Screening bacterial metabolites for inhibitory effects against Batrachochytrium dendrobatidis using a spectrophotometric assay

Certain bacteria present on frog skin can prevent infection by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), conferring disease resistance. Previous studies have used agar-based in vitro challenge assays to screen bacteria for Bd-inhibitory activity and to identify candidates for bacterial supplementation trials. However, agar-based assays can be difficult to set up and to replicate reliably. To overcome these difficulties, we developed a semi-quantitative spectrophotometric challenge assay technique. Cell-free supernatants were prepared from filtered bacterial cultures and added to 96-well plates in replicated wells containing Bd zoospores suspended in tryptone-gelatin hydrolysate-lactose (TGhL) broth medium. Plates were then read daily on a spectrophotometer until positive controls reached maximum growth in order to determine growth curves for Bd. We tested the technique by screening skin bacteria from the Australian green-eyed tree frog Litoria serrata. Of bacteria tested, 31% showed some degree of Bd inhibition, while some may have promoted Bd growth, a previously unknown effect. Our cell-free supernatant challenge assay technique is an effective in vitro method for screening bacterial isolates for strong Bd-inhibitory activity. It contributes to the expanding field of bioaugmentation research, which could play a significant role in mitigating the effects of chytridiomycosis on amphibians around the world.

Current and emerging screening methods to identify post-head-emergence frost adaptation in wheat and barley

Cereal crops can suffer substantial damage if frosts occur at heading. Identification of post-head-emergence frost (PHEF) resistance in cereals poses a number of unique and difficult challenges. Many decades of research have failed to identify genotypes with PHEF resistance that could offer economically significant benefit to growers. Research and breeding gains have been limited by the available screening systems. Using traditional frost screening systems, genotypes that escape frost injury in trials due to spatial temperature differences and/or small differences in phenology can be misidentified as resistant. We believe that by improving techniques to minimize frost escapes, such ofalse-positive' results can be confidently identified and eliminated. Artificial freezing chambers or manipulated natural frost treatments offer many potential advantages but are not yet at the stage where they can be reliably used for frost screening in breeding programmes. Here we describe the development of a novel photoperiod gradient method (PGM) that facilitates screening of genotypes of different phenology under natural field frosts at matched developmental stages. By identifying frost escapes and increasing the efficiency of field screening, the PGM ensures that research effort can be focused on finding genotypes with improved PHEF resistance. To maximize the likelihood of identifying PHEF resistance, we propose that the PGM form part of an integrated strategy to (i) source germplasm;(ii) facilitate high throughput screening; and (iii) permit detailed validation. PGM may also be useful in other studies where either a range of developmental stages and/or synchronized development are desired.

Pericarps retained in the tree canopy and stomatal abundance are components of resistance to husk spot caused by Pseudocercospora macadamiae in macadamia

Pseudocercospora macadamiae Beilharz, Mayers and Pascoe infects macadamia fruit via stomata causing husk spot disease. Information on the variability of fruit stomatal abundance, its association with diseased fruit pericarps (sticktights) that are retained in the tree canopy, and its influence on the husk spot intensity (incidence, severity and lesion number) among macadamia genotypes is lacking. We examined a total of 230 macadamia trees comprising 19 cultivars, 56 wild germplasm accessions and 40 breeding progeny, for the prevalence of sticktights and husk spot intensity over three production seasons. We observed a strong association between the prevalence of sticktights and disease intensity indicating its usefulness as a predictor of husk spot and as a useful phenotypic trait for husk spot resistance selection in breeding programmes. Similarly, stomatal abundance varied among macadamia genotypes, and a significant linear relationship (P < 0.001; 93%) was observed between fruit stomatal abundance and husk spot for all the macadamia genotypes analysed, confirming the utility of that trait for disease resistance screening. The genotypes were grouped into disease resistance groups. Correlations between fruit stomatal abundance, disease intensity and prevalence of sticktights revealed that the numbers of sticktights, and relative stomatal abundance were the main factors influencing the intensity of husk spot among macadamia genotypes. This is the first comprehensive study of natural variation of stomatal abundance in Macadamia species that reveals genetic variation, and provides relevant relationships with disease intensity and the prevalence of sticktights. The phenotypic plant traits indentified in this study may serve as selection tools for disease resistance screening in macadamia breeding programmes.

