The recombinogenic history of turnip mosaic potyvirus reveals its introduction to Japan in the 19th century.

Characterizing the detailed spatial and temporal dynamics of plant pathogens can provide valuable information for crop protection strategies. However, the epidemiological characteristics and evolutionary trajectories of pathogens can differ markedly from one country to another. The most widespread and important virus of brassica vegetables, turnip mosaic virus (TuMV), causes serious plant diseases in Japan. We collected 317 isolates of TuMV from Raphanus and Brassica plants throughout Japan over nearly five decades. Genomic sequences from these isolates were combined with published sequences. We identified a total of eighty-eight independent recombination events in Japanese TuMV genomes and found eighty-two recombination-type patterns in Japan. We assessed the evolution of TuMV through space and time using whole and partial genome sequences of both nonrecombinants and recombinants. Our results suggest that TuMV was introduced into Japan after the country emerged from its isolationist policy (1639-1854) in the Edo period and then dispersed to other parts of Japan in the 20th century. The results of our analyses reveal the complex structure of the TuMV population in Japan and emphasize the importance of identifying recombination events in the genome. Our study also provides an example of surveying the epidemiology of a virus that is highly recombinogenic.

Comparison of an Immunochromatographic Assay Kit with DAS-ELISA for Large-Scale Diagnosis and Molecular Discrimination of Satsuma Dwarf Virus Collected from Citrus Orchards.

Satsuma dwarf virus (SDV) seriously damages citrus production by reducing the quality and yield of fruit. To avoid contamination with SDV, mother trees are checked to be SDV-free in advance of nursery tree distribution. In this study, we compared an immunochromatographic assay (ICA) kit with double-antibody sandwich enzyme-linked immunosorbent assay (DASELISA) for large-scale diagnosis of SDV in orchardgrown trees in Shizuoka Prefecture, Japan. The two methods gave conflicting results for 11 of 1,705 samples, all of which were negative by DAS-ELISA but positive by ICA. The samples scored as positive by either DASELISA or ICA were analyzed by reverse transcription polymerase chain reaction and all were confirmed to be positive. These results validate the use of ICA as a screening method for large-scale diagnosis. Strain discrimination revealed that 16 of 22 isolates belonged to SDV, while citrus mosaic virus (CiMV) infection only and co-infection (SDV and CiMV) were in a minority.

Evolutionary rates and genetic diversities of mixed potyviruses in Narcissus.

There is no attempt to evaluate evolutionary rates, timescales and diversities of viruses collected from mixedly infected hosts in nature. Plants of the genus Narcissus are a monocotyledon and are susceptible to several viruses. In this study, narcissus plants (Narcissus tazetta var. chinensis) showing mosaic or striping leaves were collected in Japan, and these were investigated for potyvirus infections using potyvirus-specific primers. Individual narcissus plants were found frequently to be mixedly infected with different potyviruses, different isolates and quasispecies of same virus. The viruses were potyviruses and a macluravirus in the family Potyviridae, namely Narcissus late season yellows virus (NLSYV), Narcissus yellow stripe virus (NYSV), Narcissus degeneration virus (NDV), Cyrtanthus elatus virus A (CyEVA) and Narcissus latent virus (NLV). Genetic diversities of coat protein coding region of different virus species were different; NYSV and CyEVA were most diverse whereas NDV was least. Evolutionary rates of all five narcissus viruses were 1.33-7.15×10-3nt/site/year and were similar. The most recent common ancestors (TMRCAs) varied between virus species; NYSV and CyEVA were the oldest whereas NDV was the youngest. Thus, the oldness of TMRCAs of the viruses correlated well with the greatness of nucleotide diversities.

Effect of the Timing of Fungicide Application on Fusarium Head Blight and Mycotoxin Contamination in Wheat.

Fungicide application to control Fusarium head blight (FHB) and accompanying mycotoxin contamination in wheat is generally performed at anthesis because wheat is most susceptible to FHB around this stage. In this study, we evaluated the effect of the timing of fungicide application on FHB and mycotoxin (deoxynivalenol and nivalenol) accumulation in wheat based on our previous finding that the late period of grain development (beyond 20 days after anthesis [DAA]) is important to determine the final toxin contamination level in wheat. Thiophanate-methyl fungicide was tested under artificial inoculation conditions in which moisture and inoculum spores were provided throughout the testing period. Eight treatments differing in application timing (anthesis, 10, 20, and 30 DAA) and in the number of applications (0 to 2) were tested for 2 years. The results indicated that fungicide application timing differentially affects FHB (disease) and mycotoxin concentration. Fungicide application at 20 DAA reduced mycotoxin concentration in matured grain without reducing FHB severity, whereas application at anthesis was crucial for reducing FHB. These results and our previous findings suggest that around 20 DAA (late milk stage) is a potentially critical timing for mycotoxin control in wheat.

Differentiation of "Candidatus Liberibacter asiaticus" isolates by variable-number tandem-repeat analysis.

