Isolation and characterisation of bacteria associated with flying insects in hospitals, with particualr emphasis on Clostridum difficile

Clostridium difficile is a bacterial healthcare-associated infection, which houseflies Musca domestica may transfer due to their synanthropic nature. The aims of this thesis were to determine the ability of M. domestica to transfer C. difficile mechanically and to collect and identify flying insects in UK hospitals and classify any associated bacteria. M. domestica exposed to independent suspensions of vegetative cells and spores of C. difficile were able to mechanically transfer the bacteria on to agar for up to 4 hours following exposure. C. difficile could be recovered from fly excreta for 96hrs and was isolated from the M. domestica alimentary canal. Also confirmed was the carriage of C. difficile by M. domestica larvae, although it was not retained in the pupae or in the adults that subsequently developed. Flying insects were collected from ultra-violet light flytraps in hospitals. Flies (order Diptera) were the most commonly identified. Chironomidae were the most common flies, Calliphora vicina were the most common synanthropic fly and ‘drain flies’ were surprisingly numerous and represent an emerging problem in hospitals. External washings and macerates of flying insects were prepared and inoculated onto a variety of agars and following incubation bacterial colonies identified by biochemical tests. A variety of flying insects, including synanthropic flies (e.g. M. domestica and C. vicina) collected from UK hospitals harboured pathogenic bacteria of different species. Enterobacteriaceae were the group of bacteria most commonly isolated, followed by Bacillus spp, Staphylococci, Clostridia, Streptococci and Micrococcus spp. This study highlights the potential for M. domestica to contribute to environmental persistence and spread of C. difficile in hospitals. Also illustrated is the potential for flying insects to contribute to environmental persistence and spread of other pathogenic bacteria in hospitals and therefore the need to implement pest control as part of infection control strategies.

Study of Liquid Desiccant Air-Conditioning for High Performance Greenhouses

Greenhouses have become an invaluable source of year-round food production. Further development of viable and efficient high performance greenhouses is important for future food security. Closing the greenhouse envelope from the environment can provide benefits in space heating energy savings, pest control, and CO2 enrichment. This requires the application of a novel air conditioning system to handle the high cooling loads experienced by a greenhouse. Liquid desiccant air-conditioning (LDAC) have been found to provide high latent cooling capacities, which is perfect for the application of a humid greenhouse microclimate. TRNSYS simulations were undertaken to study the feasibility of two liquid desiccant dehumidification systems based on their capacity to control the greenhouse microclimate, and their cooling performance. The base model (B-LDAC) included a natural gas boiler, and two cooling systems for seasonal operation. The second model (HP-LDAC) was a hybrid liquid desiccant-heat pump dehumidification system. The average tCOPdehum and tCOPtotal of the B-LDAC system increased from 0.40 and 0.56 in January to 0.94 and 1.09 in June. Increased load and performance during a sample summer day improved these values to 3.5 and 3.0, respectively. The average eCOPdehum and eCOPtotal values were 1.0 and 1.8 in winter, and 1.7 and 2.1 in summer. The HP-LDAC system produced similar daily performance trends where the annual average eCOPdehum and eCOPtotal values were 1.3 and 1.2, but the sample day saw peaks of 2.4 and 3.2, respectively. The B-LDAC and HP-LDAC results predicted greenhouse temperatures exceeding 30°C for 34% and 17% of the month of July, respectively. Similarly, humidity levels increased in summer months, with a maximum of 14% of the time spent over 80% in May for both models. The percentage of annual savings in space heating energy associated with closing the greenhouse to ventilation was 34%. The additional annual regeneration energy input was reduced by 26% to 526 kWhm-2, with the implementation of a heat recovery ventilator on the regeneration exhaust air. The models also predicted an electrical energy input of 245 kWhm-2 and 305 kWhm-2 for the B-LDAC and HP-LDAC simulations, respectively.

Evidence for different QTL underlying the immune and hypersensitive responses of Eucalyptus globulus to the rust pathogen Puccinia psidii

The rust Puccinia psidii infects many species in the family Myrtaceae. Native to South America, the pathogen has recently entered Australia which has a rich Myrtaceous flora, including trees of the ecologically and economically important genus Eucalyptus. We studied the genetic basis of variation in rust resistance in Eucalyptus globulus, the main plantation eucalypt in Australia. Quantitative trait loci (QTL) analysis was undertaken using 218 genotypes of an outcross F2 mapping family, phenotyped by controlled inoculation of their open pollinated progeny with the strain of P. psidii found in Australia. QTL analyses were conducted using a binary classification of individuals with no symptoms (immune) versus those with disease symptoms, and in a separate analysis dividing plants with disease symptoms into those exhibiting the hypersensitive response versus those with more severe symptoms. Four QTL were identified, two influencing whether a plant exhibited symptoms (Ppr2 and Ppr3), and two influencing the presence or absence of a hypersensitive reaction (Ppr4 and Ppr5). These QTL mapped to four different linkage groups, none of which overlap with Ppr1, the major QTL previously identified for rust resistance in Eucalyptus grandis. Candidate genes within the QTL regions are presented and possible mechanisms discussed. Together with past findings, our results suggest that P. psidii resistance in eucalypts is quantitative in nature and influenced by the complex interaction of multiple loci of variable effect.

