Functionally associated molecular genetic marker map construction in perennial ryegrass (Lolium perenne L.)

A molecular marker-based map of perennial ryegrass (Lolium perenne L.) has been constructed through the use of polymorphisms associated with expressed sequence tags (ESTs). A pair-cross between genotypes from a North African ecotype and the cultivar Aurora was used to generate a two-way pseudo-testcross population. A selection of 157 cDNAs assigned to eight different functional categories associated with agronomically important biological processes was used to detect polymorphic EST–RFLP loci in the F1(NA6 × AU6) population. A comprehensive set of EST–SSR markers was developed from the analysis of 14,767 unigenes, with 310 primer pairs showing efficient amplification and detecting 113 polymorphic loci. Two parental genetic maps were produced: the NA6 genetic map contains 88 EST–RFLP and 71 EST–SSR loci with a total map length of 963 cM, while the AU6 genetic map contains 67 EST–RFLP and 58 EST–SSR loci with a total map length of 757 cM. Bridging loci permitted the alignment of homologous chromosomes between the parental maps, and a sub-set of genomic DNA-derived SSRs was used to relate linkage groups to the perennial ryegrass reference map. Regions of segregation distortion were identified, in some instances in common with other perennial ryegrass maps. The EST-derived marker-based map provides the basis for in silico comparative genetic mapping, as well as the evaluation of co-location between QTLs and functionally associated genetic loci.

Quantitative assessment of total and Gram-positive aerobic bacteria in fresh and ambient-temperature-stored sub-tropical marine fish

The current study was undertaken to enumerate Gram-positive bacteria in fresh sub-tropical marine fish and determine the effect of ambient storage (25°C) on the Gram-positive bacterial count. Total and Gram-positive bacteria were enumerated in the muscles, gills and gut of fresh and stored Pseudocaranx dentex, Pagrus auratus and Mugil cephalus on tryptone soya agar (TSA) and TSA with 0.25% phenylethyl alcohol (PEA), respectively. Initial studies indicated that PEA significantly reduced total aerobic bacterial count (TABC) whereas control Gram-positive bacteria were not affected by 0.25% PEA. TABC significantly increased in all fish body parts, whereas Gram-positive aerobic bacterial count (GABC) significantly increased only in the muscles and gills during ambient storage for 15 h. The TABC of the fish species increased from 4.00, 6.13 and 4.58 log cfu g-1, respectively in the muscles, gills, and gut to 6.31, 7.31 and 7.23 log cfu g-1 by the end of storage. GABC increased from 2.00, 3.52 and 2.20 log cfu g-1 to 4.70, 5.85 and 3.36 log cfu g-1. Within each species, TABC were significantly higher in the gills compared to that of muscles and gut; however, no significant differences were found in GABC between muscles and gills. This study demonstrated the potential importance of Gram-positive bacteria in sub-tropical marine fish and their spoilage.

Antimicrobial susceptibility testing of Brachyspira intermedia and Brachyspira pilosicoli isolates from Australian chickens

Susceptibilities of predominantly Australian isolates of the pathogenic intestinal spirochaetes Brachyspira intermedia (n=25) and Brachyspira pilosicoli (n=17) from chickens were tested in agar dilution against four concentrations each of the antimicrobials tiamulin, lincomycin, tylosin, metronidazole, tetracycline and ampicillin. Based on available minimum inhibitory concentration (MIC) breakpoint values for Brachyspira hyodysenteriae or other Gram-negative enteric veterinary pathogens, isolates of both species generally were susceptible to tiamulin, lincomycin, metronidazole and tetracycline. Although not classed as resistant, four isolates of B. intermedia had an elevated MIC range for tiamulin (1 to 4 mg/l), 11 isolates of B. intermedia and five of B. pilosicoli had an elevated MIC range for lincomycin (10 to 50 mg/l), one isolate of B. pilosicoli had an elevated MIC range for tetracycline (10 to 20 mg/l), and one isolate of B. intermedia and five of B. pilosicoli had an elevated MIC range for ampicillin (10 to 50 mg/l). A clear lack of susceptibility to tylosin (MIC >4 mg/l) was seen in 11 isolates each of B. intermedia and B. pilosicoli, and to ampicillin (MIC >32 mg/l) in two isolates of B. pilosicoli. These data suggest that some resistance to common antimicrobials exists among intestinal spirochetes obtained from laying hens and supports the need of MIC data for clinical isolates before any treatment is considered.

