Rapid typing of P multocida

Fowl cholera, caused by P. multocida, is a serious disease of poultry with sudden surges in mortality and an emerging disease of the free ranged poultry industries. This project will develop a more rapid and cost effective screening method for P. multocida. The impacts of this new method are manifold: It will lead to an improved understanding of the epidemiology of fowl cholera and the possible sources of entry onto the farm leading to improved biosecurity measures and control programs. Another impact is improved serotyping, which will ensure more effective and targeted vaccination programs. Improving prevention and control programs and decreasing the reliance on antibiotics will enhance the sustainability and profitability of the industry.

Barley Grains Defects (Blackpoint, Pre-harvest sprouting, Kernal staining)

Screening new and existing breeding germplasm and cultivars for grain defect tolerance for breeding programs, evaluate new methods and technologies to screen more effectively for the barley grains defects - pre-harvest sprouting, blackpoint, kernel discolouration, and investigate genetic mechanisms involved in controlling barley grain defect tolerance.

DAQ00152 Herbicide Tolerance Screening in Northern Region (Phase IV)

The previous projects (phase I - III) highlighted that northern region wheat and barley cultivars differ considerably in their sensitivity to herbicides. The new project will focus on increased screening of advanced breeding lines and new cultivars lines to commonly used herbicides, for barley, chickpea and wheat. Studies on impact of environment on herbicide x genotype responses will also be undertaken with the national team. The new information will be added to the existing information package on herbicide tolerance. Thus, adverse impacts of herbicides on productivity in northern region will be reduced, as growers and agronomists will select safer herbicides for their sown variety, or select more tolerant varieties for their important herbicides.

National screening for barley grain defects

Three defects on barley grain can impact on the price paid to grain growers. Black point (BP), kernel staining (KS) and pre-harvest sprouting (PHS) can result in malting barley being downgraded to feed. Resistance to these defects is the best option, and in this project hundreds of breeding lines grown over three years were screened for these traits. A number of lines exhibited resistance to each defect but very few had resistance to all defects. The results from the screening program have been provided to the Australian barley breeders through the Barley Breeding Australia (BBA) program.

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.

Genetic control of nodal root angle in sorghum and its implications on water extraction

Genotypic variability in root system architecture has been associated with root angle of seedlings and water extraction patterns of mature plants in a range of crops. The potential inclusion of root angle as a selection criterion in a sorghum breeding program requires (1) availability of an efficient screening method, (2) presence of genotypic variation with high heritability, and (3) an association with water extraction pattern. The aim of this study was to determine the feasibility for inclusion of nodal root angle as a selection criterion in sorghum breeding programs. A high-throughput phenotypic screen for nodal root angle in young sorghum plants has recently been developed and has been used successfully to identify significant variation in nodal root angle across a diverse range of inbred lines and a mapping population. In both cases, heritabilities for nodal root angle were high. No association between nodal root angle and plant size was detected. This implies that parental inbred lines could potentially be used to asses nodal root angle of their hybrids, although such predictability is compromised by significant interactions. To study effects of nodal root angle on water extraction patterns of mature plants, four inbred lines with contrasting nodal root angle at seedling stage were grown until at least anthesis in large rhizotrons. A consistent trend was observed that nodal root angle may affect the spatial distribution of root mass of mature plants and hence their ability to extract soil water, although genotypic differences were not significant. The potential implications of this for specific adaptation to drought stress are discussed. Results suggest that nodal root angle of young plants can be a useful selection criterion for specific drought adaptation, and could potentially be used in molecular breeding programs if QTLs for root angle can be identified. (C) 2012 Elsevier B.V. All rights reserved.

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.

Estimating Hydrogen Cyanide in Forage Sorghum (Sorghum bicolor) by Near-Infrared Spectroscopy

Hydrogen cyanide (HCN) is a toxic chemical that can potentially cause mild to severe reactions in animals when grazing forage sorghum. Developing technologies to monitor the level of HCN in the growing crop would benefit graziers, so that they can move cattle into paddocks with acceptable levels of HCN. In this study, we developed near-infrared spectroscopy (MRS) calibrations to estimate HCN in forage sorghum and hay. The full spectral NIRS range (400-2498 nm) was used as well as specific spectral ranges within the full spectral range, i.e., visible (400-750 nm), shortwave (800-1100 nm) and near-infrared (NIR) (1100-2498 nm). Using the full spectrum approach and partial least-squares (PLS), the calibration produced a coefficient of determination (R-2) = 0.838 and standard error of cross-validation (SECV) = 0.040%, while the validation set had a R-2 = 0.824 with a low standard error of prediction (SEP = 0.047%). When using a multiple linear regression (MLR) approach, the best model (NIR spectra) produced a R-2 = 0.847 and standard error of calibration (SEC) = 0.050% and a R-2 = 0.829 and SEP = 0.057% for the validation set. The MLR models built from these spectral regions all used nine wavelengths. Two specific wavelengths 2034 and 2458 nm were of interest, with the former associated with C=O carbonyl stretch and the latter associated with C-N-C stretching. The most accurate PLS and MLR models produced a ratio of standard error of prediction to standard deviation of 3.4 and 3.0, respectively, suggesting that the calibrations could be used for screening breeding material. The results indicated that it should be feasible to develop calibrations using PLS or MLR models for a number of users, including breeding programs to screen for genotypes with low HCN, as well as graziers to monitor crop status to help with grazing efficiency.

