A metagenomic investigation of phytoplasma diversity in Australian vegetable growing regions.

In this study, metagenomic sequence data was used to investigate the phytoplasma taxonomic diversity in vegetable-growing regions across Australia. Metagenomic sequencing was performed on 195 phytoplasma-positive samples, originating either from historic collections (n=46) or during collection efforts between January 2015 and June 2022 (n=149). The sampled hosts were classified as crop (n=155), weed (n=24), ornamental (n=7), native plant (n=6), and insect (n=3) species. Most samples came from Queensland (n=78), followed by Western Australia (n=46), the Northern Territory (n=32), New South Wales (n=17), and Victoria (n=10). Of the 195 draft phytoplasma genomes, 178 met our genome criteria for comparison using an average nucleotide identity approach. Ten distinct phytoplasma species were identified and could be classified within the 16SrII, 16SrXII (PCR only), 16SrXXV, and 16SrXXXVIII phytoplasma groups, which have all previously been recorded in Australia. The most commonly detected phytoplasma taxa in this study were species and subspecies classified within the 16SrII group (n=153), followed by strains within the 16SrXXXVIII group ('Ca. Phytoplasma stylosanthis'; n=6). Several geographic- and host-range expansions were reported, as well as mixed phytoplasma infections of 16SrII taxa and 'Ca. Phytoplasma stylosanthis'. Additionally, six previously unrecorded 16SrII taxa were identified, including five putative subspecies of 'Ca. Phytoplasma australasiaticum' and a new putative 16SrII species. PCR and sequencing of the 16S rRNA gene was a suitable triage tool for preliminary phytoplasma detection. Metagenomic sequencing, however, allowed for higher-resolution identification of the phytoplasmas, including mixed infections, than was afforded by only direct Sanger sequencing of the 16S rRNA gene. Since the metagenomic approach theoretically obtains sequences of all organisms in a sample, this approach was useful to confirm the host family, genus, and/or species. In addition to improving our understanding of the phytoplasma species that affect crop production in Australia, the study also significantly expands the genomic sequence data available in public sequence repositories to contribute to phytoplasma molecular epidemiology studies, revision of taxonomy, and improved diagnostics.

High-quality full genome assembly of historic Xylella fastidiosa strains from ICMP collection using a hybrid sequencing approach.

High-quality complete genomes of five Xylella fastidiosa strains were assembled by combining Nanopore and Illumina sequencing data. Among these, International Collection of Micro-organisms from Plants (ICMP) 8731, ICMP 8742 and ICMP 8745 belong to subspecies fastidiosa while ICMP 8739 and ICMP 8740 were determined as subspecies multiplex. The strains were further classified into sequence types.

Whole mitochondrial genomes reveal the relatedness of the browsing ant incursions in Australia.

Global trade and human movements outspread animal species, for example ants, from their native habitats to new areas. This causes biosecurity concerns because an exotic ant might have adverse impacts on agriculture, the environment, or health; thus, incurring economic losses. The browsing ant, Lepisiota frauenfeldi, was first detected in 2013 at the Perth Airport. Since then, more discrete browsing ant infestations have been found in Perth and at the Ports of Darwin and Brisbane. This exotic ant has been deemed a significant pest in Australia and eradication efforts are underway. However, tackling this invasion requires an understanding of how these infestations are related. Are they same or separate or a combination of both? Here, we carried out a phylogenetic analysis using high-throughput sequencing data to determine their relatedness. Our results showed that each interstate incursion was separate. Furthermore, the Western Australian incursions might have two introductions. These findings are critical in devising effective biosecurity measures. However, we discovered that this information could only be revealed by analysing the whole mitochondrial genome; not by a single mitochondrial gene as typically done for species identification. Here, we sequenced 51 whole mitogenomes including three of its congener L. incisa for the first time, for tracing future infestations.

Genome Characterisation of the CGMMV Virus Population in Australia-Informing Plant Biosecurity Policy.

The detection of cucumber green mottle mosaic (CGMMV) in the Northern Territory (NT), Australia, in 2014 led to the introduction of strict quarantine measures for the importation of cucurbit seeds by the Australian federal government. Further detections in Queensland, Western Australia (WA), New South Wales and South Australia occurred in the period 2015-2020. To explore the diversity of the current Australian CGMMV population, 35 new coding sequence complete genomes for CGMMV isolates from Australian incursions and surveys were prepared for this study. In conjunction with published genomes from the NT and WA, sequence, phylogenetic, and genetic variation and variant analyses were performed, and the data were compared with those for international CGMMV isolates. Based on these analyses, it can be inferred that the Australian CGMMV population resulted from a single virus source via multiple introductions.

