Dissertations / Theses on the topic 'Bacillus thuringiensis kurstaki'

The work presented in this thesis involves the characterization of a small plasmid, pHD2, of approximately 2 kb in size from Bacillus thuringiansis subspecies kurstaki HDl-Dipel. The plasmid was cloned and sequenced and compared to other plasmids from Gram-positive bacteria for which sequence information was available and for which replication functions had been assigned. Homology between the predicted amino acid sequence of an open reading frame within pHD2 and the rep gene products of the pT181 group of staphylococcal plasmids suggested a common method of plasmid replication. The further identification of possible plus and minus origins indicated that pHD2 was a member of a family of plasmids replicating via a single-stranded DNA intermediate. Plasmid replication control in the staphylococcal plasmids pT181, pC221 and pS194 involves a negative control circuit using countertranscripts. Replication control in the case of pHD2 may utilize an alternative system involving the gene product of a second small open reading frame with homology to RepA of pLSl in which replication control is achieved by the binding of the repA gene product to the promoter region of the replication protein repB. pHD2 is the first plasmid from a Bacillus thuringiensis isolate to which replication functions have been assigned. The work presented here suggests pHD2 to be a member of the ssDNA family of plasmids and extends the range of such plasmids which have been characterized with the suggestion that this group contains, in addition to the highly related staphylococcal plasmids, a number of more distantly related members. Additionally, chimeric plasmids containing pHD2 and pBR322 have been demonstrated to show structural instability, although not segregational instability, in an alternative Bacillus thuringiensis host. Consequently, the use of such constructs in the cloning of heterologous genes in Bacillus thuringiensis in such a system may prove impractical at this stage with further work being required in order to overcome these problems and extend the exploitation of this industrially important family of entomopathogenic bacteria.

Thesis (M.S.)--West Virginia University, 2004.
Title from document title page. Document formatted into pages; contains viii, 40 p. : ill. (some col.), map. Vita. Includes abstract. Includes bibliographical references (p. 36-39).

Thesis (M.S.)--West Virginia University, 2001.
Title from document title page. Document formatted into pages; contains x, 87 p. : ill. (some col.), map. Vita. Includes abstract. Includes bibliographical references (p. 76-82).

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Conselho Nacional de Desenvolvimento Científico e Tecnológico
The objective of this research was evaluation "in vitro" the effect of fungicides and herbicides on Bacillus thuringiensis var. kurstaki (Btk). The five fungicides: epoxiconazole, fentin hydroxide, azoxystrobin, tebuconazole and carbendazim, and five herbicides: sulfentrazone, lactofen, imazethapyr, fenoxaprop and metribuzin were tested at two concentrations, on cells and spores, in nutrient agar (NA) - solid medium (SM), and in nutrient broth (NB) - liquid medium (LM), in addition to their metabolization (MT) by Btk. For cells, the phytosanitary products in NA were tested incorporated into the medium and on filter paper disks impregnated with the products. The evaluation consisted, respectively, of the number of CFU/mL and of the formation of an inhibition halo. For cells, the Bt+products+NB mixture was incubated in Erlenmeyer flasks (30± 2°C, 150rpm, 24h)an then the NB was diluted and inoculated onto NA; the number of CFU/mL was evaluated. The MT and spore tests were performed in liquid medium (water for MT and NB for spores). The effects of the products varied with the category (fungicide and herbicide), their concentrations, technique used, contact time, and developmental stage of the pathogen. The LM test is the most indicated to evaluate the effect of the products on cells. It was observed that most fungicides were compatible with cells, and only epoxiconazole and azoxystrobin were metabolized. The herbicides, independents of the concentrations, were incompatible with cells, and only sulfentrazone and imazethapyr were metabolized. Fentin hydroxide was the only product incompatible with spore
Objetivou-se comparar técnicas para a avaliação in vitro do efeito de fungicidas e herbicidas sobre Bacillus thuringiensis var. kurstaki (Btk). Foram testados cinco fungicidas: epoxiconazole, fentin hidróxido, azoxystrobin, tebuconazole e carbendazin, e cinco herbicidas: sulfentrazone, lactofen, imazethapyr, fenoxaprop e metribuzin, em duas concentrações, sobre células e esporos, em ágar nutriente (AN) - sólido (MS) e caldo nutriente (CN) - líquido (ML), além da metabolização (MT) destes por Btk. Para células, em AN os fungicidas foram testados incorporados ao meio e em discos de papel-filtro impregnados nos produtos. Avaliou-se, respectivamente, as UFC/mL e a formação de halo de inibição. Para células, a mistura Bt+produtos+CN foi incubada em erlemmeyers (30± 2°C, 150rpm, 24h), e em seguida, o meio CN foi diluído e inoculado em AN, avaliando-se o número de UFC/mL. Os testes de MT e esporos foram realizados em líquido (água para MT e CN para esporos). O efeito dos produtos variou conforme a categoria destes (fungicida e herbicida), concentrações, técnica, tempo de contato e fase/estágio do desenvolvimento do patógeno. O teste ML é o mais indicado para a avaliação do efeito em células, sendo observado que a maioria dos fungicidas mostrou-se compatível às mesmas, e apenas epoxiconazole e azoxystrobin foram metabolizados. Os herbicidas, independentes das concentrações, foram incompatíveis com as células, e somente sulfentrazone e imazethapyr foram metabolizados. Fentin hidróxido foi o único produto incompatível com esporos

