In vitro assessment of Metarhizium anisopliae isolates to control the cattle tick Boophilus microplus

SUMMARY

Eighteen isolates of the entomopathogenic fungus Verticillium lecanii from various world-wide locations, from insect hosts and soil were bioassayed against the aphid Macrosiphoniella sanborni in the laboratory. Virulence ranged from an isolate which achieved 100% mortality and LT 50 value (adults) of 3 days ± 0· 2 at 24 ° C, compared with the least virulent isolates causing less than 10% mortality over 14 days (when treated with an inoculum of 1 × 106 conidiospores/ml). All isolates produced extracellular protease and lipase, irrespective of their virulence. A number of traits were frequently associated with the expression of virulence including fast germination, high sporulation rate, an absence of extracellular amylase activity and high extracellular chitinase activities. Large spore size was not strongly associated with virulence. There were exceptions in each variate studied, suggesting that overall expression of virulence is a result of the total complex of these and other traits still to be determined.

Traits associated with virulence to the aphid Macrosiphoniella sanborni in eighteen isolates of Verticillium lecanii.

There are no reports to date of entire gene sequences coding for chitinolytic enzymes from entomopathogenic fungi, even though these enzymes act synergistically with proteolytic enzymes to solubilize insect cuticle during the key step of host penetration, having considerable importance in the biological control of some insect pests. This paper reports the complete nucleotide sequence and analysis of the chromosomal and full-length cDNA copies of the regulated gene (chit1) coding one of the chitinases produced by the biocontrol agent Metarhizium anisopliae. Degenerated primers, encompassing conserved regions of other fungal chitinases, were used to amplify a 650-bp DNA fragment, which was used to isolate genomic and cDNA clones from M. anisopliae. Albeit at least two different chitinases are characterized in this fungus, only one chit gene was isolated. The chit1 gene is interrupted by three short typical fungal introns and has a 1,521-bp ORF, which encodes a protein of 423 amino acids with a stretch of 35 amino acid residues displaying characteristics of signal peptide. The deduced sequence of the mature protein predicts a 42-kDa protein with pI of 5.8. Southern analysis of genomic DNA indicates a single copy of chit1 in the M. anisopliae genome.

A Chitinase Encoding Gene (chit1 Gene) from the Entomopathogen Metarhizium anisopliae: Isolation and Characterization of Genomic and Full-Length cDNA

Characteristics relating to the pathogenicity of Metarhizium anisopliae toward Nilaparvata lugens

Chronological histological alterations of Metarhizium anisopliae during interaction with the cattle tick Boophilus microplus were investigated by light and scanning electron microscopy. M. anisopliae invades B. microplus by a process which involves adhesion of conidia to the cuticle, conidia germination, formation of appressoria and penetration through the cuticle. Twenty-four hours post-infection conidia are adhered and germination starts on the surface of the tick. At this time, the conidia differentiate to form appressoria exerting mechanical pressure and trigger hydrolytic enzyme secretion leading to penetration. Massive penetration is observed 72 h post-inoculation, and after 96 h, the hyphae start to emerge from the cuticle surface to form conidia. The intense invasion of adjacent tissues by hyphae was observed by light microscopy, confirming the ability of M. anisopliae to produce significant morphological alterations in the cuticle, and its infective effectiveness in B. microplus.

Y.B. Rosato

Papers

Production of extracellular enzymes by isolates of Metarhizium anisopliae