Natural Infection of Murraya paniculata and Murraya sumatrana with CLas in Java.

The phloem-limited bacterium, 'Candidatus Liberibacter asiaticus' (CLas), is the putative causal pathogen of the severe Asiatic form of huanglongbing (citrus greening) and is most commonly transmitted by the Asiatic citrus psyllid (ACP), Diaphorina citri. CLas severely affects many Citrus species and hybrids and has been recorded in the Citrus relative, orange jasmine, Murraya paniculata (L.) Jack (syn. M. exotica L.). In this study, 13 accessions of three Murraya species (M. paniculata, M. sumatrana Roxb. and M. lucida (G.Forst.) Mabb,) and the Papuan form of a putative hybrid (M. omphalocarpa Hayata) were identified morphologically and molecularly based on sequence identity of the matK-5'trnK region of the chloroplast genome, and infection on these plants under field conditions was determined by PCR and qPCR on 2-4 occasions over 14 months. CLas was repeatedly detected in leaflet midribs by PCR and qPCR on four and three accessions of M. paniculata and M. sumatrana, respectively. It was not detected in leaflet midribs of single accessions of M. lucida and M. omphalocarpa. The species identification of the CLas-positive accessions was further confirmed using all the molecular taxonomic markers consisting of the six fragments of the maternally inherited chloroplast genome and part of the nuclear-encoded ITS region. The results indicated that natural infection of M. paniculata and M. sumatrana with CLas can occur in Java. This is the first demonstration of the natural infection of M. sumatrana with CLas. Further studies are required to determine if infections persist in the absence of D. citri.

Complete Genome Sequence of Rehmannia Mosaic Virus Infecting Rehmannia glutinosa in Japan.

The complete genome sequence of isolate Jiou of rehmannia mosaic virus (ReMV) infecting Rehmannia glutinosa in Japan was obtained via Sanger sequencing. Isolate Jiou shared high nucleotide sequence identity (>94%) with other known ReMV isolates.

Alterations of Candidatus Liberibacter asiaticus-Associated Microbiota Decrease Survival of Ca. L. asiaticus in in vitro Assays.

Phloem-inhabiting bacterial phytopathogens often have smaller genomes than other bacterial phytopathogens. It is thought that they depend on both other phloem microbiota and phloem nutrients for colonization of the host. However, the mechanism underlying associations between phloem-inhabiting phytopathogens and other phloem microbiota are poorly understood. Here, we demonstrate that the survival of Candidatus Liberibacter asiaticus (CLas), a cause of huanglongbing (citrus greening disease), depends on interplay with a specific subset of CLas-associated microbiota. CLas was not susceptible to oxytetracycline in vitro. However, oxytetracycline treatment eliminated a particular sub-community dominated by the Comamonadaceae, Flavobacteriaceae, Microbacteriaceae, and Pseudomonadaceae, decreasing CLas survival. We speculate that CLas uses ecological services derived from CLas-associated microbiota to colonize the host and to construct a pathogen-associated community that stimulates disease development.

ICTV Virus Taxonomy Profile: Secoviridae.

Members of the family Secoviridae are non-enveloped viruses with mono- or bipartite (RNA-1 and RNA-2) linear positive-sense ssRNA genomes with the size of the RNAs combined ranging from 9 to 13.7 kb. They are related to picornaviruses and are classified in the order Picornavirales. The majority of known members infect dicotyledonous plants and many are important plant pathogens (e.g. grapevine fanleaf virus and rice tungro spherical virus). This is a summary of the current International Committee on Taxonomy of Viruses (ICTV) report on the taxonomy of the family Secoviridae available at www.ictv.global/report/secoviridae.

Nucleotide sequences of Japanese isolates of citrus vein enation virus.