Orientador: Mohamed Habib
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O conceito de Manejo Integrado de Pragas (MIP) busca a utilização consciente de métodos de controle ambientalmente seguros e economicamente viáveis. Neste contexto, é de extrema importância o conhecimento das interações entre os agentes de controle a serem empregados, para garantir maior eficiência na implantação de tais programas. Tanto as microondas (300 a 300.000 MHz), como o Bacillus thuringiensis varo kurstaki e o ectoparasitóide Bracon hebetor (Say, 1836) vêm sendo estudados isoladamente como promissores agentes de controle de pragas em produtos armazenados. Este trabalho teve por objetivo analisar as respostas e relações do parasitóide Bracon hebetor, do seu hospedeiro natural, o piralídeo Anagasta kühniella Zeller (1879) e do entomopatógeno, B. thuringiensis varo kurstaki (Sorotipo H­3a:3b) à radiação de Microondas (2.450 MHz). o material entomológico foi coletado em moinho de farinha em Campinas, SP, e utilizado para dar início a criações laboratoriais . As condições adotadas neste trabalho foram de 25 ::!: 2 o C, 60 ::!: 10 % de umidade relativa (UR) e 12 horas de fotoperíodo. Para os biensaios de avaliação da sensibilidade de Bacillus thuringiensis varo Iwrstaki a microondas foram utilizadas amostras do produto Dipel (Abbot @), contendo o complexo esporo - cristal desta bactéria, 16.000 UII mg de virulência, formulação em pó molhável. Todos os bioensaios, envolvendo a utilização de radiação, foram realizados sob a potência máxima de um forno microondas Panasonic NN 7954 8H I K, operando na freqüência de 2.450 MHz. Tanto o piralídeo, como como o parasitóide, apresentaram uma relação positiva entre o aumento do tempo de exposição e a mortalidade, independentemente do estágio de vida avaliado. No caso de esporos de B. thur;ng;ens;s irradiados, esta relação foi visualizada no bioensaio realizado na concentração de 0,2%, a qual é próxima à CLso das larvas de terceiro estádio. Quanto à sensibilidade das diferentes fases de desenvolvimento de Anagasta kühn;ella à microondas observou-se a existência de um gradiente de sensibilidade entre os estágios de desenvolvimento do piralídeo : larvas de último estádio (TELso = 15,63 s) > larvas de terceiro estádio (TELso = 19,34 s) > adultos (TELso = 22,61 s) > ovos (TELso = 121,67 s), provavelmente decorrente de diferenças físico- químicas. Encontrou-se diferenças significativas quanto ao impacto de microondas nos diferentes estágios de desenvolvimento dos ovos de Anagasta kühn;ella (1 e 4 dias de idade) quando usado tempos de exposição subletais (10 e 30 segundos). Os ovos de 4 dias demonstraram ser mais sensíveis à radiação que os de 1 dia de idade. Já no caso das exposições letais (45, 60 e 90 segundos) não houve indício de diferenças nas duas etapas de desenvolvimento embrionário. As larvas que eclodiram dos ovos tratados com radiação não portavam nenhum tipo de alteração morfológica. Quanto à interferência do meio no impacto causado em larvas de último estádio por exposição à radiação não se constatou diferença significativa entre as larvas expostas diretamente (TELso = 15,63 s) e em meio de farinha de trigo integral (TELso = 16,28 s). A sintomatologia apresentada após o tratamento com radiação variou em função do tempo de exposição. Observou-se desde letargia, desfunção do sistema locomotor até dessecamento, escurecimento do tegumento e morte. A análise da influência da radiação na capacidade reprodutiva e na longevidade de adultos do piralídeo sobreviventes à exposição indicou que as microondas provocam alterações negativas em ambos os casos. Entretanto, a radiação não influiu na viabilidade dos ovos colocados pelas fêmeas tratadas. Os adultos de Bracon hebetor demonstraram um nível de tolerância à radiação de microondas bem maior (TELso = 106,57 s) que as larvas (TELso = 15,63 s) e adultos (TELso = 22,61 s) do seu piralídeo hospedeiro, Anagasta kühniella, enquanto as larvas do braconídeo (TELso = 20,31 s) aparentemente apresentam o mesmo nível de sensibilidade do píralídeo praga. A exposição do produto à base de Bacillus thuringiensis varo kurstaki à radiação de microondas, nas concentrações letais (2,6%) e sub-Ietais (0,02%) não influiu no seu potencial patogênico. Entretanto, na concentração próxima à letal mediana (CLso) observou-se que o aumento do tempo de exposição à radiação diminuiu a patogenicidade da bactéria. Sendo assim, a radiação de microondas apresentou-se como um método de controle bastante promissor para Anagasta kühniella criada em farinha de trigo integral. Pode ser empregado em ecossistemas de armazemagem deste produto em associação ao ectoparasitóide Bracon hebetor e ao Bacillus thuringiensis varo kurstaki
Abstract: The application of Integrated Pest Management (IPM) means the utilization of efficient control methods which are ecologically safe and economicaly viable. Therefore, it is extremaly important to gather more information about the possible interactions between the different control agents, aimimg to optimize such a method. Microwaves (300 a 300.000 MHz), as well as Bacillus thuringiensis and the ectoparasitoid Bracon hebetor (Say, 1836) are being studied individually by some institutions, as promissing control agents of some stored product pests. The present work was developed to study and evaluate the relations among the parasite Bracon hebetor, his natural host, Anagasta kühniella Zeller (1879), the entomopathogen Bacillus thuringiensis' varo kurstaki (H- 3a:3b) and microwaves radiation. The mother culture of the pyralid and braconid species were initiated from live material collected from flour mills in Campinas municipality, state of São Paulo. The cultures were maintained and experiments were realized under controlled laboratory condítions of 25 :t 2° C, 60 :t 10 % R.H. and 12 hours of photoperiod. To evaluate the tolerance of B. thuringiensis to microwave radiation samples of the commercial product Dipel (Abbot) were utilized. This product contains 16,000 IUI mg and formulated as a wettable powder. The radiation treatments were undertaken utilizing the maximum potency of a domestic Panasonic microwave oven (NN 7954 BH/K, 2450 MHz). The mortality rate among the different developmental stages of the two insect species was observed to be positively related with the radiation exposure time. Among the Anagasta kühniella developmental stages, while the last instar larvae showed to be more sensitive to radiation (median letal exposure time, LET 50 = 15,63 s), the egg stage showed to be more resistant (LETso= 121,67 s). The third instar larvae, as well the adult stage, revealed a moderate sensitivity (LETso= 19,34 s and 22,61 s, respectively). Within the egg stage of Anagasta kühniella the full grow embryo showed to be more sensitive than newly deposited eggs. The impact of the direct exposition to microwaves on the full grow Anagasta kühniella larvae (LET so= 15,63 s) did not differ when compared with those larvae imbeded in their natural diet, whole wheat flour (LETso = 16,28 s). The sequence and intensity of the external symptoms, showed to be directly related to the duration time in which larvae of Anagasta kühniella were exposed to the microwave radiation. Letargy and locomotion desfunctions were the initial alterations. Dehydration and body wall darkening , reaching dark brown, were observed before death. The radiation also showed to be harmful to the adult stage of the same pyralidae species, reducing longevity as well as reproductive capacity. Bracon hebetor adults showed to be more resistant to radiation (LETso ::: 106,57 s) when compared to Anagasta kühniella adults (LETso = 22,61 s) and larvae (LET50 = 15,63 s). Samples of Bacillus thuringiensis var kurstaki were exposed to different times of microwaves radiation (2450 MHz) and were ofered to third instar larvae of Anagasta kühniella in three different concentrations (0.02 %, 0.2% and 2.6%). This electromagnetic wave did not present any interference in the patogenicity of the entomopathogen in the extrem concentrations (0.02 and 2.6%). However, in the 0.2% concentration, next to LCso of the larvae, the patogenicity of Bacillus thuringiensis was altered by the time of exposure to the radiation. Finally, radiation by microwaves could be considered as a promissing method, efficient and compatible with biological control ones, to participate in IPM programs of the Mediterranean Flour Moth, Anagasta kühniella
Mestrado
Parasitologia
Mestre em Ciências Biológicas

