Infection by Alternaria alternata causes discolouration of Backhousia myrtifolia foliage and flowers.

The pathogenicity of three isolates of Alternaria alternata from Backhousia myrtifolia leaves was characterised and compared. Isolate BRIP 52222 was virulent compared to isolates BRIP 52223 and BRIP 52221. A comparison of inoculation methods showed that abrasion was more effective at establishing an infection than puncture wounding. Koch's postulates were assessed to confirm the pathogenicity of A. alternata on B. myrtifolia foliage and floral tissues using a conidial suspension of the most virulent isolate. Sporulation was triggered by incubating A. alternata (BRIP 52222) at 28 degrees C for 10 d under alternating 12 h black-light/12 h dark conditions on half-strength potato dextrose agar (PDA). In contrast, incubation of A. alternata under continuous black-light on either half- or full-strength PDA did not yield conidia. Host symptoms caused by inoculation with the pathogen included a brown-black discolouration of both foliage and floral tissues. Microscopic examination of cellular structures suggested that perturbation of oil glands may contribute to the tissue discolouration in B. myrtifolia caused by A. alternata infection. Oil gland structures can be disrupted during an active A. alternata infection, causing the leakage of essential oil followed by discolouration.

Comparison of bioassay responses to the potential fungal biopesticide Metarhizium anisopliae in Rhipicephalus(Boophilus) microplus and Lucilia cuprina.

Quantal response bioassays were conducted with cattle ticks and sheep blowflies with three different isolates of Metarhizium anisopliae and different methods of inoculation. Ticks were either topically dosed with 2 mu l or immersed in the conidial preparations. Blowflies were either topically dosed with 2 mu l of the conidial preparation or fed on conidia mixed with sugar. Probit analyses were carried out on the mortality data to compare the virulence of these isolates to ticks and blowflies and look for indications of different virulence mechanisms employed by M. anisopliae isolates when invading these hosts. One isolate (ARIM16) showed high virulence to both hosts killing 95% of ticks after 2 days and 88 (+/- 2)% of blowflies after 4 days. Strikingly different mortality patterns indicated that virulence is dependent on different mechanisms in ticks and blowflies. The pattern of mortality seen with ticks suggested that the number of conidia adhering per unit area of the cuticle was more important for rapid tick death than the total number of conidia contacting the entire tick surface. Blowflies fed conidia mixed with food died rapidly after an initial lag phase regardless of dose.

Comparative analysis of Tritrichomonas foetus (Riedmuller, 1928) cat genotype, T. foetus (Riedmuller, 1928) cattle genotype and Tritrichomonas suis (Davaine, 1875) at 10 DNA loci

The parasitic protists in the genus Tritrichomonas cause significant disease in domestic cattle and cats. To assess the genetic diversity of feline and bovine isolates of Tritrichomonas foetus (Riedmuller, 1928) Wenrich and Emmerson, 1933, we used 10 different genetic regions, namely the protein coding genes of cysteine proteases 1,2 and 4-9 (CP1, 2, 4-9) involved in the pathogenesis of the disease caused by the parasite. The cytosolic malate dehydrogenase 1 (MDH1) and internal transcribed spacer region 2 of the rDNA unit (ITS2) were included as additional markers. The gene sequences were compared with those of Tritrichomonas suis (Davaine. 1875) Morgan and Hawkins, 1948 and Tritrichomonas mobilensis Culberson et al., 1986. The study revealed 100% identity for all 10 genes among all feline isolates (=T. foetus cat genotype), 100% identity among all bovine isolates (=T. foetus cattle genotype) and a genetic distinctness of 1% between the cat and cattle genotypes of T. foetus. The cattle genotype of T. foetus was 100% identical to T. suis at nine loci (CP1, 2,4-8, ITS2, MDH1). At CP9, three out of four T. suis isolates were identical to the T. foetus cattle genotype, while the T. suis isolate SUI-H3B sequence contained a single unique nucleotide substitution. Tritrichomonas mobilensis was 0.4% and 0.7% distinct from the cat and cattle genotypes of T. foetus, respectively. The genetic differences resulted in amino acid changes in the CP genes, most pronouncedly in CP2, potentially providing a platform for elucidation of genotype-specific host-pathogen interactions of T. foetus. On the basis of this data we judge T. suis and T. foetus to be subjective synonyms. For the first time, on objective nomenclatural grounds, the authority of T. suis is given to Davaine, 1875, rather than the commonly cited Gruby and Delafond, 1843. To maintain prevailing usage of T. foetus, we are suppressing the senior synomym T. suis Davaine, 1875 according to Article 23.9, because it has never been used as a valid name after 1899 and T. foetus is widely discussed as the cause of bovine trichomonosis. Thus bovine, feline and porcine isolates should all be given the name T. foetus. This promotes the stability of T. foetus for the veterinary and economically significant venereal parasite causing bovine trichomonosis. (C) 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Evidence of bat origin for Menangle virus, a zoonotic paramyxovirus first isolated from diseased pigs

