Avaliou-se em laboratório a patogenicidade de 52 isolados de fungos entomopatogênicos a Brevipalpus phoenicis e a produção, em meio sólido, de quatro isolados de Hirsutella spp.. Utilizando-se microscópio eletrônico de varredura, estudou-se as etapas do ciclo biológico de Hirsutella sp. (isolado 1269) em adultos de B. phoenicis, além da ocorrência de transmissão horizontal deste patógeno para adultos do ácaro a partir de cadáveres esporulados e de substrato contaminado. Todos os isolados de Hirsutella sp. foram patogênicos para o ácaro causando mortalidade de adultos superiores a 90% após seis dias da inoculação. Comprovou-se também que em meio de cultura completo (M.C.) que o isolado 1269 foi o que apresentou maior crescimento vegetativo e esporulação, sendo selecionado como o mais promissor. O ciclo biológico completo de Hirsutella sp., desde a adesão dos conídios ao ácaro até sua extrusão, ocorreu em 120 horas após a aplicação. A transmissão horizontal do patógeno a partir de cadáveres e do substrato contaminado ocasionou mortalidade superior a 50% em ambas as situações, comprovando que o fungo foi capaz de infectar novos hospedeiros. Contatou-se que Hirsutella sp. é um patógeno eficiente no controle de B. phoenicis podendo ser utilizado em campo em estratégias de introdução inoculativa, inundativa, incremento e conservação. / In this research, the patogenicity of 52 isolates of entomopathogenic fungi against Brevipalpus phoenicis and the production of four isolates of Hirsutella spp. in solid medium (M.C.) were evalueted under laboratory condition. The stages of the biological cycle of Hirsutella sp. (isolated 1269) on adults of B. phoenicis were evalueted with the use of an electron scan microscope, besides the occurrence of horizontal transmission of this fungi from cadavers and from an inoculate substract to new hosts. The isolates of Hirsutella sp. tested were pathogenic to the mite, with high mortality (>90%) after six days of the inoculation. The isolate 1269 showed high levels of vegetative growth and sporulation and was selected as the most promising one. The complete biological cycle of Hirsutella sp. ocurred after 120 hours of the inoculation, from adhesion to extrusion processes. The horizontal transmission of fungi from cadavers and inoculate substract ocurred by causing levels mortality superior to 50% in both cases, showing that the fungi was capable to infect new hosts in these situations. Hirsutella sp. was an efficient pathogen in the control of B. phoenicis and could be exploited in field control strategies through an inoculative introduction, increment or conservation.

O Brasil é o maior produtor de cana-de-açúcar (Saccharum officinarum L.) do mundo e a cultura continua em expansão na safra 2014/15. A expansão das áreas de plantio de cana-de-açúcar e o sistema de colheita mecanizada de cana crua tem levado ao aumento proporcional de inúmeras pragas da cultura, destacando-se o bicudo da cana, Sphenophorus levis, considerado praga primária e limitante da cultura. Inicialmente, essa espécie foi considerada restrita a região de Piracicaba, mas desde 2010 foi encontrada nos estados de São Paulo, Minas Gerais, Paraná, Mato Grosso do Sul, Mato Grosso e Goiás. Suas larvas abrem galerias nos rizomas, causando danos diretos nos tecidos dos colmos, sobretudo nas épocas mais secas do ano, o que ocasiona a morte das touceiras, falhas na rebrota, além de promover danos indiretos, como o aumento do número de plantas invasoras que competem com a cultura. Os métodos químico, mecânico, cultural e biológico já foram testados até o momento, no entanto, o sucesso no seu controle ainda não foi obtido e a população dessa praga continua crescendo e se tornando mais constante nos canaviais pelo Brasil. Dessa forma, estudar estratégias para a melhoria do manejo de S. levis é fundamental para o estabelecimento de métodos de controle eficientes no contexto do MIP. Diante disso, objetivou-se com esse trabalho: 1) Determinar a distribuição espacial e a flutuação populacional de S. levis; 2) Testar o manejo da palha pós-colheita nas linhas de plantio na ocorrência de S. levis e; 3) Avaliar a eficácia da aplicação de Beauveria bassiana na população de S. levis. Em relação à flutuação populacional de S. levis foi observada uma elevação linear da última quinzena de outubro para a primeira de novembro, com queda subsequente e, aumento populacional maior à partir da última quinzena de dezembro. O pico populacional se deu no mês de março, seguido por uma rápida redução a partir de abril. A proporção de fêmeas foi similar a de machos em todos os tratamentos. Os dados sugerem que não houve efeito dos tratamentos na distribuição de adultos do bicudo. Os resultados da flutuação populacional, da distribuição de frequência e distribuição espacial se complementam, pois, em hipótese, a distribuição dos adultos está diretamente relacionada com a flutuação populacional, uma vez que em altas densidades a distribuição foi agregada. Além disso, as estratégias de afastamento da palha da linha de plantio não interferiram nos níveis de infestação de S. levis, quando comparado ao tratamento que não houve manejo da palha e, essa tática utilizada conjuntamente à aplicação de B. bassiana também não influenciou na densidade populacional de adultos de S. levis no período avaliado. Diante disso, concluiu-se que a distribuição de S. levis em cana-de-açúcar apresenta o padrão agregado em altas densidades populacionais; o manejo da palha entre as linhas de cana-de-açúcar não reduz a densidade populacional de S. levis em locais com elevado histórico de infestação e, são necessários mais estudos para ajustar a dose de B. bassiana adequada e eficaz no controle de S. levis. / Brazil is the world\'s largest sugarcane producer and the cultivated area continues to expand in 2014/2015 season. This expansion, along with the mechanical harvesting of green sugarcane leads to a proportional increase in the number of crop pests, especially the sugarcane weevil, Sphenophorus levis, considered a primary and limiting pest in this crop. Initially, this