Orientador: Mohamed E. M. Habib
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Ephestia kuhniella é uma expressiva praga de moinhos de trigo. Seu controle é feito basicamente através de produtos químicos sintéticos. Porém, devido aos inúmeros problemas causados ao ambiente e à saúde humana por estas substâncias, novas alternativas para o manejo deste lepidóptero vêm sendo buscadas. Neste sentido, pesquisas em controle biológico, físico e mecânico, entre outros, são fundamentais para embasar novos programas de manejo integrado (MIP) desta praga. Dentre os agentes de controle biológico, produtos à base do entomopatógeno Bacillus thuringiensis var. kurstaki (Btk) e o ectoparasitóide Bracon hebetor são considerados altamente eficientes no controle de E. kuehniella.Todavia, a inserção destes dois em programas de MIP depende de estudos sobre suas interações na regulação da densidade do piralídeo. Sendo assim, este trabalho propõe avaliar o impacto e as interações dos dois agentes de mortalidade sobre este piralídeo. Para tal buscou-se avaliar a susceptibilidade de larvas de E. kuehniella ao Btk; analisar os efeitos crônicos de Btk no desenvolvimento dos piralídeos sobreviventes; avaliar o impacto da patogenia causada pelo Btk nas larvas de E. kuehniella sobre o desenvolvimento do parasitóide; analisar possíveis interações de compatibilidade, antagonismo ou sinergismo entre o parasitóide e o entomopatógeno e avaliar a capacidade de B. hebetor de distinguir entre larvas sadias e infectadas por Btk. Os estudos foram realizados no Laboratório de Entomologia Aplicada, do Departamento de Zoologia da UNICAMP, sob condições controladas (25 ± 20 C; 70 ± 10 % de umidade relativa e 12 horas de fotofase). Com os critérios adotados, observou-se que a susceptibilidade de E. kuehniella variou inversamente em função do estádio de desenvolvimento larval. Desta forma, larvas de primeiro estádio apresentaram maior susceptibilidade ao produto à base do patógeno (CL50 = 0,05%; IC = 0,04 % ¿ 0,06 %), seguidas pelas larvas de terceiro, que apresentaram um nível intermediário (CL50 = 0,16%; IC = 0,12 % ¿ 0,20 %). Já as larvas de quinto estádio foram as menos susceptíveis ao Btk (CL50 = 1,77%; IC = 1,42 % ¿ 2,23 %). Os estudos de efeitos agudos e crônicos do patógeno sobre o desenvolvimento, reprodução e longevidade de E. kuehniella elucidaram o caráter variável da resposta biológica ao tratamento em função da idade larval, da concentração do Btk e do tempo de exposição. Larvas de primeiro (L1) e de quinto estádios apresentaram reduções na viabilidade dos estágios imaturos. Adultos xix oriundos de larvas de primeiro e terceiro estádios sobreviventes a tratamento com Btk não apresentaram efeitos crônicos em termos de capacidade reprodutiva e longevidade. Porém, os adultos que se originaram de indivíduos tratados no quinto estádio larval apresentaram menor capacidade reprodutiva. Já a viabilidade dos seus ovos não diferiu do grupo testemunha. A longevidade do piralídeo foi diminuída quando estes adultos foram expostos ao contato direto com o produto microbiano. Este efeito indireto provavelmente foi decorrente do estresse provocado pelo odor do produto. O Btk também foi indiretamente responsável pela redução na capacidade de B. hebetor paralisar e parasitar larvas de E. kuehniella. Adultos do braconídeo rejeitaram larvas hospedeiras que manifestavam sintomas da bacteriose. A redução da viabilidade dos imaturos do braconídeo variou em função da concentração e do tempo de exposição ao tratamento. Todavia, a longevidade dos adultos obtidos desta F1 não foi alterada. O inseticida microbiano também provocou efeitos indiretos sobre a longevidade dos adultos do braconídeo decorrentes do odor/ contato, com este. A ingestão de mel contendo Btk também diminuiu significativamente a longevidade do ectoparasitóide. Através dos estudos de escolha e olfatometria, pode-se constatar que a localização do hospedeiro por B. hebetor não é alterada em função da infecção por Btk. Mas, a aceitação das larvas para o parasitismo variou inversamente à concentração do patógeno e ao tempo de tratamento do hospedeiro. Desta forma, a maior rejeição ocorreu nos lotes onde havia a maior proporção de larvas doentes e manifestando sintomas evidentes da patogenia. Em condições laboratoriais, a ação do patógeno, nas concentrações aqui avaliadas, associada ao parasitóide, para o controle de E. kuehniella, apresentou a mesma eficiência que o parasitóide isoladamente. Sendo assim, pode-se concluir que Btk e B. hebetor atuam de maneira complementar no controle de populações dessa traça, dependendo de quem a atinge primeiro. Se o programa de MIP visa trabalhar com introduções alternadas desses dois agentes biológicos visando controle de larvas em diferentes estádios, haveria uma vantajosa compatibilidade entre estes, que permite a não utilização de produtos químicos, evita a seleção de resistência pelo Btk na população alvo e contorna o problema gerado pelo número de partículas de insetos presentes em produtos para consumo humano, como as farinhas, decorrente da introdução contínua de parasitóides
Abstract: Ephestia kuehniella is a very important pest, attacking wheat flour in mills and stored places. Due to the negative impact of the chemical control methods, alternative agents are being studied to reach more safety and more satisfactory results. Some biological, physical and mechanical components should be adequate to be includes in integrated pest management programs (IPM) to reduce the injury of this lepidopterous pest. Within the biological agents, Bacillus thuringiensis var. kurstaki (Btk) and the braconid wasp Bracon hebetor are considered very efficient in controlling, separately, E. kuehniella populations. However, the insertion of these two agents in IPM programs needs some studies concerning the interaction between them and their natural pyralid host. The present study aimed to investigate the interaction between Btk and B. hebetor, in addition to evaluate their efficiency in controlling E. kuehniella. Within such a purpose some approaches were studied, such as: susceptibility of E. kuehniella larvae to Btk; chronic effects of Btk on the survived individuals; effect of diseased and survived E. kuehniella larvae on B. hebetor development; compatibility between the two biological agents and B. hebetor capacity to distinguish between healthy and diseased hosts. All experiments were conducted under controlled laboratory conditions of 25 ± 20C, 70 ± 10% R. H. and 12 hours of photophase (Department of Zoology, IB, UNICAMP). The pyralid larval susceptibility varied according to the age. The young larvae (L1) showed to be more susceptible than the old ones (L3 and L5). The CL50s were calculated as 0,05%; 0,16% and 1,77% for the first, third and fifth instars respectively. The acute as well as the chronic effects of Btk on E. kuehniella development varied according to the exposure time, age and pathogen concentration. Adults originated from survived E. kuehniella larvae did not show any biological or structural alterations, when the pathogen was applied against the first and thirdinstar larvae. On the other hand, adults originated from survived 5th instar larvae suffered reduction in their reproductive capacity. The egg viability in all these cases did not reveal any significant differences when compared with testimony block. The direct contact of Btk commercial product resulted in reduced longevity among E. kuehniella adults. Reduction in longevity was also detected when B. hebetor females were kept in contact with the same product. The braconid female parasite was able to distinguish between healthy and diseased host larvae, avoiding to attack the later. Parasitization capacity was reduced when the wasp female was able to deposit its eggs in pyralid larvae during the initial phase of the bacterial disease. B. hebetor adult longevity was reduced when parasitoid wasps were fed on honey containing Btk. Combinations between the two biological agents did not result in any synergistic effect under the conditions of the present study, and both of then could be recommended to be applied in wheat mills
Doutorado
Parasitologia
Doutor em Ciências Biológicas

