Rapid and sensitive detection of Avibacterium paragallinarum in the presence of other bacteria using a 5' Taq nuclease assay: A new tool for diagnosing infectious coryza

A 5' Taq nuclease assay specific for Avibacterium paragallinarum was designed and optimized for use in diagnosing infectious coryza. The region chosen for assay design was one of known specificity for Av. paragallinarum. The assay detected Av. paragallinarum reference strains representing the three Page and the eight Kume serovars, and field isolates from diverse geographical locations. No cross-reactions were observed with other Avibacterium species, with other bacteria taxonomically related to Av. paragallinarum nor with bacteria and viruses likely to be present in swabs collected from suspected infectious coryza cases. The detection limit for the assay was 6 to 60 colony-forming units per reaction. Twenty-two out of 53 swabs collected from sick birds reacted in the 5' Taq nuclease assay, whereas Av. paragallinarum was not isolated from any of the swabs. All of the 22 swabs yielded other bacteria in culture. The presence of Av. paragallinarum in the swabs was also demonstrated by sequencing, thereby confirming the ability of the assay to detect Av. paragallinarum in the presence of other bacteria. The ability to quantify bacterial load in the swabs using the 5' Taq nuclease assay was demonstrated.

Infection with Menangle virus in flying foxes (Pteropus spp.) in Australia

Objective: To examine flying foxes (Pteropus spp.) for evidence of infection with Menangle virus. Design: Clustered non-random sampling for serology, virus isolation and electron microscopy (EM). Procedure: Serum samples were collected from 306 Pteropus spp. in northern and eastern Australia and tested for antibodies against Menangle virus (MenV) using a virus neutralisation test (VNT). Virus isolation was attempted from tissues and faeces collected from 215 Pteropus spp. in New South Wales. Faecal samples from 68 individual Pteropus spp. and four pools of faeces were examined by transmission EM following routine negative staining and immunogold labelling. Results: Neutralising antibodies (VNT titres ≥ 8) against MenV were detected in 46% of black flying foxes (P. alecto), 41% of grey-headed flying foxes (P. poliocephalus), 25% of spectacled flying foxes (P. conspicillatus) and 1% of little red flying foxes (P. scapulatus) in Australia. Positive sera included samples collected from P. poliocephalus in a colony adjacent to a piggery that had experienced reproductive disease caused by MenV. Virus-like particles were observed by EM in faeces from Pteropus spp. and reactivity was detected in pooled faeces and urine by immunogold EM using sera from sows that had been exposed to MenV. Attempts to isolate the virus from the faeces and tissues from Pteropus spp. were unsuccessful. Conclusion: Serological evidence of infection with MenV was detected in Pteropus spp. in Australia. Although virus-like particles were detected in faeces, no viruses were isolated from faeces, urine or tissues of Pteropus spp.

Absolute quantitation of Marek's disease virus and Herpesvirus of turkeys in chicken lymphocyte, feather tip and dust samples using real-time PCR

The further development of Taqman quantitative real-time PCR (qPCR) assays for the absolute quantitation of Marek's disease virus serotype 1 (MDV1) and Herpesvirus of turkeys (HVT) viruses is described and the sensitivity and reproducibility of each assay reported. Using plasmid DNA copies, the lower limit of detection was determined to be 5 copies for the MDV1 assay and 75 copies for the HVT assay. Both assays were found to be highly reproducible for Ct values and calculated copy numbers with mean intra- and inter-assay coefficients of variation being less than 5% for Ct and 20% for calculated copy number. The genome copy number of MDV1 and HVT viruses was quantified in PBL and feather tips from experimentally infected chickens, and field poultry dust samples. Parallelism was demonstrated between the plasmid-based standard curves, and standard curves derived from infected spleen material containing both viral and host DNA, allowing the latter to be used for absolute quantification. These methods should prove useful for the reliable differentiation and absolute quantitation of MDV1 and HVT viruses in a wide range of samples.

Development of an immunomagnetic capture-reverse transcriptase-PCR assay for three pineapple ampeloviruses

A semi-automated, immunomagneticcapture-reverse transcription PCR(IMC-RT-PCR) assay for the detection of three pineapple-infecting ampeloviruses, Pineapple mealybug wilt-associated virus-1, -2 and -3, is described. The assay was equivalent in sensitivity but more rapid than conventional immunocapture RT-PCR. The assay can be used either as a one- or two-step RT-PCR and allows detection of the viruses separately or together in a triplex assay from fresh, frozen or freeze-dried pineapple leaf tissue. This IMC-RT-PCR assay could be used for high throughput screening of pineapple planting propagules and could easily be modified for the detection of other RNA viruses in a range of plant species, provided suitable antibodies are available.

