Using Trichogramma Westwood (Hymenoptera: Trichogrammatidae) for insect pest biological control in cotton crops: an Australian perspective.

Trichogramma Westwood egg parasitoids alone generally fail to suppress heliothine pests when released in established cotton-growing regions. Factors hindering their success include indiscriminate use of detrimental insecticides, compensation for minimal pest larval hatch due to their activity via reduced larval cannibalism or mortality in general, singly laid heliothine eggs avoiding detection and asynchronous development benefiting host over parasitoid. Yet, despite these limitations, relatively large Trichogramma pretiosum Riley populations pervade and effectively suppress Helicoverpa (Hardwick) pests in Australian Bt (Bacillus thuringiensis Berliner)-transgenic cotton, Gossypium hirsutum L., crops, especially in the Ord River Irrigation Area (ORIA) of tropical northern Australia, where their impact on the potentially resistant pest species, Helicoverpa armigera (Hubner), is considered integral to the local insecticide resistance management (IRM) strategy for continued, sustainable Bt-transgenic cotton production. When devoid of conventional insecticides, relatively warm and stable conditions of the early dry season in winter grown ORIA Bt-transgenic cotton crops are conducive to Trichogramma proliferation and biological control appears effective. Further, there is considerable scope to improve Trichogramma's biological control potential, in both the ORIA and established cotton-growing regions, via habitat manipulation. It is proposed that Trichogramma may prove equally effective in developing agricultural regions of monsoonal northern Australia, and that environmental constraints on Trichogramma survival, and those of other natural enemies, require due consideration prior to their successful application in biological control programs.

A comparison of the performance and biological efficiency of new knapsack sprayers and a controlled droplet application (CDA) sprayer for the control of the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae)

Field trials and laboratory bioassays were undertaken to compare the performance and efficacy (mortality of diamondback moth larvae) of insecticides applied to cabbages with three high volume hydraulic knapsack sprayers (NS-16, PB-20 and Selecta 12V) and a controlled droplet application (CDA) sprayer. In field experiments, the high volume knapsack sprayers (application rate 500-600 L ha-') provided better spray coverage on the upper and lower surfaces of inner leaves, the upper surfaces of middle and outer leaves, and greater biological efficacy than the CDA sprayer (application rate 20~40 L ha-'). The PB-20 provided better spray coverage on the upper surface of middle leaves and both Surfaces of outer leaves when compared with the Selecta I2V. However, its biological efficacy in the field was not significantly different from that of the other high volume sprayers. Increasing the application rate from 20 to 40 L ha - ' for the CDA sprayer significantly increased droplet density but had no impact on test insect mortality. Laboratory evaluations of biological efficacy yielded higher estimates than field evaluations and there was no significant difference between the performance of the PB-20 and the CDA sprayer. Significant positive relationships were detected between insect mortality and droplet density deposited for both the PB-20 and the CDA sprayers

Genetic variability of Tomato spotted wilt virus in Australia and validation of real time RT-PCR for its detection in single and bulked leaf samples

The potential for large-scale use of a sensitive real time reverse transcription polymerase chain reaction (RT-PCR) assay was evaluated for the detection of Tomato spotted wilt virus (TSWV) in single and bulked leaf samples by comparing its sensitivity with that of DAS-ELISA. Using total RNA extracted with RNeasy® or leaf soak methods, real time RT-PCR detected TSWV in all infected samples collected from 16 horticultural crop species (including flowers, herbs and vegetables), two arable crop species, and four weed species by both assays. In samples in which DAS-ELISA had previously detected TSWV, real time RT-PCR was effective at detecting it in leaf tissues of all 22 plant species tested at a wide range of concentrations. Bulk samples required more robust and extensive extraction methods with real time RT-PCR, but it generally detected one infected sample in 1000 uninfected ones. By contrast, ELISA was less sensitive when used to test bulked samples, once detecting up to 1 infected in 800 samples with pepper but never detecting more than 1 infected in 200 samples in tomato and lettuce. It was also less reliable than real time RT-PCR when used to test samples from parts of the leaf where the virus concentration was low. The genetic variability among Australian isolates of TSWV was small. Direct sequencing of a 587 bp region of the nucleoprotein gene (S RNA) of 29 isolates from diverse crops and geographical locations yielded a maximum of only 4.3% nucleotide sequence difference. Phylogenetic analysis revealed no obvious groupings of isolates according to geographic origin or host species. TSWV isolates, that break TSWV resistance genes in tomato or pepper did not differ significantly in the N gene region studied, indicating that a different region of the virus genome is responsible for this trait.

