Os agentes de controle biológico têm recebido grande reconhecimento, e o seu uso tem contribuído como um complemento ou substituição de agroquímicos. No entanto, existem poucos estudos sobre o controle biológico da Pinta Preta dos Citros, causada pelo fungo Guignardia citricarpa, o que impede o seu mercado in natura, além de prejudicar a sua exportação pelo uso intensivo de insumos químicos para controlar este patógeno. Os fungos do gênero Trichoderma são agentes de controle utilizados em todo o mundo contra vários fitopatógenos relevantes. Este estudo visou identificar proteínas extracelulares secretadas por T. atroviride T17 e por T.harzianum T1A, eficazes para o controle de G. citricarpa. Por eletroforese bidimensional (2D) foram obtidos perfis de proteínas secretadas por Trichoderma em meio de glicose (controle) e em meio suplementado com micélio desativado de G. citricarpa. As proteínas foram identificadas por LC-MS/MS mostrando que ambas as espécies secretam proteínas diferentes. Foram identificadas 68 proteínas das 178 diferencialmente expressas por T. harzianum, sendo a maioria relacionada aos mecanismos de biocontrole, mesmo no meio controle. Foi verificado que em contato com o patógeno a expressão de proteínas relacionadas com o metabolismo primário diminui. Por outro lado, Trichoderma atroviride mostrou uma maior expressão de proteínas relacionadas com biocontrole na presença de micélio do patógeno. Nesta espécie identificamos 59 proteínas de 116 diferencialmente expressas, principalmente proteínas relacionadas com a degradação da parede celular: α- manosidase, quitinase, mutanase, glicosidase, endoquitinase e, algumas famílias de glicoside hidrolases. Os resultados indicam que estas espécies apresentam um elevado potencial como agentes de controle de G. citricarpa. Os resultados são pioneiros em detalhar a interação de Trichoderrma com G. citricarpa, por meio da análise do secretoma. / Submitted by Ana Guimarães Pereira (agpereir@ucs.br) on 2016-05-11T14:24:21Z No. of bitstreams: 1 Tese Fernanda Blauth de Lima.pdf: 5347447 bytes, checksum: 314027a9e2af4a7cf6e30be761df9f99 (MD5) / Made available in DSpace on 2016-05-11T14:24:21Z (GMT). No. of bitstreams: 1 Tese Fernanda Blauth de Lima.pdf: 5347447 bytes, checksum: 314027a9e2af4a7cf6e30be761df9f99 (MD5) Previous issue date: 2016-05-11 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES. / Biological control agents (BCA) have received great recognition, and their use has contributed as a complement or replacement of agrochemicals. However, there are few studies on the biological control of Black spot of citrus, caused by the fungus Guignardia citricarpa, which prevents the market in natura, besides damaging their export by the intensive use of chemical inputs to control this pathogen. Trichoderma fungi are the most applied worldwide BCA against various relevant plant pathogens. The purpose of this study was to identify extracellular proteins secreted by T. atroviride T17 and of T. harzianum T1A, which are effective for the control of G. citricarpa. Bidimensional electrophoresis (2D) allowed obtaining the secreted protein profiles of Trichoderma grown in glucose medium (control) and in medium containing inactivated mycelium of G. citricicarpa. From the 178 differentially expressed proteins by T. harzianum, 68 were identified, most of them related to biocontrol mechanisms, even in the control medium. In the presence of the pathogen, the expression of proteins related to the metabolism decreases. On the other hand, Trichoderma atroviride showed higher expression of proteins related to biocontrol pathogen when grown in the presence of pathogen mycelium, when compared to the control medium. From the 116 differentially expressed proteins, 59 were identified, mainly proteins related to fungi cell wall degradation such as α-mannosidase, chitinase, mutanase, glycosidase, endochitinase and, some families of glycoside hydrolases. The results indicate that these species have a high potential as biocontrol agents of G. citricicarpa. These results are pioneers in to detail the Trichoderrma interaction with G. citricicarpa through the secretome analysis.