Four highly polymorphic simple sequence repeat (SSR) loci were selected and used to differentiate 84 Japanese isolates of "Candidatus Liberibacter asiaticus." The Nei's measure of genetic diversity values for these four SSRs ranged from 0.60 to 0.86. The four SSR loci were also highly polymorphic in four isolates from Taiwan and 12 isolates from Indonesia.

Evaluation of genetic diversity among 'Candidatus Liberibacter asiaticus' isolates collected in Southeast Asia.

The aim of this study was to investigate the genetic diversity and relationships among 'Candidatus Liberibacter asiaticus' isolates from different hosts and distinct geographical areas in Southeast Asia. Genetic diversity among 'Ca. Liberibacter asiaticus' was estimated by sequencing four well-characterized DNA fragments: the 16S ribosomal DNA (rDNA) and 16S/23S intergenic spacer regions; the outer membrane protein (omp) gene region; the trmU-tufB-secE-nusG-rplKAJL-rpoB region (gene cluster region); and the bacteriophage-type DNA polymerase region. The sequences of the 16S rDNA and 16S/23S intergenic spacer regions were identical among all 'Ca. Liberibacter asiaticus' isolates. In contrast, nucleotide substitutions were observed in both the omp gene and the gene cluster regions. However, extended bacteriophage-type DNA polymerase sequences acquired by thermal asymmetric interlaced polymerase chain reaction provided the most sequence diversity among isolates. Phylogenetic analysis of the bacteriophage-type DNA polymerase sequences revealed three clusters in the Southeast Asian 'Ca. Liberibacter asiaticus' population. All Indonesian 'Ca. Liberibacter asiaticus' isolates clustered in one group. The other clusters were not correlated with geographic distribution. The differences in genetic sequences did not reflect differences in the original citrus host (mandarin or pummelo). These results suggest that the bacteriophage-type DNA polymerase region would be useful for molecular differentiation between different Southeast Asian 'Ca. Liberibacter asiaticus' isolates.

Development of VNTR markers for three Aspergillus species used in brewing.

Using a bioinformatics approach, we developed 18 variable number of tandem repeat markers for Aspergillus oryzae for use in population genetic studies. Repeat sequences in the genome sequences of A. oryzae were identified by a tandem repeat finding program. Length polymorphisms at 18 loci were examined in 41 strains of A. oryzae. The total number of alleles per locus ranged from two to 20. Investigation of cross-species amplifications with A. sojae and A. tamarii showed success. The variable number of tandem repeat markers will be used to determine the population structure of these three Aspergillus species used in brewing.

Effect of the Timing of Fungicide Application on Fusarium Head Blight and Mycotoxin Accumulation in Closed-Flowering Barley.

Fungicide application is one measure available to reduce the risk of Fusarium head blight (FHB) and mycotoxin contamination in barley. The stage at or near anthesis, or at full head emergence, is generally thought to be optimal for fungicide application, regardless of cultivar. However, we have previously found that the most critical time for Fusarium graminearum infection and mycotoxin accumulation in barley differs among cultivars. Whereas chasmogamous (open-flowering) cultivars were most susceptible at anthesis, cleistogamous (closed-flowering) cultivars were considerably resistant at anthesis but became susceptible after 'spent' anther extrusion. Therefore, this study evaluated the effect of the timing of fungicide application on FHB and mycotoxin (deoxynivalenol and nivalenol) accumulation in cleistogamous barley. Thiophanate-methyl fungicide was applied at different developmental stages, from before anthesis to 30 days after anthesis (DAA), under artificial inoculation conditions in the field in which inoculum spores were provided throughout the testing period. As expected, the optimal timing for chemical control of FHB and mycotoxin accumulation was the time around the beginning of spent anther extrusion rather than at anthesis. Later application, as late as 30 DAA, was also effective in controlling mycotoxin accumulation, although it was not effective in controlling disease levels.

Patterns of recombination in turnip mosaic virus genomic sequences indicate hotspots of recombination.

Potyviruses have variable single-stranded RNA genomes and many show clear evidence of recombination. This report studied the distribution of recombination sites in the genomes of 92 isolates of the potyvirus Turnip mosaic virus (TuMV); 42 came from the international gene sequence databases and an additional 50 complete genomic sequences were generated from field samples collected in Europe and Asia. The sequences were examined for evidence of recombination using seven different sequence comparison methods and the exact position of each site was confirmed by sequence composition analysis. Recombination sites were found throughout the genomes, except in the small 6K1 protein gene, and only 24 of the genomes (26%) showed no evidence of recombination. Statistically significant clusters of recombination sites were found in the P1 gene and in the CI/6K2/VPg gene region. Most recombination sites were bordered by an upstream (5') region of GC-rich and downstream (3') region of AU-rich sequence of a similar length. Correlations between the presence and type of recombination site and provenance, host type and phylogenetic relationships are discussed, as is the role of recombination in TuMV evolution.

Comparisons of the genetic structure of populations of Turnip mosaic virus in West and East Eurasia.