species was restricted to the Piracicaba region, however, since 2010 it has been found in several regions of São Paulo State, and also in other states in the country, such as Minas Gerais, Paraná, Mato Grosso do Sul, Mato Grosso and Goiás. The larvae penetrate the basal part of the plants and the rhizomes, building irregular galleries and causing damage to stem tissues, mainly in the driest season, leading to the death of clumps, failures in regrowth and, consequently, indirect damage due to the increase in the incidence of invasive plants that compete with the crop. Chemical, mechanical, cultural and biological methods have already been tested, however, the success in pest control has not yet been achieved and the population of S. levis continues to grow, becoming more constant in Brazilian sugarcane fields. Thus, the study of strategies to enhance the management of S. levis is essential to adopt effective control methods in the IPM framework. In this sense, this study aims to: 1) Determine the spatial distribution and population dynamics of S. levis; 2) Evaluate the postharvest management of straw on planting rows in occurrence of S. levis and; 3) Evaluate the efficacy of B. bassiana application in the S. levis population in sugarcane. Concerning population dynamics of S. levis, it was observed a linear increase beginning in the last quarter of October until November, with a subsequent decrease and, a highest population increase was observed in the last half of December. The population peak occurred in March, followed by a fast decline in early April. The proportion of females to males was similar in all treatments. The data suggest that there was no effect of treatments on the distribution of adult weevils. The results of population dynamics, frequency distribution and spatial distribution are complementary, because, hypothetically, the distribution of adults is directly related to population dynamics, since at high densities the distribution was aggregated. In addition, strategies for straw management in the planting row did not affect the infestation levels of S. levis and this procedure along with the addition of B. bassiana did not influence population density of adults during the study period. Hence, it is concluded that the distribution of S. levis in sugarcane exhibits an aggregated pattern at high population densities. Straw management between the cane rows does not reduce population density of S. levis in areas with high historical infestation. Therefore, further studies are needed to adjust the appropriate and effective concentration of B. bassiana in the control of S. levis.

Orientador: Antonio Batista Filho / Banca: Carlos Gilberto Raetano / Banca: Edson Luiz Lopes Baldin / Banca: José Eduardo Marcondes de Almeida / Banca: Luis Garrigós Leite / Resumo: A cana-de-açúcar colhida sem queima é uma realidade em todo o Estado de São Paulo e os ataques da cigarrinha-da-raiz da cana estão cada vez mais freqüentes e intensos. O controle biológico desta praga com o fungo Metarhizium anisopliae também vem se desenvolvendo e adquirindo relevada importância. Com isso, este trabalho foi conduzido com os seguintes objetivos: avaliar a produção de conídios de diferentes isolados para o controle da cigarrinha-das-raízes, a partir de dois tipos de arroz; avaliar a eficiência dos isolados selecionados como mais produtivos em populações naturais de Mahanarva fimbriolata, na cultura da cana-de-açúcar colhida mecanicamente e; verificar o manejo da população de M. fimbriolata em áreas de cana-de-açúcar colhidas sem queima da palha, observando a influência da umidade sobre sua ocorrência. Foram avaliados 14 isolados, os quais foram produzidos em arroz tipo 1 e arroz parboilizado, em sacos de polipropileno, incubados em sala climatizada para desenvolvimento do fungo. Avaliou-se a concentração e viabilidade de cada isolado para os dois tipos de arroz. Os isolados que apresentaram maior produtividade foram aplicados em campo para avaliar a patogenicidade dos mesmos à cigarrinha-das-raízes, sendo eles: ESALQ 1037, IBCB 425, IBCB 353, IBCB 410, F 99 e IBCB 333. Foram pulverizados 2 kg/ha de arroz+fungo, contendo 1,0 x 1012 conídios/ha, além do tratamento com o inseticida tiametoxam 250 WG e a testemunha, sem aplicação. As avaliações foram realizadas aos 15, 30, 60, 90 e 120 dias após a aplicação (DAA), observando-se o número de ninfas e adultos de M. fimbriolata vivos, mortos, parasitados ou não, em cada parcela. A partir dos resultados, foi possível observar que os isolados IBCB 410 e F 99 causaram, respectivamente, mortalidades de 66,67 e 33,33% para ninfas, aos 15 DAA. Após 30 DAA, os isolados IBCB 425, IBCB 353 e IBCB 333 ...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The sugar cane is harvested without burning a reality throughout the state of Sao Paulo and the attacks of the leafhopper-root cane are increasingly frequent and intense. Biological control this pest with the Metarhizium anisopliae also has been developing and acquiring increasing importance. Therefore, this work was conducted to evaluate the production of conidial selected for the control of root speatlebug in two types of rice, evaluate the efficiency of the isolates selected as the most productive in natural populations of M. fimbriolata, the culture of cane sugar harvested mechanically, monitor the population of M. fimbriolata in areas of sugarcane harvested without burning the straw, and the influence of temperature and humidity on its occurrence. We evaluated 14 isolates, which were produced in rice type 1 and parboiled rice in polypropylene bags, incubated in a room for fungal growth and were evaluated the concentration and viability of each isolate for both types of rice. The isolates that had higher yields were applied in the field to assess the pathogenicity of the same root speatlebug, namely: ESALQ 1037, IBCB 425, IBCB 353, IBCB 410, F 99 and IBCB 333. Were sprayed 2 kg / ha of rice + fungus, containing 1,0 x 1012 conidia / ha, in addition to treatment with the insecticide tiametoxam 250 WG and the control. Evaluations were performed at 15, 30, 60, 90 and 120 days after the application, noting the number of nymphs and adults of M. fimbriolata alive in each plot, and the number of nymphs and adults dead, infected and non-parasitized. From the results, it was observed that isolates IBCB 410 and F 99 caused mortality to nymphs of 66.67 and 33.33%, respectively, at 15 days after application. After 30 days of spraying, the isolates IBCB 425, IBCB 353 and IBCB 333 had efficiencies... (Complete abstract click electronic access below) / Doutor

