A broca da cana-de-açúcar, Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae) é naturalmente infectada por diversos patógenos, sendo os mais importantes Metarhizium anisopliae (Metsch.) (Ascomycota: Clavicipitaceae), Beauveria bassiana (Bals.) (Ascomycota: Cordycipitaceae) e vírus da granulose. Esses microrganismos podem co-infectar um mesmo indivíduo em condições de campo, mas os resultados das interações desses patógenos não são conhecidos. O presente trabalho objetivou determinar a virulência desses patógenos aplicados de forma isolada e em infecções mistas na fase larval de D. saccharalis em condições de laboratório sobre dieta artificial e em plantas de milho. A maioria das combinações resultou em efeito aditivo na mortalidade dos insetos (M. anisopliae + B. bassiana, M. anisopliae + DsGV e M. anisopliae + B. bassiana + DsGV), entretanto ficou evidenciado antagonismo entre B. bassiana e o granulovírus. Todos os cadáveres oriundos das aplicações associadas apresentaram sintomas de apenas um dos patógenos envolvidos na infecção. A produção de conídios de uma espécie de fungo nos cadáveres submetidos à co-infecção foi semelhante à produção de conídios da mesma espécie na infecção isolada. Na infecção mista, os dois fungos se desenvolveram na hemolinfa do hospedeiro, entretanto em um estágio tardio do processo infectivo ocorreu a exclusão de um dos patógenos. A infecção por B. bassiana diminuiu drasticamente a densidade de hemócitos circulantes na hemolinfa das lagartas, efeito não observado para M. anisopliae. Duas populações de laboratório de D. saccharalis, uma oriunda de Piracicaba-SP e outra de Araras-SP, foram testadas quanto à suscetibilidade aos fungos M. anisopliae e B. bassiana. A população de insetos de Piracicaba foi menos suscetível em 2009 do que a mesma população em 2008 e também em relação à população de Araras. Dessa forma, foi realizado um bioensaio comparando a suscetibilidade de insetos das duas populações e da progênie (F1) dos cruzamentos diretos entre elas, criadas nas mesmas condições bióticas e abióticas, para determinar se a suscetibilidade diferencial estaria relacionada a fatores genéticos. Não foram observadas diferenças significativas na suscetibilidade das duas populações e dos cruzamentos aos fungos. A suscetibilidade diferencial observada nos dois primeiros bioensaios provavelmente esteja relacionada às variações no sistema de criação, em componentes nutricionais e antimicrobianos da dieta e não a variações entre as populações dos hospedeiros. / The sugarcane borer, Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae) is naturally infected by various pathogens, the most important are Metarhizium anisopliae (Metsch) (Ascomycota: Clavicipitaceae), Beauveria bassiana (Bals.) (Ascomycota: Cordycipitaceae) and granulovirus. These microorganisms may coinfect the same host under field conditions, but the result of these pathogens interactions is not known. This study investigated the virulence of these pathogens applied separately and together on larvae of D. saccharalis in the laboratory on artificial diet and on corn plants. Most combinations resulted in additive effect on insect mortalities (M. anisopliae + B. bassiana, M. anisopliae + DsGV and M. anisopliae + B. bassiana + DsGV), however it became apparent antagonism between B. bassiana and the granulovírus. In all cadavers resulted from mixed applications, only one pathogen sporulated or externalized their symptoms in the host. Production of conidia of one fungal species on the cadavers subjected to co-infection was similar to the production of conidia of the same species in a single infection. In mixed infection, the two fungi have developed in the hemolymph of the host, and the exclusion of one pathogen occurred at a later stage of the infective process. Infection with B. bassiana has drastically reduced the density of circulating hemocytes in the hemolymph of the larvae, which was not observed for M. anisopliae. Two laboratory populations of D. saccharalis, one coming from Piracicaba-SP and other from Araras-SP, were tested for susceptibility to M. anisopliae and B. bassiana. The insect population from Piracicaba was less susceptible to application of fungi in 2009 compared to the same population in 2008 and compared to the Araras population. Therefore, a bioassay was performed comparing the susceptibility of the two populations and the progeny (F1) of direct crossings between them, using insects reared in the same conditions. This assay was performed to determine if the differential susceptibility would be related to genetic factors. No significant differences were observed in the susceptibility of insect populations and crossings to both fungi. The differential susceptibility observed in the first two assays was probably related to variations in the rearing system and nutritional and antimicrobial components in the diet and not due to variations among host populations.

