Genomic sequence analysis reveals diversity of Australian Xanthomonas species associated with bacterial leaf spot of tomato, capsicum and chilli.

BACKGROUND: The genetic diversity in Australian populations of Xanthomonas species associated with bacterial leaf spot in tomato, capsicum and chilli were compared to worldwide bacterial populations. The aim of this study was to confirm the identities of these Australian Xanthomonas species and classify them in comparison to overseas isolates. Analysis of whole genome sequence allows for the investigation of bacterial population structure, pathogenicity and gene exchange, resulting in better management strategies and biosecurity. RESULTS: Phylogenetic analysis of the core genome alignments and SNP data grouped strains in distinct clades. Patterns observed in average nucleotide identity, pan genome structure, effector and carbohydrate active enzyme profiles reflected the whole genome phylogeny and highlight taxonomic issues in X. perforans and X. euvesicatoria. Circular sequences with similarity to previously characterised plasmids were identified, and plasmids of similar sizes were isolated. Potential false positive and false negative plasmid assemblies were discussed. Effector patterns that may influence virulence on host plant species were analysed in pathogenic and non-pathogenic xanthomonads. CONCLUSIONS: The phylogeny presented here confirmed X. vesicatoria, X. arboricola, X. euvesicatoria and X. perforans and a clade of an uncharacterised Xanthomonas species shown to be genetically distinct from all other strains of this study. The taxonomic status of X. perforans and X. euvesicatoria as one species is discussed in relation to whole genome phylogeny and phenotypic traits. The patterns evident in enzyme and plasmid profiles indicate worldwide exchange of genetic material with the potential to introduce new virulence elements into local bacterial populations.

Potato spindle tuber viroid: Stability on Common Surfaces and Inactivation With Disinfectants.

The length of time Potato spindle tuber viroid (PSTVd) remained infective in extracted tomato leaf sap on common surfaces and the effectiveness of disinfectants against it were investigated. When sap from PSTVd-infected tomato leaves was applied to eight common surfaces (cotton, wood, rubber tire, leather, metal, plastic, human skin, and string) and left for various periods of time (5 min to 24 h) before rehydrating the surface and rubbing onto healthy tomato plants, PSTVd remained infective for 24 h on all surfaces except human skin. It survived best on leather, plastic, and string. It survived less well after 6 h on wood, cotton, and rubber and after 60 min on metal. On human skin, PSTVd remained infective for only 30 min. In general, rubbing surfaces contaminated with dried infective sap directly onto leaves caused less infection than when the sap was rehydrated with distilled water but overall results were similar. The effectiveness of five disinfectant agents at inactivating PSTVd in sap extracts was investigated by adding them to sap from PSTVd-infected leaves before rubbing the treated sap onto leaves of healthy tomato plants. Of the disinfectants tested, 20% nonfat dried skim milk and a 1:4 dilution of household bleach (active ingredient sodium hypochlorite) were the most effective at inactivating PSTVd infectivity in infective sap. When reverse-transcription polymerase chain reaction was used to test the activity of the five disinfectants against PSTVd in infective sap, it detected PSTVd in all instances except in sap treated with 20% nonfat dried skim milk. This study highlights the stability of PSTVd in infective sap and the critical importance of utilizing hygiene practices such as decontamination of clothing, tools, and machinery, along with other control measures, to ensure effective management of PSTVd and, wherever possible, its elimination in solanaceous crops.

Genome sequence of a polerovirus isolated from wild oat in Australia.

We present the genome sequence of a polerovirus (family Solemoviridae) isolated from wild oat (Avena fatua) in Australia. The genome sequence consists of 5,631 nucleotides and shares 87% nucleotide identity with its closest relative, cereal yellow dwarf virus RPV isolate 010 (GenBank accession number EF521830).

First Report of Pepper chat fruit viroid in Traded Tomato Seed, an Interception by Australian Biosecurity.

