Prospects for predicting insect mortality in relation to changing phosphine concentrations

Typically, in bag-stack or silo fumigations the concentration of phosphine is not constant, and yet most of what is known about phosphine efficacy against grain insects comes from studies with fixed concentrations. Indeed, where changing concentration experiments have been performed, researchers have been unable to explain observed efficacy on the basis of data from fixed concentrations. The ability to predict insect mortality in relation to changing phosphine concentrations would facilitate the development of effective fumigation protocols. In this paper, we explore the prospects for making such predictions. After reviewing published and new results, we conclude that the commonly used concentration x time (Ct) product is unreliable for this purpose. New results, for a strongly resistant strain of Rhyzopertha dominica from Australia, suggest that the relationship Cnt = k may be useful for predicting mortality of this type of insect in changing concentrations. However, in the case of a strain of Sitophilus oryzae with a type of resistance common in Australian S. oryzae, the relationship Cnt = k proved to be less reliable.

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.

Absolute quantitation of Marek's disease virus and Herpesvirus of turkeys in chicken lymphocyte, feather tip and dust samples using real-time PCR

The further development of Taqman quantitative real-time PCR (qPCR) assays for the absolute quantitation of Marek's disease virus serotype 1 (MDV1) and Herpesvirus of turkeys (HVT) viruses is described and the sensitivity and reproducibility of each assay reported. Using plasmid DNA copies, the lower limit of detection was determined to be 5 copies for the MDV1 assay and 75 copies for the HVT assay. Both assays were found to be highly reproducible for Ct values and calculated copy numbers with mean intra- and inter-assay coefficients of variation being less than 5% for Ct and 20% for calculated copy number. The genome copy number of MDV1 and HVT viruses was quantified in PBL and feather tips from experimentally infected chickens, and field poultry dust samples. Parallelism was demonstrated between the plasmid-based standard curves, and standard curves derived from infected spleen material containing both viral and host DNA, allowing the latter to be used for absolute quantification. These methods should prove useful for the reliable differentiation and absolute quantitation of MDV1 and HVT viruses in a wide range of samples.

No-tillage and nitrogen application affects the decomposition of 15N-labelled wheat straw and the levels of mineral nitrogen and organic carbon in a Vertisol

No-tillage (NT) practice, where straw is retained on the soil surface, is increasingly being used in cereal cropping systems in Australia and elsewhere. Compared to conventional tillage (CT), where straw is mixed with the ploughed soil, NT practice may reduce straw decomposition, increase nitrogen immobilisation and increase organic carbon in the soil. This study examined 15N-labelled wheat straw (stubble) decomposition in four treatments (NT v. CT, with N rates of 0 and 75 kg/ha.year) and assessed the tillage and fertiliser N effects on mineral N and organic C and N levels over a 10-year period in a field experiment. NT practice decreased the rate of straw decomposition while fertiliser N application increased it. However, there was no tillage practice x N interaction. The mean residence time of the straw N in soil was more than twice as long under the NT (1.2 years) as compared to the CT practice (0.5 years). In comparison, differences in mean residence time due to N fertiliser treatment were small. However, tillage had generally very little effect on either the amounts of mineral N at sowing or soil organic C (and N) over the study period. While application of N fertiliser increased mineral N, it had very little effect on organic C over a 10-year period. Relatively rapid decomposition of straw and short mean residence time of straw N in a Vertisol is likely to have very little long-term effect on N immobilisation and organic C level in an annual cereal cropping system in a subtropical, semiarid environment. Thus, changing the tillage practice from CT to NT may not necessitate additional N requirement unless use is made of additional stored water in the soil or mineral N loss due to increased leaching is compensated for in N supply to crops.

