Phenotypic and genotypic characterisation of Pasteurella multocida isolated from pigs at slaughter in New Zealand

AIMS: To examine pigs at slaughter in New Zealand for the presence of Pasteurella multocida, and to determine for isolates, their biochemical profi les, somatic and capsular types, and the presence or absence of the HSB and toxA genes, associated with haemorrhagic septicaemia (HS) and progressive atrophic rhinitis (PAR), respectively. METHODS: Swabs from 173 lungs, 158 palatine tonsils and 82 nasal passages of pigs at two abattoirs in New Zealand were cultured for P. multocida using conventional techniques, and isolated colonies were subjected to biochemical tests for identi- fi cation of biovars. Somatic serotyping was conducted using an agar gel immunodiffusion (AGID) test. Polymerase chain reaction (PCR) assays were used to confi rm phenotypic identifi cation of colonies using species-specifi c primers, capsule type using serogroup-specifi c primers and multiplex PCR, and to test for the presence of HSB and toxA genes. RESULTS: Pasteurella multocida was isolated from 11/173 (6.4%) lung, 32/158 (20.2%) palatine tonsil and 5/82 (6.1 %) nasal swab samples, a total of 48 isolates from 413 samples (11.6%). Isolation rates per farm ranged from 1–53% of tissue samples collected from pigs 5–6 months of age. On phenotypic characterisation, isolates were allocated to seven main biovars, viz 1, 2, 3, 5, 9, 12, and a dulcitol-negative variant of Biovar 8, the majority (30/48) being Biovar 3. Of the 42 isolates for which somatic serotyping was conducted, 10% were Serovar 1, 79% were Serovar 3, 2% were Serovar 6,1, 2% were Serovar 12, and 7% could not be typed. All 48 isolates were confi rmed as P. multocida using a species-specifi c PCR. In the capsular multiplex PCR, 92% of isolates were Capsular (Cap) type A, 2% were Cap D, and 6% could not be typed. None of the samples were positive for the HSB or toxA genes. CONCLUSION: Serovars or capsular types of P. multocida associated with HS or PAR in pigs were not detected. Establishment of species-specifi c, capsular and toxin PCR assays allowed the rapid screening of isolates of P. multocida, while serotyping provided an additional tool for epidemiological and tracing purposes.

Isolation and characterization of novel microsatellite markers and their application for diversity assessment in cultivated groundnut (Arachis hypogaea)

Background: Cultivated peanut or groundnut (Arachis hypogaea L.) is the fourth most important oilseed crop in the world, grown mainly in tropical, subtropical and warm temperate climates. Due to its origin through a single and recent polyploidization event, followed by successive selection during breeding efforts, cultivated groundnut has a limited genetic background. In such species, microsatellite or simple sequence repeat (SSR) markers are very informative and useful for breeding applications. The low level of polymorphism in cultivated germplasm, however, warrants a need of larger number of polymorphic microsatellite markers for cultivated groundnut. Results: A microsatellite- enriched library was constructed from the genotype TMV2. Sequencing of 720 putative SSR-positive clones from a total of 3,072 provided 490 SSRs. 71.2% of these SSRs were perfect type, 13.1% were imperfect and 15.7% were compound. Among these SSRs, the GT/CA repeat motifs were the most common (37.6%) followed by GA/CT repeat motifs (25.9%). The primer pairs could be designed for a total of 170 SSRs and were optimized initially on two genotypes. 104 (61.2%) primer pairs yielded scorable amplicon and 46 (44.2%) primers showed polymorphism among 32 cultivated groundnut genotypes. The polymorphic SSR markers detected 2 to 5 alleles with an average of 2.44 per locus. The polymorphic information content (PIC) value for these markers varied from 0.12 to 0.75 with an average of 0.46. Based on 112 alleles obtained by 46 markers, a phenogram was constructed to understand the relationships among the 32 genotypes. Majority of the genotypes representing subspecies hypogaea were grouped together in one cluster, while the genotypes belonging to subspecies fastigiata were grouped mainly under two clusters. Conclusion. Newly developed set of 104 markers extends the repertoire of SSR markers for cultivated groundnut. These markers showed a good level of PIC value in cultivated germplasm and therefore would be very useful for germplasm analysis, linkage mapping, diversity studies and phylogenetic relationships in cultivated groundnut as well as related Arachis species.

