Identification of Molecular Variants in Mitochondrial DNAs of Members of the Genera Beauveria, Verticillium, Paecilomyces, Tolypocladium, and Metarhizium

A set of five mitochondrial (mt) probes derived from a strain of Beauveria bassiana was used to evaluate the similarity of mtDNAs from 15 additional isolates of this fungus and five genera of other entomopathogenic fungi. The probes and genes encoded for (shown in parentheses) were pBbmtE2 (NADI, ATP6), pBbmtE3 (ATP6, small rRNA [srRNA]), pBbmtE4 (srRNA, CO3, NAD6), pBbSE1 (NAD6, tRNA^{Val, Ile, Ser, Trp, Pro}, large rRNA [lrRNA]), and pBbXS1 (lrRNA). The probes produced identical hybridization patterns in EcoRI-digested DNA from nearly all isolates of B. bassiana and Beauveria caledonica. Similar patterns were also observed with Beauveria densa. The isolates of B. caledonica and B. densa DNAs could be differentiated from each other and from B. bassiana on the basis of a HindIII digestion and probing with pBbmtE3. Probe pBbmtE2 produced either a 5.0-kb or a 4.1-kb band in all of the B. bassiana isolates. This observation was used to categorize the mtDNA of B. bassiana into two types, designated A and B. Hybridization of the five probes produced distinct banding patterns in Beauveria brongniartii, Tolypocladium cylindrosporum, Tolypocladium nivea, Metarhizium anisopliae, Verticillium lecanii, and Paecilomyces farinosus. Hybridizations carried out with multiple probes simultaneously present produced unique patterns which characterized the B. bassiana group from all other fungi tested. These results are discussed in terms of how mtDNA polymorphisms in B. bassiana may relate to natural population structures, mt transmission in deuteromycetes, and the use of mtDNA polymorphisms in structural analysis of mtDNA.     

Age-specific dose-mortality effects of Beauveria bassiana (Deuteromycotina: Hyphomycetes) on the European corn borer,Ostrinia nubilalis (Lepidoptera: Pyralidae)

Three isolates of Beauveria bassiana were evaluated under laboratory conditions for pathogenicity against the European corn borer, Ostrinia nubilalis. Dose-mortality regressions and nonparametric statistical analyses revealed significant differences in pathogenicity between isolates and across O. nubilalis instars. First instars were found to be the most susceptible of the five larval stages. Little difference was noted for instars 2, 3, and 5 within any of the three B. bassiana isolates. Fourth instar larvae were found to be the most tolerant in all cases.     

Microsatellite markers to monitor a commercialized isolate of the entomopathogenic fungus Beauveria bassiana in different environments: Technical validation and first applications

Here, we report on the application of five previously developed microsatellite markers (simple sequence repeats, SSRs) to monitor an isolate of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuill. in different environments. Discriminatory power of these SSR markers was assessed in two commercialized B. bassiana isolates as well as in 16 B. bassiana isolates from a world-wide collection, and three of the five SSR markers were estimated to allow a confident discrimination among the given isolates. Sensitivity thresholds of 0.1 pg DNA were subsequently determined for all SSR markers in case pure genomic fungal B. bassiana DNA was used as a template for PCR assays, but threshold levels varied depending on the environment (soil, plant) of the PCR assay. Furthermore, presence of a commercialized B. bassiana isolate was monitored via these SSR markers in three different types of potting substrates over a period of 14 weeks. With two SSR markers, strain-specific products were detected up to 14 weeks after application of B. bassiana to the substrate. Infectivity of B. bassiana conidia in the respective soil samples was confirmed by the Galleria baiting technique. Together these results indicate that molecular markers like SSRs specific for commercialized strains of entomopathogenic fungi are important tools to monitor a particular fungal strain in complex environmental samples such as bulk soil or plant DNA.     

Genetic diversity among summer and winter Beauveria bassiana populations as revealed by AFLP analysis

The genetic diversity of Beauveria bassiana was investigated by comparing 40 isolates collected from summer and overwintering populations of Sunn pest from different areas in Syria and Turkey, using amplified fragment length polymorphism (AFLP) markers. Considerable genetic variability among B. bassiana isolates was revealed. The examined isolates were divided into three distinct clusters (A, B, and C). Within these clusters, the summer isolates from Syria and Turkey were grouped together in three sub-clusters (A3, A4, and B2). Also, principal coordinate analyses (PCA) showed clear separation (62.5%) between summer and winter isolates. These differences in the genetic structure may be explained by the variety of eco-geography over the sampled areas of B. bassiana isolates. This information on genetic variation among summer and winter B. bassiana isolates is helpful in designing an effective integrated pest management program for Sunn pest.     

