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Due to the rapid development of tick resistance to synthetic chemical acaricides and the potential risk posed by these chemicals to non-target species, efforts are intensifying towards the development of entomopathogenic fungi as an environmentally benign alternative tick control strategy. The main objective of this study is the development of formulations and delivery systems for control of ticks with entomopathogenic fungi. Screening of entomopathogenic fungal isolates is the first step towards the development of mycoacaricides. Twelve isolates of Metarhizium anisopliae (Metschnik.) Sorok. and three isolates of Beauveria bassiana (Bals.) Vuill. (Ascomycota: Hypocreales) were evaluated for pathogenicity against unfed Rhipicephalus pulchellus Gerstäcker (Acari: Ixodidae) adults under laboratory conditions. Out of the 15 isolates screened, 4 isolates were virulent against R. pulchellus, inducing significantly (F value = 11.86; DF = 14, 75; P < 0.0001) higher tick mortalities; i.e. ICIPE 78 (76.1 ± 5.9%), ICIPE 69 (62.6 ± 5%), ICIPE 62 (49.8 ± 5.8%) and ICIPE 60 (49.6 ± 7.7%), four weeks post-treatment compared to the others. The results of this study demonstrate that R. pulchellus ticks were susceptible to some M. anisopliae isolates. Since the process of strain selection is an important step in the development of insect pathogens for biological control, an appropriate bioassay technique for infecting different life stages of a host is of paramount importance. Bioassays were conducted in the laboratory to evaluate the efficacy of different methods of inoculation using R. pulchellus as a model. In a preliminary experiment, an oil-based formulation of M. anisopliae titred at 109 conidia ml-1 was applied to R. pulchellus adults using a Burgerjon spray tower or a microapplicator. Inoculation by microapplicator yielded poor results (25.0% tick mortality) compared to Burgerjon’s spray tower (52.3% tick mortality) , although the mean number of fungal conidia on R. pulchellus adults was lower (1.5 x 104 ± 1.1 x 103 conidia ml-1) after spraying by Burgerjon’s spray tower compared to 1 x 106 conidia ml-1 obtained with the microapplicator. Thus, inoculation by Burgerjon’s spray tower was selected for further investigations. Different modes of inoculation were tested and included direct spray of inoculum on the tick and substrate (SS), direct spray on the substrate and tick followed by transfer of the tick to clean uncontaminated Petri dish (SP) or indirect inoculation of ticks through substrate (SW). The LC50 values following contamination of nymphs (LC50 = 1.4 x 107 conidia ml-1) and adults (LC50 = 6.7 x 107 conidia ml-1) in SS were significantly lower compared to SP; nymphs (LC50 = 5.7 x 108 conidia ml-1) and adults (LC50 = 5.3 x 109 conidia ml-1) and SW; nymphs (LC50 = 5 x 108 conidia ml-1) and adults (LC50 > 1010 conidia ml-1). Although the LC50 value in SS was the lowest, it recorded the highest tick mortality among control ticks (24.2% at two weeks post-treatment) and (23.3% at three weeks post-treatment) in nymphs and adults respectively compared to SP (2.5 and 5.8% respectively) and SW (0.0 and 0.0). Results show that among the modes of contamination tested, SP was the most appropriate for contaminating R. pulchellus adults. SW and SP were identified as appropriate techniques for infecting the R. pulchellus nymphs with conidia formulated in oil. The integrated use of semiochemicals and entomopathogenic fungus (hypocreales) may optimise the performance of a fungus as a biocontrol agent against ticks. Initially, experiments were conducted to evaluate the attraction of the hunter tick Amblyomma variegatum Fabriscius (Acari: Ixodidae) to semiochemicals. In one of the experiments, the simultaneous release of 1-octen-3-ol and AAAP together with CO2 from a trap in simulated field plots attracted up to 94 ± 6% of adult ticks from a distance of 6 m, and up to 24.0 ± 5.1% from 8 m. Formulations of M. anisopliae (dry powder, oil, and emulsifiable) applied within the trap baited with AAAP and 1-octen-3-ol resulted in high levels of contamination of