Differential recognition by tick-resistant cattle of the recombinantly expressed Rhipicephalus microplus serine protease inhibitor-3 (RMS-3)

Rhipicephalus micro plus is an important bovine ectoparasite, widely distributed in tropical and subtropical regions of the world causing large economic losses to the cattle industry. Its success as an ectoparasite is associated with its capacity to disarm the antihemostatic and anti-inflammatory reactions of the host. Serpins are protease inhibitors with an important role in the modulation of host-parasite interactions. The cDNA that encodes for a R. microplus serpin was isolated by RACE and subsequently cloned into the pPICZ alpha A vector. Sequence analysis of the cDNA and predicted amino acid showed that this cDNA has a conserved serpin domain. B- and T-cell epitopes were predicted using bioinformatics tools. The recombinant R. microplus serpin (rRMS-3) was secreted into the culture media of Pichia pastoris after methanol induction at 0.2 mg l(-1) qRT-PCR expression analysis of tissues and life cycle stages demonstrated that RMS-3 was mainly expressed in the salivary glands of female adult ticks. Immunological recognition of the rRMS-3 and predicted B-cell epitopes was tested using tick-resistant and susceptible cattle sera. Only sera from tick-resistant bovines recognized the B-cell epitope AHYNPPPPIEFT (Seq7). The recombinant RMS-3 was expressed in P. pastoris, and ELISA screening also showed higher recognition by tick-resistant bovine sera. The results obtained suggest that RMS-3 is highly and specifically secreted into the bite site of R. microplus feeding on tick-resistant bovines. Capillary feeding of semi-engorged ticks with anti-AHYNPPPPIEFT sheep sera led to an 81.16% reduction in the reproduction capacity of R. microplus. Therefore, it is possible to conclude that R. microplus serpin (RMS-3) has an important role in the host-parasite interaction to overcome the immune responses in resistant cattle. (C) 2012 Elsevier GmbH. All rights reserved.

Screening Corymbia populations for resistance to Puccinia psidii

The exotic rust pathogen Puccinia psidii is now widespread along the east coast of Australia from temperate Victoria to tropical far north Queensland, with a current host range exceeding 200 species from 37 myrtaceous genera. To determine the threat P. psidii poses to plantation and native eucalypts, artificial inoculation was used to screen germplasm of spotted gum (Corymbia spp.) for resistance to the biotype of P. psidii that has become established in Australia. The objective was to characterize resistance to P. psidii within the Corymbia species complex so that management strategies for the deployment of germplasm from existing breeding programmes of these spotted gum species could be developed. Symptom development initiated 7 days after inoculation, with resistant and susceptible seedlings identified within all species, provenances and families. Inter- and intraspecific variability in rust resistance was observed among spotted gum species. There was no apparent relationship between climatic conditions at the provenance origin and disease resistance. The heritability estimates for all assessments are moderate to high and indicate a significant level of additive genetic variance for rust resistance within the populations. The results of this study clearly identify potential to select for resistance at the family level within the tested populations. While the potential for P. psidii to detrimentally impact upon Corymbia in the nursery and in young plantations was demonstrated, estimations of the heritability of resistance suggest that efforts to enhance this trait through breeding have reasonable prospects for success.

