Inconsistent Transmission of Banana Bunchy Top Virus in Micropropagated Bananas and its Implication for Germplasm Screening

Banana bunchy top virus (BBTV) was readily transmitted through tissue culture in banana (Mum sp.) cv. Lady finger (AAB) and Cavendish cv. Williams (AAA). Lines derived from infected and healthy field plants had similar in vitro multiplication rates. BBTV infected in vitro cultures displayed symptoms of stunting, leaf curling, chlorotic and green flecks, and poor root growth. Symptoms became milder with time, and were often difficult to discern in older, rapidly multiplying cultures. A triple antibody sandwich ELISA using polyclonal and monoclonal antibodies was very efficient for detecting BBTV in vitro. Symptomless, ELISA-negative plants arose in 10 out of 11 lines derived from BBTV-infected field plants and first appeared after 9 months continuous in vitro culture at a constant 28OC. Meristem tip culture or heat therapy was not used. These plants remained symptomless and ELISA-negative after planting out in the glasshouse (individual plants checked for up to 16 months). The implications of this inconsistent transmission of BBTV for germplasm indexing and exchange are discussed.

Rapid and efficient screening of a Representational Difference Analysis library using reverse Southern hybridisation: Identification of genetic differences between Haemophilus parasuis isolates

Representational Difference Analysis (RDA) is an established technique used for isolation of specific genetic differences between or within bacterial species. This method was used to investigate the genetic basis of serovar-specificity and the relationship between serovar and virulence in Haemophilus parasuis. An RDA clone library of 96 isolates was constructed using H. parasuis strains H425(P) (serovar 12) and HS1967 (serovar 4). To screen such a large clone library to determine which clones are strain-specific would typically involved separately labelling each clone for use in Southern hybridisation against genomic DNA from each of the strains. In this study, a novel application of reverse Southern hybridisation was used to screen the RDA library: genomic DNA from each strain was labelled and used to probe the library to identify strain-specific clones. This novel approach represents a significant improvement in methodology that is rapid and efficient.

Drivers for uptake of environmental management systems by pastoralists in western Queensland

Pastoralists from 37 beef cattle and sheep properties in western Queensland developed and implemented an environmental management system (EMS) over 18 months. The EMS implemented by them was customised for the pastoral industry as part of a national EMS pilot project, and staff from this project encouraged and assisted pastoralists during this trial. The 31 pastoralists surveyed at the end of the pilot project identified few benefits of EMS implementation, and these were largely associated with environmental management and sustainability. In terms of the reasons for uptake of an EMS, these pastoralists identified drivers similar to those reported in other primary industry sectors. These included improving property and environmental management, financial incentives, a range of market benefits, assistance with red tape issues, access to other training opportunities and assistance and support with the development of their EMS. However, these drivers are weak, and are not motivating pastoralists to adopt an EMS. In contrast, barriers to adoption such as the time involved in developing and implementing EMS are tangible and immediate. Given a lack of effective drivers and that pastoralists are under considerable pressure from ongoing rural adjustment processes, it is not surprising that an EMS is a low priority. It is concluded that widespread uptake and on-going use of an EMS in the pastoral industry will not occur unless pastoralists are required or rewarded for this by markets, governments, financiers, and regional natural resource management bodies.