Made available in DSpace on 2016-04-28T20:17:23Z (GMT). No. of bitstreams: 1 mestrado-Alzimiro Marcelo Conteiro Castilho.pdf: 399125 bytes, checksum: f42c772f226470430fe65c4d260420dd (MD5) Previous issue date: 2005-07-29 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / This survey was carried out under laboratory and field conditions for evaluating the efficiency of entomopathogenic fungi to control the ants Atta sexdens rubropilosa and Atta bisphaerica. The isolates from Agronomy National school (Escola Nacional de Agronomia) ENA1, ENA2, ENA3 and ENA4 of Metarhizium anisopliae as well as the isolates ENA6, ENA7, ENA13 and ENA14 of Beauveria bassiana were tested and inoculated on soldiers ants. After to have been collected, the soldiers were separated by groups of 10 individuals and wetted on a conidia suspension of 1.0 x 106 to 1.0 x 1011 conidia/ml .Each ants group was placed on a wet chamber and kept under no feeding condition at 27 ? 1 0C. Both fungi were pathogenic , although the isolates ENA 4 of Metarhizium anisopliale and ENA 6 and ENA 13 of Beauveria bassiana were considered the most virulent ones for A. bisphaerica, and for A. sexdens rubropilosa the isolates ENA 4 of Metarhizium anisopliae and ENA 14, ENA 13 and ENA 6 of Beauveria bassiana among the tested ones as well .The greatest mortality percentage was performed by these isolates with Lethal time (LT50) of 1.15 days for the isolate ENA 4, 1.39 days for ENA 6 and 1.44 days for ENA 13 ,for A. bisphaerica soldiers. For A. sexdens rubropilosa the Lethal time (LT50) was 1.37 days for the isolate ENA 4, 1.68 days for ENA 14, 1.95 days for ENA 13 and 2.14 days for ENA 6. In regard to the mortality index, the percentage of dead soldiers at the tested concentrations ranged from 45% to 71,67% . The isolates ENA 4, ENA 14, ENA 13, and ENA 6 caused the greater soldiers mortality averages in a shorter time period. The isolates ENA 4 and ENA 14 were applied by pulverization and spraying over plus 3 years old adult ants nests and 30 days after the entomopathogenic fungi application there still was some activity on the underground galleries. / Este trabalho foi conduzido em condi??es de laborat?rio e campo para se observar a efici?ncia de fungos entomopatog?nicos no controle de Atta sexdens rubropilosa e Atta bisphaerica. Foram utilizados os isolados ENA 1 (Escola Nacional de Agronomia) ENA 2, ENA 3 e ENA 4 de Metarhizium anisopliae e Beauveria bassiana, ENA 6, ENA 7, ENA 13 e ENA 14, inoculados em soldados. Ap?s coleta, exemplares de soldados foram separados em grupos de 10 indiv?duos e banhados em suspens?es de con?dios de 1,0 x 106 a 1,0 x 1011 con?dios.mL-1. Cada grupo de formiga foi transferido para c?mara ?mida e mantido sob regime de fome a 27 ? 1? C. Os dois fungos foram patog?nicos, embora os isolados ENA 4 de M. anisopliae e ENA 6 e ENA 13 de B. bassiana tenham sido os mais virulentos para A. bisphaerica e para A. sexdens rubropilosa dentre os testado. Esses isolados foram aqueles que apresentaram maior porcentagem de mortalidade confirmada, com TL50 de 1,15 dias para o isolado ENA 4, 1,39 dias para o ENA 6 e 1,44 dias para o ENA 13, para soldados de A. bisphaerica. Para A. sexdens rubropilosa a TL50 foi de 1,37 dias para o isolado ENA 4, 1,68 dias para o isolado ENA 14, 1,95 dias para o ENA 13 e 2,14 dias para o ENA 6. Quanto ao par?metro mortalidade, as concentra??es testadas proporcionaram a porcentagem de soldados mortos que variou de 45,0 a 71,67 %. Os isolados ENA 4, ENA14, ENA 13 e ENA 6 causaram as maiores m?dias de mortalidade de soldados em menor espa?o de tempo. Os isolados ENA 4 e ENA 14 foram aplicados na forma de pulveriza??o e polvilhamento, em formigueiros adultos com mais de tr?s anos de idade, e ap?s 30 dias da aplica??o dos fungos entomopatog?nicos, os olheiros ainda apresentavam ativos.

