Improvement of Akanthomyces lecanii resistance to tebuconazole through UV-C radiation and selective pressure on microbial evolution and growth arenas.

Tebuconazole (TEB) is a fungicide widely used in agriculture; however, its constant application has increased the emergence of resistant plant pathogenic fungal strains and reduced the effectiveness of fungi as biological control agents; for instance, the entomopathogenic and hyperparasitic fungus Akanthomyces lecanii, suitable for simultaneous biological control of insect pest and plant pathogenic fungi, is highly sensitive to fungicides. We carried out the induction of resistance to TEB in two wild type strains of A. lecanii by UV radiation and selective pressure in increasing fungicide gradients using a modified Microbial Evolution and Growth Arena (MEGA), to produce A. lecanii strains that can be used as biological control agent in the presence of tebuconazole. Nine UV-induced and three naturally adapted A. lecanii strains were resistant to TEB at the agriculturally recommended dose, and three irradiated strains were resistant to TEB concentration ten times higher; moreover, growth, sporulation rates, production of hydrolytic enzymes, and virulence against the hemipteran Coccus viridis, a major pest of coffee crops, were not affected in the TEB-resistant strains. These A. lecanii TEB-resistant strains would have a greater opportunity to develop and to establish themselves in fields where the fungicide is present and can be used in a combined biological-chemical strategy to improve insect and plant pathogenic fungal control in agriculture. Also, the selective pressure through modified MEGA plate methodology can be used for the adaptation of entomopathogenic filamentous fungi to withstand other chemical or abiotic stresses that limits its effectiveness for pest control.

Induction of esterase activity during the degradation of high concentrations of the contaminant di(2-ethylhexyl) phthalate by Fusarium culmorum under liquid fermentation conditions.

In this study, the induction of esterase activity during the degradation of a high concentration of di(2-ethylhexyl) phthalate (DEHP) (1500 mg l-1) by Fusarium culmorum was investigated using Ca(NO3)2 as nitrogen source under liquid fermentation conditions. Assessments of esterase activities through biochemical tests and zymographic assays, as well as fungal growth were studied. A high concentration of DEHP increased esterase activity in F. culmorum, which produces five esterase isoforms (26.4, 31.7, 43, 73.6 and 125 kDa), which were different in abundance and molecular weight to those produced constitutively in glucose-containing medium (control medium). F. culmorum showed higher µ and Y X/S values in DEHP-containing medium than those observed in the control medium. F. culmorum has great potential for use in the restoration of sites contaminated with high concentrations of DEHP and even of other phthalates with less complex structures.

Evaluation of bezafibrate, gemfibrozil, indomethacin, sulfamethoxazole, and diclofenac removal by ligninolytic enzymes.

The laccase (Lac), manganese peroxidases (MnP), and lignin peroxidase enzymes produced by basidiomycete have been studied due to their potential in bioremediation, therefore, in this study, degradation of diclofenac (DCF), sulfamethoxazole (SMX), indomethacin (IND), gemfibrozil (GFB), and bezafibrate (BZF) by enzymes produced by Trametes maxima, Pleurotus sp., and Pycnosporus sanguineus grown in culture was evaluated. The degradation of drugs can mainly be attributed to MnP because a correlation between the activity of this enzyme and the degree of removal was found. The specific activity of Lac did not show correlation with drug removal, while lignin peroxidase was not expressed. Trametes maxima showed the highest specific activity of MnP (387.6 ± 67.4 U/mg) and efficiency removal 90.2% of DCF, 72.62% of SMX, 60.76% of IND, 43.39% of GFB, and 32.59% of BZF) followed by Pleurotus sp. with specific activity of MnP of 55.9 ± 8.5 U/mg and 89.47% of DCF, 47.61% of GFB and 73% of IND were removed, P. sanguineus had the lowest specific activity of 18 ± 1.3 U/mg and was able to remove only 42% of SMX and 10.59% of IND. In order to prove that MnP remove drugs instead of Lac, the pure Lac was tested and only degraded DCF.

Biodegradation patterns of the endocrine disrupting pollutant di(2-ethyl hexyl) phthalate by Fusarium culmorum.

Di(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer, which is considered an endocrine disrupting pollutant. Growth kinetics and esterases activity by biochemical tests and polyacrylamide gel electrophoresis were characterized for Fusarium culmorum grown in DEHP-supplemented (1000 mg/L) medium as the only carbon source and in control medium with glucose. Intermediate compounds of biodegraded DEHP were identified by GC-MS. F. culmorum degraded 92% of DEHP within 36 h. DEHP was degraded to butanol, hexanal, catechol and acetic acid. It is suggested that the first two compounds would transform into butanediol and the last two would enter into the Krebs cycle and would be mineralized to CO2 and H2O. DEHP induced eight esterase isoforms, which were different to those constitutive isoforms produced in the control medium. It is suggested that five enzymes (25.7, 29.5, 31.8, 97.6 and 144.5 kDa) detected during the first 36 h be involved in the primary biodegradation of DEHP. The rest of the enzymes (45.9, 66.6 and 202.9 kDa) might be involved in the final steps for DEHP metabolism. F. culmorum has a promising practical application in the treatment of DEHP-contaminated environments because it can secrete specific esterase to breakdown high concentrations of DEHP in a short period of time. This research represents the first approach for the study of esterase involved in the DEHP degradation by fungi using this phthalate as the sole source of carbon and energy.