the ticks attracted to the traps. However, 48 hr after autoinoculation, 89.1 and 33.3% of conidia were lost in dry powder and oil formulations, respectively. Emulsifiable formulation lost the lowest number of propagules (17.1%). Samples of ticks attracted to the baited traps were transferred to plastic basins containing grass and maintained for 5 weeks. The experiment was conducted in the rainy and dry seasons. The emulsifiable formulation gave the highest relative reduction in tick numbers in both seasons: 54.7 and 46.5% in the rainy and dry seasons, respectively, followed by the oil formulation (32 and 23.8%) and the powder formulation (38.0 and 24.4%). Following the high attraction and contamination of A. variegatum under semi-field conditions, experiments were done to evaluate the efficacy of M. anisopliae-treated semiochemical-baited traps for control of A. variegatum under field conditions. Unfed A. variegatum adults (118) were seeded in each 100-m plot. An emulsifiable formulation of M. anisopliae (consisting of 49.5% sterile distilled water, fungal conidia, 49.5% corn oil [CHEF cooking oil, Premier Oil Mills LTD] and 1% Tween 80) titrated at 109 conidia ml-1- was applied in a semiochemical-baited traps (900 cm2) which were placed at 5 spots within the plot. The control and fungal treatments were repeated after 14 and 28 days soon after rotating the traps clockwise (45o) in order to cover different sections of the plot. In the control plots, traps baited with semiochemicals only were used. Six weeks after the initiation of the experiments, five semiochemical-baited traps (untreated) were deployed in each plot for 3 successive days to trap ticks in the treated and control plots. The percentage of ticks recovered in the fungus-treated plots were significantly lower (31.1 ± 5.2%) than in the control plots (85.6% ± 3%) (P < 0.001), which represented a relative tick reduction of 63.7%. Mortality of 93.8 ± 2.3% was observed among the ticks that were recovered from the field and maintained in the laboratory for two weeks; while only 3.3 ± 0.9% died from the control plots. The results of this study open up the possibility of developing an environmentally friendly, low cost product to control these economically important ticks. / Thesis (PhD)--University of Pretoria, 2009. / Paraclinical Sciences / unrestricted

Thesis (PhD (Paraclinical Sciences, Veterinary Science))--University of Pretoria, 2009. / Includes bibliographical references. Also available in print format.

Thesis (M.Sc.(Zoology and Entomology))--University of Pretoria, 2000. / Abstracts in Afrikaans and English. Includes bibliographical references (leaves 72-79).

A THESIS PRESENTED TO CAPE PENINSULA UNIVERSITY OF TECHNOLOGY IN FULFILMENT OF THE REQUIREMENTS FOR THE MASTERS OF TECHNOLOGY DEGREE IN HORTICULTURE. IN THE FACULTY OF APPLIED SCIENCES 2014 / Codling moth ([Cydia pomonella (Lepidoptera: Tortricidae]) infest pomes resulting in high production losses to fruit farmers in South Africa and globally. Many farmers are adopting biocontrol methods including the use of entomopathogens as alternatives to synthetic insecticides for sustainable management of Arthropod pests. Research activities on screening and application of entomopathogenic fungi (EPF) have intensified in recent years. This study was carried out to isolate and identify virulent indigenous entomopathogenic fungal strains from soils of selected locations in the Western Cape region of South Africa that are pathogenic against codling moth (Cydia pomonella (Linn) [Lepidoptera: Totricidae]) and to evaluate the possiblility of combining semiochemicals and fungal inoculums in a simulated attract-and-fungus contamination system for codling moth control. Soil samples were collected from 10 locations in the Western Cape, South Africa for the isolation of entomopathogenic fungi (EPF) by baiting the soil samples with 5th instar larvae of codling moths. Fungal strains were identified and characterized using light microscopy and DNA analysis (internal transcribed spacer region) and elongation factor 1-α (EF 1-α) genes of fungal cultures. Thirty-nine (39) isolates belonging