Orientador: Antonio Carlos Monteiro / Banca: Odair Aparecido Fernandes / Banca: Luis Garrigós Leite / Resumo: A broca da cana Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae) é responsável por grandes prejuízos na cultura da cana-deaçúcar. O controle tem como alvo principal somente a fase de lagarta. No entanto, a eficiência pode ser maior atingindo fases do ciclo da praga mais expostas aos agentes de controle. O objetivo deste estudo foi avaliar a patogenicidade de Metarhizium anisopliae (Metsch.) Sorok. Beauveria bassiana (Bals.) Vuill. e Isaria farinosa (Holmsk.) Fr. para ovos, lagartas, pupas e adultos de D. saccharalis e avaliar, através da análise de parâmetros biológicos, os efeitos no ciclo de vida da praga. Ovos postos no período de 24 horas, lagartas com 15 dias de desenvolvimento, pupas recém formadas e adultos com 24 horas de vida foram inoculados com os fungos nas concentrações de 108, 107 e 106 conídios mL-1. A viabilidade dos ovos foi afetada significativamente no tratamento com 108 con. mL-1 de M. anisopliae, mas as lagartas sobreviventes não foram afetadas em nenhum outro parâmetro avaliado. M. anisopliae e B. bassiana causaram mortalidade de lagartas principalmente nas concentrações de 108 e 107 con. mL-1, enquanto a viabilidade pupal e o peso de pupas fêmeas formadas a partir de lagartas tratadas foram reduzidos apenas pelos tratamentos com 108 e 107 con. mL-1 de M. anisopliae, respectivamente. I. farinosa não se mostrou patogênico para as fases de ovo e lagarta de D. saccharalis. O tratamento com 108 con. mL-1 de M. anisopliae e B. bassiana produziu 52,0 e 42,0% de mortalidade de pupas, respectivamente, afetando também a viabilidade de pupas e a longevidade de adultos emergidos. A fecundidade destes adultos foi severamente afetada por I. farinosa na concentração 108 con. mL-1 e pelos outros fungos em todas as concentrações avaliadas. Verificou-se grande mortalidade de adultos tratados com todas as concentrações de M. anisopliae... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The sugarcane borer Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae) is responsible for high losses in sugarcane. The larval stage is the main target of control. However, the efficiency can be higher by reaching different and more exposed stages with more exposure to the control agents. The aim of this study was to evaluate the pathogenicity of Metarhizium anisopliae (Metsch.) Sorok. Beauveria bassiana (Bals.) Vuill. and Isaria farinosa (Holmsk.) Fr. to eggs, larvae, pupae and adults of D. saccharalis and to evaluate the effects on pest lifecycle. Eggs with 24 hours, larvae with 15 days of development, newly formed pupae and adults with 24 hours of life were inoculated with fungi at concentrations of 108, 107 and 106 conidia mL-1. The eggs viability was significantly affected in the treatment with 108 con. mL-1 of M. anisopliae, but the survival larvae were not affected in any of the analyzed parameters. M. anisopliae and B. bassiana caused larval mortality mainly at concentrations of 108 and 107 con. mL-1, while pupae viability and female pupal weight formed from treated larvae were reduced only by treatments with 108 and 107 con. mL-1 of M. anisopliae, respectively. I. farinosa wasn't pathogenic to eggs and larvae of D. saccharalis. The treatment with 108 con. mL-1 of M. anisopliae and B. bassiana produced 52.0 and 42.0% pupae mortality respectively, also affecting the pupal viability and the longevity of adults. The fecundity of these adults was severely affected by I. farinosa in the concentration of 108 con. mL-1 and by other fungi in all evaluated concentrations. High mortality of treated adults with all concentrations of M. anisopliae and with 107 con. mL-1 was observed. The longevity and fecundity were not affected by the fungi, and the eggs viability laid by treated females was significantly reduced by treatment with 106 and 108 con. mL-1 of B. bassiana... (Complete abstract click electronic access below) / Mestre

This thesis is focused on evaluation of the compatibility of entomopathogenic fungus Isaria fumosorosea with different species of entomopathogenic fungi and mycoparasitic fungi at different temperatures. The strains of species Beauveria bassiana, Metarhizium anisopliae, Lecanicillium muscarium, Coniothyrium minitans and Clonostachys rosea f. catenulata were selected for experiments base on compatibility. The results showed that combination of I. fumosorosea with species L. muscarium is compatible. The species do not limit to each other in the environment at all temperatures (15, 23 and 25 °C). On the other side, fungus I. fumosorosea in combination with other species have affected their growth and spore production. The efficacy of entomopathogenic fungi against larvae Tenebrio molitor was evaluated. The most effective species against larvae were species I. fumosorosea, B. bassiana and M. anisopliae. On the contrary, the smallest effective was observed after infection larvae by L. muscarium. Mycoparasitic fungus C. rosea f. catenulata was not able to directly infect larvae of T. molitor. This species did not infect healthy larvae. However it is able to infect weakened individuals or is growing as saprotrophs on the cadavers.

