Genome Sequencing And Analysis Of Inonotus Hispidus And Screening Of Control Agents

Inonotus hispidus,is an important wood rot fungus that mainly grows on the trunk and main branches of broad-leaved trees such as Fraxinus mandshurica Rupr.and Populus L.It can cause major economic losses and ecological damage.This study utilized the second-generation sequencing platform Illumina combined with the third-generation sequencing platform Pac Bio to sequence and assemble the whole genome of I.hispidus.Through whole-genome sequencing,reveal the nutritional pattern and related enzyme family of I.hispidus,further clarify its pathogenic mechanism as a rot pathogen,find effective measures to control the dry rot disease of F.mandshurica,and provide basic reference data for effective control of wood rot disease.According to the predicted proteome,the genomes of I.hispidus and 24 near-source fungi were compared and analyzed,and glycoside hydrolase（GHs）,glycosyltransferases（GTs）,polysaccharide lyases（PLs）,carbohydrate esterases（CEs）The distribution of carbohydrate binding modules（CBMs）,auxiliary activities（AAs）and cytochrome P450（cytochrome P450）.On this basis,we analyzed its genes related to host selection and pathogenicity,and explored the lignocellulosic degradation ability of Inonotus hispidus at the genomic level using comparative genomics.At the same time,six fungicides were selected to carry out the indoor toxicity test and the field control effect experiment of some fungicides on the I.hispidus,providing theoretical reference for the control of ash dry rot.The main results of this study are as follows:（1）The sequencing results of the whole genome of I.hispidus showed that:The genome size was about 40.27 Mbp,and the GC content was 48.74%.4585 contigs were assembled.7807 protein-coding genes were annotated.At the same time,the annotation results of carbohydrate active enzyme（CAZyme）showed that the genome contained 383 CAZyme genes,and there was no significant difference between the distribution of CAZyme in the genome of the fungi of the order Agaricomycetes.（2）The analysis of enzymes and pathways of lignin degradation function genes showed that:A total of 9 categories of 51 lignin degradation related genes were selected,including 9categories of copper oxidase,lignin modified peroxidase,glucose methanol choline（GMC）oxidoreductase,vanillin oxidase,copper free radical oxidase,1,4-benzoquinone reductase,oligosaccharide oxidase,iron reductase,and ferulate esterase.Among them,14 genes have been annotated into three metabolic pathways,closely related to the degradation of lignin by I.hispidus.（3）Comparative genomic analysis showed that various fungi utilize multiple AA1 family copper oxidase and multiple AA2 family II peroxidase enzymes,while the number of AA1laccase families is significantly different.In fungi of the order Poromycelia,the AA2,AA3,and AA5 families related to lignin degradation have significantly expanded,while the important lignin degrading enzyme AA8 family has been missing from the genome of most fungi of the order Poromycelia.Among the four different ecological types of fungi,the average number of enzymes related to the degradation of lignocellulose is grass rot fungi>white rot fungi>brown rot fungi>symbiotic vegetative fungi.The largest number of core genes（4616）are shared by the two fungi,but they are closest to the average number of lignocellulosic degrading enzyme genes（112）in brown rot fungi.The number of CAZymes of the four trunk decay pathogens was significantly different,and CE10 in the CE family was only distributed in large numbers in I.hispidus.（4）A total of 6 chemical fungicides were selected for the indoor bacteriostatic efficacy test of I.hispidus.The results of indoor bacteriostatic tests showed that 40%difenoconazole had very high bacteriostatic activity against I.hispidus,with an EC₅₀value of 0.1277μg·m L^-1;The second is 40%fenitrazole,with an EC₅₀value of 1.0954μg·m L^-1。（5）After treatment with fungicide stress,the physiological indicators of the mycelium were measured.The results showed that 40%difenoconazole had the earliest action time and the most rapid efficacy.After treatment,the SOD enzyme activity,CAT enzyme activity,MDA content,and melanin content of the strain were increased by 31.44%,13.99%,15.32%,and130.11%,respectively,compared to the control group.However,the effect of 40%nitrionazole was relatively late,but the duration of its bacteriostatic effect was longer.Compared with the control group during the same period,the SOD enzyme activity,CAT enzyme activity,MDA content,and melanin content of the treatment group were increased by 13.91%,40.46%,23.67%,and 1.97%,respectively.Both agents have a strong inhibitory effect on the bacteria,and can be used as the first choice for controlling the bacteria.（6）The results of field control showed that the control effect of Thiophanate-Methyl was9.09%against I.hispidus,and the control effect of Chlorothalonil was 1.89%.The control effect of Thiophanate-Methyl was significantly better than Chlorothalonil.