Publicación a texto completo no autorizada por el autor
Evalúa y selecciona un agente fotoprotector y un agente fagoestimulante que permita mejorar la actividad entomotóxica de B. thuringiensis sobre larvas de S. frugiperda. Para ello, se evaluó el espectro de absorbancia de 9 agentes fotoprotectores, la compatibilidad con B. thuringiensis y además de exposición a la luz artificial del sol (UV A y UVB); seleccionándose al Ac. Ascórbico como el mejor agente fotoprotector compatible con B. thuringiensis. Para la selección del fagoestimulante se realizaron pruebas de preferencia alimenticia con larvas neonatas de Spodoptera frugiperda así como la evaluación de la compatibilidad con B. thuringiensis seleccionándose la hoja de maíz tratada. Se evaluó mediante bioensayos la efectividad de los agentes seleccionados observándose una disminución del DL50 cuando se adicionaron fagoestimulante y fotoprotector a la cepa comparándose con la cepa sola y sometiéndola a radiación UV durante 4 y 8 horas.
Tesis

The development of insecticides resistance among many insect species and the ecological damage occasionally caused by the lack of specificity in the toxic effects of insecticides have provided the impetus to seek alternative methods of insect control. This observation led to the development of bioinsecticides based on the insecticidal action Bacillus sphaericus (Bs), Bacillus turingiensis (Bt). The discovery of biolarvicidal actions of Bacillus thuringiensis and Bacillus sphaericus opened a new perspective for insect control. In the first part of the study was initiated to determine a suitable fermentation medium formulation and optimal fermentation conditions for large scale, low cost production of Bs. Bs 2362 was tested in whey and soy flour based media. These media was reformulized form of NYSM (Nutrient Broth Yeast Extract Sporulation Medium). Soy flour based medium, SYSM, gave the promising results in terms of cell yield, sporulation frequency and toxin production. In the second part of the study, fermentation productivity anlaysis of a local isolate Bacillus thuringiensis subsp. kurstaki 81 was evaluated. In order to compare different C:N ratios (1:1, 2:1, 4:1, 8:1, 10:1 20:1 and 30:1) of YSM medium. Btk 81 were run for 72 h and cell growth, sporulation and toxin protein profile of Btk 81 were determined for each. When all the quantitative toxin data for both glucose and sucrose varying C:N ratios were compared, it was determined that the crystal protein concentrations had the highest value in sucrose based medium when C:N ratio was 10:1. Regulation by C:N ratio of crystal protein biosynthesis was investigated for improving the production of this protein by our third candidate strain Bacillus thuringiensis subsp. israelensis ONR60. The experiments were performed by using TBL medium, at three different C:N ratios, 2:1, 4:1 and 8:1 respectively. In view of the cell growth characteristics and bioassy results, TBL medium designed with 2:1 C:N ratio was chosen as the best for further steps. In addition, running time of the culture determined as 60 hours as was also determined in the previous experiment. As the last step of this study, the pre-determined optimal conditions were applied to a 30L batch type fermentor for toxin production by using Bacillus thuringiensis subsp. israelensis ONR60. Unfortunately, the toxicity was not satisfactory, being much below the level of that expected as based on the results of the laboratory scale studies.