The potential synergism between chosen species of entomopathogenic nematodes (Steinernema arenarium, Steinernema feltiae) and entomopathogenic fungi (Beauveria bassiana, Isaria fumosorosea, Lecanicillium lecanii) is investigated in this study. It is theoretically possible to expect increasing of their efficiency at the collective introduction into environment. Creating of uniform laboratory method which will be possible to use at the research of this interaction system is a part of this study.

Elucidation of the primary structure of peptides and proteins de novo by mass spectrometry (MS) has become possible with the advent of tandem MS methods. The most widely used chemical method due to Edman (Edman & Begg, 1967) has shortcomings with regard to N- terminal blocked peptides, cyclic peptides and posttranslational modifications, for example phosphorylation (Metzger, 1994). However, mass spectrometric sequencing methods are increasingly becoming applicable for a variety of peptides and proteins, including N- and C- termini modified peptides and cyclic peptides (Jegorov et al., 2003; Sabareesh & Balaram, 2006; Sabareesh et al., 2007). Further, conventional and tandem mass spectrometry have proven useful in the detection of post-translational modifications (Hansson et al., 2004; Nair et al., 2006; Mandal et al., 2007). This thesis details mass spectrometric sequencing of acyclic and cyclic peptides, involving tandem MS methods carried out using both electrospray ionization (ESI) ion trap (Esquire 3000 plus, Bruker Daltonics) and matrix assisted laser desorption and ionization time-of-flight/time-of-flight (MALDI TOF/TOF) (Ultraflex TOF/TOF, Bruker Daltonics) instruments. The peptides are either chemically synthesized or isolated from diverse natural sources. Synthetically designed peptides possessing modified N- and C- termini and peptaibols from the soil fungus Trichoderma constitute the acyclic peptides. The cyclic peptides include backbone cyclized depsipeptides from the fungus Isaria and disulfide bonded peptides from the venom of marine cone snails. Chapter 1 gives an account of various concepts of mass spectrometry, tandem mass spectrometry and peptide fragmentation chemistry, providing necessary background information for the following chapters. Chapter 2 describes the fragmentation studies of [M + H]+ and [M + Na]+ adducts of six neutral peptides with blocked N- and C- termini investigated using an electrospray ion trap mass spectrometer. The N- terminus of these synthetically designed peptides is blocked with a tertiarybutyloxycarbonyl (Boc) group and the C- terminus is esterified. These peptides do not possess sidechains that are capable of complexation and hence the backbone amide units are the sole sites of protonation and metallation. The cleavage pattern of protonated adducts is strikingly different from that of sodium adducts. While the loss of the N- terminal blocking group happens quite readily in the case of MS/MS of [M + Na]+, the cleavage of C- terminal methoxy group seems to be a facile process in the case of MS/MS of [M + H]+. Fragmentation of the protonated adducts yields only bn ions, while yn and an type ions are predominantly formed from the fragmentation of sodium adducts. The an ions arising from the fragmentation of [M + Na]+ lack the N-terminal Boc group (termed as an*). MS/MS of [M + Na]+ species also yields bn ions of substantial lower intensities, that lack the N- terminal Boc group (bn*). Comparison of the fragmentation of [M + H]+ with [M + Na]+ of the peptides chosen in this study reveal that the combined use of both protonated and sodium adducts should prove useful in de novo sequencing of peptides that possess modified N- and C- termini, particularly naturally occurring neutral peptides, for example, peptaibols. Chapter 3 describes about the ESI-MS/MS investigation of an HPLC fraction from the soil fungus