Genomic analysis of the brassica pathogen turnip mosaic potyvirus reveals its spread along the former trade routes of the Silk Road.

Plant pathogens have agricultural impacts on a global scale and resolving the timing and route of their spread can aid crop protection and inform control strategies. However, the evolutionary and phylogeographic history of plant pathogens in Eurasia remains largely unknown because of the difficulties in sampling across such a large landmass. Here, we show that turnip mosaic potyvirus (TuMV), a significant pathogen of brassica crops, spread from west to east across Eurasia from about the 17th century CE. We used a Bayesian phylogenetic approach to analyze 579 whole genome sequences and up to 713 partial sequences of TuMV, including 122 previously unknown genome sequences from isolates that we collected over the past five decades. Our phylogeographic and molecular clock analyses showed that TuMV isolates of the Asian-Brassica/Raphanus (BR) and basal-BR groups and world-Brassica3 (B3) subgroup spread from the center of emergence to the rest of Eurasia in relation to the host plants grown in each country. The migration pathways of TuMV have retraced some of the major historical trade arteries in Eurasia, a network that formed the Silk Road, and the regional variation of the virus is partly characterized by different type patterns of recombinants. Our study presents a complex and detailed picture of the timescale and major transmission routes of an important plant pathogen.

d-Tartrate utilization correlates with phylogenetic subclade in Pseudomonas cichorii.

Pseudomonas cichorii is divided into two subclades based on the 16S ribosomal RNA gene sequence and core genome multilocus sequence typing. It was shown that subclade 2 strains utilize d-tartrate as a sole carbon source, whereas subclade 1 strains do not. Draft genome sequencing was performed with P. cichorii strains to identify d-tartrate utilization genes. By genome comparative and homology search studies, an ∼7.1-kb region was identified to be involved in d-tartrate utilization. The region is subclade 2 specific, and contains tarD and dctA genes, which encode a putative enzyme and transporter of d-tartrate, respectively. When the region was introduced into subclade 1 strains, the transformants were able to utilize d-tartrate. Partial fragments of tarD and dctA were amplified from all subclade 2 strains tested in this study by PCR using gene-specific primers, but not from subclade 1 strains. This is the first report on the genetic analysis of biochemical characteristics corresponding to a specific phylogenetic group in P. cichorii.

Complete Genome Sequences of Ralstonia solanacearum Strains Isolated from Zingiberaceae Plants in Japan.

Here, we report the complete genome sequences of three Ralstonia solanacearum strains isolated from Zingiberaceae plants in Japan. The total genome sizes of these strains ranged from 5.87 to 6.05 Mb. Strains MAFF 211472, MAFF 211479, and MAFF 311693 each carried one chromosome and one megaplasmid. MAFF 311693 contained an additional 71.9-kb plasmid.

Characterization and Genome Structure of Virulent Phage EspM4VN to Control Enterobacter sp. M4 Isolated From Plant Soft Rot.

Enterobacter sp. M4 and other bacterial strains were isolated from plant soft rot disease. Virulent phages such as EspM4VN isolated from soil are trending biological controls for plant disease. This phage has an icosahedral head (100 nm in diameter), a neck, and a contractile sheath (100 nm long and 18 nm wide). It belongs to the Ackermannviridae family and resembles Shigella phage Ag3 and Dickeya phages JA15 and XF4. We report herein that EspM4VN was stable from 10°C to 50°C and pH 4 to 10 but deactivated at 70°C and pH 3 and 12. This phage formed clear plaques only on Enterobacter sp. M4 among tested bacterial strains. A one-step growth curve showed that the latent phase was 20 min, rise period was 10 min, and an average of 122 phage particles were released from each absorbed cell. We found the phage's genome size was 160,766 bp and that it annotated 219 open reading frames. The genome organization of EspM4VN has high similarity with the Salmonella phage SKML-39; Dickeya phages Coodle, PP35, JA15, and Limestone; and Shigella phage Ag3. The phage EspM4VN has five tRNA species, four tail-spike proteins, and a thymidylate synthase. Phylogenetic analysis based on structural proteins and enzymes indicated that EspM4VN was identified as a member of the genus Agtrevirus, subfamily Aglimvirinae, family Ackermannviridae.