Diversity of indigenous Beauveria and Metarhizium spp. in a commercial banana field and their virulence toward Cosmopolites sordidus (Coleoptera: Curculionidae)

Metarhizium anisopliae and Beauveria bassiana sensu lato were isolated, from 7 and 41 % of soil samples from a commercial banana field, with average fungal density of 4.3 × 10³ and 8.2 × 10³ CFU g^?1 soil, respectively. Twenty-one morphologically distinct B. bassiana and four M. anisopliae sensu lato isolates from different plots within the field were further characterized. ISSR fingerprinting revealed six different clusters for B. bassiana, whereas gene sequencing revealed three M. anisopliae sensu stricto and one unclassified Metarhizium sp. Bioassays with one or more representative isolates from each Metarhizium species and B. bassiana cluster showed that all indigenous isolates had lower virulence and significantly greater ST₅₀s than reference (exotic) isolates. The data suggest that the low virulence of most indigenous isolates toward Cosmopolites sordidus adults and their relatively low density in soil samples, may help explain the low occurrence of epizootics caused by entomopathogenic fungi in populations of this pest, also known to burrow under the soil surface in banana plantations.     

Comparative laboratory studies on three fungal pathogens of the elm bark beetle Scolytus scolytus: Pathogenicity of Beauveria bassiana,metarhizium anisopliae, and Paecilomyces farinosus to larvae and adults of S. scolytus

The entomogenous fungi Beauveria bassiana (nine isolates), Metarhizium anisopliae (seven isolates), and Paecilomyces farinosus (four isolates) were tested as pathogens of larvae of the elm bark beetle, Scolytus scolytus. Single isolates of B. bassiana and M. anisopliae were also tested against adult beetles. Of the 21 isolates tested as conidial suspensions against larvae, all proved pathogenic. The three most and least virulent isolates were, respectively, isolates of B. bassiana and M. anisopliae. The other isolates fell between these two extremes, with the four P. farinosus isolates all moderately virulent. Spore retention on larvae following inoculation was estimated by washing conidia off the larvae. From the results it was possible to relate larval mortality to the approximate spore dose causing infection at different spore concentrations. Thus, application of spores of the three pathogens at a concentration of 10³ spores/ml resulted in limited mortality. At this concentration, an average of only a single spore was recovered from the inoculated larva. Adult bark beetles also proved susceptible to infection by isolates of B. bassiana and M. anisopliae. They were exposed to discs of elm bark dipped in a conidial suspension. It was estimated that a dose of less than 100 spores could cause infection of beetles following feeding on the elm bark discs.     

Natural occurrence of soil-borne entomopathogenic fungi in the Moroccan Endemic forest of <Emphasis Type="Italic">Argania spinosa</Emphasis> and their pathogenicity to <Emphasis Type="Italic">Ceratitis capitata</Emphasis>

The occurrence and abundance of entomopathogenic fungi were analysed in 203 soil samples of the Moroccan endemic forests of Argania spinosa, the world main refuge of the Mediterranean fruit fly, Ceratitis capitata. Using the Galleria baiting method and selective media, entomopathogenic fungi were isolated from 186 of the 203 (91.62%) soil samples, with only three species found: Beauveria bassiana (Balsamo) Vuillemin, Metarhizium anisopliae (Metschnikoff) Sorokin and Paecilomyces lilacinus (Thom.) Samson. B. bassiana was the most widespread entomopathogenic fungi (90.64%) in the Argan forest whereas M. anisopliae was less common (15.27%) and P. lilacinus was very rare (1.48%). This is the first report of natural occurrence of M. anisopliae and P. lilacinus in Morocco. Furthermore, 118 Moroccan B. bassiana isolates were studied for their pathogenicity to C. capitata and thermotolerance. Most of these autochtonous B. bassiana isolates were virulent (86.44%) to Medfly pupae and tolerant (55.08%) to temperature stress at 45°C for 2 h. Only 60.17% of Moroccan B. bassiana isolates might be considered as highly entomopathogenic and will serve as a source of potential biological control agents to C. capitata. The percentage of thermotolerant and pathogenic B. bassiana to C. capitata were shown to decrease significantly at winter time characterized by low temperatures and absence of any noticeable medfly in the Argan forest. The occurrence, thermotolerance and virulence of B. bassiana isolates to C. capitata seemed to be related to the sampling periods and location. Our data are discussed with respect to fungal ecology and biocontrol potential of B. bassiana isolates in relation to their habitat.     