Os agentes de controle biológico têm recebido grande reconhecimento, e o seu uso tem contribuído como um complemento ou substituição de agroquímicos. No entanto, existem poucos estudos sobre o controle biológico da Pinta Preta dos Citros, causada pelo fungo Guignardia citricarpa, o que impede o seu mercado in natura, além de prejudicar a sua exportação pelo uso intensivo de insumos químicos para controlar este patógeno. Os fungos do gênero Trichoderma são agentes de controle utilizados em todo o mundo contra vários fitopatógenos relevantes. Este estudo visou identificar proteínas extracelulares secretadas por T. atroviride T17 e por T.harzianum T1A, eficazes para o controle de G. citricarpa. Por eletroforese bidimensional (2D) foram obtidos perfis de proteínas secretadas por Trichoderma em meio de glicose (controle) e em meio suplementado com micélio desativado de G. citricarpa. As proteínas foram identificadas por LC-MS/MS mostrando que ambas as espécies secretam proteínas diferentes. Foram identificadas 68 proteínas das 178 diferencialmente expressas por T. harzianum, sendo a maioria relacionada aos mecanismos de biocontrole, mesmo no meio controle. Foi verificado que em contato com o patógeno a expressão de proteínas relacionadas com o metabolismo primário diminui. Por outro lado, Trichoderma atroviride mostrou uma maior expressão de proteínas relacionadas com biocontrole na presença de micélio do patógeno. Nesta espécie identificamos 59 proteínas de 116 diferencialmente expressas, principalmente proteínas relacionadas com a degradação da parede celular: α- manosidase, quitinase, mutanase, glicosidase, endoquitinase e, algumas famílias de glicoside hidrolases. Os resultados indicam que estas espécies apresentam um elevado potencial como agentes de controle de G. citricarpa. Os resultados são pioneiros em detalhar a interação de Trichoderrma com G. citricarpa, por meio da análise do secretoma. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES. / Biological control agents (BCA) have received great recognition, and their use has contributed as a complement or replacement of agrochemicals. However, there are few studies on the biological control of Black spot of citrus, caused by the fungus Guignardia citricarpa, which prevents the market in natura, besides damaging their export by the intensive use of chemical inputs to control this pathogen. Trichoderma fungi are the most applied worldwide BCA against various relevant plant pathogens. The purpose of this study was to identify extracellular proteins secreted by T. atroviride T17 and of T. harzianum T1A, which are effective for the control of G. citricarpa. Bidimensional electrophoresis (2D) allowed obtaining the secreted protein profiles of Trichoderma grown in glucose medium (control) and in medium containing inactivated mycelium of G. citricicarpa. From the 178 differentially expressed proteins by T. harzianum, 68 were identified, most of them related to biocontrol mechanisms, even in the control medium. In the presence of the pathogen, the expression of proteins related to the metabolism decreases. On the other hand, Trichoderma atroviride showed higher expression of proteins related to biocontrol pathogen when grown in the presence of pathogen mycelium, when compared to the control medium. From the 116 differentially expressed proteins, 59 were identified, mainly proteins related to fungi cell wall degradation such as α-mannosidase, chitinase, mutanase, glycosidase, endochitinase and, some families of glycoside hydrolases. The results indicate that these species have a high potential as biocontrol agents of G. citricicarpa. These results are pioneers in to detail the Trichoderrma interaction with G. citricicarpa through the secretome analysis.

The public concern over synthetic pesticides in foods and the environment has created an interest to find effective and safe non-fungicide means of controlling postharvest pathogens. The overall objective of this thesis was to evaluate the effect of potassium silicate, yeast antagonists and hot water dip treatment to control postharvest grey mould and blue mould of pome fruits, caused by Botrytis cinerea and Penicillium expansum, respectively. Botrytis cinerea and Penicillium expansum were isolated from infected strawberry and pear fruits, respectively. These isolates were found to be non-resistant to YieldPlus® (Anchor yeast, Cape Town, South Africa), a biofungicide containing a yeast Cryptococcus albidus. A total of 100 epiphytic yeast isolates were obtained from the fruit surface of “Golden Delicious” apples and “Packham’s Triumph” pears, and screened against B. cinerea and P. expansum. Fifteen yeast isolates reduced grey mould incidence by > 50%, when applied four hours before inoculation with B. cinerea. Similarly, seven yeast isolates reduced blue mould incidence by > 50%, when applied four hours before inoculation with P. expansum. YieldPlus® and yeast Isolate YP25 provided the best control of B. cinerea, while Isolate YP60 and YieldPlus® provided the best control of P. expansum on “Golden Delicious” apples. A mixture of YP25 and YP60 provided complete control of both B. cinerea and P. expansum, when applied to “Golden Delicious” apples before inoculation with either B. cinerea or P. expansum. Electron microscopy