Genetic control of propagation traits in a single Corymbia torelliana x Corymbia variegata family

Genetic control of vegetative propagation traits was described for a second-generation, outbred, intersectional hybrid family (N = 208) derived from two species, Corymbia torelliana (F. Muell.) K.D. Hill & L.A.S. Johnson and Corymbia variegata (F. Muell.) K.D. Hill & L.A.S. Johnson, which contrast for propagation characteristics and in their capacity to develop lignotubers. Large phenotypic variances were evident for rooting and most other propagation traits, with significant proportions attributable to differences between clones (broad-sense heritabilities 0.2-0.5). Bare root assessment of rooting rate and root quality parameters tended to have the highest heritabilities, whereas rooting percentage based on root emergence from pots and shoot production were intermediate. Root biomass and root initiation had the lowest heritabilities. Strong favourable genetic correlations were found between rooting percentage and root quality traits such as root biomass, volume, and length. Lignotuber development on a seedling was associated with low rooting and a tendency to poor root quality in cuttings and was in accord with the persistence of species parent types due to gametic phase disequilibrium. On average, nodal cuttings rooted more frequently and with higher quality root systems, but significant cutting type x genotype interaction indicated that for some clones, higher rooting rates were obtained from tips. Low germination, survival of seedlings, and rooting rates suggested strong hybrid breakdown in this family.

Potential of imidacloprid to control four species of psocids (Psocoptera: Liposcelididae) infesting stored grain

The effectiveness of the neonicotinoid insecticide imidacloprid was evaluated against four psocid pests of stored grain. This research was undertaken because of the growing importance of psocids in stored grain and the need to identify methods for their control. The mortality and reproduction of adults of Liposcelis bostrychophila Badonnel, L. entomophila (Enderlein), L. decolor (Pearman) and L. paeta Pearman in wheat treated with imidacloprid were determined. There were five application rates (0.5, 1, 2, 5 and 10 mg AI kg -1 grain) and an untreated control. There were significant effects of application rate on both adult mortality and reproduction for all four species, but the effect of imidacloprid was sometimes more pronounced on reproduction. Imidacloprid was most effective against L. bostrychophila, with 100% adult mortality after 7 d at 5 mg AI kg-1, 14 d at 2 mg AI kg-1 and 28 d at 0.5 and 1 mg AI kg-1. No live progeny were produced at 2 mg AI kg-1. For L. decolor, there was 100% adult mortality after 28 d at 10 mg AI kg-1 and no live progeny were produced at 2 mg AI kg-1. For L. entomophila, there was 100% adult mortality after 14 d at 10 mg AI kg-1 and 28 d at 2 and 5 mg AI kg-1. No live progeny were produced at 10 mg AI kg-1. At 10 mg AI kg-1 there was 100% mortality of L. paeta adults after 28 d exposure and no live progeny developed. Because reproduction at some application rates occurred only in the first 14 d of exposure, it is concluded that the application rate leading to population extinction was 1 mg AI kg-1 for L. bostrychophila, 2 mg AI kg-1 for L. decolor and L. entomophila and 5 mg AI kg -1 for L. paeta. This study shows that imidacloprid has potential as a grain protectant to control all four Liposcelis species in stored grain.

Responses of two species of mesquite to initial and follow-up applications of selected herbicides in a potted trial

Quilpie mesquite (Prosopis velutina) is an invasive woody weed that is believed to have been introduced into south-west Queensland in the 1930s. Following the withdrawal of 2,4,5-T, research on P. pallida resulted in revised recommendations for control of all Prosopis spp. in Queensland. Adoption of many of these recommendations for Quilpie mesquite control produced substandard results. Following a pilot trial, a shade-house experiment was conducted to determine the differences in susceptibility of two species of mesquite, P. velutina and P. pallida, to commonly available herbicides. It was hypothesized that P. velutina was less susceptible than P. pallida, based upon claims that the registered chemical recommendations for Prosopis spp. were not sufficiently effective on P. velutina. Nine foliar herbicide treatments were applied to potted shade-house plants. Treatment effects indicated differing susceptibility between the two species. P. velutina consistently showed less response to metsulfuron, fluroxypyr, 2,4-D/picloram and triclopyr/picloram, compared to the glyphosate formulations, where negligible differences occurred between the two species. The response to glyphosate was poor at all rates in this experiment. Re-application of herbicides to surviving plants indicated that susceptibility can decrease when follow-up application is in autumn and the time since initial application is short. The relationship between leaf structure and the volume of spray adhering to a plant was assessed across species. The herbicide captured by similar-sized plants of each species differed, with P. pallida retaining a greater volume of herbicide.