Baseline responses of Alphitobius diaperinus (Coleoptera: Tenebrionidae) to spinosad, and susceptibility of broiler populations in eastern and southern Australia

Spinosad was proposed as a potential chemical for control of lesser mealworm, Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae), in Australian broiler houses after the detection of strong cyfluthrin resistance in many beetle populations. In 2004-2006, spinosad susceptibility of 13 beetle populations from eastern and southern Australian broiler houses and a cyfluthrin/fenitrothion-resistant reference population was determined using topical application, and was compared with the susceptibility of an insecticide-susceptible reference population. Comparisons of dose-response curves and baseline data showed that all populations, including the insecticide-susceptible population, were roughly equivalent in their response to spinosad, indicating no preexisting spinosad resistance. Two field populations, including the resistant reference population, which had confirmed cyfluthrin/fenitrothion- resistance, showed no cross-resistance to spinosad. There was no significant correlation between beetle weight and LC99.9. A discriminating concentration of 3% spinosad was set to separate resistant and susceptible individuals. Considering the levels of spinosad resistance that have been recorded in other insect pests, the sustained future usefulness of spinosad as a broiler house treatment will rely on effective integrated beetle management programs combined with carefully planned chemical use strategies.

Cultivar affects browning susceptibility of freshly cut star fruit slices

Consumption of freshly-cut horticultural products has increased in the last few years. The principal restraint to using freshly-cut carambola is its susceptibility to tissue-browning, due to polyphenol oxidase-mediated oxidation of phenolic compounds present in the tissue. The current study investigated the susceptibility to browning of star fruit slices (Averrhoa carambola L.) of seven genotypes (Hart, Golden Star, Taen-ma, Nota-10, Malasia, Arkin, and Fwang Tung). Cultivar susceptibility to browning as measured by luminosity (L*) varied significantly among genotypes. Without catechol 0.05 M, little changes occurred on cut surface of any cultivars during 6 hour at 25 degrees C, 67% RH. Addition of catechol led to rapid browning, which was more intense in cvs. Taen-ma, Fwang Tung, and Golden Star, with reduction in L* value of 28.60%, 27.68%, and 23.29%, respectively. Browning was more intense in the center of the slices, particularly when treated with catechol, indicating highest polyphenol oxidase (PPO) concentration. Epidermal browning, even in absence of catechol, is a limitation to visual acceptability and indicates a necessity for its control during carambola processing. Care must be given to appropriate selection of cultivars for fresh-cut processing, since cultivar varied in browning susceptibility in the presence of catechol.

A high-density consensus map of barley linking DArT markers to SSR, RFLP and STS loci and agricultural traits

Background: Molecular marker technologies are undergoing a transition from largely serial assays measuring DNA fragment sizes to hybridization-based technologies with high multiplexing levels. Diversity Arrays Technology (DArT) is a hybridization-based technology that is increasingly being adopted by barley researchers. There is a need to integrate the information generated by DArT with previous data produced with gel-based marker technologies. The goal of this study was to build a high-density consensus linkage map from the combined datasets of ten populations, most of which were simultaneously typed with DArT and Simple Sequence Repeat (SSR), Restriction Enzyme Fragment Polymorphism (RFLP) and/or Sequence Tagged Site (STS) markers. Results: The consensus map, built using a combination of JoinMap 3.0 software and several purpose-built perl scripts, comprised 2,935 loci (2,085 DArT, 850 other loci) and spanned 1,161 cM. It contained a total of 1,629 'bins' (unique loci), with an average inter-bin distance of 0.7 ± 1.0 cM (median = 0.3 cM). More than 98% of the map could be covered with a single DArT assay. The arrangement of loci was very similar to, and almost as optimal as, the arrangement of loci in component maps built for individual populations. The locus order of a synthetic map derived from merging the component maps without considering the segregation data was only slightly inferior. The distribution of loci along chromosomes indicated centromeric suppression of recombination in all chromosomes except 5H. DArT markers appeared to have a moderate tendency toward hypomethylated, gene-rich regions in distal chromosome areas. On the average, 14 ± 9 DArT loci were identified within 5 cM on either side of SSR, RFLP or STS loci previously identified as linked to agricultural traits. Conclusion: Our barley consensus map provides a framework for transferring genetic information between different marker systems and for deploying DArT markers in molecular breeding schemes. The study also highlights the need for improved software for building consensus maps from high-density segregation data of multiple populations.