The genomic sequences of five Japanese isolates of citrus vein enation virus (CVEV) isolates that induce vein enation were determined and compared with that of the Spanish isolate VE-1. The nucleotide sequences of all Japanese isolates were 5,983 nt in length. The genomic RNA of Japanese isolates had five potential open reading frames (ORF 0, ORF 1, ORF 2, ORF 3, and ORF 5) in the positive-sense strand. The nucleotide sequence identity among the Japanese isolates and Spanish isolate VE-1 ranged from 98.0% to 99.8%. Comparison of the partial amino acid sequences of ten Japanese isolates and three Spanish isolates suggested that four amino acid residues, at positions of 83, 104, and 113 in ORF 2 and position 41 in ORF 5, might be unique to some Japanese isolates.

Changes in Variable Number of Tandem Repeats in 'Candidatus Liberibacter asiaticus' through Insect Transmission.

Citrus greening (huanglongbing) is the most destructive citrus disease worldwide. The disease is associated with three species of 'Candidatus Liberibacter' among which 'Ca. Liberibacter asiaticus' has the widest distribution. 'Ca. L. asiaticus' is commonly transmitted by a phloem-feeding insect vector, the Asian citrus psyllid Diaphorina citri. A previous study showed that isolates of 'Ca. L. asiaticus' were clearly differentiated by variable number of tandem repeat (VNTR) profiles at four loci in the genome. In this study, the VNTR analysis was further validated by assessing the stability of these repeats after multiplication of the pathogen upon host-to-host transmission using a 'Ca. L. asiaticus' strain from Japan. The results showed that some tandem repeats showed detectable changes after insect transmission. To our knowledge, this is the first report to demonstrate that the repeat numbers VNTR 002 and 077 of 'Ca. L. asiaticus' change through psyllid transmission. VNTRs in the recipient plant were apparently unrelated to the growing phase of the vector. In contrast, changes in the number of tandem repeats increased with longer acquisition and inoculation access periods, whereas changes were not observed through psyllid transmission after relatively short acquisition and inoculation access periods, up to 20 and 19 days, respectively.

Unique features of a Japanese 'Candidatus Liberibacter asiaticus' strain revealed by whole genome sequencing.

Citrus greening (huanglongbing) is the most destructive disease of citrus worldwide. It is spread by citrus psyllids and is associated with phloem-limited bacteria of three species of α-Proteobacteria, namely, 'Candidatus Liberibacter asiaticus', 'Ca. L. americanus', and 'Ca. L. africanus'. Recent findings suggested that some Japanese strains lack the bacteriophage-type DNA polymerase region (DNA pol), in contrast to the Floridian psy62 strain. The whole genome sequence of the pol-negative 'Ca. L. asiaticus' Japanese isolate Ishi-1 was determined by metagenomic analysis of DNA extracted from 'Ca. L. asiaticus'-infected psyllids and leaf midribs. The 1.19-Mb genome has an average 36.32% GC content. Annotation revealed 13 operons encoding rRNA and 44 tRNA genes, but no typical bacterial pathogenesis-related genes were located within the genome, similar to the Floridian psy62 and Chinese gxpsy. In contrast to other 'Ca. L. asiaticus' strains, the genome of the Japanese Ishi-1 strain lacks a prophage-related region.

Production of monoclonal antibodies specific for the recombinant viral coat protein of Apple stem grooving virus-citrus isolate and their application for a simple, rapid diagnosis by an immunochromatographic assay.

A simple and rapid immunochromatographic assay (ICA) for the diagnosis of Apple stem grooving virus (ASGV) in citrus was developed. Nine lines of monoclonal antibodies (mAbs) were produced by immunizing with a recombinant viral coat protein of ASGV as the antigen. According to the competitive-binding ELISA results, the 9 mAbs comprised 2 paratope groups, A and B. After screening for the most effective combination of mAbs, the two lines from different paratope groups (4A12 from group A and 6N31 from group B) were used to create a colloidal gold conjugate and for the test line, respectively, in ICA test plate preparation. The ICA detection using this test plate was accurate for positive and negative samples, and ASGV was detectable to a dilution of 1:2430 for the infected citrus sample. Furthermore, ICA was more sensitive than ELISA for the detection of ASGV isolates in citrus. The simple and sensitive ICA for ASGV provides a straightforward method for diagnosis by non-experts, including nursery workers and growers.

