| Grassland is the largest terrestrial ecosystem in China,with the functions of production,ecological services and recreation.Studying the basic features of its biological communities and the ecological environment can reflect the structure,types and ecological processes of grassland ecosystems in Inner Mongolia.At present,the degradation of grassland in Inner Mongolia is serious,mainly as follows: the effective nutrient content and the bio-diversity of grassland has been decreasing,the growth of pasture had restricted and the quality was slow down and the sustainable development of livestock husbandry was seriously threatened too.It is worthy to mention that the grassland has been degraded constantly in recent decade,showing severe deleterious symbol such as decreased available nutrient contents,reduced plant biodiversity,lower growth of pasture and the poor quality,and the sustainable development of livestock husbandry is seriously threatened.Fertilization is one of the most important soil-restoring measures in degraded steppe and has been widely used in arid steppes in recent decades.However,the differences in types of steppe as well as the soil and climate conditions cause different response of steppes to fertilization.In typical steppe with low precipitation,whether or not the fertilization can improve the grassland productivity is still uncertain.Understanding the responsive mechanisms of degraded typical steppe to nitrogen and phosphorus fertilization will provide basic theories and technical guidance for making reasonable grassland management measures.The study site was located in Xilingol,Inner Mongolia and the site was fenced at the end of June 2014,and then fertilized for consecutive three years,July in 2014 and June in 2015 and 2016.There were five fertilizations,blank treatment without fertilization(Control);(2)phosphate fertilizer application,60 kg P / ha(P);(3)Nitrogen fertilizer,100 kg N / ha(N);(4)NP 60 kg P / ha plus 100 kg N / ha(P + N),(5)organic fertilizer,4000 kg sheep manure / ha(M,equaling 16.4 kg P / ha and 81.2 kg N / ha).Inorganic phosphate fertilizer is superphosphate(14% P),nitrogen fertilizer is urea(46% N),and sheep manure is harvested from farms.Each treatment replicated three times.The plant and soil samples were collected in June,July and August in 2016.We analyzed 1)the bacterial 16 S rRNA gene and the fungi 18 S rRNA gene from soil samples;2)the diversity and abundance of soil bacteria and fungi;3)phylogenetic analysis of the arbuscular mycorrhizal fungi(AMF)and the colonization of AMF indominant plant species;4)the aboveground biomass,the soil chemical properties,and the microbial biomass nitrogen and microbial biomass carbon;and 5)the relationship between soil chemical properties and the abundance of soil microbes.The main results were as follows:(1)Fertilization being applying for three years on degraded grassland,the total vegetation in the degraded steppe was relatively stable while the aboveground biomass of the main species was insensitive to the response of fertilizers.In 2016,the proportion of Stipa kirssii was basically over 60% during the period of fertilization and the proportion of other species was small consistently.The species composition of the community was basically not affected by fertilization.(2)Fertilization being applying for three years on degraded grassland,total nutrients of 0-10 cm and 10-20 cm such as soil TC,TN,TP and soil available indicators AN,pH were no change significantly.As the increase of soil depth,the content of AN and AP showed a downward trend,while pH showed an upward trend.The application of P had significantly increased the content of AP.Because of the fertilization being applied on the surface of natural grassland,the fertilizer moving slowly to the lower layer of the soil,and the available nutrients being fixed by drought soil environment,these soil nutrients except soil available phosphorus was not significantly changed to three years fertilization.(3)These sequences data of bacteria and fungi were obtained by high-throughput MiSeq sequencing.Next,data being optimized,the result showed that the sequences of soil bacterial and fungi were about 1,120,431 and 1,129,387,respectively.Finally,according to the similarity of 97%,the number of bacteria and fungal sequences were clustered into 3907 OTU and 453 OTU,respectively.(4)Applying phosphate and organic fertilizers led to significant changes in the abundance of individual bacterial or fungal community structure of bacteria and fungi.According to the hierarchical clustering tree on OTU level,it was indicated that the structure of bacterial and fungal communities were significantly influenced by the organic fertilizer.Excepting M,the other four bacterial groups were aggregated because of similar community structure in 0-10 cm or 10-20 cm,respectively.Excepting P in0-10 cm and M in 10-20 cm,the other four fungal groups of community structure gathered a group,respectively.In addition,the difference of community between the different treatments of bacteria was greater than that of the fungal community.(5)Fertilization had significant effect on the composition and abundance of soil bacteria and fungi,especially the application of organic fertilizer.The dominant bacterial species included Actinobacteria,Proteobacteria,Acidobacteria and Chloroflexi,accounted for 84.63%,and fungal dominant species were Ascomycota(71.26%)and Basidiomycota(13.50%),accounting for 84.76% of the total number.0-10 cm,theapplication of organic manure significantly reduced the abundance of Nitrospirae(1.11%),while significantly increased the abundance of Proteobacteria(19.54%)and Ciliophora(1.83%)(P<0.05).10-20 cm,the application of organic manure significantly increased the abundance of Chloroflexi(7.55%).The P and NP fertilizer treatments significantly increased the abundance of Chytridiomycota(1.01%)by compared with no fertilizer treatments,while N and M fertilizer treatments had no use significantly.There was no significant change in the abundance of other bacterial or fugal phylum.(6)The RDP classifier Bayes algorithm was used to analyze the OTU representative sequences of 97% similar level.By comparing with the fungal database UNITE(confidence threshold 0.7),the result was species classification information corresponding to each OTU.The experiment found 7 sequences of AMF.After these AMF sequences being comparing with GenBank database,it was found that MF693140 was the closest cluster to Paraglomus occultum and the other six sequences were clustered into another population,among which MF693141 and Ambispora fennicia,MF693144 and Scutellospora specieslus,MF693143,MF693138 and Rhizophagus species,MF693139 and Diversispora species,MF693142 and Claroideglomus species were clustered into one group respectively.In degraded grassland,the abundance of AMF species had been influenced by fertilizer,especially phosphate fertilization.In comparison of seven AMF sequences,MF693138 has the 97% similarity with Rhizophagus species,which was found within the highest content in the phosphate fertilizer.There was no significant difference in the other fertilization treatments.(7)The dominant plants(Cleistogenes squarrosa;Stipa krylovii;Leymus chinensis)Arbuscular mycorrhizal fungi infection results showed that colonization(F%),mycorrhizal infection intensity(M%),root Arbuscular rate(A%)were influenced significantly by fertilization,but did not show the similar trend.(8)Soil bacteria and fungi were affected by soil physical and chemical properties.By spearman correlation Heatmap analysis between bacteria or fungal phylum species and environmental factors,these results obtained that the effect of pH on bacteria was significantly positively correlated and negatively on fungi.In conclusion,soil physical and chemical factors have a significant effect on the distribution of soil microbial communities.After long-term fertilization,the chemical properties of grassland soil changed,which may affect the grassland plant community by changing the soil microbial structure. |
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