studies showed that yeast Isolates YP25 and YP60 inhibited the mycelial growth of B. cinerea and P. expansum, respectively. Preventative and curative application of potassium silicate resulted in reduced incidence of B. cinerea or P. expansum of “Golden Delicious” apples. Electron microscopy studies indicated that potassium silicate inhibited the growth of B. cinerea and P. expansum. Furthermore, treatment of “Golden Delicious” apples with either potassium chloride or potassium hydroxide resulted in reduced incidence of both B. cinerea and P. expansum. In vivo tests showed that the disease incidence of P. expansum and B. cinerea on “Golden Delicious” apples was reduced by hot water dip treatments at 58-60°C for 60 to 120 seconds, compared with the control fruit treated with sterile distilled water, without causing skin damage. The use of potassium silicate, yeasts (Isolates YP25 and YP60), YieldPlus® and the antagonists mixture (YP25+YP60) in combination, resulted in the control of B. cinerea and P. expansum of “Golden Delicious” apples compared with Imazalil® treated fruit. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

A viticultura é uma atividade de grande importância econômica, destacando-se a sustentabilidade da pequena propriedade e o desenvolvimento territorial associados às atividades ligadas ao turismo. As plantas cultivadas se tornam vulneráveis a patógenos e insetos-praga e a videira apresenta diversas espécies consideradas pragas que reduzem sua produção e rentabilidade, entre estas, Spodoptera frugiperda, causando danos em diferentes partes da planta. Fungos entomopatogênicos podem oferecer uma alternativa aos pesticidas convencionais para o controle de pragas, pois produzem enzimas que degradam o exoesqueleto do inseto como quitinases e proteases facilitando o modo de infecção. Neste trabalho foi avaliado o potencial inseticida do fungo Nomurea rileyi, linhagem UCS03, contra S. frugiperda e o perfil eletroforético por SDS-PAGE das proteínas secretadas por N. rileyi em presença da cutícula do inseto em diferentes intervalos de tempo em gel unidimensional. O fungo N. rileyi apresentou virulência contra S. frugiperda, determinando um CL50 de 2 x 109conídios/mL com a linhagem UCS03 demonstrando atividade bionseticida. Na avaliação do perfil proteico do secretoma de N. rileyi em presença da cutícula do inseto, em diferentes tempos de cultivo, foi possível verificar um perfil altamente diferenciado. A maior concentração de proteína foi encontrada no 14° dia de incubação (0,3507 mg/mL) reduzindo a quantidade de proteínas após este período. Na análise por SDS-PAGE foi possível verificar diferentes proteínas de diferentes massas moleculares, nos intervalos de tempo considerados, sendo muitas inferiores a 75 kDa. Estas proteínas com diferentes massas moleculares podem estar envolvidas no metabolismo do fungo. Desta forma, estes resultados podem contribuir para a compreensão do processo de infecção de N. rileyi em S. frugiperda, oferecendo potencial para o desenvolvimento de novas pesquisas e aplicações destas em processos biotecnológicos. / Submitted by Ana Guimarães Pereira (agpereir@ucs.br) on 2017-03-14T12:21:21Z No. of bitstreams: 1 Dissertacao Ana Carolina Ruiz.pdf: 656327 bytes, checksum: 52c5a7db081b0ec94339972595956f52 (MD5) / Made available in DSpace on 2017-03-14T12:21:21Z (GMT). No. of bitstreams: 1 Dissertacao Ana Carolina Ruiz.pdf: 656327 bytes, checksum: 52c5a7db081b0ec94339972595956f52 (MD5) Previous issue date: 2017-03-14 / Viticulture is an activity of great economic importance with emphasis on the sustainability of small property and territorial development associated with tourism related activities. Cultivated plants become vulnerable to pathogens and insect pests and the vine has several species considered as pests that reduce their production and profitability, among them, Spodoptera frugiperda, with causes damage in different parts of the plant. Entomopathogenic fungi can provide an alternative to conventional pesticides for controlling pests, they produce enzymes that degrade the insect exoskeleton, such aschitinases and proteases facilitating the infection. In this work, the insecticide potential of the fungus Nomurea rileyi and S. frugiperda was evaluated, as well as the proteins secreted by N. rileyi in the presence of insect cuticle at different time intervals in one-dimensional gel. The fungus N. rileyi presented virulence against S. frugiperda, determining a CL50 of 2 x 109 con / mL with UCS03 strain demonstrating bionseticida activity. In assessing the protein profile of secretome N. rileyi in the presence of insect cuticle, at different times of cultivation, a highly differentiated profile was verified. The highest concentration of protein was found at day 14 of incubation (0.3507 mg / ml) reducing the amount of protein after this period. In the one-dimensional gel analysis was verified different molecular weights of proteins, in the time interval considered being many less than 75kDa. These proteins with different molecular weights may be involved in fungal metabolism. Thus, these results can contribute to the understanding of the infection process of N. rileyi in S. frugiperda, offering potential for the development of new researches and applications in biotechnological processes.

