Response Of Soil Microorganism To Nitrogen Deposition At A Young Plantation Of Cunninghamia Lanceolata And Research On Nitrogen-fixing Strains

With the development of social economy,industrialization and agricultural production,atmospheric N deposition is increasing caused by anthropogenic activities,which exceeds the amount of nature nitrogen fixation and the rate continues to increase.The researches indicated that excess N deposition had negative impacts on ecosystem health.Chinese fir(Cunninghamia lanceolata(Lamb.)Hook)is one of the main rapid-growth and high-yield evergreen trees in China.Its planting area and storage volume are the first place in artificial tree forest,so the growth affects the development of forestry ecology.The forest soil is important in sustaining Chinese fir growth,whereas soil microorganism is dominant component and its diversity is particularly sensitive to environmental change.Microorganism has important effects on soil formation,material cycling and fertility evolution.In this study,the soil microorganism was studied,which could provide scientific basis for the effects of nitrogen deposition on soil properties and fertility.In Chinese fir plantation,Composite functional strains with high efficient phosphorus,potassium and antagonistic pathogens were screened,which provide a solid theoretical foundation for the application of eco friendly microbial fertilizer.This study was carried out in Fenyi Jiangxi Province and the experiment plot was set on young Chinese Fir Plantation of Shanxia forest farm.The simulation experiment method of nitrogen deposition was the space replacement time for 2 years.The experimental design included two N forms(NH4Cl and KNO3)and five levels of N deposition 0,20,40,60,80 kg N hm-2 a-1.The effects of different nitrogen forms and doses on soil microflora,structural diversity and genetic diversity were studied by plate counting method,phospholipidfattyacid(PLFA)composition and the high-throughput pyrosequencing method.Therefore,the functions and antagonistic ability of nitrogen fixing bacteria which contained the nifH gene were studied.Finally,the functional strains were identified,which stability of antagonistic ability was studied.The effect of nitrogen deposition on the microflora showed that the total amount of culturable microorganism,bacterial number,fungi and nitrogen transformation microbial number were promoted by low nitrogen treatment,but inhibited by high nitrogen treatment.And the effect of ammonium nitrogen on the growth of fungi was greater than that of nitrate.Examination of microbial biomass showed that under ammonium nitrogen deposition,the amounts of total microbial biomass,Gram positive bacteria,Gram negative bacteria,fungi and bacterial biomass were higher compared with the control group.Meanwhile,with the increased rates of nitrate nitrogen deposition,the amount of total microbial biomass,Gram positive bacteria,Gram negative bacteria,fungi and bacterial biomass were increased initially and then decreased with increasing N addition.Ammonium nitrogen deposition had a significant effect on the bacterial and actinomycetes biomass.The high-throughput sequencing showed that N deposition of the two N forms inhibited the growth of bacteria compared with control plots,and the changing trend was related to the NH4+-N:NO3--N ratio of soil.Molecular methods showed that the total percentage of unique tags and OTUs were higher in nitrate treatments than in ammonium treatments.We observed 22 bacterial phyla,in which Proteobacteria,Acidobacteria,and Actinobacteria were dominant.Acidobacteria relative abundance decreased with increasing of ammonium dose,but increased with increasing nitrate dose.In this study,there were 103 bacterial strains containing nifH gene and the most suitable growth condition of these strains was 28 ℃,pH=7.Meanwhile,10 strains of nitrogen fixing bacteria were screened out,which were suitable in the special environment(Low temperature and alkaline,high temperature and alkaline,pH values of large span).The results of qualitative phosphate and potassium solubilization test of 103 strains showed that there were 42 strains of dissolved inorganic phosphorus,12 strains of dissolved organic phosphorus and 12 strains of releasing potassium.The results also showed that the growth characteristics of the strains were not completely correlated with their biological characteristics by growth condition cluster analysis and 16 S rDNA identification.Screening of antagonistic strains and antagonistic stability showed that there were 24 strains had strong antagonistic ability to Fusarium oxysporum Schltdl,Fusarium solani Sacc,Colletotrichum gloeosporiodes Penz,Pestalotiopsis apiculatus Huang.2024 and 7014 were selected as the potential broad spectrum antagonist by phosphorus and potassium dissolving,16 S rDNA sequencing compariso,morphological analysis.The results showed that 2024 was Bacillus subtilis subsp.subtilis and the 7014 strain was Pantoea vagans.The antagonistic abilities of 7014 to 88533 and 6913 were less affected by pH.With increasing of pH,the inhibition rate of the7014 to 5143 was increased first and then decreased.The antagonistic stability of 2024 to the 88533 was less affected by pH,but it was more affected to 6913 and 5143.The responses of 7014 and 2024 to fungi were consistent with temperature change,which maximum inhibitory ability displayed in 25-40 ℃,and the minimum inhibitory rate appeared in 100 ℃.The strongest antagonistic ability of 7014 strain appeared in 365 nm treatments,and that of 2024 appeared in dark treatments.Examination of antagonistic substance location showed that 7014 plundered nutrient to prevent fungal growth,and 2024 secreted some non-protein big molecular substances to inhibit fungal growth.