Biochemical and molecular characterization of wild-type and fused protoplasts of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis>

Protoplasts were isolated from two isolates each of Beauveria bassiana and Metarhizium anisopliae using lysing enzymes. Intra- and intergeneric protoplast fusion has been carried out using 40% polyethylene glycol. The fused protoplasts of B. bassiana and M. anisopliae have been regenerated on Czapek–Dox agar media, and a total of four fusants were selected for further studies. An increase in proteinase and chitinase enzyme activity was recorded with all fusants as compared to the wild-type isolates. To understand the nature of recombination process, random amplification of polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) were carried out on genomic DNA of fused and wild-type isolates. The present study demonstrates the scope and significance of the protoplast fusion technique as a rapid consistent method for identification of B. bassiana and M. anisopliae fused and wild-type isolates based on the banding pattern of RAPD and RFLP that can be reliably used ahead for further applications on these species.     

Preliminary study of genetic variation in Hawaiian isolates of Beauveria bassiana [Hypocreales, Cordycipitaceae

There have been no previous surveys documenting genetic diversity in Beauveria bassiana (Balsamo) Vuillemin in Hawaii. We used PCR primers and DNA sequencing to genetically characterize 14 isolates of B. bassiana collected from insects in east Hawaii island (the largest Hawaiian island, known as the ‘Big Island’) and compared these with the ‘GHA’ strain found in the commercial product BotaniGard®. Twelve of the 14 Hawaiian isolates were unique and the GHA strain was not among those isolated from the wild. Our data provides evidence that genetic diversity of B. bassiana in Hawaii is high over small spatial scales.     

Diversity analysis of Beauveria bassiana isolated from infected silkworm in southwest China based on molecular data and morphological features of colony

Beauveria bassiana is an important entomopathogenic fungus that not only often causes infection and epidemics of wild insects but some strains also show pathogenicity to the silkworm, Bombyx mori. The present study is about diversity of B. bassiana isolated from the silkworm in southwest China. Five strains of B. bassiana were isolated from infected silkworm. Two isolates, GXtr1009 and GXtr1010, were isolated from infected silkworms treated with two kinds of biological pesticides applied in Guangxi province, and three isolates, SCsk1006, YNsk1106 and GXsk1011, were collected from naturally infected silkworms from different geographical locations in Yunnan and Sichuan. All of the isolates showed highly similar conidia and conidial fructification, but the colony characteristics demonstrated great differences among the isolates. The ITS and 18S rDNA sequence analysis was sufficient to identify all five isolates as B. bassiana. However, the dendrogram, based on the ISSR data, produced two large genetic groups. GXtr1009 and GXtr1010 comprised one group, and SCsk1006, YNsk1106 and GXsk1011 converged in a different large group. The results suggested that, although all of these five B. bassiana strains were pathogenic to silkworms, strains of biological pesticides could be differentiated from strains of naturally infected silkworm via ISSR analysis.     

Cold activity of Beauveria and Metarhizium, and thermotolerance of Beauveria

Heat and cold are environmental abiotic factors that restrict the use of entomopathogenic fungi as agents for biological control of insects. The thermotolerance and cold activity of 60 entomopathogenic fungal isolates, including five species of Beauveria and one isolate of Engyodontium albus (=Beauveria alba) were examined as to tolerance of temperatures that might be encountered during field use. In addition, cold activity of eight Metarhizium spp. isolates was evaluated. The isolates were from various geographic regions, arthropod hosts or substrates. High variability in conidial thermotolerance was found among the Beauveria spp. isolates after exposure to 45 °C for 2 h, as evidenced by low (0-20%), medium (20-60%), or high germination (60-80%). The thermal death point (0% germination) for three rather thermotolerant B. bassiana isolates (CG 138, GHA and ARSEF 252) was 46 °C for 6 h. At low temperatures (5 °C), with few exceptions (e.g. CG 66, UFPE 479, CG 227, CG 02), most of the B. bassiana isolates germinated well (ca. 100%). On the other hand, only one isolate of Metarhizium sp. was cold-active (i.e. ARSEF 4343 from Macquarie Island, 54.4°S, Australia). This probably is a M. frigidum isolate. The E. albus isolate (UFPE 3138) was the most susceptible isolate to both heat and cold stress. Isolates ARSEF 252 and GHA of B. bassiana, on the other hand, presented exceptionally high thermotolerance and cold activity. Some isolates with high cold activity, however, were thermosensitive (e.g. ARSEF 1682) and others with high thermotolerance had low cold activity (e.g. CG 227). An attempt to correlate the latitude of origin with thermotolerance or cold activity indicated that B. bassiana isolates from higher latitudes were more cold-active than isolates from nearer the equator, but there was not a similar correlation for heat.     