Using Genomics to Design a Pathovar-Specific Loop-Mediated Isothermal Amplification (LAMP) Assay, for the Improved Detection of Xanthomonas citri pv. citri.

The ability to swiftly respond to pathogen incursions relies heavily on fast and accurate diagnostics. Current published assays for citrus bacterial canker do not target Xanthomonas citri pv. citri, the causative agent, with high specificity when testing Australian samples. While the current diagnostics are useful in countries where canker is endemic, the detection of canker in Australia requires an emergency response. Close relatives to X. citri pv. citri found in Australia may generate false positives with the current recommended diagnostic assays. Therefore, we developed a more specific detection tool for citrus bacterial canker to provide greater diagnostic confidence for surveillance and eradication efforts. We used genomic comparisons of 161 Xanthomonad genomes and identified and confirmed genomic regions specific for X. citri pv. citri by performing local alignments of unique regions to reference genomes. We then developed loop-mediated isothermal amplification primers and validated them against a panel of 190 isolates to confirm specificity. Our diagnostic assay showed 100% corroboration with the concurrently developed multiplex primers and represents an improved diagnostic method capable of effective citrus bacterial canker identification.

Biology and genetic diversity of phasey bean mild yellows virus, a common virus in legumes in Australia.

This study examined the natural and experimental host range and aphid and graft transmission of the tentative polerovirus phasey bean mild yellows virus (PBMYV). Eleven complete coding sequences from PBMYV isolates were determined from a range of hosts and locations. We found two genetically distinct variants of PBMYV. PBMYV-1 was the originally described variant, and PBMYV-2 had a large putative recombination in open reading frame 5 such that PBMYV-1 and PBMYV-2 shared only 65-66% amino acid sequence identity in the P5 protein. The virus was transmitted by a clonal colony of cowpea aphids (Aphis craccivora) and by grafting with infected scions but was not transmitted by a clonal colony of green peach aphids (Myzus persicae). PBMYV was found in natural infections in 11 host species with a range of symptoms and severity, including seven important grain legume crops from across a wide geographic area in Australia. PBMYV was common and widespread in the tropical weed phasey bean (Macroptilium lathyroides), but it is likely that there are other major alternative hosts for the virus in temperate regions of Australia. The experimental host range of PBMYV included the Fabaceae hosts chickpea (Cicer arietinum), faba bean (Vicia faba), pea (Pisum sativum), and phasey bean, but transmissions failed to infect several other members of the families Asteraceae, Cucurbitaceae, Fabaceae and Solanaceae. PBMYV was commonly found in grain legume crops in eastern and western Australia, sometimes at greater than 90% incidence. This new knowledge about PBMYV warrants further assessments of its economic impact on important grain legume crops.

Genetic diversity and recombination between turnip yellows virus strains in Australia.

Disease outbreaks caused by turnip yellows virus (TuYV), a member of the genus Polerovirus, family Luteoviridae, regularly occur in canola and pulse crops throughout Australia. To understand the genetic diversity of TuYV for resistance breeding and management, genome sequences of 28 TuYV isolates from different hosts and locations were determined using high-throughput sequencing (HTS). We aimed to identify the parts of the genome that were most variable and clarify the taxonomy of viruses related to TuYV. Poleroviruses contain seven open reading frames (ORFs): ORF 0-2, 3a, and 3-5. Phylogenetic analysis based on the genome sequences, including isolates of TuYV and brassica yellows virus (BrYV) from the GenBank database, showed that most genetic variation among isolates occurred in ORF 5, followed by ORF 0 and ORF 3a. Phylogenetic analysis of ORF 5 revealed three TuYV groups; P5 group 1 and group 3 shared 45-49% amino acid sequence identity, and group 2 is a recombinant between the other two. Phylogenomic analysis of the concatenated ORFs showed that TuYV is paraphyletic with respect to BrYV, and together these taxa form a well-supported monophyletic group. Our results support the hypothesis that TuYV and BrYV belong to the same species and that the phylogenetic topologies of ORF 0, 3a and 5 are incongruent and may not be informative for species demarcation. A number of beet western yellow virus (BWYV)- and TuYV-associated RNAs (aRNA) were also identified by HTS for the first time in Australia.