Convenient detection of the citrus greening (huanglongbing) bacterium 'Candidatus Liberibacter asiaticus' by direct PCR from the midrib extract.

A phloem-limited bacterium, 'Candidatus Liberibacter asiaticus' (Las) is a major pathogen of citrus greening (huanglongbing), one of the most destructive citrus diseases worldwide. The rapid identification and culling of infected trees and budwoods in quarantine are the most important control measures. DNA amplification including conventional polymerase chain reaction (PCR) has commonly been used for rapid detection and identification. However, long and laborious procedures for DNA extraction have greatly reduced the applicability of this method. In this study, we found that the Las bacterial cells in the midribs of infected leaves were extracted rapidly and easily by pulverization and centrifugation with mini homogenization tubes. We also found that the Las bacterial cells in the midrib extract were suitable for highly sensitive direct PCR. The performance of direct PCR using this extraction method was not inferior to that of conventional PCR. Thus, the direct PCR method described herein is characterized by its simplicity, sensitivity, and robustness, and is applicable to quarantine testing.

Sensitive and robust detection of citrus greening (huanglongbing) bacterium "Candidatus Liberibacter asiaticus" by DNA amplification with new 16S rDNA-specific primers.

Citrus greening disease is caused by "Candidatus Liberibacter spp.," including "Candidatus Liberibacter asiaticus (Las)." For detecting this disease, we designed new primers from the Las 16S rDNA and used a very small DNA template for PCR. More Las-infected tissues were detected with our primers than with the common primers.

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.

In planta distribution of 'Candidatus Liberibacter asiaticus' as revealed by polymerase chain reaction (PCR) and real-time PCR.

Huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide, and is caused by a phloem-limited fastidious prokaryotic alpha-proteobacterium that is yet to be cultured. In this study, a combination of traditional polymerase chain reaction (PCR) and real-time PCR targeting the putative DNA polymerase and 16S rDNA sequence of 'Candidatus Liberibacter asiaticus,' respectively, were used to examine the distribution and movement of the HLB pathogen in the infected citrus tree. We found that 'Ca. Liberibacter asiaticus' was distributed in bark tissue, leaf midrib, roots, and different floral and fruit parts, but not in endosperm and embryo, of infected citrus trees. Quantification analysis of the HLB bacterium indicated that it was distributed unevenly in planta and ranged from 14 to 137,031 cells/mug of total DNA in different tissues. A relatively high concentration of 'Ca. Liberibacter asiaticus' was observed in fruit peduncles. Our data from greenhouse-infected plants also indicated that 'Ca. Liberibacter asiaticus' was transmitted systemically from infection site to different parts of the plant. Understanding the distribution and movement of the HLB bacterium inside an individual citrus tree is critical for discerning its virulence mechanism and to develop management strategies for HLB.

Overwintering Viruliferous Frankliniella occidentalis (Thysanoptera: Thripidae) as an Infection Source of Tomato spotted wilt virus in Green Pepper Fields.

Populations of overwintering viruliferous Frankliniella occidentalis were evaluated in Tomato spotted wilt virus (TSWV)-affected green pepper fields in Bungo-Ohno City, Oita Prefecture, Japan. A survey of TSWV-infected weeds showed that the incidence of infection was low in weeds. Stellaria aquatica was infected frequently; however, the infections were considered secondary cases since S. aquatica appeared in the fields around late February to early March. In contrast, TSWV was frequently detected from green pepper fruits until they rotted. F. occidentalis primarily inhabited and reproduced on the green pepper fruits and moved to Lamium amplexicaule when the fruits rotted and subsequently spread to other weed species as young shoots or flowers appeared. The flying activity level of F. occidentalis rose in late February, and viruliferous F. occidentalis transmitted TSWV to green pepper plants. We concluded that TSWV-infected green pepper fruits discarded in greenhouses and fields are the major source of infection.