Australian Bat Lyssavirus

In Chapter 1, the literature relating to rabies virus and the rabies like lyssaviruses is reviewed. In Chapter 2, data are presented from 1170 diagnostic submissions for ABLV testing by fluorescent antibody test (Centocor FAT). All 27 non-bat submissions were ABLV-negative. Of 1143 bat accessions 74 (16%) were ABLV-positive, including 69 of 974 (7.1%) flying foxes (Pteropus spp.), 5 of 7 (71.4%) Saccolaimus flaviventris (Yellow-bellied sheathtail bats), none of 151 other microchiropteran bats, and none of 11 unidentified bats. Statistical analysis of data from 868 wild Black, Grey-headed, Little Red and Spectacled flying foxes (Pteropus alecto, P. poliocephalus, P. scapulatus, and P. conspicillatus) indicated that three factors; species, health status and age were associated with significant (p< 0.001) differences in the proportion of ABLV-positive bats. Other factors including sex, whether the bat bit a person or animal, region, year, and season submitted, were not associated with ABLV. Case data for 74 ABLV-positive bats, including the circumstances in which they were found and clinical signs, is presented. In Chapter 3, the aetiological diagnosis was investigated for 100 consecutive flying fox submissions with neurological signs. ABLV (32%), spinal and head injuries (29%), and neuro-angiostrongylosis (18%) accounted for most neurological syndromes in flying foxes. No evidence of lead poisoning was found in unwell (n=16) or healthy flying foxes (n=50). No diagnosis was reached for 16 cases, all of which were negative for ABLV by TaqMan PCR. The molecular diversity of ABLV was examined in Chapter 4 by sequencing 36 bases of the leader sequence, the entire N gene, and start of the P gene of 28 isolates from pteropid bats and 3 isolates from Yellow-bellied sheathtail (YBST) bats. Phylogenetic analysis indicated all ABLV isolates clustered together as a discrete group within the Lyssavirus genera closely related to rabies virus and European bat lyssavirus-2 isolates. The ABLV lineage consisted of two variants; one (ybst-ABLV) consisted of isolates only from YBST bats, the other (pteropid-ABLV) was common to Black, Grey-headed and Little Red flying foxes. No associations were found between the sequences and either the geographical location or year found, or individual flying fox species. In Chapter 5, 15 inocula prepared from the brains or salivary glands of naturally-infected bats were evaluated by intracerebral (IC) and footpad (FP) inoculation of Quackenbush mice in order to select and characterize a highly virulent inoculum for further use in bats (Inoculum 5). In Chapter 6, nine Grey-headed flying foxes were inoculated with 105.2 to 105.5 MICED50 of Inoculum 5 divided into four sites, left footpad, pectoral muscle, temporal muscle and muzzle. Another bat was inoculated with half this dose divided into the footpad and pectoral muscle only. Seven of 10 bats developed clinical disease of 1 to 4 days duration between PI-days 10 and 19 and were shown to be ABL-positive by FAT, HAM immunoperoxidase staining, virus isolation in mice, and TaqMan PCR. Five of the seven bats displayed overt aggression, one died during a seizure, and one showed intractable agitation, pacing, tremors, and ataxia. Viral antigen was demonstrated throughout the central and peripheral nervous systems and in the epithelial cells of the submandibular salivary glands (n=4). All affected bats had mild to moderate non-suppurative meningoencephalitis and severe ganglioneuritis. No ABLV was detected in three bats that remained well until the end of the experiment on day 82. One survivor developed a strong but transient antibody response. In Chapter 7, the relative virulence of inocula prepared from the brains and salivary glands of experimentally infected flying foxes was evaluated in mice by IC and FP inoculation and TaqMan assay. The effects in mice were correlated to the TaqMan CT value and indicated a crude association between virulence and CT value that has potential application in the selection of inocula. In Chapter 8, 36 Black and Grey-headed flying foxes were vaccinated with one (day 0) or two (+ day 28) doses of Nobivac rabies vaccine and co-vaccinated with keyhole limpet haemocyanin (KLH). All bats responded to the Nobivac vaccine with a rabies-RFFIT titer > 0.5 IU/mL that is nominally indicative of protective immunity. Plasma from bats with rabies titres >2 IU/mL had cross-neutralising ABLV titres >1:154. A specifically developed ELISA detected a strong but transient response to KLH.

The control of climbing asparagus (Asparagus africanus Lam.) in remnant Brigalow scrub in south-east Queensland

A replicated trial was conducted at Tallegalla in south-east Queensland to assess the effectiveness of a range of control methods for climbing asparagus Asparagus africanus Lam. A total of 18 treatments using mechanical, cut stump, basal bark, foliar spray and splatter gun techniques were trialled with a range of herbicides and application rates. Removing the plant and placing it above the ground surface was most effective in killing climbing asparagus. Basal bark spraying of 24 g triclopyr ester (40 mL Garlon® 600) or 10 g fluroxypyr ester (50 mL Starane® 200) L-1 diesel and the cut stump application of neat diesel or 225 g glyphosate (500 mL Glyphosate CT®) L-1 water offered the best chemical control of climbing asparagus.