Potato Virus A Isolates from Three Continents: Their Biological Properties, Phylogenetics, and Prehistory.

Forty-seven potato virus A (PVA) isolates from Europe, Australia, and South America's Andean region were subjected to high-throughput sequencing, and 46 complete genomes from Europe (n = 9), Australia (n = 2), and the Andes (n = 35) obtained. These and 17 other genomes gave alignments of 63 open reading frames 9,180 nucleotides long; 9 were recombinants. The nonrecombinants formed three tightly clustered, almost equidistant phylogroups; A comprised 14 Peruvian potato isolates; W comprised 37 from potato in Peru, Argentina, and elsewhere in the world; and T contained three from tamarillo in New Zealand. When five isolates were inoculated to a potato cultivar differential, three strain groups (= pathotypes) unrelated to phylogenetic groupings were recognized. No temporal signal was detected among the dated nonrecombinant sequences, but PVA and potato virus Y (PVY) are from related lineages and ecologically similar; therefore, "relative dating" was obtained using a single maximum-likelihood phylogeny of PVA and PVY sequences and PVY's well-supported 157 CE "time to most common recent ancestor". The PVA datings obtained were supported by several independent historical coincidences. The PVA and PVY populations apparently arose in the Andes approximately 18 centuries ago, and were taken to Europe during the Columbian Exchange, radiating there after the mid-19th century potato late blight pandemic. PVA's phylogroup A population diverged more recently in the Andean region, probably after new cultivars were bred locally using newly introduced Solanum tuberosum subsp. tuberosum as a parent. Such cultivars became widely grown, and apparently generated the A × W phylogroup recombinants. Phylogroup A, and its interphylogroup recombinants, might pose a biosecurity risk.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

First report of grapevine rupestris vein feathering virus in grapevine in Australia.

Grapevine rupestris vein feathering virus (GRVFV; tentative genus Marafivirus; family Tymoviridae ) was first detected from a Greek grapevine (Vitis vinifera), with asteroid mosaic-like symptoms (El Beaino et al. 2001; Ghanem-Sabanadzovic et al. 2003) and was also infected with grapevine fleck virus. GRVFV has been detected in the United States, South Africa, Canada, Spain, China, New Zealand, Brazil, Germany, Korea, Slovakia, Hungary and Pakistan (Cho et al. 2018; Mahmood et al. 2019).Transmission vectors are currently unknown. In 2018, nine grapevine samples were collected between May to July in South Australia (SA) and Western Australia (WA) (Table S1), were analysed by high-throughput sequencing (HTS) to characterise grapevine viruses in Australian vineyards. Total RNA or double stranded RNA was extracted from grapevine canes using RNeasy 96 QIAcube HT kit (Qiagen) with MacKenzie buffer (MacKenzie et al. 1997) or using CF-11 (Balijja et al. 2008). Libraries were prepared using the NEBNext® Ultra II RNA library Prep Kit (NEB) or TruSeq® Stranded mRNA Prep kit (Illumina) with Ribo-Zero®gold plant kit for ribosomal depletion (Illumina, San Diego, CA). Libraries were sequenced using Illumina Miseq (SA) or Hiseq (WA) technology with 2x300 (SA) or 2x100 (WA) paired end reads which were trimmed using Trim Galore! (0.4.0) or BBmap (38.20), respectively. De novo assembly, using the SPAdes (version 3.12.0) genome assembler with default settings, resulted in twelve near full length GRVFV genomes (6713-6737nt), eight sequences from the WA samples and four from the SA samples. WA samples 171 and 178 and SA sample BV each had two distinct GRVFV molecular variants. Variants 171-1 and 171-2 (GenBank accessions MT084811, MT084812) from sample 171 shared 83.39% nucleotide (nt) identity. Variants 178-1 and 178-2 (MT084813, MT084814) from sample 178 shared 83.54% nt identity. Variants BV6799 and BV8822 (MN974274, MN974275) from sample BV shared 82.85% nt identity. Only one GRVFV sequence was obtained from all other samples. The genome of SA isolate LC1 (MN974273) was confirmed by RT-PCR amplification and Sanger sequencing of overlapping genome regions. Tissue from the infected LC1 isolate has been deposited into the Victorian plant pathogen reference collection (VPRI accession No. 43698). When the genomes of all Australian isolates were compared, they had 78.94% to 94.37 % nt identity with each other. The SA isolates LC1, BV8822, BV6799, and SEL-L (MN974276), and the WA isolates 172 (MT084807), 179 (MT084808), 180 (MT084809), and 182 (MT084810) were most closely related to the Swiss isolate CHASS (KY513702; 82.87% to 85.46% nt identity). The WA isolates 171-1, 171-2, 178-1 and 178-2 were most closely related to the New Zealand isolate Ch8021 (MF000325; 83.21% to 93.87%). Grapevine leafroll-associated virus 1 (GLRaV-1), GLRaV-3, GLRaV-4 (strain 6 and 9), grapevine virus A, grapevine rupestris stem pitting associated virus, grapevine yellow speckle viroid 1 and hop stunt viroid were also identified in the sequencing data. This is the first report of GRVFV in Australia. All WA samples were collected during dormancy and symptoms were not observed. Sample LC1 from SA had Shiraz disease, the other SA samples were asymptomatic, and none had asteroid mosaic-like symptoms. Further research is required to determine its distribution and association with disease in Australia.

