| Millions of hibernating bats in the United States and Canada have died from white-nose syndrome（WNS）,an infectious disease caused by Pseudogymnoascus destructans（Pd）,and the bat diversity is now critically threatened,with some species even going extinct.Because bats are crucial for the management of agroforestry pests,seed dissemination,and plant pollination,their deaths due to WNS have resulted in severe ecological issues and financial losses.Bats in China have also been infected with Pd,although the fungal load is lower than in North America and the absence of disease suggests high resistance to Pd for bats in China.In this thesis,we selected Myotis pilosus,a bat with high resistance to Pd,as a research subject,and combined high-throughput sequencing and culture experiments to determine the skin microbiome of the bat during the late hibernation period,screened anti-Pd bacteria,detected volatile compounds,examined their effects on Pd,and revealed the reasons for the inhibition of Pd growth.The following were the key findings:During late hibernation,Myotis pilosus had a maximum Pd load of-4.36 and 52.63%prevalence.According to data from the 16S r RNA amplicon sequencing,the skin microbiome of the bat during late hibernation mainly contained Myroides,Flavobacterium,Rhodococcus,Sphingobacterium,Microbacterium,Delftia,Pseudochrobactrum,Chryseobacterium,and Arthrobacter.The skin microbiome was unaffected by sex,body weight,or roost site temperature.Only Simpson diversity for infected individuals was significantly lower than uninfected individuals(Mann-Whitney U test,Padj=0.041).For the genera with relative abundance higher than 1%in the skin microbiome of Myotis pilosus,combined with published literature,25 genera,including Pseudomonas,Arthrobacter,Erythrobacter,Sphingomonas,Comamonas,and Microbacterium were found to have specific inhibitory effects on fungal pathogens,and their relative abundance ranged from 42.16%to70.08%.The results of PICRUSt2 functional prediction analysis also indicated that the skin microbiome has potential inhibition pathways including terpene skeleton biosynthesis,neomycin biosynthesis and vancomycin antibiotics.No significant difference was found in the relative abundance of potential anti-Pd genera and inhibit pathways between infected and uninfected individuals.While,the relative abundance of Pseudomonas was significant higher in infected individuals than those in uninfected individuals(Wilcox.test,Padj=0.035).In this study,165 bacterial strains were isolated and cultured from Myotis pilosus in late hibernating.The strains with the ability to inhibit the growth of Pd were screened by co-culture experiments and were identified by 16S r RNA and rop B gene to 9 genera and 11 species,including Algoriella xinjiangensis,Pseudomonas paraversuta,Pseudomonas jessenii,Pseudomonas gessardii,Comamonas jiangduensis,Glutamicibacter bergerei,Rhodococcus qingshengii,Chryseobacterium sp.,Sphingobacterium sp.,Psychrobacter sp.and Arthrobacter sp..The relative abundance of the screened inhibitory genera in the skin microbiome was still high（33.00～79.96%）.Anti-Pd strains revealed many volatile compounds known to have inhibitory effects on fungal pathogens,such as disulfide dimethyl,trisulfide dimethyl,sulfide dimethyl and nonanal,etc.Methyl sulfide compounds accounted for a relatively high percentage（35.43～95.67%）.The compounds with high relative abundance were chosen to test the effect on inhibiting Pd.The experimental results showed that 100μL dimethyl trisulfide（1.9 mg/m L）,dimethyl disulfide（1.7 mg/m L）and propylene sulfide（1.4 mg/m L）completely inhibited the growth of Pd,andγ-decanolactone（1.7 mg/m L）and dimethyl sulfide（1.3mg/m L）had obvious inhibitory effects.In conclusion,this study demonstrated that skin microbiome of Myotis pilosus during the late hibernation period had the ability to resist the Pd fungus,which partially revealed the reason for the high resistance to Pd for bats in China.Additionally,the anti-Pd strains can provide a scientific basis for the biological control of WNS in bats. |