Menangle virus (MenPV) is a zoonotic paramyxovirus capable of causing disease in pigs and humans. It was first isolated in 1997 from stillborn piglets at a commercial piggery in New South Wales, Australia, where an outbreak of reproductive disease occurred. Neutralizing antibodies to MenPV were detected in various pteropid bat species in Australia and fruit bats were suspected to be the source of the virus responsible for the outbreak in pigs. However, previous attempts to isolate MenPV from various fruit bat species proved fruitless. Here, we report the isolation of MenPV from urine samples of the black flying fox, Pteropus alecto, using a combination of improved procedures and newly established bat cell lines. The nucleotide sequence of the bat isolate is 94% identical to the pig isolate. This finding provides strong evidence supporting the hypothesis that the MenPV outbreak in pigs originated from viruses in bats roosting near the piggery. © 2012 Printed in Great Britain.

In planta localization and interactions of impatiens necrotic spot tospovirus proteins

Impatiens necrotic spot tospovirus (INSV) is a significant pathogen of ornamentals. The tripartite negative- and ambi-sense RNA genome encodes six proteins that are involved in cytoplasmic replication, movement, assembly, insect transmission and defence. To gain insight into the associations of these viral proteins, we determined their intracellular localization and interactions in living plant cells. Nucleotide sequences encoding the nucleoprotein N, non-structural proteins NSs and NSm, and glycoproteins Gn and Gc of a Kentucky isolate of INSV were amplified by RTPCR, cloned, sequenced and transiently expressed as fusions with autofluorescent proteins in leaf epidermal cells of Nicotiana benthamiana. All proteins accumulated at the cell periphery and co-localized with an endoplasmic reticulum marker. The Gc protein fusion also localized to the nucleus. N and NSm protein self-interactions and an NSm-N interaction were observed by using bimolecular fluorescence complementation. A tospovirus NSm homotypic interaction had not been reported previously.

Electrotransformation of Haemophilus parasuis with in vitro modified DNA based on a novel shuttle vector

The objective of the present study was to establish a valid transformation method of Haemophilus parasuis, the causative agent of Glasser's disease in pigs, using a novel H. parasuis-Escherichia coli shuttle vector. A 4.2 kb endogenous plasmid pYC93 was extracted from an H. parasuis field isolate and completely sequenced. Analysis of pYC93 revealed a region approximately 800 bp showing high homology with the defined replication origin oriV of pLS88, a native plasmid identified in Haemophilus ducreyi. Based on the origin region of pYC93, E. coli cloning vector pBluescript SK(+) and the Tn903 derived kanamycin cassette, a shuttle vector pSHK4 was constructed by overlapping PCR strategy. When electroporation of the 15 H. parasuis serovar reference strains and one clinical isolate SH0165 with pSHK4 was performed, only one of these strains yielded transformants with an efficiency of 8.5 x 10(2) CFUhlg of DNA. Transformation efficiency was notably increased (1.3 x 10(5) CFU/mu g of DNA) with vector DNA reisolated from the homologous transformants. This demonstrated that restriction-modification systems were involved in the barrier to transformation of H. parasuis. By utilizing an in vitro DNA modification method with cell-free extracts of the host H. parasuis strains, 15 out of 16 strains were transformable. The novel shuttle vector pSHK4 and the established electrotransformation method constitute useful tools for the genetic manipulation of H. parasuis to gain a better understanding of the pathogen. (C) 2011 Elsevier B.V. All rights reserved.

Confirmed case of encephalitis caused by Murray Valley encephalitis virus infection in a horse

A 5-year-old Australian stock horse in Monto, Queensland, Australia, developed neurological signs and was euthanized after a 6-day course of illness. Histological examination of the brain and spinal cord revealed moderate to severe subacute, nonsuppurative encephalomyelitis. Sections of spinal cord stained positively in immunohistochemistry with a flavivirus-specific monoclonal antibody. Reverse transcription polymerase chain reaction assay targeting the envelope gene of flavivirus yielded positive results from brain, spinal cord, cerebrospinal fluid, and facial nerve. A flavivirus was isolated from the cerebrum and spinal cord. Nucleotide sequences obtained from amplicons from both tissues and virus isolated in cell culture were compared with those in GenBank and had 96-98% identity with Murray Valley encephalitis virus. The partial envelope gene sequence of the viral isolate clustered into genotype 1 and was most closely related to a previous Queensland isolate.