Rhipicephalus (Boophilus) microplus tick in vitro feeding methods for functional (dsRNA) and vaccine candidate (antibody) screening

Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) ticks cause economic losses for cattle industries throughout tropical and subtropical regions of the world estimated at $US2.5 billion annually. Lack of access to efficacious long-lasting vaccination regimes and increases in tick acaricide resistance have led to the investigation of targets for the development of novel tick vaccines and treatments. In vitro tick feeding has been used for many tick species to study the effect of new acaricides on the transmission of tick-borne pathogens. Few studies have reported the use of in vitro feeding for functional genomic studies using RNA interference and/or the effect of specific anti-tick antibodies. In particular, in vitro feeding reports for the cattle tick are limited due to its relatively short hypostome. Previously published methods were further modified to broaden optimal tick sizes/weights, feeding sources including bovine and ovine serum, optimisation of commercially available blood anti-coagulant tubes, and IgG concentrations for effective antibody delivery. Ticks are fed overnight and monitored for ∼5–6 weeks to determine egg output and success of larval emergence using a humidified incubator. Lithium-heparin blood tubes provided the most reliable anti-coagulant for bovine blood feeding compared with commercial citrated (CPDA) and EDTA tubes. Although >30 mg semi-engorged ticks fed more reliably, ticks as small as 15 mg also fed to repletion to lay viable eggs. Ticks which gained less than ∼10 mg during in vitro feeding typically did not lay eggs. One mg/ml IgG from Bm86-vaccinated cattle produced a potent anti-tick effect in vitro (83% efficacy) similar to that observed in vivo. Alternatively, feeding of dsRNA targeting Bm86 did not demonstrate anti-tick effects (11% efficacy) compared with the potent effects of ubiquitin dsRNA. This study optimises R. microplus tick in vitro feeding methods which support the development of cattle tick vaccines and treatments.

Status, challenges and tools for identification and diagnosis of Peronosclerospora and Sclerophthora of regulatory concern for graminicolous crops

Graminicolous Downy Mildew (GDM) diseases caused by the genera Peronosclerospora (13 spp.) and Sclerophthora (6 spp. and 1 variety) are poorly studied but destructive diseases of major crops such as corn, sorghum, sugarcane and other graminoids. Eight of the 13 described Peronosclerospora spp. are able to infect corn. In particular, P. philippinensis (= P. sacchari), P. maydis, P. heteropogonis, and S. rayssiae var. zeae cause major losses in corn yields in tropical Asia. In 2012 a new species, P. australiensis, was described based on isolates previously identified as P. maydis in Australia; this species is now a pathogen of major concern. Despite the strong impact of GDM diseases, there are presently no reliable molecular methods available for their detection. GDM pathogens are among the most difficult Oomycetes to identify using molecular tools, as their taxonomy is very challenging, and little genetic sequence data are available for development of molecular tools to detect GDM pathogens to species level. For example, from over 15 genes used in identification, diagnostics or phylogeny of Phytophthora, only ITS1 and cox2 show promise for use with GDM pathogens. Multiplex/multigene conventional and qPCR assays are currently under evaluation for the detection of economically important GDM spp. Scientists from the USA, Germany, Canada, Australia, and the Philippines are collaborating on the development and testing of diagnostic tools for these pathogens of concern.

Screening and breeding for genetic resistance to anthracnose in mango

Postharvest diseases remain a significant constraint to the transport, storage and marketing of mangoes. The two main ones are anthracnose and stem end rot. Anthracnose caused by Colletotrichum gloeosporioides is the more wide-spread of the two. Varieties within Mangifera indica are known to vary in their level of reactions to anthracnose; however, the best tolerance in current commercial cultivars is not sufficient to eliminate the need for pre- and postharvest fungicides treatments. A screening program was initiated in mango accessions in the Australian National Mango Genebank to look for any significant resistance to C. gloeosporioides in fruit as they ripened. Screening was conducted by rating reactions to natural infection of anthracnose and reactions to artificially inoculating fruit with virulent isolates of C. gloeosporioides. A range of reactions to the pathogen were identified, with strong resistance found in one accession of the species M. laurina. This accession was used as the pollen parent in a controlled crossing program with a M. indica hybrid from the Australian Mango Breeding Program (AMBP). Sixty successful hybrids between the species have been generated. The hybrid population will be screened for resistance to anthracnose and used for gene discovery investigations to identify markers for anthracnose resistance.

Screening Eucalyptus cloeziana and E. argophloia populations for resistance to Puccinia 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. 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.

