Metagenomic Research Of Nitrifying Microorganisms And Nitrification In Purple Soils With Different PH

Purple soil as a characteristic soil resources in China,widely distributed in Sichuan Basin.The purple soil in this area with rich mineral elements is an important local agricultural soil resources,and accounted for more than 90% of the total area of purple soil around the country.It is well known that nitrification in soil is a biochemical process,closely related to the agricultural environment pollution,and is highly sensitive to pH.pH is considered to be a key factor of nitrification and nitrifying microorganisms.As the pH has a widespread influence on physical,chemical and biological process in the soil,its effect on soil nitrification is integrated.It's very important to find out the influence mechanism of soil pH on nitrification for improving nitrogen utilization efficiency and reducing the environmental pollution.Firstly,three purple soils with different pH in Sichuan Basin were used as the object of study(acid purple soil pH 5.7;neutral purple soil pH 7.3;alkaline purple soil pH 8.0),artificially adding acid or alkali to three kinds of purple soil with different initial pH to obtained three short-term justified subsamples of each kind of purple.All the samples were added 8 mmol N kg-1 ammonium sulfate and incubated at 60% of field moisture capacity at 28? in the dark,sampled regularly to determinate soil nitrate,ammonium,pH and abundances of ammonia oxidizers,and finally to uncover the influence of pH long-term and short-term change on soil nitrification and ammonia oxidizers.The results are as follows:(1)The average net nitrification rate during 9 day's incubation in a descending order as: alkaline purple soil(7.02 mg kg-1 d-1)> neutral purple soil(2.42 mg kg-1d-1)> acid purple soil(0.23 mg kg-1d-1),the nitrification was significantly positively related to soil pH.And the AOA/AOB ratio from high to low in order was: acid purple soil(126)> neutral purple soil(1.55)> alkaline purple soil(0.6),indicating that AOA dominated in number in acid purple soil,AOB dominated in alkaline purple soil,but in neutral purple soil,AOA and AOB were equal in number,both may play a part in the process of nitrification.(2)The average net nitrification rate of three subsamples of acid purple soil were:0.23,0.22,0.23 mg kg-1 d-1;the average net nitrification rate of three subsamples of neutral purple soil(pH 6.5-H2SO4,pH 7.3,pH 8.1-NaOH)were 1.23,2.42,3.27 mg kg-1d-1,respectively;the average net nitrification rate of three subsamples of alkaline purple soil were: 7.05,7.04,7.02 mg kg-1 d-1.These results indicated that short-term pH change did not significantly affect the nitrification in acid and alkaline purple soils,but in neutral purple soil,short-term increase or reduce the soil pH correspondingly increase or decrease the nitrification.The abundances of AOA and AOB among three subsamples of each purple soil had no significant differences at day 0,and they were with the same change trend during the incubation,implying that short-term pH change had no significant effect on the number of AOA and AOB.(3)The two-factor variance analysis found that the effect of long-term pH change on nitrification reached a highly significant(F=569.547,P<0.001),short-term pH change just had a significant effect on nitrification(F=4.723,P=4.723),the effect of long-term pH change on nitrification is greater than that of short-term pH change.According to the result of incubation experiment,we presumed that the influence mechanisms of long-term and short-term pH changes on nitrification were different,the long-term pH change affected nitrification by affecting nitrifying microorganisms.To prove this,the total microbial DNA was extracted from three purple soils with different pH,and Hiseq high-throughput sequencing technology was used to reveal the effects of long-term pH change on soil microbial community structure and functional genes.The results are as follows:(1)The Hiseq high-throughput sequencing found a total of 89 phyla,222 classes,527 orders,1009 families,2769 genera,14354 species.Ten kinds of the same advantage group were detected in all soil samples.The neutral purple soil had an unique advantage group Nitrospirae,and the alkaline purple soil lacked an advantage group Thaumarchaeota.Proteobacteria accounted for most in all three kinds of purplesoil.(2)Selecting the functional genes of all sequences,the proportions of nxrA gene in all of soils were the largest,followed by nxrB;functional genes related to AMO accounted for the biggest in neutral soil,followed by alkaline soil and acidic soil;no hao gene was detected in acidic soil,but it was highest in alkaline soil.(3)Correlation analysis of microbial community and soil properties found that the soil pH,organic matter,total nitrogen,ammonium nitrogen,nitrate nitrogen and field water capacity had significant correlations with soil microbial community structure,and the soil pH had the biggest effects on microbial community(r2=0.9985,P=0.9985);redundancy analysis found that there was highly significantly difference of the microbial community structure among three purple soils with different pH,total nitrogen,water content,organic matter and ammonia is the main factor affecting acid purple soil microbial community,while soil pH and nitrate has an obvious influence on the microbial community structure of neutral and alkaline purple soils,respectively.Above all,we found that the microbial community structure of three purple soils with different pH are highly different,nitrification is significantly positively related to soil pH.Long-term pH change affect nitrification by changing the community structure and function of nitrifying microorganisms,but short-term pH change changing the effectiveness of the substrate to influence soil nitrification.