Nutrient uptake in tropical turfgrasses growing in winter in southern Queensland

The effects of fertilisers on 8 tropical turfgrasses growing in 100-L bags of sand were studied over winter in Murrumba Downs, just north of Brisbane in southern Queensland (latitude 27.4°S, longitude 153.1°E). The species used were: Axonopus compressus (broad-leaf carpetgrass), Cynodon dactylon (bermudagrass 'Winter Green') and C. dactylon x C. transvaalensis hybrid ('Tifgreen'), Digitaria didactyla (Queensland blue couch), Paspalum notatum (bahiagrass '38824'), Stenotaphrum secundatum (buffalograss 'Palmetto'), Eremochloa ophiuroides (centipedegrass 'Centec') and Zoysia japonica (zoysiagrass 'ZT-11'). Control plots were fertilised with complete fertilisers every month from May to September (72 kg N/ha, 31 kg P/ha, 84 kg K/ha, 48 kg S/ha, 30 kg Ca/ha and 7.2 kg Mg/ha), and unfertilised plots received no fertiliser. Carpetgrass and standard bermudagrass were the most sensitive species to nutrient supply, with lower shoot dry weights in the unfertilised plots (shoots mowed to thatch level) compared with the fertilised plots in June. There were lower shoot dry weights in the unfertilised plots in July for all species, except for buffalograss, centipedegrass and zoysiagrass, and lower shoot dry weights in the unfertilised plots in August for all species, except for centipedegrass. At the end of the experiment in September, unfertilised plots were 11% of the shoot dry weights of fertilised plots, with all species affected. Mean shoot nitrogen concentrations fell from 3.2 to 1.7% in the unfertilised plots from May to August, below the sufficiency range for turfgrasses (2.8-3.5%). There were also declines in P (0.45-0.36%), K (2.4-1.5%), S (0.35-0.25%), Mg (0.24-0.18%) and B (9-6 mg/kg), which were all in the sufficiency range. The shoots in the control plots took up the following levels (kg/ha.month) of nutrients: N, 10.0-27.0; P, 1.6-4.0; K, 8.2-19.8; S, 1.0-4.2; Ca, 1.1-3.3; and Mg, 0.8-2.2, compared with applications (kg/ha.month) of: N, 72; P, 31; K, 84; S, 48; Ca, 30; and Mg, 7.2, indicating a recovery of 14-38% for N, 5-13% for P, 10-24% for K, 2-9% for S, 4-11% for Ca and 11-30% for Mg. These results suggest that buffalograss, centipedegrass and zoysiagrass are less sensitive to low nutrient supply than carpetgrass, bermudagrass, blue couch and bahiagrass. Data on nutrient uptake showed that the less sensitive species required only half or less of the nitrogen required to maintain the growth of the other grasses, indicating potential savings for turf managers in fertiliser costs and the environment in terms of nutrients entering waterways.

Screening selected myrtaceous tree species for production of essential oils in northern Queensland

Trials to identify alternative cropping options to Melaleuca alternifolia for northern Queensland essential oil growers were established at Dimbulah and Innot Hot Springs in 2001. Seed sources of Asteromyrtus symphyocarpa (1,8-cineole form), Eucalyptus staigeriana (citral), Melaleuca cajuputi subsp. cajuputi (trans-nerolidol), M. ericifolia (d-linalool), M. quinquenervia (trans-nerolidol and viridiflorol forms) and M. viridiflora (methyl cinnamate) with potential to produce commercial foliar oils were evaluated. Information was gathered on their adaptability, growth and oil yields over 49 months and 52 months (two harvests) from planting at Dimbulah and Innot Hot Springs, respectively. Of the species and chemotypes evaluated, M. quinquenervia showed potential for commercial production of trans-nerolidol, a compound used in perfumery. It had a very high survival rate (96%) and yields could be expected to improve dramatically from the average 100 kg/ha per harvest achieved in these trials with further research into selection of seed source, control of insect damage and breeding for genetic improvement. M. cajuputi subsp. cajuputi gave a similar performance to M. quinquenervia. The rarity of the trans-nerolidol form of this species and remoteness of its natural occurrence are impediments to further planting and research. E. staigeriana, with second harvest yields of ~600 kg/ha, performed exceptionally well on both sites but potential for development is limited by the ready availability of competitively priced E. staigeriana oil produced in South America. Survival of M. ericifolia ranged from 62% to 82% at 32 months (second harvest) at Innot Hot Springs and was deemed a failure at Dimbulah with poor growth and low survival, raising a major question about the suitability of this species for cultivation in the seasonally dry tropics. Planting of this species on a wider scale in northern Queensland cannot be recommended until more is known about factors affecting its survival. A. symphyocarpa and M. viridiflora were too slow-growing to warrant further consideration as potential oil-producing species at this time.

