Partial biological and molecular characterization of a novel citrivirus from Nandina domestica.

We report the complete genome sequence of a novel virus isolated from Nandina domestica 'Firepower' in Auckland, New Zealand. It was mechanically transmitted to Nicotiana species, although all of these infections were symptomless. The complete genome of the new virus is 8892 nucleotides (nt) long, excluding the 3' poly(A) tail, contains three open reading frames (ORF), and is most closely related to citrus leaf blotch virus (CLBV) Actinidia isolate (CLBV-Act; 72% nt sequence identity), a member of the genus Citrivirus. Replicase and coat proteins, encoded by genome ORFs 1 and 3 respectively, shared 81-83% and 76-79% amino acid (aa) sequence identity, respectively, with CLBV-Act. Computer-based analysis suggests that this novel virus is the result of recombination between CLBV-Act and an unknown virus, highlighting the importance of this phenomenon for betaflexivirus evolution.

Characterization of the bacterial communities of psyllids associated with Rutaceae in Bhutan by high throughput sequencing.

BACKGROUND: Several plant-pathogenic bacteria are transmitted by insect vector species that often also act as hosts. In this interface, these bacteria encounter plant endophytic, insect endosymbiotic and other microbes. Here, we used high throughput sequencing to examine the bacterial communities of five different psyllids associated with citrus and related plants of Rutaceae in Bhutan: Diaphorina citri, Diaphorina communis, Cornopsylla rotundiconis, Cacopsylla heterogena and an unidentified Cacopsylla sp. RESULTS: The microbiomes of the psyllids largely comprised their obligate P-endosymbiont 'Candidatus Carsonella ruddii', and one or two S-endosymbionts that are fixed and specific to each lineage. In addition, all contained Wolbachia strains; the Bhutanese accessions of D. citri were dominated by a Wolbachia strain first found in American isolates of D. citri, while D. communis accessions were dominated by the Wolbachia strain, wDi, first detected in D. citri from China. The S-endosymbionts from the five psyllids grouped with those from other psyllid taxa; all D. citri and D. communis individuals contained sequences matching 'Candidatus Profftella armatura' that has previously only been reported from other Diaphorina species, and the remaining psyllid species contained OTUs related to unclassified Enterobacteriaceae. The plant pathogenic 'Candidatus Liberibacter asiaticus' was found in D. citri but not in D. communis. Furthermore, an unidentified 'Candidatus Liberibacter sp.' occurred at low abundance in both Co. rotundiconis and the unidentified Cacopsylla sp. sampled from Zanthoxylum sp.; the status of this new liberibacter as a plant pathogen and its potential plant hosts are currently unknown. The bacterial communities of Co. rotundiconis also contained a range of OTUs with similarities to bacteria previously found in samples taken from various environmental sources. CONCLUSIONS: The bacterial microbiota detected in these Bhutanese psyllids support the trends that have been seen in previous studies: psyllids have microbiomes largely comprising their obligate P-endosymbiont and one or two S-endosymbionts. In addition, the association with plant pathogens has been demonstrated, with the detection of liberibacters in a known host, D. citri, and identification of a putative new species of liberibacter in Co. rotundiconis and Cacopsylla sp.

The complete nucleotide sequence and genome organisation of a novel member of the family Betaflexiviridae from Actinidia chinensis.

We report the complete genome sequence of a novel virus, tentatively named "actinidia seed-borne latent virus" (ASbLV), isolated from Actinidia chinensis in Auckland, New Zealand. The complete genome of ASbLV is 8,192 nucleotides long, excluding the 3' poly(A) tail, contains four open reading frames, and is most closely related to Caucasus prunus virus (56% nucleotide sequence identity), a member of the genus Prunevirus. Based on the demarcation criteria of the family Betaflexiviridae, ASbLV is a new member of the genus Prunevirus.

Comparison of the complete genome sequence of two closely related isolates of 'Candidatus Phytoplasma australiense' reveals genome plasticity.

