Entomopathogenic fungi (EPF) are the one of the potential cause of the morbidity and mortality of insects. In agro-forestry uses, they are applied mainly in the form of conidial preparations in dry, aqueous or oil formulations. This approach, while practical, works in a hit and miss fashion leading to a frustrating dilemma of why successes and failure perpetuate. The fundamental solution is to bridge gaps in our knowledge about conidia of EPF in varied environments where they confront a diversity of insect hosts to start their pathogenesis.<p>This thesis was undertaken to examine the effects of hydration and the regulatory role of ambient pH on proteases which are the primary pathogenicity determinants in Beauveria bassiana and Metarhizium anisopliae. The approaches used were those of biochemical, proteomics and functional proteomics. <p>Novel aspects of pH regulation/homeostasis during the soaking of conidia in water, (type II water, which had a maximum electrical conductivity of 1ìS/ cm at 298K/ 25° C) were identified. Hydrated conidia showed swelling in type II water as assessed by (Multisizer IIITM (Coulter CounterTM). Release of proteases, metabolic activity through liberation of ammonia and citrate and synthesis of protein, RNA and DNA was established. It was deduced that conidial enzymes are either attached by loose hydrogen bonding or were associated to the spore membranes. Water soaked or hydrated conidia can secrete citrate and ammonia to modify the ambient pH and maximize the activity of secreted proteases. <p>Pr1- and- Pr2-like proteases were liberated by washing conidia in tween (Tw), water (Ww) and buffer. The washing of conidia in buffers (pH 4-10) affected the release/activity of Pr1 and Pr2. The thesis shows a newly designed native IPG strip zymography to identify the release of 4 and 8 isoforms of proteases, respectively from conidia. The 2-DE zymography (copolymerized gelatin) of protease from Tw of <i>B. bassiana</i> and <i>M. anisopliae </i> indicated one band (Mr 70 kDa; pI 6.3) and six isozymes (Mr 115-129 kDa; pI 3.7-9.0), respectively, which were identified using mass spectrometry (MALDI-TOF) as a serine-like protease. <p>Six metalloprotease isozymes from <i>M. anisopliae</i> but only one from <i>B. bassiana</i> was documented by 1-DE native zymography combined with 2-D spot densitometry scans. Cationic PAGE native zymography separated two basic protease isozymes from Tw extract of M. anisopliae depending upon the pH of the incubation buffer. However, one activity band was identified from <i>B. bassiana</i>. Furthermore, only one activity band was apparent during 1st and 2nd Ww up to day 2 for both EPF. SDS PAGE (non-dissociating) zymogram of secreted protease isozymes from Tw of <i>B. bassiana</i> revealed three bands of Mr100, 60, and 36.3 kDa. The isozymes observed at day 2 and 3 had a Mrs of 35.4 and 25 kDa, and 24.7 and 20.3 kDa at day 4. The SDS PAGE zymograms for <i>M. anisopliae</i> indicated two isozymes of Mr 103 and 12 kDa, respectively. During the 1st Ww and incubation of spores at day 2 and 3, a 12 kDa band was observed. These results confirm the presence of diversity of proteases and their isozymes with unique molecular sizes.<p>This thesis research discovered and characterized a diversity of proteins/enzymes not previously reported from any other fungi. A newly designed enzyme overlay membrane (EOM) technique revealed three isoforms of Pr1-like subtilisin from Tw of <i>M. anisopliae </i>(pI 8.1-9.7) and <i>B. bassiana</i> (pI 8.4-9.7). Conversely, only one isoform of Pr2-like trypsin was identified from <i>M. anisopliae</i> and no Pr2-like activity was observed from <i>B. bassiana</i>. Use of metalloprotease (MEP) inhibitors in conjunction with EOM analysis revealed their release during treatment in Tw. In <i>M. anisopliae</i> four activities (pI 4.4-7.5) of thermolysin-like MEP were observed. However, Tw of <i>B. bassiana</i> showed one activity band (pI 5.5). In addition, an isozyme of neutral MEP containing Zinc from <i>M. anisopliae </i>(pI 6.1) and one from <i>B. bassiana</i> (pI 6.5-7.6), respectively, was identified. MALDI-TOF and Q-TOF analysis revealed the presence of proteins