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

Encephalomyocarditis virus infection in a splenectomised calf

Background Encephalomyocarditis (EMC) caused by EMC virus (EMCV) was diagnosed in a 5-month-old splenectomised calf, which died suddenly on an experimental farm that had a high infestation of rodents. Results At postmortem examination, the lungs were dark purple and diffusely congested. On histological examination, the calf had severe necrotising myocarditis. EMCV was isolated from the heart. The polyprotein gene of the EMCV isolate was amplified by PCR and had 85–91% identity with published EMCV sequences, including 89% identity with isolates from Queensland. On phylogenetic analysis, the polyprotein gene had highest sequence identity with South Korean EMCV strain, CBNU. Conclusion This is the first report of naturally occurring EMC in cattle in Australia and the first report of naturally occurring bovine EMC from which EMCV has been isolated.

Pathogenic variation of Alternaria species associated with leaf blotch and fruit spot of apple in Australia

Four Alternaria species groups (A. longipes, A. arborescens, A. alternata/A. tenuissima and A. tenuissima/A. mali) are associated with leaf blotch and fruit spot of apple in Australia. There is no information on the variability of pathogenicity among the species and isolates within each species causing leaf blotch or fruit spot. We used a detached leaf assay and an in planta fruit inoculation assay to determine the pathogenicity and virulence of the four Alternaria species. Our results showed that isolates within the same species were not specific to either leaf or fruit tissue and showed great variability in pathogenicity and virulence, indicating cross-pathogenicity, which may be isolate dependent rather than species dependent. Generally, virulence of A. tenuissima and A. alternata isolates on leaf and fruit was higher than other species. Isolates of all species groups were pathogenic on leaves of different cultivars, but pathogenicity on fruit of different cultivars varied among isolates and species. Implications of our findings on prevalence of the diseases in different apple-producing regions in Australia and the development of targeted disease management of the diseases are discussed

Large-chamber methane and nitrous oxide measurements are comparable to the backward Lagrangian stochastic method

Measurement of individual emission sources (e.g., animals or pen manure) within intensive livestock enterprises is necessary to test emission calculation protocols and to identify targets for decreased emissions. In this study, a vented, fabric-covered large chamber (4.5 × 4.5 m, 1.5 m high; encompassing greater spatial variability than a smaller chamber) in combination with on-line analysis (nitrous oxide [N2O] and methane [CH4] via Fourier Transform Infrared Spectroscopy; 1 analysis min-1) was tested as a means to isolate and measure emissions from beef feedlot pen manure sources. An exponential model relating chamber concentrations to ambient gas concentrations, air exchange (e.g., due to poor sealing with the surface; model linear when ≈ 0 m3 s-1), and chamber dimensions allowed data to be fitted with high confidence. Alternating manure source emission measurements using the large-chamber and the backward Lagrangian stochastic (bLS) technique (5-mo period; bLS validated via tracer gas release, recovery 94-104%) produced comparable N2O and CH4 emission values (no significant difference at P < 0.05). Greater precision of individual measurements was achieved via the large chamber than for the bLS (mean ± standard error of variance components: bLS half-hour measurements, 99.5 ± 325 mg CH4 s-1 and 9.26 ± 20.6 mg N2O s-1; large-chamber measurements, 99.6 ± 64.2 mg CH4 s-1 and 8.18 ± 0.3 mg N2O s-1). The large-chamber design is suitable for measurement of emissions from manure on pen surfaces, isolating these emissions from surrounding emission sources, including enteric emissions. © © American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America.

High-throughput prediction of eucalypt lignin syringyl/guaiacyl content using multivariate analysis: a comparison between mid-infrared, near-infrared, and Raman spectroscopies for model development