Development of a New Zealand database of plant virus and virus-like organisms.

The recent 8th Australasian plant virology workshop in Rotorua, New Zealand, discussed the development of a New Zealand database of plant virus and virus-like organisms. Key points of discussion included: (i) the purpose of such a database; (ii) who would benefit from the information in a database; (iii) the scope of a database and its associated collections; (iv) database information and format; and (v) potential funding of such a database. From the workshop and further research, we conclude that the preservation and verification of specimens within the collections and the development of a New Zealand database of plant virus and virus-like organisms is essential. Such a collection will help to fulfil statutory requirements in New Zealand and assist in fulfilling international obligations under the International Plant Protection Convention. Sustaining such a database will assist New Zealand virologists and statutory bodies to undertake scientifically sound research. Establishing reliable records and an interactive database will help to ensure accurate and timely diagnoses of diseases caused by plant viruses and virus-like organisms. Detection of new incursions and their diagnosis will be further enhanced by the use of such reference collections and their associated database. Connecting and associating this information to similar overseas databases would assist international collaborations and allow access to the latest taxonomic and diagnostic resources. Associated scientists working in the areas of plant breeding, export phytosanitary assurance and in the area of the conservation estate would also benefit from access to verified specimens of plant viruses and virus-like organisms. We conclude that funding of a New Zealand database of virus and virus-like organisms and its associated collections should be based partly on Crown funds, as it is a nationally significant biological resource.

Diagnosis of a naturally occurring dual infection of layer chickens with fowlpox virus and gallid herpesvirus 1 (infectious laryngotracheitis virus).

An outbreak of acute respiratory disease in layers was diagnosed as being of dual nature due to fowlpox and infectious laryngotracheitis using a multidisciplinary approach including virus isolation, histopathology, electron microscopy and polymerase chain reaction (PCR). The diagnosis was based on virus isolation of gallid herpesvirus 1 (GaHV-1) in chicken kidney cells and fowlpox virus (FWPV) in 9-day-old chicken embryonated eggs inoculated via the chorioallantoic membrane. The histopathology of tracheas from dead birds revealed intra-cytoplasmic and intra-nuclear inclusions suggestive of poxvirus and herpesvirus involvement. The presence of FWPV was further confirmed by electron microscopy, PCR and histology. All FWPV isolates contained the long terminal repeats of reticuloendotheliosis virus as demonstrated by PCR. GaHV-1 isolates were detected by PCR and were shown to have a different restriction fragment length polymorphism pattern when compared with the chicken embryo origin SA2 vaccine strain; however, they shared the same pattern with the Intervet chicken embryo origin vaccine strain. This is a first report of dual infection of chickens with GaHV-1 and naturally occurring FWPV with reticuloendotheliosis virus insertions. Further characterization of the viruses was carried out and the results are reported here.

Two novel mastreviruses from chickpea (Cicer arietinum) in Australia

Two novel mastreviruses (genus Mastrevirus; family Geminiviridae), with proposed names chickpea chlorosis virus (CpCV) and chickpea redleaf virus, are described from chickpea (Cicer arietinum) from eastern Australia. The viruses have genomes of 2,582 and 2,605 nucleotides, respectively, and share similar features and organisation with typical dicot-infecting mastreviruses. Two distinct strains of CpCV were suggested by phylogenetic analysis. Additionally, a partial mastrevirus Rep sequence from turnip weed (Rapistrum rugosum) indicated the presence of a distinct strain of Tobacco yellow dwarf virus (TYDV). In phylogenetic analyses, isolates of Bean yellow dwarf virus, Chickpea chlorotic dwarf Pakistan virus and Chickpea chlorotic dwarf Sudan virus from southern and northern Africa and south-central and western Asia clustered separately from these three viruses from Australia. An Australian, eastern Asian, or south-eastern Asian origin for the novel mastreviruses and TYDV is discussed.

Bromus catharticus striate mosaic virus: a new mastrevirus infecting Bromus catharticus from Australia.