Conhecer os efeitos dos produtos fitossanitários naturais (PFN) sobre insetos praga e organismos não alvo é fundamental no contexto do Manejo Integrado de Pragas. Esse trabalho objetivou avaliar a atividade de PFN sobre Anticarsia gemmatalis (Lepidoptera: Noctuidae), esporos e cristais de Bacillus thuringiensis subesp. kurstaki (Btk) e a seletividade sobre Apis mellifera L. (Hymenoptera: Apidae). Foram utilizados os extratos de pitanga (Eugenia uniflora), pimenta (Capsicum baccatum var. pendulum), trombeta (Brugmansia suaveolens) e uva-do-japão (Hovenia dulcis), à 5%, Natuneem®, Natualho®, Pironat®, Rotenat® e Calda Bordalesa (CB), na concentração recomendada pelo fabricante. Para esporos, os tratamentos Btk + PFN e Btk, isoladamente, foram incubados em agitador horizontal (150 rpm, 30 ± 2 ºC, 2 h) e, inoculados em meio de cultura ágar nutriente (AN), em placas de Petri. Foram preparadas quatro placas (repetições), acondicionadas em câmara climatizada (30 ± 2 ºC, 18 h), quantificando-se as unidades formadoras de colônias (UFC)/mL. Para cristais, os PFN e Btk, isoladamente, água destilada esterilizada (testemunha) e Btk + PFN, após incubação, nas condições descritas acima, foram aplicados sobre cubos de dieta artificial para A. gemmatalis, em placas de Petri, que receberam 20 lagartas de segundo ínstar, cada. Foram preparadas quatro placas (repetições), acondicionadas em câmara climatizada (26 ± 2 ºC, UR: 70%±10 % fotofase 14 h). Avaliou-se a mortalidade após, 24, 48, 72, 96 e 120 h, o percentual de empupamento e emergência e o período ovo-adulto. A seletividade sobre A. mellifera foi avaliada pulverizando-se os tratamentos sobre operárias e adicionando-os a pasta Cândi. Como testemunha utilizou-se respectivamente água destilada esterilizada e pasta Cândi pura. Em ambos os experimentos utilizou-se 30 operárias por tratamento (repetição), individualizadas em tubos de vidro (10 cm × 2,5 cm), acondicionados em câmara climatizada (25 ± 2 °C, U.R. 70% ± 10%, fotofase de 12 h) e a mortalidade, avaliada a uma; duas; três; quatro; cinco; seis; nove; 12; 15; 18; 21; 24; 30; 36; 42; 48; 60; 72 e 96 h. Verificou-se que os extratos de pitanga, pimenta e uva-do-japão reduziram significativamente as UFC/mL. O extrato de pitanga foi o único que afetou negativamente a toxicidade dos cristais de Btk. Nenhum extrato vegetal afetou a mortalidade, não havendo diferença entre os tempos de avaliação, nem o empupamento, emergência e período ovo-adulto. Na mistura com Btk o extrato de pitanga retardou a mortalidade e o extrato de pimenta acelerou a mortalidade. Nos experimentos com A. mellifera, houve redução significativa, apenas para o extrato de uva-do-japão, quando pulverizado. Os produtos Natuneem® e Rotenat® aumentaram significativamente as UFC/mL. Somente a CB afetou negativamente a toxicidade dos cristais de Btk. Nos demais parâmetros, observou-se redução significativa do percentual de empupamento para CB e Natualho®e percentual de emergência para CB e Natuneem®. Entre os tempos de avaliação, não houve influência dos produtos isolados, na mistura com Btk Natualho®, Pironat®, Rotenat® aceleraram a mortalidade e a CB influenciou negativamente. Não se verificou atividade dos produtos sobre o período ovo-adulto. Para A. mellifera, em ambos os experimentos, os produtos não afetaram significativamente a longevidade.
Knowing the effects of natural products phytosanitary (NPP) on pests and organisms target is not essential in the context of Integrated Pest Management. This study aimed to evaluate the activity of PFN on Anticarsia gemmatalis Hubner (Lepidoptera: Noctuidae), spores and crystals of Bacillus thuringiensis subsp. kurstaki (Btk) and selectivity of Apis mellifera L. (Hymenoptera: Apidae). We used extracts of cherry (Eugenia uniflora), pepper (Capsicum baccatum var. pendulum), trumpet (Brugmansia suaveolens) and Blueberry japan (Hovenia dulcis), to 5%, Natuneem®, Natualho®, Pironat®, Rotenat® Bordeaux and Syrup (SB), the concentration recommended by the manufacturer. For spores, the treatments Btk + NFP and Btk alone were incubated on horizontal shaker (150 rpm, 30 ± 2 ° C, 2 h) and inoculated onto nutrient agar plates (NA), in petri dishes. We prepared four plates (repetitions), placed in a climatic chamber (30 ± 2 º C, 18 h), quantifying the colony forming units (CFU) / mL. To crystals, the PFN and Btk alone sterilized distilled water (control) and Btk + NFP after incubation under the conditions described above were applied on artificial diet cubes for A. gemmatalis, in Petri dishes, which received 20 second instar larvae of each. We prepared four plates (repetitions), placed in a climatic chamber (26 ± 2 º C, RH 70% ± 10% photophase 14 h), evaluating mortality after 24, 48, 72, 96 and 120 h, the percentage empupament and the emergence and egg-adult period. The selectivity of A. mellifera was evaluated by spraying treatments on workers and adding them to pasta Candi. As a control we used respectively sterile distilled water and paste Candi pure. In both experiments, we used 30 workers per treatment (repeat), individually in glass tubes (10 cm × 2.5 cm), packed in a climatic chamber (25 ± 2 ° C, RH 70% ± 10%, photophase 12 h) mortality, assessed by one, two, three, four, five, six, nine, 12, 15, 18, 21, 24, 30, 36, 42, 48, 60, 72 and 96 h. It was found that the extracts of cherry, pepper and Blueberry japan significantly reduced CFU / mL. The cherry extract was the only negatively affecting toxicity of Btk crystals. No plant extract affected the mortality, there was no difference between the time of evaluation, nor empupament, emergence and egg-adult period. In mixture with Btk cherry extract delayed the mortality and pepper extract accelerated mortality. In experiments with A. mellifera, was significantly reduced only to extract Blueberry japan, when sprayed. Natuneem® products and Rotenat® were the only ones who have significantly increased CFU / mL. Only the SB negatively affected the toxicity of Btk crystals. Ns other parameters, there was significant reduction in the percentage of empupament for SB and Natualho® and percentage of emergency and SB Natuneem®. Between the time of evaluation, no influence of individual products, the mix with Btk Natualho®, Pironat®, Rotenat® accelerated mortality and SB negatively influenced. There was no activity of the products on the egg-adult period. To A. mellifera, in both experiments, the products did not significantly affect longevity.