to six species were obtained; Purpureocillium lilacinum (8 isolates), Fusarium oxysporum (five isolates), Fusarium polyphialidicum (two isolates), Beauveria pseudo-bassiana (one isolate), Aspergillus flavus (three isolates) and Metarhizium robertii (20 isolates). Generally, M. robertsii was the most frequently encountered species representing 51% of the total number of isolates collected from the soil samples. This is the first report of the isolation of M. robertsii in southern Africa. A screening test to identify the most virulent fungal strains against codling moth was carried out on 39 isolates by immersing 5th instar larvae of C. pomonella into aqueous spore suspension 1 x 108 conidia ml-1. Mortality data from the immersion bioassay indicated that the 39 fungal isolates were pathogenic against fifth instar larvae of codling moth inducing 47-85% insect mortality at an infective concentration of 1 x108 spores’ ml-1, 5 days post-treatment. Two fungal strains MTL151 and GW461 induced over 85% larval mortality and were selected for further evaluations. The effect of MTL151 and GW461 on egg hatchability of 0-day old eggs was evaluated by exposing freshly laid eggs on wax papers that were pre-treated with fungal spores ranging from 103 -108 spores/ml. Egg hatchability reduced significantly from 93-71% (GW461) and 95-66% (MTL151) as spore concentration increases from1 x 103 to 1 x 108 spores ml-1, respectively. The highest spore concentration significantly inhibited egg hatchability resulting in lower egg hatchability compared to that obtained with the cypermethrin containing commercial insecticide (Fruitfly [registered by Kombat (PTY) Ltd] ) tested at the recommended dose of 0.25 ml/250 ml of water. The potential of the two M. robertsii strains to protect apples from infestation by codling moth neonates was assessed in an apple fruit bioassay under laboratory and field bioassay. Codling moth neonates were exposed to apples that were sprayed topically with varied conidia concentrations (103 - 108 sporesml-1). The mean number of participating apple fruits having developing/developed larvae in the core/flesh significantly reduced from 5.3 to 1 and 7.6 to 1 for MTL151 and GW461, respectively as spore concentrations increased from 1 x103 to 1x 108 spores/ml-1. A concurrent decrease in apple fruit rot as conidia concentration increased was observed. Up to 90% of apples treated with 1 x 108 spores/ml-1 had no larva present in their cores and this result compared favourably with the commercial pesticide (Fruitfly) used at a recommended dose of 0.25g/250 ml of water. No significant difference was found between the EC50 values of 1.2 x 103 (CI=1.2 x 102 -1 x 105) (MTL151) and 1.1 x 105 (CI =7 x103 – 5.6x105) (GW461) spores ml-1. However, neither the two M. robertsii strains nor the insecticide deterred the neonates from feeding on the surface of the fruits. The attraction responses of male and female adult codling moths to butyl hexanoate and codlemone blend; butyl hexanoate (0.025 mg / ml of hexane) and codlemone (0.1 mg / ml of hexane) in a ratio of 1:1 (0.5 ml: 0.5 ml from both solutions) were assessed in a one-choice wind tunnel olfactory bioassay during scoto-phase in the absence of fungal spores. The blend attracted more females (69%) than males (66.7%), however these responses were not significantly different (P > 0.5). The prospect of integrated use of entomopathogenic fungal conidia and semiochemical blend for control of C. pomonella adults was assessed in a wind tunnel containing the combo lure and M. robertsii (MTL151) spores formulated as a powder (250 mg) or aqueous spore suspension (3.1 x 108 spores m-1 ± 7 x 102 sporesml-1). Insect attraction, mean number of spores picked per insect and insect mortality was assessed. No significant difference (P > 0.5) was observed in the number of inoculi picked by unsexed adult moths in the two conidial formulations tested; spore powder (3.1 x 103 ± 1 x 103 spores ml-1) and aqueous spore suspension (3.1 x 103 ± 7 x 102 spores ml-1). No mortality was recorded among fungus or control-treated moths. Both M. robertsii (MTL151) spore formulations