Pepper chat fruit viroid (PCFVd), a species of Pospiviroid, was first discovered in a capsicum crop in the Netherlands in 2006 (4) and was then reported only in Thailand (2) and Canada. The mechanism of international spread was not known, but movement with traded seed was suspected. PCFVd is transmissible through capsicum seed (4) and very probably through tomato seed, like other pospiviroids. The viroid causes disease in capsicum and tomato and experiments by others indicate a capacity to cause disease in potato. It poses a biosecurity threat to crops internationally. PCFVd was intercepted by the Australian Government Department of Agriculture, Fisheries, and Forestry (DAFF) in five shipments of tomato seed (Solanum lycopersicum) exported from Israel and Thailand in September and October 2012. Batches of up to 20,000 seeds were sampled from each seed lot in a shipment and total nucleic acids were extracted from sub-samples, each of about 400 seeds, following a method similar to Hoshino et al. (1). PCFVd was initially detected when reverse transcription PCR using the generic pospiviroid primers Pospi1-FW and Pospi1-RE (3) produced amplicons of 189 bp, which were then sequenced. The PCFVd specific primers AP FW1 and AP RE2 (4) were used to amplify the remainder of the viroid genome, which was directly sequenced. Overlapping sequences were aligned to produce complete sequences of 349 bases, one from seed from Thailand and two from seed from Israel (GenBank: KC762952, KC762953, KC762954). Searches of the GenBank nucleotide non-redundant database indicated close matches with sequences from PCFVd isolates from tomato in Thailand (2); alignments generated by BLAST showed the sequences differed from those from Thailand at only 2 to 18 nucleotide positions, equating to 95 to 99% identity. PCFVd sequences from seed from Thailand were almost identical (>99%) to the sequences from seed from Israel. Many sub-samples were negative, indicating that the number of contaminated seeds was very small in some shipments. The positive sub-samples as a proportion of the total number of sub-samples tested from the five shipments was 1/1, 1/5, 1/1, 12/50, and 7/50. Tomato and capsicum seed are produced in many countries and often traded through second countries. The infected tomato seed shipments intercepted by DAFF were destroyed or re-exported following Australian regulations. Other countries were informed through the International Plant Protection Convention. This pest viroid has not been intercepted by Australian authorities before and has not been detected in recent Australian survey work (data not shown). References: (1) S. Hoshino et al. Res. Bull. Plant Prot. Japan 42:75, 2006. (2) K. Reanwarakorn et al. New Dis. Rep. 24:6, 2011 (3) J. Th. J. Verhoeven et al. EJPP 110:823, 2004. (4) J. Th. J. Verhoeven et al. Virus Res. 144:209, 2009.

Oxford Nanopore Sequencing Reveals an Exotic Broad bean mottle virus Genome within Australian Grains Post-Entry Quarantine.

A Broad bean mottle virus (BBMV) isolate (S52) obtained from an infected Vicia faba leaf sample from Syria was sequenced using Oxford Nanopore long-read sequencing at the Australian border. The genome had 95.6%, 98.2%, and 93.4% nucleotide sequence identity to BBMV strains RNA1 (Bawden), RNA2 (Mo), and RNA3 (Bawden).

Transcriptome Sequencing Reveals the Genome Sequence of Pea Early Browning Virus from a 29-Year-Old Faba Bean Sample.

Pea early browning virus (PEBV) is transmitted by soil-inhabiting trichodorid nematodes and via seeds. The transcriptome sequencing method, followed by de novo assembly, revealed the PEBV Libyan isolate LyV66-91 genome. Its RNA1 resembled that of UK isolate SP5 with 93.91% nucleotide identity, and its RNA2 had 63.32% nucleotide identity to that of Dutch isolate E116.

Genome Sequence and Phylogeny of a Bean Yellow Mosaic Virus Isolate Obtained from a 14-Year-Old Australian Lentil Sample.

