalis and Frankliniella fusca (Thysanoptera: Thripidae) and as reservoirs for tomato spotted wilt Tospovirus in North Carolina

In North Carolina, Tomato spotted wilt tospovirus (family Bunyaviridae, genus Tospovirus, TSWV) is vectored primarily by the tobacco thrips, Frankliniella fusca (Hinds), and the western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae). TSWV overwinters in winter annual weeds from which it is spread to susceptible crops in spring. Because most susceptible crops are destroyed after harvest before winter weeds emerge in the fall, infected summer weeds are thought to be the principal source for spread of TSWV to winter annual weeds in fall. A survey of summer weeds associated with TSWV-susceptible crops in the coastal plain of North Carolina conducted between May and October revealed that relatively few species were commonly infected with TSWV and supported populations of F. fusca or F. occidentalis. F. occidentalis made up > 75% of vector species collected from 15 summer weed species during 2002. The number of F. occidentalis and F. fusca immatures collected from plant samples varied significantly among plant species. Ipomoea purpurea (L.) Roth, Mollugo verticillata L., Cassia obtusifolia L., and Amaranthus palmeri S. Wats supported the largest numbers of immature F. occidentalis. Richardia scabra L., M. verticillata, and Ipomoea hederacea (L.) supported the largest numbers of F. fusca immatures. TSWV was present at 16 of 17 locations, and naturally occurring infections were found in 14 of 29 weed species tested. Five of the TSWV-infected species have not previously been reported as hosts of TSWV (A. palmeri, Solidago altissima L., Ipomoea lacunosa L., I. purpurea, and Phytolacca americana L.). Estimated rates of infection were highest in I. purpurea (6.8%), M. verticillata (5.3%), and I. hederacea (1.9%). When both the incidence of infection by TSWV and the populations of F. occidentalis and F. fusca associated with each weed species are considered, the following summer weed species have the potential to act as significant sources for spread of TSWV to winter annual weeds in fall: I. purpurea, I. hederacea, M. verticillata, A. palmeri, C. obtusifolia, R. scabra, Ambrosia artemisiifolia L., Polygonum pensylvanicum L., and Chenopodium album L.     

Analysis of Tospovirus NSs Proteins in Suppression of Systemic Silencing

RNA silencing is a sequence-specific gene regulation mechanism that in plants also acts antiviral. In order to counteract antiviral RNA silencing, viruses have evolved RNA silencing suppressors (RSS). In the case of tospoviruses, the non-structural NSs protein has been identified as the RSS. Although the tomato spotted wilt virus (TSWV) tospovirus NSs protein has been shown to exhibit affinity to long and small dsRNA molecules, its ability to suppress the non-cell autonomous part of RNA silencing has only been studied to a limited extent. Here, the NSs proteins of TSWV, groundnut ringspot virus (GRSV) and tomato yellow ring virus (TYRV), representatives for three distinct tospovirus species, have been studied on their ability and strength to suppress local and systemic silencing. A system has been developed to quantify suppression of GFP silencing in Nicotiana benthamiana 16C lines, to allow a comparison of relative RNA silencing suppressor strength. It is shown that NSs of all three tospoviruses are suppressors of local and systemic silencing. Unexpectedly, suppression of systemic RNA silencing by NSs^TYRV was just as strong as those by NSs^TSWV and NSs^GRSV, even though NSs^TYRV was expressed in lower amounts. Using the system established, a set of selected NSs^TSWV gene constructs mutated in predicted RNA binding domains, as well as NSs from TSWV isolates 160 and 171 (resistance breakers of the Tsw resistance gene), were analyzed for their ability to suppress systemic GFP silencing. The results indicate another mode of RNA silencing suppression by NSs that acts further downstream the biogenesis of siRNAs and their sequestration. The findings are discussed in light of the affinity of NSs for small and long dsRNA, and recent mutant screen of NSs^TSWV to map domains required for RSS activity and triggering of Tsw-governed resistance.     