similar to ROD 1, Ü- and â-glucanases, elastase, lipase 5 and galectin 7, which are important during the initial phase of germination and pathogenesis. <p>In addition subtilisin (Pr1-like), trypsin (Pr2-like) and NAGase synthesis from the germinating conidia and mycelia under the supply of different carbon and nitrogen (C/ N) sources was studied. The regulation of the synthesis of cuticle-degrading enzymes (CDE) from germinating conidia and mycelia was hypothesized to be controlled through regulatory derepression and nutritional starvation. Pr1 and Pr2 are regulated in a different manner in conidia and mycelia. Both enzymes are regulated through a multiple control mode. It was concluded that C/ N repression occurs only when it is necessary for infective structures to establish a nutritional relationship with the host cuticular structures. In addition, C/ N sources have a significant effect upon pH modulation, ammonia production and protease secretion. Furthermore, the synthesis of Pr1 and Pr2 from germinating conidia was affected by the (inducer pH) pHi of the growth media. Growing mycelia of <i>B. bassiana</i> under acidic (4.0), neutral (7.0) and basic (11.0) pH conditions produce ammonia which modifies the pH thereby creating environments suitable for protease. Growth, morphology, radial extension rate and conidiation at different pHi revealed that both EPF modify the pH of growth medium effectively as opposed to the saprophytic fungus, <i>Aspergillus nidulans</i>. <p>The presence of MEPs and Pr2-like trypsin suggests that these enzymes can act as a back up system for Pr1 to breach the cuticle and facilitate penetration before appressoria formation. The diversity of isozymes released from conidia suggests that the EPF are pre-adapted to pathogenic mode of life style, further contributing complexity to their interaction with host insects. Such isozymes can circumvent protease inhibitors present in the insect cuticle and the hemolymph. In addition, these isozymes may offer selective advantages in exploring new habitats (substrates) either as pathogen or saprophyte.

Entomopathogenic fungi (EPF) are the one of the potential cause of the morbidity and mortality of insects. In agro-forestry uses, they are applied mainly in the form of conidial preparations in dry, aqueous or oil formulations. This approach, while practical, works in a hit and miss fashion leading to a frustrating dilemma of why successes and failure perpetuate. The fundamental solution is to bridge gaps in our knowledge about conidia of EPF in varied environments where they confront a diversity of insect hosts to start their pathogenesis.<p>This thesis was undertaken to examine the effects of hydration and the regulatory role of ambient pH on proteases which are the primary pathogenicity determinants in Beauveria bassiana and Metarhizium anisopliae. The approaches used were those of biochemical, proteomics and functional proteomics. <p>Novel aspects of pH regulation/homeostasis during the soaking of conidia in water, (type II water, which had a maximum electrical conductivity of 1ìS/ cm at 298K/ 25° C) were identified. Hydrated conidia showed swelling in type II water as assessed by (Multisizer IIITM (Coulter CounterTM). Release of proteases, metabolic activity through liberation of ammonia and citrate and synthesis of protein, RNA and DNA was established. It was deduced that conidial enzymes are either attached by loose hydrogen bonding or were associated to the spore membranes. Water soaked or hydrated conidia can secrete citrate and ammonia to modify the ambient pH and maximize the activity of secreted proteases. <p>Pr1- and- Pr2-like proteases were liberated by washing conidia in tween (Tw), water (Ww) and buffer. The washing of conidia in buffers (pH 4-10) affected the release/activity of Pr1 and Pr2. The thesis shows a newly designed native IPG strip zymography to identify the release of 4 and 8 isoforms of proteases, respectively from conidia. The 2-DE zymography (copolymerized gelatin) of protease from Tw of <i>B. bassiana</i> and <i>M. anisopliae </i> indicated one band (Mr 70 kDa; pI 6.3) and six isozymes (Mr 115-129 kDa; pI 3.7-9.0), respectively, which