Using RNA strand-specific sequencing followed by de novo assembly, a Bean yellow mosaic virus (BYMV) genome was obtained from a lentil sample (Aus14BY) collected in Victoria, Australia, in 2005. When compared with 51 BYMV genomes, it closely resembled the Western Australian isolate PN83A (Lupinus angustifolius), with 98.4% nucleotide identity.

The role of plant biosecurity in preventing and controlling emerging plant virus disease epidemics.

A number of research strategies have been initiated over the last decade to enhance plant biosecurity capacity at the pre-border, border and post-border frontiers. In preparation for emerging plant virus epidemics, diagnostic manuals for economically important plant viruses that threaten local industries have been developed and validated under local conditions. Contingency plans have also been prepared that provide guidelines to stakeholders on diagnostics, surveillance, survey strategies, epidemiology and pest risk analysis. Reference collections containing validated positive virus controls have been expanded to support a wide range of biosecurity sciences. Research has been conducted to introduce high throughput diagnostic capabilities and the design and development of advanced molecular techniques to detect virus genera. These diagnostic tools can be used by post entry quarantine agencies to detect known and unknown plant viral agents. Pre-emptive breeding strategies have also been initiated to protect plant industries if and when key exotic viruses become established in localized areas. With the emergence of free trade agreements between trading partners there is a requirement for quality assurance measures for pathogens, including viruses, which may occur in both the exporting and importing countries. These measures are required to ensure market access for the exporting country and also to minimize the risk of the establishment of a damaging virus epidemic in the importing country.

Validation of Genotyping by Sequencing Using Transcriptomics for Diversity and Application of Genomic Selection in Tetraploid Potato.

Potato is an important food crop due to its increasing consumption, and as a result, there is demand for varieties with improved production. However, the current status of breeding for improved varieties is a long process which relies heavily on phenotypic evaluation and dated molecular techniques and has little emphasis on modern genotyping approaches. Evaluation and selection before a cultivar is commercialized typically takes 10-15 years. Molecular markers have been developed for disease and pest resistance, resulting in initial marker-assisted selection in breeding. This study has evaluated and implemented a high-throughput transcriptome sequencing method for dense marker discovery in potato for the application of genomic selection. An Australian relevant collection of commercial cultivars was selected, and identification and distribution of high quality SNPs were examined using standard bioinformatic pipelines and a custom approach for the prediction of allelic dosage. As a result, a large number of SNP markers were identified and filtered to generate a high-quality subset that was then combined with historic phenotypic data to assess the approach for genomic selection. Genomic selection potential was predicted for highly heritable traits and the approach demonstrated advantages over the previously used technologies in terms of markers identified as well as costs incurred. The high-quality SNP list also provided acceptable genome coverage which demonstrates its applicability for much larger future studies. This SNP list was also annotated to provide an indication of function and will serve as a resource for the community in future studies. Genome wide marker tools will provide significant benefits for potato breeding efforts and the application of genomic selection will greatly enhance genetic progress.

Metagenomic Analysis Reveals a Nearly Complete Genome Sequence of Alfalfa Mosaic Virus from a Field Pea in Australia.

Here, we report the first nearly complete genome sequence of Alfalfa mosaic virus (AMV) obtained from a symptomatic field pea sample (Aus295) in Australia. Its genome RNA1 and RNA2 segments resembled those of the Argentinian isolate Manfredi, with 99.4% and 96.7% nucleotide (nt) identity, respectively; its RNA3 segment resembled that of Chinese isolate AMV-Gyn, with 99.6% nt identity.

Coding-Complete Sequences of Barley Virus G Isolates from Australia, Obtained from a 34-Year-Old and a 1-Year-Old Sample.

Coding-complete sequences of two barley virus G isolates from Australia were obtained from a 34-year-old oat sample (isolate Aus8) and a 1-year-old barley sample (isolate Aus17N). The Aus8 and Aus17N isolates share 96.3% nucleotide identity with each other and 95.7 to 95.8% nucleotide identity with the South Korean isolate Uiseong.

