Studies On Comparative Genomics And Endobacteria Of Esteya Vermicola

Pine wilt disease caused by pine wood nematode(PWN)gives rise to devastating ecological damage and economic loss in Asia.Nematophagous fungi which can kill nematodes,is an important ecological component of the natural ecosystem.The endoparasitic fungus Esteya vermicola(Ev)is able to infect and kill PWN in a short time,and its high infectivity and lethality to PWN makes Ev a high potential biocontrol strain.But its evolutionary history and parasitic adaptation mechanism is not clear,the molecular basis of the nematophagous fungal lifestyle is poorly understood,and the similarity and difference between different nematophagous fungi are also unknown.We first discovered the biocontrol strain Ev concealing the symbiotic bacteria in the study.Symbiosis plays a key role in the biological,ecological and evolutionary processes,and symbiotic bacteria affect the host's biology in a number of ways.The study of comparative genome and symbiotic bacteria of Ev helps to reveal its pathogenic mechanism,and lay an important theoretical basis for biological control of PWN.The main results of sequencing using the PacBio RS II platform and annotation of Ev were as follows.The sequence depth of Ev was 104.66? coverage.The reads were assembled into 50 contigs and then further 42 scaffolds with an average coverage of 68.59?.The genome was 34.2 Mb size with 49.26% GC content and 8,424 protein coding genes.8,144 genes were annotated,accounting for 96.68% of the total gene.A total of 329 t RNAs,27 5.8S r RNA,6 28 S rRNA and 7 18 S r RNA were identified respectively.Repeated structural analysis showed that the genome had 12,778 simple sequence repeats(SSRs).The mitochondrial genome is 47,282 bp in length with a GC content of 24.85%.Annotated genes include 14 conserved protein-coding genes,1 large and 1 small rRNA subunit(rnl and rns)and 27 t RNAs.Comparative genomic analysis showed that the three groups of pathogenic fungi were consistent with their ability to invade the host,i.e.,the ecological adaptability.The nematophagous fungi encoded the highest number of adhesion proteins,the subtilases involved in invasion and lethal activity,the anchored repeat protein,the leucine repeat protein,tyrosinase,while insect pathogenic fungi encoded more chitinase,peptidase,protein kinase and toxins,and plant pathogenic fungi are more abundant in the content of carbohydrate enzyme and peroxidase,especially hemicellulase,pectate lyase,cutinase and P450.In particular,the endoparasitic fungal Ev highlights the highest amount of iron transporters,vitamin transporters and allantoin transporters,and the potential for transporting urea and ammonium is also strong in Ev,indicating that microbial pathogenesis is difference from other nematophagous fungi.The widespread presence of antibiotic resistance genes in 21 pathogenic fungi indicates the adaptability of pathogenic fungi in ecological competition which is conducive to their colonization in their host.Secondary metabolic analysis showed that two parasitic fungi,Ev and Hirsutella minnesotensis,had richer secondary metabolites,and their secondary metabolites could contribute to colonization in host nematodes.Further secreted protein predictions indicate that nematode fungi are rich in pathogenic-related extracellular secretory proteins such as subtilisin-like protease,lectin,chitinase,aspartic protease and tyrosine protease.Ev genome encodes more extracellular secreted proteins such as aspartic protease,glucanase,endoglucanase,laccase,cellobiose dehydrogenase,P450 and urea transporter.Based on the genomic level of phylogenetic and evolutionary analysis,the age of the split between Ev and its sister-group Grosmannia clavigera was 128.2 million years ago.Many transporter families of Ev have undergone expansion,accounting for 13.2% of the total expanded gene family,much higher than other pathogenic fungi.The proportion of species-specific gene family(17.0% and 21.0%,respectively)and species-specific expansion gene family(84.0% and 89.7%,respectively)in Ev and H.minnesotensis is also the highest among the nematophagous fungi group,suggesting that unknown functional diversity of endoparasitic fungi is worthy of further study.Among the nematophagous fungi,some important pathogenesis-related genes have been expanded,such as subtilisin-like,protease S41,aspartate peptidase and chitinase.Important orthologous genes such as nitrogen regulatory genes,subtilisin,aspartic protease and P450 have undergone positive selection in nematophagous fungi.The nematophagous fungi have evolutionary convergence in attacking nematodes,but their genetic structures and expansion gene families are different.The PCR of 16S rRNA and bacterial endotoxin detection showed that there were endobacteria in 4 strains Ev from different geographical regions.Fluorescence In Situ Hybridization(FISH)further confirmed the existence of endobacteria.Based on the 16 S rRNA phylogenetic analysis,the endobacteria of 4 strains of Ev fungi were clustered in monophyletic group.The endobacteria belonged to Gammaproteobacteria and was closely related to Pseudomonas stutzeri.The endobacteria could not be artificially cultured.At the same time,the non-symbiotic Ev fungi could not were obtained by antibiotic treatment.The results showed that the Ev host and its endobacteria were closely related in life.FISH,transmission electron microscopy and scanning electron microscopy showed that the symbiotic bacteria were spherical.The cell diameter of endobacteria is between 50 nm and 2 ?m.The cell wall thickness of the bacteria of different sizes is very different.The combination of ultrastructure and nucleic acid staining showed that the different growth stage of endobacteria were different in cell size,cell wall thickness and reproduction degree.The study of the basic biology of endobacteria will help to reveal the roles of endobacteria on the evolution and ecology of their fungal host.