Polychaete-assisted sand filters – prawn farm wastewater remediation trial

Medium bedding sand which is commonly available in coastal sedimentary deposits, and a marine polychaete-worm species from Moreton Bay recently classified as Perinereis helleri (Nereididae), were deployed in a simple low-maintenance sand filter design that potentially has application at large scale. Previous work had shown that this physical and biological combination can provide a new option for saline wastewater treatment, since the worms help to prevent sand filter blocking with organic debris and offer a profitable by-product. To test the application of this new concept in a commercial environment, six 1.84 m2 Polychaete-assisted sand filters were experimentally tested for their ability to treat wastewater from a semi-intensive prawn culture pond. Polychaetes produced exclusively on the waste nutrients that collected in these gravity-driven sand filters were assessed for their production levels and nutritional contents. Water parameters studied included temperature, salinity, pH, dissolved oxygen (DO), oxidation/ reduction potential (redox), suspended solids, chlorophyll a, biological oxygen demand (BOD), and common forms of nitrogen and phosphorus. Pond water which had percolated through the sand bed had significantly lower pH, DO and redox levels compared with inflow water. Suspended solids and chlorophyll a levels were consistently more than halved by the process. Reductions in BOD appeared dependant on regular subsurface flows. Only marginal reductions in total nitrogen and phosphorus were documented, but their forms were altered in a potentially useful way: dissolved forms (ammonia and orthophosphate) were generated by the process, and this remineralisation also seemed to be accentuated by intermittent flow patterns. Flow rates of approximately 1,500 L m-2 d-1 were achieved suggesting that a 1 ha polychaete bed of this nature could similarly treat the discharge from a 10 ha semi-intensive prawn farm. Sixteen weeks after stocking sand beds with one-month-old P. helleri, over 3.6 kg of polychaete biomass (wet weight) was recovered from the trial. Production on a sand bed area basis was 328 g m-2. Similar (P>0.05) overall biomass production was found for the two stocking densities tested (2000 and 6000 m-2; n = 3), but survival was lower and more worms were graded as small (<0.6 g) when produced at the higher density (28.2 ± 1.5 % and approx. 88 %, respectively) compared with the lower density (46.8 ± 4.4 % and approx. 76 %, respectively). When considered on a weight for weight basis, about half of the worm biomass produced was generally suitable for use as bait. The nutritional contents of the worms harvested were analysed for different stocking densities and graded sizes. These factors did not significantly affect their percentages of dry matter (DM) (18.23 ± 0.57 %), ash (19.77 ± 0.80 % of DM) or gross energy 19.39 ± 0.29 MJ kg-1 DM) (n = 12). Although stocking density did not affect the worms’ nitrogen and phosphorus contents, small worms had a higher mean proportion of nitrogen and phosphorus (10.57 ± 0.17 % and 0.70 ± 0.01 % of DM, respectively) than large worms (9.99 ± 0.12 % and 0.65 ± 0.01 % of DM, respectively) (n = 6). More lipid was present in large worms grown at the medium density (11.20 ± 0.19 %) compared with the high density (9.50 ± 0.31 %) and less was generally found in small worms (7.1-7.6 % of DM). Mean cholesterol and total phospholipid levels were 5.24 ± 0.15 mg g-1 and 13.66 ± 2.15 mg g-1 DM, respectively (n = 12). Of the specific phospholipids tested, phosphatidyl-serine or sphingomyelin were below detection limits (<0.05 mg g-1), whilst mean levels of phosphatidyl-ethanolamine, phosphatidyl-inositol, phosphatidyl-choline and lysophosphatidyl-choline were 6.89 ± 1.09, 0.89 ± 0.26, 4.04 ± 1.17 and 1.84 ± 0.37 mg g-1, respectively (n = 12). Culture density generally had a more pronounced effect on phospholipid contents than did size of worms. By contrast, worm size had a more pronounced effect on total fatty acid contents, with large worms containing significantly higher (P<0.001) levels on a DM basis (46.88 ± 2.46 mg g-1) than smaller worms (27.76 ± 1.28 mg g-1). A very broad range of fatty acids were detected with palmitic acid being the most heavily represented class (up to 14.23 ± 0.49 mg g-1 DM or 27.28 ± 0.22 % of total fatty acids). Other heavily represented classes included stearic acid (7.4-8.8 %), vaccenic acid (6.8-7.8 %), arachidonic acid (3.5-4.4 %), eicosapentaenoic acid (9.9-13.8 %) and docosenoic acid (5.7-7.0 %). Stocking density did not affect (P>0.05) the levels of amino acids present in polychaete DM, but there was generally less of each amino acid tested on a weight per weight basis in large worms than in small worms. This difference was significant (P<0.05) for the most heavily represented classes being glutamic acid (73-77 mg g-1), aspartic acid (50-54 mg g-1), and glycine (46-53 mg g-1). These results demonstrate how this polychaete species can be planted and sorted at harvest according to various strategies aimed at providing biomass with specific physical and nutritional qualities for different uses.

