Study On Diversity And Pathogenicity Of Ophiostomatoid Fungi Associated With Stem-boring Pests In Coniferous Forests

Herbivores and microbes have coexisted in coniferous forests since the Mesozoic era.Fungi associated with beetles have been a research hotspot for many years because of the variety of insect hosts,serious damages and strong virulences of the associated fungi.Fungi interacted with bark beetles and co-evolved each other.In most cases,associated fungi can provide nutrients to herbivores,change their feeding strategies,synergistically overcome host resistance,and help herbivores successfully invade the host,while fungi also benefit from bark beetles to spread to and inhabit in a new environment.Three pine shoot beetles(Tomicus yunnanensis,T.minor,T.brevipilosus)in Yunnan Province,Ips subelongatus and Monochamus alternatus/pathogenic pine wood nematode are the important pests and diseases endangering coniferous forests in China.The damage of herbivores is serious and lasts for a long time,which directly causes the death of the forests and brings irreversible losses to local economic development and ecological security once a large-scale outbreak.In this study,high-throughput sequencing,the traditional culture-dependent method,and chemical ecology methods were used to analyze the diversity and communities of associated fungi of three pine shoot beetles,Ips subelongatus and Monochamus alternatus/pine wood nematode.Phylogeny and pathogenicity of associated ophiostomatoid were analyzed further in details.The main results obtained are as follows:Seven hundred and seventy-two strains of ophiostomatoid fungi were isolated from the three pine shoot beetles and their galleries in Yunnan Province(Anning,Qujing,Yuxi,Xiangyun,Pu'er)used traditional culture-dependent method.The representative strains were identified based on comparisons of multiple DNA sequences,including the nuclear ribosomal large subunit(LSU)region,the internal transcribed spacer regions 1 and 2 together with the intervening 5.8S gene(ITS),the partial genes of ?-tubulin(tub-2),elongation factor 1?(tef-1?),and calmodulin(CAL).Phylogenetic analyses were performed using maximum parsimony(MP)as well as maximum likelihood(ML).Combinations of culture features,morphological characters,and temperature-dependent growth rates were also employed for speciesidentification.Eleven species belonging to five genera were identified.These included six known species,Esteya vermicola,Leptographium yunnanense,Ophiostoma brevipilosi,O.canum,O.minus,and O.tingens,and four novel taxa,described formally as Graphilbum anningense sp.nov.,O.aggregatum sp.nov.,Sporothrix pseudoabietina sp.nov.,and S.macroconidia sp.nov..A residual species was left unidentified as Ophiostoma sp.1.The overall ophiostomatoid community was by far dominated by three species,representing 87.3 %of the total isolates;these were O.canum,O.brevipilosi,and O.minus;and they were the dominated ophiostomatoid fungus of Tomicus minor,T.brevipilosus and T.yunnanensis,respectively;A new ophiostomatoid species,Ophiostoma olgensis,isolated from Larix olgensis and Larix gmelini infested by Ips subelongatus in northern China.Morphological characteristics and phylogenetic analysis showed that O.olgensis was most similar to O.kryptum;The aims of our study were to investigate and identify the ophiostomatoid communities associated with the epidemic pine wood nematode and the pine sawyer in Pinus massoniana and P.thunbergii forests in Shandong and Zhejiang Provinces,China.Two hundred and forty eight strains,six ophiostomatoid species were identified on the basis of morphological,physiological,and molecular data..The ophiostomatoid species included one known species,Ophiostoma ips,three undescribed species,viz.Ophiostoma album,O.massoniana,and Sporothrix zhejiangensis,and two species whose identity still is uncertain,Ophiostoma cf.deltoideosporum and Graphilbum cf.rectangulosporium.Ophiostoma ips was the most frequently isolated species,accounting for over 90% of all Ophiostomatales strains.In this study,we employed Illumina Mi Seq PE300 platform,internal transcribed spacer 1(ITS1)ribosomal DNA(r DNA)libraries to process 45 samples pooled from 1260 beetle individuals(each sample including 25–30 beetles,depending on the number of beetles collected)which were collected from five locations at both branch and trunk infectious periods of the beetles across five locations in Yunnan Province.A total of 1,326,579 reads were generated;the 97% sequence similarity cutoff produced 6 phyla,29 classes,76 orders,142 families,271 genera,416 species and 948 OTUs.641,604 and 261 OTUs were confined to T.yunnanensis,T.minor and T.brevipilosus,respectively.Venn diagram and Heat-map indicatedthat the fungal communities associated to T.yunnanensis and T.minor,were remarkably more diverse than that to T.brevipilosus,and the former two structures,associated to T.yunnanensis and T.minor,were similar to each other but distinct to the third one.Principal Coordinate Analysis suggested that epibiotic fungal community structures of the three Tomicus spp.conditioned by beetle species,hosts,infectious periods and locations.And fungal community structures of the three Tomicus spp.conditioned strongly by hosts and locations but weakly by beetle species and infected periods.Nine ophiostomatoid fungi were selected out and two strains of each species were tested on 5-year-old seedlings of Pinus yunnanensis to elucidate their pathogenicities.Preliminary results showed that O.aggregatum and L.yunnanense were the most pathogenic;followed by O.brevipilosi,O.minus,O.tingens,S.pseudoabietina,S.macroconidium;Gra.anningense and O.canum were the weakest.Subsequently,the volatiles from phloem of lesion area acaused by 9 species inoculation on Pinus yunnanensis and from adjacent healthy area were analyzed.It was found that the content of ?-pinene in lesion area was significantly higher than that in healthy area,while the content of ?-pinene and other compounds in phloem of Pinus yunnanensis did not change significantly.?-pinene and ?-pinene were selected to analyze the effects of different concentrations of the standard products on the growth rate of the fungi.It was found that low concentration of pinene has little inhibitory effect on the growth of the ophiostomatoid fungi,and even promotes the growth of some strains.However,high concentration of pinene significantly inhibited the growth of the fungi;the effect of ?-pinene was more obvious than ?-pinene.In conclusion,18 ophiostomatoid fungi were isolated and obtained,including eight new species,seven reported species and three unidentified species.High-throughput sequencing analysis showed that epibiotic fungal community structures of the three Tomicus spp.conditioned by beetle species,hosts,infectious periods and locations.Pathogenic test of ophiostomatoid fungi associated with three Tomicus spp.showed that the pathogenicity of ophiostomatoid fungi was different,but they could cause lesion to host pine trees.Recent years,there have been increasing studies on the biodiversity,biogeography and ecology ofophiostomatoid fungi in China.The results of ophiostomatoid fungi associated with three pine shoot beetles in Yunnan Province,Ips subelongatus and pathogenic pine wood nematodes showed that ophiostomatoid fungi have high diversity in China,which is helpful to compare with ophiostomatoid fungi associated with other bark beetles or pine wood nematode distribution areas,and to better understand the mechanism of these outbreaks of diseases and insect pests.It can also provide guidance for effective pest management and quarantine measures,and reduce the ecological and economic losses after pest outbreaks.