Response Of Functional Groups Of Nitrifiers And Denitrifiers To Different Grazing Intensities In Qinghai-Tibetan Alpine Meadows Soil

Qinghai-Tibetan Plateau,where the geographical environment is quite unique,is very sensitive to global climate changes.In recent years,human disturbances(e.g.overgrazing)have contributed greatly to the severe degradation of alpine meadows,the main vegetation in this area.Grazing is a widely spread type of land use in grassland,which influences plant species diversity,drives community successions,and can stimulate below ground nutrient cycling and energy flows.In particular,grazing often alters soil nitrogen cycling and influences the aboveground primary production.Soil microorganisms are the key drivers of soil nitrogen available to plants and preventing undesirable nitrogen loss from ecosystems.Understanding how grazing influences microbial communities is thus of paramount importance to predict ecological effects of human disturbances.Here,we investigated the community structure and abundance of ammonia-oxidizing archaea(AOA)and ammonia-oxidizing bacteria(AOB)as well as the associated environmental variables under different grazing managements in Qinghai-Tibetan alpine meadows by using q-PCR and PCR-clone library analysis.The concept of functional response groups will be used to identify response groups of nitrifiers and denitrifiers to grazing disturbance and the driving soil environmental factors.The results are given as follows:1.The effects of grazing on the abundances of nitrifiers and denitrifiersThe results of real-time PCR assays,targeting amoA gene,showed that both the abundances of AOA and AOB were increased by grazing(P<0.05).The abundances of AOA and AOB amoA gene copies ranged from 2.1×107 to 7.9×107 copies g*1 dry soil and 7.7×105-3.2×107 copies g-1 dry soil,respectively.Nirk gene copies increased significantly in response to grazing(P<0.05),ranging fom 2.1-5.8×105 copies g-1 dry soil.The abundance of nirS gene was two orders of magnitude higher than that of nirK,ranging from 3.9×107 to 9.7×107 copies g-1 dry soil.Unlike nirK,nirS gene copies tended to decrease with grazing intensities(P = 0.094).Generally,grazing increased AOB and AOA abundances up to 42 fold and 3.7 fold,respectively,and increased the percentage of AOB within total ammonia oxidizers from 3.1%to 10.8%and increased the percentage of nirK-like relative to total nitrite reducers from 0.2%to 1.5%.2.The effects of grazing on the community structures of nitrifiers and denitrifiers,and theirrelationships with environmental variablesThe community structures of nitrifiers and denitrifiers were changed by grazing.Changes in the AOA community structure were correlated to changes in C/N(P = 0.032),whereas changes in the AOB community structure were not correlated to any of the environmental factors studied.The nirK community strucuture significantly correlated to soil total C and NO3-concentrations(P =0.014;P = 0.027).In contrast,the community structure of nirS-like denitrifier was significantly related to soil C/N ratio(P = 0.021).3.Response groups of nitrifiers and denitrifiers to grazing,and the driving environmental factorsThe abundance of AOA belonging to M2,M4,M5 and arable soil sub-groups increased with grazing intensity.In contrast,the abundance of AOA belonging to M1 decreased with grazing,while the abundance of AOA belinging to M3 and marine cluster was not affected by grazing.The abundance of AOB belonging to cluster 3a significantly with grazing,no changes were detected for the abundance of AOB belonging to clusters 1-2,3b,ME,4 and 6.The abundances of nirK-like denitrifiers belonging to clusters Ⅰ-3,Ⅱ-1,and Ⅲ significantly increased with grazing,and the abundance of nirK-like denitrifiers belonging to cluster Ⅰ-1 significantly decreased with grazing,while the abundance of nirK-like denitrifiers belonging to Ⅱ-2,Ⅳ-1 and Ⅴ did not response to grazing.The abundance of nirS-like denitrifiers belonging to clusters Ⅲ-1 and Ⅲ-4 significantly decreased with grazing,while abundance of denitrifiers from clusters III-2,IV and V were not affected by grazing.The abundances of the 4 AOA groups(M2,M4,M5 and arable)and the AOB cluster 3a that positively responed to grazing were all positively related to soil NO3-concentration.In contrast,the abundance of nitrifiers belonging to the M1 cluster-responding negtively to grazing-was negatively related to soil NO3-concentration and total nitrogen,and positively related to total soil carbon concentration and C/N.For 4(Ⅰ-2,Ⅱ-1,Ⅱ-3 and Ⅱ)among the 5 groups of nirK-like denitrifers(Ⅰ-2,Ⅰ-3,Ⅱ1,Ⅱ-3 and Ⅲ)that positively responded to grazing,abundance was positively related to soil NO3-concentration,while the abundance of 3 of these groups(Ⅰ-3,Ⅱ-3 and Ⅲ was negatively correlated to total carbon concentration.In contrast,the abundance of denitrifiers belonging to each of the 4 groups responding negtively to grazing(nirK:Ⅰ-1;nirS:Ⅱ,Ⅲ-1 and Ⅲ-4)was positively related to soil moisture.For three of these groups(nirK:Ⅰ-1;nirS:Ⅱand Ⅲ-1),abundance was also positively related to total soil carbon concentration.In conclusion,AOB and nirK-harbouring denitrifiers responded more positively to grazing than AOA and nirS-harbouring denitrifiers.In general,sub-groups within these four broad functional groups of nitrifiers and denitrifiers responsed differently to grazing disturbance,which implied functional diversity exists within each group.Soil nitrate was the main driver of the abundance of denitrifier sub-groups(nirK or nirS)positively responding to grazing,while soil moisture and carbon concentration were the main drivers of the abundance of denitrifier sub-groups negatively responding to grazing.Our results demonstrate that significant functional diversity exists within each group,and groups that responded consistently to disturbance might shared similar functional traits and environmental drivers.The responses of nitrifiers and denitrifiers to a certain disturbance may actually be the sum of the responses of sub-groups that responded differently to these disturbances.