The entomopathogenic fungus Beauveria bassiana and the bacterium Bacillus thuringiensis var. israelensis (Bti) have been widely studied for their role in biocontrol against many arthropods and extensively exploited for insect pest control. The purpose of this study was to evaluate the effect of four B. bassiana and two Bti formulations and their respective combinations, for the biological control of the common house fly, Musca domestica L., a major pest in poultry facilities. In vitro screening was undertaken to select the best B. bassiana isolates from 34 B. bassiana isolates and two Paecilomyces isolates. All the isolates of B. bassiana were found to be effective against adult house flies, but were marginally effective in controlling fly larvae. The Paecilomyces isolates were non-pathogenic towards both adult house flies and larvae. The best four isolates R444, 7320, 7569 and 7771 caused >90% mortality within 2d and were subjected to dose-mortality bioassays. Microscopic studies using light and scanning electron microscopy indicated the different durations of the lifecycle of B. bassiana development on the house fly. High temperature was found to delay conidial germination. Spore germination and mycelial growth were also inhibited by high adjuvant concentrations. Laboratory baseline bioassay data established, a dose-time response relationship using a waterdispersible granules (WDG) Bti formulation that demonstrated that the susceptibility of M. domestica larvae to a given concentration of Bti increased as the duration of exposure increased. In the laboratory studies, the LC50 and LC90 values of Bti for the larvae ranged between 65 - 77.4 and 185.1 - 225.9?g ml-1, respectively. LT50 and LT90 values were 5.5 and 10.3d respectively. In the field, a concentration of 10g Bti kg-1 (bran formulation) of feed resulted in 90% reduction of larvae for 4wk post-treatment. A higher concentration (2g L-1) of Bti in spray (WDG) applications was not significantly more effective than the lower concentration of 1g L-1. Thus, adding Bti to chicken feed has potential for the management and control of house flies in cagedpoultry facilities. The impact of oral feed applications of a bran formulation of Bti and a commercial chemical larvicide, Larvadex®, were compared with respect to their efficacy on the control of house fly 3 larval populations in poultry manure. The sublethal effects were manifested in terms of decreasing emergence of adult house flies. Although Larvadex® reduced larval density and caused significant reductions in emergence of adult house flies, it generally exhibited weaker lethal effects than Bti. The reduction levels achieved as a result of feeding 250mg Bti kg-1 at 5wk were similar to those achieved as a result of feeding twice the amount of Larvadex® at 4wk to the layers. From both an efficiency and economic perspective, comparisons to assess the impact of combining different concentrations of the two Bti formulations were carried out to evaluate their success in controlling house fly larvae and adults in poultry houses. The percentage mortality of larvae accomplished as a result of using a combination of 250mg kg-1 Bti in feed and 2g L-1 spray applications was equivalent to that obtained as a result of combining 500mg kg-1 Bti in feed and 1g L-1 spray application. The cost-benefit analysis (expressed in terms of mortality of larvae) indicated that the most effective combination for control of house fly larvae and fly emergence was the 500mg kg-1 in feed and 2g L-1 spray application combination that resulted in 67% larval mortality and 74% inhibition of adult house fly emergence. This study presents commercial users with possible combinations of applications of the two Bti formulations. Comparisons of larval mortalities and house fly emergence resulting from the Bti - B. bassiana treatments with those from Larvadex® - B. bassiana treatments, showed better control levels compared to any of the individual agents alone. The Bti treatments were more effective at controlling larval populations and inhibiting the emergence of house flies than Larvadex®, even when Larvadex® was applied together with B. bassiana. The effects of the Bti - B. bassiana and the Larvadex® - B. bassiana interactions were additive. These trials suggest that the efficacy of Bti in the control of house fly larvae may be improved with frequent applications of B. bassiana. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