O Brasil é o maior produtor de citros, sendo um dos seus principais produtos de exportação da cadeia agroindustrial. Um dos fatores que afetam a qualidade e a produção do produto final obtido é a Mancha Marrom de Alternaria, causado pelo fungo Alternaria alternata. A doença provoca desfolha, seca de ramos e queda de frutos em tangerinas e seus híbridos. Para controle dessa doença são utilizados fungicidas, porém, a mancha marrom é uma doença de difícil controle podendo ser necessárias de 7 a 10 aplicações do fungicida, aparecimento de resistência dos fungos, intoxicação nas pessoas e contaminação do meio ambiente devido ao emprego de fungicidas. Métodos alternativos como o controle biológico com aplicações de micro-organismos antagonistas e a nutrição das plantas são opções de menor impacto ao meio ambiente. Sendo assim, o objetivo deste trabalho foi avaliar o potencial de Trichoderma spp., Bacillus subtilis e fertilizante organomineral, no controle da Mancha Marrom de Alternaria em plantas cítricas de tangerina Ponkan. O experimento foi realizado com delineamento casualizado, constituído de oito tratamentos com quatro repetições. Os experimentos foram avaliados quanto a incidência da doença. Os dados mostraram que o melhor controle foi o tratamento químico, porém os tratamentos com Trichoderma spp., Trichoderma spp. + fertilizante e Trichoderma spp. + Bacillus subtilis apresentaram ótimas taxas de controle, podendo inclusive substituir o tratamento químico no controle desta doença. / Submitted by Ana Guimarães Pereira (agpereir@ucs.br) on 2017-06-13T14:28:19Z No. of bitstreams: 1 Dissertacao Bianca Luzardo Porto.pdf: 1070166 bytes, checksum: 4e890666fce3c2b0d8ce25ef2c71241f (MD5) / Made available in DSpace on 2017-06-13T14:28:19Z (GMT). No. of bitstreams: 1 Dissertacao Bianca Luzardo Porto.pdf: 1070166 bytes, checksum: 4e890666fce3c2b0d8ce25ef2c71241f (MD5) Previous issue date: 2017-06-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES. / Brazil is the largest citrus producer, being one of its main exports of agro-industrial chain. One of the factors that affect the quality and production of the final product is the Brown Spot Alternaria, caused by the fungus Alternaria alternata. The disease causes leaf drop, dry branches and fruit drop in tangerines and their hybrids. To control this disease fungicides are used, however, the brown spot is a difficult to control disease, may be required from 7 to 10 applications of fungicide, appearance of resistance of fungi, intoxication in people and environment contamination owing the application of fungicides. Alternative methods such as biological control with applications antagonistic microorganisms and plant nutrition are options of smaller impact to the environment. Therefore, the objective of this work was to evaluate the potential of Trichoderma spp., Bacillus subtilis and fertilizer, in control of Alternaria Brown Spot in citrus plants Ponkan tangerine. The experiment was carried out in a randomized design, consisting of eight treatments with four replicates. The experiments were evaluated for the incidence of the disease. The data showed that the best control was the chemical treatment, but the treatments with Trichoderma spp., Trichoderma spp. + Fertilizer and Trichoderma spp. + Bacillus subtilis presented excellent control rates, and may even replace chemical treatment in the control of this disease.

A viticultura é uma atividade de grande importância econômica, destacando-se a sustentabilidade da pequena propriedade e o desenvolvimento territorial associados às atividades ligadas ao turismo. As plantas cultivadas se tornam vulneráveis a patógenos e insetos-praga e a videira apresenta diversas espécies consideradas pragas que reduzem sua produção e rentabilidade, entre estas, Spodoptera frugiperda, causando danos em diferentes partes da planta. Fungos entomopatogênicos podem oferecer uma alternativa aos pesticidas convencionais para o controle de pragas, pois produzem enzimas que degradam o exoesqueleto do inseto como quitinases e proteases facilitando o modo de infecção. Neste trabalho foi avaliado o potencial inseticida do fungo Nomurea rileyi, linhagem UCS03, contra S. frugiperda e o perfil eletroforético por SDS-PAGE das proteínas secretadas por N. rileyi em presença da cutícula do inseto em diferentes intervalos de tempo em gel unidimensional. O fungo N. rileyi apresentou virulência contra S. frugiperda, determinando um CL50 de 2 x 109conídios/mL com a linhagem UCS03 demonstrando atividade bionseticida. Na avaliação do perfil proteico do secretoma de N. rileyi em presença da cutícula do inseto, em diferentes tempos de cultivo, foi possível verificar um perfil altamente diferenciado. A maior concentração de proteína foi encontrada no 14° dia de incubação (0,3507 mg/mL) reduzindo a quantidade de proteínas após este período. Na análise por SDS-PAGE foi possível verificar diferentes proteínas de diferentes massas moleculares, nos intervalos de tempo considerados, sendo muitas inferiores a 75 kDa. Estas proteínas com diferentes massas moleculares podem estar envolvidas no