Although monocotyledonous-plant-infecting mastreviruses (in the family Geminiviridae) are known to cause economically significant crop losses in certain areas of the world, in Australia, they pose no obvious threat to agriculture. Consequently, only a few Australian monocot-infecting mastreviruses have been described, and only two have had their genomes fully sequenced. Here, we present the third full-genome sequence of an Australian monocot-infecting mastrevirus from Bromus catharticus belonging to a distinct species, which we have tentatively named Bromus catharticus striate mosaic virus (BCSMV). Although the genome of this new virus shares only 57.7% sequence similarity with that of its nearest known relative, Digitaria didactyla striate mosaic virus (DDSMV; also from Australia), it has features typical of all other known mastrevirus genomes. Phylogenetic analysis showed that both the full genome and each of its probable expressed proteins group with the two other characterised Australian monocot-infecting mastreviruses. Besides the BCSMV genome sequence revealing that Australian monocot-infecting mastrevirus diversity rivals that seen in Africa, it has enabled us, for the first, to time detect evidence of recombination amongst the Australian viruses. Specifically, it appears that DDSMV possesses a short intergenic region sequence that has been recombinationally derived from either BCSMV or a close relative that has not yet been identified.

Detection of Polymyxa graminis in a barley crop in Australia.

Polymyxa graminis was detected in the roots of barley plants from a field near Wondai, Queensland, in 2009. P. graminis was identified by characteristic sporosori in roots stained with trypan blue. The presence of P. graminis f. sp. tepida (which is hosted by wheat and oats as well as barley) in the roots was confirmed by specific PCR tests based on nuclear ribosomal DNA. P. graminis is the vector of several damaging soil-borne virus diseases of cereals in the genera Furovirus, Bymovirus and Pecluvirus. No virus particles were detected in sap extracts from leaves of stunted barley plants with leaf chlorosis and increased tillering. Further work is required to determine the distribution of P. graminis in Australian grain crops and the potential for establishment and spread of the exotic soil-borne viruses that it vectors.

Commercial application of polychaete sand filters for wastewater remediation and broodstock feeds