CAPES; CNPq / Thaumastocoris peregrinus (Hemiptera: Thaumastocoridae) é um inseto originário da Austrália que está causando sérios danos à cultura do eucalipto ao redor do mundo. Ao alimentar-se da seiva das folhas, causa seu bronzeamento, podendo levar à desfolha. Medidas de controle estão sendo estudadas e a mais promissora é o parasitoide de ovos Cleruchoides noackae (Hymenoptera: Mymaridae). Produtos alternativos a base de compostos provenientes de plantas com potencial inseticida também poderiam ser uma ferramenta importante, e talvez serem utilizados concomitantemente com o parasitoide, visando um controle mais efetivo. Desta forma, o objetivo deste trabalho foi verificar a ação dos extratos aquosos de Matricaria chamomilla, Echinodorus grandiflorus, Punica granatum, Maytenus ilicifolia e Origanum majorana a 5% sobre T. peregrinus. Além disso, estudar sua possível toxicidade contra C. noackae e Gallus domesticus L., tendo em vista que estes compostos podem ter efeito negativo indesejado sobre organismos não alvo. Em uma primeira etapa, cromatografia líquida de alta eficiência (HPLC) foi utilizada para verificar os compostos fenólicos presentes nos extratos. Os extratos, então, foram testados sobre percevejo adultos, em confinamento (para verificar a ação inseticida) e teste de livre escolha (para verificar a ação repelente). Os três extratos que mostraram melhores resultados foram selecionados para os testes com os organismos não alvo. Com relação à C. noackae, testes pré e pós-parasitismo, de confinamento e de livre-escolha foram realizados para verificar se os extratos afetariam a escolha do hospedeiro pelas fêmeas ou o desenvolvimento das fases imaturas do parasitoide. Para verificar se os extratos seriam tóxicos a G. domesticus, estes foram adicionados à dieta de aves juvenis por cinco dias. Parâmetros como peso, consumo de alimento, quantificação de enzimas séricas e análise histopatológica foram realizados. Por meio das análises cromatográficas, foram detectados os ácidos gálico, ferúlico, cafeico, cumárico e vanílico. Os extratos levaram à mortalidade de 100% dos insetos em até 49% do tempo, quando comparados com a testemunha, mas E. grandiflorus, Matricaria chamomilla e Maytenus ilicifolia destacaram-se mostrando efeito repelente, sendo selecionados para a próxima etapa. Nenhum destes afetou a escolha do hospedeiro pela fêmea ou a emergência dos parasitoides, quando comparado com a testemunha. Além disso, os extratos não causaram alterações em G. domesticus, em nenhum dos parâmetros avaliados. Desta forma, verificou-se que E. grandiflorus, Matricaria chamomilla e Maytenus ilicifolia têm potencial para serem utilizados no controle de T. peregrinus, bem como mostraram-se seguros para C. noackae e G. domesticus. / Thaumastocoris peregrinus (Hemiptera: Thaumastocoridae ) is an insect from Australia which is causing severe damage to eucalyptus crops around the world. When feeding from the leaves sap, it causes bronzening, and in extreme cases, may lead to the tree death. Control methods have been studied and the most promising so far is the egg parasitoid Cleruchoides noackae (Hymenoptera: Mymaridae). Alternative products from plants with insecticidal properties could also be a viable option, and they might even be used concomitantly with C. noackae, aiming for a most effective control, but still safe for the environment. Thus, the objective of this work was to verify the action of 5% aqueous plant extracts of Matricaria chamomilla, Echinodorus grandiflorus, Punica granatum, Maytenus ilicifolia a n d Origanum majorana on T. peregrinus. In addition, we aimed to study the extracts potential toxicity to C. noackae and Gallus domesticus L., since the plant compounds might have negative effect upon the non-target organisms. At first, HPLC (High Performance Liquid Chromatography) was used to verify which phenolic compounds would be found in the plant extracts. These were tested on bronze bug adults, in confinement test (to verify the insecticidal action of the extracts) and free-choice test (to verify the repellency). The extracts that showed better results were selected for further tests with non-target organisms. Regarding C. noackae, pre-parasitism and post-parasitism, confinement and free-choice tests were performed to verify if the extracts would affect the host-choosing by the female or the development of the immature stages of the parasitoid. To verify if the extracts would be toxic to G. domesticus, the plant extracts were added to young birds feed for five days. Parameters such as weight gain, food intake, quantification of serum enzymes and histopathological analysis were carried out. HPLC analysis detected gallic, ferulic, vanillic, caffeic and cumaric acid in the extracts samples. All plant extracts tested reduced T. peregrinus survival, but E. grandiflorus, Matricaria chamomilla Maytenus ilicifolia had also a repellent effect, and were tested on the non-target organisms. None of these extracts affected neither the host choice by C. noackae nor adult emergency, when compared to the control group. In addition, the extracts did not cause alterations in any of the studied parameters. Thus, we verified that E. grandiflorus, Matricaria chamomilla and Maytenus ilicifolia have potential to be used to control T. peregrinus and are safe to C. noackae and G. domesticus.