O objetivo da pesquisa foi selecionar isolados de Metarhizium anisopliae patogênicos para cigarrinha-da-raiz, Mahanarva fimbriolata, e caracterizá-los morfológica e geneticamente, por meio de técnicas de análise de DNA (RAPD). A seleção foi feita em laboratório, utilizando ninfas coletadas a campo que foram pulverizadas com o fungo e mantidas nas raízes de mudas de cana-de-açúcar. A mortalidade corrigida, ao quinto dia após a inoculação, variou de 10,5 a 60%. Verificou-se que todos os isolados apresentaram coloração das colônias variando de verde acinzentado ao verde escuro, com crescimento de 28mm para o isolado IBCB-353 à 38mm para o isolado IBCB-348. O comprimento dos conídios não teve influência na patogenicidade e variou de 5,465µm para o isolado IBCB-345 a 7,970µm para o isolado ESALQ 1301 sendo que todos pertencem à subespécie anisopliae. Constatou-se a presença de RNA de fita dupla em onze dos vinte isolados, mas não houve relação entre a presença desta banda e sua patogenicidade para M. fimbriolata. Foi possível separar os isolados em dois grupos (A e B) com 72,5% de similaridade, sendo observada a composição de dois subgrupos (B1 e B2), com 77,5% de similaridade, dentro do grupo B. A alta similaridade entre os dois grupos e dentro de cada um deles, indicou que os isolados pertencem à mesma subespécie, reforçando o que foi concluído com a caracterização morfológica. O método utilizado confirma a grande diversidade genética da espécie M. anisopliae, porém, não reflete sua similaridade de patogenicidade a ninfas de M. fimbriolata, pois os dois isolados mais patogênicos (IBCB-384 e ICBC-348) foram dispostos em grupos diferentes. Não se observou um padrão específico de agrupamento entre isolados oriundos da mesma região ou hospedeiro, ou seja, a diversidade genética parece ser independente do local de origem do fungo, como seria esperado com um patógeno que, em geral, tem revelado alto grau de especialização ao hospedeiro. / This research was carried out to evaluate the pathogenicity of isolates of the entomopatogenic fungus Metarhizium anisopliae against the spittlebug, Mahanarva fimbriolata, and to characterize them morphologically and genetically through RAPD method. The selection was made under laboratory condition, using nymphs collected at field. The fungus was sprayed on the nymphs by a Potter tower (15 pounds/pol2) and then, they were maintained in roots of sugar-cane seedlings. The mortality was evaluated 5 days after the inoculation, ranging from 10.5 to 60%. The colonies’ color varied from grayish green to dark green and the colonies diameter ranged from 28mm (isolate IBCB-353) to 38mm (isolate IBCB-348). The conidia length ranged from 5.465µm for the isolate IBCB-345 to 7.970µm for the isolate ESALQ 1301. With those results it could be concluded that all the studied strains belong to the subspecies anisopliae. The size did not have influence in the pathogenicity of the isolate. It was evidenced presence of double-stranded RNA (virus) in eleven out of the twenty isolates tested, but it did not have relation between the presence of the virus and its pathogenicity for M. fimbriolata. It was possible to separate isolates in two groups (A and B) with 72.5% of similarity, being observed the composition of two sub-groups (B1 and B2), with 77.5% of similarity, inside of group B. The high similarity between the two groups and inside of each one indicated that the isolates belong to the same subspecies, confirming what it was concluded with the morphologic characterization. The used method confirms the great genetic diversity of species M. anisopliae, however, does not reflect its similarity of pathogenicity the nymphs of M. fimbriolata, therefore the two isolated most pathogenic (IBCB-384 and ICBC-348) were located in different fenetic groups. In the present study a specific standard of grouping between isolated deriving of the same region or host was not observed, or either, the genetic diversity seems to be independent on the fungus origin, as it would be expected from a pathogen that, in general, shows high degree of specialization to the host.

