Diagnostic Molecular Markers for Phosphine Resistance in U.S. Populations of Tribolium castaneum and Rhyzopertha dominica

Stored product beetles that are resistant to the fumigant pesticide phosphine (hydrogen phosphide) gas have been reported for more than 40 years in many places worldwide. Traditionally, determination of phosphine resistance in stored product beetles is based on a discriminating dose bioassay that can take up to two weeks to evaluate. We developed a diagnostic cleaved amplified polymorphic sequence method, CAPS, to detect individuals with alleles for strong resistance to phosphine in populations of the red flour beetle, Tribolium castaneum, and the lesser grain borer, Rhyzopertha dominica, according to a single nucleotide mutation in the dihydrolipoamide dehydrogenase (DLD) gene. We initially isolated and sequenced the DLD genes from susceptible and strongly resistant populations of both species. The corresponding amino acid sequences were then deduced. A single amino acid mutation in DLD in populations of T.castaneum and R.dominica with strong resistance was identified as P45S in T.castaneum and P49S in R.dominica, both collected from northern Oklahoma, USA. PCR products containing these mutations were digested by the restriction enzymes MboI and BstNI, which revealed presence or absence, respectively of the resistant (R) allele and allowed inference of genotypes with that allele. Seven populations of T.castaneum from Kansas were subjected to discriminating dose bioassays for the weak and strong resistance phenotypes. Application of CAPS to these seven populations confirmed the R allele was in high frequency in the strongly resistant populations, and was absent or at a lower frequency in populations with weak resistance, which suggests that these populations with a low frequency of the R allele have the potential for selection of the strong resistance phenotype. CAPS markers for strong phosphine resistance will help to detect and confirm resistant beetles and can facilitate resistance management actions against a given pest population.

Ecological Networks in Stored Grain: Key Postharvest Nodes for Emerging Pests, Pathogens, and Mycotoxins

Wheat is at peak quality soon after harvest. Subsequently, diverse biota use wheat as a resource in storage, including insects and mycotoxin-producing fungi. Transportation networks for stored grain are crucial to food security and provide a model system for an analysis of the population structure, evolution, and dispersal of biota in networks. We evaluated the structure of rail networks for grain transport in the United States and Eastern Australia to identify the shortest paths for the anthropogenic dispersal of pests and mycotoxins, as well as the major sources, sinks, and bridges for movement. We found important differences in the risk profile in these two countries and identified priority control points for sampling, detection, and management. An understanding of these key locations and roles within the network is a new type of basic research result in postharvest science and will provide insights for the integrated pest management of high-risk subpopulations, such as pesticide-resistant insect pests.

Managing resistance to chemical treatments in stored products pests

This review focuses on key trends in resistance to chemical treatments in stored product pests, and advances in resistance management, with an emphasis on resistance to the fumigant phosphine. Findings: Phosphine resistance continues to be a major concern. In particular, phosphine resistance in Cryptolestes ferrugineus has emerged as a serious issue, with some populations exhibiting the strongest level detected so far for this fumigant. In response, a 'quick knock down test' has been established to deliver industry and scientists 'same day' advice on the resistance status of field samples; sulfuryl fluoride is being developed as a 'resistance breaker' and phosphine dosages are being revised to manage this problem. There has been major progress in identifying the genes responsible for phosphine resistance and the development of molecular resistance diagnostics for key pests. Several studies on Rhyzopertha dominica have demonstrated that molecular screening can be used to determine the frequency of resistance alleles in samples collected from farm storages. Despite on-going research in several pests, there is no definitive answer to the question of whether there is a fitness cost associated phosphine resistance, with some studies showing a clear cost and others none. Evidence continues to emerge of resistance to grain protectants, including the juvenile hormone analogue methoprene. The development and adoption of spinosad, as a next generation 'green' treatment, and the use of protectant combinations provides opportunities to counter the problem of protectant resistance.Directions for future research: A uniform set of protocols should be developed for phosphine resistance detection for all major species. It should combine 'quick tests' and molecular diagnostics to be adopted internationally. Research is required on the establishment of a decision making system that integrates newly developed grain protectants and fumigants, other alternative control methods, as well as an accurate and rapid resistance detection system for early warning of the emergence of new resistances.

First report of Tomato spotted wilt virus in chickpea (Cicer arietinum) in Australia

Tomato spotted wilt virus (genus Tospovirus) is recorded on chickpea (Cicer arietinum) in Australia for the first time. It caused shoot tip symptoms of wilting, necrosis, bunching and chlorosis, followed by premature death of plants.

First records of the papaya strain of Papaya ringspot virus (PRSV-P) in French Polynesia and the Cook Islands

The papaya strain of Papaya ringspot virus (PRSV-P), the cause of papaya ringspot disease, was confirmed in French Polynesia and the Cook Islands by double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA). In French Polynesia, the virus has probably been on the islands of Tahiti and Moorea for several years, but appears not to have spread to eight other islands. In contrast, PRSV-P has only recently appeared in the Cook Islands and is now the subject of an eradication campaign.