Submitted by Erika Demachki (erikademachki@gmail.com) on 2015-02-27T17:03:09Z No. of bitstreams: 2 Dissertação - Glennyha Fernandes Duarte - 2014.pdf: 1764909 bytes, checksum: fa26338a2793bde550bdf59fd0986bc1 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2015-02-27T17:03:21Z (GMT) No. of bitstreams: 2 Dissertação - Glennyha Fernandes Duarte - 2014.pdf: 1764909 bytes, checksum: fa26338a2793bde550bdf59fd0986bc1 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-02-27T17:03:21Z (GMT). No. of bitstreams: 2 Dissertação - Glennyha Fernandes Duarte - 2014.pdf: 1764909 bytes, checksum: fa26338a2793bde550bdf59fd0986bc1 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-07-17 / Biomphalaria glabrata snails are aquatic and inhabit often ephemeral basins. They have high reproductive capacity, and egg masses are laid on submerged substrates on the banks of breeding sites exposed to desiccation and pathogens like fungi. Virtually nothing is known about the viability of egg masses in breeding and transient sites and their pathogens. This information is important to control this intermediate host of Schistosoma mansoni, which causes schistosomiasis. This study describes the feasibility of egg masses in water films and susceptibility infection with to Beauveria bassiana and Metarhizium ansiopliae. Egg masses were exposed to water film formed in two different devices, one with a stone clay in a cup with water and another one with water agar at different concentrations of agar (0.3% - 3%) in Petri dishes in different relative humidity (RH > 98% and 75%). Conidia or blastospores of B. bassiana (ARSEF 9588) or M. anisopliae (IP 46) (2 x 107 conidia or blastospores/egg mass) were inoculated in egg masses exposed in the water film in stone clay or submerged (2 x 107 conidia/ml or blastospores/ml). Egg masses exposed in the water film in stone clay or in water agar (agar < 1%) at RH > 98% were viable. The eclosion and the displacement of juveniles in the films were observed regardless of the tested device (RH > 98%, agar < 1%), except at 75% RH and glass without water film. Mycelium and conidia developed on egg masses treated with conidia or blastospores of M. anisopliae in the water film in stone clay (RH > 98%), and there was no eclosion of juveniles from egg masses treated with blastospores of M. anisopliae. In egg masses submerged treated with blastospores of M. anisopliae or B. bassiana no eclosion of juveniles and egg masses treated with conidia accumulated average onset was < 52.5%. The feasibility of egg masses and the displacement of juveniles in both devices on RH > 98% was due to the formation of stable water film. Blastospores may have released toxic metabolites that inhibited the eclosion of juvenile B. glabrata. Both devices are promising for testing egg masses in the water film. The feasibility of egg masses in water under laboratory conditions film should be considered to control B. glabrata. Blastospores of M. anisopliae and B. bassiana are promising against B. glabrata. / Biomphalaria glabrata são caramujos aquáticos e habitam coleções hídricas frequentemente efêmeras. Possuem alta capacidade reprodutiva, e massas ovígeras são postas em substratos submersos nas margens dos criadouros expostas ao ressecamento e a patógenos como fungos. Praticamente nada é sabido sobre a viabilidade de massas ovígeras em criadouros transitórios e a sua susceptibilidade a fungos. Estas informações são importantes para o controle desse caramujo hospedeiro intermediário de Schistosoma mansoni, causador de esquistossomose. O presente trabalho relata a viabilidade de massas ovígeras em filme de água e a susceptibilidade a Metarhizium ansiopliae e Beauveria bassiana. Massas ovígeras foram expostas em filme de água formado em dois dispositivos diferentes confeccionados, um com pedra de argila em copo com água e outro com meio ágar água com diferentes concentrações de ágar (0,3% - 3%) em placas de Petri em diferentes umidades relativas (UR > 98% e 75%). Conídios ou blastosporos de B. bassiana (ARSEF 9588) ou M. anisopliae (IP 46) (2 x 107 conídios ou blastosporos/massa ovígera) foram inoculados em massas ovígeras expostas em filme de água em pedra de argila ou submersas (2 x 107 conídios/ml ou blastosporos/ml). As massas ovígeras expostas em filme de água em pedra de argila ou em meio ágar água (ágar < 1%) em UR > 98% foram viáveis. A eclosão e o deslocamento de juvenis nos filmes foram observados independentemente do dispositivo testado (UR > 98%, ágar < 1%), exceto em UR 75% e em vidro sem filme de água. Micélio e conídios desenvolveram sobre massas ovígeras tratadas com conídios ou blastosporos de M. anisopliae em filme de água em pedra de argila (UR > 98%), e não houve eclosão de juvenis a partir de massas ovígeras tratadas com blastosporos de M. anisopliae. Em massas ovígeras submersas tratadas com blastosporos de M. anisopliae ou B. bassiana não houve eclosão de juvenis e em massas ovígeras tratadas com conídios a eclosão média acumulada foi < 52,5%. A viabilidade de massas ovígeras e o deslocamento de juvenis em ambos os dispositivos em UR > 98% foi devido à formação de filme de água estável. Blastosporos podem ter liberado metabólitos tóxicos que inibiram a eclosão de juvenis de B. glabrata. Ambos os dispositivos são promissores para testes com massas ovígeras em filme de água. A viabilidade de massas ovígeras em filme de água em condições laboratoriais deve ser considerada no controle de B. glabrata. Blastosporos de M. anisopliae e B. bassiana são promissores contra B. glabrata.

