The Establishment and Spread of a Newly Introduced Begomovirus in a Dry Tropical Environment Using Tomato Yellow Leaf Curl Virus as a Case Study

Early detection of tomato yellow leaf curl virus (TYLCV) in a previously unaffected tomato production district in Australia allowed its spread to be evaluated spatially and temporally. The population dynamics of the TYLCV vector, Bemisia argentifolii (silverleaf whitefly, SLW), were also evaluated. The district is a dry tropical environment with a clear break to commercial production during the summer wet season. The incidence of TYLCV within crops and its prevalence through the district was influenced by weather, location, vector movements, and the use of Ty-1 virus-resistant hybrids. Rainfall had an important influence, with late summer and early autumn rain suppressing the levels of SLW and, by contrast, a dry summer supporting faster population growth. The use of Ty-1 hybrids appears to have reduced the incidence of TYLCV in this district. There was limited use of Ty-1 hybrids during 2013, and by season end, crops had moderate levels of SLW and high virus incidence. The 2015 and early 2016 season had high SLW populations, but TYLCV incidence was lower than in 2013, possibly due to the widespread adoption of the Ty-1 hybrids reducing virus spread. This study provides valuable epidemiology data for future incursions of begomoviruses, and other viruses spread by SLW

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

Comparative genomics and genomic diversity of Pseudomonas syringae clade 2b-a in Australia

A zucchini disease outbreak with unusual symptoms associated with Pseudomonas syringae clade 2b was identified in Bundaberg, Australia during autumn 2016. To investigate the genetic diversity of the 11 Australian isolates obtained from the outbreak, the genomes were compared to the publicly available P. syringae strains in phylogroup 2

Cowpea mild mottle virus, a sometimes problem for French bean crops

A carlavirus, closely related to cowpea mild mottle virus (CPMMV) and spread by silverleaf whitefly (SLW) was reported affecting fresh market beans in a major Australian growing district in 2016. Further investigations of this virus were completed through regular surveys of crops, weeds and SLW in this district from 2016–2019. Sequencing of the 3'end of the virus genome from a range of samples detected four variants, referred to as CPMMV:A:FB5288 and CPMMV:A:S1 to S3. The distribution of these four variants in survey samples showed the dominant variant in French bean crops as CPMMV:A:FB5288. The surveys also showed disease impacts were limited to autumn and varied over time. This variation is attributed to the influence of rainfall on adult insect vector levels. The experimental host range of CPMMV:A:FB5288 was shown to be limited to the Phaseoleae plant tribe and included the Australian native species, Glycine canescens. French bean varieties showed a range of susceptibilities to this dominant sequence variant from highly tolerant to very susceptible. The tolerant varieties provide the local industry with some options for disease management where previously there were none. Genetic diversity studies further highlight the need for taxonomic reform of the species referred to as CPMMV

Detection and Distribution of Viruses Infecting Garlic Crops in Australia

The distribution of viruses in eastern Australian field garlic was evaluated. Detection assays were developed that involved generic RT-PCR for viruses in the Allexivirus, Carlavirus and Potyvirus genera followed by virus-specific colorimetric dot-blot hybridization. Assays targeted the potyviruses (onion yellow dwarf virus (OYDV), shallot yellow stripe virus (SYSV), and leek yellow stripe virus (LYSV)), the carlaviruses (garlic common latent virus (GCLV) and shallot latent virus (SLV)), and the allexiviruses (garlic viruses A, B, C, X (GarVA, -B, -C, -X) and shallot virus X (ShVX)). Virus incidence in crops was consistently high, with most plants infected with at least one virus from each genus. OYDV, LYSV, SLV, and GCLV were commonly detected. Three of the four allexiviruses were in all districts surveyed but varied in incidence, whereas ShVX and SYSV were not detected. There was no association between virus species complement and bulb size, indicating size is not a good predictor of the virus status of planting material. The variation of virus incidence across different Australian growing districts and in different cultivars implies multiple introductions of viruses rather than spread within the country. The genetic diversity observed within coat protein sequences of some virus species also supports multiple separate introductions

Development of a rapid, accurate, and field deployable LAMP-CRISPR-Cas12a integrated assay for Xylella fastidiosa detection and surveillance

Xylella fastidiosa is an aggressive plant pathogenic bacterium of significant quarantine concern. Accurate and reliable detection tools are essential to minimise the risk of the pathogen’s spread and for outbreak control, as limited post-infection management strategies are possible. Here, we report the development of a specific and potentially field-deployable assay combining a pre-existing Loop-Mediated Isothermal Amplification (LAMP) assay and a Cas12a-based DNA Endonuclease-Targeted (DETECTR) Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) trans reporter for X. fastidiosa detection. The LAMP-CRISPR-Cas12a integrated assay detected the amplified target region of the X. fastidiosa specific rimM gene at the low femto-molar range within 10 min of initiation. The assay detected varied X. fastidiosa sub-species in a range of naturally infected and economically relevant host material, with no non-target amplification recorded. The results show integration of LAMP with CRISPR-based detection is a specific, sensitive and a potentially field-adaptable strategy for the detection of X. fastidiosa and has the potential for further operationally focused improvements

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

Detection of a glitch in the pulsar J1709-4429

We report the detection of a glitch event in the pulsar J1709$-$4429 (also known as B1706$-$44) during regular monitoring observations with the Molonglo Observatory Synthesis Telescope (UTMOST). The glitch was found during timing operations, in which we regularly observe over 400 pulsars with up to daily cadence, while commensally searching for Rotating Radio Transients, pulsars, and FRBs. With a fractional size of $\Delta\nu/\nu \approx 52.4 \times10^{-9}$, the glitch reported here is by far the smallest known for this pulsar, attesting to the efficacy of glitch searches with high cadence using UTMOST.Comment: 3 pages, 1 figur

Rapid Antigen-Capture Assay To Detect West Nile Virus in Dead Corvids

The utility of the VecTest antigen-capture assay to detect West Nile virus (WNV) in field-collected dead corvids was evaluated in Manitoba and Ontario, Canada, in 2001 and 2002. Swabs were taken from the oropharynx, cloaca, or both of 109 American Crows, 31 Blue Jays, 6 Common Ravens, and 4 Black-billed Magpies from Manitoba, and 255 American Crows and 28 Blue Jays from Ontario. The sensitivity and specificity of the antigen-capture assay were greatest for samples from American Crows; oropharyngeal swabs were more sensitive than cloacal swabs, and interlaboratory variation in the results was minimal. The sensitivity and specificity of the VecTest using oropharyngeal swabs from crows were 83.9% and 93.6%, respectively, for Manitoba samples and 83.3% and 95.8%, respectively, for Ontario birds. The VecTest antigen-capture assay on oropharyngeal secretions from crows is a reliable and rapid diagnostic test that appears suitable for incorporation into a WNV surveillance program