The Effect Of Long-term Fertilization On Soil Microbial Diversity And The Accumulation Of Antibiotic Resistance Genes

It is well documented that fertilization has varied effects on soil microorganisms.However,the effects of long-term applying with different fertilizers on soil microbial diversity are still not clear.And there is still lack of evidence that long-term manure fertilization contributed the accumulation of antibiotic resistance genes(ARGs)in the soil.Based on three long-term fertilization experiments in China and Canada,we analyzed the soil microbial community composition and ARGs abundance by Roche 454 or Illumina Miseq high-through sequencing technology and real-time qPCR technology.The results are listed as follows:1.We determined soil bacterial diversity and community structure after 7-years(2007-2014)of applying different fertilizers to acidic,near-neutral or alkaline soil.In alkaline soil,nutrient sources did not affect bacterial richness or Shannon diversity index.In contrast,bacterial richness and diversity index were significantly lower in acidic and near-neutral soils under NPK than manure or their combination,which corresponded with changes in soil pH.The PCoA ordination patterns indicated the effect was less distinct among nutrient sources in alkaline than in acidic and near-neural soils.Distance-based redundancy analysis showed that bacterial community structures were significantly altered by soil pH in acidic and near-neutral soils,but not by any soil chemical properties in alkaline soil.We conclude that after 7-years of fertilization,soil bacterial diversity and community structure were shaped more by changes in soil pH rather than the direct effect of nutrient addition.2.We selected soils in a wheat-rice rotation that was fertilized for 23 years(1991 to 2013)with or without inorganic P fertilizer to determine soil microbial diversity.Fungal ?-diversity was not altered while bacterial richness index,and archaeal richness and diversity index were significantly greater without inorganic P fertilizer than soil fertilized with inorganic P.Principal component and redundancy analyses showed that fungal community structures were different in soil fertilized with inorganic P than those receiving no inorganic P fertilizer.Chloroflexi had significantly greater relative abundance with inorganic P fertilization,while soil methanogenic archaea,Methanocellales,and the mycorrhizal fungi,Basidiomycota and Glomeromycota,were significantly more abundant without inorganic P fertilization.3.We analyzed soil prokaryotic and fungal communities in soils with varying histories of cattle manure application.The manure treatments were i)annual application for 43 years(MF),ii)annual application for 14 years followed by 29 years without application(MF14),and iii)annual application for 30 years followed by 13 years without application(MF30).Distinct prokaryotic and fungal community structures were observed in MF compared with other treatments across fall,spring and summer seasons.The MF significantly increased the relative abundance of Firmicutes,Gammaproteobacteria,and Gemmatimonadetes,while significantly decreasing the relative abundance of Acidobacteria.In the fall,the soil prokaryotic and fungal community structures with MF30 were significantly different than the other fertilization treatments.Soil with MF30 in fall had a greater relative abundance of Microascaceae,Acidimicrobiia,Ellin6529,and Thaumarchaeota than control soil.4.We analyzed the archived soil by real-time qPCR to determine the effect of different manure fertilization doses on the accumulation of soil ARGs.The results showed that,soil total ARGs/16 S rRNA abundances were enhanced with the increasing of manure applying doses,the relative anundance of ARGs had a positive linear relationship with time series.For each of the aminoglycosides and sulfonamides,total ARGs didn‘t show significant relationship with time series.However,total ARGs of erythromycins and tetracyclines significantly logarithm and linear increased with the manure fertilization years,respectively.This study demonstrated that when stop applying manure in 2003,the total ARGs anbundance,except ARGs of erythromycins,were all decreased at 2003 and than more less in 2013.This study offered a evidence that long-time fertiliztion with manure have negative effect on soil ARGs and stop manure application may be a effctive way to control the accumulation of ARGs in soil.This study showed that pH shapes soil microbial community diversity other than soil nutrition when soil pH is changed by long-term fertilization.Soil acidification will decrease soil richness and diversity index.When soil pH doesn‘t change by fertilization,soil phosphorus nutrition will be the major factor to control soil microorganism.This study showed that application with manure could maintain and enhace soil microbial richness and diversity,while induce the accumulation of soil ARGs at the same time.