En las zonas productoras de papa (Solanum tuberosum Linneo) del Perú, una especie de polilla de la papa (PTM): Symmetrischema tangolias Gyen (Lepidoptera: Gelechiidae) viene desplazando en predominancia a Phthorimaea operculella Zeller (Lepidoptera: Gelechiidae). El objetivo de esta investigación fue desarrollar una fórmula bioinsecticida eficaz contra ambas plagas, basado en PoGV, Btk o la combinación de ambos. El tratamiento de tubérculos de papa (cv. “Peruanita”) con fórmulas de patógenos fue por inmersión o impregnación; e inoculadas con larvas neonatales de ambas especies. Los bioensayos se desarrollaron a: 25°C (P. operculella), 20°C (S. tangolias) y condiciones naturales en almacenes de papa. Las fórmulas en talco mejoraron la eficacia de los patógenos, alcanzando niveles CL50 con menores concentraciones de PoGV (16 veces) y Btk (5-6 veces) al compararse con sus respectivas aplicaciones acuosas (1,55×107 gránulos/mL; IC95%: 1,26×107-2,14×107 y 1321 mg/L; IC95%: 1084-1555) obtenidas contra P. operculella; asi mismo fueron 2-4 veces menores respecto a aplicaciones acuosas de Btk (3095 mg/L; IC95% 1711 - 3575) en S. tangolias. La interacción PoGV-Btk contra P. operculella fue mixta, tendiendo al antagonismo ante concentraciones altas de ambos patógenos y sinérgicos a bajas concentraciones. Dos fórmulas: Btk y Btk+PoGV, desarrollados y aplicados en almacenes de papa, permitieron incrementos poblacionales, 90 días después, solo en S. tangolias (3,0 y 2,7%) (larvas-pupas/100 tubérculos); luego de disminuirlos a niveles de 1,7% (30 días). No resulta conveniente utilizar fórmulas combinadas de Btk+PoGV por no conseguirse sinergísmos importantes; recomendándose el producto Btk en talco, por su efectividad contra ambas especies de PTM.
In the areas of potato productions (Solanum tuberosum Linneo) from Peru, a species of potato tuber moth (PTM): Symmetrischema tangolias Gyen (Lepidoptera: Gelechiidae) it comes displacing in predominance to Phthorimaea operculella Zeller (Lepidoptera: Gelechiidae). The objective of this investigation was to develop an effective bioinsecticide formule against both pests, based on PoGV, Btk or the combination of both. The treatment of the potato tubers (cv. “Peruanita”) was realized by immersion when was used one solution formulated or by covers when the powder formulation was, over, which was, inoculated the neonatal larvae. The laboratory bioassays were developed at control condition 25°C (P. operculella), 20°C (S. tangolias) and at room condition for the potato storage. Due the effects of some inert material over the mortality, the talcum formulation of both pathogens tested showed better efficacy than aqueous solution formulated. Reaching the CL50 level using less concentration of PoGV (16 times) and Btk (5 to 6 times) in comparison with the respective aqueous applications (1,55×107 granules/mL; IC95%: 1,26×107- 2,14×107 and 1321 mg/L; IC95%: 1084-1555) against P. operculella. The difference was 2 to 4 time minor in S. tangolias concerning aqueous applications of Btk (3095 mg/L; IC95% 1711-3575). The PoGV-Btk interaction against P. operculella results mixed, reaching to the antagonism in front of high concentrations of both pathogens and synergistic to low concentrations. Two formulations: Btk and Btk+PoGV was developed and applied in potato storage, permitting increments populations 90 days later, only in S. tangolias (3,0 2,7%) (larvae and pupae/100 tubers), then of diminishing them to levels of 1,7% (30 days). With this results is not recommended the use formulations combined of Btk+PoGV by not getting important synergism; recommending the use of the talcum Btk product, for their high affectivity against both species of potato tuber moth.
Tesis

Depuis plus de 20 ans, l'insecticide microbien Bacillus thuringiensis var. kurstaki (Btk) est appliqué au Québec afin de réduire les impacts négatifs de la tordeuse des bourgeons de l'épinette (TBE) (Choristoneura fumiferana (Clem.)) sur la ressource ligneuse. Mais, qu'en est-il de sa rentabilité ? En période épidémique, le territoire forestier québécois ne peut être protégé en totalité en raison de sa grande superficie et de la récurrence annuelle des attaques de l'insecte ce qui impliquerait des coûts de protection cumulatifs très élevés. Dans le contexte forestier actuel et devant l'éventuel retour d'une épidémie de la tordeuse des bourgeons de l'épinette, il devient primordial d'identifier les aires à traiter sur la base des pertes anticipées en incluant les effets de l'insecte et des traitements sur la croissance. Ce questionnement est abordé dans ce projet de recherche, plus spécifiquement sur le sapin baumier, l'essence de prédilection de la tordeuse des bourgeons de l'épinette. Dans le sud-ouest du Québec, l'accroissement annuel de trois catégories (traités, non-traités et sans TBE) de peuplements mélangés de sapin baumier (Abies balsamea (L.) Mill) a été mesuré pendant une période de neuf années consécutives (1994-2002), alors qu'à différents intervalles de temps, des traitements au Btk ont été appliqués afin de réduire la défoliation causée par la tordeuse des bourgeons de l'épinette. Cette recherche démontre que les opérations de pulvérisations aériennes d'insecticide biologique s'avèrent rentables en protégeant 66% de la capacité photosynthétique. Également, grâce à ces opérations aériennes, les peuplements traités ont conservé des niveaux de croissance et de mortalité d'arbres similaires à ceux sans TBE.