In der vorliegenden Arbeit wurden anhand von Labor- und praxisnahen Flugversuchen die Möglichkeiten der biologischen Bekämpfung der Dörrobstmotte, Plodia interpunctella HÜBNER, und der Mehlmotte, Ephestia kuehniella (ZELLER), mit entomopathogenen Pilzen der Ordnung Hyphomycetales untersucht. Getestet wurden Pilzstämme von Metarhizium anisopliae, Beauveria bassiana, Paecilomyces farinosus und Paecilomyces fumosoroseus. Hauptgegenstand der experimentellen Arbeit war die Untersuchung verschiedener Einflußfaktoren auf die Wirksamkeit der Pilze unter Lagerbedingungen unter besonderer Berücksichtigung des Auftretens von Wechselwirkungen zwischen Erreger, Wirtsinsekt und Umwelt. Die Stammeigenschaften der getesteten Pilze wurden neben den herrschenden Umweltbedingungen als ein Hauptfaktor identifiziert, der die Wirksamkeit des Einsatzes entomopathogener Pilze gegen vorratsschädliche Motten entscheidend beeinflußt. Darüber hinaus sind der Ernährungszustand der Pilze, die Anfälligkeit der Wirtsinsekten sowie die Inokulumdichte verantwortlich für das Zustandekommen bedeutsamer Bekämpfungseffekte. Für eine praktische Nutzung wurden als mögliche Anwendungstechnologien die Kontamination von Verpackungen, die Behandlung von Fraß- bzw. Brutsubstrat unter Ausnutzung deren Lockwirkung sowie die Verwendung pheromonbeköderter Kontaminationsherde untersucht. Herausgestellt werden konnte, daß für nachhaltige kurative Bekämpfungserfolge die Dichte von P. interpunctella im Lager gering sein muß. Die Verwendung von pheromonbeköderten Kontaminatinsherden führte bei über 50% der Weibchen und etwa 80% der Männchen von P. interpunctella zur Inokulation und kann somit die Effizienz entomopathogener Pilze im Vorratsschutz erhöhen. / Strains of the entomopathogenic fungi Metarhizium anisopliae, Beauveria bassiana, Paecilomyces farinosus and Paecilomyces fumosoroseus were tested against the stored product infesting moths Plodia interpunctella HÜBNER, and Ephestia kuehniella (ZELLER). The potential of the strains was assessed by conducting experiments investigating the different factors influencing their effectiveness under stored conditions. The quality of virulence and the environmental conditions are the mean factors for a successful use of entomopathogenic fungi. Furthermore, the nutritional state of the fungi, the susceptibility of the moths and the density of inoculum influence the effect of a fungi treatment. The contamination of packing, the treatment of food and the use of contaminated TDA pheromone places was assessed for a practical method. The control of P. interpunctella is successful with slow population density of moths. The inoculation of Plodia interpunctella males and females showed that the combined action of pheromone and fungi spores could serve as potential method in the biological control of stored product infesting moths.

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.

O percevejo-bronzeado do eucalipto, Thaumastocoris peregrinus, é um inseto fitófago que ocasiona o amarelecimento das folhas de Eucalyptus e, em casos severos, a desfolha e morte das plantas. Buscam-se formas de controle natural e/ou biológico para o controle deste inseto. Nesta perspectiva, o controle biológico vem ganhando espaço e o emprego de fungos entomopatogênicos torna-se viável, uma vez que já ocorre naturalmente no ambiente. Nesse sentido, objetivou-se avaliar a ação do fungo entomopatogênico B. bassiana, isolado IBCB 66 sobre diferentes fases de desenvolvimento de T. peregrinus, em condições de laboratório. Os tratamentos foram: Água destilada esterilizada (ADE), Água destilada esterilizada + Tween® 80 (0,01%) (ADET) e B. bassiana isolado IBCB 66 (1,0 x 108 conídios. mL-1). Esses tratamentos foram avaliados em três bioensaios em delineamento inteiramente casualizado. 1- Ação de B. bassiana sobre ovos de T. peregrinus: Folhas de Eucalyptus benthamii foram recortadas em discos de 2,4cm Ø, alocados no interior de tubos de vidro chato (2,5cm Ø e 10 cm h). Cartelas contendo em média 10 ovos de T. peregrinus foram imersas na solução correspondente a cada tratamento e colocadas sobre os discos foliares, totalizando 20 repetições/tratamento, sendo mantidos em câmara climatizada BOD (27±2° C, 14 h de fotofase e UR de 70 ± 10%) (procedimento padrão para todos os bioensaios). Foram avaliados o período de incubação e a viabilidade dos ovos de T. peregrinus. 