Host plant affiliations of aphid vector species found in a remote tropical environment.

The Ord River Irrigation Area (ORIA) produces annual crops during the dry season (April to October), and perennial crops all-year-round, and is located in tropical northwestern Australia. Sandalwood plantations cover 50 % of the ORIA's cropping area. Aphids cause major crop losses through transmission of viruses causing debilitating diseases and direct feeding damage. During 2016-2017, in both dry and wet seasons a total of 3320 leaf samples were collected from diverse types of sites on cultivated and uncultivated land and 1248 (38 %) of them were from aphid-colonized plants. In addition, aphids were found at 236 of 355 sampling sites. The 62 plant species sampled came from 23 families 19 of which contained aphid-colonized species. Aphid hosts included introduced weeds, Australian native plants, and volunteer or planted crop plants. Six aphid species were identified by light microscopy and CO1 gene sequencing, but there was no within species nucleotide sequence diversity. Aphis nerii, Hysteroneura setariae, Rhopalosiphum maidis and Schoutedenia ralumensis each colonized 1-3 plant species from a single plant family. A. craccivora colonized 14 species in five plant families. A. gossypii was the most polyphagous species colonizing 19 species in 11 plant families. A. gossypii, A. craccivora, A. nerii and S. ralumensis were found in both wet and dry seasons. Because of A. craccivora's prevalence and high incidences on understory weeds and host trees, sandalwood plantations were important reservoirs for aphid spread to wild and crop plant hosts growing in cultivated and uncultivated land. Alternative hosts growing in rural bushland, irrigation channel banks, vacant or fallow land, and orchard plantation understories also constituted significant aphid reservoirs. This study provides new knowledge of the ecology of aphid vector species not only in the ORIA but also in tropical northern Australia generally. It represents one of relatively few investigations on aphid ecology in tropical environments worldwide.

Genetic diversity and SNP's from the chloroplast coding regions of virus-infected cassava.

Cassava is a staple food crop in sub-Saharan Africa; it is a rich source of carbohydrates and proteins which currently supports livelihoods of more than 800 million people worldwide. However, its continued production is at stake due to vector-transmitted diseases such as Cassava mosaic disease and Cassava brown streak disease. Currently, the management and control of viral diseases in cassava relies mainly on virus-resistant cultivars of cassava. Thus, the discovery of new target genes for plant virus resistance is essential for the development of more cassava varieties by conventional breeding or genetic engineering. The chloroplast is a common target for plant viruses propagation and is also a potential source for discovering new resistant genes for plant breeding. Non-infected and infected cassava leaf samples were obtained from different locations of East Africa in Tanzania, Kenya and Mozambique. RNA extraction followed by cDNA library preparation and Illumina sequencing was performed. Assembling and mapping of the reads were carried out and 33 partial chloroplast genomes were obtained. Bayesian phylogenetic analysis from 55 chloroplast protein-coding genes of a dataset with 39 taxa was performed and the single nucleotide polymorphisms for the chloroplast dataset were identified. Phylogenetic analysis revealed considerable genetic diversity present in chloroplast partial genome among cultivated cassava of East Africa. The results obtained may supplement data of previously selected resistant materials and aid breeding programs to find diversity and achieve resistance for new cassava varieties.

Epidemiology of Zucchini yellow mosaic virus in cucurbit crops in a remote tropical environment.