Identification of viral and non-viral reverse transcribing elements in pineapple ( Ananas comosus ), including members of two new badnavirus species

A previously published partial sequence of pineapple bacilliform virus was shown to be from a retrotransposon (family Metaviridae) and not from a badnavirus as previously thought. Two newly discovered sequence groups isolated from pineapple were associated with bacilliform virions and were transmitted by mealybugs. Phylogenetic analyses indicated that they were members of new badnavirus species. A third caulimovirid sequence was also amplified from pineapple, but available evidence suggests that this DNA is not encapsidated, but more likely derived from an endogenous virus.

The genetic diversity of ampeloviruses in Australian pineapples and their association with mealybug wilt disease

Pineapple mealybug wilt-associated virus 1 (PMWaV-1), 2 (PMWaV-2) and -3 (PMWaV-3) have been detected in Australian commercial pineapple crops, along with a previously undescribed ampelovirus, for which the name Pineapple mealybug wilt-associated virus 5 (PMWaV-5) is proposed. Partial sequences extending from open reading frame 1b through to the heat shock protein homologue were obtained for PMWaV-1, -3 and -5. Phylogenetic analyses of selected regions of these sequences indicated that PMWaV-5 is a distinct species and most closely related to PMWaV-1. The amino acid sequence variation observed in the RNA-dependent RNA polymerase region of PMWaV-1 isolates was 95.8–98.4% and of PMWaV-3 isolates was 92.2–99.5%. In surveys of mealybug wilt disease (MWD) affected crops, none of the four viruses was clearly associated with the disease at all survey sites. A statistically significant association (P < 0.001) between the presence of PMWaV-2 and symptoms was observed at one survey site (site 3), but the virus was at a low incidence at the remaining three survey sites. By contrast, although PMWaV-1 and -3 were equally distributed between symptomless and MWD-affected plants at site 3, there was a statistically significant (P < 0.001) association between each of these two viruses and MWD at sites 1 and 4. At site 2, there was a statistically significant (P < 0.001) association only between PMWaV-3 and MWD. PMWaV-1 was the most commonly found of the four viruses and conversely PMWaV-5 was only occasionally found. Australian isolates of PMWaV-1, -2 and -3 were transmitted by the mealybug species Dysmicoccus brevipes.

Detecting quantitative trait loci affecting beef tenderness on bovine chromosome 7 near calpastatin and lysyl oxidase

From a study of 3 large half-sib families of cattle, we describe linkage between DNA polymorphisms on bovine chromosome 7 and meat tenderness. Quantitative trait loci (QTL) for Longissimus lumborum peak force (LLPF) and Semitendonosis adhesion (STADH) were located to this map of DNA markers, which includes the calpastatin ( CAST) and lysyl oxidase (LOX) genes. The LLPF QTL has a maximum lodscore of 4.9 and allele substitution of approximately 0.80 of a phenotypic standard deviation, and the peak is located over the CAST gene. The STADH QTL has a maximum lodscore of 3.5 and an allele substitution of approximately 0.37 of a phenotypic standard deviation, and the peak is located over the LOX gene. This suggests 2 separate likelihood peaks on the chromosome. Further analyses of meat tenderness measures in the Longissimus lumborum, LLPF and LL compression (LLC), in which outlier individuals or kill groups are removed, demonstrate large shifts in the location of LLPF QTL, as well as confirming that there are indeed 2 QTL on bovine chromosome 7. We found that both QTL are reflected in both LLPF and LLC measurements, suggesting that both these components of tenderness, myofibrillar and connective tissue, are detected by both measurements in this muscle.

Cyanide poisoning in cattle from Dysphania glomulifera (red crumbweed): Using the internet for rapid plant identification and diagnostic advice: Case Report

A 300-strong Angus-Brahman cattle herd near Springsure, central Queensland, was being fed Acacia shirleyi (lancewood) browse during drought and crossed a 5-hectare, previously burnt area with an almost pure growth of Dysphania glomulifera subspecies glomulifera (red crumbweed) on their way to drinking water. Forty cows died of cyanide poisoning over 2 days before further access to the plant was prevented. A digital image of a plant specimen made on a flat-bed scanner and transmitted by email was used to identify D glomulifera. Specific advice on the plant's poisonous properties and management of the case was then provided by email within 2 hours of an initial telephone call by the field veterinarian to the laboratory some 600 km away. The conventional method using physical transport of a pressed dried plant specimen to confirm the identification took 5 days. D glomulifera was identified in the rumen of one of two cows necropsied. The cyanogenic potential of D glomulifera measured 4 days after collection from the site of cattle deaths was 18,600 mg HCN/kg in dry matter. The lethal dose of D glomulifera for a 420 kg cow was estimated as 150 to 190 g wet weight. The plant also contained 4.8% KNO3 equivalent in dry matter, but nitrate-nitrite poisoning was not involved in the deaths.