A simplified PCR test for early detection of dwarf off-types in micropropagated Cavendish bananas (Musa spp. AAA)

The dwarf somaclonal variant is a major problem affecting micropropagation of the banana cultivar Williams (Musa spp. AAA; subgroup Cavendish). This problem arises from genetic changes that occur during the tissue culture process. Early identification of this problem is difficult and propagators must wait until plants are ex vitro in order to visualise the dwarfism phenotype. In this study, we have improved a SCAR-based molecular diagnostic technique, developed by Damasco et al. [Acta Hortic. 461 (1997) 157], for the early identification of dwarf off-types. We have included a positive internal control in a multiplex PCR and adapted the technique for use with small amounts of fresh in vitro leaf material as PCR template. The control product is a 500 bp fragment from 18S rRNA and is amplified in all tissues irrespective of phenotype. The use of small in vitro leaf material removing the need for genomic DNA extraction.

Characterisation and early detection of an offtype from micropropagated Lady Finger bananas

An offtype has been identified from micropropagated Lady Finger bananas (Musa spp., AAB group, Pome subgroup) that is characterised by its slow growth and poor bunch size. Bunch weights were approximately 25% those of normal Lady Finger plants and all of the fruit produced was unmarketable. This particular offtype is the most commonly encountered from micropropagated Lady Finger plants and, in 2 instances, blocks of 3000 and 1500 plants were entirely comprised of this single offtype. Detection of offtype plants was possible during establishment and growth of plants in the glasshouse by the presence of chlorotic streaks in the leaves. In more severe cases the streaks coalesced into chlorotic patches that developed thin, necrotic areas that eventually produced holes or splits in the leaves. Symptom expression was not ameliorated by the addition of fertiliser and even though symptoms were similar to severe Ca and B deficiency, both normal and offtype plants had similar levels of these elements in the leaves. The offtype plants were also slow growing in the glasshouse and produced significantly (P<0.05) smaller pseudostems and leaves than normal plants. Offtype plants could be readily detected after 4 weeks deflasking using the presence of chlorotic streaks in the leaves as the main selection criterion. Maximum discrimination was possible between weeks 5–7 and at the 6-leaf stage when all of the offtypes could be detected.

Pasteurella multocida detection by 5' Taq nuclease assay: a new tool for use in diagnosing fowl cholera

A 5′ Taq nuclease assay utilising minor groove binder technology and targeting the 16S rRNA gene was designed to detect Pasteurella multocida (the causative agent of fowl cholera) in swabs collected from poultry. The assay was first evaluated using pure cultures. The assay correctly identified four P. multocida taxonomic type strains, 18 P. multocida serovar reference strains and 40 Australian field isolates (17 from poultry, 11 from pigs and 12 from cattle). Representatives of nine other Pasteurella species, 26 other bacterial species (18 being members of the family Pasteurellaceae) and four poultry virus isolates did not react in the assay. The assay detected a minimum of approximately 10 cfu of P. multocida per reaction. Of 79 poultry swabs submitted to the laboratory for routine bacteriological culture, 17 were positive in the 5′ Taq nuclease assay, but only 10 were positive by culture. The other 62 swabs were negative for P. multocida by both 5′ Taq nuclease assay and culture. The assay is suitable for use in diagnosing fowl cholera, is more rapid than bacteriological culture, and may also have application in diagnosing P. multocida infections in cattle and pigs.