Real-Time PCR Assays for the Detection of Puccinia psidii

Puccinia psidii (Myrtle rust) is an emerging pathogen that has a wide host range in the Myrtaceae family; it continues to show an increase in geographic range and is considered to be a significant threat to Myrtaceae plants worldwide. In this study, we describe the development and validation of three novel real-time polymerase reaction (qPCR) assays using ribosomal DNA and β-tubulin gene sequences to detect P. psidii. All qPCR assays were able to detect P. psidii DNA extracted from urediniospores and from infected plants, including asymptomatic leaf tissues. Depending on the gene target, qPCR was able to detect down to 0.011 pg of P. psidii DNA. The most optimum qPCR assay was shown to be highly specific, repeatable, and reproducible following testing using different qPCR reagents and real-time PCR platforms in different laboratories. In addition, a duplex qPCR assay was developed to allow coamplification of the cytochrome oxidase gene from host plants for use as an internal PCR control. The most optimum qPCR assay proved to be faster and more sensitive than the previously published nested PCR assay and will be particularly useful for high-throughput testing and to detect P. psidii at the early stages of infection, before the development of sporulating rust pustules.

Lantana: current management status and future prospects.

Lantana camara L. is a significant weed of which there are some 650 varieties in over 60 countries or island groups. It has been the focus of biological control attempts for a century, yet still poses major problems in many regions. Lantana has a significant impact on economic and environmental areas and is difficult to control. The key to good management of lantana is constant vigilance. Repeated control of new regrowth is critical to success. Control of new infestations should be a priority because the species is able to expand its range during good seasons, but does not die out during poor conditions. This book is a resource for land managers and researchers on methods of lantana control, particularly biocontrol.

BYGUM – a new tool for BarnYard Grass Understanding and Management

Weed management has become increasingly challenging for cotton growers in Australia in the last decade. Glyphosate, the cornerstone of weed management in the industry, is waning in effectiveness as a result of the evolution of resistance in several species. One of these, awnless barnyard grass, is very common in Australian cotton fields, and is a prime example of the new difficulties facing growers in choosing effective and affordable management strategies. RIM (Ryegrass Integrated Management) is a computer-based decision support tool developed for the south-western Australian grains industry. It is commonly used there as a tool for grower engagement in weed management thinking and strategy development. We used RIM as the basis for a new tool that can fulfil the same types of functions for subtropical Australian cotton-grains farming systems. The new tool, BYGUM, provides growers with a robust means to evaluate five-year rotations including testing the economic value of fallows and fallow weed management, winter and summer cropping, cover crops, tillage, different herbicide options, herbicide resistance management, and more. The new model includes several northernregion- specific enhancements: winter and summer fallows, subtropical crop choices, barnyard grass seed bank, competition, and ecology parameters, and more freedom in weed control applications. We anticipate that BYGUM will become a key tool for teaching and driving the changes that will be needed to maintain sound weed management in cotton in the near future.

Single gene resistance to Fusarium oxysporum f. sp. cubense Race 4 in the wild banana Musa acuminata subsp. malaccensis

Fusarium wilt of banana, caused by the fungal pathogen Fusarium oxysporum f. sp. cubense (Foc), is one of the most destructive diseases of banana. A particularly virulent strain of the pathogen, tropical race 4 (TR4), presents an emerging threat to banana producing regions throughout the world. No commercially acceptable banana cultivar is resistant to TR4 and, as with all strains of the Fusarium wilt pathogen, there is no effective chemical control. Genetic resistance to TR4 has been observed in the diploid wild banana Musa acuminata subsp. malaccensis, which has consequently received attention as a potential source of Fusarium resistance genes. The aim of this research was to determine the pattern of inheritance of the resistance trait by screening plants for resistance to Foc subtropical race 4 (SR4) and TR4. Our results showed that the F1 progeny of self-fertilized malaccensis plants challenged in pot trials against SR4 (VCGs 0120, 0129, 01211) and TR4 (VCG 01213/16) segregated for resistance according to a Mendelian ratio of 3:1 which is consistent with a single dominant gene hypothesis.