The performance of broilers offered sorghum - compared to wheat-based diets: a large sacle experiment.

Sufficient evidence tended to indicate that at least four factors can negatively influence broiler performance when offered sorghum-based diets; in particular energy utilisation of sorghum in young birds. It was proposed that mainly CT would further influence sorghum grain AME values when consumed by young chicks (0-7 and 7-14 d old). Overall, birds consuming sorghum-based diets during the starter phase (0-21 d), did not match the performance of birds offered wheat-based diets. The use of phytase enzymes in sorghum-based diets tended to improve bird performance. However, reducing the obtained AME of sorghum grains by -0.8 MJ during the 0-21 d period appears to be a practical solution.

Controlled pollination methods for creating Corymbia hybrids.

Inter-specific Corymbia hybrids are of increasing interest to plantation forestry, yet there is little knowledge of the most suitable controlled pollination methods for this genus. Inter-specific crosses were made between C. torelliana [CT(maternal parent)] and C. citriodora subsp. variegata (CCV), C. henryi (CH) and C. citriodora subsp. citriodora (CCC) using conventional pollination, one-stop pollination (OSP) and artificially-induced protogyny on yellow buds (AIP Y) pollination methods. Additional treatments included AIP on green buds (AIP G) and the use of exclusion bags for the OSP and AIP methods. Inter-specific hybrids (CT x CCV, CT x CH and CT x CCC) were successfully created using all three pollination methods. The AIP Y treatment provided the highest seed yields and achieved time savings of >41% over the conventional and OSP methods, resulting in up to five-fold increases in operator productivity. However, the AIP Y treatment also had the highest C. torelliana contamination levels (9.3–13.2%). The use of exclusion bags with the AIP method had minimal effect on contamination rates, indicating a high proportion of selfpollen contamination. Contamination rates varied between maternal parents, suggesting variation in selfcompatibility for C. torelliana individuals. AIP using semi-ripe green buds was not effective at reducing selfing and had low operator productivity. The AIP method is suitable for use in a large-scale hybrid breeding program for C. torelliana. When self-pollination effects are managed, it could greatly reduce the costs associated with the production of seed of elite family crosses for commercial forestry deployment.

Detection and differentiation of bovine herpesvirus 1 and 5 using a multiplex real-time polymerase chain reaction.

A multiplex real-time PCR was developed for the detection and differentiation of two closely related bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5). The multiplex real-time PCR combines a duplex real-time PCR that targets the DNA polymerase gene of BoHV-1 and BoHV-5 and a real-time PCR targeting mitochondrial DNA, as a house-keeping gene, described previously by Cawthraw et al. (2009). The assay correctly identified 22 BoHV-1 and six BoHV-5 isolates from the Biosecurity Sciences Laboratory virus collection. BoHV-1 and BoHV-5 were also correctly identified when incorporated in spiked semen and brain tissue samples. The detection limits of the duplex assay were 10 copies of BoHV-1 and 45 copies of BoHV-5. The multiplex real-time PCR had reaction efficiencies of 1.04 for BoHV-1 and 1.08 for BoHV-5. Standard curves relating Ct value to template copy number had correlation coefficients of 0.989 for BoHV-1 and 0.978 for BoHV-5. The assay specificity was demonstrated by testing bacterial and viral DNA from pathogens commonly isolated from bovine respiratory and reproductive tracts. The validated multiplex real-time PCR was used to detect and differentiate BoHV-1 and BoHV-5 in bovine clinical samples with known histories.