Production of Conidia by the Fungus Metarhizium anisopliae Using Solid-State Fermentation.

This chapter describes the production of conidia by Metarhizium anisopliae using solid-state fermentation. Before production of conidia, procedures for strains conservation, reactivation, and propagation are essential in order to provide genetic stability of the strains. The strain is conserved in freeze-dried vials and then reactivated through insect inoculation. Rice is used as a substrate for the conidia production in two different bioreactors: plastic bags and tubular bioreactor. The CO2 production in the tubular bioreactors is measured with a respirometer; this system allows calculating indirect growth parameters as lag time (tlag) (25-35 h), maximum rate of CO2 production (rCO2 max) (0.5-0.7 mg/gdm h), specific rate of CO2 production (µ) (0.10-0.15 1/h), and final CO2 production (CO2) (100-120 mg/gdm). Conidial yield per gram of dry substrate (gdm) should be above 1 × 10(9) conidia/gdm after 10 days of incubation. Germination and viability of conidia obtained after 10 days of incubation should be above 80 % and 75 %, respectively. Bioassays using of Tenebrio molitor as a host insect should yield a final mortality above 80 %.

Considerations on the use of exogenous fibrolytic enzymes to improve forage utilization.

Digestion of cell wall fractions of forage in the rumen is incomplete due to the complex links which limit their degradation. It is therefore necessary to find options to optimize the use of forages in ruminant production systems. One alternative is to use exogenous enzymes. Exogenous fibrolytic enzymes are of fungal or bacterial origin and increase nutrient availability from the cell wall, which consists of three fractions in different proportions depending on the species of forage: digestible, potentially digestible, and indigestible. The response to addition of exogenous enzymes varies with the type of forage; many researchers infer that there are enzyme-forage interactions but fail to explain the biological mechanism. We hypothesize that the response is related to the proportion of the potentially digestible fraction. The exogenous enzyme activity depends on several factors but if the general conditions for enzyme action are available, the potentially digestible fraction may determine the magnitude of the response. Results of experiments with exogenous fibrolytic enzymes in domestic ruminants are inconsistent. This, coupled with their high cost, has made their use unattractive to farmers. Development of cheaper products exploring other microorganisms with fibrolytic activity, such as Fomes fomentarius or Cellulomonas flavigena, is required.

Actividad fibrolítica de enzimas producidas por Trametes sp. EUM1, Pleurotus ostreatus IE8 y Aspergillus niger AD96.4 en fermentación sólida / Fibrolytic activity of enzymes produced by Trametes sp. EUM1, Pleurotus ostreatus IE8 and Aspergillus niger AD96.4 in soild fermentation

Se estudió la actividad de enzimática (UI·g-1·MS) y la actividad específica (UI·mg-1 proteína) de xilanasas, celulasas y lacasas de Trametes sp. EUM1, Pleurotus ostreatus IE8 y Aspergillus niger AD96.4, a los 14 y 19 días de fermentación sólida en bagazo de caña de azúcar. Las xilanasas y celulasas producidas por Trametes sp. EUM1 mostraron la mayor actividad (P<0,01) expresando 141,77UI·g-1 MS y 1073,8UI·mg-1 proteína las primeras, y 9,04UI·g-1 MS y 69,16UI·mg-1 proteína las celulasas, sin diferencias significativas entre 14 y 19 días. La actividad de estas enzimas de P. ostreatus y A. niger fue menor (P<0,01) que Trametes sp., pero similar entre ellos y en ambos tiempos de fermentación. La mayor (P<0,01) actividad de lacasas fue expresada por P. ostreatus con promedios de 15,54UI·g-1 MS y 128,75UI·mg-1 proteína a los 14 días de fermentación, mayores (P<0,01) que a los 19 días (11,75UI·g-1 MS y 102,88UI·mg-1 proteína). En Trametes sp., la actividad de lacasas fue menor (P<0,01) que en P. ostreatus y similar en ambos tiempos de fermentación. En A. niger la actividad de lacasas fue menor (P<0,01) con respecto a Trametes sp. y P. ostreatus. La actividad de las enzimas fibrolíticas del Trametes sp. muestra potencial para su posible utilización en aplicaciones biotecnológicas.