Genetic variability of Tomato spotted wilt virus in Australia and validation of real time RT-PCR for its detection in single and bulked leaf samples

The potential for large-scale use of a sensitive real time reverse transcription polymerase chain reaction (RT-PCR) assay was evaluated for the detection of Tomato spotted wilt virus (TSWV) in single and bulked leaf samples by comparing its sensitivity with that of DAS-ELISA. Using total RNA extracted with RNeasy® or leaf soak methods, real time RT-PCR detected TSWV in all infected samples collected from 16 horticultural crop species (including flowers, herbs and vegetables), two arable crop species, and four weed species by both assays. In samples in which DAS-ELISA had previously detected TSWV, real time RT-PCR was effective at detecting it in leaf tissues of all 22 plant species tested at a wide range of concentrations. Bulk samples required more robust and extensive extraction methods with real time RT-PCR, but it generally detected one infected sample in 1000 uninfected ones. By contrast, ELISA was less sensitive when used to test bulked samples, once detecting up to 1 infected in 800 samples with pepper but never detecting more than 1 infected in 200 samples in tomato and lettuce. It was also less reliable than real time RT-PCR when used to test samples from parts of the leaf where the virus concentration was low. The genetic variability among Australian isolates of TSWV was small. Direct sequencing of a 587 bp region of the nucleoprotein gene (S RNA) of 29 isolates from diverse crops and geographical locations yielded a maximum of only 4.3% nucleotide sequence difference. Phylogenetic analysis revealed no obvious groupings of isolates according to geographic origin or host species. TSWV isolates, that break TSWV resistance genes in tomato or pepper did not differ significantly in the N gene region studied, indicating that a different region of the virus genome is responsible for this trait.

Soil sorption-desorption of phosphorus from piggery effluent compared with inorganic sources

The leaching of phosphorus (P) within soils can be a limiting consideration for the sustainable operation of intensive livestock enterprises. Sorption curves are widely used to assist estimation of P retention, though the effect of effluent constituents on their accuracy is not well understood. We conducted a series of P-sorption-desorption batch experiments with an Oxic Haplustalf (soil 1), Haplusterts (soils 2 and 3), and a Natrustalf (soil 4). Phosphorus sources included effluent, orthophosphate-P in a matrix replicating the effluent's salt constituents (the reference solution), and an orthophosphate-P solution. Treated soils were incubated for up to 193 days before sequential desorption extraction. Effluent constituents, probably the organic or particulate components, temporarily increased the vulnerability of sorbed-P to desorption. The increase in vulnerability was removed by 2-113 days of incubation (25 degrees C). Despite vigorous extraction for 20 consecutive days, some P sorbed as part of the treatments of soils 1 and 2 was not desorbed. The increased vulnerability due to effluent constituents lasted a maximum of about one cropping season and, for all other treatments, adsorption curves overestimated vulnerability to desorption. Therefore, adsorption curves provide a conservative estimate of vulnerability to desorption where effluent is used in continued crop production in these soils.

Concordant molecular and phenotypic data delineate new taxonomy and conservation priorities for the endangered mountain yellow-legged frog

The mountain yellow-legged frog Rana muscosa sensu lato, once abundant in the Sierra Nevada of California and Nevada, and the disjunct Transverse Ranges of southern California, has declined precipitously throughout its range, even though most of its habitat is protected. The species is now extinct in Nevada and reduced to tiny remnants in southern California, where as a distinct population segment, it is classified as Endangered. Introduced predators (trout), air pollution and an infectious disease (chytridiomycosis) threaten remaining populations. A Bayesian analysis of 1901 base pairs of mitochondrial DNA confirms the presence of two deeply divergent clades that come into near contact in the Sierra Nevada. Morphological studies of museum specimens and analysis of acoustic data show that the two major mtDNA clades are readily differentiated phenotypically. Accordingly, we recognize two species, Rana sierrae, in the northern and central Sierra Nevada, and R. muscosa, in the southern Sierra Nevada and southern California. Existing data indicate no range overlap. These results have important implications for the conservation of these two species as they illuminate a profound mismatch between the current delineation of the distinct population segments (southern California vs. Sierra Nevada) and actual species boundaries. For example, our study finds that remnant populations of R. muscosa exist in both the southern Sierra Nevada and the mountains of southern California, which may broaden options for management. In addition, despite the fact that only the southern California populations are listed as Endangered, surveys conducted since 1995 at 225 historic (1899-1994) localities from museum collections show that 93.3% (n=146) of R. sierrae populations and 95.2% (n=79) of R. muscosa populations are extinct. Evidence presented here underscores the need for revision of protected population status to include both species throughout their ranges.

