Virus identification and development of long-term management strategies for the rhubarb industry

The purpose of this report is to present the final results of all activities conducted under HAL Project VG05053 ‘Virus identification and development of long-term management strategies for the rhubarb industry’. The report provides a summary of project findings, a description of technology transfer activities, and recommendations arising from the outcomes of the project. The overall objective of this project was to devise a strategy for the control of rhubarb decline disease through 1) knowledge of the viruses present and their epidemiology, 2) production of virus-free planting material via tissue culture, and 3) formation of a national grower group to represent industry.

DAQ339A Pest and disease identification and management in herbs

The major objective is to produce an educational tool for growers and research/extension personnel to allow accurate identification of a range of pests and diseases encountered in herbs. To a lessor extent develop both a mechanism to manage beneficial insects in field crops pre-harvest and to identify some common seed borne diseases in herbs.

Identification and analysis in sweet corn

Identification and analysis of allelic variation in carotenoid biosynthesis genes present in sweet corn germplasm for eye health.

Single kernel NIR assessment for QTL identification

Development and evaluation of a single kernel NIR assessment method for improving baley malting quality QTL identification.

Comparison of the reproductive ecology of two sympatric blacktip sharks (Carcharhinus limbatus and Carcharhinus tilstoni) off north-eastern Australia with species identification inferred from vertebral counts

Precaudal vertebral counts were used to distinguish between 237 morphologically similar Carcharhinus limbatus and Carcharhinus tilstoni and were congruent with differences in reproductive ecology between the species. In addition to differing lengths at maturity and adult body size, the two species had asynchronous parturition, were born at different sizes and the relative frequencies of neonates differed in two coastal nursery areas. Despite evidence that hybridization can occur, these differences suggest the species are largely reproductively isolated.

Identification of diverse full-length endogenous betaretroviruses in megabats and microbats

Background: Betaretroviruses infect a wide range of species including primates, rodents, ruminants, and marsupials. They exist in both endogenous and exogenous forms and are implicated in animal diseases such as lung cancer in sheep, and in human disease, with members of the human endogenous retrovirus-K (HERV-K) group of endogenous betaretroviruses (βERVs) associated with human cancers and autoimmune diseases. To improve our understanding of betaretroviruses in an evolutionarily distinct host species, we characterized βERVs present in the genomes and transcriptomes of mega- and microbats, which are an important reservoir of emerging viruses.Results: A diverse range of full-length βERVs were discovered in mega- and microbat genomes and transcriptomes including the first identified intact endogenous retrovirus in a bat. Our analysis revealed that the genus Betaretrovirus can be divided into eight distinct sub-groups with evidence of cross-species transmission. Betaretroviruses are revealed to be a complex retrovirus group, within which one sub-group has evolved from complex to simple genomic organization through the acquisition of an env gene from the genus Gammaretrovirus. Molecular dating suggests that bats have contended with betaretroviral infections for over 30 million years.Conclusions: Our study reveals that a diverse range of betaretroviruses have circulated in bats for most of their evolutionary history, and cluster with extant betaretroviruses of divergent mammalian lineages suggesting that their distribution may be largely unrestricted by host species barriers. The presence of βERVs with the ability to transcribe active viral elements in a major animal reservoir for viral pathogens has potential implications for public health. © 2013 Hayward et al.; licensee BioMed Central Ltd.

Identification of a new Polerovirus (family Luteoviridae) associated with cotton bunchy top disease in Australia

Cotton bunchy top (CBT) disease has caused significant yield losses in Australia and is now managed by control of its vector, the cotton aphid (Aphis gossypii). Its mode of transmission and similarities in symptoms to cotton Blue Disease suggested it may also be caused by a luteovirus or related virus. Degenerate primers to conserved regions of the genomes of the family Luteoviridae were used to amplify viral cDNAs from CBT-affected cotton leaf tissue that were not present in healthy plants. Partial genome sequence of a new virus (Cotton bunchy top virus, CBTV) was obtained spanning part of the RNA-dependent-RNA-polymerase (RdRP), all of the coat protein and part of the aphid-transmission protein. CBTV sequences could be detected in viruliferous aphids able to transmit CBT, but not aphids from non-symptomatic plants, indicating that it is associated with the disease and may be the causal agent. All CBTV open-reading frames had their closest similarity to viruses of the genus Polerovirus. The partial RdRP had 90 % amino acid identity to the RdRP of Cotton leafroll dwarf virus (CLRDV) that causes cotton blue disease, while other parts of the genome were more similar to other poleroviruses. The sequence similarity and genome organization of CBTV suggest that it should be considered a new member of the genus Polerovirus. This partial genome sequence of CBTV opens up the possibility for developing diagnostic tests for detection of the virus in cotton plants, aphids and weeds as well as alternative strategies for engineering CBT resistance in cotton plants through biotechnology. © 2012 Australasian Plant Pathology Society Inc.