O Brasil é o país líder no uso do fungo entomopatogênico Metarhizium anisopliae para o controle pragas agrícolas. Pouco se conhece sobre a diversidade e o impacto dos micovírus em M. anisopliae sensu stricto. Este trabalho mostra a riqueza dos micovírus associados a isolados de M. anisopliae da coleção de entomopatógenos da Universidade de São Paulo, usinas sucroalcooleiras e produtos microbianos à base do entomopatógeno. RNAfd foram encontrados em 55% dos 36 isolados de Metarhizium e apresentaram 16 diferentes padrões eletroforéticos consistindo de 3 a 18 bandas de RNAfd em gel de poliacrilamida. RNAfd não foram detectados em nenhum dos produtos comerciais utilizados no presente estudo. Os diferentes padrões de vírus encontrados nos isolados aqui estudados, aparentemente, não têm relação com os locais onde foram coletados. O inibidor de síntese protéica ciclohexamida não foi eficiente na eliminação dos vírus de RNAfd em nenhum dos isolados de fungos testados. Colônias dos isolados de M. anisopliae ESALQ 866, M. anisopliae ESALQ 1256 e M. anisopliae CTC F8 foram curadas por meio do cultivo monoconidial ou isolamento de ponta de hifas. Alguns segmentos do isolado M. anisopliae PL26 foram perdidos após o subcultivo de ponta das hifas. Colônias de M. anisopliae ESALQ PL26 obtidas por meio do cultivo monoconidial ou subcultivo de ponta de hifas apresentaram grande variabilidade morfológica, no entanto, essa variação não foi correlacionada com a presença de micovírus. Colônias isogênicas de M. anisopliae ESALQ 1256 mostraram diferenças no crescimento, na produção de conídios e na virulência, no entanto, essas diferenças não foram associadas à presença de RNAfd. Não foram observadas diferenças na tolerância aos raios ultravioleta e ao calor entre as colônias de M. anisopliae ESALQ 1256, com e sem vírus de RNAfd. Colônias oriundas de setores formados no isolado M. anisopliae PL26 produziram menor quantidade de conídios e apresentaram menor quantidade de vírus RNAfd em comparação com as colônias oriundas de outras regiões da colônias originais com características normais. A repicagem sucessiva dos isolados de M. anisopliae ESALQ PL26, ESALQ 1256, CTC F8 e CTC F15, infectados por diferentes vírus de RNAfd, nos meios de cultura BDA, SDAY e meio de arroz, bem como a passagem dos fungos em larvas de Tenebrio molitor, em geral, não afetaram a replicação dos micovírus. / Brazil is the leading country in the use of the entomopathogenic fungus Metarhizium anisopliae against agricultural pests. Little is known about the diversity and the impact of mycovirus in M. anisopliae sensu stricto. This study shows the richness of mycovirus associated with M. anisopliae isolates from the collections of entomopathogens of the University of São Paulo, from sugar-alcohol factories and microbial based products. dsRNA were found in 55% of the 36 Metarhizium isolates and showed 16 different electrophoretic patterns consisting of 3 to 18 dsRNA bands in polyacrylamide gels. dsRNA was not detected in any of the commercial products used in this study. The different viral patterns found in the isolates studied here apparently have no relation to the locations where they were collected. The inhibitor of protein synthesis cycloheximide culture was efficient in eradicating dsRNA virus from fungi. Colonies of isolates of M. anisopliae ESALQ 866, M. anisopliae ESALQ 1256 and M. anisopliae F8 were cured by monoconidial or by hyphal tip isolation of. Some segments of the M. anisopliae PL26 isolate were lost following hyphal tip subculture. Colonies both from monoconidial culture and hyphal tip subculture of M. anisopliae ESALQ PL26 showed great morphological variability; however, this variation was not correlated with the presence of mycoviruses. Isogenic colonies of M. anisopliae ESALQ 1256 showed differences in growth, conidia production and virulence; however, these differences were not associated to the presence of the dsRNA. No difference was observed regarding tolerance to ultraviolet rays and heat among the colonies of M. anisopliae ESALQ 1256, with and without the dsRNA virus. Sectors formed in the isolate M. anisopliae PL26 produced a smaller number of conidia and fewer dsRNA virus in comparison to the original colonies with normal characteristics. The repeated subculture of isolates of M. anisopliae ESALQ PL26, ESALQ 1256, CTC F8 and CTC F15, infected by different virus of dsRNA, in the PDA, SDAY culture media and in rice, as well as the fungi passage in larvae of Tenebrio molitor, in general, did not affect the replication of the mycovirus.

