Genetic, morphological, and virulence characterization of the entomopathogenic fungus Verticillium lecanii

Department of Agro-environmental Science, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, Obihiro 080-8555, Japan.
Journal of Invertebrate Pathology (Impact Factor: 2.11). 04/2003; 82(3):176-87. DOI: 10.1016/S0022-2011(03)00014-4
Source: PubMed

ABSTRACT In order to clarify relationships among genetic diversity, virulence, and other characteristics of conidia, 46 isolates of Verticillium lecanii from various hosts and geographical locations were examined. The internal transcribed spacer (ITS) and intergenic spacer (IGS) regions of ribosomal DNA (rDNA), mitochondrial small subunit rDNA (mt-SrDNA) and beta-tubulin were analyzed by PCR-RFLP. PCR-single stranded conformational polymorphism (SSCP) was performed on regions of the mitochondrial large subunit rDNA, mt-SrDNA, beta-tubulin and histone 4. There were no relationships among the results of RFLP, SSCP, isolation source, and location. However, amplified product size of IGS did have relationships with conidia size and sporulation. Six isolates with 4.0-kb IGS products had large conidia dimensions, and yielded low numbers of conidia compared with other isolates. Three out of the six isolates were high virulence (over 90%) against green peach aphids. Furthermore, double-stranded RNA (dsRNA) was detected in 22 out of 35 V. lecanii isolates and related with the amplicon sizes of IGS, though not with virulence or isolation location. Isolates containing dsRNA were divided into six distinct types based on banding pattern. These data demonstrate the level of genetic diversity of V. lecanii, and suggest relations among the genetic properties and conidial morphology.