Damage potential of an introduced biological control agent Agonosoma trilineatum (F.) on bellyache bush (Jatropha gossypiifolia L.)

The seed-feeding jewel bug, Agonosoma trilineatum (F.), is an introduced biological control agent for bellyache bush, Jatropha gossypiifolia L. To quantify the damage potential of this agent, shadehouse experiments were conducted with individual bellyache bush plants exposed to a range of jewel bug densities (0, 6 or 24 jewel bugs/plant). The level of abortion of both immature and mature seed capsules and impacts on seed weight and seed viability were recorded in an initial short-term study. The ability of the jewel bug to survive and cause sustained damage was then investigated by measuring seed production, the survival of adults and nymph density across three 6-month cycles. The level of seed capsule abortion caused by the jewel bug was significantly affected by the maturity status of capsules and the density of insects present. Immature capsules were most susceptible and capsule abortion increased with jewel bug density. Similarly, on average, the insects reduced the viability of bellyache bush seeds by 79% and 89% at low and high densities, respectively. However, sustaining jewel bug populations for prolonged periods proved difficult. Adult survival at the end of three 6-month cycles averaged 11% and associated reductions in viable seed production ranged between 55% and 77%. These results suggest that the jewel bug has the potential to reduce the number of viable seeds entering the soil seed bank provided populations can be established and maintained at sufficiently high densities.

Detection of Arcobacter spp. in piggery effluent and effluent-irrigated soils in southeast Queensland

Aims: To investigate the occurrence and levels of Arcobacter spp. in pig effluent ponds and effluent-treated soil. Methods and Results: A Most Probable Number (MPN) method was developed to assess the levels of Arcobacter spp. in seven pig effluent ponds and six effluent-treated soils, immediately after effluent irrigation. Arcobacter spp. levels in the effluent ponds varied from 6.5 × 105 to 1.1 × 108 MPN 100 ml-1 and in freshly irrigated soils from 9.5 × 102 to 2.8 × 104 MPN g-1 in all piggery environments tested. Eighty-three Arcobacter isolates were subjected to an abbreviated phenotypic test scheme and examined using a multiplex polymerase chain reaction (PCR). The PCR identified 35% of these isolates as Arcobacter butzleri, 49% as Arcobacter cryaerophilus while 16% gave no band. All 13 nonreactive isolates were subjected to partial 16S rDNA sequencing and showed a high similarity (>99%) to Arcobacter cibarius. Conclusions: A. butzleri, A. cryaerophilus and A. cibarius were isolated from both piggery effluent and effluent-irrigated soil, at levels suggestive of good survival in the effluent pond. Significance and Impact of the Study: This is the first study to provide quantitative information on Arcobacter spp. levels in piggery effluent and to associate A. cibarius with pigs and piggery effluent environments.

Detection of Chlamydiaceae DNA in veterinary specimens using a family-specific PCR

Aims: The aim of this work was to develop a rapid molecular test for the detection of the Chlamydiaceae family, irrespective of the species or animal host. Methods and Results: The method described herein is a polymerase chain reaction targeting the 16S rRNA gene of the Chlamydiaceae family, and the results demonstrate that the test reacts with five reference Chlamydiaceae but none of the 19 other bacterial species or five uninfected animal tissues tested. The results also indicate the enhanced sensitivity of this test when compared with conventional culture or serology techniques. This is demonstrated through parallel testing of six real clinical veterinary cases and confirmatory DNA sequence analysis. Conclusions, Significance and Impact of the Study: This test can be used by veterinary diagnostic laboratories for rapid detection of Chlamydiaceae in veterinary specimens, with no restriction of chlamydial species or animal host. The test does not differentiate chlamydial species, and if required, speciation must be carried out retrospectively using alternate methods. However, for the purpose of prescribing therapy for chlamydiosis, this test would be an invaluable laboratory tool.