Σκοπός της παρούσας εργασίας ήταν να μελετηθεί η επίδραση των εντομοπαθογόνων μυκήτων Beauveria bassiana Balsamo (Vuillemin) (Hypocreales: Cordycipitaceae), Metarhizium robertsii (Metchnikoff) Sorokin (Hypocreales: Clavicipitaceae) και Isaria fumosorosea (Wize) Brown & Smith (Hypocreales: Clavicipitaceae), πουαπομονώθηκαν από ελληνικά εδάφη, επί του κυριότερου και πιο επιβλαβούς εχθρού της καλλιέργειας του γλυκού σόργου, του λεπιδόπτερου Sesamia nonagrioides (Lefebvre) (Lepidoptera: Noctuidae). Μελετήθηκε στο εργαστήριο η επίδραση έξι στελεχών των παραπάνω μυκήτων στην επιβίωση των προνυμφών του εντόμου. Τα στελέχη των μυκήτων προκάλεσαν υψηλή θνησιμότητα στις προνύμφες του εντόμου, που κυμάνθηκε σε ποσοστά από 78%-98%. Αποτελεσματικότερα στελέχη ήταν τα B. bassiana (ΙΓΕ3), M. robertsii (Elateridae) και I. fumosorosea (Άγιος Στέφανος). Αυτές οι τρείς απομονώσεις επιλέχθηκαν προκειμένου να εξεταστεί η επίδραση της πυκνότητας των κονιδιακών εναιωρημάτων στη θνησιμότητα των προνυμφών. Καταγράφηκαν ποσοστά θνησιμότητας 7%-93% σε σχέση με την πυκνότητα των κονιδιακών εναιωρημάτων επί των προνυμφών. Διαπιστώθηκε ότι η ικανότητα που παρουσιάζουν οι εντομοπαθογόνοι μύκητες στον έλεγχο της επιβίωσης προνυμφών του εντόμου ήταν ανάλογη της χρησιμοποιημένης πυκνότητας των κονιδιακών εναιωρημάτων. Επίσης, μελετήθηκε η αλληλεπίδραση των εντομοπαθογόνων μυκήτων B. bassiana (ΙΓΕ3) και M. robertsii (Elateridae) με εμπορικό σκεύασμα Bacillus thuringiensis subsp. kurstaki όσον αφορά τη θανάτωση προνυμφών του εντόμου S. nonagrioides. Η θνησιμότητα των προνυμφών καταγράφηκε καθημερινά για δεκαέξι ημέρες και κυμάνθηκε από 56-100% για τον M robertsii μαζί με το B thuringiensis subsp. kurstaki και 54-100% για τον B. bassiana μαζί με το βάκιλο. Επίσης, η συνδυασμένη δράση των μικροοργανισμών υπολογίστηκε για την όγδοη, δέκατη τρίτη και τη δέκατη έκτη μέρα και βρέθηκε προσθετική στους περισσότερους συνδιασμούς για την δέκατη τρίτη και δέκατη έκτη μέρα ενώ την όγδοη μέρα η συνδυαστική δράση των παθογόνων βρέθηκε αρνητική στους περισσότερους συνδιασμούς και χαρακτηρίζεται ως ανταγωνιστική. Η ανάπτυξη των εντομοπαθογόνων μυκήτων όταν καλλιεργήθηκαν σε θρεπτικό υπόστρωμα που περιείχε υδατικό διάλυμα B. thuringiensis subsp. kurstaki, δεν επηρεάστηκε από την παρουσία του βακτηρίου. Ο συνδυασμός του B. thuringiensis subsp. kurstaki με τους εντομοπαθογόνους μύκητες που εξετάσαμε είχε ως αποτέλεσμα να αυξηθεί η θνησιμότητα των προνυμφών του S. nonagrioides. Τα στελέχη ΙΓΕ3, Elateridae και Άγιος Στέφανος των εντομοπαθογόνων μυκήτων B. bassiana, M. robertsii και I. fumosorosea αντίστοιχα, δοκιμάστηκαν επί των προνυμφών του S. nonagrioides σε τρεις κονιδιακές συγκεντρώσεις και μελετήθηκε η επίδρασή τους στην πρόσληψη τροφής, στην κατανάλωση τροφής και στην ανάπτυξη των προνυμφών. Παρατηρήθηκε μείωση της πρόσληψης τροφής από προνύμφες 3ης προνυμφικής ηλικίας σε ποσοστά 41-81%, μείωση του βάρους τους κατά 29-45% και μείωση των απορριμάτων κατά 46-81%. Παρατηρήθηκε επίσης ελάττωση της σχετικής ταχύτητας ανάπτυξης των προνυμφών κατά 31-72% και της σχετικής ταχύτητας κατανάλωσης τροφής κατά 12-35%. Τέλος, ο δείκτης πεπτικότητας, ο δείκτης μετατροπής της προσληφθείσας τροφής και ο δείκτης μετατροπής της αφομοιωμένης τροφής ήταν υψηλότεροι σε σχέση με τους αντίστοιχους του μάρτυρα. Τα παραπάνω αποτελέσματα υποδεικνύουν ενδεχομένως ότι η μόλυνση από τους εντομοπαθογόνους μύκητες προκαλεί επιδείνωση σε ορισμένες τροφικές διεργασίες της προνύμφης που θα μπορούσαν να οδηγήσουν στην υπονόμευσή της από ασιτία. Επίσης πραγματοποιήθηκαν πειράματα στο πεδίο προκείμενου να μελετηθεί η επίδραση της ενδοφυτικής παρουσίας των εντομοπαθογόνων μυκήτων B. bassiana, M. robertsii και I. fumosorosea στις προνύμφες του εντόμου S. nonagrioides in planta. Kαι οι τρείς μύκητες έδρασαν αποτρεπτικά απέναντι στην προσπάθεια των προνυμφών να εισέλθουν στο στέλεχος του φυτού. Οι προνύμφες της 4ης προνυμφικής ηλικίας που πέτυχαν να εγκατασταθούν στο στέλεχος των φυτών δημιούργησαν στοές μικρότερου μήκους παρουσία των μυκήτων, και η θνησιμότητά τους ήταν μεγαλύτερη, επιβεβαιώνοντας τα αποτελέσματα των in vitro πειραμάτων. Ως προς τις επιπτώσεις της παρουσίας των ενδοφύτων στο φυτό ξενιστή, η επίδραση της παρουσίας των μυκήτων στην ανάπτυξη, την τελική βιομάζα και την κατανομή της βιομάζας σε υπέργειους ή υπόγειους ιστούς των φυτών καταγράφηκε ως ουδέτερη. Η συγκέντρωση της χλωροφύλλης και η περιεκτικότητα σε νερό δεν παρουσίασαν μεταβολές που μπορούν να αποδοθούν στην παρουσία των ενδοφύτων. Ωστόσο, ορισμένες παράμετροι της φωτοσυνθετικής διαδικασίας (καθαρή φωτοσύνθεση, ταχύτητα ροής ηλεκτρονίων, η πιθανότητα μετατροπής της συλληφθείσας ενέργειας σε ροή ηλεκτρονίων, η πιθανότητα μεταφοράς ηλεκτρονίων από τους ενδιάμεσους φορείς μέχρι την αναγωγή των τελικών υποδοχέων στο PSI και ο δείκτης ζωτικότητας της φωτοσυνθετικής συσκευής PItotal) επηρεάστηκαν αρνητικά μετά την προσβολή των στελεχών του σόργου από τις προνύμφες του εντόμου. Η ενδοφυτική παρουσία των μυκήτων B. bassiana και M. robertsii απέτρεψε τις παραπάνω αρνητικές