Summary Prototype sand-worm filtration beds were constructed at two prawn farms and one fish farm to assess and demonstrate their polychaete (marine worm) production and wastewater remediation capacities at semi-commercial scale. Wastewater treatment properties were monitored and worms produced were assessed and either sold for bait or used by the farms’ hatcheries as broodstock (prawn or fish breeder) feed. More than 34 megalitres of prawn- and fish-pond water was beneficially treated in the 116-319-d trial. The design of the polychaete-assisted sand filters (PASFs) constructed at each farm affected their water handling rates, which on average ranged from 315 to 1000 L m-2 d-1 at the three farms. A low profile design incorporating shallow bunded ponds made from polyethylene liner and timber stakes provided the easiest method of construction. This simple design applied at broad scale facilitated the highest quantities of treated water and the greatest worm production. Designs with higher sides increased the head pressure above the sand bed surface, thus increasing the amount of water that could be treated each day. Most water qualities were affected in a similar way to that demonstrated in the previous tank trials: dissolved oxygen, pH, total suspended solids and chlorophyll a levels were all consistently significantly lowered as pond water percolated through the sand bed, and dissolved forms of nitrogen and phosphorus were marginally increased on several occasions. However, unlike the previous smaller-scale tank trials, total nitrogen (TN) and total phosphorus (TP) levels were both significantly lowered by these larger-scale PASFs. The reasons for this are still unclear and require further research. Maximum TN and TP removals detected in the trial were 48.8% and 67.5%, respectively, and average removals (in unfed beds) at the three farms ranged from 20.0 to 27.7% for TN and from 22.8 to 40.8% for TP. Collectively, these results demonstrate the best suspended solids, chlorophyll and macronutrient removal capacities so far reported for any mariculture wastewater treatment methodology to date. Supplemental feeding of PASFs with fish meal was also investigated at one farm as a potential means of increasing their polychaete biomass production. Whilst fed beds produced higher biomass (152 ± 35 g m-2) compared with unfed beds (89 ± 17 g m-2) after 3.7 months of operation, the low number of replicates (2) prevented statistically significant differences from being demonstrated for either growth or survival. At harvest several months later, worm biomass production was estimated to be similar to, or in slight excess of, previously reported production levels (300-400 g m-2). Several qualities of filtered water appear to have been affected by supplemental feeding: it appeared to marginally lower dissolved oxygen and pH levels, and increased the TN and TP levels though not so much to eliminate significant beneficial water treatment effects. Periodic sampling during an artificial-tide demonstrated the tendency for treated-water quality changes during the first hour of filtration. Total nitrogen and ammonia peaked early in the tidal flow and then fell to more stable levels for the remainder of the filtration period. Other dissolved nutrients also showed signs of this sand-bed-flushing pattern, and dissolved oxygen tended to climb during the first hour and become more stable thereafter. These patterns suggest that the routine sampling of treated water undertaken at mid-inflow during the majority of the wider study would likely have overestimated the levels of TN and dissolved nutrients discharged from the beds, and hence underestimated the PASFs treatment efficacies in this regard. Analyses of polychaete biomass collected from each bed in the study revealed that the worms were free from contamination with the main prawn viruses that would create concerns for their feeding to commercial prawn broodstock in Australia. Their documented proximal and nutritional contents also provide a guide for hatchery operators when using live or frozen stock. Their dry matter content ranged from 18.3 to 22.3%, ash ranged from 10.2 to 14.0%, gross energy from 20.2 to 21.5 MJ kg-1, and fat from 5.0 to 9.2%. Their cholesterol levels ranged from 0.86 to 1.03% of dry matter, whilst total phospholipids range from 0.41 to 0.72%. Thirty-one different fatty acids were present at detectable (≥0.005% of dry matter) levels in the sampled worm biomass. Palmitic acid was by far the most prevalent fatty acid detected (1.21 ± 0.18%), followed by eicosapentaenoic (EPA) (0.48 ± 0.03%), stearic (0.46 ± 0.04%), vaccenic (0.38 ± 0.05%), adrenic (0.35 ± 0.02%), docosadienoic (0.28 ± 0.02%), arachidonic (AA) (0.22 ± 0.01%), palmitoleic (0.20 ± 0.04%) and 23 other fatty acids with average contents of less than 0.2% of dry matter. Supplemental feeding with fish meal at one farm appeared to increase the docosahexaenoic acid (DHA) content of the worms considerably, and modify the average AA : EPA : DHA from 1.0 : 2.7 : 0.3 to 1.0 : 2.0 : 1.1. Consistent with previous results, the three most heavily represented amino acids in the dry matter of sampled worms were glutamic acid (8.5 ± 0.2%), aspartic acid (5.5 ± 0.1%) and glycine (4.9 ± 0.5%). These biomass content results suggest that worms produced in PASF systems are well suited to feeding to prawn and fish broodstock, and provide further strong evidence of the potential to modify their contents for specific nutritional uses. The falling wild-fishery production of marine bloodworms in Queensland is typical of diminishing polychaete resources world-wide and demonstrates the need to develop sustainable production methods here and overseas. PASF systems offer the dual benefits of wastewater treatment for environmental management and increased productivity through a valuable secondary crop grown exclusively on waste nutrients.

Cultivar-specific effects of pathogen testing on storage root yield of sweetpotato, Ipomoea batatas.

The accumulation and perpetuation of viral pathogens over generations of clonal propagation in crop species such as sweetpotato, Ipomoea batatas, inevitably result in a reduction in crop yield and quality. This study was conducted at Bundaberg, Australia to compare the productivity of field-derived and pathogen-tested (PT) clones of 14 sweetpotato cultivars and the yield benefits of using healthy planting materials. The field-derived clonal materials were exposed to the endemic viruses, while the PT clones were subjected to thermotherapy and meristem-tip culture to eliminate viral pathogens. The plants were indexed for viruses using nitrocellulose membrane-enzyme-linked immunosorbent assay and graft-inoculations onto Ipomoea setosa. A net benefit of 38% in storage root yield was realised from using PT materials in this study. Conversely, in a similar study previously conducted at Kerevat, Papua New Guinea (PNG), a net deficit of 36% was realised. This reinforced our finding that the response to pathogen testing was cultivar dependent and that the PNG cultivars in these studies generally exhibited increased tolerance to the endemic viruses present at the respective trial sites as manifested in their lack of response from the use of PT clones. They may be useful sources for future resistance breeding efforts. Nonetheless, the potential economic gain from using PT stocks necessitates the use of pathogen testing on virus-susceptible commercial cultivars. .

