Fusarium species associated with citrus blackfly (Aleurocanthus woglumi) from an agroecological polyculture in Brazil, including an augmented description of F. volatile.

The objectives of this study were to report Fusarium species associated with Aleurocanthus woglumi (Hemiptera: Aleyrodidae) collected from citrus leaves from an agroecological polyculture in Brazil, assess sexual reproductive mode of the species with unknown sexual stages, and provide an augmented description of F. volatile, for which we discovered a sexual stage. Nineteen Fusarium isolates were recovered from A. woglumi. These fungi belong to three species complexes, i.e., the F. chlamydosporum species complex (FCSC), the F. fujikuroi species complex (FFSC), and the F. incarnatum-equiseti species complex (FIESC). Based on multilocus phylogenetic analyses, the species were identified as F. annulatum, F. chlamydosporum, F. pernambucanum, F. sulawesiense, F. verticillioides, and F. volatile. Our results suggest that three species whose sexual stages are unknown (F. chlamydosporum, F. sulawesiense, and F. volatile) are also heterothallic. Intraspecific crosses of F. sulawesiense and F. volatile produced protoperithecia, whereas 66.7% of F. volatile crosses produced fertile perithecia. We provide an augmented description of the latter species to include characteristics of its sexual morph and those observed in the asexual morph that had not yet been described for the species. This study highlights the potential of researching insect-associated fungi to increase knowledge about the diversity, taxonomy, and versatility of Fusarium in ecosystems.

Molecular identification of Brazilian Fusarium strains: sources of proteases with milk-clotting properties.

Fusarium is a genus of ubiquitous fungi that comprises mycotoxigenic animal and plant pathogens. These fungi have the ability to exploit a wide range of substrates and hosts, indicating their great potential for enzyme production; however, this aspect is understudied. Therefore, the present study aimed for revaluating the identity of twenty-three Fusarium strains maintained in the University Recife Mycology (URM) culture collection, Brazil, and to evaluate their potential for proteases production and the milk-clotting activity of these proteases. According to phylogenetic analysis of translation elongation factor 1-alpha (TEF1) gene partial sequences, these strains belonged to 12 species representing four species complexes: Fusarium concolor, F. fujikuroi, F. incarnatum-equiseti, and F. oxysporum. Four of these species are putatively novel to science. Notably, novel associations of Fusarium spp. with certain hosts/substrates were documented. The proteolytic activity ranged from 1.67 U ml-1 to 22.03 U ml-1 among the evaluated fungal isolates, with specific proteolytic activity reaching 205.86 U mg-1. The values for coagulant activity and specific activity were up to 157.14 U ml-1 and 1,424.11 U mg-1, respectively. These results indicate the potential of URM Fusarium strains as a source for the production of enzymes of industrial interest. Additionally, they reinforce the importance of applying DNA-based methods for reviewing the identification of fungal strains preserved in biodiversity repositories.

Evaluation of Mycotoxin Production and Phytopathogenicity of the Entomopathogenic Fungi Fusarium caatingaense and F. pernambucanum from Brazil.

Fusarium incarnatum-equiseti species complex (FIESC) is considered as one of the richest insecticolous species. Fusarium species synthesize toxic secondary metabolites that are not fully understood. Mycotoxin production and pathogenicity on germinating seeds, seedlings, and leaves must be carefully studied for the use of Fusarium species in the biological control of insect pests. In this study, we evaluated the mycotoxin production and phytopathogenic potential of entomopathogenic strains of Fusarium sulawesiensis (1), F. pernambucanum (3), and F. caatingaense (23). The phytopathogenicity tests of F. caatingaense (URM 6776, URM 6777, URM 6778, URM 6779, and URM 6782) were performed during the development of bean (Phaseolus vulgaris, Vigna unguiculata, and Phaseolus lunatus), and corn (Zea mays) seedlings, using four treatments (soil infestation with the inoculum, spraying on leaves, root dip, and negative control). The mycotoxins, monoacetyl-deoxynivalenols (AcDON), deoxynivalenol (DON), beauvericin (BEA), fusarenone-X (FUS), T-2 toxin (T2), diacetoxyscirpenol (DAS), and zearalenone (ZEA), were detected in the study; BEA (detected in 25 strains) and FUS (detected in 21 strains) were found to be predominant. None of the strains showed any ability to cause disease or virulence in beans and corn. The FIESC strains showed a highly variable production of mycotoxins without the potential to be used as phytopathogenic agents for the cultures tested.

Morphology, phylogeny, and sexual stage of Fusarium caatingaense and Fusarium pernambucanum, new species of the Fusarium incarnatum-equiseti species complex associated with insects in Brazil.