CAPES; CNPq / Thaumastocoris peregrinus (Hemiptera: Thaumastocoridae) é um inseto originário da Austrália que está causando sérios danos à cultura do eucalipto ao redor do mundo. Ao alimentar-se da seiva das folhas, causa seu bronzeamento, podendo levar à desfolha. Medidas de controle estão sendo estudadas e a mais promissora é o parasitoide de ovos Cleruchoides noackae (Hymenoptera: Mymaridae). Produtos alternativos a base de compostos provenientes de plantas com potencial inseticida também poderiam ser uma ferramenta importante, e talvez serem utilizados concomitantemente com o parasitoide, visando um controle mais efetivo. Desta forma, o objetivo deste trabalho foi verificar a ação dos extratos aquosos de Matricaria chamomilla, Echinodorus grandiflorus, Punica granatum, Maytenus ilicifolia e Origanum majorana a 5% sobre T. peregrinus. Além disso, estudar sua possível toxicidade contra C. noackae e Gallus domesticus L., tendo em vista que estes compostos podem ter efeito negativo indesejado sobre organismos não alvo. Em uma primeira etapa, cromatografia líquida de alta eficiência (HPLC) foi utilizada para verificar os compostos fenólicos presentes nos extratos. Os extratos, então, foram testados sobre percevejo adultos, em confinamento (para verificar a ação inseticida) e teste de livre escolha (para verificar a ação repelente). Os três extratos que mostraram melhores resultados foram selecionados para os testes com os organismos não alvo. Com relação à C. noackae, testes pré e pós-parasitismo, de confinamento e de livre-escolha foram realizados para verificar se os extratos afetariam a escolha do hospedeiro pelas fêmeas ou o desenvolvimento das fases imaturas do parasitoide. Para verificar se os extratos seriam tóxicos a G. domesticus, estes foram adicionados à dieta de aves juvenis por cinco dias. Parâmetros como peso, consumo de alimento, quantificação de enzimas séricas e análise histopatológica foram realizados. Por meio das análises cromatográficas, foram detectados os ácidos gálico, ferúlico, cafeico, cumárico e vanílico. Os extratos levaram à mortalidade de 100% dos insetos em até 49% do tempo, quando comparados com a testemunha, mas E. grandiflorus, Matricaria chamomilla e Maytenus ilicifolia destacaram-se mostrando efeito repelente, sendo selecionados para a próxima etapa. Nenhum destes afetou a escolha do hospedeiro pela fêmea ou a emergência dos parasitoides, quando comparado com a testemunha. Além disso, os extratos não causaram alterações em G. domesticus, em nenhum dos parâmetros avaliados. Desta forma, verificou-se que E. grandiflorus, Matricaria chamomilla e Maytenus ilicifolia têm potencial para serem utilizados no controle de T. peregrinus, bem como mostraram-se seguros para C. noackae e G. domesticus. / Thaumastocoris peregrinus (Hemiptera: Thaumastocoridae ) is an insect from Australia which is causing severe damage to eucalyptus crops around the world. When feeding from the leaves sap, it causes bronzening, and in extreme cases, may lead to the tree death. Control methods have been studied and the most promising so far is the egg parasitoid Cleruchoides noackae (Hymenoptera: Mymaridae). Alternative products from plants with insecticidal properties could also be a viable option, and they might even be used concomitantly with C. noackae, aiming for a most effective control, but still safe for the environment. Thus, the objective of this work was to verify the action of 5% aqueous plant extracts of Matricaria chamomilla, Echinodorus grandiflorus, Punica granatum, Maytenus ilicifolia a n d Origanum majorana on T. peregrinus. In addition, we aimed to study the extracts potential toxicity to C. noackae and Gallus domesticus L., since the plant compounds might have negative effect upon the non-target organisms. At first, HPLC (High Performance Liquid Chromatography) was used to verify which phenolic compounds would be found in the plant extracts. These were tested on bronze bug adults, in confinement test (to verify the insecticidal action of the extracts) and free-choice test (to verify the repellency). The extracts that showed better results were selected for further tests with non-target organisms. Regarding C. noackae, pre-parasitism and post-parasitism, confinement and free-choice tests were performed to verify if the extracts would affect the host-choosing by the female or the development of the immature stages of the parasitoid. To verify if the extracts would be toxic to G. domesticus, the plant extracts were added to young birds feed for five days. Parameters such as weight gain, food intake, quantification of serum enzymes and histopathological analysis were carried out. HPLC analysis detected gallic, ferulic, vanillic, caffeic and cumaric acid in the extracts samples. All plant extracts tested reduced T. peregrinus survival, but E. grandiflorus, Matricaria chamomilla Maytenus ilicifolia had also a repellent effect, and were tested on the non-target organisms. None of these extracts affected neither the host choice by C. noackae nor adult emergency, when compared to the control group. In addition, the extracts did not cause alterations in any of the studied parameters. Thus, we verified that E. grandiflorus, Matricaria chamomilla and Maytenus ilicifolia have potential to be used to control T. peregrinus and are safe to C. noackae and G. domesticus.