Correlation between NIRS generated and chemically measured feed quality data in barley (Hordeum vulgare), and potential use in QTL analysis identification

A study was performed to investigate the value of near infrared reflectance spectroscopy (NIRS) as an alternate method to analytical techniques for identifying QTL associated with feed quality traits. Milled samples from an F6-derived recombinant inbred Tallon/Scarlett population were incubated in the rumen of fistulated cattle, recovered, washed and dried to determine the in-situ dry matter digestibility (DMD). Both pre- and post-digestion samples were analysed using NIRS to quantify key quality components relating to acid detergent fibre, starch and protein. This phenotypic data was used to identify trait associated QTL and compare them to previously identified QTL. Though a number of genetic correlations were identified between the phenotypic data sets, the only correlation of most interest was between DMD and starch digested (r = -0.382). The significance of this genetic correlation was that the NIRS data set identified a putative QTL on chromosomes 7H (LOD = 3.3) associated with starch digested. A QTL for DMD occurred in the same region of chromosome 7H, with flanking markers fAG/CAT63 and bPb-0758. The significant correlation and identification of this putative QTL, highlights the potential of technologies like NIRS in QTL analysis.

Identification of intersectional Corymbia hybrids based on seedling morphology improves with parental divergence

Differences in morphology have provided a basis for detecting natural interspecific hybridisation in forest trees for decades but have come to prominence again more recently as a means for directly measuring gene flow from planted forests. Here we examined the utility of seedling morphology for hybrid discrimination in three hybrid groups relevant to the monitoring of gene flow from plantings of Corymbia (L.D. Pryor & L.A.S. Johnson ex Brooker) taxa in subtropical Australia. Thirty leaf and stem characters were assessed on 907 8-month old seedlings from four parental and six hybrid taxa grown in a common garden. Outbred F1 hybrids between spotted gums (Corymbia citriodora subspecies variegata, C. citriodora subspecies citriodora and Corymbia henryi) tended to more closely resemble their maternal Corymbia torelliana parent and the most discriminating characters were the ratio of blade length to maximum perpendicular width, the presence or absence of a lignotuber, and specific leaf weight. Assignment of individuals into genealogical classes based on a multivariate model limited to a set of the more discriminating and independent characters was highest in the hybrid group, where parental taxa were genetically most divergent. Overall power to resolve among outbred F1 hybrids from both parental taxa was low to moderate, but this may not be a limitation to its likely major application of identifying hybrids in seedlots from native spotted gum stands. Advanced generation hybrids (outbred F2 and outbred backcrosses) were more difficult to resolve reliably due to the higher variances of hybrid taxa and the tendency of backcrosses to resemble their recurrent parents. Visual assessments of seedling morphology may provide a filter allowing screening of the large numbers needed to monitor gene flow, but will need to be combined with other hybrid detection methods to ensure hybrids are detected.

Pheromone of the banana-spotting bug, Amblypelta lutescens lutescens Distant (Heteroptera: Coreidae): Identification, synthesis, and field bioassay

The banana-spotting bug, Amblypelta lutescens lutescens Distant (Heteroptera: Coreidae), is one of the principal pests of tree fruits and nuts across northern and eastern Australia. Apart from visual damage assessment, there are currently no reliable methods for monitoring bug activity to aid management decisions. An attractant pheromone for this species that could be used as a trap lure could potentially fill this void. Earlier, two male-specific compounds were identified in airborne extracts from A. lutescens lutescens, (E,E)-α-farnesene and (R,E)-nerolidol; an unknown compound with a molecular weight 220 was also detected. We now report the identification of this hitherto unknown compound as (R,E,E)-α-farnesene-10,11-oxide. Synthesis of this epoxide was conducted using a regioselective asymmetric dihydroxylation of a sulfolene. A blend mimicking the natural proportions of (E,E)-α-farnesene, (R,E)-nerolidol, and (R,E,E)-α-farnesene-10,11- oxide attracted male and female A. lutescens lutescens as well as nymphs in the field, verifying that the aggregation pheromone comprises or is contained within this group of compounds. Copyright © 2012 Ashot Khrimian et al.