Molecular Characterization and Virulence of Beauveria spp. from the Pine Processionary Moth, Thaumetopoea pityocampa (Lepidoptera: Thaumetopoeidae)

The pine processionary moth Thaumetopoea pityocampa (Den. & Schiff.) is one of the most harmful pests to pine species in Mediterranean countries including Turkey. Caterpillars of T. pityocampa are not only significantly harmful to forest trees but also responsible for various allergic reactions in humans and animals. In this study, in order to find a more effective and safe biological control agent against T. pityocampa, we investigated fungal pathogens of T. pityocampa in the Black Sea Region of Turkey and tested their pathogenicity on it. Five different fungi were isolated and identified based on their morphological and molecular characteristics including ITS and partial sequence of EF1-[alpha]. Based on these characteristics, four isolates were identified as Beauveria bassiana cf. Clade C (Rehner and Buckley in Mycologia 97:84–98, 2005) and one isolate was identified as Beauveria bassiana. Among these isolates, B. bassiana KTU-24, B. bassiana cf. Clade C KTU-66 and KTU-67 showed the highest virulence with 100% mortality within 10 days after application. B. bassiana isolate KTU-24 produced the highest mycosis value with %100. Consequently, B. bassiana KTU-24 seems to be good candidate for further investigation as a possible biological control agent against this pest.     

Hypocrealean fungi associated with populations of <Emphasis Type="Italic">Ips typographus</Emphasis> in West Carpathians and selection of local <Emphasis Type="Italic">Beauveria</Emphasis> strains for effective bark beetle control

In Slovakia, a diversity of entomopathogenic fungi (Ascomycota, Hypocreales) associated with outbreaks of Ips typographus was studied in 81 localities and as many as 113 in vitro cultures of five entomopathogenic species were isolated from infected individuals: Beauveria bassiana (87 isolates), B. pseudobassiana (14 isolates), B. caledonica (6 isolates), Lecanicillium lecanii (4 isolates) and Isaria farinosa (2 isolates). B. pseudobassiana is recorded in natural populations of I. typographus for the first time. Biological properties of selected Beauveria isolates, including colony growth, biomass production, conidia yield and pathogenicity to I. typographus adults, were studied in a series of laboratory bioassays and much intra- and interspecific variability was detected. B. bassiana isolates produced biomass or conidia at significantly higher rate than B. pseudobassiana and B. caledonica isolates. Two B. bassiana isolates were selected as the most virulent to bark beetle adults, demonstrating a mean LC₅₀ ranging from 0.72 to 2.05?×?10⁶ conidia ml^?1, and were qualified as promising candidates for biocontrol of I. typographus. Their virulence was significantly higher than that of the mycoinsecticides Boverol®, which was used as a reference strain in the virulence bioassays.     

Susceptibility of preimaginal western cherry fruit fly, Rhagoletis indifferens (Diptera: Tephritidae) to Beauveria bassiana (Balsamo) Vuillemin Clavicipitaceae (Hypocreales)

Last-instar larvae of the western cherry fruit fly, Rhagoletis indifferens, were subjected to Beauveria bassiana GHA incorporated into sterile sand and non-sterile orchard soil. Mycosis in the pupal stage was observed in >20% of buried R. indifferens pupae and >80% of larvae entering sand treated with either of two B. bassiana isolates. When pre-pupal larvae burrowed into conidium-treated non-sterile cherry orchard soil, the incidence of mycosis, on both the puparia and internally developing pupae, increased with dose. Internal pupal tissues were found to contain B. bassiana. Increasing the soil moisture level from 20% to 35% water holding capacity did not have an effect on the percentage of mycosed pupae. This is the first evidence that the preimaginal stages of R. indifferens are susceptible to infection by B. bassiana.     