Fourteen spore-forming bacterial strains were isolated and screened for entomopathogenic activity. Five displayed toxicity towards the common mealworm, Tenebrio molitor L., (Coleoptera: Tenebrionidae). The majority of the isolates were obtained from insect larvae and insect rich environments. The three bacterial species identified were Bacillus thuringiensis Berliner, Brevibacillus laterosporus Laubach and Bacillus cereus Frankland and Frankland. Bioassays were conducted using T. molitor larvae. The one isolate of B. cereus required the highest concentration of bacterial cells to achieve its LC50, whereas one of the isolates of B. laterosporus required the lowest cell concentration to achieve its LC50. Dose response curves were generated for the five best isolates, which showed that the isolate of B. laterosporus (NDR2) was substantially more toxic than the other isolates. / Thesis (Ph.D.) - University of KwaZulu-Natal, Pietermaritzburg, 2009.

Lantana camara L. (Verbenaceae) remains a highly invasive and ecologically damaging weed in South Africa, despite some 50 years of biological control efforts. Lack of success has been ascribed to varietal differences, climate and predation of agents but these have not been tested. In this study, the effects of ant predation were tested on populations of three biological control agents for L. camara. Colonies of two species, Crematogaster sp. 1 and 2 were investigated. Crematogaster sp. 1 colonies were offered no choice between immature stages of the agents Hypena laceratalis Walker (Lepidoptera: Noctuidae), Falconia intermedia Distant (Hemiptera: Miridae) or Teleonemia scrupulosa Stål (Hemiptera: Tingidae) on lantana shoots. Density-dependent predation on F. intermedia and T. scrupulosa nymphs on lantana shoots was tested using Crematogaster sp. 2 colonies. In choice experiments Crematogaster sp. 2 colonies were offered F. intermedia or T. scrupulosa nymphs on potted lantana plants. Preliminary food trials confirmed that colonies foraged for protein, thereby validating results of no-choice experiments. Crematogaster sp.1 foragers removed 50% of F. intermedia nymphs, followed by 45% of H. laceratalis larvae and only 9% of T. scrupulosa nymphs. Foragers recruited most actively to H. laceratalis larvae and significantly more H. laceratalis biomass was removed than either F. intermedia or T. scrupulosa. A trade-off existed in prey size selection because larger larvae provided considerably more biomass but required forager cooperation and a longer time to subdue than did smaller prey. This increases both forager energy expense and mortality risk by other predators. This study showed that all Crematogaster sp. 1 colonies removed small (≤10mm) H. laceratalis larvae more frequently than larvae larger than 10mm. Thus, of these biological control agents, predators probably prefer small H. laceratalis larvae. Significantly more F. intermedia than T. scrupulosa nymphs were removed by Crematogaster sp. 1, while Crematogaster sp. 2 colonies removed comparable numbers of both agent species. Falconia intermedia nymphs' fast movement triggered a predatory response by these ant species. In contrast, the relatively immobile behaviour of T. scrupulosa nymphs was identified as a highly effective predator avoidance strategy. Since T. scrupulosa nymphs are unable to escape predators by moving, they appear to depend on the presence of alternative prey attracting predator attention. At high agent and/or forager density, T. scrupulosa nymphs attempted escape, but foragers identified them as prey once they moved and caught them. Predation on F. intermedia was also density dependent in that at high nymph and/or forager densities, escape routes were congested and nymphs were more easily caught. Survival of F. intermedia and T. scrupulosa nymphs in particular was low on ant-accessed shrubs in choice experiments and high on ant-excluded shrubs. It is likely that ants significantly depress F. intermedia populations in the field since besides predation, ant foragers probably interrupt F. intermedia feeding and ovipositioning. The combination of parasitism and predation on early instar larvae may explain why H. laceratalis occurs across lantana's range in South Africa but populations remain low. It is unlikely that T. scrupulosa nymphs are habitually preyed on by ant species unless they attract attention by being mobile. Although biological control of L. camara is influenced by climate and physiological defence mechanisms, this study has shown that predation by two ant species severely impacts leaf-feeding agents for L. camara. Thus, it is recommended that future selection of additional agents to control lantana should exclude leaf-feeding .