A hairy-leaf gene, BLANKET LEAF, of wild Oryza nivara increases photosynthetic water use efficiency in rice.

BACKGROUND: High water use efficiency is essential to water-saving cropping. Morphological traits that affect photosynthetic water use efficiency are not well known. We examined whether leaf hairiness improves photosynthetic water use efficiency in rice. RESULTS: A chromosome segment introgression line (IL-hairy) of wild Oryza nivara (Acc. IRGC105715) with the genetic background of Oryza sativa cultivar 'IR24' had high leaf pubescence (hair). The leaf hairs developed along small vascular bundles. Linkage analysis in BC5F2 and F3 populations showed that the trait was governed by a single gene, designated BLANKET LEAF (BKL), on chromosome 6. IL-hairy plants had a warmer leaf surface in sunlight, probably due to increased boundary layer resistance. They had a lower transpiration rate under moderate and high light intensities, resulting in higher photosynthetic water use efficiency. CONCLUSION: Introgression of BKL on chromosome 6 from O. nivara improved photosynthetic water use efficiency in the genetic background of IR24.

Unique clade of alphaproteobacterial endosymbionts induces complete cytoplasmic incompatibility in the coconut beetle.

Maternally inherited bacterial endosymbionts in arthropods manipulate host reproduction to increase the fitness of infected females. Cytoplasmic incompatibility (CI) is one such manipulation, in which uninfected females produce few or no offspring when they mate with infected males. To date, two bacterial endosymbionts, Wolbachia and Cardinium, have been reported as CI inducers. Only Wolbachia induces complete CI, which causes 100% offspring mortality in incompatible crosses. Here we report a third CI inducer that belongs to a unique clade of Alphaproteobacteria detected within the coconut beetle, Brontispa longissima This beetle comprises two cryptic species, the Asian clade and the Pacific clade, which show incompatibility in hybrid crosses. Different bacterial endosymbionts, a unique clade of Alphaproteobacteria in the Pacific clade and Wolbachia in the Asian clade, induced bidirectional CI between hosts. The former induced complete CI (100% mortality), whereas the latter induced partial CI (70% mortality). Illumina MiSeq sequencing and denaturing gradient gel electrophoresis patterns showed that the predominant bacterium detected in the Pacific clade of B. longissima was this unique clade of Alphaproteobacteria alone, indicating that this endosymbiont was responsible for the complete CI. Sex distortion did not occur in any of the tested crosses. The 1,160 bp of 16S rRNA gene sequence obtained for this endosymbiont had only 89.3% identity with that of Wolbachia, indicating that it can be recognized as a distinct species. We discuss the potential use of this bacterium as a biological control agent.

Species-Specific Detection of Mycosphaerella polygoni-cuspidati as a Biological Control Agent for Fallopia japonica by PCR Assay.

The ascomycete fungus Mycosphaerella polygoni-cuspidati has been undergoing evaluation as a potential classical biological control agent for the invasive weed Fallopia japonica (Japanese knotweed), which has become troublesome in Europe and North America. In advance of the potential release of a biocontrol agent into a new environment, it is crucial to develop an effective monitoring system to enable the evaluation of agent establishment and dispersal within the target host population, as well as any potential attacks on non-target species. Therefore, a primer pair was designed for direct, rapid, and specific detection of the Japanese knotweed pathogen M. polygoni-cuspidati based on the sequences of the internal transcribed spacer regions including the 5.8S rDNA. A PCR product of approximately 298 bp was obtained only when the DNA extracted from mycelial fragments of M. polygoni-cuspidati was used. The lower limit of detection of the PCR method was 100 fg of genomic DNA. Using the specific primer pair, M. polygoni-cuspidati could be detected from both naturally and artificially infected Japanese knotweed plants. No amplification was observed for other Mycosphaerella spp. or fungal endophytes isolated from F. japonica. The designed primer pair is thus effective for the specific detection of M. polygoni-cuspidati in planta.

A single amino acid at N-terminal region of the 2b protein of cucumber mosaic virus strain m1 has a pivotal role in virus attenuation.