Screening of new lines of cereal grains for inclusion in Near Infra Red Scan calibrations

The project will provide enough data for a reliable and robust NIRs. It will more fully develop the in vitro method to enable less costly assessment of grains in the future. It will also provide a reliable assessment for DE which is the most expensive component of pig feed.

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.

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.

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.

Cu2+ inhibition of gel secretion in the xylem and its potential implications for water uptake of cut Acacia holosericea stems

Maintaining a high rate of water uptake is crucial for maximum longevity of cut stems. Physiological gel/tylosis formation decreases water transport efficiency in the xylem. The primary mechanism of action for post-harvest Cu2+ treatments in improving cut flower and foliage longevity has been elusive. The effect of Cu2+ on wound-induced xylem vessel occlusion was investigated for Acacia holosericea A. Cunn. ex G. Don. Experiments were conducted using a Cu2+ pulse (5 h, 2.2 mM) and a Cu2+ vase solution (0.5 mM) vs a deionized water (DIW) control. Development of xylem blockage in the stem-end region 10 mm proximal to the wounded stem surface was examined over 21 days by light and transmission electron microscopy. Xylem vessels of stems stood into DIW were occluded with gels secreted into vessel lumens via pits from surrounding axial parenchyma cells. Gel secretion was initiated within 1-2 days post-wounding and gels were detected in the xylem from day 3. In contrast, Cu2+ treatments disrupted the surrounding parenchyma cells, thereby inhibiting gel secretion and maintaining the vessel lumens devoid of occlusions. The Cu2+ treatments significantly improved water uptake by the cut stems as compared to the control. © 2013 Scandinavian Plant Physiology Society.

Drought adaptation of stay-green sorghum is associated with canopy development, leaf anatomy, root growth, and water uptake

Stay-green sorghum plants exhibit greener leaves and stems during the grain-filling period under water-limited conditions compared with their senescent counterparts, resulting in increased grain yield, grain mass, and lodging resistance. Stay-green has been mapped to a number of key chromosomal regions, including Stg1, Stg2, Stg3, and Stg4, but the functions of these individual quantitative trait loci (QTLs) remain unclear. The objective of this study was to show how positive effects of Stg QTLs on grain yield under drought can be explained as emergent consequences of their effects on temporal and spatial water-use patterns that result from changes in leaf-area dynamics. A set of four Stg near-isogenic lines (NILs) and their recurrent parent were grown in a range of field and semicontrolled experiments in southeast Queensland, Australia. These studies showed that the four Stg QTLs regulate canopy size by: (1) reducing tillering via increased size of lower leaves, (2) constraining the size of the upper leaves; and (3) in some cases, decreasing the number of leaves per culm. In addition, they variously affect leaf anatomy and root growth. The multiple pathways by which Stg QTLs modulate canopy development can result in considerable developmental plasticity. The reduction in canopy size associated with Stg QTLs reduced pre-flowering water demand, thereby increasing water availability during grain filling and, ultimately, grain yield. The generic physiological mechanisms underlying the stay-green trait suggest that similar Stg QTLs could enhance post-anthesis drought adaptation in other major cereals such as maize, wheat, and rice.

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.

Pratylenchus thornei populations reduce water uptake in intolerant wheat cultivars

Pratylenchus thornei is a major pathogen of wheat crops in the northern grain region of Eastern Australia with an estimated annual yield loss of $38 million. Damaged crops show symptoms of water and nutrient stress that suggest uptake is significantly affected. In order to understand the mechanisms involved in reducing water uptake and consequently plant yield, detailed measurements of water extraction and leaf area were conducted on a range of wheat cultivars with differing levels of tolerance and resistance to P. thornei.