Cydia pomonella (Linnaeus) (Family: Tortricidae), the codling moth, is considered one of the most significant pests of apples and pears worldwide, causing up to 80% crop loss in orchards if no control measures are applied. Cydia pomonella is oligophagous feeding on a number of alternate hosts including quince, walnuts, apricots, peaches, plums and nectarines. Historically the control of this pest has been achieved with the use of various chemical control strategies which have maintained pest levels below the economic threshold at a relatively low cost to the grower. However, there are serious concerns surrounding the use of chemical insecticides including the development of resistance in insect populations, the banning of various insecticides, regulations for lowering of the maximum residue level and employee and consumer safety. For this reason, alternate measures of control are slowly being adopted by growers such as mating disruption, cultural methods and the use of baculovirus biopesticides as part of integrated pest management programmes. The reluctance of growers to accept baculovirus or other biological control products in the past has been due to questionable product quality and inconsistencies in their field performance. Moreover, the development and application of biological control products is more costly than the use of chemical alternatives. Baculoviruses are arthropod specific viruses that are highly virulent to a number of lepidopteran species. Due to the virulence and host specificity of baculoviruses, Cydia pomonella granulovirus has been extensively and successfully used as part of integrated pest management systems for the control of C. pomonella in Europe and around the world, including South Africa. Commercial formulations have been typically based on the Mexican strain of CpGV. However due to long-term multiple applications of CpGV and the reliance on CpGV in organic farming practices in Europe, resistance to the CpGV-M strain has developed in a number of field populations of C. pomonella. This study aimed to identify and characterize novel isolates of CpGV in South Africa and compare their virulence with the commercial standard CpGV-M. Secondly, since C. pomonella is difficult to culture on a large scale, an alternate method of CpGV production was investigated in order to determine if CpGV could be produced more efficiently and at a reduced cost without negatively impacting the quality of the product. Several isolates of CpGV were recovered either from field collected larvae or from a laboratory-reared C. pomonella colony. Characterisation of DNA profiles using a variety of restriction enzymes revealed that only a single isolate, CpGV-SA, was genetically different from the Mexican strain of the virus used in the commercially available CpGV based products in South Africa. In dose-response bioassays using CpGV-SA, LC₅₀ and LC₉₀ values for neonate C. pomonella larvae were 3.18 x 10³ OBs/ml and 7.33 x 10⁴ respectively. A comparison of these values with those of CpGV-M indicated no significant difference in the virulence of the two isolates under laboratory conditions. This is a first report of a genetically distinct CpGV isolate in South Africa. The biological activity and novelty of CpGV-SA makes this isolate a potentially important tool for CpGV resistance management in South Africa. In order to justify production of CpGV in an alternative host, studies on the comparative biological performance of C. pomonella and T. leucotreta based on oviposition, time to hatch, larval developmental times and rearing efficiency as well as production costs were performed. Thaumatotibia leucotreta was found to be more fecund and to have significantly shorter egg and larval developmental times. In addition, larval production per unit of artificial diet was significantly higher than for C. pomonella. This resulted in T. leucotreta being more cost effective to produce with implications for reduced insectary space, sanitation practices as well as the labour component of production. Virus yield data generated by inoculation both C. pomonella and T. leucotreta with nine concentrations of CpGV resulted in comparable virus yields, justifying the continuation of the research into production of CpGV in T. leucotreta. It was important to determine the LC and LT values required for mass production of CpGV in late instar T. leucotreta larvae. Dose- and time-response bioassays with CpGV-M were conducted on artificial diet to determine these values. Fourth instar LC₅₀ and LC₉₀ values were 5.96 x 10³ OBs/ml and 1.64 x 10⁵ OBs/ml respectively. LT50 and LT90 values were 81.10 hours and 88.58 hours respectively. Fifth instar LC₅₀ and LC₉₀ values were 6.88 x 10⁴ OBs/ml and 9.78 x 10⁶ OBs/ml respectively. LT₅₀ and LT₉₀ values were 111.56 hours and 137.57 hours respectively. Virus produced in fourth instar T. leucotreta larvae was bioassayed against C. pomonella neonate larvae and compared to CpGV-M to establish if production in the heterologous host negatively affected the virulence of the isolate. No significant difference in virulence was observed between virus produced in T. leucotreta and that produced in C. pomonella. The data generated in the bioassays was used in CpGV mass production trials to evaluate production. All production methods tested produced acceptable virus yields. To examine the quality of the virus product, genomic DNA was extracted from larval cadavers and subjected to REN analysis with HindIII. The resulting DNA profiles indicated that the virus product was contaminated with the homologous virus, CrleGV. Based on the above results, the use of T. leucotreta as an alternate host for the in vivo production of CpGV on a commercial basis is not at this stage viable and requires further investigation before this production methodology can be reliable used to produce CpGV. However, this study has shown that CpGV can be produced in a homologous host, T. leucotreta and significant strides have been made towards developing a set of quality control standards that are essential for further development of successful production methodology. Finally a novel isolate of CpGV has been identified with comparable virulence to the CpGV-M. This is an important finding as it has broad reaching implications for resistance management of CpGV products in South Africa.

