Claudio Luiz Messias

Effects of UVB Irradiance on Conidia and Germinants of the Entomopathogenic Hyphomycete Metarhizium anisopliae: A Study of Reciprocity and Recovery¶

We tested the effects of irradiances of 920 and 1200 mW m−2 (weighted irradiance) on the conidia and germinants of the entomopathogenic Hyphomycete Metarhizium anisopliae. The conidia were exposed to the two irradiances for 1, 2, 4, 6, 7 or 8 h. Increased exposure decreased relative percent culturability. The inactivation provoked by the irradiance of 1200 mW m−2 was higher than for the 920 mW m−2, with a reduction in the 50% lethal time (LT50) from 6 h 40 min to 4 h 26 min. Reciprocity was not observed when conidia in water suspension and germinants in different stages of the germinative process were exposed to a 17.3 kJ m−2 total dose at both irradiance levels. Although nonreciprocity was observed in all situations, its magnitude varied as a function of metabolic state and/or cell‐cycle phase in which the conidia were at the exposure time. The least difference between the effects of the two irradiance levels was observed when nongerminating conidia in suspension were exposed, and the greatest was observed when conidia were exposed during an advanced germination phase. Doses of 6.6 and 17.3 kJ m−2 supplied through the two irradiance levels delayed the germination of the surviving conidia. At both doses, delay was greater during exposure to the higher irradiance. Nonreciprocity was higher for the 17.3 kJ m−2 dose. Nonreciprocity magnitude, in addition to depending on the conidial physiological state, also depended on dose. The results demonstrate the importance of evaluating the impact of the increase in irradiance during the different stages of the fungal life cycle, especially during the stages which are more sensitive to UV, and not simply in dormant conidia.

Effect of UV-B on conidia and germlings of the entomopathogenic hyphomycete Metarhizium anisopliae

The effects of two uv irradiances (920 and 1200 mW m −2 weighted irradiance) on the conidia and germlings of the ARSEF 2575 and ARSEF 23 strains of M. anisopliae were studied. Conidia were exposed to the two irradiance levels for 1, 2, 3, 4, 6, or 8 h. The 30% increase (from 920 to 1200 mW m −2 ) in uv irradiance caused a significant decrease in culturability following all periods of exposure and reduced the 50% lethal time (TL 50 ) 36% for strain 2575 and 48% for strain 23. Exposure to uv radiation of only 1 h caused a delay of several hours in the germination of surviving conidia. Longer periods of exposure delayed germination for days, demonstrating that, depending on the dose, the fungus may require a long period of time to recover and to resume germination. The results demonstrated the inability of the fungus to germinate during direct exposure to the uv-B portion of simulated sunlight. Both strains showed a transitory increase in uv tolerance during germination. The beginning of germination increased uv tolerance. However, starting on the 6th hour of germination, a decrease in tolerance was observed, indicating that uv tolerance varies as a function of physiological state and cell-cycle phase.

Production of extracellular enzymes by isolates of Metarhizium anisopliae

Abstract Extracellular enzyme production in solid culture media was analyzed in order to determine the variability among different Metarhizium anisopliae isolates. Using specific substrates, amylase, lipase, chitinase, and protease production was tested in 11 isolates from different regions of Brazil. Enzyme production was determined by the formation of a halo around the colony, and the diameters of both halo and colony were measured. The enzymatic index was expressed by the colony diameter/halo diameter ratio. In general, the isolates from the same region had similar enzymatic indexes, although similar indexes were also found for isolates from geographically distinct regions. The different isolates were tentatively grouped according to index similarity.

Electrophoretic variation in esterases and phosphatases in eleven wild-type strains ofMetarrhizium anisopliae

11 wild-type strains ofMetarrhizium anisopliae were electrophoretically tested for phosphatases and esterases. Phosphatase was homogeneous for all strains. For esterase it was possible to distinguish 5 different patterns with which it is possible to characterize the strains analyzed.

Protease production during growth and autolysis of submerged Metarhizium anisopliae cultures

The growth and autolysis of two strains of the entomopathogenic deuteromycete fungus Metarhizium anisopliae var. anisopliae were evaluated in medium containing casein or glucose as carbon source. Parameters such as economic coefficient and degree of autolysis were determined for each strain. Protease production was determined throughout the growth and autolysis phases of the cultures on medium under conditions of protease induction (in the presence of casein as sole source of carbon and nitrogen). The fungus was shown to utilize casein as a carbon/energy source in a more efficient manner than glucose. The autolysis shown by the strains was intense under both types of growth conditions, reaching up to 62.7% of the dry mass produced and started soon after the depletion of the exogenous carbon source. The relationship between the proteolytic activities of the two strains evaluated varied significantly (a maximum of 19.78 on the 5th day and a minimum of 2.03 on the 16th day of growth) during the various growth and autolysis phases, clearly showing that the difference between the growth curves and the difference in the kinetics of enzyme production may decisively affect the process of strain selection for protease production.