Trichoderma, which aided in identification of microheterogeneous trichotoxin peptaibols in that fraction. Dramatic differences were noted between the fragmentation spectra of [M + H]+ and [M + Na]+ species. While b-type ions were noted from the former, the latter yielded a-, b-and y- type ions (the same feature was noted in the cases presented in the previous chapter). Inspection of the isotope pattern of b-ions yielded from the dissociation of H+ species, clearly revealed the presence of three microheterogeneous trichotoxin sequences; two isobars (1718 Da), each possessing one Glu residue and another completely neutral peptide (1717 Da). The microheterogeneity is due to Gly ↔ Ala, Iva ↔ Aib and Gln ↔ Glu replacements and exchanges (Iva: DIva: R-Isovaline; Aib: α-aminoisobutyric acid). The MS/MS of [M + Na]+ adduct predominantly yielded product ions from the neutral peptaibol. Further, the fragmentation patterns of H+ and Na+ adducts of two N-acetyl peptide esters were found to be very similar to that of the neutral peptaibol component. The results presented in this chapter establish that under the electrospray ion trap conditions, the fragmentation patterns of the H+ and Na+ adducts of model peptides that possess modified N- (Boc and acetyl) and C- termini are indeed very similar to that of the neutral trichotoxin. Chapter 4 delineates the applicability of liquid chromatography coupled to conventional and tandem electrospray ionization mass spectrometry (LC-ESI-MS, LC-ESI-MS/MS, LC-ESI-MS3) for the screening of novel cyclic hexadepsipeptide metabolites directly from the crude hyphal extract of the fungus Isaria. The fungal strain was grown on a solid medium (potato carrot agar), which yields aerial hyphae growing erect from the basal mycelial colony (Ravindra et al., 2004). A total of ten microheterogeneous components were identified to belong to the isariin class of cyclodepsipeptides from the LC-ESI-MS and LC-ESI-MS/MS analysis of the crude hyphal extract. Out of ten, six are determined to be new and the remaining four are previously reported isariins A-D. The primary structures of isariins A-D were from the fungi Isaria cretacea and Isaria felina (Vining & Taber 1962; Deffieux et al., 1981) and the fungal strain used in this study resembles Isaria felina (Sabareesh et al., 2007). Isariins are backbone cyclized hexadepsipeptides composed of a D-β-hydroxy acid possessing a hydrocarbon sidechain and five α-amino acids; one of the α-amino acids is a D-amino acid (Vining & Taber 1962; Deffieux et al., 1981). The detection of fragment ions due to loss of CO concomitant with the loss of H2O from the protonated precursor ion ([M + H]+) ascertained the cyclic depsipeptide nature of both the known and the new components. The fragmentation behavior of the [M + H]+ of known isariins facilitated sequence determination of the new components. Therefore, the configuration of the amino acids and the β-hydroxy acid of the new components is assumed to be same as that of the reported peptides. The microheterogeneity of the ten sequences is due to changes in the D-β-hydroxy acid (residue 1) and the adjoining α-amino acid (residue 6), whose carbonyl is linked to the hydroxyl function by an ester linkage. The number of methylene units ((-CH2)n) in the hydrocarbon sidechain of the residue 1 differs between 2 and 8 and the variability of the residue 6 is limited to Ala/Val. The ester oxygen atom was chosen as the preferable site of protonation causing ring-opening, based on the observed distribution of the fragment ions. Chapter 5 demonstrates the utility of the LC-ESI-MS and LC-ESI-MS/MS methods in the identification and characterization of six microheterogeneous backbone cyclized hexadepsipeptides, isaridins, directly from the crude hyphal extract of the fungus Isaria. Among the six components, four were found to be novel. The other two peptides, isaridins A and B were