En las zonas productoras de papa (Solanum tuberosum Linneo) del Perú, una especie de polilla de la papa (PTM): Symmetrischema tangolias Gyen (Lepidoptera: Gelechiidae) viene desplazando en predominancia a Phthorimaea operculella Zeller (Lepidoptera: Gelechiidae). El objetivo de esta investigación fue desarrollar una fórmula bioinsecticida eficaz contra ambas plagas, basado en PoGV, Btk o la combinación de ambos. El tratamiento de tubérculos de papa (cv. “Peruanita”) con fórmulas de patógenos fue por inmersión o impregnación; e inoculadas con larvas neonatales de ambas especies. Los bioensayos se desarrollaron a: 25°C (P. operculella), 20°C (S. tangolias) y condiciones naturales en almacenes de papa. Las fórmulas en talco mejoraron la eficacia de los patógenos, alcanzando niveles CL50 con menores concentraciones de PoGV (16 veces) y Btk (5-6 veces) al compararse con sus respectivas aplicaciones acuosas (1,55×107 gránulos/mL; IC95%: 1,26×107-2,14×107 y 1321 mg/L; IC95%: 1084-1555) obtenidas contra P. operculella; asi mismo fueron 2-4 veces menores respecto a aplicaciones acuosas de Btk (3095 mg/L; IC95% 1711 - 3575) en S. tangolias. La interacción PoGV-Btk contra P. operculella fue mixta, tendiendo al antagonismo ante concentraciones altas de ambos patógenos y sinérgicos a bajas concentraciones. Dos fórmulas: Btk y Btk+PoGV, desarrollados y aplicados en almacenes de papa, permitieron incrementos poblacionales, 90 días después, solo en S. tangolias (3,0 y 2,7%) (larvas-pupas/100 tubérculos); luego de disminuirlos a niveles de 1,7% (30 días). No resulta conveniente utilizar fórmulas combinadas de Btk+PoGV por no conseguirse sinergísmos importantes; recomendándose el producto Btk en talco, por su efectividad contra ambas especies de PTM.
In the areas of potato productions (Solanum tuberosum Linneo) from Peru, a species of potato tuber moth (PTM): Symmetrischema tangolias Gyen (Lepidoptera: Gelechiidae) it comes displacing in predominance to Phthorimaea operculella Zeller (Lepidoptera: Gelechiidae). The objective of this investigation was to develop an effective bioinsecticide formule against both pests, based on PoGV, Btk or the combination of both. The treatment of the potato tubers (cv. “Peruanita”) was realized by immersion when was used one solution formulated or by covers when the powder formulation was, over, which was, inoculated the neonatal larvae. The laboratory bioassays were developed at control condition 25°C (P. operculella), 20°C (S. tangolias) and at room condition for the potato storage. Due the effects of some inert material over the mortality, the talcum formulation of both pathogens tested showed better efficacy than aqueous solution formulated. Reaching the CL50 level using less concentration of PoGV (16 times) and Btk (5 to 6 times) in comparison with the respective aqueous applications (1,55×107 granules/mL; IC95%: 1,26×107- 2,14×107 and 1321 mg/L; IC95%: 1084-1555) against P. operculella. The difference was 2 to 4 time minor in S. tangolias concerning aqueous applications of Btk (3095 mg/L; IC95% 1711-3575). The PoGV-Btk interaction against P. operculella results mixed, reaching to the antagonism in front of high concentrations of both pathogens and synergistic to low concentrations. Two formulations: Btk and Btk+PoGV was developed and applied in potato storage, permitting increments populations 90 days later, only in S. tangolias (3,0 2,7%) (larvae and pupae/100 tubers), then of diminishing them to levels of 1,7% (30 days). With this results is not recommended the use formulations combined of Btk+PoGV by not getting important synergism; recommending the use of the talcum Btk product, for their high affectivity against both species of potato tuber moth.
Tesis

Pine processionary moth (PPM, Thaumetopoea pityocampa) is an active range expanding pest species having significant economic impact in terms of tree growth and public health. A lot is known about its natural history although one important aspect of its life history, diapause, has been so far neglected in spite of its acknowledged importance in pest establishment and dynamics. A few questions related to diapause have been addressed in this PhD project. Although the existence of prolonged diapause (PD) was reported long ago, no study provided its maximum duration. We provide evidence of existence of at least 7 years of PD in one mountain population of PPM, and establish the importance of PD individuals in the sustenance of population density. This study underscores the importance of long-term surveillance of population for understanding the dynamics of PD. Although several studies have been conducted on the incidence of PD in PPM across it natural range, they were often published as forest service or forest health reports and not available to the large public. A collection of these reports, their validation and comparison with data from papers allowed to make a synthesis that included most of the pest range, including the sibling species Thaumetopoea wilkinsoni in the Near East. We suggest that winter conditions at the larval stage influence this decision. Data analysis suggests a strong U-shaped relationship between the incidence of PD and average winter temperature and a linear positive relationship between mortality and proportion of individuals in PD. The result contributes to the advancement of knowledge on the ecology, population dynamics and distribution of the PPM. Previous phenological models of PPM lack the description of dormant prepupal stage and its significance in synchronization of emergence. Besides, no study reported on the occurrence of diapause in PPM in a stage other than pupa. We show how with a weekly sampling during the two-month long procession period of pine processionary prepupae, it was possible to discover that prepupae differentially regulate their development time in such a way that moth emergence of short-lived adults resulted to be concentrated and synchronized in less than one month. Early descending individuals don’t pupate immediately, rather wait as diapausing prepupae for their late counterparts, whereas late-descending prepupae develop into pupae much faster. The finding of prepupal diapause and its significance in synchronization of emergence may improve the existing model of phenology and provide managers with a new tool to handle this pest. Diapause termination mechanism in both univoltine and prolonged diapausing individuals of PPM has been previously hypothesized based on the occurrence of a key period during the pupal stage. By using three proxies of metabolic activity, such as body temperature, O2 consumption, and weight loss, we confirm the hypothesis of existence of a key period (termination) in univoltine and prolonged diapausing individuals of PPM. This finding is a starting point for the study of diapause development in PPM from an ecological point of view. Compared to simple phenomenon of diapause, field of prolonged diapause in insects per se is not vast. A lot of questions regarding the regulation of prolonged diapause are still unresolved. When we consider PPM, the gap of knowledge in this regard is gigantic. Being an important pest of Mediterranean forests, PPM deploys diapause for its success. We know almost nothing about the regulation of diapause in this species. Despite that, this thesis attempted to start the effort of answering a few of thousands of questions on the regulation of diapause in PPM. This project has prepared the ground for several possible future works. Among them, some are: testing the findings in other populations, understanding genetic regulation of diapause and prolonged diapause, understanding the importance of climate change in regulating diapause, improving phenology models and using for predicting the effects of climate change under different scenarios.
La processionaria del pino (PPM, Thaumetopoea pityocampa) è una specie in fase di espansione dell’areale e che causa perdite importanti di crescita degli alberi nonché problemi sanitari all’uoma e altri animali. Nonostante molti studi siano stati condotti su questa specie, alcuni aspetti relativi alla diapausa sono stati finora trascurati anche se riconosciuti come importanti per l’affermazione della specie. Questa tesi si pone l’obiettivo di chiarirne alcuni. La presenza di diapausa prolungata (PD) in Th. pityocampa è nota da tempo ma mancano dati precisi sull’effettiva durata del fenomeno. Con questo studio si è potuto dimostrare che in una valle alpina al limite dell’areale la diapausa si estende fino a 7 anni, con ripercussioni significative sulla dinamica di popolazione. Ciò indica la necessità di una sorveglianza estesa delle popolazioni dell’insetto. Nell’areale della processionaria del pino sono stati svolti vari studi in cui la presenza di diapausa è stata accertata e quantificata, e alcuni di questi sono stati inclusi in rapporti interni di difficile reperibilità. L’accesso a questa informazione e la sua verifica puntuale hanno consentito di produrre una sintesi dei risultati, includendo anche la specie sorella Th. wilkinsoni diffusa in Asia Minore. I risultati mostrano che un fattore importante è rappresentato dalle temperature invernali cui sono esposte le larve. E’ stata infatti trovata una relazione a U tra temperatura invernale e frequenza della diapausa, associata a una maggiore mortalità per gli individui diapausanti. Tale risultato rappresenta un passo in avanti nella documentazione dell’effetto dei fattori ecologici sull’ecologia e sulla dinamica dell’insetto. I modelli di sviluppo disponibili per la processionaria del pino non hanno mai considerato lo stadio di prepupa e l’esistenza di una quiescenza/diapausa che porti a una migliore sincronizzazione degli sfarfallamenti. Grazie a un campionamento settimanale durante il periodo delle processioni di interramento è stato possibile accertare che il periodo di discesa è lungo circa il doppio rispetto allo sfarfallamento, e che le prepupe presentano una diapausa mirata a sincronizzare l’uscita degli adulti e quindi gli accoppiamenti. Il meccanismo si basa su un arresto dello sviluppo nei primi individui che scendono al suolo, che si riduce progressivamente durante il periodo. Queste nuove conoscenze sono di notevole importanza nella definizione di modelli di sviluppo e di previsione delle popolazioni dell’insetto. La conclusione della diapausa pupale in individui univoltini o in diapausa prolungata è stata associata alla presenza di un periodo chiave durante il quale l’individuo decide se proseguire nello sviluppo ad adulto o se rimanere in diapausa. Grazie a misure di alcune variabili non distruttive (temperatura superficiale, consumo di ossigeno, peso corporeo) è stato possibile mettere in evidenza per la prima volta l’esistenza di tale periodo in individui mantenuti a varie condizioni ambientali. Questo risultato rappresenta un enorme passo in avanti nelle conoscenza sulla regolazione della diapausa in questa specie. Nonostante la diapausa degli insetti sia in generale ben nota, le informazioni sulla diapausa prolungata sono scarse e molte domande giacciono irrisolte. Nel caso specifico della processionaria del pino questo ritardo è notevole e allo stesso tempo importante per la regolazione della dinamica di popolazione e quindi dei danni a piante e animali. Questo lavoro contribuisce a chiarire alcune aspetti importanti e apre la strada a studi mirati a conoscere la regolazione genetica della diapausa, gli effetti del cambiamento climatico, e lo sviluppo di modelli in grado di prevedere con affidabilità l’andamento demografico.