2- Ação de B. bassiana sobre ninfas de T. peregrinus: Discos de E. benthamii de 2,4cm Ø, previamente tratados foram alocados no interior de tubos de vidro que recebeu, na sequência, uma ninfa de 3° ínstar de T. peregrinus, totalizando 50 tubos/repetições por tratamento. Neste bioensaio foram avaliadas a duração e a viabilidade dos ínstares ninfais. 3- Ação de B. bassiana sobre adultos de T. peregrinus: este bioensaio foi realizado conforme metodologia descrita para ninfas. Cada tubo de vidro recebeu um adulto de T. peregrinus, totalizando 50 tubos/repetições por tratamento. Neste bioensaio foi avaliada a longevidade diariamente, até a mortalidade total dos insetos. Alguns exemplares de adultos mortos que entraram em contato com B. bassiana foram coletados, para análise histológica, conforme metodologia padrão. O fungo B. bassiana não interferiu no período de incubação e viabilidade de ovos de T. peregrinus. O mesmo foi observado sobre ninfas de 3° ínstar e adultos, que não foram afetados pelo fungo. A duração média e a viabilidade foram reduzidas em ninfas de 4° e 5° ínstar, demonstrando que o fungo B. bassiana é patogênico nessa fase de desenvolvimento. Na análise histológica de adultos de T. peregrinus verificou-se a presença de hifas, conídios e conidióforos nos tecidos do sistema digestivo e muscular. / The bronze bug of eucalyptus, Thaumastocoris peregrinus, is a phytophagous insect that causes yellowness of leaves of Eucalyptus, and in severe cases, death, and defoliation of plants. Forms of natural control and/or biological control for this insect are aimed. In this perspective, biological control has been increasing and the use of entomopathogenic fungi is feasible, as already occurs naturally in the environment. In this sense, it aimed to evaluate the action of entomopathogenic fungus B. bassiana, isolated IBCB 66 on different developmental stages of T. peregrinus in laboratory conditions. The treatments were: sterilized distilled water (SDW), Distilled water Tween 80 (0.01%) (DWT) and B. bassiana IBCB 66 (1.0 x 108 conidia mL-1). These treatments were evaluated in three bioassays in experimental design. 1: B. bassiana action on eggs of T. peregrinus: Eucalyptus benthamii leaves were cut into disks of 2.4 cm Ø, allocated within flat glass tubes (2.5 cm Ø and 10 cm h). Cards containing on average 10 eggs of T. peregrinus were immersed in the solution corresponding to each treatment and placed on leaf discs a total of 20 replications / treatment, and kept in a climatized chamber BOD (27 ± 2 ° C, 14 h photoperiod and RH 70 ± 10%) (standard procedure for all bioassays). They evaluated the incubation period and the viability of T. peregrinus eggs. 2: B. bassiana action on nymphs of T. peregrinus: E. benthamii discs of 2.4 cm Ø, pretreated were placed inside glass tubes that received following a nymph 3° instar T. peregrinus totaling 50 tubes / replications. In this bioassay were evaluated duration and viability of the nymphal instars. 3: B. bassiana action on adults of T. peregrinus: This bioassay was performed as described methodology for nymphs. Each glass tube received an adult T. peregrinus, totaling 50 tubes / replications per treatment. In this bioassay was assessed daily longevity to the total mortality of insects. Some adult specimens dead who came into contact with B. bassiana were collected for histologic analysis as standard methodology. B. bassiana did not affect the incubation period and viability of eggs of T. peregrinus. The same was observed on nymphs of 3° instar and adults who have not had the studied biological parameters affected by the fungus. The average duration and viability were reduced by 4 ° to 5 ° instar nymphs , indicating that the pathogen is B. bassiana this development phase. Histological examination of the adults of T. peregrinus verified the presence of hyphae, conidia and conidiophores in the digestive system and muscle tissues.

