Gene cloning, molecular modeling, and phylogenetics of serine protease P32 and serine carboxypeptidase SCP1 from nematophagous fungi Pochonia rubescens and Pochonia chlamydosporia

The fungi Pochonia chlamydosporia and Pochonia rubescens are parasites of nematode eggs and thus are biocontrol agents of nematodes. Proteolytic enzymes such as the S8 proteases VCP1 and P32, secreted during the pathogenesis of nematode eggs, are major virulence factors in these fungi. Recently, expression of these enzymes and of SCP1, a new putative S10 carboxypeptidase, was detected during endophytic colonization of barley roots by these fungi. In our study, we cloned the genomic and mRNA sequences encoding P32 from P. rubescens and SCP1 from P. chlamydosporia. P32 showed a high homology with the serine proteases Pr1A from the entomopathogenic fungus Metarhizium anisopliae and VCP1 from P. chlamydosporia (86% and 76% identity, respectively). However, the catalytic pocket of P32 showed differences in the amino acids of the substrate-recognition sites compared with the catalytic pockets of Pr1A and VCP1 proteases. Phylogenetic analysis of P32 suggests a common ancestor with protease Pr1A. SCP1 displays the characteristic features of a member of the S10 family of serine proteases. Phylogenetic comparisons show that SCP1 and other carboxypeptidases from filamentous fungi have an origin different from that of yeast vacuolar serine carboxypeptidases. Understanding protease genes from nematophagous fungi is crucial for enhancing the biocontrol potential of these organisms.

Biological control of sheep parasite nematodes by nematode-trapping fungi: In vitro activity and after passage through the gastrointestinal tract

The main method used for the control of gastrointestinal nematodes in sheep production is the application of chemotherapeutic agents, which often lead to the selection of parasites resistant to given active principles. Biological control can be considered a promising alternative, contributing to an increase in the efficacy of verminous control. We determined the in vitro activity and in situ survival of the predatory fungi Arthrobotrys musiformis and Arthrobotrys conoides during passage through the gastrointestinal tract of sheep after oral administration of conidia in microencapsulated form and as a liquid in natura. Initial in vitro tests showed that both fungi were efficient in the predation of trichostrongylid L3 larvae present in the faeces of sheep naturally infected with gastrointestinal nematodes. The fungi presented high nematophagous activity, which was 99.3% for A. conoides and 73.7% for A. musiformis. A. conoides did not survive passage through the gastrointestinal tract under the conditions of the present experiment. On the other hand, A. musiformis was reisolated after administration in either microencapsulated or liquid form, suggesting that this species is a promising alternative for the control of nematodes in sheep since it survives without any protection (in natura). © Springer 2005.

Crescimento e esporulação de duas espécies de arthrobotrys corda em diferentes meios de cultura e dois ambientes

The researches on the biological control of nematodes with nematophagous fungi has been intensified in recent years. The knowledge of the ecological conditions for the growth and sporulation of these fungi is a prerequisite for attainment of pure cultures needed to attend the demand for formulation of these organisms. With the objective to evaluate the micelial growth and sporulation of Arthrobotrys musiformis and A. oligospora in two environments (B.O.D at 25 +/- 1 degrees C and the environment of the Laboratory), 20 cultures media prepared with common materials found in the communities and industrialized media such as mycological agar, PDA and CMA were evaluated. The media were tested in Petri dishes, being the micelial growth of the fungi evaluated daily, during six days. The measured sporulation at the end of the experiment was done by estimation of the number of conidia/Petri dish. The experiment was carried out in a random design following a factorial arrangement of 20 x 2 x 2, corresponding to 20 media, two fungi and two environments, with five replicates. The variance analysis of the data evidenced significant statistical difference by the F Test, at 1% probability, among media x fungi x environment interaction. Fifty percent of the tested media provided the adequate micelial growth of A. musiformis and there was no statistical difference among them, namely: cassava meal (FM), sweet starch (PD), "corn meal agar" (CMA), oat in fine flakes (AFF), agar-water + dextrose (AA+D), mycological agar (AM), potato dextrose agar (BDA), meal of maize (FMI), flour of wheat (FT) and wheat for kibble (TK). In relation to A. oligospora, 75% of the tested media promoted the maximum growth of the fungus, which are: AFF, AM, FM, PD, CMA, AA+D, BDA, FT, TK, the water from the decoction of rice (AAZ), rice in grains (AZG), triturated rice (AZT), thread flour (FR), oats flour (FA), oats in thick flakes (AFG) and flour of maize (FU). In relation to the sporulation the media that had better role for A. musiformis, in decreasing order, were: FR, TK, AFG, BDA, FA, AFF, AM, FMI, AZT and FM, varying between 1,01 x 10(6) and 1,4 x 10(4) conidia/Petri dish. For the A. oligospora sporulation, the CMA medium provided the maximum level with an estimated average of 5,7 x 10(6) conidia/Petri dish. In the general, the best media for the micelial growth and sporulation of A. musiformis had also been the best for A. oligospora. However, some that had been the best for the A. oligospora did not had been efficient for the micelial growth or the sporulation of A. musiformis, indicating that the isolate of A. musiformis in case is more demanding than that A. oligospora one. The evidences from the study indicate that, in Jaboticabal, São Paulo state, the growth and the sporulation of these fungi do not demand special chambers. Some adaptations of an environment at the laboratory, enough to obliterate the light are sufficient.