identified earlier from this laboratory (Ravindra et al., 2004). The isaridins are characterized by the presence of unusual amino acids such as N-methylated residues, β-methylproline (β-MePro) and hydroxyleucine (HyLeu) (Ravindra et al., 2004). The cyclic nature of both the known and the new peptides were confirmed from the observation of peaks due to loss of CO and H2O from the protonated precursor ion ([M + H]+). However, unlike isariins (Chapter 4), the intensity of the peak corresponding to [M + H - H2O]+ was noted to be of very low intensity, in the case of isaridins. Detection of product ion peak due to [M + H - CO2]+ suggests an additional dissociation pathway involving cleavage at the depsipeptide linkage and is supportive of the cyclic depsipeptide nature (Eckart, 1994). The sequencing of the newly detected components was enabled by understanding the fragmentation mechanism of the known isaridins. The tertiary amide nitrogens of the N-methylated residues were regarded as the preferable sites of protonation leading to ring-opening, as noted from the fragmentation spectra. The microheterogeneity in the sequences was identified using the diagnostic product ions obtained from the protonated precursor of the known isaridins. The microheterogeneity can be attributed to the variations of two residues; Pro ↔ β-MePro and N-MePhe ↔ N-MeLxx (Lxx: Leu, Ile, alloIle). The recently reported ‘isarfelins’ from the fungus Isaria felina (Guo et al., 2005) were reassigned as ‘isaridins’. The reassignment was based on very similar fragmentation profiles observed for the [M + Na]+ adduct of isaridins and isarfelins; further, the fungal strain used in this study resembles Isaria felina (Sabareesh et al., 2007). Chapter 6 presents mass spectrometric sequencing of disulfide bonded peptides from marine cone snails (conopeptides), using the MALDI LIFT MS/MS method. Lo959, a single disulfide bonded octapeptide isolated from Conus loroisii, was identified to belong to the class of contryphans (Sabareesh et al., 2006). Contryphans are small single disulfide bonded conopeptides, whose length is in the range of 7-11 residues and are rich in tryptophan. A significant feature of the contryphans is the presence of conserved DTrp (DW) at the 3rd residue within the disulfide loop (Sabareesh et al., 2006). Lo959 displays an unusual behavior under reverse phase chromatographic conditions, typical of the DW containing contryphans (Jacobsen et al., 1998). It undergoes slow conformational interconversion on the chromatographic time scale exhibiting two distinct peaks. The presence of DW at the 4th position in Lo959 was established by comparing the chromatographic profiles of natural peptide with that of two chemically synthesized peptides, one containing LW (4) and another possessing DW (4). De novo sequencing of the two peptides Ar1446 and Ar1430 from Conus araneosus established that they belonged to M-superfamily of conotoxins, in particular m-2 branch. M-superfamily conotoxins are three-disulfide bonded peptides characterized by the consensus cysteine framework, CC…C…C…CC (Corpuz et al., 2005). Ar1446 and Ar1430 are fourteen residue long peptides, each possessing three disulfide bonds. The peptides have the cysteine scaffold typical of the M-superfamily, as shown above. Specifically, the peptides belong to m-2 branch of M-superfamily, where the fourth and fifth cysteines are separated by two residues (Corpuz et al., 2005). The sequences of the peptides were derived following chemical and enzymatic modifications. The carboxamidomethylation reaction established the presence of three disulfide bonds. Indeed, the sequences were deduced from the MALDI LIFT MS/MS of [M + H]+ of the tryptic peptides. The sequences of the two peptides are almost identical and they differ only at residue 12; hydroxyproline in Ar1446, proline in Ar1430.

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.