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.

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.

Bycatch and strandings programs as ecological indicators for data-limited cetaceans

An integrated approach of using strandings and bycatch data may provide an indicator of long-term trends for data-limited cetaceans. Strandings programs can give a faithful representation of the species composition of cetacean assemblages, while standardised bycatch rates can provide a measure of relative abundance. Comparing the two datasets may also facilitate managing impacts by understanding which species, sex or sizes are the most vulnerable to interactions with fisheries gear. Here we apply this approach to two long-term datasets in East Australia, bycatch in the Queensland Shark Control Program QSCP, 1992–2012) and strandings in the Queensland Marine Wildlife Strandings and Mortality Program StrandNet, 1996–2012). Short-beaked common dolphins, Delphinus delphis, were markedly more frequent in bycatch than in the strandings dataset, suggesting that they are more prone to being incidentally caught than other cetacean species in the region. The reverse was true for humpback whales, Megaptera novaeangliae, bottlenose dolphins, Tursiops spp.; and species predominantly found in offshore waters. QSCP bycatch was strongly skewed towards females for short-beaked common dolphins, and towards smaller sizes for Australian humpback dolphins, Sousa sahulensis. Overall, both datasets demonstrated similar seasonality and a similar long-term increase from 1996 until 2008. Analysis on a species-by-species basis was then used to explore potential explanations for long-term trends, which ranged from a recovering stock (humpback whales) to a shift in habitat use (short-beaked common dolphins).

Bycatch and strandings programs as ecological indicators for data-limited cetaceans

An integrated approach of using strandings and bycatch data may provide an indicator of long-term trends for data-limited cetaceans. Strandings programs can give a faithful representation of the species composition of cetacean assemblages, while standardised bycatch rates can provide a measure of relative abundance. Comparing the two datasets may also facilitate managing impacts by understanding which species, sex or sizes are the most vulnerable to interactions with fisheries gear. Here we apply this approach to two long-term datasets in East Australia, bycatch in the Queensland Shark Control Program (QSCP, 1992–2012) and strandings in the Queensland Marine Wildlife Strandings and Mortality Program (StrandNet, 1996–2012). Short-beaked common dolphins, Delphinus delphis, were markedly more frequent in bycatch than in the strandings dataset, suggesting that they are more prone to being incidentally caught than other cetacean species in the region. The reverse was true for humpback whales, Megaptera novaeangliae, bottlenose dolphins, Tursiops spp.; and species predominantly found in offshore waters. QSCP bycatch was strongly skewed towards females for short-beaked common dolphins, and towards smaller sizes for Australian humpback dolphins, Sousa sahulensis. Overall, both datasets demonstrated similar seasonality and a similar long-term increase from 1996 until 2008. Analysis on a species-by-species basis was then used to explore potential explanations for long-term trends, which ranged from a recovering stock (humpback whales) to a shift in habitat use (short-beaked common dolphins).

High-throughput Phenotyping of Wheat Seminal Root Traits in a Breeding Context

Water availability is a major limiting factor for wheat (Triticum aestivum L.) in rain-fed agricultural systems worldwide. Root architecture has important functional implications for the timing and extent of soil water extraction, yet selection for root traits in wheat breeding programs has been largely limited due to the lack of suitable phenotyping methods. The aim of this research was to develop a low-cost high-throughput phenotyping method to facilitate selection for desirable root traits. We developed a method to assess ‘seminal root angle’ and ‘seminal root number’ in seedlings – two proxy traits associated to root architecture of mature wheat plants (1). The method involves measuring the angle between the first pair of seminal roots and the number of roots of wheat seedlings grown in transparent pots (Figure 1). Images captured at 5 to 10 days after sowing are analyzed to calculate seminal root angle and number. Performing this technique under “speed breeding” conditions (plants grown at a density of 600 plants / m2, under controlled temperature and constant light) allows the selection based on the desired root traits of up to 5 consecutive generations within 12 months. Alternatively, when focusing only on germplasm screening, up to 52 successive phenotypic assays can be conducted within 12 months. This approach has been shown to be highly reproducible, it requires little resource (time, space, and labour) and can be used to rapidly enrich breeding populations with desirable alleles for narrow root angle and a high number of seminal roots to indirectly target the selection of deeper root system with higher branching at depth. Such root characteristics are highly desirable in wheat to cope with the climate model projections, especially in summer rainfall dominant regions including some Australian, Indian, South American and African cropping regions, where winter crops mainly rely on deep stored water.