| White-nose syndrome（WNS）is an infectious disease of bats caused by a cryophilic fungus,Pseudogymnoascus destructans（Pd）,which primarily infects the skin tissue of bats during hibernation.It is currently responsible for one of the highest infectious disease related mortality rates ever in mammals and has a worldwide distribution.Over the past decade,millions of insectivorous bats have died in North America,reducing the control effect of bats on agricultural pests and causing severe economic losses,and there is no targeted drug or available vaccine to date.However,bats do not show mortality but exhibit resistance to the disease in Eurasia,probably due to multiple factors that contribute to bats persistence with this fungus.Considering that skin microbiota have important functions in defense against pathogen invasion,this thesis intends to study the bat skin microbial community by non-culture and culture methods,to reveal the microecological mechanism of bats resisting Pd infection,to elucidate the mechanism of microbes against Pd infection,and to discover key active substances.In order to show the prevalence of WNS in China and explore the role of bat skin microbiota under Pd invasion,the following studies were conducted:1.Investigation and analysis of the prevalence of WNS in bats in ChinaThe prevalence of WNS in bats was investigated during the bat hibernation period for 5 consecutive years from December 2017 to April 2021 in central and northeastern China.A probe-based q PCR method tested Pd load（infection intensity）from the collected cotton swabs.The results showed that the highest Pd prevalence of bats in 2021 was 74.13%,and the highest Pd load was-4.34±1.21;The lowest Pd prevalence of bats in 2020 was 17.24%,and the lowest Pd load was-5.55±1.04,with significant differences in Pd load among different years.The Pd prevalence of Myotis petax and Myotis ricketti was the highest,and the Pd load of M.petax reached-3.47±1.38,with significant differences in Pd load among species.The highest prevalence of Pd in bats was 82.89%in Fangshan district of Beijing and the fungal load was-3.78±1.37,with significant differences in Pd load between regions.Both Pd prevalence and fungal load were highest in the late hibernation period.However,there was no significant difference in fungal load between different genders.2.Skin microbial community and function differences of bat speciesBat skin bacteria of four species were collected from central and northeastern China.Using 16S r RNA sequencing,we found significant differences in the skin microbial community composition of four bat species in central and northeastern China.The dominant genera of Rhinolophus ferrumequinum were Brackiella,Corynebacterium and Staphylococcus.Murina leucogaster had a rich complement of dominant genera,such as Pseudomonas,Sphingobacterium and Flavobacterium.M.petax had communities dominated by the genera Citrobacter,families Micrococcaceae and Pasteurellaceae.However,the dominant genera of Rhinolophus pusillus were Mycobacterium and Kaistobacter.Skin microbial community diversity（Shannon diversity and OTU richness）differed significantly among bat species,and sites and body mass index significantly affected the diversity variation.Meanwhile,there were also significant differences in the skin microbial community structure of different bat species,and environmental temperature was significantly correlated with the changes of the skin microbial community of bat species.Although there were significant differences in alpha（OTU richness）and beta diversity among populations of Mu.leucogaster,no factors were found to influence them.Moreover,significant differences in microbial community structure existed between bats and the environment,and bat skin microbial communities were enriched with bacterial taxa that had low relative abundances in the environment.It was also found that most of the potential functions of the interspecies and interpopulation skin microbiota of bat species were associated with metabolism and secreted a variety of active substances that may potentially resist the infection of Pd.3.Screening of anti-Pd strains and whole genome sequencing analysisA total of 90 bacterial strains were isolated and cultured from 25 skin swabs collected from R.ferrumequinum and M.petax in central and northeastern China in March and April 2018,and three bacterial strains were successfully screened with ability to inhibit the growth of Pd.These strains were identified by morphology and molecular biology as Pseudomonas yamanorum GZD14026,Pseudomonas fragi GZD14479 and Pseudomonas brenneri XRD11711.The strongest inhibitory effect was found in GZD14026 strain,with an average inhibition rate of 71.33%.Whole genome sequencing analysis was performed on GZD14026 strain,and the raw data was filtered to remove low quality data,a total of 1,158,109,772 bp clean data was obtained.The assembled genome is 7,159,252 bp in length,encoding 6,997 genes.A total of 10 secondary metabolite gene clusters were predicted:7 non-ribosomal peptide synthases（NRPS）,1 terpene and 1 phenazine gene clusters.4.The study on mechanism of P.yamanorum GZD14026 against PdThe fermentation components and conditions of the Pd inhibitory active substances produced by GZD14026 strain were optimized by monofactorial test and orthogonal test,and the culture optimum components and conditions:glucose 0.5 g,tryptone 1.5 g,yeast extract 0.75 g,Na Cl 1.5 g,water 1000 m L;temperature 9℃,inoculation amount 1%,p H 6.5,rotation speed 220 r/min,and fermentation time 84 h,the inhibition rate increased to 85.84%.The effect of strain GZD14026 on the growth and development of Pd mycelium was investigated by scanning electron microscopy,which showed that the surface of the mycelium was rough,irregular,malformed,short and branches.The liquid fermentation broth of GZD14026 strain was extracted with methanol and dialyzed,the active substance was a non-protein small molecule ingredient with a molecular weight less than 1000 Da.After the fermentation broth was extracted by dichloromethane,the anti-Pd activity was the strongest,and the separated crude extract was extracted by column chromatography on silica gel with dichloromethane:methanol（100:5）as eluent to obtain a yellow active substance.The active substance was identified by high performance liquid chromatography,UV absorption spectroscopy,mass spectrometry and nuclear magnetic resonance and proved to be phenazine-1-carboxylic acid（PCA）with molecular formula C13H8N2O2,and the minimum inhibitory concentration of 50.12μg/m L against Pd.Based on sequence alignment,the whole genome of this strain was found to have PCA biosynthetic phz gene cluster.In addition,the volatile organic compounds produced by GZD14026 strain were also found to inhibit the growth of Pd.A total of 16compounds were detected by headspace solid-phase micro-extraction coupled with gas chromatography-mass spectrometry,and their activities were detected by co-culture tests of the above compounds with Pd.The results showed that three compounds inhibited the growth of Pd,namely,octanoic acid,3-methyl-3-buten-1-ol and 3-tert-butyl-4-hydroxyanisole.In summary,this study assessed the prevalence of WNS in China and elucidated the factors that may influence the skin microbiota of bat species and the roles of potential defense against pathogens,and successfully isolated a potential biocontrol strain of P.yamanorum.The results reveal the internal mechanism of high WNS resistance of bats in China from the perspective of skin microbes,and provide an important scientific theoretical basis for the spread of biological control of WNS. |