επιδράσεις. Τα αποτελέσματα της παρούσας διατριβής προσφέρουν ισχυρές ενδείξεις για επιτυχή χρησιμοποίηση ορισμένων στελεχών των εντομοπαθογόνων μυκήτων στον έλεγχο των προνυμφών του εντόμου S. nonagrioides τόσο στο εργαστήριο όσο και στο πεδίο. Επίσης, θα ήταν ενδιαφέρουσα η περαιτέρω μελέτη της χρήσης αυτών τωνεντομοπαθογόνων μυκήτων κάτω από συνθήκες μειωμένης διαθεσιμότητας νερού. / The aim of this study was to investigate the effect of entomopathogenic fungi Beauveria bassiana Balsamo (Vuillemin) (Hypocreales: Cordycipitaceae), Metarhizium robertsii (Metchnikoff) Sorokin (Hypocreales: Clavicipitaceae) and Isaria fumosorosea (Wize) Brown & Smith (Hypocreales: Clavicipitaceae), isolated from Greek soils against the main and most damaging pest of sweet sorghum, Mediterranean corn borer Sesamia nonagrioides (Lefebvre) (Lepidoptera: Noctuidae). Laboratory studies were performed to determine the effect of six strains of the above fungi on survival of larvae of S. nonagrioides. All strains of fungi causing high mortality among larvae, killed 78 - 98% of exposed larvae. The most effective were B. bassiana (IGE3), M. robertsii (Elateridae) and I. fumosorosea (Agios Stefanos). These three isolates were selected to examine the effect several conidial suspensions on the mortality of larvae. Recorded mortality was from 7% - 93% depending on the density of conidial suspensions used on larvae. From the above results was found that the capability provided by the entomopathogenic fungi to control the insect was according by the density of the conidial suspensions. Also, the interactions between commercial B. thuringiensis subsp. kurstaki and two entomopathogenic fungi were examined on larvae of S. nonagrioides in 8, 13 and 16 days post-treatment intervals. An overall positive interaction between the pathogens was observed and larval mortality at 16 days was 56–100% exposed to M. robertsii combined with B. thuringiensis subsp. kurstaki, whereas B. bassiana combined with B. thuringiensis subsp. kurstaki killed 54–100 % of exposed larvae. After 8 days, in 6 of the combinations, we found an additive relationship between the pathogens, whereas, a negative interaction was observed in 10 of them. In contrast, after 13 days, we observed a positive interaction in two combinations, which could be considered as synergistic, additive in ten combinations and negative in only four combinations. Finally, after 16 days, in eleven combinations we found an additive interaction, whereas negative interaction was recorded in five of them. We conclude that application of combined pathogens may be more effective for S.nonagrioides control. The strains IGE3, Elateridae and Agios Stefanos of B. bassiana, M. robertsii and I. fumosorosea respectively, were tested for their effectiveness on larval food intake, food consumption and larvae growth at three conidial concentrations. We observed a decrease in food intake by the larvae ranged between 41 and 81%, reduced larvae weight from 29 to 45% and reduced production of wastes from 46 to 81%. We also recorded significant differences in the relative growth rate of larvae, the relative consumption rate of larvae, the index digestibility, the conversion ratio of ingested food and the conversion ratio of assimilated food between infected and control individuals. Therefore, it is possible that infection of larvae by the fungi has an adverse effect on physiological and metabolic processes, which results in larva starvation. Subsequently, we performed experiments in the field, in order to investigate the plant–endophyte association behavior using B. bassiana, M. robertsii and I. fumosorosea, as well as the impact of these associations on larva activity when in planta and under environmental conditions. All three fungal species significantly prevented larvae from entering into plants’ stalk. Statistically significant differences were also observed in the length of tunnels created by larvae in the stem of the plant and in their mortality for individuals infected by the entomopathogenic fungi. These results confirm our findings of the in vitro experiments. Regarding the effect of endophytes on the host plant, a neutral impact was recorded for the development, final biomass and distribution of biomass to the aboveground and underground plant tissues. Temporary changes of chlorophyll concentration and leaf water content stability throughout the experimental period should not be attributed to the presence of endophytes. However, some aspects of the photosynthetic process (net photosynthesis, electron transport rate, as well as parameters of the photosynthetic machinery relative to electron flow round the two photosystems) negatively affected after infection of plants by the larvae. The presence of B. bassiana and M. robertsii ameliorated these adverse effects. The results of this study are strongly indicative of successful usage of certain fungi strains for biological control of S. nonagrioides larvae both in laboratory conditions and in the field.

