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.

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.

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.

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.

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.

Unusual occurrence of a DAG motif in the Ipomovirus Cassava brown streak virus and implications for its vector transmission.

Cassava is the main staple food for over 800 million people globally. Its production in eastern Africa is being constrained by two devastating Ipomoviruses that cause cassava brown streak disease (CBSD); Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), with up to 100% yield loss for smallholder farmers in the region. To date, vector studies have not resulted in reproducible and highly efficient transmission of CBSV and UCBSV. Most virus transmission studies have used Bemisia tabaci (whitefly), but a maximum of 41% U/CBSV transmission efficiency has been documented for this vector. With the advent of next generation sequencing, researchers are generating whole genome sequences for both CBSV and UCBSV from throughout eastern Africa. Our initial goal for this study was to characterize U/CBSV whole genomes from CBSD symptomatic cassava plants sampled in Kenya. We have generated 8 new whole genomes (3 CBSV and 5 UCBSV) from Kenya, and in the process of analyzing these genomes together with 26 previously published sequences, we uncovered the aphid transmission associated DAG motif within coat protein genes of all CBSV whole genomes at amino acid positions 52-54, but not in UCBSV. Upon further investigation, the DAG motif was also found at the same positions in two other Ipomoviruses: Squash vein yellowing virus (SqVYV), Coccinia mottle virus (CocMoV). Until this study, the highly-conserved DAG motif, which is associated with aphid transmission was only noticed once, in SqVYV but discounted as being of minimal importance. This study represents the first comprehensive look at Ipomovirus genomes to determine the extent of DAG motif presence and significance for vector relations. The presence of this motif suggests that aphids could potentially be a vector of CBSV, SqVYV and CocMov. Further transmission and ipomoviral protein evolutionary studies are needed to confirm this hypothesis.

First Complete Genome Sequence of Cucumber green mottle mosaic virus Isolated from Australia.

We present here the first complete genome sequence of the tobamovirus Cucumber green mottle mosaic virus (CGMMV) from Australia, obtained from an infected cucumber plant. Compared with other CGMMV genomes, its closest nucleotide identities were 99.6% to KP772568, 99.3% to KF155229, and 99.1% to DQ767631 from Canada, Israel, and India, respectively.

Zucchini yellow mosaic virus Populations from East Timorese and Northern Australian Cucurbit Crops: Molecular Properties, Genetic Connectivity, and Biosecurity Implications.

Zucchini yellow mosaic virus (ZYMV) isolates from cucurbit crops growing in northern Australia and East Timor were investigated to establish possible genetic connectivity between crop viruses in Australia and Southeast Asia. Leaves from symptomatic plants of pumpkin (Cucurbita moschata and C. maxima), melon (Cucumis melo), and zucchini (C. pepo) were sampled near Broome, Darwin, and Kununurra in northern Australia. Leaves from symptomatic plants of cucumber (C. sativus) and pumpkin sampled in East Timor were sent to Australia on FTA cards. These samples were subjected to high-throughput sequencing and 15 complete new ZYMV genomic sequences obtained. When their nucleotide sequences were compared with those of 48 others from GenBank, the East Timorese and Kununurra sequences (three per location) and single earlier sequences from Singapore and Reunion Island were all in major phylogroup B. The seven Broome and two Darwin sequences were in minor phylogroups I and II, respectively, within larger major phylogroup A. When coat protein (CP) nucleotide sequences from the 15 new genomes and 47 Australian isolates sequenced previously were compared with 331 other CP sequences, the closest genetic match for a sequence from Kununurra was with an East Timorese sequence (95.5% nucleotide identity). Analysis of the 63 complete genomes found firm recombination events in 12 (75%) and 2 (4%) sequences from northern Australia or Southeast Asia versus the rest of the world, respectively; therefore, the formers' high recombination frequency might reflect adaptation to tropical conditions. Both parents of the recombinant Kununurra sequence were East Timorese. Phylogenetic analysis, nucleotide sequence identities, and recombination analysis provided clear evidence of genetic connectivity between sequences from Kununurra and East Timor. Inoculation of a Broome isolate to zucchini and watermelon plants reproduced field symptoms observed in northern Australia. This research has important biosecurity implications over entry of damaging viral crop pathogens not only into northern Australia but also moving between Australia's different agricultural regions.

Cassava brown streak virus has a rapidly evolving genome: implications for virus speciation, variability, diagnosis and host resistance.

Cassava is a major staple food for about 800 million people in the tropics and sub-tropical regions of the world. Production of cassava is significantly hampered by cassava brown streak disease (CBSD), caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). The disease is suppressing cassava yields in eastern Africa at an alarming rate. Previous studies have documented that CBSV is more devastating than UCBSV because it more readily infects both susceptible and tolerant cassava cultivars, resulting in greater yield losses. Using whole genome sequences from NGS data, we produced the first coalescent-based species tree estimate for CBSV and UCBSV. This species framework led to the finding that CBSV has a faster rate of evolution when compared with UCBSV. Furthermore, we have discovered that in CBSV, nonsynonymous substitutions are more predominant than synonymous substitution and occur across the entire genome. All comparative analyses between CBSV and UCBSV presented here suggest that CBSV may be outsmarting the cassava immune system, thus making it more devastating and harder to control.

