Improvements in strains of entomopathogenic fungi have been attempted through selection as well as molecular methods. Selection of fungal strains with altered acyclic sugar alcohol (polyol) and trehalose content of the conidia may improve the endogenous reserves to enhance viability and desiccation tolerance. Cultures of B. bassiana, M. anisopliae, and P. farinosus grown under different conditions to obtain conidia with a modified polyol and trehalose content resulted in conidia with increased intracellular levels of glycerol and erythritol that germinated more quickly than unselected conidia and at lower water activity (Hallsworth and Magan 1995). Conidia with increased trehalose germinated more slowly but stored for longer than unselected conidia. Another approach is to use genetic modification to "improve" strains and overcome limitations. This type of approach is in its infancy for entomopathogenic fungi, but there have been some interesting studies indicating the utility of the process. Two of the more promising studies on the potential of biotechnology to improve entomopathogenic fungi were published by Couteaudier et al. (1996) and Vaiud et al. (1998). They demonstrated that protoplast fusion between a strain of B. bassiana from Leptinotarsa decemlineata with an insecticidal toxin-producing strain of B. sulfurescens resulted in recovery of some di-auxotrophic mutants with enhanced activity (faster kill) against L. decemlineata and the caterpillar Ostrinia nubilalis. The stability of the virulence following passage through the insect-host and stability of molecular structure for two of the fusion products suggested that asexual genetic recombination by protoplast fusion may provide an attractive method for the genetic improvement of biocontrol efficiency in entomopathogenic fungi (Vaiud et al. 1998).
The most studied genes in the entomopathogenic fungi are the protease genes of M. anisopliae, particularly the Pr1 gene. This was the first protease gene from an entomopathogenic fungi implicated in disease and was isolated by St Leger et al. (1992). Pr1 has sequence similarity to proteinase K, but was more effective than that enzyme at degrading cuticle. It is similar to the subtilisin subclass of serine endopeptidases. Modification of pr1 gene expression in M. anisopliae resulted in melanisation and cessation of feeding 25-30h earlier than wild-type disease in caterpillars (St Leger et al. 1996). V. lecanii, B. bassiana, Tolypocladium niveum, and P. farinosus also produced Pr1-type enzymes during nutrient deprivation (St Leger et al. 1991). Southern analysis demonstrated that genes with significant homologies to Metarhizium pr1 were present in the entomopathogens A. flavus and V. lecanii but not Z. radicans (St Leger et al. 1992). More recently, 11 subtilisin proteases (Pr1s) were identified from one strain of M. anisopliae (St Leger et al. 2001). Recently, intended field release of a modified M. anisopliae strain was reported (St Leger 2001). The strain has the pr1 cuticle degrading protease gene under control of a constitutive promoter. The gene overproduction did not alter the host range, but resulted in a strain with a reduced median lethal time to kill. It also reduced the ability of transformants to sporulation. The pr1 gene expression was under dual control of a general carbon catabolite repression/depression mechanism and a carbon source induction mechanism to control expression. Overexpression of extracellular chitinase, an enzyme important in the cuticular penetration of insects by entomopathogenic fungi, has also been demonstrated for M. anisopliae var. anisopliae (Screen et al. 2001). They expressed a chitinase gene from M. anisopliae var. acridium under control of an Aspergillus regulatory element to express in noninducible conditions. While successful expression was achieved, there was no altered virulence to the caterpillar, Manduca sexta, compared to the wildtype fungus. Genetic manipulation of entomopathogenic fungi has a long way to go before transgenic pest control strains become available, if such technology is ever acceptable to regulators and the community. However, strain modification continues to provide a wealth of data on disease processes.
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