Analysis On Active Microorganisms In Air-Dried Agricultural Soils Based On Propidium Monoazid Incorporating High-Throughput Sequencing

Total soil microbial DNA is mainly derived from extracellular DNA as well as active and inactive microbiota.Therefore,studies based solely on total soil microbial DNA do not reflect the true active microbiota in soil and are highly prone to overestimation of soil microbial abundance and community diversity,but there is no accurate and feasible method to screen for active soil microbiota.This project aims to use propidium monoazide(PMA)for the detection of active microorganisms in soil samples under air-drying preservation strategy,according to its ability to specifically bind intracellular DNA and free DNA of damaged cells,and to reveal its effectiveness in detecting the structure and diversity of active microbial communities in air-dried soils.This study provides new theory and method for the research on the ecology of soil active microorganisms,as well as providing an important reference for the application of air-drying preservation strategy for soil samples in microbial ecology.The main results of this study are as follows:(1)Air-drying and preservation reduced the nutrient content of the soil,but raised the nitrate nitrogen content of the soil,and fertilizer elevated the nutrients of the agricultural soil.Chemical fertilizers reduced soil pH,which continued to fall from 5.08 to 5.02 after air-dry preservation,while pH increased in the remaining treatments,with the organic fertilizer treatment rising from 5.97 to6.17;in-situ soil ammonium and nitrate nitrogen contents were highest with chemical fertilizer alone and organic fertilizer alone,respectively,and the highest retention effect of both nutrients was observed in soil with chemical fertilizer and organic fertilizer after air-dry preservation,with the highest contents;total nitrogen and total carbon content were best in the in-situ soil with chemical fertilizer treatment alone and with organic fertilizer treatment alone,respectively,and although the content decreased after air-drying and preservation,it was still the highest among the fertilizer treatments.(2)The structure of the microbial community composition in the soils of the four fertilizer treatments preserved by air-drying and supplemented with PMA was significantly different.The highest bacterial richness,diversity and phylogenetic diversity were found in the treatment groups with organic fertilizer alone,control and organic fertilizer alone;as for fungi,they were found in the control,organic fertilizer alone and control groups.The relative abundance of Proteobacteria was highest in the control and organic fertilizer alone treatment groups,while the relative abundance of Actinobacteriota was highest in the control and organic fertilizer alone and organic fertilizer treatment groups,while the relative abundance of Firmicutes was higher than in the other two treatment groups;the active bacterial genera were dominated by unclassified＿f＿＿Micrococcaceae,Massilia and Bacillus,with the highest relative abundance of Massilia in the group treated with organic fertilizer alone and the highest relative abundance of unclassified＿f＿＿Micrococcaceae in the other three groups;Ascomycota and Basidiomycota dominated the active fungal phylum,with Ascomycota having the highest relative abundance in all four treatment groups;Didymella and Solicoccozyma dominated the active fungal genus,with Didymella having the highest relative abundance in all four treatment groups,and Cryptococcus＿f＿＿Cryptococcaceae and Rhodosporidiobolus only survived in the treatment groups with chemical and organic fertilizers,and their relative abundance was higher than that of Solicoccozyma.PCo A and Per MANOVA analyses based on Bray-Curtis distances showed that the structural composition of the microbial community was similar in the control and organic fertilizer alone treatment groups,and in the chemical fertilizer alone and in the treatment groups with chemical and organic fertilizer.(3)The presence of relic DNA in air-dried soils interferes with the composition and diversity of the active microbial community.A comparison with PMA-added air-dried soils showed that relic DNA inhibited the abundance of bacteria and the diversity of bacteria and fungi in air-dried soils and that fertilization alleviated these inhibitory effects;The relic DNA increased the abundance of fungi,but this was exacerbated by the fertilization treatment.Fertilizer application exacerbated the overestimation of the relative abundance of Massilia in Proteobacteria,with the highest increase in the fertilizer and organic fertilizer treatments,and fertilizer application caused an underestimation of the relative abundance of unclassified＿f＿＿Micrococcaceae in Actinobacteriota,with fertilizer application alone being the most significant.Fertilizer alone exacerbated the overestimation of the relative abundance of Didymella in Ascomycota,and fertilizer alone and with chemical and organic fertilizers slowed the overestimation,with the best effect of fertilizer alone,and organic fertilizer alone exacerbated the underestimation of the relative abundance of Solicoccozyma in Basidiomycota,with fertilizer alone and with chemical and organic fertilizers slowing the the latter had the best effect.The combination of PCo A and Per MANOVA tests showed that the bacterial community structure of the air-dried soil before and after the addition of PMA differed significantly in the treatment groups with chemical fertilizer alone and with organic fertilizer alone,and the fungi differed significantly in the control group and in the treatment groups with chemical and organic fertilizer.(4)PMA detected efficiently microorganisms in air-dried preserved soils.Microbial community richness,diversity and phylogenetic diversity were significantly reduced in in-situ soils after airdry preservation,however,the best improvement in bacteria was found in the control group for all four fertilizer conditions after the addition of PMA,and in fungi for the groups treated with chemical and organic fertilizers,the control group and chemical fertilizers alone.The in-situ soil is dominated by the bacterial phyla Proteobacteria,Acidobacteriota and Actinobacteriota,the genera Massilia and unclassified＿f＿＿Comamonadaceae,and the fungal phyla Ascomycota and Basidiomycota,the genera Solicoccozyma,Chaetomium and Didymella.DESeq2 results showed that more genera under Acidobacteriota died and more genera under Firmicutes survived among bacteria and more genera under Ascomycota died among fungi after air-dry preservation,but Didymella survived best and occupied the highest relative abundance.In addition,the mantel test showed that air-drying and preservation reduced the positive correlation between pH and the main phyla of soil microorganisms,and PMA could properly restore the positive correlation.(5)Bacterial and bacterial-fungal co-occurrence networks are non-random and modular in character.Soil-specific ASVs were distributed in different network modules,which could reflect differences in different soil treatments;the microbial networks of all four fertilization treatments contained modules with a high number of in situ soil-specific ASVs,as well as a module with a relatively high abundance of air-dried soil-specific ASVs.The first two modules contained more ASVs of Acidobacteriota and the third contained more ASVs of Firmicutes;in addition,the third module contained only Ascomycota and Basidiomycota.The microbial network of the single organic fertilizer treatment group of the four fertilizer treatments was well buffered against air-drying preservation and the intervention of PMA.