were identified using mass spectrometry (MALDI-TOF) as a serine-like protease. <p>Six metalloprotease isozymes from <i>M. anisopliae</i> but only one from <i>B. bassiana</i> was documented by 1-DE native zymography combined with 2-D spot densitometry scans. Cationic PAGE native zymography separated two basic protease isozymes from Tw extract of M. anisopliae depending upon the pH of the incubation buffer. However, one activity band was identified from <i>B. bassiana</i>. Furthermore, only one activity band was apparent during 1st and 2nd Ww up to day 2 for both EPF. SDS PAGE (non-dissociating) zymogram of secreted protease isozymes from Tw of <i>B. bassiana</i> revealed three bands of Mr100, 60, and 36.3 kDa. The isozymes observed at day 2 and 3 had a Mrs of 35.4 and 25 kDa, and 24.7 and 20.3 kDa at day 4. The SDS PAGE zymograms for <i>M. anisopliae</i> indicated two isozymes of Mr 103 and 12 kDa, respectively. During the 1st Ww and incubation of spores at day 2 and 3, a 12 kDa band was observed. These results confirm the presence of diversity of proteases and their isozymes with unique molecular sizes.<p>This thesis research discovered and characterized a diversity of proteins/enzymes not previously reported from any other fungi. A newly designed enzyme overlay membrane (EOM) technique revealed three isoforms of Pr1-like subtilisin from Tw of <i>M. anisopliae </i>(pI 8.1-9.7) and <i>B. bassiana</i> (pI 8.4-9.7). Conversely, only one isoform of Pr2-like trypsin was identified from <i>M. anisopliae</i> and no Pr2-like activity was observed from <i>B. bassiana</i>. Use of metalloprotease (MEP) inhibitors in conjunction with EOM analysis revealed their release during treatment in Tw. In <i>M. anisopliae</i> four activities (pI 4.4-7.5) of thermolysin-like MEP were observed. However, Tw of <i>B. bassiana</i> showed one activity band (pI 5.5). In addition, an isozyme of neutral MEP containing Zinc from <i>M. anisopliae </i>(pI 6.1) and one from <i>B. bassiana</i> (pI 6.5-7.6), respectively, was identified. MALDI-TOF and Q-TOF analysis revealed the presence of proteins similar to ROD 1, Ü- and â-glucanases, elastase, lipase 5 and galectin 7, which are important during the initial phase of germination and pathogenesis. <p>In addition subtilisin (Pr1-like), trypsin (Pr2-like) and NAGase synthesis from the germinating conidia and mycelia under the supply of different carbon and nitrogen (C/ N) sources was studied. The regulation of the synthesis of cuticle-degrading enzymes (CDE) from germinating conidia and mycelia was hypothesized to be controlled through regulatory derepression and nutritional starvation. Pr1 and Pr2 are regulated in a different manner in conidia and mycelia. Both enzymes are regulated through a multiple control mode. It was concluded that C/ N repression occurs only when it is necessary for infective structures to establish a nutritional relationship with the host cuticular structures. In addition, C/ N sources have a significant effect upon pH modulation, ammonia production and protease secretion. Furthermore, the synthesis of Pr1 and Pr2 from germinating conidia was affected by the (inducer pH) pHi of the growth media. Growing mycelia of <i>B. bassiana</i> under acidic (4.0), neutral (7.0) and basic (11.0) pH conditions produce ammonia which modifies the pH thereby creating environments suitable for protease. Growth, morphology, radial extension rate and conidiation at different pHi revealed that both EPF modify the pH of growth medium effectively as opposed to the saprophytic fungus, <i>Aspergillus nidulans</i>. <p>The presence of MEPs and Pr2-like trypsin suggests that these enzymes can act as a back up system for Pr1 to breach the cuticle and facilitate penetration before appressoria formation. The diversity of isozymes released from conidia suggests that the EPF are pre-adapted to pathogenic mode of life style, further contributing complexity to their interaction with host insects. Such isozymes can circumvent protease inhibitors present in the insect cuticle and the hemolymph. In addition, these isozymes may offer selective advantages in exploring new habitats (substrates) either as pathogen or saprophyte.