The genetic structure of populations of Turnip mosaic virus in Eurasia was assessed by making host range and gene sequence comparisons of 142 isolates. Most isolates collected in West Eurasia infected Brassica plants whereas those from East Eurasia infected both Brassica and Raphanus plants. Analyses of recombination sites (RSs) in five regions of the genome (one third of the full sequence) showed that the protein 1 (P1 gene) had recombined more frequently than the other gene regions in both subpopulations, but that the RSs were located in different parts of the genomes of the subpopulations. Estimates of nucleotide diversity showed that the West Eurasian subpopulation was more diverse than the East Eurasian subpopulation, but the Asian-BR group of the genes from the latter subpopulation had a greater nonsynonymous/synonymous substitution ratio, especially in the P1, viral genome-linked protein (VPg) and nuclear inclusion a proteinase (NIa-Pro) genes. These subpopulations seem to have evolved independently from the ancestral European population, and their genetic structure probably reflects founder effects.

The dual role of the potyvirus P3 protein of Turnip mosaic virus as a symptom and avirulence determinant in brassicas.

Two isolates of the potyvirus Turnip mosaic virus (TuMV), UK 1 and CDN 1, differ both in their general symptoms on the susceptible propagation host Brassica juncea and in their ability to infect B. napus lines possessing a variety of dominant resistance genes. The isolate CDN 1 produces a more extreme mosaic in infected brassica leaves than UK 1 and is able to overcome the resistance genes TuRB01, TuRB04, and TuRB05. The resistance gene TuRB03, in the B. napus line 22S, is effective against CDN 1 but not UK 1. The nucleic acid sequences of the UK 1 and CDN 1 isolates were 90% identical. The C-terminal half of the P3 protein was identified as being responsible for the differences in symptoms in B. juncea. A single amino acid in the P3 protein was found to be the avirulence determinant for TuRB03. Previous work already has identified the P3 as an avirulence determinant for TuRB04. Our results increase the understanding of the basis of plant-virus recognition, show the importance of the potyviral P3 gene as a symptom determinant, and provide a role in planta for the poorly understood P3 protein in a normal infection cycle.

Mutations in Turnip mosaic virus P3 and cylindrical inclusion proteins are separately required to overcome two Brassica napus resistance genes.

The Brassica napus differential line 165 is resistant to infection by Turnip mosaic virus (TuMV) isolates belonging to pathotypes 1 and 3. Nucleotide sequences of resistance-breaking mutants of pathotype 1 (UK 1), pathotype 3 (CHN 12), and wild-type isolates have been determined. When the mutations identified were introduced into an infectious clone of UK 1, a single mutation in the viral P3 protein induced a hypersensitive (necrotic) response in inoculated leaves of line 165 plants. Full systemic nonnecrotic infection was only possible when another mutation (in the cylindrical inclusion protein) was introduced. Tests on segregating populations derived from line 165 indicated that the two viral genes were pathogenicity determinants for two different resistance genes in line 165. One gene responsible for an extreme form of resistance (no symptoms seen) was epistatic to a second responsible for the hypersensitive reaction. These results help to explain the relative stability of the resistance in line 165 and to further define the genetic basis of the TuMV pathotyping system.

Molecular evolution of Turnip mosaic virus: evidence of host adaptation, genetic recombination and geographical spread.

Turnip mosaic virus (TuMV), a species of the genus Potyvirus, occurs worldwide. Seventy-six isolates of TuMV were collected from around the world, mostly from Brassica and Raphanus crops, but also from several non-brassica species. Host tests grouped the isolates into one or other of two pathotypes; Brassica (B) and Brassica-Raphanus (BR). The nucleotide sequences of the first protein (P1) and coat protein (CP) genes of the isolates were determined. One-tenth of the isolates were found to have anomalous and variable phylogenetic relationships as a result of recombination. The 5'-terminal 300 nt of the P1 gene of many isolates was also variable and phylogenetically anomalous, whereas the 380 nt 3' terminus of the CP gene was mostly conserved. Trees calculated from the remaining informative parts of the two genes of the non-recombinant sequences by neighbour-joining, maximum-likelihood and maximum-parsimony methods were closely similar, and so these parts of the sequences were concatenated and trees calculated from the resulting 1150 nt. The isolates fell into four consistent groups; only the relationships of these groups with one another and with the outgroup differed. The "basal-B" cluster of eight B-pathotype isolates was most variable, was not monophyletic, and came from both brassicas and non-brassicas from southwest and central Eurasia. Closest to it, and forming a monophyletic subgroup of it in most trees, and similarly variable, was the "basal-BR" group of eight BR pathotype Eurasian isolates. The third and least variable group, the "Asian-BR" group, was of 22 BR-pathotype isolates, all from brassicas, mostly Raphanus, and all from east Asia mostly Japan. The fourth group of 36 isolates, the "world-B" group, was from all continents, most were isolated from brassicas and most were of the B-pathotype. The simplest of several possible interpretations of the trees is that TuMV originated, like its brassica hosts, in Europe and spread to the other parts of the world, and that the BR pathotype has recently evolved in east Asia.