Based on morphological and molecular phylogenetic markers and the fertility of sexual crosses, two novel species of Fusarium associated with Dactylopius opuntiae (Hemiptera: Dactylopiidae) and Aleurocanthus woglumi (Hemiptera: Aleyrodidae) from northeastern Brazil are described as Fusarium caatingaense and F. pernambucanum. Partial sequences of five loci were generated for 29 entomopathogenic Fusarium isolates. Multilocus phylogenetic analyses demonstrated that F. caatingaense and F. pernambucanum belong to the Incarnatum clade of the Fusarium incarnatum-equiseti species complex (FIESC). These species displayed common morphological characters such as the production of various types of aerial conidia formed on monophialides and polyphialides and differ from each other mainly in the dimensions and morphology of their sporodochial conidia. Mating type polymerase chain reaction (PCR) revealed 17 MAT1-1 isolates and 12 MAT1-2 isolates, all of them heterothallic. Fertile perithecia were produced in 4.2% of infraspecific crosses of F. caatingaense and in 13.3% of infraspecific crosses of F. pernambucanum after 2-3 wk. Crosses between F. caatingaense and F. pernambucanum did not result in fertile perithecia. We demonstrate the existence of a sexual stage in species of the Incarnatum clade and describe the morphological characters of these sexual morphs for the first time. These results suggest that previously unknown sexual cycles contribute to the high genetic diversity within FIESC.

Differential expression of the pr1A gene in Metarhizium anisopliae and Metarhizium acridum across different culture conditions and during pathogenesis.

The entomopathogenic fungi of the genus Metarhizium have several subtilisin-like proteases that are involved in pathogenesis and these have been used to investigate genes that are differentially expressed in response to different growth conditions. The identification and characterization of these proteases can provide insight into how the fungus is capable of infecting a wide variety of insects and adapt to different substrates. In addition, the pr1A gene has been used for the genetic improvement of strains used in pest control. In this study we used quantitative RT-PCR to assess the relative expression levels of the pr1A gene in M. anisopliae and M. acridum during growth in different culture conditions and during infection of the sugar cane borer, Diatraea saccharalis Fabricius. We also carried out a pathogenicity test to assess the virulence of both species against D. saccharalis and correlated the results with the pattern of pr1A gene expression. This analysis revealed that, in both species, the pr1A gene was differentially expressed under the growth conditions studied and during the pathogenic process. M. anisopliae showed higher expression of pr1A in all conditions examined, when compared to M. acridum. Furthermore, M. anisopliae showed a greater potential to control D. saccharalis. Taken together, our results suggest that these species have developed different strategies to adapt to different growing conditions.

Biological insect control using Metarhizium anisopliae: morphological, molecular, and ecological aspects / Controle biológico de insetos utilizando Metarhizium anisopliae: aspectos morfológicos, moleculares e ecológicos

Microbial control of insects is based on the rational use of pathogens to maintain environmentally balanced pest population levels, and Metarhizium anisopliae has been the most studied and most utilized fungal species for that purpose. The natural genetic variability of entomopathogenic fungi is considered one of the principal advantages of microbial insect control. The inter- and intraspecific variability and the genetic diversity and population structures of Metarhizium and other entomopathogenic fungi have been examined using ITS-RFLP, ISSR, and ISSP molecular markers. The persistence of M. anisopliae in the soil and its possible effects on the structures of resident microbial communities must be considered when selecting isolates for biological insect control.

O controle microbiano consiste na utilização racional de patógenos, visando à manutenção da população de insetos em equilíbrio no ambiente. Metarhizium anisopliae é a espécie mais estudada e utilizada no controle biológico de insetos. A variabilidade genética dos fungos entomopatogênicos pode ser considerada uma das principais vantagens no controle microbiano de insetos e pode ser detectada por meio de marcadores moleculares, como ITS-RFLP, ISSR e ISSP. Esses marcadores são usados para a caracterização inter e intraespecífica de Metarhizium e outros fungos entomopatogênicos e poderão auxiliar na compreensão da diversidade genética e da estrutura das populações destes fungos. A persistência de M. anisopliae no solo e seu possível efeito na estrutura da comunidade microbiana deste solo são características importantes e pouco estudadas, que devem ser consideradas no processo de seleção de isolados para o controle biológico de insetos.

Differential pathogenicity of Metarhizium anisopliae and the control of the sugarcane root spittlebug Mahanarva fimbriolata

In order to assess the effectiveness of Metarhizium anisopliae var. anisopliae (Metschnikoff) Sorokin isolates in controlling the sugarcane root spittlebug Mahanarva fimbriolata (Stal) (Hemiptera: Cercopidae), nine isolates obtained from a single geographical region were studied. 'Confirmed cumulative' and 'corrected cumulative' spittlebug mortality rates were measured for each of the isolates. Based on the confirmed mortality curve, the isolates URM5946, URM5951 and URM6033 were considered to be potentially the most effective in a biological control program for M. fimbriolata.