The Russian wheat aphid, Diuraphis noxia is one of the most destructive exotic invaders of South Africa, capable of reducing individual wheat plant yields by up to 90%. Entomopathogenic fungi are important natural mortality factors associated with this aphid in its Eurasian endemic habitats as well as in the United States and Canada; their impact often exceeding that of predators and parasitoids. The principal objectives of this study included (1) baseline characterization of the aphid-pathogenic flora associated with aphids from South Africa, with special reference to six common cereal aphids, (2) quantification of the comparative impact of the different fungal species on the cereal-aphid complex in three different wheat producing regions of South Africa, (3) field evaluation of the Hyphomycete Beauveria bassiana against D. noxia on resistant wheat, (4) screening of six fungicides for their potential usage in managing entomophthoralean epizootics within greenhouse rearings of the Russian wheat aphid, and (5) development and evaluation of a novel bioassay protocol for screening entomopathogenic Hyphomycetes against D. noxia. A total of nine species of fungi known to infect and kill aphid hosts were collected, including the six entomophthoraleans, Pandora neoaphidis, Conidiobolus thromboides, Conidiobolus obscurus, Entomophthora planchoniana, Conidiobolus coronatus and Neozygites fresenii, and three Hyphomycetes, Beauveria bassiana, Verticillium lecanii, and Paecilomyces farinosus. The former four entomophthoraleans are considered first reports from this country. For the first time, morphological characteristics of these nine South African-collected species are visually depicted and techniques for their isolation and in vitro culture discussed. Seven species of fungi were recorded from D. noxia, of which P. neoaphidis was the most important, causing up to 50% mortality during the late season under dryland conditions in the summer-rainfall region. Mycoses at epizootic levels, together with the large diversity of fungal species recorded from this host, indicated a high level of susceptibility to fungal infection. In contrast, infection of the oat aphid Rhopalosiphum padi remained < 5% despite favourable numbers of hosts and apparently suitable environmental conditions. This phenomenon strongly suggests some level of low susceptibility to fungal infection in this species. Under irrigated conditions m KwaZulu-Natal, the rose-gram aphid Metopolophium dirhodum was the predominant aphid but remained below economical injury levels. Field surveys revealed that this aphid was effectively targeted by P. neoaphidis and C. obscurus, and findings suggest that in some areas of South Africa entomophthoralean fungi effectively suppress this aphid, negating the need for insecticide applications. On average, ca. 61% control of D. noxia on resistant wheat was observed following an application of B. bassiana (5 x 10¹³ conidia per hectare) during the early flag-leaf stage of the wheat. Efficacy of B. bassiana applications on younger plants appeared to be influenced by the level of aphid activity, possibly explained by secondary pick-up of inoculum by D. noxia. These findings accentuate the importance of understanding the tritrophic relationship between host plant, pest and pathogen. The fungicides copper oxychloride, mancozeb + oxadixyl, captab + metalaxyl, bittertanol, iprodione, and mancozeb at a rate of 0.1% active ingredient moderately to strongly inhibited C. thromboides vegetative growth (mean inhibition 81.1 %). Mancozeb at concentrations of 10.0, 2.0, 1.25, 0.5, 0.08, and 0.016% was further evaluated in vitro. The fungus growth cut-off point, midway between 1.25 and 2.0% mancozeb, was calculated and a rate of 1.625% active ingredient per litre of water was used to decontaminate the fungus-infected D. noxia cultures. A novel bioassay protocol was developed, employing live host plants for rearing aphids post inoculation. Using this design against D. noxia, an average LC₅₀ estimate of 85 conidia per mm² for B. bassiana strain GHA was calculated. Control mortality was restricted to levels below 4%. The data indicated high precision due to an average coefficient of variation for slope of less than 20%, and an average chi-squared value of 5.46 ± 2.74 (n = 10 assays). The design will accommodate the use of cereal aphid species other than D. noxia, while live host plants will facilitate tritrophic studies on the effect of host-plant resistance on fungus-induced mortality of D. noxia. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2002.