In the remote Ord River Irrigation Area (ORIA) in tropical northwest Australia, severe Zucchini yellow mosaic virus (ZYMV) epidemics threaten dry season (April-October) cucurbit crops. In 2016-2017, wet season (November-March) sampling studies found a low incidence ZYMV infection in wild Cucumis melo and Citrullus lanatus var. citroides plants, and both volunteer and garden crop cucurbits. Such infections enable its persistence in the wet season, and act as reservoirs for its spread to commercial cucurbit crops during the dry season. Tests on 1019 samples belonging to 55 species from 23 non-cucurbitaceous plant families failed to detect ZYMV. It was also absent from wild cucurbit weeds within sandalwood plantations. The transmission efficiencies of a local isolate by five aphid species found in the ORIA were: 10 % (Aphis craccivora), 7% (A. gossypii), 4% (A. nerii), and 0% (Rhopalosiphum maidis and Hysteroneura setariae). In 2016-2017, in all-year-round trapping at five representative sites, numbers of winged aphids caught were greatest in July-August (i.e. mid growing season) but varied widely between trap sites reflecting local aphid host abundance and year. Apart from one localised exception in 2017, flying aphid numbers caught and ZYMV spread in data collection blocks during 2015-2017 resembled what occurred commercial cucurbit crops. When ZYMV spread from external infection sources into melon blocks, its predominant spread pattern consisted of 1 or 2 plant infection foci often occurring at their margins. In addition, when plants of 29 cucurbit cultivars were inoculated with an ORIA isolate and two other ZYMV isolates and the phenotypes elicited were compared, they resembled each other in overall virulence. However, depending upon isolate-cultivar combination, differences in symptom expression and severity occurred, and one isolate caused a systemic hypersensitive phenotype in honeydew melon cvs Estilo and Whitehaven. When the new genomic RNA sequences of 19 Australian isolates were analysed, all seven ORIA isolates fitted within ZYMV phylogroup B, which also included two from southwest Australia, whereas the remaining 10 isolates were all within minor phylogroups A-I or A-II. Based on previous research and the additional knowledge of ZYMV epidemic drivers established here, an integrated disease management strategy targeting ZYMV spread was devised for the ORIA's cucurbit industry.

Tree Lab: Portable genomics for Early Detection of Plant Viruses and Pests in Sub-Saharan Africa.

In this case study we successfully teamed the PDQeX DNA purification technology developed by MicroGEM, New Zealand, with the MinION and MinIT mobile sequencing devices developed by Oxford Nanopore Technologies to produce an effective point-of-need field diagnostic system. The PDQeX extracts DNA using a cocktail of thermophilic proteinases and cell wall-degrading enzymes, thermo-responsive extractor cartridges and a temperature control unit. This closed system delivers purified DNA with no cross-contamination. The MinIT is a newly released data processing unit that converts MinION raw signal output into nucleotide base called data locally in real-time, removing the need for high-specification computers and large file transfers from the field. All three devices are battery powered with an exceptionally small footprint that facilitates transport and setup. To evaluate and validate capability of the system for unbiased pathogen identification by real-time sequencing in a farmer's field setting, we analysed samples collected from cassava plants grown by subsistence farmers in three sub-Sahara African countries (Tanzania, Uganda and Kenya). A range of viral pathogens, all with similar symptoms, greatly reduce yield or destroy cassava crops. Eight hundred (800) million people worldwide depend on cassava for food and yearly income, and viral diseases are a significant constraint to its production. Early pathogen detection at a molecular level has great potential to rescue crops within a single growing season by providing results that inform decisions on disease management, use of appropriate virus-resistant or replacement planting. This case study presented conditions of working in-field with limited or no access to mains power, laboratory infrastructure, Internet connectivity and highly variable ambient temperature. An additional challenge is that, generally, plant material contains inhibitors of downstream molecular processes making effective DNA purification critical. We successfully undertook real-time on-farm genome sequencing of samples collected from cassava plants on three farms, one in each country. Cassava mosaic begomoviruses were detected by sequencing leaf, stem, tuber and insect samples. The entire process, from arrival on farm to diagnosis, including sample collection, processing and provisional sequencing results was complete in under 3 h. The need for accurate, rapid and on-site diagnosis grows as globalized human activity accelerates. This technical breakthrough has applications that are relevant to human and animal health, environmental management and conservation.

Application of Loop-Mediated Isothermal Amplification in an Early Warning System for Epidemics of an Externally Sourced Plant Virus.