Molecular and Serological Characterization of Cucumber mottle virus, a New Cucurbit-Infecting Tobamo-like Virus.

A new tobamo-like virus was isolated from a greenhouse-grown cucumber that showed severe mosaic distortion on leaves and fruit, in the southern part of Japan. The virus was tentatively designated Cucumber mottle virus (CuMoV) and further characterized. The size and antigenicity of the coat protein (CP) and the complete sequence of the genome were compared with those of the known cucurbit-infecting tobamoviruses: the W and SH strains of Cucumber green mottle mosaic virus (CGMMV), the C and Y strains of Kyuri green mottle mosaic virus (KGMMV), Cucumber fruit mottle mosaic virus (CFMMV), and Zucchini green mottle mosaic virus (ZGMMV). The CP of CuMoV migrated more slowly than those of CGMMV-W and -SH and KGMMV-C and -Y in sodium dodecyl sulfate polyacrylamide gel electrophoresis. In Western blot analysis, the CP of CuMoV cross-reacted weakly with antisera against CGMMV-W and did not react with antisera against KGMMV-Y. The overall nucleotide sequence of CuMoV had 62.5 to 63.5% identity with those of CGMMV-W, -SH, KGMMV-Y, CFMMV, and ZGMMV. The genome organization was characteristic of tobamoviruses, encoding a 131-kb protein, a 188-kb protein, a movement protein (MP), and CP in 5' to 3' order. In the phylogenetic analyses of the CP, CuMoV was placed in a separate lineage from CGMMV-W, -SH, KGMMV-C, -Y, CFMMV, and ZGMMV. The results indicate that CuMoV is a distinct tobamovirus species which represents a third sub-subgroup in the cucurbit-infecting tobamoviruses.

Characterization of the tufB-secE-nusG-rplKAJL-rpoB Gene Cluster of the Citrus Greening Organism and Detection by Loop-Mediated Isothermal Amplification.

Thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was performed to amplify the uncharacterized regions adjacent to the nusG-rplKAJL-rpoB gene cluster of citrus greening organism (GO) isolates from different locations in Japan and Indonesia. Conventional PCR was used to amplify the internal nusG-rplKAJL-rpoB gene cluster of these isolates, and the complete sequence of this 6.1-kb fragment was determined. Comparisons with other bacterial sequences showed that the fragment is the tufB-secE-nusG-rplKAJL-rpoB gene cluster. The organization of this gene cluster is similar to that of the homologous cluster found in Escherichia coli. Except for three nucleotide changes, the sequence was identical among Japanese and Indonesian isolates. A loop-mediated isothermal amplification (LAMP) assay based on the conserved sequence of the nusG-rplKAJL-rpoB gene cluster was developed for the detection of the GO. The LAMP product was rapidly detected on nylon membranes by staining with AzurB. LAMP could detect as low as about 300 copies of the nusG-rplKAJL-rpoB fragment of the Japanese and Indonesian isolates of GO. The LAMP-based detection method, which does not depend upon a thermal cycler and electrophoresis apparatus, will be useful for under-equipped laboratories, including those found in extension centers and quarantine offices.

Tolerance to Citrus mosaic virus in Transgenic Trifoliate Orange Lines Harboring Capsid Polyprotein Gene.

Trifoliate orange plants (Poncirus trifoliata) were transformed with a binary vector containing the capsid polyprotein (pCP) gene of Citrus mosaic virus (CiMV) via Agrobacterium tumefaciens LBA4404. Transformation was performed on the epicotyl segments obtained from a young seedling that was grown in the dark. Southern blot hybridization analysis showed that the transgene was stable in the transgenic lines after regeneration and propagation by grafting. Transgenic lines were screened for tolerance to CiMV by mechanical inoculation. Infection was monitored 30, 60, 90, and 120 days after inoculation by reverse transcription-polymerase chain reaction. The transgenic line 24 had the lowest infection rate (7.1%) at 60 days after inoculation, in contrast to that of nontransgenic plants (65.1%).The response of other lines to inoculation ranged from susceptibility to moderate tolerance.