The use of high resolution melting (HRM) to map single nucleotide polymorphism markers linked to a covered smut resistance gene in barley

Using an established genetic map, a single gene conditioning covered smut resistance, Ruh.7H, was mapped to the telomere region of chromosome 7HS in an Alexis/Sloop doubled haploid barley population. The closest marker to Ruh.7H, abg704 was 7.5 cM away. Thirteen loci on the distal end of 7HS with potential to contain single nucleotide polymorphisms (SNPs) were identified by applying a comparative genomics approach using rice sequence data. Of these, one locus produced polymorphic co-dominant bands of different size while two further loci contained SNPs that were identified using the recently developed high resolution melting (HRM) technique. Two of these markers flanked Ruh.7H with the proximal marker located 3.8 cM and the distal marker 2.7 cM away. This is the first report on the application of the HRM technique to SNP detection and to rapid scoring of known cleaved amplified polymorphic sequence (CAPS) markers in plants. This simple, precise post-PCR technique should find widespread use in the fine-mapping of genetic regions of interest in complex cereal and other plant genomes.

A detached leaf bioassay to screen Durian cultivars for susceptibility to Phytophthora palmivora

A detached-leaf bioassay was developed and used to screen five durian (Durio zibethinus) cultivars against Phytophthora palmivora isolates from a trunk canker, root and fruit. The fruit isolate was less aggressive than the canker and root isolates. The bioassay using the canker isolate was later used to determine the variation in resistance of D. macarantha and nineteen cultivars of D. zibethinus. The cultivars displayed a range of responses with Parung and Gob being most tolerant, with Gaan Yaow, Chanee and Penang 88 being susceptible. The remaining germplasm fell between Gaan Yaow and Penang 88 in susceptibility. The leaf bioassay was found to be a rapid and reliable method for assessing the susceptibility of durian cultivars.

A consensus genetic map of sorghum that integrates multiple component maps and high-throughput Diversity Array Technology (DArT) markers

Background: Sorghum genome mapping based on DNA markers began in the early 1990s and numerous genetic linkage maps of sorghum have been published in the last decade, based initially on RFLP markers with more recent maps including AFLPs and SSRs and very recently, Diversity Array Technology (DArT) markers. It is essential to integrate the rapidly growing body of genetic linkage data produced through DArT with the multiple genetic linkage maps for sorghum generated through other marker technologies. Here, we report on the colinearity of six independent sorghum component maps and on the integration of these component maps into a single reference resource that contains commonly utilized SSRs, AFLPs, and high-throughput DArT markers. Results: The six component maps were constructed using the MultiPoint software. The lengths of the resulting maps varied between 910 and 1528 cM. The order of the 498 markers that segregated in more than one population was highly consistent between the six individual mapping data sets. The framework consensus map was constructed using a "Neighbours" approach and contained 251 integrated bridge markers on the 10 sorghum chromosomes spanning 1355.4 cM with an average density of one marker every 5.4 cM, and were used for the projection of the remaining markers. In total, the sorghum consensus map consisted of a total of 1997 markers mapped to 2029 unique loci ( 1190 DArT loci and 839 other loci) spanning 1603.5 cM and with an average marker density of 1 marker/0.79 cM. In addition, 35 multicopy markers were identified. On average, each chromosome on the consensus map contained 203 markers of which 58.6% were DArT markers. Non-random patterns of DNA marker distribution were observed, with some clear marker-dense regions and some marker-rare regions. Conclusion: The final consensus map has allowed us to map a larger number of markers than possible in any individual map, to obtain a more complete coverage of the sorghum genome and to fill a number of gaps on individual maps. In addition to overall general consistency of marker order across individual component maps, good agreement in overall distances between common marker pairs across the component maps used in this study was determined, using a difference ratio calculation. The obtained consensus map can be used as a reference resource for genetic studies in different genetic backgrounds, in addition to providing a framework for transferring genetic information between different marker technologies and for integrating DArT markers with other genomic resources. DArT markers represent an affordable, high throughput marker system with great utility in molecular breeding programs, especially in crops such as sorghum where SNP arrays are not publicly available.

