Entomopathogenic fungi are important controllers of pest-insects populations in agricultural production systems and in natural environment. These fungi have enzymatic machinery which involve since the recognition and adherence of spores in their hosts culminating with infection and death of these insects. The main objective of this study was to analyzed extracellular enzyme production of the fungi strains Beauveria bassiana, Metarhizium anisopliae and Paecilomyces sp when cultured on substrates. These fungi were grown in minimal media containing specific substrates for the analysis of different enzymes such as amylases, cellulases, esterases, lipases, proteases (gelatin and caseinase), pectinases and cuticles of Musca domestica larvae and adults. All the assays were performed with and without the presence of dextrose in the culture media. The quantification of enzyme activity was performed by the ratio of halo / colony (H/C) and the results subjected to variance analysis level of 5% (ANOVA) followed by post-Tukey test. All strains were positive for lipase and also they showed a high significant enzyme production for gelatin at concentrations of 4 and 1%. B. bassiana and Paecilomyces sp. were positive for amylase, pectinase and caseinase, and only Paecilomyces sp. showed cellulase activity.

Spot blotch disease, caused by Bipolaris sorokiniana, is one of the major diseases of wheat and is responsible for large losses of wheat crops worldwide. We used polymerase chain reaction (PCR) with universal rice primers (URP) for molecular characterization of 60 monosporic B. sorokiniana isolates from Brazil and other countries, and evaluated the diversity of the samples. PCR amplification generated 232 different DNA fragments ranging in size from 100 to 2018 bp. The primers URP-4R, URP-2R, and URP-1F generated greater numbers of amplified fragments (36, 30, and 25, respectively) from the single-spore isolates, and some diversity was observed among the isolates generated using these primers. The primers URP-2F, URP-6R, URP-17R, URP-30F, and URP-38F produced a pattern of monomorphic fragments and 73% of the isolates showed an average of 44 different DNA-amplified fragments. Primer URP-2F generated a 578-bp fragment that was common to 83.7% of the isolates; primer URP-6R generated a 548-bp fragment and primer URP-38F generated a 650-bp product that was common to 89.1% and 80% of the isolates, respectively. The URP-PCR primers provided important information about the genetic profiles of the monosporic cultures, which showed intraspecific variability among the monoconidial isolates and among the monosporic cultures that originated from the same polysporic strain. Our results indicate that URP's are sensitive and give reproducible results for assaying the genetic variability of B. sorokiniana.

Spot blotch caused by Bipolaris sorokiniana is a destructive disease of wheat in warm and humid wheat-growing regions of the world. This fungus shows a high genetic diversity and morphological and physiologic variability. In this study, 19 polysporic and 57 monosporic isolates of B. sorokiniana were characterized using universal rice primers-URP-PCR. The results obtained when the dendrogram was constructed with all the data produced with the amplification products showed very distinct clusters. However, the similarity among the isolates was low where 37 and 26.3 % of the monosporic and polysporic isolates, respectively, showed similarity above 70 %. All primers amplified multiple DNA fragments of polysporic as well as the monosporic isolates. Isolates fingerprints were constructed based on binary characters revealed by the three primers. An amplified fragment of approximately 750 bp was observed among 40 % of the isolates, when primer URP-1F was used. When primers URP-4R and URP-2R were used, a fragment of 450 and 400 bp was present in 31.5 and 29 % of the isolates, respectively. It was expected a higher similarity among the isolates since the monosporic cultures were originated from the polysporic. The dendrogram did not enable the separation of B. sorokiniana isolates by their geographic origin. This low correlation suggests that gene transfer may have occurred by parasexual combination in this fungus population. However, in spite of the research efforts for that end, it has not been possible to establish patterns that characterize the profile of B. sorokiniana.

Actinobacteria secrete substances that limit or inhibit the growth of plant pathogenic fungi and may be used in the biocontrol of these microorganisms. The aim of this study was to characterize physiological and enzymatic activity of endophytic actinobacteria, evaluate their antifungal activity against Bipolaris sorokiniana root colonization, and evaluate their efficiency in promoting the growth of wheat seedlings. Antibiosis was analyzed using the double-layer method, the agar well diffusion test, and volatile metabolites. Physiological and enzymatic activity was evaluated through chitinase, glucanase, siderophores, indole-3-acetic acid, nitrogen fixation and phosphate solubilization tests. In vivo assays were evaluated by root colonization, biocontrol test and efficiency to promote the growth of wheat seedlings. From all isolates tested, 69.6% of them presented antifungal activity against at least one B. sorokiriana isolate. Among these, 17% of the isolates produced bioactive metabolites in the supernatant when grown in submerging culture. The highest production of bioactive metabolites was at 30°C, between 72 and 96 h of incubation. Three isolates produced volatile compounds, chitinase, glucanase, siderophores and exhibited nitrogen fixation, produced indole-3-acetic acid, efficiently colonized the root system of seedlings of two wheat cultivars. The best isolate [6(2)] showed, under the greenhouse, the capacity to promote an increased biomass and tillers per wheat plant.

Bipolaris sorokiniana may present considerable genetic diversity and highly variable pathogenicity and virulence. The pathogenicity of 99 B. sorokiniana isolates (27 polysporic and 72 monosporic isolates) from Brazil and other countries was assessed. Based on aborted germination, black point of seed, leaf spot, and coleoptile lesion, the principal component analysis (PCA) was used to evaluate the similarity patterns between isolates considering the variables of pathogenicity. Polysporic isolates presented higher virulence (over 60%), when compared with the monosporic isolates (43%) for all variables, except coleoptile injury. Of all isolates used to infect seeds, 8% were highly virulent, and the score obtained was over 75%, for all variables analyzed. The correlation of B. sorokiniana isolates with pathogenicity variables demonstrated that polysporic isolates were more virulent, especially upon seeds, as compared to aerial plant parts.