Genetic divergence among Bradyrhizobium strains nodulating wild and cultivated Kummerowia spp. in China

Distribution of rhizobial species is affected by geographical isolation and selected by leguminous hosts, however, little is known about the molecular evolution of rhizobia nodulating the same legume in different eco-environments. In present study, the microevolution of Bradyrhizobium associated with the leguminous grass Kummerowia grown in exurban areas and cultivated in urban areas in China was investigated. Total 14 genospecies, including seven new groups, were identified based on a concatenated sequence analysis of taxonomic markers (SMc00019, truA and thrA) for 94 representative strains. Results demonstrated that lower levels of nucleotide diversity were found in the strains isolated from urban areas compared with those isolated from exurban areas, based on the evolutional analyses of three housekeeping genes (atpD, glnII and recA), two symbiosis-related genes (nodC and nifH), and the taxonomic markers. Moreover, compared with urban areas, gene exchange and recombination occurred more frequently among the genospecies isolated from exurban areas, regardless of the geographical distribution. Finally, the evolutionary lineage of Bradyrhizobium strains isolated from urban areas was independent of that of the strains isolated from exurban areas. In summary, the evolutionary history of Kummerowia bradyrhizobia may have been gradually segregated to different evolutionary lineages, irrespective of distinct biogeography.     

The molecular ecology of microbial eukaryotes unveils a hidden world.

In spite of the great success of small-subunit ribosomal RNA (SSU rRNA)-based studies for the analysis of environmental prokaryotic diversity, this molecular approach has seldom been applied to microbial eukaryotes. Recent molecular surveys of the smallest eukaryotic planktonic fractions at different oceanic surface regions and in deep-sea Antarctic samples revealed an astonishing protist diversity. Many of the phylotypes found in the photic region affiliate with photosynthetic groups that are known to contain picoeukaryotic representatives in the range 1-2 microm. Surprisingly, a vast diversity of presumably heterotrophic or mixotrophic lineages is also found. Among these, several novel lineages of heterokonts, and a large diversity of alveolates clustering in two major groups (Groups I and II), are present at all depths in the water column. Many of these new phylotypes appear biogeographically ubiquitous. These initial studies suggest that a wide diversity of small eukaryotes remains to be discovered not only in the ocean but also in other environments. For both ecology and evolutionary studies, it is predicted that environmental molecular identification of eukaryotes will have a profound impact in the immediate future.     

A Survey of Tospoviruses in Bulgaria

A survey of tospoviruses present in Bulgaria was conducted during three vegetation periods. A total of 258 different virus isolates were obtained from tobacco, tomato, weeds and ornamentals in the open fields and from glasshouses. Virus isolates were identified by inoculation to test plants and serology as Tomato spotted wilt virus (TSWV). No indications were obtained that other Tospovirus species were present in Bulgaria. In addition the transmission ability and efficiency of several Bulgarian populations of Thrips tabaci (Lind.) and Frankliniella occidentalis (Perg.) was determined. The experimental results indicated that Bulgarian TSWV isolates are transmitted with high efficiency by F. occidentalis and that only arrhenotokous and not thelytokouse populations of T. tabaci could transmit TSWV.     

Parasite diversity declines with host evolutionary distinctiveness: A global analysis of carnivores

Evolutionarily distinctive host lineages might harbor fewer parasite species because they have fewer opportunities for parasite sharing than hosts having extant close relatives, or because diverse parasite assemblages promote host diversification. We evaluate these hypotheses using data from 930 species of parasites reported to infect free‐living carnivores. We applied nonparametric richness estimators to estimate parasite diversity among well‐sampled carnivore species and assessed how well host evolutionary distinctiveness, relative to other biological and environmental factors, explained variation in estimated parasite diversity. Species richness estimates indicate that the current published literature captures less than 50% of the true parasite diversity for most carnivores. Parasite species richness declined with evolutionary distinctiveness of carnivore hosts (i.e., length of terminal ranches of the phylogeny) and increased with host species body mass and geographic range area. We found no support for the hypothesis that hosts from more diverse lineages support a higher number of generalist parasites, but we did find evidence that parasite assemblages might have driven host lineage diversification through mechanisms linked to sexual selection. Collectively, this work provides strong support for host evolutionary history being an essential predictor of parasite diversity, and offers a simple model for predicting parasite diversity in understudied carnivore species.     

Possible causes of the emergence of tospovirus diseases

Tomato spotted wilt virus, type species of the genus Tospovirus, ranks among the top ten of economically most important plant viruses. From 1980 on, a rapid emergence and geographic expansion of this, and some other tospoviruses, has occurred. This contribution will provide evidence that the recent emergence of tospoviruses, has two major causes. Firstly, these plant viruses have relatively recently evolved from the (animal-infecting) bunyaviruses and, secondly, one of their major vectors, the Western flower thrips, has dramatically expanded due to changes in agricultural practices and increase in transcontinental trading.     