Host responses to infection by a mild strain of cucumber mosaic virus, termed CMV-m1, were re-examined in several plant species in comparison with those by a severe strain CMV-Y. Mild systemic symptoms were developed on the six plant species inoculated with CMV-m1. Virus titer in the Nicotiana benthamiana plants infected with CMV-m1 was significantly lower than those infected with CMV-Y, although infection by CMV-m1 interfered with further infection by CMV-Y in the plants. Subsequently, the attenuated virulence of CMV-m1 was analyzed by reassortment and recombination analyses between CMV-m1 and CMV-Y RNAs. The results suggested that the 2b protein of CMV-m1 (m1-2b) is involved in the formation of mild symptoms in N. benthamiana. Furthermore, site-directed mutagenesis demonstrated that Thr18 of m1-2b is responsible for formation of mild symptoms. Local RNA silencing suppressor activity of m1-2b was a little lower than that of severe strain CMV-Y. We discuss the relationship between attenuation of CMV-m1 and the features of m1-2b.

Induction of antiviral responses by acibenzolar-s-methyl against cucurbit chlorotic yellows virus in Melon.

Cucurbit chlorotic yellows virus (CCYV) (family Closteroviridae, genus Crinivirus) is an emerging virus which causes severe diseases on melon (Cucumis melo) plants. CCYV-infected melon plants display yellowing, mottling, chlorosis, or chlorotic spots on leaves. To develop a new control strategy, the potential for 1,2,3-benzothiadiazole-7-thiocarboxylic acid-S-methyl-ester (ASM) to suppress CCYV infection was evaluated. ASM treatment on melon plants greatly increased the expression levels of pathogenesis-related 1a gene, a marker gene for systemic acquired resistance. ASM treatment on melon plants before inoculation of CCYV suppressed systemic symptoms and decreased CCYV accumulation. ASM treatment on melon even after inoculation of CCYV reduced disease severity and accumulation levels of CCYV. The results show the potential for ASM treatment on attenuation of the CCYV disease symptoms.

Multiple and independent origins of short seeded alleles of GS3 in rice.

GRAIN SIZE 3 (GS3) is a cloned gene that is related to seed length. Here we report the discovery of new deletion alleles at the GS3 locus, each of which confer short seed. We selected ten short seeded cultivars from a collection of 282 diverse cultivars. Sequence analysis across the GS3 gene in these ten cultivars identified three novel alleles and a known allele that contain several independent deletion(s) in the fifth exon of GS. These independent deletion variants each resulted in a frameshift mutation that caused a premature stop codon, and they were functionally similar to one another. Each coded for a truncated gene product that behaved as an incomplete dominant allele and conferred a short seeded phenotype. Haplotype analysis of these sequence variants indicated that two of the variants were of japonica origin, and two were from indica. Transformation experiments demonstrated that one of the deletion alleles of GS3 decrease the cell number in the upper epidermis of the glume, resulting in a significant reduction in seed length. The multiple and independent origins of these short seeded alleles indicate that farmers and early breeders imposed artificial selection favoring short seeds.

Endophytic fungi associated with Fallopia japonica (Polygonaceae) in Japan and their interactions with Puccinia polygoni-amphibii var. tovariae, a candidate for classical biological control.

Fallopia japonica (Polygonaceae), or Japanese knotweed, is now spreading globally, causing serious problems in Europe and North America in both natural and urban habitats. There is an urgent need for alternative management solutions, and classical biological control, using coevolved natural enemies found in the native range, is currently being investigated. Here, we isolated fungal endophytes from F. japonica in Japan, its natural habitat, to find endophytes that might increase the virulence of a coevolved rust pathogen, Puccinia polygoni-amphibii var. tovariae. A total of 1581 fungal endophytes were recovered from F. japonica and classified into 15 taxa. Five genera (Colletotrichum, Pestalotiopsis, Phoma, Phomopsis, and Alternaria) were dominant as endophytes in F. japonica. A greenhouse study of the dominant endophyte-pathogen interactions revealed three types of reactions: suppressive, synergistic, and neutral. In particular, one Phomopsis isolate--closely related to Diaporthe medusaea, based on ITS sequences--promoted the pathogenic aggressiveness of P. polygoni-amphibii var. tovariae and, therefore, this interaction is potentially useful to increase the effectiveness of the rust fungus as a biological control agent of F. japonica in its invasive range.