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Available from: Masanori Koike, May 09, 2014
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    • "Mitochondrial DNA (mtDNA), due to its properties to evolve faster than the nuclear DNA, to contain introns and mobile elements and to exhibit extensive polymorphisms, has been increasingly used to examine genetic diversity within fungal populations [24-26]. In other mitosporic entomopathogenic fungi, such as Metarhizium [27], Lecanicillium [28] and Nomurea [29], mtDNA data compared favourably to data based on ITS combined with a single nuclear gene, for applications in phylogeny, taxonomy and species or strain -identification. "
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    ABSTRACT: The entomopathogenic fungi of the genus Beauveria are cosmopolitan with a variety of different insect hosts. The two most important species, B. bassiana and B. brongniartii, have already been used as biological control agents of pests in agriculture and as models for the study of insect host - pathogen interactions. Mitochondrial (mt) genomes, due to their properties to evolve faster than the nuclear DNA, to contain introns and mobile elements and to exhibit extended polymorphisms, are ideal tools to examine genetic diversity within fungal populations and genetically identify a species or a particular isolate. Moreover, mt intergenic region can provide valuable phylogenetic information to study the biogeography of the fungus. The complete mt genomes of B. bassiana (32,263 bp) and B. brongniartii (33,920 bp) were fully analysed. Apart from a typical gene content and organization, the Beauveria mt genomes contained several introns and had longer intergenic regions when compared with their close relatives. The phylogenetic diversity of a population of 84 Beauveria strains -mainly B. bassiana (n = 76) - isolated from temperate, sub-tropical and tropical habitats was examined by analyzing the nucleotide sequences of two mt intergenic regions (atp6-rns and nad3-atp9) and the nuclear ITS1-5.8S-ITS2 domain. Mt sequences allowed better differentiation of strains than the ITS region. Based on mt and the concatenated dataset of all genes, the B. bassiana strains were placed into two main clades: (a) the B. bassiana s. l. and (b) the "pseudobassiana". The combination of molecular phylogeny with criteria of geographic and climatic origin showed for the first time in entomopathogenic fungi, that the B. bassiana s. l. can be subdivided into seven clusters with common climate characteristics. This study indicates that mt genomes and in particular intergenic regions provide molecular phylogeny tools that combined with criteria of geographic and climatic origin can subdivide the B. bassiana s.l. entomopathogenic fungi into seven clusters with common climate characteristics.
    BMC Microbiology 06/2010; 10(1):174. DOI:10.1186/1471-2180-10-174 · 2.73 Impact Factor
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    • "PCR-single strand conformation polymorphism, PCR-denaturing gradient gel electrophoresis, and PCR-temperature gradient gel electrophoresis SSCP (PCR-single strand conformation polymorphism ), DGGE (PCR-denaturing gradient gel electrophoresis ), and TGGE (PCR-temperature gradient gel electrophoresis) are used to detect genetic differences in PCR products obtained from SCL. SSCP relies on differences in secondary structure of single stranded DNA assessed by gel electrophoresis under non-denaturing conditions (Schwieger and Tebbe 1998), whereas DGGE and TGGE detect differences in DNA double-strand stability assessed by gel electrophoresis through a denaturant or temperature gradient (Muyzer and Smalla 1998). SSCP analysis have allowed discrimination of isolates of Lecanicillium lecanii (Ascomycota: Hypocreales) (Sugimoto et al. 2003), B. bassiana (Hegedus and Khachatourians 1996) or Nomuraea riley (Ascomycota: Hypocreales ) (Devi et al. 2007) by targeting loci coding for the mitochondrial small and large subunit rRNA, btubulin , or histon 4. DGGE has been used to discriminate B. bassiana isolates based on amplification products obtained from the ITS region (Pantou et al. 2003). TGGE has not been applied to entomopathogenic fungi, however it has a similar potential to SSCP or DGGE for identification of genotypes as demonstrated by studies on different yeast (Hernan- Gomez et al. 2000; Manzano et al. 2005) or bacterial species (Wagner-Dobler et al. 2000; Tominaga 2006). "
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    ABSTRACT: The power of molecular genetic techniques to address ecological research questions has opened a distinct interdisciplinary research area collectively referred to as molecular ecology. Molecular ecology combines aspects of diverse research fields like population and evolutionary genetics, as well as biodiversity, conservation biology, behavioural ecology, or species-habitat interactions. Molecular techniques detect specific DNA sequence characteristics that are used as genetic markers to discriminate individuals or taxonomic groups, for instance in analyses of population and community structures, for elucidation of phylogenetic relationships, or for the characterization and monitoring of specific strains in the environment. Here, we summarize the PCR-based molecular techniques used in molecular ecological research on fungal entomopathogens and discuss novel techniques that may have relevance to the studies of entomopathogenic fungi in the future. We discuss the flow chart of the molecular ecology approaches and we highlight some of the critical steps involved. There are still many unresolved questions in the understanding of the ecology of fungal entomopathogens. These include population characteristics and relations of genotypes and habitats as well as host-pathogen interactions. Molecular tools can provide substantial support for ecological research and offer insight into this far inaccessible systems. Application of molecular ecology approaches will stimulate and accelerate new research in the field of entomophathogen ecology. KeywordsCultivation-dependent analysis-Cultivation-independent analysis-Genotyping-Monitoring-Genetic diversity
    02/2010: pages 17-37;
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    • "Lecanicillium lecanii ARSEF 6543, with homologies from 100% to 99% with T. confragosa isolates (IFO 8579 and UASWS 0012), appear close to some L. muscarium isolates, whereas the Mycotal isolate, identified as L. muscarium, appears in another different branch far from the rest (Fig. 1). Also Sugimoto et al. (2003) have found some similar ambiguities. All seems to indicate that the ITS region alone can fail to resolve some phylogenetic relationships, and is insufficient to place some isolates in their appropriate Lecanicillium branches in phylogenetic studies. "
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    ABSTRACT: Seven isolates of Lecanicillium lecanii (Zimmermann) Zare & Gams isolated in Spain from infected aphids were characterized using sequences of the Internal Transcribed Spacer (ITS) regions and also based on morphological and physiological characteristics. Four of these seven L. lecanii isolates were selected to assess their virulence against nymphs of Myzus persicae (Sulzer), Nasonovia ribisnigri (Mosley), Macrosiphum euphorbiae (Thomas) and Aphis gossypii Glover. Mortality (%), lethal concentration 50 (LC50) and lethal time 50 (TC50) were calculated. The analysis of the sequences of ITS region confirmed that the new isolates were clearly Lecanicillium lecanii. The set of isolates had similar radial growth (51.5–54.0mm), except for ICAL1 (39mm). The germination time 50 (GT50) varied between 10.7h (ICAL3) and 13h (ICAL5). The isolate ICAL6 showed the highest value for conidial production (3.4×108conml−1) and also produced the highest mortality for M. persicae (95%) and was more virulent than the commercial product Vertalec® (91.6%).
    BioControl 12/2009; 54(6):825-835. DOI:10.1007/s10526-009-9218-9 · 1.69 Impact Factor
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