Research On Molecular Taxonomy Of Metarhizium Lineage In China

Since Metchnkov first used Metarhizium anisopliae to prevent anisoplia austriaca Herbst successfully in 1873, Metarhizium were widely studied as fungi insecticides. When Sorokin use Metarhizium anisopliae as type species to establish the genus Metarhizum in 1883, the research on Metarhizum enter to the time of classification based on morphological characteristics. With the development of molecular techniques and genetic engineering, molecular data have been widely used for phylogenetic studies. And it is possible to make the phylogenetic analysis as the appearence of PCR technology and the improving of DNA sequencing technology. Recently the method of phylogenetic analysis is increasingly perfect, multi gene phylogenetic relationship analysis has been boardly applied to reflcet the evolutionary history of species.This experiment mainly study the classification of Metarhizium species in China. We first use the meathod of dead insects separating to isolate Metarhizium strains, with using fine needle to pick green spores of dead insects on SDAY medium to screen and separate Metarhizium isolates. And for soil samples, we choose dodine selective culture medium to screen Metarhizium strains. When the colonies on these two medium have generated spores, colony morphology characters like the color of hypha and spores should be observed. Also the shape and size of conidia should be recorded under light microscope. After the colonies were confirmed belong to Metarhizium, the targret colonies would be transferred to SDAY medium to purify. Combined with the Metarhizium strains from the Control Key Laboratory of Anhui Agriculture University Library(RCEF), a total of 134 strains of Metarhizium isolates should make molecular identification and morpholigical observation.According to Kepler & Rehner[1], Mz IGS3 gene is good to identify Metarhizium anisopliae complex species. In order to verify the effects of Mz IGS3 gene, we choose Mz IGS3 gene to preliminary identify 134 Metarhizium isolates, with EF-1a gene to added identified preliminarily. Combine with three gene sequencing, we exactly identify the strains that are inaccuratly in Mz IGS3 gene and EF-1a gene analysis.1. This experiment use Mz IGS3 gene to amplify to preliminary identify 134 Metarhizium strains and get 100 sequences. Based on the Mz IGS3 phylogenetic tree constructed with 100 sequences, the 100 isolated can be divided into eight branches Clade I to Clade VIII. With Mz IGS3 phylogenetic tree, branch A, B, C constitude the branch Clade I. Branch C is identified Metarhizium pingshaense, strains in branch A and branch B are fuzzy strains. Clade II is identified Metarhizium robertsii. Clade III is identified Metarhizium anisopliae.Clade IV is probably Metarhizium brunneum. Branch G, H, I are made of Clade II, and the strains of these branches are all inaccuratly and should identified furtherly. Clade VI is identified Metarhizium majus, Clade VII is identified Metarhizium lepidiotae, Clade VIII is identified Metarhizium acridum.2. EF-1a gene was choosed to added identified preliminarily and 10 isolates are belong to Metarhizium anisopliae complex species. 4 isolates are belong to Metarhizium cylindrospora. 13 isolates are belong to Metarhizium pemphigi. 4 isolates are fuzzy strains. 1 isolate is belong to Metarhizium frigidum. 1 isolate is belong to Metarhizium rileyi and isolate Ma123 is unknown.3. Multiple genes analysis can accurately identify species. Phylogenetic tree constructed with multiple genes is closer to the real species tree and can actually reflect the classification status of different species. Combine with the three genes analysis, we identify the fuzzy strains of Mz IGS3 gene phylogenetic tree furtherly.In Clade I, Ma106 and Ma141 are identified Metarhizium anisopliae, and others are identified Metarhizium pingshaense. Clade IV is identified Metarhizium brunneum. For Clade V, Mf03 of branch G is identified Metarhizium pingshaense and others are identified Metarhizium guizhouense. Branch H is identified Metarhizium majus and branch I is identified Metarhizium cylindrospora. For the fuzzy isolates of EF-1a gene phylogenetic tree furtherly, 4 fuzzy isolates are identified Metarhizium pemphigi, and the unknown isolate Ma123 is identiied a new species, Metarhizium anhuiense.According to the three genes analysis and combine with morphological characters, 134 strains in this experi ment can be divided 13 species, namely, Metarhizium anisopliae, Metarhizium brunneum, Metarhizium pingshaense, Metarhizium robertsii, Metarhizium guizhouense, Metarhizium majus, Metarhizium lepidiotae, Metarhizium acridum, Metarhizium cylindrospora, Metarhizium pemphigi, Metarhizium frigidum, Metarhizium rileyi and Metarhizium anhuiense.These species include three chinese new recorded species and one new species. Three chinese new recorded species are separately Metarhizium brunneum, Metarhizium robertsii and Metarhizium frigidum. One new species is Metarhizium anhuiense.4. Strain Ma123 is identified a new species as Metarhizium anhuiense. Colony on SDAY/4 medium attaining a diameter of 15 to 17 mm within 7 days and 33-52 mm within 14 days at 25?. Vegetative hyphae villous, initially white. Spores generate from center to periphery. Spores first pigment with yellow, then gradually become green. Cultures sometimes generate purple metabolites droplet. Vegetative hyphae septate, transparent, smooth-walled,1.2-2.6?m wide. Phialides elongate to column and up to 1-3 in a whorl, normally 6.4-18.3×2.0-3.5 ?m. Conidia one-celled, ellipsoidal, short, 6.6-4.8×3.7-1.5?m. Many conidia form a chain structure when released from the phialide.