Η παρούσα μεταπτυχιακή εργασία εκπονήθηκε στο πλαίσιο του Διατμηματικού Προγράμματος Μεταπτυχιακών Σπουδών «Ιατρική Χημεία: Σχεδιασμός και Ανάπτυξη Φαρμακευτικών Προϊόντων». Σκοπός αυτής της έρευνας ήταν η διερεύνηση της επίδραση του εκχυλίσματος κρόκου τόσο στην αύξηση όσο και στην αποτελεσματικότητα των εντομοπαθογόνων μυκήτων επί των προνυμφών του λεπιδοπτέρου Sesamia nonangrioides. Στην παρούσα μελέτη χρησιμοποιήθηκαν οι εντομοπαθογόνοι μύκητες Beauveria bassiana Balsamo (Vuillemin) (Hypocreales: Cordycipitaceae), Metarhizium robertsii (Metchnikoff) Sorokin (Hypocreales: Clavicipitaceae) και Isaria fumosorosea (Wize) Brown & Smith (Hypocreales: Clavicipitaceae) από την συλλογή του Μπενακείου Φυτοπαθολογικού Ινστιτούτου των οποίων η διατήρηση έγινε στο εργαστήριο Φυσιολογίας Φυτών του τμήματος Βιολογίας με την μέθοδο των ημιεκλεκτικών υποστρωμάτων (Strasser et al. 1996). Στύλοι του καλλιεργούμενου Crocus sativus εκχυλίστηκαν με διάλυμα 50% v/v μεθανόλης σε νερό (3 mL/50 mg ξηρής δρόγης) για 4 h απουσία φωτός. Οι παραπάνω εντομοπαθογόνοι μύκητες καλλιεργήθηκαν σε τρυβλία petri με θρεπτικό υλικό Sabouraud Dextrose Agar (S.D.A.) για διάστημα 5 ημερών παρουσία (3,0% v/v) και απουσία (100% S.D.A. αλλά και 98,5% S.D.A. - 1,5% μεθανόλης) εκχυλίσματος. Μετά το πέρας, διαπιστώθηκε ότι η παρουσία της μεθανόλης σε αυτήν τη συγκέντρωση δεν επηρέασε την ανάπτυξη κανενός μύκητα. Περαιτέρω, οι εντομοπαθογόνοι μύκητες B. bassiana και I. fumosorosea δεν επηρεάστηκαν από την παρουσία του εκχυλίσματος κρόκου στο υπόστρωμα ανάπτυξης, ενώ ο εντομοπαθογόνος μύκητας M. robertsii επηρεάστηκε θετικά (P<0,05, τεστ Bonferoni) από την παρουσία του εκχυλίσματος. Επιπροσθέτως μελετήθηκε η επίδραση του εκχυλίσματος κρόκου στην αποτελεσματικότητα των εντομοπαθογόνων μυκήτων επί των προνυμφών του εντόμου S. nonangrioides.. Για κάθε εντομοπαθογόνο μύκητα παρασκευάστηκαν τρεις συγκεντρώσεις (106, 107, 108 κονίδια/mL) ενώ η θνησιμότητα των προνυμφών καταγραφόταν καθημερινά για 7 ημέρες. Oι εντομοπαθογόνοι μύκητες προκάλεσαν υψηλά ποσοστά θνησιμότητας στις προνύμφες του εντόμου ιδιαίτερα ο I. fumosorosea (υπόστρωμα μόνο S.D.A) προκάλεσε θνησιμότητα του S. nonangrioides που κυμάνθηκε από 80 έως 93%. Η παρουσία του εκχυλίσματος κρόκου στο υπόστρωμα αύξησε σημαντικά (P<0,05, test Bonferoni) τη θνησιμότητα του εντόμου (80-96%) ένεκα του εντομοπαθογόνου μύκητα M. robertsii. Η σημαντική θετική επίδραση του εκχυλίσματος κρόκου στην αύξηση και την αποτελεσματικότητα του εντομοπαθογόνου μύκητα M. robertsii επί των προνυμφών του S. nonangrioides χρήζουν περαιτέρω διερεύνησης ως προς τους μηχανισμούς με τους οποίους επετεύχθη αλλά και ανοίγουν νέους δρόμους ως προς την ανάπτυξη νέων μεθόδων βιολογικής καλλιέργεια. / This thesis was produced as part of the Interdepartmental Graduate Program entitled “Medicinal Chemistry: Design and Development of Medicinal Products”. The intention of this thesis was to study the effect of saffron extract at the growth of entomopathogenic fungi in semi selective substrates and at the efficacy of entomopathogenic fungi on larvae of the insect S. nonangrioides. This study used Beauveria bassiana Balsamo (Vuillemin) (Hypocreales: Cordycipitaceae), Metarhizium robertsii (Metchnikoff) Sorokin (Hypocreales: Clavicipitaceae) and Isaria fumosorosea (Wize) Brown & Smith (Hypocreales: Clavicipitaceae) from the collection of the Benaki Phytopathological Institute and maintain with the selective medium method in laboratory of Plant Physiology at the Department of Biology (Strasser et al. 1996). Saffron (stigmas of Crocus sativus) was extracted with methanol:water 1:1, v/v (3 mL/50 mg dry plant material) for 4 h in the absence of light. Entomopathogenic fungi were cultured in petri dishes with Sabouraud Dextrose Agar (S.D.A.) for 5 days in the presence (3.0%, v/v) and absence (100% S.D.A. and 98,5% S.D.A. -1,5% methanol) of saffron extract. Methanol in the culture medium did not affect fungal growth at the particular concentration. After the five days, entomopathogenic fungi B. bassiana and I. fumosorosea