Bovine cysticercosis-Development of a real-time PCR to enhance classification of suspect cysts identified at meat inspection

Laboratory confirmation methods are important in bovine cysticerosis diagnosis as other pathologies can result in morphologically similar lesions resulting in false identifications. We developed a probe-based real-time PCR assay to identify Taenia saginata in suspect cysts encountered at meat inspection and compared its use with the traditional method of identification, histology, as well as a published nested PCR. The assay simultaneously detects T. saginata DNA and a bovine internal control using the cytochrome c oxidase subunit 1 gene of each species and shows specificity against parasites causing lesions morphologically similar to those of T. saginata. The assay was sufficiently sensitive to detect 1 fg (Ct 35.09 +/- 0.95) of target DNA using serially-diluted plasmid DNA in reactions spiked with bovine DNA as well as in all viable and caseated positive control cysts. A loss in PCR sensitivity was observed with increasing cyst degeneration as seen in other molecular methods. In comparison to histology, the assay offered greater sensitivity and accuracy with 10/19 (53%) T. saginata positives detected by real-time PCR and none by histology. When the results were compared with the reference PCR, the assay was less sensitive but offered advantages of faster turnaround times and reduced contamination risk. Estimates of the assay's repeatability and reproducibility showed the assay is highly reliable with reliability coefficients greater than 0.94. Crown Copyright (C) 2013 Published by Elsevier B.V. All rights reserved.

Strategic tillage in no-till farming systems in Australia’s northern grains-growing regions: II. Implications for agronomy, soil and environment

In semi-arid sub-tropical areas, a number of studies concerning no-till (NT) farming systems have demonstrated advantages in economic, environmental and soil quality aspects over conventional tillage (CT). However, adoption of continuous NT has contributed to the build-up of herbicide resistant weed populations, increased incidence of soil- and stubble-borne diseases, and stratification of nutrients and organic carbon near the soil surface. Some farmers often resort to an occasional strategic tillage (ST) to manage these problems of NT systems. However, farmers who practice strict NT systems are concerned that even one-time tillage may undo positive soil condition benefits of NT farming systems. We reviewed the pros and cons of the use of occasional ST in NT farming systems. Impacts of occasional ST on agronomy, soil and environment are site-specific and depend on many interacting soil, climatic and management conditions. Most studies conducted in North America and Europe suggest that introducing occasional ST in continuous NT farming systems could improve productivity and profitability in the short term; however in the long-term, the impact is negligible or may be negative. The short term impacts immediately following occasional ST on soil and environment include reduced protective cover, soil loss by erosion, increased runoff, loss of C and water, and reduced microbial activity with little or no detrimental impact in the long-term. A potential negative effect immediately following ST would be reduced plant available water which may result in unreliability of crop sowing in variable seasons. The occurrence of rainfall between the ST and sowing or immediately after the sowing is necessary to replenish soil water lost from the seed zone. Timing of ST is likely to be critical and must be balanced with optimising soil water prior to seeding. The impact of occasional ST varies with the tillage implement used; for example, inversion tillage using mouldboard tillage results in greater impacts as compared to chisel or disc. Opportunities for future research on occasional ST with the most commonly used implements such as tine and/or disc in Australia’s northern grains-growing region are presented in the context of agronomy, soil and the environment.

Sorption and desorption of sulfuryl fluoride by wheat, flour and semolina

Sulfuryl fluoride (SF) has been developed as a fumigant for control of insect pests in stored grain. However, there is very limited information on the sorption behaviour of this fumigant, which can be critical to its bioactivity, application and potential for residues. We undertook a comprehensive laboratory study of the sorption and desorption of SF by wheat (bread and durum), flour and semolina at 15, 25 and 35 °C, moisture contents 12% and 15%, and concentration × time combinations at CT = 1500 mgh/L (4.167 mg/L × 360 h, 8.928 mg/L × 168 h and 31.25 mg/L × 48 h). At each dosage, sorption rate increased as commodity temperature and moisture content increased. The highest rates of sorption occurred at 35 °C and 15% m.c., and lowest rates at 15 °C and 12% m.c., and the rate was independent of initial concentration. Sorption followed first order reaction kinetics described by the exponential decay equation, Ct = C0·e−k*t, where k is the sorption rate constant. The most important factors determining the rate of sorption were commodity particle size (exposed surfaces) and temperature. Little sorption of fumigant occurred within the first 24 h whereas longer fumigation times resulted in significant sorption. Unbound SF was rapidly lost from the commodity upon aeration with no further desorption detected under any of the test conditions.