| A large number of antibiotics have been used in intensive animal husbandry in China.Resistant bacteria or antibiotic resistance genes(ARGs)are produced,which cause soil pollution by applying organic manure from livestock and poultry manure to farmland,and pose a potential threat to the production of green agricultural products in our province.In order to understand the effect of long-term application of manure and organic fertilizer on antibiotic resistance genes in black soil,real-time fluorescence quantitative PCR was used to analyze the abundance changes and distribution patterns of 34 antibiotic resistance genes in six categories of fixed-site quantitative fertilization soils in Shuangcheng and Helen regions of Heilongjiang Province.1.Five types of antibiotic resistance genes,including tetracycline,sulfonamide,macrolactone,beta-lactam and efflux,were detected in soils collected from Shuangcheng experimental site,which accounted for 50%,11.54%,15.38%,11.54%and 11.54% of the total antibiotic resistance genes,respectively.2.The types,abundance and diversity of antibiotic resistance genes in the soil with manure changed significantly compared with that without manure.The types of resistance genes detected in soil treated with chicken feces were approximately similar to those detected in fresh chicken feces samples,but the gene abundance varied significantly.The types and abundance of antibiotic resistance genes in the soil of cow manure and pig manure were significantly lower than that of uncomposted cow manure and pig manure.3.9,10 and 5 antibiotic resistance genes were detected in soil treated with chicken manure,pig manure and cow manure,respectively.Principal component analysis showed that the application of pig manure,chicken manure,cow manure and inorganic fertilizer in the soil significantly changed the structure of antibiotic resistance genes in the soil,and PC1 explained the structural change of antibiotic resistance genes by 56.6%.4.In the soils applied with different kinds of organic fertilizer,it was verified by fluorescence quantitative PCR that the abundance of blaTEM and tetracycline tetG2 was the highest in soil with beta lactam antibiotic resistance(beta lactam).Tetracycline tetX1 and integrin-integrase intl1 were significantly moreabundant than other resistance genes in soil treated with chicken manure,and tetracycline antibiotic resistance genes were still resistance genes with high detection rate in soil samples of chicken manure,tetracycline antibiotic resistance genes tetL1,tetC and tetX2 were significantly higher than other resistance genes in soils treated with cow manure organic manure.5.Four antibiotic resistance genes were detected in different fertilization system plots of Helen test site,including sulfonamide resistance gene sul2,transposon resistance gene intl1,beta-lactam resistance gene blaTEM and blaCTX-M.Except sulfonamide antibiotic resistance gene sul2,there was no significant difference among the four treatments.The abundances of transposon resistance gene intl1,beta-lactam resistance gene blaTEM and blaCTX-M in the treatments of 30 t/ha cow manure organic fertilizer and 15 t/ha cow manure organic fertilizer were significantly higher than those of single fertilizer(CF)and non-fertilizer(N0F)treatments.6.Four resistance genes were detected in the plot test of different fertilization systems in Helen test site,and the detection rate of sulfanilamide resistance gene sul2 in different treatments reached 100%,and the gene abundance was up to about 8.5108.The number of copies of the beat Lactam resistance gene blaTEM per gram of soil was 1.34 106~6.14 106,the abundance of sulfonamide antibiotic resistance gene sul2 was within the range of 7.18 108~8.50 108,and the abundance of the genetic element intl1 was within the range of 6.4 107~9.18 108.The abundance of blaCTX-M was within the range of 0.02 106~7.77 106. |
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