were not affected by the presence of saffron extract in the growing medium, whereas M. robertsii growth was influenced positively (P<0.05, Bonferoni test) by the presence of saffron extract. In addition, the effect of saffron extract on the efficacy of entomopathogenic fungi on larvae of S. nonangrioides was studied. For each entomopathogenic fungus three concentrations (106, 107, 108 conidia/mL) were used, while the mortality of larvae was recorded daily for 7 days. Entomopathogenic fungi caused high mortality in larvae of the insect; particularly I. fumosorosea (substrate SDA) induced mortality ranging from 80 to 93%. Saffron in the culture medium of M. robertsii significantly (P<0.05, Bonferoni test) increased insect mortality (80 to 96%). The significant positive effect of saffron in the semi-selective substrate on the growth and effectiveness of M. robertsii on S. nonangrioides larvae demand further investigation of the mechanisms of action and pose new dimension in the use of biological agents.

Made available in DSpace on 2014-06-11T19:35:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-05-25Bitstream added on 2014-06-13T19:06:00Z : No. of bitstreams: 1 gassen_mh_dr_botfca.pdf: 1151986 bytes, checksum: d10ca5877ac7bff31ebce19ef7356faf (MD5) / 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... / 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)

Orientador: Antonio Carlos Monteiro / Banca: Inajá Marchizeli Wenzel / Banca: Elisângela de Souza Loureiro / Resumo: No manejo integrado da cana-de-açúcar são utilizados fungos entomopatogênicos para o controle de insetos e também diversos agroquímicos visando aumento de produtividade. Porém, vários destes agroquímicos podem interferir na sobrevivência de Beauveria bassiana e Metarhizium anisopliae. Portanto, o objetivo deste trabalho foi avaliar a toxicidade dos agroquímicos mais utilizados no manejo da cana-de-açúcar para B. bassiana e M. anisopliae em experimentos feitos com a utilização em meio de cultura e de solo. Nos ensaios com meio de cultura, os fungos foram inoculados em meio de batata, dextrose e ágar (BDA) contendo diversos inseticidas (4), herbicidas (7) e maturadores (3). Avaliou-se o crescimento, a esporulação e a viabilidade, e com base nesses parâmetros fez-se a classificação toxicológica dos agroquímicos. Para verificar se o efeito encontrado em meio de cultura se mantém no ambiente do solo, foram usados os agroquímicos que apresentaram maior toxicidade no ensaio com meio de cultura. Nesta etapa os fungos foram inoculados nos solos argiloso e arenoso esterilizados, contendo os agroquímicos nas doses recomendadas pelos fabricantes, observando-se as seguintes formas de aplicação: T1 - inoculação do fungo no solo 1 hora antes da aplicação do agroquímico; T2 - inoculação do fungo no solo 1 hora após a aplicação do agroquímico; T3 - inoculação do fungo no solo 48 horas após a aplicação do agroquímico. Avaliou-se a sobrevivência dos fungos por um período de sete dias através das unidades formadoras de colônias (UFC) em placas de Petri. Nos ensaios com meios de cultura, os inseticidas à base de Fipronil (Regente®) e Thiametoxan (Actara®) foram compatíveis com os fungos, mas Aldicarbe (Temik®) foi considerado tóxico. A maior parte dos herbicidas avaliados foram classificados como tóxicos aos entomopatógenos, mas aqueles formulados com Imazapir... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In the management of sugarcane are used entomopathogenic fungi to control insects; and various agrochemicals aimed at increasing productivity. However, several of these agrochemicals may interfere with survival of Beauveria bassiana and Metarhizium anisopliae. Therefore, this research had the objective to evaluate the toxicity of agrochemicals used in handling of sugarcane to B. bassiana and M. anisopliae in experiments in culture medium and soil. In tests in culture medium, the fungi were inoculated on potato dextrose agar (PDA) containing various insecticides (4), herbicides (7) and ripeners (3). It were