did not significantly (P > 0.05) affect the attraction responses of the insects to the combo lure; powder (73.3 ± 3.3%) and aqueous (68.6 ± 2.9%) compared to the control treatment (without the fungal spores) (70 ± 0.5 %). These results suggest that the fungus did not inhibit the attraction of moths by semiochemicals. Despite the successful autoinoculation of moths with fungal spores, the level of contamination was too low to induce mortality among adult moths. This study opens up opportunities for research in attract-and-fungus contamination delivery systems. This is the first report of the occurrence of M. robertsii in the southern Africa. Further, results from this study indicates that C. pomonella is susceptible to indigenous South African entomopathogenic fungi and 2 indigenous M. anisopliae strains, MTL151 and GW461 provided adequate protections against codling moth larval infestations of apple fruits. Future research endeavours to improve adherence of conidia to adult moth cuticle is recommended. Key words: Entomopathogenic fungi (EPF), codling moth, B. pseudobassiana, M. robertsii, larvae, pathogenicity and biological control, coddlemone, butyl hexanoate, attraction, semiochemicals.

Thesis (Ph. D.) in Biological Sciences--University of Maine, 2001. / Includes vita. Advisory Committee: Eleanor Groden, Assoc. Prof. of Entomology, Advisor; Francis Drummond, Prof. of Insect Ecology; Seanna Annis, Asst. Prof. of Mycology; David Lambert, Assoc. Prof. of Plant Pathology; John D. Vandenberg, Research Entomology, USDA-ARS. Includes bibliographical references (leaves 158-175).

Thesis (M.S.)--University of Missouri-Columbia, 2008. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on July 9, 2009) Includes bibliographical references.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2003. / Bibliography: leaves 75-86.

Entomopathogenic fungi are highly susceptible to the damaging effects of solar radiation. Attempts to protect entomopathogenic fungi from solar radiation have been, for the most part, unsuccessful. A new strategy for formulating entomopathogenic fungi for protection from solar radiation and desiccation has been developed tested using the acridid entomopathogen Metarhizium anisopliae var acridum (IMI 330189). This strategy involves coating spores with water-soluble materials that provide protection from solar radiation and enhance spore survival during drying. Development of this formulation involved the following: 1) production of an infective spore-type in liquid culture that could survive drying; 2) coating spores during an air-drying process; 3) reducing formulation particle size for oil suspension; 4) testing the effects spore coating on spore-tolerance simulated sunlight; and 6) testing the effects of spore coating infectivity to Schistocerca americana. Aerial conidia, submerged conidia, and blastospores produced in a high-osmolality liquid medium all had high desiccation tolerance relative to blastospores produced in Adamek's media. Blastospores produced in high osmolality medium were the most infective to S. americana in an aqueous 20% molasses solution followed by submerged conidia and aerial conidia, with LT50 values (95% C.I.) at 1 x 106 spores/insect of 7.8 d (6.7 to 9.0 d), 10.5 d (9.5 to 11.6 d), 14.6 d (11.9 to 18.0 d), respectively. Comparisons were made among cell-wall characteristics of these spore-types, including cell-wall thickness, lectin-binding, charge, and hydrophobicity. An optimal spore-coating formulation was selected on the basis of spore survival and germination over time after air-drying, particle-size reduction, and storage at 28 Ë C. This spore-coating formulation, consisting of skim milk, Kraft lignin (Curan 10®) and glycerol, greatly improved the tolerance of aerial conidia and submerged conidia to simulated sunlight, increasing the LT50 (95% C.I.) of aerial conidia from 4.0 hr (3.1-5.1) to 17.0 hr (12.5-23.0). The spore coating formulation decreased the infectivity of spores in oil to adult S. americana; reducing the LT50 values of aerial conidia at a dose of 1 x 105 spores / insect from 5.8 d (4.9-6.9 d) to 8.2 d (7.3-9.3 d). / Ph. D.

