The Taxon-specific Degradation Rates Of Soil Extracellular 16S RDNA And ITS And Their Influencing Factors

Soil microbial diversity is critical to maintaining the stability and health of soil ecosystems,and is also a potentially promising indicator for assessing soil ecosystem health.It has become a hot research area in the field of environment.Soil microbial research mainly rely on total DNA extraction and amplification of high-throughput sequencing technology.The large amount of extracellular DNA exists in soil,which significantly influences the analysis of alive microbial abundance and diversity,as well as the relibilty of soil environmental risk assessment.The influence intensity of soil extracellular DNA on microbial diversity analysis largely depends on whether it has taxon-specific degradation.However,the research on extracellular DNA degradation is only carried out targeting a few sequences.There is still a large gap in the understanding of the taxon-specific degradation rates of extracellular 16S rDNA and ITS,and their influencing factors.In addition,the standard of soil loading weights for DNA extraction is not consistent,which may significantly affect the accuracy of microbial community research.In this study,soils were collected from desert,meadow,forest,and cropland ecosystems to examine the effects of soil loading weights(i.e.,0.010,0.025,0.250,0.500,and 1.000 g)on microbial community analysis.Then,a method was established to determine the taxon-specific degradation rates of extracellular DNA,based on primer labeling,qPCR,and high-throughput sequencing.With the methods developed in this study,the taxon-specific degradation rates of extracellular 16S rDNA and ITS in 30 soils collected across China were determined.The main influencing factors of taxon-specific degradation rates were determined through soil property measurements,as well as the abundance and diversity analysis of soil microbial community.Finally,based on the extraction of total and intracelluar DNA,the effect of taxon-specific degradation of extracellular DNA on microbial community analysis was examined.The main findings of this thesis are as follows:(1)The results showed that soil loading weights for DNA extraction did not significantly affect microbial abundance determination.In contrast,microbial richness,community composition and functional prediction significantly varied with different soil loading weights for DNA extraction.Microbial richness obtained from 0.010 and 0.025 g of soils was significantly lower than that obtained from 0.250-1.000 g of soils.In addition,microbial community composition significantly differed among the technical replicates using 0.010 or 0.025 g of soil for DNA extraction.However,when the soil loading weights for DNA extraction exceeded 0.500 g,the microbial community composition and predicted functional profiles were no longer affected by soil loading weights,and the variations among technical replicates significantly decreased.(2)After 48 days of incubation,the soil residual exogenous extracellular 16S rDNA and ITS were only 0.2-3.1%and 0.4-4.75%of the initial amounts,respectively.In addition,the richness of prokaryotes indicated by the exogenous extracellular DNA after incubation was only about 60%of that at the beginning of the incubation,and the lowest was only about 32%.The fungal richness indicated by the exogenous extracellular DNA also showed significant differences with incubation time.The overall degradation rates of extracellular 16S rDNA were positively correlated with soil water content,annual precipitation and annual temperature,but negatively correlated with soil pH value and altitude.The overall degradation rates of extracellular ITS DNA also showed a significant positive correlation with soil water content and a negative correlation with altitude.The composition of residual exogenous microbial communities in some sites showed significant differences with the extension of incubation time,which confirmed that there were significant differences in the taxonspecific degradation rates of soil microbial extracellular DNA.Specifically,the phyla with fast degradation rates were Crenarchaeota,Bacteroidetes,Chytridiomycota,and Glomerobacteria.The taxon-specific degradation rates of soil microbial extracellular DNA in different locations also showed some differences,and showed a significant correlation with soil physicochemical properties such as pH,available phosphorus,nitrate nitrogen and ammonium nitrogen,as well as environmental factors such as altitude and annual average temperature.The most obvious correlation was the annual average temperature.(3)The existence of extracellular DNA will lead to overestimation of microbial abundance and richness,and have a significant effect on the interpretation of microbial community composition.The research showed that the intracellular DNA accounted for about 40%of the total DNA,and the species richness of living microorganisms accounted for about 88%of the total microorganisms.After removing extracellular DNA,there are 40%differences in community structure between living microorganisms and overall microorganisms.The taxa leading to these differences were Chloroflexi,Firmicutes and their corresponding taxa under the phylum level.The relative abundance of Chloroflexi,Latescibacterota,Firmicutes and Patescibacteria in the overall microbial community was significantly higher than that in the living microbial community.However,Abditibacteriota and Badabacteria showed an opposite trend.Interestingly,microbial groups with higher rates of extracellular DNA degradation showed greater differences between living and total microbial communities.In addition,the community structure of living microbial community and total microbial community showed significant correlation with soil pH value,average annual temperature and average annual precipitation,but their relationship with environmental factors was also different.There was a significant correlation between the living microbial community structure and the richness ratio between total microorganisms and living microorganisms,as well as the Bray-Curtis distance.The total microbial community was significantly correlated with the ammonium nitrogen content of soil and the degradation rate of extracellular DNA.The findings in this thesis will contribute to optimizing the research strategy of soil microorganisms and examining the comparability and reliability evaluation of microbial research.It also provides critical information for the selection of soil microbial research methods.The methods developed in the current study can be also used to determine the extracellular DNA degradation rates of other markers.