Multihost experimental evolution of a plant RNA virus reveals local adaptation and host-specific mutations

For multihost pathogens, adaptation to multiple hosts has important implications for both applied and basic research. At the applied level, it is one of the main factors determining the probability and the severity of emerging disease outbreaks. At the basic level, it is thought to be a key mechanism for the maintenance of genetic diversity both in host and pathogen species. Using Tobacco etch potyvirus (TEV) and four natural hosts, we have designed an evolution experiment whose strength and novelty are the use of complex multicellular host organism as hosts and a high level of replication of different evolutionary histories and lineages. A pattern of local adaptation, characterized by a higher infectivity and virulence on host(s) encountered during the experimental evolution was found. Local adaptation only had a cost in terms of performance on other hosts in some cases. We could not verify the existence of a cost for generalists, as expected to arise from antagonistic pleiotropy and other genetic mechanisms generating a fitness trade-off between hosts. This observation confirms that this classical theoretical prediction lacks empirical support. We discuss the reasons for this discrepancy between theory and experiment in the light of our results. The analysis of full genome consensus sequences of the evolved lineages established that all mutations shared between lineages were host specific. A low degree of parallel evolution was observed, possibly reflecting the various adaptive pathways available for TEV in each host. Altogether, these results reveal a strong adaptive potential of TEV to new hosts without severe evolutionary constraints.     

Low haemosporidian diversity and one key-host species in a bird malaria community on a mid-Atlantic island (São Miguel, Azores)

When host species colonize new areas, the parasite assemblage infecting the hosts might change, with some parasite species being lost and others newly acquired. These changes would likely lead to novel selective forces on both host and its parasites. We investigated the avian blood parasites in the passerine bird community on the mid-Atlantic island of S?o Miguel, Azores, a bird community originating from continental Europe. The presence of haemosporidian blood parasites belonging to the genera Haemoproteus, Plasmodium, and Leucocytozoon was assessed using polymerase chain reaction. We found two Plasmodium lineages and two Leucocytozoon lineages in 11 bird species (84% of all breeding passerine species) on the island. These lineages were unevenly distributed across bird species. The Eurasian Blackbird (Turdus merula) was the key-host species (total parasite prevalence of 57%), harboring the main proportion of parasite infections. Except for Eurasian Blackbirds, all bird species had significantly lower prevalence and parasite diversity compared to their continental populations. We propose that in evolutionary novel bird communities, single species may act as key hosts by harboring the main part of the parasite fauna from which parasites "leak" into the other species. This would create very different host-parasite associations in areas recently colonized by hosts as compared to in their source populations.     

Host and geographic barriers shape the competition,coexistence, and extinction patterns of influenza A (H1N1) viruses

The influenza virus mutates and spreads rapidly, making it suitable for studying evolutionary and ecological processes. The ecological factors and processes by which different lineages of influenza compete or coexist within hosts through time and across geographical space are poorly known. We hypothesized that competition would be stronger for influenza viruses infecting the same host compared to different hosts (the Host Barrier Hypothesis), and for those with a higher cross‐region transmission intensity (the Geographic Barrier Hypothesis). Using available sequences of the influenza A (H1N1) virus in GenBank, we identified six lineages, twelve clades, and several replacement events. We found that human‐hosted lineages had a higher cross‐region transmission intensity than swine‐hosted lineages. Co‐occurrence probabilities of lineages infecting the same host were lower than those infecting different hosts, and human‐hosted lineages had lower co‐occurrence probabilities and genetic diversity than swine‐hosted lineages. These results show that H1N1 lineages infecting the same host or with high cross‐region transmission rates experienced stronger competition and extinction pressures than those infecting different hosts or with low cross‐region transmission. Our study highlights how host and geographic barriers shape the competition, extinction, and coexistence patterns of H1N1 lineages and clades.     

Ectomycorrhizal fungal diversity and community structure on three co-occurring leguminous canopy tree species in a Neotropical rainforest

? The ectomycorrhizal (ECM) symbiosis was historically considered restricted to the temperate zones, but recent studies have shown the importance of this symbiosis across the tropics. We examined ECM fungal diversity, host plant phylogeny and ECM host preferences in a rainforest dominated by the leguminous host plants Dicymbe corymbosa, Dicymbe altsonii and Aldina insignis. ? Ectomycorrhizal fungi were identified by internal transcribed spacer rDNA sequencing and host species were verified with chloroplast trnL sequencing. To test whether Dicymbe and Aldina represent independent gains of the ECM symbiosis, we constructed a Fabaceae phylogeny using MatK and trnL. We identified four independent ECM lineages within the Fabaceae. ? We detected a diverse community of 118 ECM species dominated by the /clavulina, /russula-lactarius, /boletus, and /tomentella-thelephora lineages. Ectomycorrhizal species in Agaricales, Atheliales and Polyporales may represent previously unrecognized tropical-endemic ECM lineages. Previous studies suggested that ECM fungi did not diversify in the tropics, but the /clavulina lineage appears to have a center of diversity in tropical South America. ? Dicymbe and Aldina represent independent gains of the ECM symbiosis in Fabaceae but their fungal symbionts showed no host preferences. Spatial factors are more important than hosts in structuring the ECM fungal community in this ecosystem.     