Atividade proteolítica de isolados de Metarhizium anisopliae sobre substratos cuticulares e não-cuticulares / Proteolytic activity of Metarhizium anisopliae isolates on cuticular and noncuticular substrates

Cigarrinhas são as principais pragas da pastagem e da cana-de-açúcar, destacando-se os gêneros Deois e Mahanarva. Essas pragas vêm causando sérios danos a estas culturas na região de Tangará da Serra, MT, Brasil. O fungo Metarhizium anisopliae é considerado um eficiente agente para o controle biológico de insetos, possuindo vários fatores de virulência, incluindo a produção de proteases, consideradas fundamentais no processo de penetração através da cutícula do inseto. Este trabalho tem como objetivos analisar a produção de proteases extracelulares a partir de isolados de M. anisopliae, utilizando substratos cuticulares e não-cuticulares, e verificar a influência desses substratos na expressão dessas enzimas. Foram utilizados 4 isolados de M. anisopliae obtidos na região de Tangará da Serra e o isolado R3 BB 40, empregado para o controle biológico das cigarrinhas da cana-de-açúcar pelas Usinas Itamarati (Nova Olímpia / MT). A atividade enzimática foi determinada através do índice de Relação Enzimática (IRE) em Meio Mínimo (MM), sem glicose, acrescido de caseína, gelatina e cutícula de adultos de Mahanarva fimbriolata e Deois flavopicta. Os isolados UNEMAT 03, UNEMAT 04, UNEMAT 05, UNEMAT 06 e R3-BB-40 apresentaram valores de IRE que variaram de 1,82 ± 0,062 a 2,16 ± 0,027 frente ao substrato gelatina e de 1,60 ± 0,076 a 2,08 ± 0,063 em caseína. Para os substratos cuticulares, houve crescimento das colônias, porém não apresentaram revelação de halo (IRE.= 1). Todos os isolados apresentaram IRE frente ao substrato gelatina>caseína>cutícula, destacando-se os isolados R3-BB-40 e UNEMAT 05. Existe uma variabilidade genética entre os isolados quanto à produção de proteases extracelulares e, sob tais condições, as cutículas não demonstraram ser substrato adequado para a detecção de atividade proteolítica.

Froghoppers are the main pests of pasture and sugarcane, among which the genera Deois and Mahanarva stand out. These pests have been causing serious losses in the cultures of the region of Tangará da Serra, state of Mato Grosso, Brazil. The fungus Metarhizium anisopliae is considered an efficient agent for the biological control of insects, possessing various factors of virulence, including the production of proteases considered essential in the process of penetration through the insectÆs cuticle. The purpose of this work is to analyze the production of extracellular proteases from isolates of M. anisopliae, using cuticular and noncuticular substrates to ascertain their influence on the expression of these enzymes. Four M. anisopliae isolates collected in the region of Tangará and the isolate R3 BB 40 used for the biological control of froghoppers in the sugarcane plantations of the Itamarati sugar mills (Nova Olimpia, MT, Brazil) were used. The enzymatic activity was determined from the Enzyme Relation Index (ERI) in Minimum Medium (MM) without glucose, with the addition of casein, gelatin and cuticle from adult Mahanarva fimbriolata and Deois flavopicta. The UNEMAT 03, UNEMAT 04, UNEMAT 05, UNEMAT 06 and R3-BB-40 isolates presented ERI values varying from 1.82 ± 0.062 to 2.16 ± 0.027 with gelatin substrate and from 1.60 ± 0.076 to 2.08 ± 0.063 with casein. The cuticular substrates showed colony growth, but revealed no halo (ERI = 1). All the isolates presented ERI with gelatin>casein>cuticle substrate, with the highest values going to the R3-BB-40 and UNEMAT 05 isolates. Isolates display natural variability in terms of extracellular protease production and under these conditions the cuticles proved to be an inadequate substrate for the detection of proteolytic activity.

Effects of double-stranded RNA in Metarhizium anisopliae var. acridum and Paecilomyces fumosoroseus on protease activities, conidia production, and virulence.

Isogenic strains (with and without dsRNA) of the entomogenous fungi Metarhizium anisopliae var. acridum and Paecilomyces fumosoroseus were investigated for correlation between the presence of dsRNA and the production of cuticle-degrading proteases that play an important role in host parasitism, total secreted protein, and conidia production. Similar levels of cuticle-degrading subtilisin-like (Pr1) protease were observed for isogenic strains of M. anisopliae var. acridum after growth in medium supplemented with the cuticle of the grasshopper Rhammatocerus schistocercoides. Similarly, no statistical differences were observed for protease production, detected using the chromogenic substrate azocasein. For P. fumosoroseus isogenic strains, no significant differences in protease activity were observed after growth in the presence of either Euschistus heros or Nezara viridula (Hemiptera: Pentatomidae) cuticle. Similarly, no statistical differences were observed in virulence against E. heros. A comparison of mean conidia production showed a significantly higher production in the dsRNA-free isogenic strains of M. anisopliae var. acridum. Although, for most of the fungal phenotypes analysed, no overt effects were associated with the presence of these dsRNA infections, the reduction in conidia production by the isogenic strains of M. anisopliae var. acridum with dsRNA suggested that it may not be entirely accurate to describe these infections as latent.