Susceptibility of source plants to sugarcane Fiji disease virus influences the acquisition and transmission of the virus by the planthopper vector Perkinsiella saccharicida

Fiji leaf gall (FLG) caused by Sugarcane Fiji disease virus (SCFDV) is transmitted by the planthopper Perkinsiella saccharicida. FLG is managed through the identification and exploitation of plant resistance. The glasshouse-based resistance screening produced inconsistent transmission results and the factors responsible for that are not known. A series of glasshouse trials conducted over a 2-year period was compared to identify the factors responsible for the erratic transmission results. SCFDV transmission was greater when the virus was acquired by the vector from a cultivar that was susceptible to the virus than when the virus was acquired from a resistant cultivar. Virus acquisition by the vector was also greater when the vector was exposed to the susceptible cultivars than when exposed to the resistant cultivar. Results suggest that the variation in transmission levels is due to variation in susceptibility of sugarcane cultivars to SCFDV used for virus acquisition by the vector.

Speciation of Gram-positive bacteria in fresh and ambient-stored sub-tropical marine fish.

This study identified Gram-positive bacteria in three sub-tropical marine fish species: Pseudocaranx dentex (silver trevally), Pagrus auratus (snapper) and Mugil cephalus (sea mullet). It further elucidated the role played by fish habitat, fish body part and ambient storage on the composition of the Gram-positive bacteria. A total of 266 isolates of Gram-positive bacteria were identified by conventional biochemical methods, VITEK, PCR using genus- and species-specific primers and/or 16S rRNA gene sequencing. The isolates were found to fall into 13 genera and 30 species. In fresh fish, Staphylococcus epidermidis and Micrococcus luteus were the most frequent isolates. After ambient storage, S. epidermidis, S. xylosus and Bacillus megaterium were no longer present whereas S. warned, B. sphaericus, Brevibacillus borstelensis, Enterococcus faecium and Streptococcus uberis increased in frequency. Micrococcus luteus and S. warned were the most prevalent isolates from P. dentex, while E. faecium and Strep. uberis were the most frequent isolates from P. auratus and M. cephalus. With respect to different parts of the fish body. E. faecium, Strep. uberis and B. sphaericus were the most frequent isolates from the muscles, E. faecium, Strep. uberis from the gills and M. luteus from the gut. This study showed a diversity of Gram-positive bacteria in sub-tropical marine fish; however, their abundance was affected by fish habitat, fish body part and ambient storage.

Host Susceptibility of Citrus Cultivars to Queensland Fruit Fly (Diptera: Tephritidae)

Citrus crops are considered to be relatively poor hosts for Queensland fruit fly, Bactrocera tryoni (Froggatt), as for other tephritid species. Australian citrus growers and crop consultants have reported observable differences in susceptibility of different citrus cultivars under commercial growing conditions. In this study we conducted laboratory tests and field surveys to determine susceptibility to B. tryoni of six citrus cultivars [(Eureka lemon (Citrus limon (L.) Osbeck); Navel and Valencia oranges (C. sinensis (L.) Osbeck); and Imperial, Ellendale, and Murcott mandarins (C. reticulata Blanco)]. The host susceptibility of these citrus cultivars was quantified by a Host Susceptibility Index, which is defined as the number of adult flies produced per gram of fruit infested at a calculated rate of one egg per gram of fruit. The HSI was ranked as Murcott (0.083) > Imperial (0.052) ≥ Navel (0.026) ≥ Ellendale (0.020) > Valencia (0.008) ≥ Eureka (yellow) (0.002) > Eureka (green) (0). Results of the laboratory study were in agreement with the level of field infestation in the four citrus cultivars (Eureka lemon, Imperial, Ellendale, and Murcott mandarins) that were surveyed from commercial orchards under baiting treatments against fruit flies in the Central Burnett district of Queensland. Field surveys of citrus hosts from the habitats not subject to fruit fly management showed that the numbers of fruit flies produced per gram of fruit were much lower, compared with the more susceptible noncitrus hosts, such as guava (Psidium guajava L.), cherry guava (P. littorale Raddi), mulberry (Morus nigra L.), loquat (Eriobotrya japonica (Thunb.) Lindl.), and pear (Pyrus communis L.). Therefore, the major citrus crops commercially cultivated in Australia have a relatively low susceptibility to B. tryoni, with Eureka lemons being a particularly poor host for this tephritid fruit fly.