Restricting Turnip yellows virus (TuYV) spread in canola (Brassica napus) crops often relies upon the application of systemic insecticides to protect young vulnerable plants from wide-scale green-peach aphid (GPA; Myzus persicae) colonization and subsequent virus infection. For these to be applied at the optimal time to ensure they prevent epidemics, growers would need to be forewarned of incoming viruliferous aphid migration and colonization. This study was conducted to field validate a loop-mediated isothermal amplification (LAMP) protocol designed to detect TuYV in aphids caught on traps and develop an early warning system for TuYV epidemics. Double-sided yellow sticky traps were deployed at 30 sites sown with canola over a two-year period in the south-west Australian grainbelt. Using LAMP, the percentage (%) of trap sides with TuYV-carrying aphids was measured and related to TuYV infection incidence in the adjacent crop. When TuYV was detected in aphids on >30% trap sides in a six-week period from pre-emergence to GS15 (five-leaf stage), TuYV reached >60% crop incidence by GS30 (beginning of stem elongation). Whereas, TuYV detection in aphids on ≤15% trap sides during this period was associated with ≤6% TuYV incidence by GS30. Furthermore, when large numbers of aphids, including GPA, were caught during this period but no TuYV was detected in them, minimal TuYV spread (≤5%) occurred in the crop by GS30. Therefore, the LAMP TuYV protocol can be used in an early warning system for TuYV epidemics by providing detection of initial viruliferous aphid migration into a canola crop before they establish colonies throughout the crop and spread virus. This would enable proactive, non-prophylactic, and thereby more effective systemic insecticide applications to minimize seed yield and quality losses due to early season TuYV infection.

First report of Cassava brown streak viruses on wild plant species in Mozambique.

Cassava brown streak disease (CBSD) caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) is the main constraint to cassava (Manihot esculenta Crantz) production in Mozambique. Using RT-PCR to amplify partial coat protein nucleotide sequences, we detected for the first time the occurrence of CBSV in two non-cassava perennial wild plant species: Zanha africana (Radlk.) Exell. and Trichodesma zeylanicum (Burm.f.) R.Br., that occur widely within and near cassava fields in Nampula, Zambezia, Niassa and Cabo Delgado provinces. In addition, we also detected CBSV and UCBSV in Manihot carthaginensis subsp. glaziovii (Müell-Arg.) Allem., a wild cassava relative. These findings were verified in biological assays through mechanical inoculation of CBSV to T. zeylanicum, albeit at low rates of infection. Phylogenetic analysis clustered the CBSV isolates from the non-cassava plant species with those from cultivated cassava, with high sequence homology among CBSV (91.0-99.6%) and with UCBSV (84-92%) isolates. These results provide definitive evidence of a wider host range for CBSV and UCBSV in Mozambique, indicating that these viruses are not restricted to cultivated cassava. Our findings are key to understanding the epidemiology of CBSD and will aid in the development of sustainable management strategies for the disease.

A metagenomic study of DNA viruses from samples of local varieties of common bean in Kenya.

Common bean (Phaseolus vulgaris L.) is the primary source of protein and nutrients in the majority of households in sub-Saharan Africa. However, pests and viral diseases are key drivers in the reduction of bean production. To date, the majority of viruses reported in beans have been RNA viruses. In this study, we carried out a viral metagenomic analysis on virus symptomatic bean plants. Our virus detection pipeline identified three viral fragments of the double-stranded DNA virus Pelargonium vein banding virus (PVBV) (family, Caulimoviridae, genus Badnavirus). This is the first report of the dsDNA virus and specifically PVBV in legumes to our knowledge. In addition two previously reported +ssRNA viruses the bean common mosaic necrosis virus (BCMNVA) (Potyviridae) and aphid lethal paralysis virus (ALPV) (Dicistroviridae) were identified. Bayesian phylogenetic analysis of the Badnavirus (PVBV) using amino acid sequences of the RT/RNA-dependent DNA polymerase region showed the Kenyan sequence (SRF019_MK014483) was closely matched with two Badnavirus viruses: Dracaena mottle virus (DrMV) (YP_610965) and Lucky bamboo bacilliform virus (ABR01170). Phylogenetic analysis of BCMNVA was based on amino acid sequences of the Nib region. The BCMNVA phylogenetic tree resolved two clades identified as clade (I and II). Sequence from this study SRF35_MK014482, clustered within clade I with other Kenyan sequences. Conversely, Bayesian phylogenetic analysis of ALPV was based on nucleotide sequences of the hypothetical protein gene 1 and 2. Three main clades were resolved and identified as clades I-III. The Kenyan sequence from this study (SRF35_MK014481) clustered within clade II, and nested within a sub-clade; comprising of sequences from China and an earlier ALPV sequences from Kenya isolated from maize (MF458892). Our findings support the use of viral metagenomics to reveal the nascent viruses, their viral diversity and evolutionary history of these viruses. The detection of ALPV and PVBV indicate that these viruses have likely been underreported due to the unavailability of diagnostic tools.