Comparison of host range and pathogenicity of isolates of Pythium myriotylum and Pythium zingiberis

Apart from morphology and genetic characteristics, species status of Pythium zingiberis and P. myriotylum may also be confirmed based on their pathogenicity and host range. An Australian putative P. zingiberis isolate and imported type isolates of P. myriotylum and P. zingiberis were subject to both in vitro and in vivo pathogenicity tests. In vitro tests were carried out on excised carrot, ginger, potato, radish, and sweet potato tuber/root sections, and on seeds and seedlings of cucumber, cauliflower, millet, rye, sweet corn, tomato, and wheat. In all assays conducted, the Australian isolate was found to be the most pathogenic, followed by type specimen of P. zingiberis (UOP 275), and then the type specimen P. myriotylum (CBS 254.70). An in vivo experiment on ginger plants at 35°C (with 10 h day light) in quarantine conditions showed that the ginger plants inoculated with the Australian isolate and also the type specimen of P. zingiberis died at 21 days after inoculation, whereas those inoculated with P. myriotylum CBS 254.70 were still green and healthy. Along with cardinal growth rate, the Australian isolate was confirmed to be closely related to P. zingiberis. This is also the first direct comparison in pathogenicity of P. zingiberis and P. myriotylum.

Avaliação da toxicidade aguda do inseticida metomil e o seu efeito sobre a atividade da acetilcolinesterase do peixe Danio rerio

Methomyl (Lannate®) is an insecticide from the carbamate group, frequently used in pest control in various types of crops. This compound works inhibiting the activity of the enzyme acetylcholinesterase. The use of physicochemical and ecotoxicological analysis is the most efficient strategy for the correct characterization and control of residues of metomil. The main objectives of this study were to evaluate the acute toxicity of methomyl in 96 hours of exposure and, through a sublethal assay of 5 hours, to assess its effect on the activity of acetylcholinesterase present in brain and squeletic dorsal muscle of the Danio rerio fish. The results showed that the LC50-96 found to D. rerio was 3.4 mg/L and it was found through the average of four definitive tests. In vitro assays were used to test the inhibitory action of methomyl directly over soluble AChE, extracted from the squeletic dorsal muscle, with maximum inhibition of 68.57% to the insecticide concentrations of 0.2 mg/L. In sublethal tests with D. rerio, inhibitory effect of methomyl was found over the soluble form of AChE in the squeletic dorsal muscle, both in one and five hours of fish exposure to the insecticide. In both period, the average values of inhibition were around 61%. In the same condition, no significant inhibitory effect of methomyl soluble and membrane AChE of the D. rerio was observed in the 0.42, 0.85, 1.70 and 2.50 mg/L concentrations and in both times of fish exposure

Protein feeding of Queensland fruit fly Bactrocera tryoni and cucumber fly Zeugodacus cucumis (Diptera: Tephritidae) on non-host vegetation: effect of plant species and bait height

Perimeter-baiting of non-crop vegetation using toxic protein baits was developed overseas as a technique for control of melon fly, Zeugodacus (Zeugodacus) cucurbitae (Coquillett) (formerly Bactrocera (Zeugodacus) cucurbitae), and evidence suggests that this technique may also be effective in Australia for control of local fruit fly species in vegetable crops. Using field cage trials and laboratory reared flies, primary data were generated to support this approach by testing fruit flies' feeding response to protein when applied to eight plant species (forage sorghum, grain sorghum, sweet corn, sugarcane, eggplant, cassava, lilly pilly and orange jessamine) and applied at three heights (1, 1.5 and 2 m). When compared across the plants, Queensland fruit fly, Bactrocera tryoni (Froggatt), most commonly fed on protein bait applied to sugarcane and cassava, whereas more cucumber fly, Zeugodacus (Austrodacus) cucumis (French) (formerly Bactrocera (Austrodacus) cucumis), fed on bait applied to sweet corn and forage sorghum. When protein bait was applied at different heights, B. tryoni responded most to bait placed in the upper part of the plants (2 m), whereas Z. cucumis preferred bait placed lower on the plants (1 and 1.5 m). These results have implications for optimal placement of protein bait for best practice control of fruit flies in vegetable crops and suggest that the two species exhibit different foraging behaviours.

Research into crown rot and wilt diseases of strawberries in Queensland

Plant losses due to fungal diseases in strawberry (Fragaria × ananassa Duch.) can potentially cause total loss of production. Three fungal pathogens, Fusarium oxysporum f. sp. fragariae, Colletotrichum gloeosporioides and Macrophomina phaseolina, cause similar crown rot and wilt symptoms in strawberries in Queensland. Since the withdrawal of methyl bromide in 2005, no effective chemical control of any of the three pathogens has been available. This study aims at identifying sources of plant genetic resistance that can be used in the breeding program to develop resistant cultivars for use as part of an integrated disease management plan in commercial strawberry production. Results from glasshouse pathogenicity and screening trials on the three pathogens showed that when breeding for resistance against a pathogen, resistance to other pathogens also needs to be considered, e.g., cultivar 'Festival' is resistant to F. oxysporum f. sp. fragariae, but is highly susceptible to C. gloeosporioides. The cultivars 'Earlisweet', 'Kabarla' and 'Phenomenal', two seedling clones and four DAFF breeding lines were resistant to C. gloeosporioides. Cultivar 'Suncoast Delight' showed the most promising level of resistance to M. phaseolina. These cultivars, breeding lines and seedling selections have been made available for incorporation into the crossing program to support the Queensland strawberry breeding program.