Phylogenetic considerations for predicting the host range of Ustilago sporoboli-indici, a potential biological control agent for Sporobolus species in Australia

The ribosomal DNA internal transcribed spacer region was amplified and sequenced from a selection of specimens of the Sporobolus smut Ustilago sporoboli-indici. Phylogenetic comparison with other Ustilago and Sporisorium species revealed strong support for an evolutionary radiation of Ustilago species infecting the Chloridoideae and Pooideae, of which U. sporoboli-indici forms a major lineage. Comparisons are made with other groups of plant pathogenic fungi, and it is concluded that phylogenetic analyses of potential biocontrol agents are useful for identifying pathogens that are derived from evolutionary lineages that parasitize a wide range of unrelated plants. Such pathogens are less desirable as biocontrol agents as they may have a greater likelihood of infecting plants outside their normal host ranges.

Validation of a Competitive Enzyme-Linked Immunosorbent Assay for Detection of Babesia bigemina Antibodies in Cattle

A competitive enzyme-linked immunosorbent assay (cELISA) based on a broadly conserved, species-specific, B-cell epitope within the C terminus of Babesia bigemina rhoptry-associated protein 1a was validated for international use. Receiver operating characteristic analysis revealed 16% inhibition as the threshold for a negative result, with an associated specificity of 98.3% and sensitivity of 94.7%. Increasing the threshold to 21% increased the specificity to 100% but modestly decreased the sensitivity to 87.2%. By using 21% inhibition, the positive predictive values ranged from 90.7% (10% prevalence) to 100% (95% prevalence) and the negative predictive values ranged from 97.0% (10% prevalence) to 48.2% (95% prevalence). The assay was able to detect serum antibody as early as 7 days after intravenous inoculation. The cELISA was distributed to five different laboratories along with a reference set of 100 defined bovine serum samples, including known positive, known negative, and field samples. The pairwise concordance among the five laboratories ranged from 100% to 97%, and all kappa values were above 0.8, indicating a high degree of reliability. Overall, the cELISA appears to have the attributes necessary for international application.

Biological control of parthenium (Parthenium hysterophorus) in Australian rangeland translates to improved grass production.

Biological control of parthenium, a major weed in grazing areas in Australia, was initiated in the mid 1970s. Since then, nine species of insects and two rust fungi have been introduced. Evaluation using pesticide exclusion at two sites (Mt. Panorama and Plain Creek) in Queensland, Australia, revealed that classical biological control had a significant negative effect on the target weed, but the impact varied between years. In this study, I quantified the effects of biological control of parthenium on grass production. Grass production declined with the increase in parthenium biomass. Significant increase in grass production due to biological control was observed, but only in 1 of 4 yr at Mt. Panorama and 2 of 4 yr at Plain Creek. At Mt. Panorama, there was a 40% increase in grass biomass in 1997 because of defoliation by Zygogramma bicolorata and galling by Epiblema strenuana. At Plain Creek, grass biomass increased by 52% in 1998 because of E. strenuana and by 45% in 2000 because of combined effects of E. strenuana and the summer rust Puccinia melampodii. This study provides evidence on the beneficial effects of biological control of parthenium in areas under limited grazing.

Detection of equine herpesvirus type 1 using a real-time polymerase chain reaction.

Equid herpesvirus 1 (EHV1) is a major disease of equids worldwide causing considerable losses to the horse industry. A variety of techniques, including PCR have been used to diagnose EHV1. Some of these PCRs were used in combination with other techniques such as restriction enzyme analysis (REA) or hybridisation, making them cumbersome for routine diagnostic testing and increasing the chances of cross-contamination. Furthermore, they involve the use of suspected carcinogens such as ethidium bromide and ultraviolet light. In this paper, we describe a real-time PCR, which uses minor groove-binding probe (MGB) technology for the diagnosis of EHV1. This technique does not require post-PCR manipulations thereby reducing the risk of cross-contamination. Most importantly, the technique is specific; it was able to differentiate EHV1 from the closely related member of the Alphaherpesvirinae, equid herpesvirus 4 (EHV4). It was not reactive with common opportunistic pathogens such as Escherichia coli, Klebsiella oxytoca, Pseudomonas aeruginosa and Enterobacter agglomerans often involved in abortion. Similarly, it did not react with equine pathogens such as Streptococcus equi, Streptococcus equisimilis, Streptococcus zooepidemicus, Taylorella equigenitalis and Rhodococcus equi, which also cause abortion. The results obtained with this technique agreed with results from published PCR methods. The assay was sensitive enough to detect EHV1 sequences in paraffin-embedded tissues and clinical samples. When compared to virus isolation, the test was more sensitive. This test will be useful for the routine diagnosis of EHV1 based on its specificity, sensitivity, ease of performance and rapidity.