Evolutionary insights of Bean common mosaic necrosis virus and Cowpea aphid-borne mosaic virus.

Plant viral diseases are one of the major limitations in legume production within sub-Saharan Africa (SSA), as they account for up to 100% in production losses within smallholder farms. In this study, field surveys were conducted in the western highlands of Kenya with viral symptomatic leaf samples collected. Subsequently, next-generation sequencing was carried out to gain insights into the molecular evolution and evolutionary relationships of Bean common mosaic necrosis virus (BCMNV) and Cowpea aphid-borne mosaic virus (CABMV) present within symptomatic common bean and cowpea. Eleven near-complete genomes of BCMNV and two for CABMV were obtained from western Kenya. Bayesian phylogenomic analysis and tests for differential selection pressure within sites and across tree branches of the viral genomes were carried out. Three well-supported clades in BCMNV and one supported clade for CABMNV were resolved and in agreement with individual gene trees. Selection pressure analysis within sites and across phylogenetic branches suggested both viruses were evolving independently, but under strong purifying selection, with a slow evolutionary rate. These findings provide valuable insights on the evolution of BCMNV and CABMV genomes and their relationship to other viral genomes globally. The results will contribute greatly to the knowledge gap involving the phylogenomic relationship of these viruses, particularly for CABMV, for which there are few genome sequences available, and inform the current breeding efforts towards resistance for BCMNV and CABMV.

Phylogenomic relationship and evolutionary insights of sweet potato viruses from the western highlands of Kenya.

Sweet potato is a major food security crop within sub-Saharan Africa where 90% of Africa production occurs. One of the major limitations of sweet potato production are viral infections. In this study, we used a combination of whole genome sequences from a field isolate obtained from Kenya and those available in GenBank. Sequences of four sweet potato viruses: Sweet potato feathery mottle virus (SPFMV), Sweet potato virus C (SPVC), Sweet potato chlorotic stunt virus (SPCSV), Sweet potato chlorotic fleck virus (SPCFV) were obtained from the Kenyan sample. SPFMV sequences both from this study and from GenBank were found to be recombinant. Recombination breakpoints were found within the Nla-Pro, coat protein and P1 genes. The SPCSV, SPVC, and SPCFV viruses from this study were non-recombinant. Bayesian phylogenomic relationships across whole genome trees showed variation in the number of well-supported clades; within SPCSV (RNA1 and RNA2) and SPFMV two well-supported clades (I and II) were resolved. The SPCFV tree resolved three well-supported clades (I-III) while four well-supported clades were resolved in SPVC (I-IV). Similar clades were resolved within the coalescent species trees. However, there were disagreements between the clades resolved in the gene trees compared to those from the whole genome tree and coalescent species trees. However the coat protein gene tree of SPCSV and SPCFV resolved similar clades to the genome and coalescent species tree while this was not the case in SPFMV and SPVC. In addition, we report variation in selective pressure within sites of individual genes across all four viruses; overall all viruses were under purifying selection. We report the first complete genomes of SPFMV, SPVC, SPCFV, and a partial SPCSV from Kenya as a mixed infection in one sample. Our findings provide a snap shot on the evolutionary relationship of sweet potato viruses (SPFMV, SPVC, SPCFV, and SPCSV) from Kenya as well as assessing whether selection pressure has an effect on their evolution.

The first transcriptomes from field-collected individual whiteflies ( Bemisia tabaci, Hemiptera: Aleyrodidae): a case study of the endosymbiont composition.

