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.

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.[ ] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

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 single-step 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 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 set up. To evaluate and validate capability of the system for unbiased pathogen identification by realtime 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 completely destroy cassava crops. 800 million people worldwide depend on cassava for food and yearly income, and viral diseases are a significant constraint on its production ( https://cassavavirusactionproject.com ). 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 4 hours. 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.

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.

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.