BACKGROUND: 'Candidatus Phytoplasma australiense' is associated with at least nine diseases in Australia and New Zealand. The impact of this phytoplasma is considerable, both economically and environmentally. The genome of a NZ isolate was sequenced in an effort to understand its pathogenicity and ecology. Comparison with a closely related Australian isolate enabled us to examine mechanisms of genomic rearrangement. RESULTS: The complete genome sequence of a strawberry lethal yellows (SLY) isolate of 'Candidatus Phytoplasma australiense' was determined. It is a circular genome of 959,779 base pairs with 1126 predicted open reading frames. Despite being 80 kbp larger than another 'Ca. Phytoplasma australiense' isolate PAa, the variation between housekeeping genes was generally less than 1% at a nucleotide level. The difference in size between the two isolates was largely due to the number and size of potential mobile units (PMUs), which contributed to some changes in gene order. Comparison of the genomes of the two isolates revealed that the highly conserved 5' UTR of a putative DNA-directed RNA polymerase seems to be associated with insertion and rearrangement events. Two types of PMUs have been identified on the basis of the order of three to four conserved genes, with both PMUs appearing to have been present in the last common ancestor of 'Ca. Phytoplasma asteris' and 'Ca. Phytoplasma australiense'. Comparison with other phytoplasma genomes showed that modification methylases were, in general, species-specific. A putative methylase (xorIIM) found in 'Ca. Phytoplasma australiense' appeared to have no analogue in any other firmicute, and we believe has been introduced by way of lateral gene transfer. A putative retrostransposon (ltrA) analogous to that found in OY-M was present in both isolates, although all examples in PAa appear to be fragments. Comparative analysis identified highly conserved 5' and 3' UTR regions of ltrA, which may indicate how the gene is excised and inserted. CONCLUSIONS: Comparison of two assembled 'Ca. Phytoplasma australiense' genomes has shown they possess a high level of plasticity. This comparative analysis has yielded clues as to how rearrangements occur, and the identification of sets of genes that appear to be associated with these events.

Application of Oxford Nanopore Technology to Plant Virus Detection.

The adoption of Oxford Nanopore Technologies (ONT) sequencing as a tool in plant virology has been relatively slow despite its promise in more recent years to yield large quantities of long nucleotide sequences in real time without the need for prior amplification. The portability of the MinION and Flongle platforms combined with lowering costs and continued improvements in read accuracy make ONT an attractive method for both low- and high-scale virus diagnostics. Here, we provide a detailed step-by-step protocol using the ONT Flongle platform that we have developed for the routine application on a range of symptomatic post-entry quarantine and domestic surveillance plant samples. The aim of this methods paper is to highlight ONT's feasibility as a valuable component to the diagnostician's toolkit and to hopefully stimulate other laboratories towards the eventual goal of integrating high-throughput sequencing technologies as validated plant virus diagnostic methods in their own right.

A New 'Candidatus Liberibacter' Species Associated with Diseases of Solanaceous Crops.

A new disease of glasshouse-grown tomato and pepper in New Zealand has resulted in plant decline and yield loss. Affected plants are characterized by spiky, chlorotic apical growth, curling or cupping of the leaves, and overall stunting. Transmission electron microscopy revealed the presence of phloem-limited bacterium-like organisms in symptomatic plants. The strategy used to identify the bacterium involved using specific prokaryote polymerase chain reaction (PCR) primers in combination with universal 16S rRNA primers. Sequence analysis of the 16S rRNA gene, the 16S/23S rRNA spacer region, and the rplKAJL-rpoBC operon revealed that the bacterium shared high identity with 'Candidatus Liberibacter' species. Phylogenetic analysis showed that the bacterium is distinct from the three citrus liberibacter species previously described and has been named 'Candidatus Liberibacter solanacearum'. This is the first report of a liberibacter naturally infecting a host outside the Rutaceae family. A specific PCR primer pair was developed for its detection.

Phylogenetic Analysis of "Candidatus Phytoplasma australiense" Reveals Distinct Populations in New Zealand.

ABSTRACT The phytoplasma "Candidatus Phytoplasma australiense" has been reported from New Zealand and Australia, where it has been associated with a range of host plants, especially since the 1970s. Partial tuf gene sequences of 36 New Zealand (NZ) isolates from four different host genera revealed nine different variants, which clustered into two distinct groups without any obvious correlation with host or geographic region. Phylogenetic analysis of these sequences, together with those available from Australian isolates, revealed three distinct clades: one found solely in Australia, one found solely in NZ, and a third with representatives from both countries. These divisions are consistent with differences observed in the 16-23S rRNA internal transcribed spacer region; therefore, we conclude that they represent three distinct subgroups: tuf 1, tuf 2, and tuf 3. We estimated a time of divergence for the three clades based on a synonymous substitution rate calculated by comparing the complete tuf gene sequence from the Loofah witches'-broom phytoplasma and "Candidatus Phytoplasma australiense". Using a calibration date of 110 million years, the estimated time to a common ancestor for all clades (6 to 9 million years ago) suggests divergence during the Miocene, well after the geological separation of NZ and Australia.