metabolismo do fungo. Desta forma, estes resultados podem contribuir para a compreensão do processo de infecção de N. rileyi em S. frugiperda, oferecendo potencial para o desenvolvimento de novas pesquisas e aplicações destas em processos biotecnológicos. / Viticulture is an activity of great economic importance with emphasis on the sustainability of small property and territorial development associated with tourism related activities. Cultivated plants become vulnerable to pathogens and insect pests and the vine has several species considered as pests that reduce their production and profitability, among them, Spodoptera frugiperda, with causes damage in different parts of the plant. Entomopathogenic fungi can provide an alternative to conventional pesticides for controlling pests, they produce enzymes that degrade the insect exoskeleton, such aschitinases and proteases facilitating the infection. In this work, the insecticide potential of the fungus Nomurea rileyi and S. frugiperda was evaluated, as well as the proteins secreted by N. rileyi in the presence of insect cuticle at different time intervals in one-dimensional gel. The fungus N. rileyi presented virulence against S. frugiperda, determining a CL50 of 2 x 109 con / mL with UCS03 strain demonstrating bionseticida activity. In assessing the protein profile of secretome N. rileyi in the presence of insect cuticle, at different times of cultivation, a highly differentiated profile was verified. The highest concentration of protein was found at day 14 of incubation (0.3507 mg / ml) reducing the amount of protein after this period. In the one-dimensional gel analysis was verified different molecular weights of proteins, in the time interval considered being many less than 75kDa. These proteins with different molecular weights may be involved in fungal metabolism. Thus, these results can contribute to the understanding of the infection process of N. rileyi in S. frugiperda, offering potential for the development of new researches and applications in biotechnological processes.

O Brasil é o maior produtor de citros, sendo um dos seus principais produtos de exportação da cadeia agroindustrial. Um dos fatores que afetam a qualidade e a produção do produto final obtido é a Mancha Marrom de Alternaria, causado pelo fungo Alternaria alternata. A doença provoca desfolha, seca de ramos e queda de frutos em tangerinas e seus híbridos. Para controle dessa doença são utilizados fungicidas, porém, a mancha marrom é uma doença de difícil controle podendo ser necessárias de 7 a 10 aplicações do fungicida, aparecimento de resistência dos fungos, intoxicação nas pessoas e contaminação do meio ambiente devido ao emprego de fungicidas. Métodos alternativos como o controle biológico com aplicações de micro-organismos antagonistas e a nutrição das plantas são opções de menor impacto ao meio ambiente. Sendo assim, o objetivo deste trabalho foi avaliar o potencial de Trichoderma spp., Bacillus subtilis e fertilizante organomineral, no controle da Mancha Marrom de Alternaria em plantas cítricas de tangerina Ponkan. O experimento foi realizado com delineamento casualizado, constituído de oito tratamentos com quatro repetições. Os experimentos foram avaliados quanto a incidência da doença. Os dados mostraram que o melhor controle foi o tratamento químico, porém os tratamentos com Trichoderma spp., Trichoderma spp. + fertilizante e Trichoderma spp. + Bacillus subtilis apresentaram ótimas taxas de controle, podendo inclusive substituir o tratamento químico no controle desta doença. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES. / Brazil is the largest citrus producer, being one of its main exports of agro-industrial chain. One of the factors that affect the quality and production of the final product is the Brown Spot Alternaria, caused by the fungus Alternaria alternata. The disease causes leaf drop, dry branches and fruit drop in tangerines and their hybrids. To control this disease fungicides are used, however, the brown spot is a difficult to control disease, may be required from 7 to 10 applications of fungicide, appearance of resistance of fungi, intoxication in people and environment contamination owing the application of fungicides. Alternative methods such as biological control with applications antagonistic microorganisms and plant nutrition are options of smaller impact to the environment. Therefore, the objective of this work was to evaluate the potential of Trichoderma spp., Bacillus subtilis and fertilizer, in control of Alternaria Brown Spot in citrus plants Ponkan tangerine. The experiment was carried out in a randomized design, consisting of eight treatments with four replicates. The experiments were evaluated for the incidence of the disease. The data showed that the best control was the chemical treatment, but the treatments with Trichoderma spp., Trichoderma spp. + Fertilizer and Trichoderma spp. + Bacillus subtilis presented excellent control rates, and may even replace chemical treatment in the control of this disease.

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.