| Pine shoot beetles(Tomicus spp.)are important forest pests in Southwestern China,which have caused large-scale damage to Yunnan pine(Pinus yunnanensis).It is assumed widely that the fungi associated with those beetles had positive effect for their invasion and colonization.In this study,fungi associated with Tomicus spp.were investigated in Southwestern China,their colonization ability and the roles in Tomicus damage were researched.Furthermore,the population genetics and affecting factors of main fungal species were analyzed.The main conclusions were as follows:1.In the study of fungi associated with Tomicus,10 Ophiostomatoid fungi have been obtained by molecular biology and morphological identification,including Ophiostoma canum,O.ips,Leptographium yunnanense,L.conjunctum,L.sinense,L.pini-densiflorae,three new species Leptographium wushenense sp.nov.,L.panxianense sp.nov.,L.puerense sp.nov.,and a new record species Ophiostoma tingens.Furthermore,Ophiostoma canum was the main fungal species in southwestern China,especially in Yunnan Province,accounting for 75.6% of the total number of isolates.The fungi associates distributed in the forest stands of Pinus yunnanensis,P.amandii,P.kesiya and P.massoniana,and had closely biological association with Tomicus yunnanensis,T.minor,T.brevipilosus and P.amandii.The results revealed that Southwestern China was one of the areas with the most abundant associated fungi in the world.Moreover,important clues for explaining the co-evolutian of Tomicus spp.and fungal associates were provided.2.For the colonization ability of the Ophiostomatoid fungi,different fungi showed different degree damage to P.yunnanensis after artificial inoculation.Among them,L.puerense,L.conjunctum and L.panxianense induced the longer phloem lesion lengths,on the contrary,the lesion length after inoculation with O.canum was shorter.In the induced resistance response of P.yunnanensis,some species such as L.wushanense and L.sinense could elicit hosts significant changes of antioxidant enzymes activities and monoterpenes concentrations,and had strong colonization ability to the host.3.For the study of non-Ophiostomatoid fungi,14 strains collected from Qujing,Yongsheng and Ninglang in Yunnan were identified as Sydowia polyspora based on ITS sequence data and morphological characteristics.Inoculation experiment verified that this fungus was able to colonize stem phloem and pine needles of P.yunnanensis,and cause the phloem lesion,and part needles chlorisis or discoloration.Therefore,it was speculated that S.polyspora might cause secondary damages to this host after being spread with bark beetles.4.Basec on the combined gene analysis of ITS1-5.8S-ITS2,EF-1? and GFDH,O.canum collected from 9 sampling sties in Yunnan have not showed obvious genetic differentiation at molecular level.Meanwhile,there was not a significant correlation between genetic distance and geographic distance.Furthermore,clonal expansion of O.canum was confirmed by linkage and recombination tests.Therefore,it was presumed this fungus was a new invasive species.This finding had important scientific value for confirming the origin of the fungus and explaining the causes of fungal diversity in the area.5.Based on the analysis of environmental suitability,temperature annual range and precipitation of wettest quarter were the key environmental factors that affecting the distribution of O.canum.Predicted by Max Ent model,the potential distribution of O.canum was located in the high altitude and low latitude areas of the Central Yunnan Plateau,where the elevation was above 2000 meters,annual precipitation was less than 1100 mm,and the annual mean temperature was below 15°C.This provided important clues for the further study of the geographical distribution of fungal associates in the region.6.In the study of the roles of fungal associates in Tomicus damage,we discovered that the fungus could be introduce into P.yunnanensis by Tomicus beetle carrying and existing in the invasion hole.The indoor experiment verified the success rate invading and reproductive rate of T.yunnannesis associated with L.pini-densiflorae were significant higher than those without the fungus.In addition,Tomicus spp.showed high reproductive rate in the galleries with O.canum than those without O.canum.Therefore,it was suggested that fungal associates had a positive effect on the invasion and reproduction of Tomicus.This study will provide scientific basis for further understanding the formation and development of the fungi associates,assessing their potential damage to P.yunnanensis and revealing the invasion mechanism of Tomicus in this area. |
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