Laboratory bioassay was used to determine the pathogenicity of nine isolates of fungi of the genera Metarhizium, Beauveria and Clonostachys to the parasitic mite, Varroa destructor. All nine isolates were pathogenic to V. destructor with Metarhizuim anisopliae UAMH 9198, Clonostachys rosea UAMH 9161 and Beauveria bassiana GHA being the most pathogenic within their respective species. Metarhizium anisopliae UAMH 9198 was more lethal to V. destructor than B. bassiana GHA and C. rosea UAMH 9161 with LC50 values of 1.6 x 10(5), 9.6 x 10(6) and 5.4 x 10(6) conidia/mL, respectively. Metarhizium anisopliae and B. bassiana significantly affected brood and adult honey bee survivorship and their immune responses. They were lethal to the bees with LC50s of 3.70 x 10(6) and 2.62 x 10()5 conidia/mL, respectively. The effect of temperature and thymol on conidia germination, production and colony growth of the fungal isolates was determined. Temperature significantly affected conidia germination, production and colony growth (P < 0.05) but thymol did not. Efficacy of the two most promising isolates (M. anisopliae UAMH 9198 and B. bassiana GHA) as potential bio-control agents against V. destructor in hives was evaluated. Fungal inocula were applied as dry formulation, with corn flour as carrier, using dispenser tray and dusting applications. Treatments were either applied alone or in combination with thymol to determine any synergistic effects. All treatments significantly increased mite mortality (P < 0.05), however, the mite control efficacy varied between fungal treatments and application methods. Combined treatments of fungi and thymol caused significantly higher mite mortality than single fungal treatments, which showed control levels of ≤61%. Significant differences in mite mortality were found between the two delivery methods with the differences depending on the fungal isolate. The results suggest that M. anisopliae UAMH 9198 would be a more effective bio-control agent for the management of V. destructor in honey bee colonies than the other isolates tested when dispensed continuously in hives using delivery methods such as a dispenser tray. However, future research is needed to improve delivery methods and investigate the effect of carriers used in the formulation on the efficacy of such entomopathogenic fungi. / Ontario Ministry of Agriculture and Food (OMAF), University of Guelph

Entomopathogenic fungi, including Beauveria bassiana are being developed as an alternative to chemical insecticides. Their effectiveness can be enhanced through understanding of the mechanisms of response to environmental stresses and conditions. An aspect of repair of ultraviolet radiation induced DNA damage and response to high temperature were studied here. A region of the photolyase gene (phr-1), encoding cyclobutane pyrimidine dimer photolyase (CPD-PHR), an enzyme pivotal to DNA repair, was cloned, sequenced and identified for species of the genera Beauveria, Isaria, Lecanicillium, Metarhizium and Tolypocladium. The DNA and deduced amino acid sequences were analysed using several in silico methods and annotated for functionality. The data suggested that the DNA encoded a protein with conserved residues known in CPD-PHR function, which had structural homology with other CPD-PHRs and molecular phylogeny that was generally consistent amongst this group of fungi. These results are the first for a phr-1 from the genera Isaria, Lecanicillium and Tolypocladium. In bacteria and yeasts, tolerance to environmental stress was shown to be aided through an inducible phenomenon that involves extracellular sensory component (ESC) proteins in Escherichia coli, which have yet to be purified or sequenced. The presence of an ESC-like factor (ELF) was examined in cell-free filtrate (CFF) from B. bassiana cultures. It was revealed that the tolerance of conidiospores and blastospores (BS) to ultraviolet radiation or heat could be increased by preheated CFF, respectively, but not after pretreating the CFF with trypsin. Several novel polyacrylamide-based in situ and binding bioassays were developed to screen for and characterize ELF candidate (EC) proteins. Two were detected (EC1 and EC2) and EC1 was found to interact with BS, while bioassays with purified ECs showed that EC1 could increase heat tolerance in BS. De novo peptide sequencing revealed that the ECs were the same protein, but differed by EC1 being glycosylated. An expressed sequence tag from B. bassiana, encoding six peptides that were also found in the ECs, was identified in the public data base. This sequence information was exploited to amplify the remaining coding regions of the suspected ELF gene (elf) using polymerase chain reactions. Through this a 741 nucleotide open reading frame was cloned and sequenced. Structure-function analyses of the amino acid sequence encoded by the open reading frame revealed features that were consistent with the ECs, such as eight shared peptides, its nascent derived size (26 kDa), potential glycosylation sites and secretion signal peptide. In addition, other features such as the high proportion of cysteine residues and internal amino acid repeats will be discussed. The elf gene was inserted into an expression vector and introduced into the methylotrophic yeast Pichia pastoris for its controlled over-expression. Heterologously expressed ELF conferred elevated tolerance to heat in BS to similar levels produced by ELF synthesized by B. bassiana. Several functional and molecular features of the ELF system have certain commonalities with many agonist-receptor systems involved in signal transduction, but remain to be detailed. This is the first report of the cloning and functional analyses of elf and ELF, respectively, from any organism.