BACKGROUND: In order to rapidly and efficiently screen potential biofuel feedstock candidates for quintessential traits, robust high-throughput analytical techniques must be developed and honed. The traditional methods of measuring lignin syringyl/guaiacyl (S/G) ratio can be laborious, involve hazardous reagents, and/or be destructive. Vibrational spectroscopy can furnish high-throughput instrumentation without the limitations of the traditional techniques. Spectral data from mid-infrared, near-infrared, and Raman spectroscopies was combined with S/G ratios, obtained using pyrolysis molecular beam mass spectrometry, from 245 different eucalypt and Acacia trees across 17 species. Iterations of spectral processing allowed the assembly of robust predictive models using partial least squares (PLS). RESULTS: The PLS models were rigorously evaluated using three different randomly generated calibration and validation sets for each spectral processing approach. Root mean standard errors of prediction for validation sets were lowest for models comprised of Raman (0.13 to 0.16) and mid-infrared (0.13 to 0.15) spectral data, while near-infrared spectroscopy led to more erroneous predictions (0.18 to 0.21). Correlation coefficients (r) for the validation sets followed a similar pattern: Raman (0.89 to 0.91), mid-infrared (0.87 to 0.91), and near-infrared (0.79 to 0.82). These statistics signify that Raman and mid-infrared spectroscopy led to the most accurate predictions of S/G ratio in a diverse consortium of feedstocks. CONCLUSION: Eucalypts present an attractive option for biofuel and biochemical production. Given the assortment of over 900 different species of Eucalyptus and Corymbia, in addition to various species of Acacia, it is necessary to isolate those possessing ideal biofuel traits. This research has demonstrated the validity of vibrational spectroscopy to efficiently partition different potential biofuel feedstocks according to lignin S/G ratio, significantly reducing experiment and analysis time and expense while providing non-destructive, accurate, global, predictive models encompassing a diverse array of feedstocks.

Variable-number tandem repeats genotyping used to aid and inform management strategies for a bovine Johne's disease incursion in tropical and subtropical Australia

The application of variable-number tandem repeats (VNTR) genotyping of Mycobacterium avium subsp. paratuberculosis isolates to assist in investigating incidents of bovine Johne’s disease in a low-prevalence region of Australia is described in the current study. Isolates from a response to detection of bovine Johne’s disease in Queensland were compared with strains from national and international sources. The tandem application of mycobacterial interspersed repetitive unit (MIRU) and multilocus short sequence repeats (MLSSR) genotyping identified 2 strains, 1 that infected cattle on multiple properties with trace-forward histories from a common infected property, and 1 genotypically different strain recovered from a single property. The former strain showed an identical genotype to an isolate from India. Neither strain showed a genotypic link to regions of Australia with a higher prevalence of the disease. Genotyping has indicated incursions from 2 independent sources. This intelligence has informed investigations into potential routes of entry and the soundness of ongoing control measures, and supported strategy and policy decisions regarding management of Mycobacterium avium subsp. paratuberculosis incursions for Queensland.

Infection and pathology in Queensland grouper, Epinephelus lanceolatus, (Bloch), caused by exposure to Streptococcus agalactiae via different routes

Since 2007, 96 wild Queensland groupers, Epinephelus lanceolatus, (Bloch), have been found dead in NE Australia. In some cases, Streptococcus agalactiae (Group B Streptococcus, GBS) was isolated. At present, a GBS isolate from a wild grouper case was employed in experimental challenge trials in hatchery-reared Queensland grouper by different routes of exposure. Injection resulted in rapid development of clinical signs including bilateral exophthalmia, hyperaemic skin or fins and abnormal swimming. Death occurred in, and GBS was re-isolated from, 98% fish injected and was detected by PCR in brain, head kidney and spleen from all fish, regardless of challenge dose. Challenge by immersion resulted in lower morbidity with a clear dose response. Whilst infection was established via oral challenge by admixture with feed, no mortality occurred. Histology showed pathology consistent with GBS infection in organs examined from all injected fish, from fish challenged with medium and high doses by immersion, and from high-dose oral challenge. These experimental challenges demonstrated that GBS isolated from wild Queensland grouper reproduced disease in experimentally challenged fish and resulted in pathology that was consistent with that seen in wild Queensland grouper infected with S. agalactiae.

ScreeningCorymbiapopulations for resistance toPuccinia psidii

Disease screening to determine the threat Puccinia psidii poses to plantation and native eucalypts in Australia was undertaken in half-sib families of two contrasting eucalypt species, Eucalyptus cloeziana and E. argophloia. Artificial inoculation with a single-lesion isolate of P. psidii was used to screen these species for resistance to the biotype of P. psidii established in Australia. The objective was to characterize resistance to P. psidii within these two distinct species: E. argophloia, a vulnerable species with a narrow distribution, and E. cloeziana, a species with a broad and extensive distribution in Queensland. Results for E. cloeziana indicate that inland provenances are more resistant to P. psidii infection than provenances from coastal regions. Heritability estimates for the two assessment systems used (resistance on a 1-to-5 ordinal scale verses resistance on a 0-to-1 binomial scale) were low to high (0.24 to 0.63) for E. argophloia and moderate to high (0.4 to 0.91) for E. cloeziana, indicating a significant level of additive genetic variance for rust resistance within the populations. This study demonstrates the potential to select resistant families within the tested populations and indicates that P. psidii could detrimentally affect these species in native forests, nurseries, and plantations.