Background:Bemisia tabaci species ( B. tabaci), or whiteflies, are the world's most devastating insect pests. They cause billions of dollars (US) of damage each year, and are leaving farmers in the developing world food insecure. Currently, all publically available transcriptome data for B. tabaci are generated from pooled samples, which can lead to high heterozygosity and skewed representation of the genetic diversity. The ability to extract enough RNA from a single whitefly has remained elusive due to their small size and technological limitations. Methods: In this study, we optimised a single whitefly RNA extraction procedure, and sequenced the transcriptome of four individual adult Sub-Saharan Africa 1 (SSA1) B. tabaci. Transcriptome sequencing resulted in 39-42 million raw reads. De novo assembly of trimmed reads yielded between 65,000-162,000 Contigs across B. tabaci transcriptomes. Results: Bayesian phylogenetic analysis of mitochondrion cytochrome I oxidase (mtCOI) grouped the four whiteflies within the SSA1 clade. BLASTn searches on the four transcriptomes identified five endosymbionts; the primary endosymbiont Portieraaleyrodidarum and four secondary endosymbionts: Arsenophonus, Wolbachia, Rickettsia, and Cardinium spp. that were predominant across all four SSA1 B. tabaci samples with prevalence levels of between 54.1 to 75%. Amino acid alignments of the NusG gene of P. aleyrodidarum for the SSA1 B. tabaci transcriptomes of samples WF2 and WF2b revealed an eleven amino acid residue deletion that was absent in samples WF1 and WF2a. Comparison of the protein structure of the NusG protein from P. aleyrodidarum in SSA1 with known NusG structures showed the deletion resulted in a shorter D loop. Conclusions: The use of field-collected specimens means time and money will be saved in future studies using single whitefly transcriptomes in monitoring vector and viral interactions. Our method is applicable to any small organism where RNA quantity has limited transcriptome studies.

The Biology and Phylogenetics of Potato virus S Isolates from the Andean Region of South America.

Biological characteristics of 11 Potato virus S (PVS) isolates from three cultivated potato species (Solanum spp.) growing in five Andean countries and 1 from Scotland differed in virulence depending on isolate and host species. Nine isolates infected Chenopodium quinoa systemically but two others and the Scottish isolate remained restricted to inoculated leaves; therefore, they belonged to biologically defined strains PVSA and PVSO, respectively. When nine wild potato species were inoculated, most developed symptomless systemic infection but Solanum megistacrolobum developed systemic hypersensitive resistance (SHR) with one PVSO and two PVSA isolates. Andean potato cultivars developed mostly asymptomatic primary infection but predominantly symptomatic secondary infection. In both wild and cultivated potato plants, PVSA and PVSO elicited similar foliage symptoms. Following graft inoculation, all except two PVSO isolates were detected in partially PVS-resistant cultivar Saco, while clone Snec 66/139-19 developed SHR with two isolates each of PVSA and PVSO. Myzus persicae transmitted all nine PVSA isolates but none of the three PVSO isolates. All 12 isolates were transmitted by plant-to-plant contact. In infective sap, all isolates had thermal inactivation points of 55 to 60°C. Longevities in vitro were 25 to 40 days with six PVSA isolates but less than 21 days for the three PVSO isolates. Dilution end points were 10-3 for two PVSO isolates but 10-4 to 10-6 with the other isolates. Complete new genome sequences were obtained from seven Andean PVS isolates; seven isolates from Africa, Australia, or Europe; and single isolates from S. muricatum and Arracacia xanthorhiza. These 17 new genomes and 23 from GenBank provided 40 unique sequences; however, 5 from Eurasia were recombinants. Phylogenetic analysis of the 35 nonrecombinants revealed three major lineages, two predominantly South American (SA) and evenly branched and one non-SA with a single long basal branch and many distal subdivisions. Using least squares dating and nucleotide sequences, the two nodes of the basal PVS trifurcation were dated at 1079 and 1055 Common Era (CE), the three midphylogeny nodes of the SA lineages at 1352, 1487, and 1537 CE, and the basal node to the non-SA lineage at 1837 CE. The Potato rough dwarf virus/Potato virus P (PVS/PRDV/PVP) cluster was sister to PVS and diverged 5,000 to 7,000 years ago. The non-SA PVS lineage contained 18 of 19 isolates from S. tuberosum subsp. tuberosum but the two SA lineages contained 6 from S. tuberosum subsp. andigena, 4 from S. phureja, 3 from S. tuberosum subsp. tuberosum, and 1 each from S. muricatum, S. curtilobum, and A. xanthorrhiza. This suggests that a potato-infecting proto-PVS/PRDV/PVP emerged in South America at least 5,000 years ago, became endemic, and diverged into a range of local Solanum spp. and other species, and one early lineage spread worldwide in potato. Preventing establishment of the SA lineages is advised for all countries still without them.