Infection dynamics in viral spread and interference under the synergism between Cucumber mosaic virus and Turnip mosaic virus.

Mixed infection of Cucumber mosaic virus (CMV) and Turnip mosaic virus (TuMV) induced more severe symptoms on Nicotiana benthamiana than single infection. To dissect the relationships between spatial infection patterns and the 2b protein (2b) of CMV in single or mixed infections, the CMV vectors expressing enhanced green fluorescent or Discosoma sp. red fluorescent proteins (EGFP [EG] or DsRed2 [Ds], respectively were constructed from the same wild-type CMV-Y and used for inoculation onto N. benthamiana. CMV2-A1 vector (C2-A1 [A1]) has a functional 2b while CMV-H1 vector (C2-H1 [H1]) is 2b deficient. As we expected from the 2b function as an RNA silencing suppressor (RSS), in a single infection, A1Ds retained a high level of accumulation at initial infection sites and showed extensive fluorescence in upper, noninoculated leaves, whereas H1Ds disappeared rapidly at initial infection sites and could not spread efficiently in upper, noninoculated leaf tissues. In various mixed infections, we found two phenomena providing novel insights into the relationships among RSS, viral synergism, and interference. First, H1Ds could not spread efficiently from vasculature into nonvascular tissues with or without TuMV, suggesting that RNA silencing was not involved in CMV unloading from vasculature. These results indicated that 2b could promote CMV to unload from vasculature into nonvascular tissues, and that this 2b function might be independent of its RSS activity. Second, we detected spatial interference (local interference) between A1Ds and A1EG in mixed infection with TuMV, between A1Ds (or H1Ds) and TuMV, and between H1Ds and H1EG. This observation suggested that local interference between two viruses was established even in the synergism between CMV and TuMV and, again, RNA silencing did not seem to contribute greatly to this phenomenon.

Method for simple and rapid enumeration of total epiphytic bacteria in the washing solution of rice plants.

The phyllosphere is one of the most common habitats for terrestrial bacteria. However, little is known about the populations of bacteria, including unculturable bacteria, that thrive on plant surfaces. Here, we developed a fluorescent nuclear staining technique to easily and rapidly observe and enumerate populations of total and living epiphytic bacteria, with particular emphasis on the concentration by centrifugation and fixation of the epiphytic bacteria. An investigation on the optimal conditions for centrifugation and fixation revealed that centrifugation at 20 400g for 2 min and fixation with 0.5% glutaraldehyde solution were the optimum conditions for observation of the bacteria. Using this technique, we assessed the populations of the total and living bacteria on the surface of rice plants. When epiphytic bacteria were recovered from rice seeds (Oryza sativa 'Koshihikari'), the number of total and living bacterial cells was 7.36 and 6.85 log10·g⁻¹ (fresh mass) in the seed washing, respectively. In contrast, the numbers of total and living bacterial cells in the leaf sheath washings were 5.5-5.8 and 5.3-5.7 log10·g⁻¹, respectively. Approximately 5%-30% of the total bacteria in the washing solution of rice plant were culturable. The usefulness of the enumeration method and the amount of bacteria on the plant surfaces are discussed.

Impact of a defective RNA 3 from cucumber mosaic virus on helper virus infection dynamics.

D RNA 3Yalpha (D3Yalpha), a defective (D) RNA 3 derived from the Y strain of cucumber mosaic virus (CMV-Y), was further characterized in combination with different helper viruses in the genus Cucumovirus. Interestingly, Nicotiana benthamiana plants inoculated with CMV-D8 and D3Yalpha developed systemic symptoms which were different from those induced by CMV-D8. To elucidate the potential effects of D RNA 3 on virus infection on the basis of the original combination of CMV-Y and D3Yalpha, a point mutation was made in the coat protein gene, which determined symptoms, of either CMV-Y RNA 3 (Y3) or D3Yalpha. Symptoms induced on N. benthamiana and N. tabacum plants, and semi-quantitative RT-PCR revealed that the ratio of RNA 3 to D RNA 3 was associated with the differences of symptoms in the leaf tissues. Furthermore, analysis of in situ hybridization suggested that there were spatial effects between coat proteins of Y3 and D3Yalpha in the infected leaves.

