Estudo da atividade enzimática e patogênica de isolados brasileiros de Metarhizium anisopliae s.l. visando o uso no controle biológico de Rhipicephalus (Boophilus) microplus

Abstract

The fungus Metarhizium anisopliae sensu lato (s.l.) is one of the most promising micro-organisms to be used in the biological control of ticks. This fungus has been studied worldwide showing accurate acaricidal potential, especially in in vitro tests. However, a large variation on the virulence of different fungal isolates has been observed. From this perspective, the present study aimed to verify the pathogenic action of different fungal formulations (oil-based and aqueous) on ticks and also analyze the enzymatic action of M. anisopliae s.l. isolates from different Brazilian geographical regions in order to select the most virulent isolates for further use in programs of biological control of ticks. The presence of dsRNA viral particles (mycovirus) associated to the fungal enzymatic activities was also studied. Among the studied enzymes was the subtilisin Pr 1; whose enzymatic activity was associated with the percentage of tick control caused by the fungi aiming establish virulence markers specific for Rhipicephalus microplus ticks. Five M. anisopliae s.l. isolates from different Brazilian states were analyzed. First, a bioassay was performed on R. microplus engorged females to test the virulence of these fungal isolates. Concomitant assays were performed to quantify the enzymatic activity of lipases, proteases and chitinases secreted by the same M. anisopliae s.l. isolates. Fungi were grown in liquid medium containing ticks’ cuticle and cholesteryl stearate; fluorescent and chromogenic substrates were used to quantify the enzymatic activity. Mycovirus survey was carried out following steps of total nucleic acid extraction before an endonuclease S1 and RNAse A digestion and consecutive visualization on agarose gel. Furthermore, a bioassay was conducted using different oil formulations (10 % vegetable oil and 10% mineral oil). In this experiment, R. microplus engorged females were immersed in the respective formulations for three minutes. The efficacy of the treatments was assessed through analysis in the females’ reproductive parameters. The present study showed that there is variation in the virulence among M. anisopliae s.l. isolates against R. microplus, moreover, it was evident that the isolates that yielded the highest tick control percentages were also those which had the highest lipolytic, proteolytic and chitinolytic activities. Pr1 is known to be involved in the infection process of M. anisopliae s.l.; accordingly the present results demonstrate that this enzyme can be used as a molecular marker for R. microplus, since the isolates that showed higher Pr 1 activity were also the most virulent for this tick species. However, there was no evident analogy between the infection with dsRNA virulent particles and the enzymatic activity levels of the five isolates tested, considering that only one was not infected. Regarding the bioassay with oil-based formulations, it was clear that formulation containing mineral oil were more effective than those containing vegetable oil which provided low percentage of tick control. Furthermore, it can be concluded that the isolates GC 148 and GC 629 have a high potential to control R. microplus engorged females when formulated in mineral oil