| Beauveria bassiana is an ubiquitous entomopathogenic fungus, with broad host range. It has been reported having high natural variability and its populations have great heterogeneity. Since the end of the 20th century, rapid development of molecular markers has made it possible to reveal evolutionary relationships by use of these markers.The objective of this study was to elucidate the genetic structure of large samples consisting of over 2000 B. bassiana isolates obtained from all typical parts of China. By ISSR analysis on the isolates,150 strains representing various groups at different levels were selected for sequencing Bloc gene, so as to trace the phylogeny of Chinese population of B. bassiana and to understand the genetic diversity of different areas in China. In addition, molecular tracing was made for tracing the origins of the white muscardine in sericultural areas of north and south China, according to ISSR phylogeny based on the genetic diatance between subpopulations of over 300 isolates from pine caterpillars and over 400 isolates from silkworms. Based on analysis on gene flow and genetic differentiation, the possibility was investigated of host range expansion and virulence enhancement of the pine caterpillar isolates by gene exchange. It will provide theoretical basis for more reasonably use of B. bassiana in forest pest management.The main contents and conclusions are as follows:1.For the metapopulation consisting of 2124 B. bassiana isolates of China, the percentage of polymorphic loci (PPL) of eight ISSR primers showed a high polymorphism (100%). A total of 235 fragments were amplified. Nei’s genetic diversity (h) was 0.250, and Shannon’s information index (Is) was 0.388. While the diversity indices of different provincial subpopulations of B. bassiana varied differently:the PPL varied within 22.98%-95.32%, Nei’s genetic diversity varied within 0.082-0.226, and Is varied within 0.124-0.357. All these diversity indices showed a high level of genetic diversity and population heterogeneity, the northeast subpopulation showed highest genetic diversity and highest heterogeneity, and on the contrary, the northwest subpopulation showed the lowest.2. An AMOVA analysis showed the southwest subpopulation had the highest genetic differentiation, while the northwest population had the lowest one. Genetic differentiation and gene flow among the provincial subpopulations of B. bassiana in China and among different areas of provinces were analyzed:The mean genetic differentiation (Gst) of China was 0.139; the gene flow (Nm) was 1.549, and the genetic differentiation in different geographic areas ranged from 0.118to 0.277. It was revealed that the bigger of a study area, the more abundant the genetic differentiation was. The same situation was detected in the genetic distance and genetic identity.3. One hundred and fifty representative B. bassiana isolates of the Chinese population selected from ISSR analysis on all the 2124 isolates of the Chinese population were studied based on bloc gene analysis, the southwest subpopulation showed highest genetic diversity and highest heterogeneity, and the northwest subpopulation showed the lowest. The clustering dendrogram of the isolates could be divided into two clades, one originated in southwest and the another originated in northeast. Some isolates from neighboring provinces clustered together, but some were not. In general, there was no obvious genetic lineage correlated with geographic distribution.4. A total of 489 B. bassiana isolates were obtained from cadavers of the silkworm, Bombyx mori, in 12 different provinces of China. Analysis of the genetic structure of the causal agent populations by ISSR technique was used to trace the origin of the muscardine. AUPGMA dendrogram revealed that the 489 B. bassiana isolates were heterogenic and polyphyletic, and displayed typical regional distribution, although the phylogenetic lineages were not associated with geographic distance. The genetic differentiation (Gst) among populations reached 0.406, with the maximum Gst between populations reaching 0.647. The general gene flow (Nm) was low at 0.366, with the lowest at only 0.137, suggesting a high degree of differentiation that maintained the unique genetic variations of the populations and avoided convergence. These data indicated that the white muscardine in local silkworm populations was predominantly caused by a indigenous group of B. bassiana, rather than by any exotic elements, such as biological control agents of B. bassiana.5. The metapopulation genetic structure of B. bassiana occurring in the masson’s pine caterpillar, Dendrolimus punctatua, was investigated for all the 327 isolates from nine provinces, the result showed considerable genetic variations at population level. The phylogenetic tree constructed by UPGMA method and the 2-d principal component analysis (PCA) of the overall isolates both revealed that all the populations were polyphyletic. Anhui population showed the highest genetic diversity and population heterogeneity, while Fujian population showed the lowest ones.69.3% of the variation was found within various populations and 30.7% exited among populations. The low gene flow (0.566) indicated that there was an obstacle against genetic exchanges among different populations. In general, a certain degree of geographic differentiation and regional distribution existed in the phylogeny of the Chinese population of pine caterpillar isolates of B. bassiana, but the phylogeny was not associated with geographic distance.6. By the phylogenous evidences of B. bassiana in grdually expanded populations, the phylogeny of an integrated multi-host population of B. bassiana did not show regional distribution and host distribution, while the population of the isolates from a single host or a few hosts was characteristic of obvious regional and host distribution. Consequently, it was concluded that when large amount phylogenies, clear but with some difference, are integrated and overlaid together, the regional and host distributions will be concealed due to mutually penetration and mixture.7. Four hundred and eighty-eight B. bassiana isolates obtained from silkworm, Bombyx mori, collected from 13 Chinese provinces, and 327 B. bassiana isolates obtained from pine caterpillars collected from 9 provinces, were analyzed for population genetic structure using the ISSR technique based on genetic distance. A UPGMA dendrogram clustered them into three independent clades:two silkworm clades and one pine caterpillar clade. A 3-D principal component analysis further divided them into two completely independent host groups, revealing high host-specificity. This evidence suggested that white muscardine occurring in silkworm populations throughout southern China was not caused by any B. bassiana strain, either naturally prevailing in pine caterpillar populations or by any strain artificially released as a fungal insecticide against pine caterpillars. genetic differentiation and gene flow were investugated between silkworm-pathogenic and pine caterpillar-pathogenic B. bassiana isolates from all silkworm rering areas of China and from five provinces inhabited by both silkworms and pine caterpillars. The genetic differentiation coefficient Gst value across China was computed as 0.410, while the values of the five provinces ranged from 0.508 to 0.689; all above 0.25, the threshold for significant genetic differentiation. This suggested that B. bassiana strains isolated from the two different hosts maintained their respective heredity without a convergent homogenization trend, and denied the possibility that the host range of the caterpillar isolates is expanded and their virulence is enhancedto B. mori. These findings suggest that the use of B. bassiana did not caused silkworm muscardine and will not threaten the safety of sericulture. |
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