Characterization of Apricot pseudo-chlorotic leaf spot virus, A Novel Trichovirus Isolated from Stone Fruit Trees.

ABSTRACT A trichovirus closely related to Apple chlorotic leaf spot virus (ACLSV) was detected in symptomatic apricot and Japanese plum from Italy. The Sus2 isolate of this agent cross-reacted with anti-ACLSV polyclonal reagents but was not detected by broad-specificity anti- ACLSV monoclonal antibodies. It had particles with typical trichovirus morphology but, contrary to ACLSV, was unable to infect Chenopodium quinoa and C. amaranticolor. The sequence of its genome (7,494 nucleotides [nt], missing only approximately 30 to 40 nt of the 5' terminal sequence) and the partial sequence of another isolate were determined. The new virus has a genomic organization similar to that of ACLSV, with three open reading frames coding for a replication-associated protein (RNA-dependent RNA polymerase), a movement protein, and a capsid protein, respectively. However, it had only approximately 65 to 67% nucleotide identity with sequenced isolates of ACLSV. The differences in serology, host range, genome sequence, and phylogenetic reconstructions for all viral proteins support the idea that this agent should be considered a new virus, for which the name Apricot pseudo-chlorotic leaf spot virus (APCLSV) is proposed. APCLSV shows substantial sequence variability and has been recovered from various Prunus sources coming from seven countries, an indication that it is likely to have a wide geographical distribution.

Identification and Characterization of Banana Bract Mosaic Virus in India.

We have identified banana bract mosaic potyvirus (BBMV) in banana plants growing in the Coimbatore and Tiruchchirappalli regions of southern India based on symptomatology, particle morphology, sequence homology, and nucleic acid hybridization assays. Potyvirus-like particles typical of BBMV also were detected in sap dips from banana plants growing in Maharashtra State. Sequence comparisons of the C terminus of the coat protein-coding and 3' untranslated regions revealed that the Indian isolates of BBMV had greater than 96.6 and 97.2% homology with a Philippines isolate at the nucleotide and amino acid levels, respectively. BBMV-infected banana cultivars from the Coimbatore region showed the characteristic mosaic on the bract of the banana inflorescence. In contrast, infected plants growing in the Tiruchchirappalli region and Maharashtra State displayed symptoms similar to those associated with cucumber mosaic cucumovirus and not the characteristic bract mosaic symptom. These results indicate that BBMV is more widespread than previously thought.

Characterization and expression of the coat protein-coding region of banana bract mosaic potyvirus, development of diagnostic assays and detection of the virus in banana plants from five countries in southeast Asia.

We have sequenced the entire coat protein (CP)-coding region and 5' 162 nucleotides of the 3' untranslated region (UTR) of nine different isolates of banana bract mosaic virus (BBrMV) from five different countries. Further, we have sequenced the 3' 621 nucleotides of the NIb-coding region of a Philippines isolate. This is the first report of BBrMV in Thailand, Vietnam and Western Samoa. When the sequences of the CP-coding region and 3' UTR were compared to each other, variability of between 0.3% and 5.6%, and 0.3% and 4. 3%, was observed at the nucleotide and amino acid levels, respectively. Phylogenetic analysis of the BBrMV isolates did not reveal any relationship between the geographic location of the isolates. The BBrMV CP was expressed in Escherichia coli as a fusion protein and the purified recombinant protein was used to produce a high titre BBrMV-specific polyclonal antiserum. This antiserum was used to develop a F(ab')(2) indirect double antibody sandwich ELISA and compared with immuno-capture PCR (IC-PCR) and reverse transcription PCR (RT-PCR) assays for BBrMV detection. RT-PCR was shown to be the most sensitive test followed by ELISA and IC-PCR. http://link.springer. de/link/service/journals/00705/bibs/9144009/91441725.htm