Virus identification and development of long-term management strategies for the rhubarb industry

The purpose of this report is to present the final results of all activities conducted under HAL Project VG05053 ‘Virus identification and development of long-term management strategies for the rhubarb industry’. The report provides a summary of project findings, a description of technology transfer activities, and recommendations arising from the outcomes of the project. The overall objective of this project was to devise a strategy for the control of rhubarb decline disease through 1) knowledge of the viruses present and their epidemiology, 2) production of virus-free planting material via tissue culture, and 3) formation of a national grower group to represent industry.

Developing and communicating strategies for controlling virus diseases in vegetable cucurbit crops

Virus diseases cause serious yield and quality losses in field grown cucurbit crops worldwide. In Australia, the main viruses of cucurbits are Papaya ringspot virus (PRSV), Squash mosaic virus (SqMV), Watermelon mosaic virus (WMV) and Zucchini yellow mosaic virus (ZYMV). Plants infected early have severely distorted fruit. High infection incidences, of ZYMV and PRSV in crops cause losses of marketable fruit of up to 100% and infected crops are often abandoned. Two new alternative hosts of ZYMV were identified, the native cucurbit Cucumis maderaspatanus and wild legume Rhyncosia minima. No new alternative hosts of PRSV, SqMV or WMV were found in Western Australia or Queensland. Seed transmission of ZYMV (0.7%) was found in seedlings grown from ZYMV-infected fruit of zucchini but not of pumpkin. None was detected with PRSV or SqMV in zucchini or pumpkin seedlings, respectively. ZYMV spread to pumpkins by aphids was greater downwind than upwind of a virus source. Delaying sowing by 2 weeks decreased ZYMV spread. Millet non-host barriers between pumpkin plantings slowed ZYMV infection. Host resistance gene (zym) in cucumber cultivars was effective against ZYMV. Pumpkin cultivars with resistance gene (Zym) became infected under high virus pressure but leaf symptoms were milder and infected plants higher yielding with more market-acceptable fruit than those without Zym. Most zucchini cultivars with Zym developed severe leaf and fruit symptoms. ZYMV, PRSV, WMV and SqMV spread readily from infected to healthy cucurbit plants by direct leaf contact. ZYMV survives and remains infective on diverse surfaces for up to 6 hours but can be inactivated by some disinfectants. Phylogenetic analysis indicates at least three separate introductions of ZYMV into Australia, with new introductions rarely occurring. ZYMV isolates clustered into three groups according to collection location i) Kununurra, ii) Northern Territory and iii) Carnarvon, Qld and Vic. A multiplex Real-Time PCR was developed which distinguished between the three groups of Australian isolates. Integrated disease management (IDM) strategies for virus diseases of vegetable cucurbit crops grown in the field were improved incorporating the new information gathered. These strategies are aimed at causing using minimal extra expense, labour demands and disruption to normal practices.

Cross-industry preparedness for Cotton leaf curl disease

Cotton leaf curl disease (CLCuD) is a major biosecurity threat to the Australian cotton industry. This proposal seeks cross-industry investment from the cotton (CRDC) and horticulture (HAL) industries to address the threat of exotic whitefly-transmitted viruses. Testing of silverleaf whitefly, the vector of CLCuD, could provide an alternative, cheaper strategy for early warning disease surveillance compared to surveys for disease symptoms. Control of whitefly-transmitted viruses in Australia and overseas will be reviewed to produce an integrated management package for their control in Australia. This will also involve a workshop with key stakeholders and selected overseas participants, to develop a working party to help formulate this package.

CRC20093 Increasing Diagnostic Capacity in Thailand

Accurate identification of pests is essential for practically all aspects of agricultural development and is critical to the operations of biosecurity that safeguard agricultural integrity and facilitate trade. Diagnostic capability is at the forefront of and complementary to, activities such as border protection, incursion management, surveillance and pest and disease certification. The efficiency of a biosecurity system therefore depends largely on the feedback between these activities and diagnostics. Australian scientists will train Thai scientists in diagnostics and surveillance to provide the Thai DOA with skills that will aid in the development of a Thai Diagnostic Network. The skills will be taught using a range of pests, including some which have particular biosecurity importance for both Australia and Thailand such as citrus canker, potato viruses and fruit flies.