False codling moth (FCM), Thaumatotibia leucotreta is an extremely important pest of citrus in South Africa and with the shift away from the use of chemicals, alternate control options are needed. One avenue of control which has only recently been investigated against the soil-borne life stages of FCM is the use of entomopathogenic fungi (EPF). In 2009, 12 entomopathogenic fungal isolates collected from South African citrus orchards showed good control potential during laboratory conducted bioassays. The aim of this study was to further analyse the potential of these isolates through concentration-dose and exposure-time response bioassays. After initial re-screening, concentration-dose response and exposure-time response sandconidial bioassays, three isolates were identified as exhibiting the greatest control potential against FCM in soil, Metarhizium anisopliae var. anisopliae (G 11 3 L6 and FCM Ar 23 B3) and Beauveria bassiana (G Ar 17 B3). Percentage mycosis was found to be directly related to fungal concentration as well as the amount of time FCM 5th instar larvae were exposed to the fungal conidia. LC50 values for the three isolates were not greater than 1.92 x 10⁶ conidia.ml⁻ₑ and at the LC₅₀, FCM 5th instar larvae would need to be exposed to the fungus for a maximum of 13 days to ensure a high mortality level. These isolates along with two commercially available EPF products were subjected to field persistence trials whereby net bags filled with a mixture of autoclaved sand and formulated fungal product were buried in an Eastern Cape citrus orchard. The viability of each isolate was measured on a monthly basis for a period of six months. All isolates were capable of persisting in the soil for six months with the collected isolates persisting far better than the commercially used isolates. Two of the isolates, G 11 3 L6 and G Ar 17 B3, were subjected to small scale laboratory application trials. Two formulations were investigated at two concentrations. For each isolate, each formulation and each concentration, FCM 5th instar larvae were applied and allowed to burrow into the soil to pupate before fungal application or after fungal application. Contact between fungi and FCM host is essential as, in contrast to pre-larval treatments, percentage mortality in post-larval treatments was low for both formulations and both isolates. For isolate G Ar 17 B3, a conidial suspension applied as a spray at a concentration of 1 x 10⁷ conidia.ml⁻ₑ obtained the highest percentage mortality (80 %). For isolate G 11 3 L6 however, both formulations performed equally well at a high, 1 x10⁷ conidia.ml⁻ₑ concentration (conidial suspension: 60 %; granular: 65 %) The results obtained thus far are promising for the control of FCM in citrus, but if these EPFs are to successfully integrate into current FCM control practices more research, some of which is discussed, is essential

The biology of key citrus pests Thaumatotibia leucotreta Meyrick (Lepidoptera: Tortricidae), Ceratitis capitata Wiedemann (Diptera: Tephritidae) and Ceratitis rosa Karsch (Diptera: Tephritidae) includes their dropping from host plants to pupate in the soil below citrus trees. Since most EP fungi are soil-borne microorganisms, the development and formulation of alternative control strategies using these fungi as subterranean control agents, targeted at larvae and pupae in the soil, can potentially benefit existing IPM management of citrus in South Africa. Thus, a survey of occurrence of entomopathogenic fungi was undertaken on soils from citrus orchards and natural vegetation (refugia) on conventionally and organically managed farms in the Eastern Cape Province in South Africa. A method for baiting soil samples with citrus pest T. leucotreta and C. capitata larvae, as well as with the standard bait insect, Galleria mellonella Linnaeus (Lepidoptera: Pyralidae), was implemented. Sixty-two potentially useful entomopathogenic fungal isolates belonging to four genera were collected from 288 soil samples, an occurrence frequency of 21.53%. The most frequently isolated entomopathogenic fungal species was Beauveria bassiana (Balsamo) Vuillemin (15.63%), followed by Metarhizium anisopliae var. anisopliae (Metschnikoff) Sorokin (3.82%). Galleria mellonella was the most effective insect used to isolate fungal species (χ2=40.13, df=2, P≤ 0.005), with a total of 45 isolates obtained, followed by C. capitata with 11 isolates, and T. leucotreta with six isolates recovered. There was a significantly (χ2=11.65, df=1, P≤ 0.005) higher occurrence of entomopathogenic fungi in soil samples taken from refugia compared to cultivated orchards of both organically and conventionally managed farms. No significant differences were observed in the recovery of fungal isolates when soil samples from both farming systems were compared. The physiological effects and host range of 21 indigenous fungal isolates obtained in the Eastern Cape were investigated in the laboratory to establish whether these isolates could be effectively used as biological control agents against the subterranean life stages of C. rosa, C. capitata and T. leucotreta. When these pests were treated with a fungal concentration of 1 x 10⁷ conidia ml⁻¹, the percentage of T. leucotreta adults which emerged in fungal treated sand ranged from 5 to 