Development of a Novel Tissue Blot Hybridization Chain Reaction for the Identification of Plant Viruses

Assays for the high throughput screening of crops for virus monitoring need to be quick, easy, and low cost. One method involves using tissue blot immunoassays (TBIA), where plant stems are blotted onto nitrocellulose membrane and screened with available antibodies against a range of viruses. TBIAs are inexpensive but limited by antibody availability and specificity. To circumvent the antibody limitations, we developed the tissue blot hybridization chain reaction (TB-HCR). As with TBIA, plant stems are blotted onto a nitrocellulose membrane, however, TB-HCR involves using nucleic acid probes instead of antibodies. We demonstrated for the first time that TB-HCR can be used for plant viruses by designing and testing probes against species from several virus genera including begomovirus, polerovirus, luteovirus, cucumovirus, and alfamovirus. We also explored different hairpin reporter methods such as biotin/streptavidin-AP and the Alexa Fluor-488 Fluorophore. TB-HCR has applications for low-cost diagnostics for large sample numbers, rapid diagnostic deployment for new viruses, and can be performed as a preliminary triage assay prior to downstream applications

A Rapid and Cost-Effective Identification of Invertebrate Pests at the Borders Using MinION Sequencing of DNA Barcodes

The rapid and accurate identification of invertebrate pests detected at the border is a challenging task. Current diagnostic methods used at the borders are mainly based on time consuming visual and microscopic examinations. Here, we demonstrate a rapid in-house workflow for DNA extraction, PCR amplification of the barcode region of the mitochondrial cytochrome oxidase subunit I (COI) gene and Oxford Nanopore Technologies (ONT) MinION sequencing of amplified products multiplexed after barcoding on ONT Flongle flow cells. A side-by-side comparison was conducted of DNA barcode sequencing-based identification and morphological identification of both large (>0.5 mm in length) and small (<0.5 mm in length) invertebrate specimens intercepted at the Australian border. DNA barcode sequencing results supported the morphological identification in most cases and enabled immature stages of invertebrates and their eggs to be identified more confidently. Results also showed that sequencing the COI barcode region using the ONT rapid sequencing principle is a cost-effective and field-adaptable approach for the rapid and accurate identification of invertebrate pests. Overall, the results suggest that MinION sequencing of DNA barcodes offers a complementary tool to the existing morphological diagnostic approaches and provides rapid, accurate, reliable and defendable evidence for identifying invertebrate pests at the border

Phytoplasma disease outbreaks in multiple crops in eastern Australia

Phytoplasma disease outbreaks were common and widespread in grain legume crops including mung bean (Vigna radiata), soybean (Glycine max) and peanut (Arachis hypogaea) in late 2016 and early 2017. Mung bean crops were affected in all major production areas spanning a distance of over 1,200 km from north to south with numerous crops having greater than 40% disease incidence near Dalby. Several soybean crops from Cecil Plains were also affected by phytoplasma in late autumn 2016. Almost 100% of plants were affected in some paddocks and virtually no yield. During the 2016/17 summer, some vegetable and pawpaw crops also had damaging outbreaks of disease. Several tomato crops in south-east Queensland had incidences of “big-bud” disease in excess of 50%. Capsicum and eggplant crops also had phytoplasma at higher than normal levels. In coastal Queensland, pawpaw (Carica papaya) crops had about 30% of plants affected by yellow crinkle disease but up to 80% in some younger plants. The P1/P7 region of the 16S gene was amplified from several different crop types. Resulting sequences had close to 100% nucleotide identity to pigeon pea little leaf phytoplasma, Candidatus Phytoplasma aurantifolia (16Sr-II group), which has been previously reported in Australia from pigeon pea (Canjanus cajan) and stylosanthes. A known leaf hopper vector of phytoplasma, Orosius orientalis, was collected from some affected mung bean crops but it is not certain it was associated with these disease outbreaks. To our knowledge, this is the most significant, widespread outbreak of phytoplasma in broad acre crops to occur in this region of Australia. It is unclear what the underlying reasons are for this sudden increase in disease incidence. We are continuing further studies to determine: the diversity of phytoplasma across crop and weed hosts and geographical range, which insect species are vectors, and possible management options

Genomic variation in pepper vein yellows viruses in Australia, including a new putative variant, PeVYV-10

Since the first identification and full sequence of the polerovirus pepper vein yellows virus in Australia in 2016, virus surveys of crops and weeds have sporadically identified PeVYV in different hosts and locations. Genomic comparisons of 14 PeVYV-like isolates using RT-PCR products spanning the 3’ end of the RdRp region (ORF 2), the intergenic region, ORF 3a, ORF 4, and ORF 3 (1388 nt) showed that four of the PeVYV isolates might be a new variant or PeVYV-like virus. From six PeVYV-positive plants, eight PeVYV-like sequences were obtained by high-throughput sequencing, as two hosts, 5352 and 5634, contained two slightly different PeVYV-like isolates. Three of the PeVYV-like isolates were most closely related to PeVYV-6 and PeVYV-5, and two isolates were closely related to PeVYV-9 and PeVYV-2. The other three isolates shared only 69-74% nucleotide sequence identity across the whole genome with any of the other PeVYVs, despite sharing 73-98%, 87-91%, and 84-87% amino acid sequence identity in ORF 3a, ORF 3, and the RdRp (ORF 2), respectively, suggesting that this virus is a new PeVYV-like virus, which we have tentatively called PeVYV-10. This is also the first report of a PeVYV-like virus infecting garlic