evaluated the growth, sporulation and viability, and based on these parameters became the toxicological classification of agrochemicals. To examine whether the effect found in the culture medium remains in the soil environment, were used agrochemicals that showed higher toxicity in culture medium assays. At this stage the fungi were inoculated in clay and sandy soils sterilized, containing the chemicals in the recommended dosages, observing the following application forms: T1 - inoculation of the fungi in soil 1 hour before to herbicide application, T2 - inoculation fungi in the soil 1 hour after herbicide application, T3 - inoculation of the fungi in the soil 48 hours after herbicide application. It was evaluated the survival of the fungi for a period of seven days through the colony forming units (CFU) in Petri dishes. Insecticides based on Fipronil (Regente®) and Thiamethoxan (Actara®) are compatible with the fungi, but Aldicarbe (Temik®) proved to be toxic. Most of the herbicides evaluated were classified as toxic to entomopathogenic fungi, but those made with Imazapir (Contain®), Glyphosate (Glifosato®) and Metribuzin (Sencor®) were considered compatible. Among the ripeners only Glyphosate (Round up®) showed compatibility with B. bassiana and M. anisopliae... (Complete abstract click electronic access below) / Mestre

Orientadora: Janete Apparecida Desidério / Banca: Agda Paula Facincani / Banca: Irlan Leite de Abreu / Resumo: Como uma alternativa para diminuir as agressões constantes que o ecossistema vem sofrendo, devido à grande quantidade de produtos químicos utilizados no controle de pragas, pesquisas envolvendo microrganismos capazes de promover o controle biológico tem se intensificado. Dentre estes microrganismos a bactéria Bacillus thuringiensis tem se destacado. Essa bactéria caracteriza-se pela produção de proteínas tóxicas a representantes de diversas ordens de insetos. Em particular, as proteínas Vip3A, estão em amplo estudo devido a sua especificidade, alto potencial ativo e como alternativa para o controle da resistência de insetos às proteínas Cry. Diante disto, o objetivo deste trabalho foi selecionar, a partir de 1080 isolados de diferentes regiões brasileiras, aqueles portadores de genes vip3A e obter a sequência de nucleotídeos completa dos mesmos. As linhagens padrão B. thuringiensis var. kurstaki HD1, B. thuringiensis var. tolworthi HD125 e o isolado I187 tiveram seus DNAs amplificados com oligonucleotídeos baseados na sequência de genes vip3A, descritos no banco de dados de B. thuringiensis e, a partir dos amplicons obtidos, a sequência completa de nucleotídeos dos mesmos foi determinada, utilizando-se da estratégia de "primer walking". A proteína Vip3Aa43 (GenBank: [HQ594534]) da linhagem HD1 demonstrou ser 100% idêntica às proteínas Vip3Aa já descritas. Já as proteínas Vip3Aa42 (GenBank: [HQ587048]) da linhagem HD125 e Vip3Ag5 (GenBank: [HQ542193]) do isolado I187 demonstraram similaridade de 99% com as proteínas descritas Vip3Aa35 e Vip3Ag2, respectivamente, demonstrando serem duas novas proteínas Vip3A, devido às substituições de aminoácidos ocorridas. Os três genes vip3A obtidos poderão ser utilizados na produção de plantas Bt, piramidadas ou não, visando ao manejo da resistência dos insetos praga / Abstract: As an alternative to decrease the constant aggressions that the ecosystem has suffered due to the large amount of chemical products used in pest control, researches involving microorganisms able to promoting biological control have been intensified. Among these microorganisms the bacterium Bacillus thuringiensis has been stood out. This bacterium is characterized by the production of toxic proteins to representatives of several insect orders. In particular, the Vip3A proteins are in large study due to its specificity, and high active potential as an alternative to control of insect's resistance to Cry proteins. According to this, the aim of this work was to select from 1080 isolates in different Brazilian regions, those carrying vip3A genes and