60% (F=33.295; df=21; P=0.0001) depending on fungal isolate and the percentage of pupae with visible signs of mycosis ranged from 21 to 93% (F= 96.436; df=21; P=0.0001). Based on fungal isolates, the percentage adult survival in C. rosa and C. capitata ranged from 30 to 90% and 55 to 86% respectively. The percentage of C. rosa and C. capitata puparia with visible signs of mycosis ranged from 1 to 14% and 1 to 11% respectively. Deferred mortality due to mycosis in C. rosa and C. capitata adult flies ranged from 1 to 58% and 1 to 33% respectively, depending on fungal isolate. Entomopathogenic fungal isolates had a significantly greater effect on the adults of C. rosa and C. capitata than they did on the puparia of these two fruit fly species. Further, C. rosa and C. capitata did not differ significantly in their response to entomopathogenic fungi when adult survival or adult and pupal mycosis were considered. The relative potency of the four most virulent Beauveria isolates as well as the commercially available Beauveria bassiana product, Bb Plus® (Biological Control Products, South Africa), were compared against one another as log-probit regressions of mortality against C. rosa, C. capitata and T. leucotreta which all exhibited a dose-dependent response. Against fruit flies the estimated LC50 values of all five Beauveria isolates ranged from 5.5 x 10¹¹ to 2.8 x 10¹² conidia/ml⁻¹. There were no significant differences between the relative potencies of these five fungal isolates. When T. leucotreta was considered, isolates: G Moss R10 and G 14 2 B5 and Bb Plus® were significantly more pathogenic than G B Ar 23 B3 and FCM 10 13 L1. The estimated LC₅₀ values of the three most pathogenic isolates ranged from 6.8 x 10⁵ to 2.1 x 10⁶ conidia/ml⁻¹, while those of the least pathogenic ranged from 1.6 x 10⁷ to 3.7 x 10⁷ conidia/ml⁻¹. Thaumatotibia leucotreta final instar larvae were exposed to two conidial concentrations, at four different exposure times (12, 48, 72 and 96 hrs) and showed an exposure time-dependant relationship (F=5.43; df=3; P=0.001). At 1 x 10⁷conidia/ml⁻¹ two Beauveria isolates: G Moss R10 and G 14 2 B5 were able to elicit a response in 50% of test insects at 72 hrs (3 days) exposure. Although a limited amount of mycosis was observed in the puparia of both fruit fly species, deferred adult mortality due to mycosis was high. The increased incidence of adult mortality suggests that post emergence mycosis in adult fruit flies may play a more significant role in field suppression than the control of fruit flies at the pupal stage. The increased incidence of pupal mortality, as well as the relatively low concentrations of conidia required to elicit meaningful responses in T. leucotreta pupae may suggest that pre-emergent control of false codling moth will play a more significant role in field suppression than the control of adult life stages using indigenous isolates of entomopathogenic fungi. Various entomopathogenic fungal application techniques targeted at key insect pests within integrated pest management (IPM) systems of citrus are discussed.

The two-spotted spider mite (TSM), Tetranychus urticae Koch, is an important pest of many greenhouse and field crops worldwide. The development of resistance in TSM populations to chemical acaricides, allied with public health concerns about pesticide residues, has motivated the search for alternative control measures to suppress the pest. Hyphomycetous fungi are promising agents for mite control and the fungus Beauveria bassiana (Bb) (Balsamo) Vuillemin was investigated in this study as a biocontrol agent. The principal objectives of this study comprised: a) screening Bb strains for their pathogenicity against T. urticae; b) testing the effect of adjuvants on the efficacy of Bb; c) studying the effect of plant type on persistence of Bb and the efficacy of control of Bb against T. urticae; d) evaluating the field efficacy of Bb applications against T. urticae; e) testing the compatibility of Bb with selected fungicides; and f) assessing the synergy between Bb and soluble silicon for T. urticae control. Screening bioassays of sixty-two strains of Bb identified the two most effective strains, PPRI 7315 (R289) and PPRI 7861 (R444), that caused mortality levels of more than 80% of adult mites at 9 d post-inoculation with 2 × 108 conidia ml-1. These strains performed significantly better than the Bb commercial strain PPRI 5339, in laboratory bioassays. The two strains also attacked mite eggs, causing 53.4% and 55.5% reduction in egg hatchability at 2 × 108 conidia ml-1 respectively. However, PPRI 7861 showed relatively higher production of conidia in culture and was, therefore, selected for further trials under greenhouse and field conditions. Greenhouse evaluations of the effects of two adjuvants (Break-thru® and a paraffin oil-based emulsion) on efficacy of Bb demonstrated a higher efficacy of the biocontrol agent (BCA) when it was applied with Break-thru® or the oil solution than with water alone. Moreover, Bb conidia applied in Break-thru® solution resulted in greater control of TSM than conidia applied in the mineral oil. There was also a dose-response effect and the control of TSM by Bb increased when the concentration of conidia was increased. The control of TSM by Bb in beans (Phaseolus vulgaris L), cucumber (Cucumis