Effect of fire and tree-grass patches on soil nitrogen in Australian tropical savannas

Fire is an important driver of nutrient cycling in savannas. Here, we determined the impact of fire frequency on total and soluble soil nitrogen (N) pools in tropical savanna. The study sites consisted of 1-ha experimental plots near Darwin, Australia, which remained unburnt for at least 14 years or were burnt at 1-, 2- or 5-year intervals over the past 6 years. Soil was analysed from patches underneath tree canopies and in inter-canopy patches at 1, 12, 28, 55 and 152 days after fire. Patch type had a significant effect on all soil N pools, with greater concentrations of total and soluble (nitrate, ammonium, amino acids) N under tree canopies than inter-canopy patches. The time since the last fire had no significant effect on N pools. Fire frequency similarly did not affect total soil N but it did influence soluble soil N. Soil amino acids were most prominent in burnt savanna, ammonium was highest in infrequently burnt (5-year interval) savanna and nitrate was highest in unburnt savanna. We suggest that the main effect of fire on soil N relations occurs indirectly through altered tree-grass dynamics. Previous studies have shown that high fire frequencies reduce tree cover by lowering recruitment and increasing mortality. Our findings suggest that these changes in tree cover could result in a 30% reduction in total soil N and 1060% reductions in soluble N pools. This finding is consistent with studies from savannas globally, providing further evidence for a general theory of patchiness as a key driver of nutrient cycling in the savanna biome.

Implications of methoprene resistance for managing Rhyzopertha dominica (F.) in stored grain

Commercial formulations of methoprene have been used in a number of countries such as Australia and the USA to provide long-term protection to grain from a range of storage pests. The level of resistance in Rhyzopertha dominica (F.), the lesser grain borer, was investigated in laboratory experiments by direct exposure of adults on treated wheat. Adults of a reference homozygous resistant strain of R.dominica were exposed to treatments of 0, 2, 3, 5, 10, 20 and 40kg-1 of s-methoprene for 7 days. Mortality and progeny production were dose dependent with 98.7% mortality and complete progeny suppression at 40mgkg-1, which is 67 times the registered rate at which s-methoprene is applied as a grain protectant in Australia (0.6mgkg-1) and eight times the rate which has been used in the USA (5mgkg-1). This strain was also tested by adding adults to wheat treated at 0, 1, 3, 10 and 30mgkg-1 and determining the number of adults (progeny plus original parental adults) after 6, 8, 10 or 12 weeks of continuous exposure. The effect of s-methoprene was consistent regardless of the number of weeks of continuous exposure and average population suppression was 99.5% at 30mgkg-1. Screening of 162 field samples collected from southeast Australia in 2009 showed that 93% of samples reproduced when exposed to wheat treated with the Australian registered rate of 0.6mgkg-1. When four unselected resistant field samples from this region were tested, progeny production but not mortality was dose dependent, and all four samples produced live progeny at the highest dose of 30mgkg-1. Our results show that methoprene resistance is a serious threat to the management of R.dominica, and that strategies need to be developed to minimise the further development and spread of resistance. © 2013.

Field measurement of beef pen manure methane and nitrous oxide reveals a surprise for inventory calculations

Few data exist on direct greenhouse gas emissions from pen manure at beef feedlots. However, emission inventories attempt to account for these emissions. This study used a large chamber to isolate N2O and CH4 emissions from pen manure at two Australian commercial beef feedlots (stocking densities, 13-27 m(2) head) and related these emissions to a range of potential emission control factors, including masses and concentrations of volatile solids, NO3-, total N, NH4+, and organic C (OC), and additional factors such as total manure mass, cattle numbers, manure pack depth and density, temperature, and moisture content. Mean measured pen N2O emissions were 0.428 kg ha(-1) d(-1) (95% confidence interval [CI], 0.252-0.691) and 0.00405 kg ha(-1) d(-1) (95% CI, 0.00114-0.0110) for the northern and southern feedlots, respectively. Mean measured CH4 emission was 0.236 kg ha(-1) d(-1) (95% CI, 0.163-0.332) for the northern feedlot and 3.93 kg ha(-1) d(-1) (95% CI, 2.58-5.81) for the southern feedlot. Nitrous oxide emission increased with density, pH, temperature, and manure mass, whereas negative relationships were evident with moisture and OC. Strong relationships were not evident between N2O emission and masses or concentrations of NO3- or total N in the manure. This is significant because many standard inventory calculation protocols predict N2O emissions using the mass of N excreted by the animal.