Characterization of Metarhizium anisopliae using amplifed ribossomal DNA restriction analysis (ARDRA) and internal transcribed spacer (ITS) sequence analysis

Abstract

Molecular methods have revolutionized systematic entomology in the genus Metarhizium. In Brazil, the importance of these entomopathogenic fungi in agriculture is critical to the control of the sugar cane spittlebug (Mahanarva posticata) (Homoptera: Cercopidae). Other important factors for the selection of isolates of Metarhizium anisopliae for use in the biological control of particular pests are virulence, reproductive characteristics and the media of artificial cultures. In addition, the genetic variability of these entomopathogenic fungi indicates a great potential for pest control and a lack of a link between isolates from specific host sites and fungal virulence. We studied five Metarhizium hosts including the sugar cane spittlebug (IPA213, IPA215, IPA219 and IPA216) and the grasshopper (IPA217). It is noteworthy that these five isolates are used as biological control for growing sugar cane on an industrial scale for the Brazilian Northeast. Hence, the importance to genetically characterize these isolates of Metarhizium cannot be over emphasized. Amplifed ribossomal DNA restriction analysis (ARDRA) did not differentiate any of the isolates that were studied following digestion with three endonuclease namely; HindIII, HinfI and HpaI. The data that were generated using ITS1 and ITS4 markers revealed that although these isolates infect the same host, they are genetically distinct perhaps due to the unknown origin of the hosts. However, the isolate IPA217, which was isolated from the grasshopper, did not behave differently from the others. These results show that the M. anisopliae complex is monophyletic and it was concluded that the use of markers and morphological studies yielded results that may corroborate the idea that investigation of the phenotypic and molecular characteristics of this genus may indentify a new species of Metarhizium.

Key words: Cordyceps brittebankisoides, genetic identity, ITS1, ITS4, phylogeny, sugar cane spittlebug.