The use of mt DNA gene probes for the molecular typing and differentiation of Verticillium lecanii Isolates

摘要

The genus Verticillium contains a large, heterogeneous group of asexual fungi (7). Verticillium lecanii (Zimm.) Viegas is an entomopathogenic species of considerable economic importance. This species can act not only as a potential biocontrol agentof arthropod pests, but also as a biological control agent of rust diseases (7, 10). The need for accurate, highly sensitive and fast identification of the isolates used, is more than imperative, if commercial considerations, such as identification of existing or novel isolates, quality control of product and patent protection, are to be addressed. Additionally, critical environmental issues concerning persistence, dissemination and effect of applicants on inherent biodiversity and recovery would definitely be answered (5). Conventional strain typing in Verticillium, as well as other deuteromycetous fungi is usually based on classical taxonomic criteria, such as morphological characteristics and physiological or biochemical properties. In this regard,differences in spore sizes (4), mycelial form and colour, isoenzyme profiles (3, 7) and enzymatic activities (7) have been used previously to differentiate either Verticillium species or V. lecanii. However, when classification is based on the above criteria, it is subject to inaccuracies associated with differential gene expression due to environmental and physiological conditions (6). Recently, several molecular approaches have been utilised to characterise different species or isolates within the species with considerable success, e.g. random amplification of polymorphic DNA (RAPD) (1), restriction fragment length polymorphism (RFLP) analysis, based on multiple copy DNA, such as the ribosomal rRNA gene complex (rDNA), or mitochondrial DNA (mtDNA), (8, 11, 16), and amplification of ribosomal intergenic sequences (12). In recent years, mtDNA and rDNA have been used as molecular tools because they represent a wide range of rates of evolution and levels of divergence. Hence, they allow differentiationand detection at a wide range of specificities. MtDNA is known to have a higher rate of evolution than nuclear DNA (10-100 times faster) (2, 15), and possess several unique traits, such as high AT content, relatively small size, high copy number, simplerestriction enzyme patterns and absence of methylation (6). The aim of this work was to assess mtDNA as a potential tool for the characterization and identification of V. lecanii isolates. RFLP analysis of the mitochondrial genome, hybridization with specific homologous mitochondrial probes, and PCR amplification of mitochondrial genes for 33 V. lecanii isolates were employed.