Os fungos Batkoa sp. e Furia sp.(Entomophthorales), embora ocasionalmente ocorrendo de forma epizoótica em populações das cigarrinhas-das-pastagens e cana-deaçúcar, nunca foram avaliados quanto ao nível de incidência e potencial de controle biológico. Esse estudo teve por objetivo avaliar a ocorrência natural, produção "in vitro" e preservação de micélio seco desses fungos. Nos trabalhos de produção incluiu-se como padrão o fungo Neozygites floridana, representante de outra família de Entomophthorales. O estudo sobre a ocorrência natural foi realizado na região de Pindamonhangaba, SP, no período de 22 de janeiro a 9 de fevereiro de 1998 com o objetivo de avaliar a incidência de Batkoa sp. na população de Mahanarva fimbriolata em pastagem de capim napier e de Furia sp. sobre Deois schach em pastagem de Brachiaria sp. O estudo sobre produção foi realizado em condições de laboratório com o objetivo de desenvolver meios líquidos para a produção de micélio ou corpos hifais de Batkoa sp., Furia sp. e N. floridana, tendo como base o meio líquido para cultura de tecido de inseto Grace’s suplementado, o qual permite bom crescimento do último patógeno. Finalmente, estudou-se o efeito da combinação de dois dessecantes (sílica e glicerol) com dois redutores de oxigênio (vácuo e Agelessâ ZPT-200) na preservação de micélio seco de Batkoa sp. e Furia sp. sob temperaturas de 3°C e 23°C. Concluiu-se que: Batkoa sp. e Furia sp. causam doença na população de M. fimbriolata e D. schach, respectivamente; Furia sp. possui maior potencial epizoótico que Batkoa sp.; Batkoa sp., Furia sp. e N. floridana se assemelham quanto ao crescimento em meios adicionados de sais, vitaminas e aminoácido, e em meio completo; A adição de sais ao meio básico proporciona um aumento significativo na produção das três espécies de fungos, e maior do que a adição de vitaminas e aminoácidos; A mistura dos sais CaCl2.2H2O (0,06%), KCl (0,28%), MgCl2.6H2O (0,16%), MgSO4.7H2O (0,2%), NaHCO3 (0,03%) e NaH2PO4.H2O (0,1%) proporciona maior produção dos fungos do que os sais avaliados isoladamente na concentração de 0,83%; O sulfato de magnésio (MgSO4) pode ser utilizado como único sal, na concentração de 0,83%, visando a produção de Batkoa sp., Furia sp. e N. floridana; Esses fungos se assemelham quanto ao crescimento em meios contendo diferentes fontes de carbono, mas se diferenciam em meios com diferentes fontes de nitrogênio; O meio contendo 2,66% de glucose proporciona uma produção das três espécies de fungos significativamente maior do que o meio com 2,66% de sacarose; A adição de 0,1% de monossacarídeos no meio com 2,66% de sacarose não aumenta significativamente a produção dos fungos; O extrato de levedura proporciona maior produção de Batkoa sp.; A concentração de 0,5% de extrato de levedura é a mais adequada para a produção vegetativa de Batkoa sp.; A combinação de extrato de levedura + extrato de carne + leite desnatado proporciona a maior produção de Furia sp.; A combinação de extrato de levedura + leite desnatado proporciona o segundo maior rendimento de Furia sp., sendo mais adequada para a produção do fungo devido ao menor custo; A concentração de 1% da mistura dessas fontes de nitrogênio é adequada para a produção vegetativa de Furia sp.; A combinação de extrato de levedura + peptona + leite desnatado proporciona a maior produção de N. floridana; A concentração de 3% da mistura dessas fontes de nitrogênio é adequada para a produção vegetativa de N. floridana; A combinação de sílica + Ageless® prolonga a sobrevivência desses dois fungos formulados como micélio seco até 90 dias, armazenados a 3°C e 23°C; O uso de vácuo como redutor de oxigênio não prolonga a sobrevivência desses dois fungos, mesmo em associação com dessecantes; A temperatura de 3°C provoca uma ligeira queda no potencial de crescimento de Batkoa sp., porém prolonga a sobrevivência