Seleção de isolados de Metarhizium anisopliae s.l. para o controle biológico de Rhipicephalus microplus a partir da caracterização morfológica e molecular e testes de patogenicidade

Abstract

Aiming to decrease the chemicals acaricide use and their damages, new alternatives for ticks control has been studied. Metarhizium anisopliae s.l. is one of the most studied fungi in agricultural pest management programs, since it has great acaricide potential. Therefore, this study aimed to characterize molecular and morphologically, as well as evaluate the virulent potential of 30 M. anisopliae s.l. isolates from different geographical regions, hosts or substrates allowing the selection of virulent isolates in order to be further investigated for field programs of microbial control of pests. Initially, the analyses of morphological characterizations of the isolates were made to confirm their identification. Each isolate had its conidial potential production evaluated. The colonies studied showed morphological characteristics consistent with those described in the literature. The colonies diameter varied between 29.66 mm and 51.33 mm among isolates. There was both length and width variation in the conidia and phialides in the same isolate, as well as the presence of grouped and solitary phialides. The conidial production potential was variable among isolates, but both conidial size and colonies diameter did not influence the conidial production; isolates with low conidial production showed similar colony size in comparison to isolates with high potential. In a second stage of the study, the virulence of these isolates was evaluated to Rhipicephalus microplus larvae treated with one of the four different conidial concentrations (105, 106, 107 or 108 conidia.mL-1). The lethal action of Brazilian M. anisopliae s.l isolates to R. microplus larvae were confirmaded with high mortality among the isolates, which in general was proportional to the conidia concentration of the treatments. Most isolates killed larvae population with 107 conidia.mL-1 concentration, however the most virulent isolates presented lethal concentration of 106 conidia.mL-1 with main percentages of mortality nearly 100% at day 20 after treatment. In addition, the genetic variability of these isolates was performed to evaluate their relationship with other species of Metarhizium sp. through RFLP-PCR analysis and ITS1-5.8S-ITS2 rDNA sequencing. No specificity pattern was observed when isolates from the same region, host or substrate were grouped. Low genetic variability was observed among isolates, which were basically grouped into two groups. The CG 344 isolate was shown to be genetically distant from the remaining Brazilian isolates studied, but according to the “GenBank” sequences comparison, it was related to the Metarhizium genus. It is suggested that this variation occured owing the lack of procedures that could generated morphological and molecular changes, which probably contribute to this low genetic variability. The present study allowed the detection of M. anisopliae s.l. isolates with highly virulence to R. microplus larvae, that may be considered potential biocontrol agents for this tick species, emphasizing the importance of molecular tools for identification and characterization of fungal isolates, ensuring the product quality, their success implement and the environmental track of the fungi at field biological control programs.