Commercial-scale Alternaria brown spot resistance screening as the first step in breeding new mandarins for Australia

Rapid screening tests and an appreciation of the simple genetic control of Alternaria brown spot (ABS) susceptibility have existed for many years, and yet the application of this knowledge to commercial-scale breeding programs has been limited. Detached leaf assays were first demonstrated more than 40 years ago and reliable data suggesting a single gene determining susceptibility has been emerging for at least 20 years. However it is only recently that the requirement for genetic resistance in new hybrids has become a priority, following increased disease prevalence in Australian mandarin production areas previously considered too dry for the pathogen. Almost all of the high-fruit-quality parents developed so far by the Queensland-based breeding program are susceptible to ABS necessitating the screening of their progeny to avoid commercialisation of susceptible hybrids. This is done effectively and efficiently by spraying 3-6 month old hybrid seedlings with a spore suspension derived from a toxin-producing field isolate of Alternaria alternate, then incubating these seedlings in a cool room at 25°C and high humidity for 5 days. Susceptible seedlings show clear disease symptoms and are discarded. Analysis of observed and expected segregation ratios loosely support the hypothesis for a single dominant gene for susceptibility, but do not rule out the possibility of alternative genetic models. After implementing the routine screening for ABS resistance for three seasons we now have more than 20,000 hybrids growing in field progeny blocks that have been screened for resistance to the ABS disease.

Assessment of functional forms of crop yield loss models of invasive plant species applied in decision support tools and bioeconomic modelling

AbstractObjectives Decision support tools (DSTs) for invasive species management have had limited success in producing convincing results and meeting users' expectations. The problems could be linked to the functional form of model which represents the dynamic relationship between the invasive species and crop yield loss in the DSTs. The objectives of this study were: a) to compile and review the models tested on field experiments and applied to DSTs; and b) to do an empirical evaluation of some popular models and alternatives. Design and methods This study surveyed the literature and documented strengths and weaknesses of the functional forms of yield loss models. Some widely used models (linear, relative yield and hyperbolic models) and two potentially useful models (the double-scaled and density-scaled models) were evaluated for a wide range of weed densities, maximum potential yield loss and maximum yield loss per weed. Results Popular functional forms include hyperbolic, sigmoid, linear, quadratic and inverse models. Many basic models were modified to account for the effect of important factors (weather, tillage and growth stage of crop at weed emergence) influencing weed–crop interaction and to improve prediction accuracy. This limited their applicability for use in DSTs as they became less generalized in nature and often were applicable to a much narrower range of conditions than would be encountered in the use of DSTs. These factors' effects could be better accounted by using other techniques. Among the model empirically assessed, the linear model is a very simple model which appears to work well at sparse weed densities, but it produces unrealistic behaviour at high densities. The relative-yield model exhibits expected behaviour at high densities and high levels of maximum yield loss per weed but probably underestimates yield loss at low to intermediate densities. The hyperbolic model demonstrated reasonable behaviour at lower weed densities, but produced biologically unreasonable behaviour at low rates of loss per weed and high yield loss at the maximum weed density. The density-scaled model is not sensitive to the yield loss at maximum weed density in terms of the number of weeds that will produce a certain proportion of that maximum yield loss. The double-scaled model appeared to produce more robust estimates of the impact of weeds under a wide range of conditions. Conclusions Previously tested functional forms exhibit problems for use in DSTs for crop yield loss modelling. Of the models evaluated, the double-scaled model exhibits desirable qualitative behaviour under most circumstances.

Investigation of an emerging bacterial disease in wild Queensland groper, marine fish and stingrays with production of diagnostic tools to reduce the spread of disease to other states of Australia : final report

This project describes how Streptococcus agalactiae can be transmitted experimentally in Queensland grouper. The implications of this research furthers the relatedness between Australian S. agalactiae strains from animals and humans. Additionally, this research has developed diagnostic tools for Australian State Veterinary Laboratories and Universities, which will assist in State and National aquatic animal disease detection, surveillance, disease monitoring and reporting