do fungo até 90 dias, independentemente do tratamento. / The fungi Batkoa sp. and Furia sp. although occasionally occurring at epizootic levels in populations of spittlebug pests of pasture and sugarcane, have never been evaluated as to natural incidence level nor biological control potential. This study was designed to evaluate the natural occurrence, in vitro production and dry mycelium preservation of these fungi. Neozygites floridana, a representative of another family of Entomophthorales, was included as a standard in the production studies. The study on natural occurrence was done in Pindamonhangaba county, São Paulo State, from January 22 to February 9, 1998, to evaluate the incidence of Batkoa sp. in the Mahanarva fimbriolata population on pasture of napier grass, and Furia sp. in Deois schach, on Brachiaria sp. pasture. The study on production was done in laboratory conditions with the purpose to develop liquid media for the production of mycelium or hyphal bodies of Batkoa sp. Furia sp. and N. floridana, using as a base a liquid medium for insect tissue culture [Grace’s supplemented (HyQ® TNM-FH, #SH30280.02)], which allowed good growth of the latter pathogen. Finally, the effect of combinations of two desiccants (silica and glycerol) with two oxygen reducers (vacuum and Ageless® ZPT-200) on the preservation of dry mycelium of Batkoa sp. and Furia sp. under temperatures of 3º C and 23º C was studied. It is concluded that: Batkoa sp. and Furia sp. cause disease in M. fimbriolata and D. schach sp. populations, respectively. Furia sp. has higher epizootic potential than Batkoa sp. Batkoa sp., Furia sp. and N. floridana are similar concerning their growth patterns in media with added salts, vitamins and amino acids, as well as in the complete one. The addition of salts to the basic medium causes a significant increase in biomass production of the three fungal species, and higher than that from the addition of vitamins and amino acids. The mixture of the salts CaCl2.2H2O (0,06%), KCl (0,28%), MgCl2.6H2O (0,16%), MgSO4.7H2O (0,2%), NaHCO3 (0,03%) and NaH2PO4.H2O (0,1%) afforded higher production of the three fungi than the salts solely evaluated, at the concentration of 0,83%. MgSO4 can be used as sole salt for the production of Batkoa sp., Furia sp. and N. floridana. These fungi are similar concerning their growth patterns in media with different sources of carbone, but different in media with different sources of nitrogen. The production of the three fungal species is significantly higher in medium containing 2.66% glucose than in 2.66% sucrose. The addition of 0.1% monossacarides to media containing 2.66% sucrose does not increase significantly biomass production. Yeast extract allows the highest production of Batkoa sp. The concentration of 0.5% yeast extract is the most suitable for vegetative growth of Batkoa sp. The combination of yeast extract + beef extract + skim milk allows the highest production of Furia sp. The combination of yeast extract + skim milk allows the second highest production of Furia sp., and is the most suitable for mass production due to the lower cost. The 1% concentration of the mixture of these nitrogen sources is suitable for vegetative growth of Furia sp. The combination of yeast extract + peptone + skim milk allows the highest production of N. floridana. The 3% concentration of the mixture of these nitrogen sources is suitable for the vegetative growth of N. floridana. The combination of silica + Ageless® extends the survival of these two fungi formulated as dry mycelium for 90 days, stored at 3º C and 23º C. The use of vacuum to reduce oxygen does not extend the survival of these fungi, even in association with desiccants. Storage at 3º C causes a slight decrease in growth potential of Batkoa sp., but it extends fungus survival for 90 days, independent of other treatments.