Effectiveness of Metarhizium anisopliae against immature stages of Anastrepha fraterculus fruitfly (Diptera : Tephritidae)

The study evaluated the effectiveness of Metarhizium anisopliae var. anisopliae (Hyphomycetes : Moniliales) strain E9, isolated from the pasture spittlebug Deois flavopicta (Hemiptera : Cercopidae), against larvae, prepupae and pupae stage and emergent adults of Anastrepha fraterculus, the South American fruitfly. The bioassay was carried out simulating field conditions, on autoclaved (AS) and non-autoclaved (NAS) soil from typical citrus orchards in the State of Sao Paulo, Southeastern region of Brazil. Various concentrations of conidia were incorporated into the soil the mortality, calculated based on the percentage of adult emergence, was 86% for the highest conidia concentrations: 2.52 x 1010 for AS and 2.52 x 1010 for NAS. The lethal concentration (LC50), expressed as conidia concentration, was 8.44 x 109 conidia/g of soil (S) for AS and 12.23 x 109 conidia/g of soil for NAS.

Total lipids and fatty acids of strains of Metarhizium anisopliae

Dois estagios de desenvolvimento a saber: conidio (C) e micelio (M) em 10 linhagens de Metarhizium anisopliae var. anisopliae e 2 M. anisopliae var. majus, foram estudados em dois diferentes meios de cultivos, minimo (MM) e completo (MC). As linhagens apresentaram-se similares quanto a porcentagem de lipidios totais, porem submetidas ao teste de Tukey, as medias dos conteudos de lipidios do conidio (C) e do micelio (M) cultivados em meio MM e CM respectivamente, mostraram diferenca significativa ao nivel de 5% , indicando assim variabilidade para a producao de lipidios totais. As linhagens 5 e 7, ambas da var. majus, nao apresentam diferencas mensuraveis da variedade anisopliae. Com os acidos graxos C18:1 e C18:2 oleico e linoleico respectivamente, as diferencas foram todas altamente significativas (p= 1%) com a mais alta media de C18:1 para conidio e C18:2 para micelio.

Estimates of genetic parameters related to chitinase production by the entomopathogenic fungus Metarhizium anisopliae

Chitinolytic activity and dry mass production were determined in culture filtrates from 17 Metarhizium anisopliae strains grown in liquid medium containing chitin as the only carbon source. The objectives were to estimate parameters such as genetic variance among strains, heritability and expected gain from selection, as well as correlations between tested traits. Wide genotypic variability was observed among strains in chitinolytic activity, permitting the exploitation of this property in selection. The high heritability suggests that progress can be made through phenotypic selection. The genotypic correlation coefficient between dry mass production and chitinolytic activity detected in the filtrates was negative (= - 0.588). One of the isolates was also investigated for variation in the two traits as a function of culture growth time. The results showed an increase in enzyme activity up to the 8th (and last) day of the experiment and a decrease in dry mass from the 4th day on.

Virulence of mutants and revertants of Metarhizium anisopliae var. anisopliae toward Rhodnius prolixus

Abstract The derivation of mutants of Metarhizium anisopliae var. anisopliae which had lost or showed reduced ability to produce extracellular amylases, lipases, and proteases was studied by the enzymatic index. Reversion induced by ultraviolet light was also studied. The virulence of mutants and revertant strains was evaluated in bioassays against third-instar nymphs of Rhodnius prolixus. The mutant strains for amylase and lipase showed enzymatic indices equal to one, but this was not the case for the proteolytic mutants. The revertant strains were phenotypically similar to the parental strains. The virulence of amylolytic and lipolytic mutants was reduced in relation to the parental strain, whereas the proteolytic mutants did not show any significant reduction. Virulence was restored in all revertant strains.

Quantitation of organic acids in wine and grapes by direct infusion electrospray ionization mass spectrometry

Several organic acids such as malic, tartaric and citric acids are key grape and wine constituents. They greatly contribute to the organoleptic properties of grapes and wines and are responsible for their acidity, a crucial wine property that is known to considerably vary as a function of grape variety, environmental conditions and viticulture, as well as during wine maturation. A rapid and simple method, requiring minimum sample pre-treatment and no chromatographic separation, based on direct infusion electrospray ionization mass spectrometry (ESI-MS) was developed and validated to quantitate organic acids in wines and grapes. This method was also demonstrated to be able to monitor a common wine adulteration procedure, that is, addition of citric acid for pH adjustment. ESI-MS data when treated via principal component analysis (PCA) was also found to group wine samples based on their profiles and concentrations of organic acids.