Multiplex real-time PCR for the detection and differentiation of equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4).

A multiplex real-time PCR was designed to detect and differentiate equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4). The PCR targets the glycoprotein B gene of EHV-1 and EHV-4. Primers and probes were specific to each equine herpesvirus type and can be used in monoplex or multiplex PCRs, allowing the differentiation of these two closely related members of the Alphaherpesvirinae. The two probes were minor-groove binding probes (MGB?) labelled with 6-carboxy-fluorescein (FAM?) and VIC® for detection of EHV-1 and EHV-4, respectively. Ten EHV-1 isolates, six EHV-1 positive clinical samples, one EHV-1 reference strain (EHV-1.438/77), three EHV-4 positive clinical samples, two EHV-4 isolates and one EHV-4 reference strain (EHV-4 405/76) were included in this study. EHV-1 isolates, clinical samples and the reference strain reacted in the EHV-1 real-time PCR but not in the EHV-4 real-time PCR and similarly EHV-4 clinical samples, isolates and the reference strain were positive in the EHV-4 real-time PCR but not in the EHV-1 real-time PCR. Other herpesviruses, such as EHV-2, EHV-3 and EHV-5 were all negative when tested using the multiplex real-time PCR. When bacterial pathogens and opportunistic pathogens were tested in the multiplex real-time PCR they did not react with either system. The multiplex PCR was shown to be sensitive and specific and is a useful tool for detection and differentiation of EHV-1 and EHV-4 in a single reaction. A comprehensive equine herpesvirus disease investigation procedure used in our laboratory is also outlined. This procedure describes the combination of alphaherpesvirus multiplex real-time PCR along with existing gel-based PCRs described by other authors.

Utilizing the assassin bug, Pristhesancus plagipennis (Hemiptera: Reduviidae), as a biological control agent within an integrated pest management programme for Helicoverpa spp. (Lepidoptera: Noctuidae) and Creontiades spp. (Hemiptera: Miridae) in cotton

Helicoverpa spp. and mirids, Creontiades spp., have been difficult to control biologically in cotton due to their unpredictable temporal abundance combined with a cropping environment often made hostile by frequent usage of broad spectrum insecticides. To address this problem, a range of new generation insecticides registered for use in cotton were tested for compatibility with the assassin bug, Pristhesancus plagipennis (Walker), a potential biological control agent for Helicoverpa spp. and Creontiades spp. Indoxacarb, pyriproxifen, buprofezin, spinosad and fipronil were found to be of low to moderate toxicity on P. plagipennis whilst emamectin benzoate, abamectin, diafenthiuron, imidacloprid and omethaote were moderate to highly toxic. Inundative releases of P. plagipennis integrated with insecticides identified as being of low toxicity were then tested and compared with treatments of P. plagipennis and the compatible insecticides used alone, conventionally sprayed usage practice and an untreated control during two field experiments in cotton. The biological control provided by P. plagipennis nymphs when combined with compatible insecticides provided significant (P<0.001) reductions in Helicoverpa and Creontiades spp. on cotton and provided equivalent yields to conventionally sprayed cotton with half of the synthetic insecticide input. Despite this, the utilization of P. plagipennis in cotton as part of an integrated pest management programme remains unlikely due to high inundative release costs relative to other control technologies such as insecticides and transgenic (Bt) cotton varieties.