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.

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.

Salmonella Virchow and Salmonella Weltevreden in a Random Survey of the Asian House Gecko, Hemidactylus frenatus, in Houses in Northern Australia.

Background: Salmonella enterica serotype Virchow is the most common cause of invasive nontyphoid salmonellosis in North Queensland, particularly in infants, but the zoonotic source is unknown. This study aimed at determining (i) the prevalence of the introduced Asian house gecko, Hemidactylus frenatus, in houses in North Queensland and (ii) whether they were a potential source of Salmonella Virchow. Methods: Asian house geckos were collected in a random survey of houses in Townsville, North Queensland. Gut contents underwent microbiological analysis within 2 h of removal using both direct plating and enrichment broth methods. Any organism found to be a presumptive Salmonella spp. was then sent to a reference lab for confirmation of genus/species, serotyping, and phage typing if indicated. Results: One hundred Asian house geckos were collected from 57 houses. Geckos were present in 100% of houses surveyed, and prevalence of Salmonella in large intestinal contents was 7% (95% confidence interval 2, 12%). Three serotypes were found: Virchow (phage type 8), Weltevreden, and an untypable subspecies 1 serotype 11:-:1,7. Conclusion: Since Salmonella Virchow (phage type 8) is associated with invasive disease, the introduced Asian house gecko may play a significant role in the epidemiology of sporadic salmonellosis in places invaded by these peridomestic reptiles. These results justify more detailed epidemiological studies on the role of the Asian house gecko in sporadic salmonellosis and development of evidence-based strategies to decrease this potential zoonotic hazard.

Molecular characterisation of dicot-infecting mastreviruses from Australia

Monocotyledonous and dicotyledonous plant infecting mastreviruses threaten various agricultural systems throughout Africa, Eurasia and Australasia. In Australia three distinct mastrevirus species are known to infect dicotyledonous hosts such as chickpea, bean and tobacco. Amongst 34 new "dicot-infecting" mastrevirus full genome sequences obtained from these hosts we discovered one new species, four new strains, and various variants of previously described mastrevirus species. Besides providing additional support for the hypothesis that evolutionary processes operating during dicot-infecting mastrevirus evolution (such as patterns of pervasive homologous and non-homologous recombination, and strong purifying selection acting on all genes) have mostly mirrored those found in their monocot-infecting counterparts, we find that the Australian dicot-infecting viruses display patterns of phylogeographic clustering reminiscent of those displayed by monocot infecting mastrevirus species such as Panicum streak virus and Maize streak virus.

Australian monocot-infecting mastrevirus diversity rivals that in Africa

Monocotyledonous plant infecting mastreviruses (family Geminiviridae) have been found in the Old World. The greatest diversity of these viruses has been found in Africa but this may simply reflect the more extensive sampling that has been done there. To provide a better understanding of mastrevirus diversity in Australia. we have sequenced the genomes of 41 virus isolates found in naturalised and native grasses and identified four new species in addition to the four previously characterised species. Two of these species, which were recovered from a single Sporobolus plant, are highly divergent and are most closely related to the African streak viruses. This, coupled with the discovery of divergent dicotyledonous plant infecting mastreviruses in Australia brings into question the hypothesis that mastreviruses may have originated in Africa. We found that the patterns of inter- and intra-species recombination and the recombination hotspots mirror those found in both their African monocot-infecting counterparts and dicot-infecting mastrevirus. (C) 2012 Elsevier B.V. All rights reserved.

An outbreak of Potato spindle tuber viroid in tomato is linked to imported seed

In 2011, an outbreak of the quarantine-regulated pathogen Potato spindle tuber viroid (PSTVd) occurred in a commercial glasshouse-grown tomato crop in Queensland, Australia. Phylogenetic studies showed that the genotype of this isolate grouped in a cluster of PSTVd genotypes from tomato and Physalis peruviana, and exhibited an interesting mutation (U257→A) that has previously been linked to lethal symptom expression in tomato. Transmission studies showed that the viroid could be mechanically transmitted from crushed fruit sap, but not from undamaged fruits. A low rate of asymptomatic infection was determined for plants in the affected glasshouse, demonstrating the efficacy of using symptoms to detect PSTVd infections in tomato. No PSTVd infections were detected in solanaceous weeds located outside of the infected glasshouse, excluding them from playing a role in the viroid epidemiology. Monitoring and subsequent testing of new tomato crops grown in the facility demonstrated successful eradication of the pathogen. A trace-back analysis linked the outbreak of PSTVd to an infected imported tomato seed-lot, indicating that PSTVd is transmitted internationally through contaminated seed