Heritability estimates for growth in the tropical abalone Haliotis asinina using microsatellites to assign parentage

The tropical abalone Haliotis asinina is a wild-caught and cultured species throughout the Indo-Pacific as well as being an emerging model species for the study of haliotids. H. asinina has the fastest recorded natural growth rate of any abalone and reaches sexual maturity within one year. As such, it is a suitable abalone species for selective breeding for commercially important traits such as rapid growth. Estimating the amount of variation in size that is attributable to heritable genetic differences can assist the development of such a selective breeding program. Here we estimated heritability for growth-related traits at 12 months of age by creating a single cohort of 84 families in a full-factorial mating design consisting of 14 sires and 6 dams. Of 500 progeny sampled, 465 were successfully assigned to their parents based on shared alleles at 5 polymorphic microsatellite loci. Using an animal model, heritability estimates were 0.48 ± 0.15 for shell length, 0.38 ± 0.13 for shell width and 0.36 ± 0.13 for weight. Genetic correlations were > 0.98 between shell parameters and weight, indicating that breeding for weight gains could be successfully achieved by selecting for shell length.

Reduced efficacy of macrocyclic lactone treatments in controlling gastrointestinal nematode infections of weaner dairy calves in subtropical eastern Australia.

Faecal Egg Count Reduction Tests (FECRTs) for macrocyclic lactone (ML) and levamisole (LEV) drenches were conducted on two dairy farms in the subtropical, summer rainfall region of eastern Australia to determine if anthelmintic failure contributed to severe gastrointestinal nematode infections observed in weaner calves. Subtropical Cooperia spp. were the dominant nematodes on both farms although significant numbers of Haemonchus placei were also present on Farm 2. On Farm 1, moxidectin pour-on (MXD) drenched at 0.5 mg kg-1 liveweight (LW) reduced the overall Cooperia burden by 82% (95% confidence limits, 37-95%) at day 7 post-drench. As worm burdens increased rapidly in younger animals in the control group (n = 4), levamisole was used as a salvage drench and these calves withdrawn from the trial on animal welfare grounds after sample collection at day 7. Levamisole (LEV) dosed at 6.8 mg kg-1 LW reduced the worm burden in these calves by 100%, 7 days after drenching. On Farm 2, MXD given at 0.5 mg kg-1 LW reduced the faecal worm egg count of cooperioids at day 8 by 96% (71-99%), ivermectin oral (IVM) at 0.2 mg kg-1 LW by 1.6% (-224 to 70%) and LEV oral at 7.1 mg kg-1 LW by 100%. For H. placei the reductions were 98% (85-99.7%) for MXD, 0.7% (-226 to 70%) for IVM and 100% for LEV. This is the first report in Australia of the failure of macrocyclic lactone treatments to control subtropical Cooperia spp. and suspected failure to control H. placei in cattle.

Positive results in a real-time PCR for type A influenza associated with the use of an inactivated vaccine.

A real-time reverse transcription polymerase chain reaction (qRT-PCR) test for the matrix gene of type A influenza viruses was used during the 2007 Australian equine influenza (EI) outbreak in order to confirm diagnosis and, later, eradication of the virus. During the EI outbreak, horses being exported required vaccination and individual proof of freedom from EI. At the end of the outbreak, positive results were obtained from four horses destined for export, because of contamination of the samples with the vaccine. This report highlights the need for EI testing and vaccination to occur on separate days and with the collection of swabs for testing to precede vaccination.

Finding genes for economically important traits: Brahman cattle puberty.

Age at puberty is an important component of reproductive performance in beef cattle production systems. Brahman cattle are typically late-pubertal relative to Bos taurus cattle and so it is of economic relevance to select for early age at puberty. To assist selection and elucidate the genes underlying puberty, we performed a genome-wide association study (GWAS) using the BovineSNP50 chip (similar to 54 000 polymorphisms) in Brahman bulls (n = 1105) and heifers (n = 843) and where the heifers were previously analysed in a different study. In a new attempt to generate unbiased estimates of single-nucleotide polymorphism (SNP) effects and proportion of variance explained by each SNP, the available data were halved on the basis of year and month of birth into a calibration and validation set. The traits that defined age at puberty were, in heifers, the age at which the first corpus luteum was detected (AGECL, h(2) = 0.56 +/- 0.11) and in bulls, the age at a scrotal circumference of 26 cm (AGE26, h(2) = 0.78 +/- 0.10). At puberty, heifers were on average older (751 +/- 142 days) than bulls (555 +/- 101 days), but AGECL and AGE26 were genetically correlated (r = 0.20 +/- 0.10). There were 134 SNPs associated with AGECL and 146 SNPs associated with AGE26 (P < 0.0001). From these SNPs, 32 (similar to 22%) were associated (P < 0.0001) with both traits. These top 32 SNPs were all located on Chromosome BTA 14, between 21.95 Mb and 28.4 Mb. These results suggest that the genes located in that region of BTA 14 play a role in pubertal development in Brahman cattle. There are many annotated genes underlying this region of BTA 14 and these are the subject of current research. Further, we identified a region on Chromosome X where markers were associated (P < 1.00E-8) with AGE26, but not with AGECL. Information about specific genes and markers add value to our understanding of puberty and potentially contribute to genomic selection. Therefore, identifying these genes contributing to genetic variation in AGECL and AGE26 can assist with the selection for early onset of puberty.