sativus L.), eggplant (Solanum melongena L.), maize (Zea mays L.) and tomato (Solanum lycopersicum L.) was tested in greenhouse trials. On these crops, the persistence of conidia declined over time. The rate of decline was significantly higher on maize. However, TSM mortality was positively correlated with the amount of conidia deposited on leaves immediately after spraying, rather than their persistence over time. Higher levels of mortality of TSM due to Bb application were observed on beans, cucumber and eggplants, suggesting that the type of crop must be taken into consideration when Bb is applied as a BCA. Field efficacy of Bb against mites was evaluated in two trials on eggplants. Based on assessment of population densities of mites and leaf damage assessments; both trials showed that the strain PPRI 7861 controlled TSM in the field. Two commonly used fungicides, azoxystrobin and flutriafol, were investigated in vitro tests on culture medium and laboratory bioassays on detached bean leaves (Phaseolus vulgaris L.) for their effects on Bb. Azoxystrobin (a strobilurin) was less harmful to Bb while flutriafol was found to be inhibitory. Another important finding of this study was the substantial enhancement of Bb efficacy by soluble silicon. When Bb was combined with soluble Si, the control of TSM was better than when either of the two products was applied alone. Moreover, application of soluble Si as a plant fertilizer in hydroponic water nutrient increased accumulation of peroxidase, polyphenoloxidase and phenylalanine ammonia-lyase enzymes in leaves of plants infested with TSM. Increased activity of these defense enzymes in leaves deters feeding behaviour of mites. We suggested that feeding stress renders them susceptible to Bb infection, which would explain the synergy observed between the two agents. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

The use of entomopathogenic fungi to control agriculturally important pests, both in greenhouses and in the field, has been demonstrated by various authors for a number of years. This has been brought about by the development of resistance in certain pest species to chemical applications and a growing public awareness of the safety implications of residual insecticides. Several entomopathogenic fungi were tested against four insect pests found in the Republic of South Africa (RSA), the greenhouse whitefly, Trialeurodes vaporariorum, the rice weevil, Sitophilus oryzae, the grapevine mealybug, Planococcusficus and the sugarcane stem borer, Eldana saccharina. Further concentration, temperature and humidity studies were conducted with selected isolates on the rice weevil, S. oryzae. Sitophilus oryzae is considered one ofthe most important pests of stored grain. Several fungal isolates were tested against the rice weevil, four of which, B1, PPRI 6690, PPRI 6864 and PPRI 7067, were selected for further testing based on the mortality results over a 21 d period. Varying conidial concentrations were applied and at high doses of 1x10 -6 conidia ml -1 with mortality rates of to 84% achieved. LT 50 values ranged from 6 - 68d. Increased spore concentration resulted in an increase in overall mortality. Temperature and humidity was found to affect the infection potential of the four isolates tested. Four temperatures ranging from 15 - 30°C were tested. The highest mortality rates were obtained at 25°C where mortality ranged from 46 - 65% in 14d. Mortality rates decreased with decreasing temperature, and no mortality was recorded at 30°C. Temperature was found to significantly alter the LT 50 values, increasing the LT 50 with decreasing temperatures. Decreasing the humidity resulted in an increased LT 50 and a reduction in the overall mortality rates. The mortality of S. oryzae ranged according to the RH and isolate. Isolates Bland PPRI 6690 resulted in the highest mortalities of 80 and 83% at 92.5% RH, with LT 50's of 6.3d and 6.4d, respectively. Several entomopathogenic fungi were tested against T vaporariorum, P. ficus and E. saccharina, three key pests of South African crops. Nine fungal isolates were tested against the greenhouse whitefly, T vaporariorum, with mortalities ranging from 26.7 - 74.7% over 14d. Beauveria bassiana Isolates Bl and PPRl 6690 produced the highest mortality rates and were recommended for further pathogenicity testing against T. vaporariorum. Planococcus ficus is a common pest ofvineyards in the Western Cape Province, South Africa. Nine entomopathogenic fungi were screened against P.ficus, only two of which produced mortality. Eldana saccharina is a stalk borer, which infests sugarcane in large areas of Southern Africa. Five isolates were tested against second and third instar larvae, three of which, B1, PPRl 6864 and PPRl 6690 resulted in mortalities. Mean percentage mortality was low for all three isolates. From the study it was evident that two of the isolates tested, Bland PPRI 6690 (B. bassiana), showed potential against three of the four pests, and two isolates of Lecanicillium lecanii caused mortality in P. ficus. Further research and understanding of the effect of environmental conditions, spore concentration and epizootic potential would result in the further development of these isolates as future biological control agents. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.