Genetic Variability of Beauveria bassiana and a DNA Marker for Environmental Monitoring of a Highly Virulent Isolate Against Cosmopolites sordidus

The banana weevil Cosmopolites sordidus (Germar) is one of a number of pests that attack banana crops. The use of the entomopathogenic fungus Beauveria bassiana as a biological control agent for this pest may contribute towards reducing the application of chemical insecticides on banana crops. In this study, the genetic variability of a collection of Brazilian isolates of B. bassiana was evaluated. Samples were obtained from various geographic regions of Brazil, and from different hosts of the Curculionidae family. Based on the DNA fingerprints generated by RAPD and AFLP, we found that 92 and 88 % of the loci were polymorphic, respectively. The B. bassiana isolates were attributed to two genotypic clusters based on the RAPD data, and to three genotypic clusters, when analyzed with AFLP. The nucleotide sequences of nuclear ribosomal DNA intergenic spacers confirmed that all isolates are in fact B. bassiana. Analysis of molecular variance showed that variability among the isolates was not correlated with geographic origin or hosts. A RAPD-specific marker for isolate CG 1024, which is highly virulent to C. sordidus, was cloned and sequenced. Based on the sequences obtained, specific PCR primers BbasCG1024F (5′-TGC GGC TGA GGA GGA CT-3′) and BbasCG1024R (5′-TGC GGC TGA GTG TAG AAC-3′) were designed for detecting and monitoring this isolate in the field.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-012-0292-9) contains supplementary material, which is available to authorized users.     

Detection and Identification of Bursaphelenchus Species with DNA Fingerprinting and Polymerase Chain Reaction

We have evaluated the potential of DNA-based methods to identify and differentiate Bursaphelenchus spp. and isolates. The isolation of a DNA probe, designated X14, and development of a DNA fingerprinting method for the identification and differentiation of Bursaphelenchus species and strains is described. Polymerase chain reaction (PCR) amplification of DNA isolated from Bursaphelenchus species using two primers derived from the sequence of the cloned repetitive DNA fragment X14 resulted in multiple band profiles. A 4-kb fragment thus amplified from B. xylophilus DNA was not amplified from B. mucronatus or B. fraudulentus DNA. In addition to this fragment, several other fragments are amplified from the three species. The banding patterns obtained allowed species identification and may have value in determining taxonomic affinities.     

Potential of Metarhizium anisopliae and Beauveria bassiana to control Dinoderus porcellus (Coleoptera: Bostrychidae) infesting yam chips

The beetle Dinoderus porcellus Lesne is a serious storage insect pest that causes important losses by destroying stocks of yam chips. In the aim to found an alternative control method to the use of synthetic insecticides for its management, the virulence of the entomopathogenic fungi Beauveria bassiana (Balsamo) Vuillemin (isolate Bb115) and Metarhizium anisopliae (Metschnikoff) Sorokin (isolate Met 31) against adults of D. porcellus was evaluated under laboratory conditions (25 ± 2 °C and 70 ± 5% RH). Then, the effectiveness of the most virulent entomopathogenic fungus as biological agent against D. porcellus was assessed under farmer storage conditions. For each entomopathogenic fungus isolate, four conidial concentration (0, 10⁵, 10⁷, and 10⁹ conidia/mL) at the dose of 1 µL were inoculated topically on D. porcellus adults (3–5 days old). Observations focused on insect mortality, cadaver sporulation and weight loss of yam chips. Lethal dose and lethal time values were estimated using probit analysis. Both fungal isolates at all conidial dose caused more than 50% mortality on day 7, with the highest mortality (94.44%) achieved using B. bassiana at the 10⁹ conidia/mL. LT₅₀ values for B. bassiana and M. anisopliae isolates were 2.63 and 3.35 days, respectively, while their LT₉₀ values were 6.15 and 9.87 days, respectively. Yielding the lower LD₉₀ values and the highest rates of cadaver sporulation, B. bassiana isolate appeared as the most virulent against D. porcellus. After 3 months of storage, comparatively to the control, the B. bassiana isolate at the highest conidial dose (10⁹ conidia/mL) significantly reduced D. porcellus populations, and weight loss of yam chips. This study revealed the potential of B. bassiana and M. anisoplae isolates as biological control agent against D. porcellus for yam chips protection.     