Control of the citrus pest, false codling moth (FCM), Thaumatotibia leucotreta Meyrick (Lepidoptera: Tortricidae) is crucial for the South African citrus industry. The economic losses and phytosanitary status of this pest, coupled with increased consumer awareness and demands, has created a need for effective, IPM-compatible control measures for use against the soil-dwelling life stages of FCM. Promising developments in the field of microbial control through the use of entomopathogenic fungi (EPF) and entomopathogenic nematodes (EPNs) have highlighted the need for research regarding pupation biology, behaviour and survival of FCM, as a good understanding of biology of the target organism is an important component of any biological control programme. The aim of this study was to improve the current understanding of FCM pupation habits through the manipulation of soil texture class, ground cover, shading, soil compaction, air temperature, and soil moisture in the laboratory. These findings would then be used to aid the biological control programmes using EPF and EPNs against FCM in the soil. Three soil texture classes (sandy loam, silt loam and silty clay loam) were obtained from orchards for use in the study. FCM larvae were allowed to drop into the soil of their own accord and the pupation behaviour that followed was then captured on film with pupae formed in the soil being kept in order to measure adult eclosion. In general, very few abiotic factors had a clear influence on FCM pupation. Larval wandering time and distance was short, but also variable between individuals. Distance did increase when soils were moist. Pupation depth was shallow, with pupal cocoons generally being formed on the soil surface. Depth of pupation was less than one centimetre for all abiotic conditions, with little burrowing into soil. Eclosion success was higher for sandier soils when these were dry and uncompacted, but the addition of both moisture and soil compaction increased FCM eclosion success. FCM was sensitive to desiccation when the soils were dry and temperature limits of 15 °C and 32 °C had a strongly negative impact on eclosion success. Preferences for particular abiotic conditions were limited to only certain moisture conditions when interacting with soil texture class and a preference for pupating in soil when it is available. Limited preference was found for particular soil textures despite this having a strong influence on eclosion success, but individuals did appear to pupate in close proximity to one another. Viable direct habitat manipulation for FCM control could not be identified. These results and all of the abiotic variables measured have important implications for EPF and EPN application, survival and persistence in the soil in order to improve the ability of these biological control agents to control FCM. These are discussed in each chapter.

My diploma thesis deals with genetic variability of entomopathogenic fungi of the Isaria genus encountered in the Czech Republic. Individual representative of the genus can be found in soil where they attack all developmental stages of insects, giving preference to larvae and pupae. The Isaria fungi find application first and foremost where plants have to be provided biological protection. In case of mitosporic fungi is the precise identification very difficult, taxonomy is often unclear in many genera, including the genus Paecilomyces/Isaria to demonstrate their polyphyletic nature. The fungi are classified primarily with reliance on morphological studies. The most common markers used to identify fungi are the shapes and sizes of their conidia and the biological properties (germination of spores, tests of biological efficiency). Identification made in consideration of the morphological markers is inaccurate and very variable. To overcome those accuracies, there are very useful molecular DNA markers, which can be relevant in ecology, biology and in fungi genetics. This paper relies on applying the ITS region (Internal Transcribed Spacer) as a molecular marker. ITS regions are partial constituent rDNA carrying no code - that is why the regions are likely to accumulate evolutionary changes in the DNA sequence, which makes them suitable for extensive use in taxonomic analyses of many organisms. The study results in a phylogenetic trees constructed by comparing different sequences of ITS regions obtained from the samples of entomopathogenic fungi of the Isaria genus gathered in the Czech Republic during the monitoring stage 2013 to 2014. Thereunder detection of Isaria sp. occurring in the Czech Republic.

This diploma theses focuses on comparison of natural and intentionally inducated supressiveness of environment induced by application of entomopathogenic fungi Beauveria bassiana, Metarhizium anisopliae, Isaria fumosorosea a Lecanicillium muscarium. In tests were evaluated the in vitro parameters as well as the effectiveness in vivo biotests on insect host larva Tenebrio molitor. The species of entomopatoghenic fungi were applied in suspensions, single strains and also in combination of two strains. In the in vitro conditions the possibilities of objective evaluation of the supresivity level were tested by using the CFU test (Colony Forming Units) on three different nutrient media (PDA, PDA + A , PDA + D), as one of the basic evaluation parameters. Further the germination tests were evaluated according to GI (Germination Index), determination of radial growth (comparison of median cultures) and interaction of strain suspensions on nutrient media PDA. In the in vivo biotests were watched the epizooties from suspensions of these entomopathogenic fungi on insect larva Tenebrio molitor in competitive test of strains according to FDI (Fungl development index) evaluation scala. Chosen larva covered by fully sporulating mycelium from epizootie were further evaluated in CFU test. The result were dominant strain/s on the larva from applied suspensions.

In laboratory bioassays, the efficacy of the entomopathogenic fungus Beauveria bassiana against the yellow mealworm (Tenebrio molitor) was tested under various temperature conditions. Six different strains of fungus B. bassiana was investigated. The evaluation was based on vitality bioassays including germination and growth index assessment and the bioassay of virulence based on target organism T. molitor was also assessed growth and yield of conidia different strains of fungus B. bassiana on natural substrates and artificial nutrient substrates.