A proposal to rationalize within-species plant virus nomenclature: benefits and implications of inaction.

Current approaches used to name within-species, plant virus phylogenetic groups are often misleading and illogical. They involve names based on biological properties, sequence differences and geographical, country or place-association designations, or any combination of these. This type of nomenclature is becoming increasingly unsustainable as numbers of sequences of the same virus from new host species and different parts of the world increase. Moreover, this increase is accelerating as world trade and agriculture expand, and climate change progresses. Serious consequences for virus research and disease management might arise from incorrect assumptions made when current within-species phylogenetic group names incorrectly identify properties of group members. This could result in development of molecular tools that incorrectly target dangerous virus strains, potentially leading to unjustified impediments to international trade or failure to prevent such strains being introduced to countries, regions or continents formerly free of them. Dangerous strains might be missed or misdiagnosed by diagnostic laboratories and monitoring programs, and new cultivars with incorrect strain-specific resistances released. Incorrect deductions are possible during phylogenetic analysis of plant virus sequences and errors from strain misidentification during molecular and biological virus research activities. A nomenclature system for within-species plant virus phylogenetic group names is needed which avoids such problems. We suggest replacing all other naming approaches with Latinized numerals, restricting biologically based names only to biological strains and removing geographically based names altogether. Our recommendations have implications for biosecurity authorities, diagnostic laboratories, disease-management programs, plant breeders and researchers.

Plant virology and next generation sequencing: experiences with a Potyvirus.

Next generation sequencing is quickly emerging as the go-to tool for plant virologists when sequencing whole virus genomes, and undertaking plant metagenomic studies for new virus discoveries. This study aims to compare the genomic and biological properties of Bean yellow mosaic virus (BYMV) (genus Potyvirus), isolates from Lupinus angustifolius plants with black pod syndrome (BPS), systemic necrosis or non-necrotic symptoms, and from two other plant species. When one Clover yellow vein virus (ClYVV) (genus Potyvirus) and 22 BYMV isolates were sequenced on the Illumina HiSeq2000, one new ClYVV and 23 new BYMV sequences were obtained. When the 23 new BYMV genomes were compared with 17 other BYMV genomes available on Genbank, phylogenetic analysis provided strong support for existence of nine phylogenetic groupings. Biological studies involving seven isolates of BYMV and one of ClYVV gave no symptoms or reactions that could be used to distinguish BYMV isolates from L. angustifolius plants with black pod syndrome from other isolates. Here, we propose that the current system of nomenclature based on biological properties be replaced by numbered groups (I-IX). This is because use of whole genomes revealed that the previous phylogenetic grouping system based on partial sequences of virus genomes and original isolation hosts was unsustainable. This study also demonstrated that, where next generation sequencing is used to obtain complete plant virus genomes, consideration needs to be given to issues regarding sample preparation, adequate levels of coverage across a genome and methods of assembly. It also provided important lessons that will be helpful to other plant virologists using next generation sequencing in the future.

Split personality of a Potyvirus: to specialize or not to specialize?

Bean yellow mosaic virus (BYMV), genus Potyvirus, has an extensive natural host range encompassing both dicots and monocots. Its phylogenetic groups were considered to consist of an ancestral generalist group and six specialist groups derived from this generalist group during plant domestication. Recombination was suggested to be playing a role in BYMV's evolution towards host specialization. However, in subsequent phylogenetic analysis of whole genomes, group names based on the original hosts of isolates within each of them were no longer supported. Also, nine groups were found and designated I-IX. Recombination analysis was conducted on the complete coding regions of 33 BYMV genomes and two genomes of the related Clover yellow vein virus (CYVV). This analysis found evidence for 12 firm recombination events within BYMV phylogenetic groups I-VI, but none within groups VII-IX or CYVV. The greatest numbers of recombination events within a sequence (two or three each) occurred in four groups, three which formerly constituted the single ancestral generalist group (I, II and IV), and group VI. The individual sequences in groups III and V had one event each. These findings with whole genomes are consistent with recombination being associated with expanding host ranges, and call into question the proposed role of recombination in the evolution of BYMV, where it was previously suggested to play a role in host specialization. Instead, they (i) indicate that recombination explains the very broad natural host ranges of the three BYMV groups which infect both monocots and dicots (I, II, IV), and (ii) suggest that the three groups with narrow natural host ranges (III, V, VI) which also showed recombination now have the potential to reduce host specificity and broaden their natural host ranges.

A proposal to help resolve the disagreement between naming of potato virus Y strain groups defined by resistance phenotypes and those defined by sequencing.

The complete coat protein (CP) nucleotide sequences of nine historical (1943-1984) potato virus Y (PVY) isolates belonging to resistance strain groupings Y(C), Y(Z) or Y(O), and nine new Australian isolates from potato and tomato were compared with those of 85 others. New potato isolate BL was in resistance group Y(O). On phylogenetic analysis, the historical potato isolates fitted within phylogenetic groups C1 or C2 (Y(C)), 'Y(O)' (Y(Z), Y(O)) or N-Wi (Y(Z)), while the new isolates were in phylogenetic groups C1 (tomato) or 'Y(O)' (potato, tomato). Substitution of the designation (Y(Q)) for the current phylogenetic 'Y(O)' grouping is proposed for consideration.