球孢白僵菌种内比较线粒体基因组学分析

【目的】明确球孢白僵菌种内线粒体基因组的分化程度。【方法】从GenBank下载已知的球孢白僵菌6个菌株线粒体基因组序列,详细分析基因组的组成结构,比较外显子区、内含子区和基因间区的碱基变异情况,分析菌株间的系统发育关系。【结果】球孢白僵菌不同菌株的线粒体基因组大小为28.8–32.3 kb,都有14个常见的核心蛋白编码基因、2个rRNA基因和25个tRNA基因,具有很强的共线性关系。但是,不同菌株含有的线粒体内含子数目存在差异(2–5个/菌株),在cox1、cox2和nad1基因中表现出内含子插入/缺失多态性,这是导致线粒体基因组大小变化的主要因素。对外显子、内含子和基因间区的碱基变异情况进行分析,发现内含子和基因间区相对变异较大,而外显子区相对变异较小。系统发育分析发现,这些球孢白僵菌菌株以很高的支持度聚在一起,具有相同内含子分布规律的菌株也具有较近的聚类关系。【结论】本研究首次报道球孢白僵菌因内含子数目不同、插入缺失突变和单核苷酸变异等在线粒体基因组上表现出较大程度的遗传分化,为认识真菌种内线粒体基因组分化提供了新的证据。     

Genetic nature, stability, and improved virulence of hybrids from protoplast fusion in Beauveria

Genetic improvement of two different strains of the entomopathogenic fungus Beauveria bassiana for more effective control of Ostrinia nubilalis and Leptinotarsa decemlineata was obtained by crosses with the insecticidal toxin-producing strain Beauveria sulfurescens. Protoplast fusion between diauxotrophic mutants resulted in the recovery of some stable prototrophic fusion products. The low levels of virulence of the wild type strain B. bassiana 28 isolated originally from L. decemlineata were enhanced both on L. decemlineata and O. nubilalis for one of the hybrids obtained (FP 8) from the cross B. bassiana 28 × B. sulfurescens 2. Fusion product 25 obtained from the cross between B. sulfurescens and the highly pathogenic strain B. bassiana 147 showed a three-day reduction in the LT₅₀ towards O. nubilalis. Southern blot hybridization with nine probe-enzyme combinations were conducted on genomic DNAs from the original wild strains, parental mutant strains, and fusion products. Additive banding patterns or unique banding pattern of either parental strain was observed in five hybrids, indicating their status as recombinant and/or partially diploïd. Combination of RFLP markers indicative of both parental genomes was never observed with fusion product FP 25. The stability of the virulence following passage through insect-host and stability of molecular structure for the fusion products FP 8 and FP 25 suggest that asexual genetic recombination by protoplast fusion may provide an attractive method for the genetic improvement of biocontrol efficiency in entomopathogenic fungi.     

Natural populations of the entomopathogenic fungus Beauveria bassiana in Chinese forest ecosystems are diverse and reveal equal frequencies of mating types within phylogenetic species

Beauveria bassiana is an important entomopathogenic fungus with widespread application in the biological control of harmful insect pests. This species is widely distributed as an anamorph while only two teleomorph specimens have been found in eastern China. However, little is known about the ecological conditions for sexual reproduction in natural populations of B. bassiana. Here, we collected 488 isolates of Chinese B. bassiana sensu stricto from five sites, in which teleomorph or anamorph occurred, and used molecular phylogenetic and haplotype information to determine phylogenetic diversity, mating types, and sexual reproductive potential in these populations. Molecular identification based on denaturing gradient gel electrophoresis (DGGE) and combined data of the nuclear intergenic region Bloc and translation elongation factor-1a (TEF) assemblage resolved five B. bassiana s.s phylogenetic species labeled according to their geographic origin: Europe/N. Africa 1, Asia 3, Asia 4, AFNEO_1, and N. America 2. In Guniujiang and Manshuihe collection sites, teleomorph isolates RCEF 0771 and RCEF 0382 were both identified as Europe/N. Africa 1 phylogenetic species. In addition, more than half of the isolates in five representative sites belonged to Europe/N. Africa 1. However, the teleomorph of B. bassiana s.s. was not detected in Kuankuoshui while isolates within Europe/N. Africa 1 were present at this site, and isolates belonging to Europe/N. Africa 1 were not found in either Jingyuetan or Dinghushan collection sites. Distribution of MAT1 and MAT2 mating type idiomorphs in Europe/N. Africa 1 were 51:69, 37:24, and 15:15 in Guniujiang, Manshuihe, and Kuankuoshui, respectively. The presence of teleomorph and roughly equal frequencies of opposite mating types indicate regular sexual reproduction in B. bassiana natural populations. The data offer a better understanding of the ecological conditions of sexual reproduction in natural populations of B. bassiana. These results also yield insights into the potential for sexual reproduction in other supposedly ‘asexual’ fungi.