Cosmid cloning and sample sequencing of the genome of the uncultivable mollicute, Western X-disease phytoplasma, using DNA purified by pulsed-field gel electrophoresis.

Western X-disease (WX) phytoplasma is an uncultivable, intracellular pathogen of plants and insects with an AT-rich genome of 670 kb. As part of the genome sequencing project of WX phytoplasma, we have cloned approximately 50% of its genome into the pcosRW2 cosmid vector using DNA purified from pulsed-field gels. One of the cosmid clones with an insert of 24.6 kb was sequenced, which along with the cosmid end sequences, represents 60 kb of unique sequence from the WX phytoplasma genome. The putative genes identified in this sequence represent a wide variety of functions and many had not been previously identified in a phytoplasma.

Phytoplasma plasmid DNA extraction.

Phytoplasma plasmids have generally been detected from DNA extracted from plants and insects using methods designed for the purification of total phytoplasma DNA. Methods include extraction from tissues that are high in phytoplasma titre, such as the phloem of plants, with the use of CsCl-bisbenzimide gradients that exploit the low G+C content of phytoplasma DNA. Many of the methods employed for phytoplasma purification have been described elsewhere in this book. Here we describe in detail two methods that are specifically aimed at isolating plasmid DNA.

'Candidatus Liberibacter solanacearum', associated with plants in the family Solanaceae.

A liberibacter (isolate NZ082226) was detected in a symptomatic tomato plant and subsequently in five other members of the family Solanaceae: capsicum, potato, tamarillo, cape gooseberry and chilli. Phylogenetic analyses of the 16S rRNA gene sequence, the deduced amino acid sequence of the rplJ gene and a partial nucleotide sequence of the beta operon indicated that isolate NZ082226 represents a novel candidate species of 'Candidatus Liberibacter', for which the name 'Candidatus Liberibacter solanacearum' is proposed.

Comparative analysis of the plasmids from two isolates of "Candidatus Phytoplasma australiense".

Two plasmids from the plant-pathogenic mollicute "Candidatus Phytoplasma australiense" were completely sequenced from two isolates derived from different plant hosts. Plasmid pPAPh2 (3607bp) was obtained from Phormium showing Phormium yellow leaf symptoms and pPASb11 (3635bp) from strawberry showing strawberry lethal yellows symptoms. The plasmids varied in their copy number and nucleotide sequence yet contained the same four open reading frames (ORFs). The deduced amino acid sequence derived from ORF1 shares similarity with hypothetical proteins encoded on the plasmids from onion yellows and beet leafhopper-transmitted virescence agent phytoplasmas. The deduced amino acid sequences of both ORF2 and ORF3 share similarity with functionally unknown proteins on the chromosome of onion yellows phytoplasma. An ORF with a similar sequence to ORF2 is also present on the chromosome of "Ca. P. australiense." The deduced amino acid sequence derived from ORF4 is most similar to replication proteins encoded by other phytoplasma plasmids and by geminiviruses, the only protein on the plasmids for which a putative function can be assigned. The identities of the deduced amino acid sequences of ORF1, ORF2, ORF3, and ORF4 between pPAPh2 and pPASb11 were 89, 68, 91, and 68%, respectively; the differences being consistent with the subgroup status of the parental phytoplasmas.

Sequence analysis of two plasmids from the phytoplasma beet leafhopper-transmitted virescence agent.

The complete nucleotide sequences of the two plasmids from the phytoplasma beet leafhopper-transmitted virescence agent (BLTVA) have been determined. The larger plasmid, pBLTVA-1, was 10 785 nt in length and contained 11 putative ORFs, almost all of which were duplicated or triplicated on the plasmid due to the presence of large repeated regions. The sequence contained a series of tandem repeats, the largest of which was 338 nt long. The sequences of ORFs 4 and 11 showed homology with the replication genes of plasmids from other phytoplasmas and from geminiviruses. ORF9, the only ORF present as a single copy, showed homology with DNA primase genes from bacterial chromosomes and contained the conserved zinc finger and topoisomerase/primase domains. None of the other eight ORFs showed homology with known sequences in the GenBank database. pBLTVA-2 was 2587 nt in length, and all of its sequence was nearly identical to sequences from pBLTVA-1, most of which spanned ORFs 10 and 11, including the 338 nt tandem repeat. Analysis of 30 strains of BLTVA showed that most of the 11 putative ORFs were present, but the size of the plasmids varied in these strains.