| Larix spp. are distributed widely in China. Due to the characteristics, e.g. drought resistance, cold tolerance, rapid growth and high value of timber, larches have became the fast-growing and high-yield timber forest tree species. Ips subelongatus is the main pest infected the Larix spp. in eastnorther Asia, including China, Korea, Japan and Far east of Russia. It has been reported that I. subelongatus mainly happened in northeast and north of China. I. subelongatus was a secondary pest threatening the larch plantations in China. Current researches showed that the bark-beetles and fungi formed a stable association in nature. Generally, the associated fungi are of remarkable benefit for the symbiosis integrated to destruct the defense devices of the hosts. They also help bark beetles infringe upon trees and conserve population density. The present study aimed at providing theoretical basis for controling the composite damages of I. subelongatus from the viewpoint of associated fungus pathogens.In this research, The dominant species of ophiostomatoid fungi community in the beetle habits in China were tested for their comparative pathogenicities and possible host differentiations through artificial inoculations to mature larch trees. Three forest plots with different beetle attack intensities were chosen for experiments at two inoculum densities. In this research, we separated and purified the associated fungi from the bodies of I. subelongatus infecting three allopatric larchs: L. olgensis in Heilongjiang, L. prineipis-rupprechtii in Inner Mongolia and L. gmelini in Heilongjiang, Inner Mongolia and their gallery tissues. In order to inventory the Graphium and Leptographium fungi associated with the I. subelongatus, colonies and morphology observation, multiple DNA sequences comparisons and phylogenetic analyses were conducted.The pathogenicity of dominant ophiostomatoid species obtained in this study was tested on mature Larix trees. The results showed that O. kryptum produced the largest lesions on the inner bark around the inoculation point while O. brunneo-ciliatum produ-ced the shortest. Observations of the comparisons of pathogenicity for O. kryptum which isolates from L. decidua and O. kryptum which isolates from L. olgensis confirmed that O. kryptum probably had pathogenicity differentiation. The research reveals that apparent difference in pathogenicity of strain with the host of Larix gmelini and strain with the host of L. prineipis-rupprechtii exists when inoculating L. prineipis-rupprechtii, which demonstrates that E. fujiensis had pathogenicity differentiation. The comparisons of pathogenicity for different hosts strains of O. kryptum confirmed that the pathogeni-city of O. kryptum was identical, no matter with the strain origin. However, the pathogenicity of E. fujiensis was not identical, the strains that isolated from L. prineipis-rupprechtii was the most strongly pathogenic to three allopatric larchs and the strains that isolated from L. gmelini was weakest. It indicates that E. fujiensis had host differentiation. Combing with the multi-gene sequences characteristic indicates that E. fujiensis may exists subspecies differentiation under host stress for long-term. Further research reveals that strains isolated from China was strongly pathogenic for L. kaempferi, but weak pathogenic for Larix in China, which demonstrates that E. fujiensis were strong virulent to L. kaempferi. Compared with the susceptibility of L. kaempferi, confirmed that Larix in China formed stable resistance in the process of co-evolution of Larix in China and pathogen.Observations of colony, morphology characteristic, DNA sequence comparisons for ITS2-partial LSU rDNA region, as well as the β-tubulin and EF-1α gene regions indicated that three Leptographium spp. fungi were collected: L. taigense and two previously undescribed species, one belonging to the L. piceiperda-complex, for which the name Leptographium zhangense Liu, Lu & Zhang sp. nov. is provided; another closely related to L. taigense, for which the name Leptographium innermongoliaae Liu, Lu & Zhang sp. nov. is provided. Moreover, L. taigense was a new record species from China. L. zhangense and L. innermongoliaae can be distinguished from resembling species with some morphological characters, for example the size and shape of condia. All those three Leptographium spp. were isolated from L. gmelini in Heilongjiang and Inner Mongolia, which formed new association with I. subelongatus and the larch.Observations of colony, morphology characteristic, and DNA sequence comparisons indicate that two species of genus Graphium were obtained, G. laricis and G. carbonarium, which can associated with I. subelongatus. Moreover, G. laricis is a new record species from China.G. laricis and G. carbonarium are firstly isolated from I. subelongatus, and forms a new association with the beetle. The result in this paper adds two new host species to the fungus and extends the distribution of its host as well.Based on the study of the associated fungi flora composition and pathogenicity, O. kryptum and E. fujiensis were chosen for developing a method of rapid and direct detection from wood and beetle body using species-specific primers PCR(SS-PCR). A pair of specific primers(OK1/OK2) was selected from the 12 pairs of primers of designed for O. kryptum. An amplicon of 248 bp was generated by using the specific primers(OK1/OK2) for O. kryptum. The specific primers(CFU1/CFU2) was designed for O. kryptum. An amplicon of 251 bp was generated by using the specific primers(CFU1/CFU2) for E. fujiensis. However, no amplicon was obtained from other Ophiostoma and Endoconidiophora fungi. The SS-PCR method has some advantages: short-time, high efficiency, saving cost and using easily in practice. |
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