Characterization of a defective RNA derived from RNA 3 of the Y strain of cucumber mosaic virus.

A defective (D) RNA 3 naturally generated from the Y strain of cucumber mosaic virus (CMV-Y) was characterized using a biologically active cDNA clone. Sequencing of the clone revealed that the D RNA 3, named D RNA 3Yalpha, was derived from CMV-Y RNA 3 and contained a 10 nt deletion in the 5' untranslated region and a 162 nt deletion within the 3a open reading frame. Co-inoculation of D RNA 3Yalpha with CMV-Y derived from in vitro transcripts facilitated propagation of CMV-Y containing D RNA 3Yalpha in Nicotiana benthamiana or Nicotiana tabacum plants. CMV-Y, however, did not produce deletion mutants upon serial mechanical passages in the plants.

Phylogenetic study and multiplex PCR-based detection of Burkholderia plantarii, Burkholderia glumae and Burkholderia gladioli using gyrB and rpoD sequences.

In order to develop a detection method for the rice pathogens Burkholderia plantarii, Burkholderia glumae and Burkholderia gladioli, the phylogeny of six plant-pathogenic Burkholderia species was analysed using the combined nucleotide sequences of gyrB and rpoD. B. plantarii, B. glumae and B. gladioli formed tight monophyletic branches supported by high bootstrap probabilities. The high sequence similarity revealed a close phylogenetic relationship between B. glumae and B. plantarii. B. plantarii strains were divided into three subclusters comprising rice strains, whereas the single Vanda strain occupied a unique position in the phylogenetic tree. The gyrB and rpoD sequences of all B. glumae strains examined were highly conserved. In contrast, B. gladioli strains demonstrated a far greater sequence diversity, but this diversity did not correlate with pathovar, host plant or geographical origin of the strains. A multiplex-PCR protocol using specific primers from the gyrB sequences was designed that allowed the specific detection and identification of B. plantarii, B. glumae and B. gladioli in rice seeds infected with these pathogenic species.

The glycosphingolipid receptor for Vibrio trachuri in the red sea bream intestine is a GM4 ganglioside which contains 2-hydroxy fatty acids.

Three major glycosphingolipids (tentatively designated IGL-1, 2, and 3) were isolated from the intestine of red sea bream (Pagrus major) and were subjected to a TLC-overlay assay with (35)S-labeled Vibrio trachuri which causes vibriosis of fish. The bacteria adhered to IGL-2, which was determined to be a GM4 ganglioside (NeuAcalpha2-3Galbeta1-ceramide). The fatty acid portion of IGL-2 was composed of 2-hydroxy C22:0, C24:0, and C24:1, in addition to the non-hydroxy C16:0 and C18:0, while the sphingoid base was composed exclusively of sphingenine (d18:1). Among glycosphingolipids tested, V. trachuri adhered to GM4 the most strongly followed by adherence to GM3 and GalCer, but the bacteria did not adhere to GM1a, GM2, LacCer, or GlcCer. V. trachuri was found to aggregate with the erythrocytes coated with GM4, but not with those coated with GM1a or GM2, thus indicating that specific adhesion occurs on intact cells. Interestingly, the dynamics for adhesion of V. trachuri to glycosphingolipids was defined by the structure of not only the sugar moiety but also the ceramide moiety, since the bacteria adhered to GM4 which contained 2-hydroxy fatty acids much more strongly than to that which contained non-hydroxy fatty acids.

Spatial analysis for exclusive interactions between subgroups I and II of Cucumber mosaic virus in cowpea.

The dynamics of virus interference in Cucumber mosaic virus (CMV) infection in cowpea were investigated by tissue-blotting and in situ hybridization. Using co-inoculation assays, we discovered that spatial competition between CMV-LE (subgroup I) and CMV-m2 (subgroup II) occurred in the inoculated leaves. Interestingly, competitive interactions between the two viruses also could be observed in the non-inoculated upper leaf tissues of the plants. Furthermore, the pattern of exclusive distribution was observed between challenge and protecting viruses in the serially inoculated leaves. Taken together, it is suggested that the dynamics of competitive interactions between the two subgroups could be characterized by exclusive infection and multiplication of the individual viruses in cowpea plants.