Complete Genome Sequence of Mulberry Vein Banding Associated Virus,a New Tospovirus Infecting Mulberry

Mulberry vein banding associated virus (MVBaV) that infects mulberry plants with typical vein banding symptoms had been identified as a tentative species of the genus Tospovirus based on the homology of N gene sequence to those of tospoviruses. In this study, the complete sequence of the tripartite RNA genome of MVBaV was determined and analyzed. The L RNA has 8905 nucleotides (nt) and encodes the putative RNA-dependent RNA polymerase (RdRp) of 2877 aa amino acids (aa) in the viral complementary (vc) strand. The RdRp of MVBaV shares the highest aa sequence identity (85.9%) with that of Watermelon silver mottle virus (WSMoV), and contains conserved motifs shared with those of the species of the genus Tospovirus. The M RNA contains 4731 nt and codes in ambisense arrangement for the NSm protein of 309 aa in the sense strand and the Gn/Gc glycoprotein precursor (GP) of 1,124 aa in the vc strand. The NSm and GP of MVBaV share the highest aa sequence identities with those of Capsicum chlorosis virus (CaCV) and Groundnut bud necrosis virus (GBNV) (83.2% and 84.3%, respectively). The S RNA is 3294 nt in length and contains two open reading frames (ORFs) in an ambisense coding strategy, encoding a 439-aa non-structural protein (NSs) and the 277-aa nucleocapsid protein (N), respectively. The NSs and N also share the highest aa sequence identity (71.1% and 74.4%, respectively) with those of CaCV. Phylogenetic analysis of the RdRp, NSm, GP, NSs, and N proteins showed that MVBaV is most closely related to CaCV and GBNV and that these proteins cluster with those of the WSMoV serogroup, and that MVBaV seems to be a species bridging the two subgroups within the WSMoV serogroup of tospoviruses in evolutionary aspect, suggesting that MVBaV represents a distinct tospovirus. Analysis of S RNA sequence uncovered the highly conserved 5’-/3’-ends and the coding regions, and the variable region of IGR with divergent patterns among MVBaV isolates.     

Experimental adaptation of an RNA virus mimics natural evolution

Identification of virulence determinants of viruses is of critical importance in virology. In search of such determinants, virologists traditionally utilize comparative genomics between a virulent and an avirulent virus strain and construct chimeras to map their locations. Subsequent comparison reveals sequence differences, and through analyses of site-directed mutants, key residues are identified. In the absence of a naturally occurring virulent strain, an avirulent strain can be functionally converted to a virulent variant via an experimental evolutionary approach. However, the concern remains whether experimentally evolved virulence determinants mimic those that have evolved naturally. To provide a direct comparison, we exploited a plant RNA virus, soybean mosaic virus (SMV), and its natural host, soybean. Through a serial in vivo passage experiment, the molecularly cloned genome of an avirulent SMV strain was converted to virulent variants on functionally immune soybean genotypes harboring resistance factor(s) from the complex Rsv1 locus. Several of the experimentally evolved virulence determinants were identical to those discovered through a comparative genomic approach with a naturally evolved virulent strain. Thus, our observations validate an experimental evolutionary approach to identify relevant virulence determinants of an RNA virus.     

VIRUSES OF COWPEA, VIGNA UNGUICULATA L. (WALP.), IN NIGERIA

The cowpea strain of tobacco mosaic virus was isolated from a range of leguminous hosts at Ibadan, but was rare in cultivated crops. Systemic symptoms in species infected experimentally are described.
A new virus of cowpea was also found in Nigeria. The physical properties (thermal inactivation point 56° C., dilution end-point 1/50,000 and longevity in vitro 4 days at 25° C.) differ from those for cowpea viruses reported elsewhere and the name cowpea yellow mosaic virus is proposed. This virus produces local lesions in French bean ( Phaseolus vulgaris L.) and local and systemic lesions in Bengal bean ( Mucuna aterrima Holland), but does not infect other leguminous hosts. The virus was purified and an antiserum prepared against it.
Both viruses are transmitted by a beetle ( Ootheca mutabilis Sahlb.) which loses infectivity within 48 hr. of leaving plants infected with either or both viruses.