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.

Rapid genome wide mapping of phosphine resistance loci by a simple regional averaging analysis in the red flour beetle, Tribolium castaneum

Background Next-generation sequencing technology is an important tool for the rapid, genome-wide identification of genetic variations. However, it is difficult to resolve the ‘signal’ of variations of interest and the ‘noise’ of stochastic sequencing and bioinformatic errors in the large datasets that are generated. We report a simple approach to identify regional linkage to a trait that requires only two pools of DNA to be sequenced from progeny of a defined genetic cross (i.e. bulk segregant analysis) at low coverage (<10×) and without parentage assignment of individual SNPs. The analysis relies on regional averaging of pooled SNP frequencies to rapidly scan polymorphisms across the genome for differential regional homozygosity, which is then displayed graphically. Results Progeny from defined genetic crosses of Tribolium castaneum (F4 and F19) segregating for the phosphine resistance trait were exposed to phosphine to select for the resistance trait while the remainders were left unexposed. Next generation sequencing was then carried out on the genomic DNA from each pool of selected and unselected insects from each generation. The reads were mapped against the annotated T. castaneum genome from NCBI (v3.0) and analysed for SNP variations. Since it is difficult to accurately call individual SNP frequencies when the depth of sequence coverage is low, variant frequencies were averaged across larger regions. Results from regional SNP frequency averaging identified two loci, tc_rph1 on chromosome 8 and tc_rph2 on chromosome 9, which together are responsible for high level resistance. Identification of the two loci was possible with only 5-7× average coverage of the genome per dataset. These loci were subsequently confirmed by direct SNP marker analysis and fine-scale mapping. Individually, homozygosity of tc_rph1 or tc_rph2 results in only weak resistance to phosphine (estimated at up to 1.5-2.5× and 3-5× respectively), whereas in combination they interact synergistically to provide a high-level resistance >200×. The tc_rph2 resistance allele resulted in a significant fitness cost relative to the wild type allele in unselected beetles over eighteen generations. Conclusion We have validated the technique of linkage mapping by low-coverage sequencing of progeny from a simple genetic cross. The approach relied on regional averaging of SNP frequencies and was used to successfully identify candidate gene loci for phosphine resistance in T. castaneum. This is a relatively simple and rapid approach to identifying genomic regions associated with traits in defined genetic crosses that does not require any specialised statistical analysis.

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.

Pythium soft rot of ginger: Detection and identification of the causal pathogens, and their control

Ginger is considered by many people to be the outstanding member among 1400 other species in the family Zingiberaceae. Not only it is a valuable spice used by cooks throughout the world to impart unique flavour to their dishes but it also has a long track record in some Chinese and Indian cultures for treating common human ailments such as colds and headaches. Ginger has recently attracted considerable attention for its anti-inflammatory, antibacterial and antifungal properties. However, ginger as a crop is also susceptible to at least 24 different plant pathogens, including viruses, bacteria, fungi and nematodes. Of these, Pythium spp. (within the kingdom Stramenopila, phyllum Oomycota) are of most concern because various species can cause rotting and yield loss on ginger at any of the growth stages including during postharvest storage. Pythium gracile was the first species in the genus to be reported as a ginger pathogen, causing Pythium soft rot disease in India in 1907. Thereafter, numerous other Pythium spp. have been recorded from ginger growing regions throughout the world. Today, 15 Pythium species have been implicated as pathogens of the soft rot disease. Because accurate identification of a pathogen is the cornerstone of effective disease management programs, this review will focus on how to detect, identify and control Pythium spp. in general, with special emphasis on Pythium spp. associated with soft rot on ginger.