La voie de synthèse non-ribosomique représente une source importante de molécules d’intérêt. Mettant en jeu de larges complexes multi-enzymatiques, les Synthétases de Peptides Non Ribosomiques (NRPS), ce mode de production aboutit à la formation de peptides originaux d’une grande diversité structurale et fonctionnelle. Les problématiques de recherche de nouvelles molécules d’origines naturelles pour une utilisation dans différents domaines industriels font des molécules d’origine non-ribosomique de parfaites candidates pour la découverte des médicaments, surfactants ou antibiotiques des prochaines années. Les travaux décrits ont pour but l’étude du mécanisme de production de deux familles de molécules issues de la synthèse non-ribosomique, la kurstakine, un lipopeptide produit par Bacillus thuringiensis, et les PPCC (PentaPeptides Cycliques Chlorés), regroupant plusieurs molécules d’origine eucaryote. L’étude de la production de kurstakine par une série de mutants de B. thuringiensis a permis d’identifier plusieurs facteurs limitant la production de cette molécule, et d’ouvrir la voie à la mise en place de procédé de production en milieu liquide de cette molécules. L’étude des PPCC a permis d’identifier plusieurs gènes NRPS potentiellement impliqués dans la production de ces familles de molécules dans le génome de deux moisissures
The Non Ribosomal Synthesis pathway is a very important source of molecules of interest. Based on large multi-enzymatic complexes, the Non Risosomal Peptide Synthetases (NRPS), this production mode leads to the formation of original peptides, with a large structural and activities diversity. The need of new molecules from natural sources, turns the non-ribosomal molecules into a promising source of drugs, surfactant or antibiotics for the future. This work aim to the understanding of the production mechanism of two families of non-ribosomal molecules, the kurstakin, a lipopeptide produced by Bacillus thuringiensis, and the CCPPs (Chlorinated Cyclic Penta Peptides), regrouping several eukaryotic molecules. The study of the kurstakin production using a series of B. thuringiensis mutants led to the identification of several limitating factors for the production of this molecule, and opened the possibility to the setting up of production process in liquid medium of this molecule. The study of CCPPs led to the identification of several genes, potentially implied on the production of these molecules in the genome of two fungal strains

碩士
國立中興大學
昆蟲學系
81
Bacillus thuringiensis var. kurstaki HD-1 (Btk),when examined by scanning electron microscope (SEM), showed two observable forms of parasporal crystal; one is bipyramidal crystal, called P1 toxin, and the other cuboidal crystal, P2 toxin. The electrophoretic analysis of their crystal proteins found that the molecular weight of P1 is 135 KDa and t hat of P2 63 KDa. When activated with trypsin, P1 produced the toxic proteins with molecular weights of ca. 56-63 KDa. The lawn assay devised with cultured cells in agar plate and trypan blue layewed on the top surface,could rapidly detect the toxicity of the B. thuringiensis (Bt) toxic proteins. The concentration of a Plutella xylostella cell line (PX-1187) used in this assay had to be over 6x10 cell/dish ( 6 cm dia.) to produce the detectable reaction. On the other hand, when the cell concentration was lower than 2x10 cells/dish, no normal reaction was obtained. Using this lawn assay with the PX-1187 cell line, it was also found that the threshold dose of P1 against this line was 875 ng, while that of the activated P1 toxin was 245 ng, and P2 toxin 417 ng. Three different subclones of PX-1187, namely D、E、G, showed similiar degree of sensitivity to the Btkδ-endotoxin in comparison with their parental line. This assay system was not suitable for detecting the enhancing effect of some compounds, i.e., 0.125 % K CO , 0.5 % EDTA, 0.1 % ZnSO , 0.1 % CaCO , to Btk P1, when added to the assaying plates.

Thesis deals with advanced protection options against plum fruit mothCydia funebrana in organic and integrated production. A separate chapter is devoted to the characteristics, bionomy, the hosts plants and the importance of plum fruit moth. There are possibilities of monitoring and detection of the pest described in third chapter. The experimental part is devoted to comparison of productswith the active substance Bacillus thuringiensis var. kurstaki. There are two preparations compared Lepinox Plus and Biobit XL. The experiment was conducted in 2014 in a production orchard set in Brno Starý Lískovec. Product Lepinox Plusreached the highest efficiency.