House flies (Musca domestica L.) are common pests affecting horses and their owners. Control of house flies in stable yards is currently based on the use of pesticides. However, the development of resistance by these flies to most pesticide groups has motivated horse owners to seek alternative methods of fly control. An entomopathogenic fungus, Beauveria bassiana (Bb) and an entomopathogenic bacterium, Bacillus thuringiensis var. israelensis (Bti) are two biological agents known to have activity against house flies. The broad objective of this study was to evaluate the effect of these two biological control agents on house flies in an equine environment. Using a structured questionnaire, presented in Chapter 2, thirty horse owners in KwaZulu-Natal were asked about the nuisance value of house flies, their current control measures, the potential market for biocontrol agents against house flies, and each owner’s perception of biocontrol methods. The horse owners were using three methods of house fly control namely, physical, chemical and biological. Most horse owners (97%) wanted access to effective biocontrol agents for control of house flies. Most horse owners (80%) stabled their horses at night, some or all of the time. The resultant manure piles in the stable yard were considered to be the primary cause of house fly problems. About 64% of the horse owners were dissatisfied with the currently available methods of controlling house flies in this situation. Chapter 3 covers two observational trials in which varying doses of Bacillus thuringiensis var. israelensis (Bti) were fed to horses, in order to identify a baseline dosage to give to horses in order to adequately control house fly populations growing in horse manure. The bacterium Bti, grown on wheat bran, was fed to six miniature horses at doses of 0, 0.125, 0.25, 0.5, 0.75, and 1.0 g per meal in Trial 3a, and at 0, 0.5, 1, 2, 4 and 8 g per meal in Trial 3b. Faeces were collected three times a week for 11 weeks and placed in incubation trays to allow the number of emerging adult house flies and closed pupae to be counted. In Trial 3a, there was a significant reduction in the number of closed pupae with an increase in Bti in the feed. The regression equation suggests that there will be 3.1 times as many closed pupae in the faeces when horses are fed 1 g of Bti in their feed, than when horses are fed no Bti. This dosage is the minimum baseline dosage for future trials. v Using manure from horses dosed in Trials 3a and 3b, the survival of the bacterium through the gut of horses was evaluated using a standard isolation technique. The growth of Bt colonies on the manure after the Bt isolation technique showed that some of the bacterial cells survived transition through the digestive tract of the horse. This study was qualitative in nature and did not attempt to quantify the level of Bti spore survival. These two observations suggest that Bacillus thuringiensis var. israelensis has the potential to be used as a biocontrol agent, applied via horse feed, for the control of house flies in stable yards. Future clinical trials, with appropriate replication, should be conducted using 1 g Bti/meal as the lowest test dosage. The objective of Chapter 4 was to determine whether spraying Bti or Bb on to horse manure is effective in the control of house flies. Over a six week period, two spraying trials were conducted in which increasing doses of Bb and Bti were sprayed on to 500 g samples of horse manure. Counts of house fly pupae and adults were taken. The doses of Bb and Bti tested were 0, 1, 2, 4 g in Trial 4a, and 0, 4, 8 and 12 g in Trial 4b. The research reported in Chapter 4 was characterized by the unexpectedly high levels of biological variation in egg, larvae and pupae numbers that were found in samples of horse manure, taken from the same skip two days apart. The statistical design of the two trials conducted was inadequate to cope with the high level of variation about treatment means for fly and larval counts. However, despite the lack of significant differences between treatment means, there is observational evidence that suggests that both Bb and Bti do have an effect on house fly survival. A simplified statistical model, which compared the number of hatched house flies on untreated manure, with the number on manure treated with any level of Bb (1 to 4 g /250 ml water), found a significant reduction in the number of hatched flies on treated manure. There was no significant corresponding reduction in the number of closed pupae, which suggests that Bb acts primarily before the larva pupates. The optimal dose of Bb and Bti to be sprayed on to manure could not be determined because of the high variation about treatment means. It is suggested that, in future trials similar dosages for Bb could be tested, but that higher dosages of Bti (starting at 2 g/250 ml water) should be used. Trial periods should be extended and replication increased dramatically to reduce variation about treatment means. Transformation of data before analysis may also be necessary to equalize variation about treatment means. / M.Sc.Agric. University of KwaZulu-Natal, Pietermaritzburg 2013.

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xv, 126 p.; also includes graphics (some col.). Includes bibliographical references (p. 96-107). Available online via OhioLINK's ETD Center