obtain the complete nucleotide sequence of the same. The standard strains B. thuringiensis var. kurstaki HD1, B. thuringiensis var. tolworthi HD125 and the isolate I187 had their DNA amplified with primers based on vip3A gene sequence described in database of B. thuringiensis, and from the amplicons obtained, the full sequence of nucleotides was determined, by the use of the strategy of "primer walking". The protein Vip3Aa43 (GenBank: [HQ594534]) of HD1 strain showed to be 100% identical to Vip3Aa proteins already described. However, the proteins Vip3Aa42 (GenBank: [HQ587048]) of HD125 strain and Vip3Ag5 (GenBank: [HQ542193]) of the isolate I187 showed 99% similarity with the Vip3Aa35 and Vip3Ag2 proteins described, respectively, showing been two new Vip3A proteins due to amino acid substitutions occurred. The three vip3A genes obtained can be used in the production of Bt crops, pyramidal or not, aiming to resistance management of pest insects / Mestre

Biological plant protection against insect pests is an important alternative to chemical protection. One of the most important group used in the biological plant protection against insect pests are the entomopathogenic fungi. Entomopathogenic fungi are microscopic fungi that are able to induce a primary disease to insect pests. It is a very heterogeneous group of species. Worldwide there were isolated and described more than 750 species of entomopathogenic fungi. Genus Beauveria is considered one of the most important genera of entomopathogenic fungi. In the Czech Republic there has been confirmed species B. bassiana, B. brongniartii and now, on the basis of this work, B. caledonica. This study is aimed to confirm the occurrence of B. caledonica in National Park Šumava. To confirm this occurrence, I used analyzes based on the methods of molecular markers. Molecular markers are an indispensable part of science in the field of mycology, for example the strain characterization, population genetics, detection and identification of fungi, phylogenetic studies and evolutionary biology. For this study there were used sequence analysis of ITS, EF1- and LSU regions. The output data of these analyzes were used to create phylogenetic trees. The result of my thesis is taxonomical classification of studied isolates on species level.

Entomopathogenic fungus \kur{Metarhizium anisopliae} is one of the most common species used in biological control against pests. The thesis is analyzing effectiveness of original strains and continuously passaged strains of \kur{M. anisopliae} through nutrient substrates and different developmental stages mealworm \kur{(Tenebrio molitor)}. For original and continuously passaged strains were also evaluated the growth and spore production at different temperatures cultivation. In this thesis was investigated the efficacy of the original strains of \kur{M. anisopliae} on selected economically important pests. Strains were tested on populations of adults Pollen beetles \kur{(Meligethes aeneus)} and Cabbage seedpod weevil \kur{(Ceutorhynchus obstrictus)} and the eggs of Colorado potato beetle \kur{(Leptinotarsa decemlineata)} in laboratory conditions. Other entomopathogenic fungi were tested on the eggs of Colorado potato beetle eggs.

During the evolution some groups of organisms have become coevolutionary associated with other groups, as is the case of host symbiont systems. To explore coevolutionary history of hosts and their associated symbionts, phylogenetic reconstruction of symbionts and phylogenetic reconstruction of hosts are usually compared. Coevolution is described by coevolutionary events (cospeciation, host switch, duplication, failure to diverge events and linage sorting events). The aim of this work was to test the suitability of MLST method for the complex of entomopathogenic nematodes from the genus Steinernema (with detailed analysis of Steinernema feltiae) and their symbiotic bacteria Xenorhabdus bovienii and subsequently use cophylogenetic comparative analysis to determine their level of cospeciation.