Freeze-drying of ovalbumin loaded mesoporous silica nanoparticle vaccine formulation increases antigen stability under ambient conditions

Amino functionalised mesoporous silica nanoparticles (AM-41) have been identified as a promising vaccine delivery material. The capacity of AM-41 to stabilise vaccine components at ambient temperature (23–27 °C) was determined by adsorbing the model antigen ovalbumin (OVA) to AM-41 particles (OVA-41). The OVA-41 was successfully freeze-dried using the excipients 5% trehalose and 1% PEG8000. Both the immunological activity of OVA and the nanoparticle structure were maintained following two months storage at ambient temperature. The results of immunisation studies in mice with reconstituted OVA-41 demonstrated the induction of humoral and cell-meditated immune responses. The capacity of AM-41 particles to facilitate ambient storage of vaccine components without loss of immunological potency will underpin the further development of this promising vaccine delivery platform.

Susceptibility of Coconut Wood to Damage by Subterranean Termites (Isoptera: Mastotermitidae, Rhinotermitidae)

Two field trials were conducted with untreated coconut wood (“cocowood”) of varying densities against the subterranean termites Coptotermes acinaciformis (Froggatt) and Mastotermes darwiniensis Froggatt in northern Queensland, Australia. Both trials ran for 16 weeks during the summer months. Cocowood densities ranged from 256 kg/m3 to 1003 kg/m3, and the test specimens were equally divided between the two termite trial sites. Termite pressure was high at both sites where mean mass losses in the Scots pine sapwood feeder specimens were: 100% for C. acinaciformis and 74.7% for M. darwiniensis. Termite species and cocowood density effects were significant. Container and position effects were not significant. Mastotermes darwiniensis fed more on the cocowood than did C. acinaciformis despite consuming less of the Scots pine than did C. acinaciformis. Overall the susceptibility of cocowood to C. acinaciformis and M. darwiniensis decreases with increasing density, but all densities (apart from a few at the high end of the density range) could be considered susceptible, particularly to M. darwiniensis. Some deviations from this general trend are discussed as well as implications for the utilisation of cocowood as a building resource.

Use of a proposed antimicrobial susceptibility testing method for Haemophilus parasuis

The aim of this study was to examine the antimicrobial susceptibility of 97 Haemophilus parasuis cultured from Australian pigs. As there is no existing standard antimicrobial susceptibility technique available for H. parasuis, methods utilising the supplemented media, BA/SN for disc diffusion and test medium broth (TMB) for a microdilution technique, were initially evaluated with the reference strains recommended by the Clinical and Laboratory Standards Institute. The results of the media evaluation suggested that BA/SN and TMB can be used as suitable media for susceptibility testing of H. parasuis. The proposed microdilution technique was then used with 97 H. parasuis isolates and nine antimicrobial agents. The study found that Australian isolates showed elevated minimum inhibitory concentrations (MICs) for ampicillin (1%), penicillin (2%), erythromycin (7%), tulathromycin (9%), tilmicosin (22%), tetracycline (31%) and trimethoprim-sulfamethoxazole (40%). This study has described potential antimicrobial susceptibility methods for H. parasuis and has detected a low percentage of Australian H. parasuis isolates with elevated antimicrobial MICs.