SUSCEPTIBILITY TO BRUCHIDS AMONG COMMON BEANS IN UGANDA

The bean bruchids, Acanthoscelides obtectus Say and Zabrotes subfasciatus Boheman (Coleoptera: Bruchidae), are cosmopolitan pests of stored dry common beans ( Phaseolus vulgaris L. ), causing damage through reduction of grain quality and seed germination. Biological resistance to these bruchids was definitively established in non-cultivated bean accessions, and has been introgressed into a range of drybean market classes. However, existing resistance to bruchids in Uganda’s common bean germplasm has not been systematically studied. In this study, 45 bean genotypes from the National Bean-Breeding Programme (25 genotypes) and agroecologically diverse bean growing areas in Uganda (20 genotypes), were evaluated for postharvest bruchid resistance. None of the evaluated bean genotypes expressed resistance to either bruchid species, with all the 45 bean genotypes supporting bruchid development, reproduction and feeding. All genotypes were severely damaged by bruchids feeding, resulting in significant (P<0.05) reduction of seed germination. Reduction in seed germination was related to the number of emergence holes and seed size; small bean seeds damaged by up to 2 bruchid emergence holes had a 7.1% reduction in germination, while large bean seeds with a similar number of emergence holes showed a 25% reduction in germination. Whereas this study further confirms bruchids as important storage pests of beans causing direct loss through consumption of the seed and indirect loss through viability deterioration, the resistance to bruchids in the evaluated range of Uganda’s dry bean germplasm is inadequate for direct exploitation in a breeding programme.

Regulamentação do intercâmbio internacional de agentes de controle biológico no Brasil.

2009

Anti-anxiety effect of methanol extract of Pericarpium zanthoxyli using a strychnine-sensitive glycine receptor model

Purpose: To determine if the methanol extract of Pericarpium zanthoxyli exerts anti-anxiety effects and also to explore any probable anti-anxiety mechanism in vivo. Methods: The staircase test, elevated plus maze test, rota-rod treadmill test and convulsions induced by strychnine and picrotoxin on mice were tested to identify potential mechanism of anti-anxiety activity of the plant extract. Results: The plant extract (10 mg/kg, p.o.) significantly reduced rearing numbers in the staircase test while it increased the time spent in the open arms as well as the number of entries to the open arms in the elevated plus maze test, suggesting that it has significant anti-anxiety activity. Furthermore, the extract inhibited strychnine-induced convulsion. However, it had little effect on picrotoxin-induced convulsion, suggesting that its anti-anxiety activity may be linked to strychnine-sensitive glycine receptor and not GABA receptor. Conclusion: These results suggest that the Pericarpium zanthoxyli extract may be beneficial for the control of anxiety.

Avaliação da toxicidade aguda do inseticida metomil e o seu efeito sobre a atividade da acetilcolinesterase do peixe Danio rerio

Methomyl (Lannate®) is an insecticide from the carbamate group, frequently used in pest control in various types of crops. This compound works inhibiting the activity of the enzyme acetylcholinesterase. The use of physicochemical and ecotoxicological analysis is the most efficient strategy for the correct characterization and control of residues of metomil. The main objectives of this study were to evaluate the acute toxicity of methomyl in 96 hours of exposure and, through a sublethal assay of 5 hours, to assess its effect on the activity of acetylcholinesterase present in brain and squeletic dorsal muscle of the Danio rerio fish. The results showed that the LC50-96 found to D. rerio was 3.4 mg/L and it was found through the average of four definitive tests. In vitro assays were used to test the inhibitory action of methomyl directly over soluble AChE, extracted from the squeletic dorsal muscle, with maximum inhibition of 68.57% to the insecticide concentrations of 0.2 mg/L. In sublethal tests with D. rerio, inhibitory effect of methomyl was found over the soluble form of AChE in the squeletic dorsal muscle, both in one and five hours of fish exposure to the insecticide. In both period, the average values of inhibition were around 61%. In the same condition, no significant inhibitory effect of methomyl soluble and membrane AChE of the D. rerio was observed in the 0.42, 0.85, 1.70 and 2.50 mg/L concentrations and in both times of fish exposure