Molecular characterization of dsRNA segments 2 and 5 and electron microscopy of a novel reovirus from a hypovirulent isolate, W370, of the plant pathogen Rosellinia necatrix.

A hypovirulent isolate, W370, of the white root rot fungus Rosellinia necatrix has previously been shown to harbour 12 dsRNA segments. In this study, complete nucleotide sequences of segments 2 and 5 of W370 dsRNAs were determined. The nucleotide sequence of genome segment 2 was 3773 bases long with a single long open reading frame (ORF) encoding 1226 amino acid residues with a predicted molecular mass of approximately 138.5 kDa. The nucleotide sequence of segment 5 was 2089 bases long with a single long ORF, whose deduced polypeptide contained 646 amino acid residues with a predicted molecular mass of about 72 kDa. Comparative analysis showed that the deduced protein sequence of segment 2 had significant homology with the putative VP2 of Colorado tick fever virus (CTFV) and European Eyach virus (EYAV) in the genus Coltivirus, but the deduced protein sequence of segment 5 had no similarity with other virus proteins. Double-shelled spherical particles approximately 80 nm in diameter associated with W370 dsRNAs were observed in a preparation from the mycelial tissue of isolate W370. The results demonstrated that the virus associated with W370 dsRNAs is a novel reovirus of the family Reoviridae. The virus was named Rosellinia anti-rot virus (RArV).

Cloning and characterization of a totivirus double-stranded RNA from the plant pathogenic fungus, Helicobasidium mompa Tanaka.

Virus-like particles (VLPs, named HmTV1-17), about 40 nm in diameter were found in the violet root rot fungus Helicobasidium mompa Tanaka strain No. 17, which had been isolated from an apple tree. Purified preparations of HmTV1-17 contained two species of double-stranded RNA (dsRNA), designated 17L and 17S. cDNAs were constructed from HmTV1-17 genomic dsRNAs purified using CF-11 cellulose column chromatography. The sequences of 17L and 17S cDNA comprised 5,207 and 2,096 bp, respectively. Although 17S has no large open reading flame (ORF) on either strand, 17L has two large overlapping ORFs. The 5' located ORF1 encodes the coat protein (CP, 788 amino acids), whereas the gene product of ORF2, which is in the -1 frame relative to ORF1, shows the typical features of a RNA dependent RNA polymerase (RDRP, 845 amino acids). Phylogenetic analysis based on RDRP showed that HmTV1-17 is closely related to Sphaeropsis sapinea SsRV1, a member of the genus Totivirus from filamentous fungus S. sapinea.

Detection of a double-stranded RNA virus from a strain of the violet root rot fungus Helicobasidium mompa Tanaka.

Three double-stranded (ds) RNA species (ca. 1.30, 1.27 and 1.23 x 106) were isolated by CF-11 cellulose chromatography from a strain of the violet root rot fungus Helicobasidium mompa recovered from apple roots. Purified virion preparations contained isometric particles about 25 nm in diameter, and also the same three species of dsRNA isolated from total extracts by CF-11 cellulose chromatography. The molecular mass of the coat protein was about 67 K when estimated by SDS-PAGE. The largest dsRNA (referred to as dsRNA1) contains a single, long open reading frame of 1794 nucleotides that encodes a putative polypeptide containing 598 amino acid residues with a molecular mass of 69.9 K. This polypeptide contains amino acid sequence motifs conserved in putative RNA-dependent RNA polymerases of RNA viruses. Phylogenetic analysis revealed similarities to RNA-dependent RNA polymerases from Atkinsonella hypoxylon 2H virus, a member of the family Partitiviridae.