Fungal Planet description sheets: 128-153

Novel species of microfungi described in the present study include the following from Australia: Catenulostroma corymbiae from Corymbia, Devriesia stirlingiae from Stirlingia, Penidiella carpentariae from Carpentaria, Phaeococcomyces eucalypti from Eucalyptus, Phialophora livistonae from Livistona, Phyllosticta aristolochiicola from Aristolochia, Clitopilus austroprunulus on sclerophyll forest litter of Eucalyptus regnans and Toxicocladosporium posoqueriae from Posoqueria. Several species are also described from South Africa, namely: Ceramothyrium podocarpi from Podocarpus, Cercospora chrysanthemoides from Chrysanthemoides, Devriesia shakazului from Aloe, Penidiella drakensbergensis from Protea, Strelitziana cliviae from Clivia and Zasmidium syzygii from Syzygium. Other species include Bipolaris microstegii from Microstegium and Synchaetomella acerina from Acer (USA), Brunneiapiospora austropalmicola from Rhopalostylis (New Zealand), Calonectria pentaseptata from Eucalyptus and Macadamia (Vietnam), Ceramothyrium melastoma from Melastoma (Indonesia), Collembolispora aristata from stream foam (Czech Republic), Devriesia imbrexigena from glazed decorative tiles (Portugal), Microcyclospora rhoicola from Rhus (Canada), Seiridium phylicae from Phylica (Tristan de Cunha, Inaccessible Island), Passalora lobeliaefistulosis from Lobelia (Brazil) and Zymoseptoria verkleyi from Poa (The Netherlands). Valsalnicola represents a new ascomycete genus from Alnus (Austria) and Parapenidiella a new hyphomycete genus from Eucalyptus (Australia). Morphological and culture characteristics along with ITS DNA barcodes are also provided. © 2012 Nationaal Herbarium Nederland & Centraalbureau voor Schimmelcultures.

Variation in water extraction with maize plant density and its impact on model application

The use of maize simulation models to determine the optimum plant population for rainfed environments allows the evaluation of plant populations over multiple years and locations at a lower cost than traditional field experimentation. However the APSIM maize model that has been used to conduct some of these 'virtual' experiments assumes that the maximum rate of soil water extraction by the crop root system is constant across plant populations. This untested assumption may cause grain yield to be overestimated in lower plant populations. A field experiment was conducted to determine whether maximum rates of water extraction vary with plant population, and the maximum rate of soil water extraction was estimated for three plant populations (2.4, 3.5 and 5.5 plants m(-2)) under water limited conditions. Maximum soil water extraction rates in the field experiment decreased linearly with plant population, and no difference was detected between plant populations for the crop lower limit of soil water extraction. Re-analysis of previous maize simulation experiments demonstrated that the use of inappropriately high extraction-rate parameters at low plant populations inflated predictions of grain yield, and could cause erroneous recommendations to be made for plant population. The results demonstrate the importance of validating crop simulation models across the range of intended treatments. (C) 2013 Elsevier E.V. All rights reserved.

Phyllosticta species on orchids (Orchidaceae), introducing Phyllosticta speewahensis sp. nov. (Phyllostictaceae, Ascomycota) from northern Australia

Several species of Phyllosticta (syn. Guignardia) have been described from orchids worldwide. A new species, Phyllosticta speewahensis, is proposed for a specimen isolated from leaf spots on a hybrid Vanda orchid in northern Queensland, Australia. Phylogenetic analysis of the nrDNA internal transcribed spacer region (ITS) and partial translation elongation factor 1-alpha (TEF1) gene sequences showed that P. speewahensis is most closely related to P. hostae. The likelihood that orchids harbour further cryptic species of endophytic and pathogenic Phyllosticta species is discussed.

Molecular diversity of Chickpea chlorotic dwarf virus in Sudan: High rates of intra-species recombination – a driving force in the emergence of new strains

In Sudan Chickpea chlorotic dwarf virus (CpCDV, genus Mastrevirus, family Geminiviridae) is an important pathogen of pulses that are grown both for local consumption, and for export. Although a few studies have characterised CpCDV genomes from countries in the Middle East, Africa and the Indian subcontinent, little is known about CpCDV diversity in any of the major chickpea production areas in these regions. Here we analyse the diversity of 146 CpCDV isolates characterised from pulses collected across the chickpea growing regions of Sudan. Although we find that seven of the twelve known CpCDV strains are present within the country, strain CpCDV-H alone accounted for ∼73% of the infections analysed. Additionally we identified four new strains (CpCDV-M, -N, -O and -P) and show that recombination has played a significant role in the diversification of CpCDV, at least in this region. Accounting for observed recombination events, we use the large amounts of data generated here to compare patterns of natural selection within protein coding regions of CpCDV and other dicot-infecting mastrevirus species.