Patogenicidade in vitro de Monacrosporium robustum a Rotylenchulus reniformis

O nematóide reniforme (Rotylenchulus reniformis) é um importante patógeno de importantes culturas brasileiras, tais como soja, algodão, feijão, caupi, maracujazeiro, mamoeiro, tomate e alface, entre outras. Dentre os fungos nematófagos com potencial de utilização comercial como agentes do controle biológico de nematóides, destacam-se espécies do gênero Monacrosporium. No presente estudo, avaliou-se, in vitro, a capacidade predatória e de parasitismo de Monacrosporium robustum ao nematóide reniforme. em períodos de 24, 48 e 72 horas após, constatou-se que a percentagem de predação do nematóide pelo fungo foi de, respectivamente, 81,3, 99,9 e 100.

Microscopia eletrônica de varredura de fungos nematófagos associados a Tylenchulus Semipenetrans e Pratylenchus Jaehni

Nematophagous fungi have been studied as a promising alternative for the management of nematodes of citrus. This study had the objective of documenting by scanning electron microscopy the outslading morphological structures for the identification of the main species of nematophagous fungi involved in the study and proving the pathogenicity of the isolated ones associated with T. semipenetrans and P. jaehni, Isolated came from in citrus orchards, mainly in São Paulo State, were studied in the Laboratory of Scanning Electron Microscopy of FCAV/UNESP - Campus de Jaboticabal-SP. The data obtained in this study have confirmed that the scanning electron microscopy is an effective tool to assist in the understanding of way of action of the nematophagous fungi: Arthrobotrys oligospora, A. conoides, A. robusta, A. musiformis, Dactylella leptospora, Monacrosporium eudermatum and M. elegans and to reveal details of their reproductive structures and the capture, to confirm the pathogenicity of the isolated to T. semipenetrans and P. jaehni, as well as to confirm the identification of some isolated.

Controle de nematóides parasitos gastrintestinais por Arthrobotrys musiformis em ovinos naturalmente infestados mantidos em pastagens

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efficacy of Duddingtonia flagrans and Arthrobotrys robusta in controlling sheep parasitic gastroenteritis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Estabelecimento de fungos nematófagos aplicados no campo para o controle dos nematoides-chaves dos citros no Brasil

Monitoring the survival of nematophagous fungi is needed to establish periods of reapplication of formulations of nematophagous fungi to control the citrus nematode in the field. We monitored the survival of fungi: Arthrobotrys robusta, A. oligospora, A. musiformis Dactylella leptospora and Monacrosporium eudermatum in plots treated with 1, 2, 4, 6 liters of the formulation of fungi/plant or witness without the application, during the period of nine months with the first assessment six months after application and the other with intervals of three months after the first evaluation. The fungus D. leptospora was found only in the evaluation of 6 months after treatment application, indicated a short survival time in the soil. However, the isolated A. robusta, A. musiformis and A. oligospora were recovered in all evaluations and especially in plots treated with higher doses of the formulation and witness. Monacrosporium eudermatum was recovered in all experimental periods and even in assessing the witness portion of nine months after application. The fact of the presence of species of Arthrobotrys and M. eudermatum in control plots indicates that native species that were already orchard.

Flutuação populacional de Meloidogyne mayaguensis em pomar de goiabeira e estudo do controle biológico com fungos nematófagos associado a culturas de cobertura

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudo do controle biológico dos nematóides dos citros no Estado de São Paulo

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Estudo do controle biológico de fitonematóides com fungos nematófagos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Fungos nematófagos em diferentes solos e caracterização filosófica de Arthrobotrys oligospora

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Formulações de fungos nematófagos associadas ao controle químico e matéria orgânica no manejo dos nematoides dos citros (Tylenchulus semipenetrans e Pratylenchus jaehni)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Agressividade comparada de Pratylenchus brachyurus com P. zeae e eficácia de métodos de controle de nematoides em cana-de-açúcar

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Eficácia dos fungos nematófagos Duddingtonia flagrans e Arthrootrys robusta na profilaxa das infecções naturais por nematódeos gastrointestinais em ovinos

Pós-graduação em Medicina Veterinária - FMVZ