| Soil organic matter is a key component in soils.It is the driver of soil biological processes,such as promoting the formation of soil aggregates and structure,providing energy and raising soil biota,thereby plays important ecosystem service function in driving and regulating nutrient,water and energy cycle in soils.Therefore,the relationships among soil organice carbon,soil aggregates,soil microbial communities and soil ecosystem functions under soil development are the essential problem in soil science.Many studies have shown that soil organic carbon has significant accumulated and sequestration potential under prolonged rice cultivation.Rice paddy soil has been recognized as a unique type of hydroagric Stagnic Anthrosol due to long term rice cultivation with frequent flooding/submerging for rice growth.In China,rice has been cultivated for 7000-10000 years.Long term rice cultivation and the management practices have contributed to build-up of paddy soils with particular morphology and organo-mineral intercations though derive from various kinds of parent soils.While paddy soils are known to have high organic carbon storage and sequestration potential,the interaction of organic carbon,mineral components and soil mcirobes both of bulk soil and at aggregate level are still not fully addresed.In the eastern coastal area of Zhejiang Province,China,new agricultural land has been created through consecutive land reclamation by protective dikes over the past 2000 years.Under prolonged agricultural use,large amounts of tidal wetlands were converted into farmland for rice cultivation.The construction ages of the dikes were estimated according to historically written record and provide a chronosequence of paddy soil formation.This soil chronosequence,derived from calcareous marine/estuarine sediments,provides a model system to evaluate the effect of the rice cultivation over long time periods on the evolution and distribution of soil carbon and nitrogen during pedogenesis.Understanding how soil could change under long-term agricultural use would provide scientific bases for making wise land use strategies and help to predict the dynamics of agroecosystem development.The influence of different types of changes in land use on microbial communities has been studied with growing interest in recent years due to the importance of soil microbes in geochemical nutrient turnover and soil health.However,the consequences of prolonged rice cultivation for soil microbial communities are poorly understood.Therefore,to explore how basic soil properties and microbial communities in bulk soil and different particle size fractions(2000-200?m,200-20?m,20-2?m,<2?m)together to drive soil development,we selected the cultivation age from 50 to 700 years(P50-P700)compared to a tidal wetland(0 years,P0)for this study.Main soil physicochemical properties were analyzed to understand the basic pedogenetic processes.Bacterial,fungal and archaeal abundances and community structures were investigated based on BIOLOG,quantitative real-time PCR(qPCR),terminal-restriction fragment length polymorphism(T-RFLP),denaturing gradient gel electrophoresis(DGGE)and 454 Roche pyrosequencing,combined with greenhouse gases production and soil enzyme activity along the soil chronosequence.The main results obtained were as follows:1.Comparison of physic-chemical properties and microbial activity in paddy soils of different agesSoil physicochemical properties have significantly changed with time since tidal marsh reclamation,and clear patterns of soil development were observed.Major pedogenetic processes included increase in the content of coarse sand fraction(from 2.78%to 7.63%)and improvements of soil aggregation,accumulation of soil organic carbon and total nitrogen(from 6.32 to 21.70 g kg-1 and from 0.79 to 2.14 g kg-1 respectively)and decrease in soil pH(from 8.62 to 6.65)after 700 years of soil development.Total DNA content,total P,dissolved organic carbon(DOC),microbial biomass carbon(SMBC)and CEC in bulk soil increased rapidly within 50-100 years after reclamation and then remain relatively stable along soil development.Under 30d waterlogged incubation,long term rice cultivation significantly increased total greenhouse gases emissions(CH4 and CO2)by 3.81 and 3.89 folds.The analysis of the functional diversity of microbial communities suggested that average well color development(AWCD),H'BIOLOG,SBIOLOG increased with 100 years rice cultivation and be stable thereafter.In different particle size fractions,DNA content,SOC and total N were divergently distributed.These properties with coarse sand and clay fractions were significantly higher compared to other PSFs.Generally,the DNA,SOC and total N contents followed an order of coarse sand fraction>clay fraction>fine sand and silt fractions in a single soil.Nevertheless,C/N ratio was significantly higher in the coarse sand fraction than in other fractions across the soils.2.Effects of prolonged rice cultivation on soil bacterial communitiesWith increased duration of management after land embankment,bacterial 16S rRNA gene abundance significantly increased from 3.99×108 to 1.83×1010 copies g-1 dry soil.Meanwhile,significant effects were observed on most bacterial functional groups and potential pathogens with prolonged rice cultivation.Bacteroidetes abundance was highest in tidal marsh(29.02%),but decreased to 1.52%within 100 years and remained around low level thereafter.In contrast,Acidobacteria increased with time and peaked in the 300 years old site(from 3.44 to17.91%).Nitrospirae,Deltaproteobacteria,Chloroflexi and Gemmatimonadetes increased with time during the 700 years,but Actinobacteria,Alphaproteobacteria,Gammaproteobacteria showed an overall decrease along soil development.However,the temporal trends of Betaproteobacteria were not clear.The OTU numbers,Shannon,Chaol and phylogenetic diversity(PD)were used to estimate and compare bacterial diversity and richness with soil development.We found paddy soils(P50-P700)had higher diversity and richness than tidal marsh(P0),and all indices of bacterial diversities reached the plateau when soil development time was over 100 year.Succession of bacterial community structure was mainly associated with changes in pH,SOC,TN and SMBC.Of all of the environmental variables examined,SOC showed the highest correlation with the community composition(r = 0.76,p<0.001).3.Effects of prolonged rice cultivation on soil archaeal and methanogenic communitiesThe 16S rRNA gene and the mcrA gene abundance increased from 6.70×106 and 1.53×105 copies g-1 dry soil in P0 respectively to 2.89×108 and 2.73×107 copies g-1 dry soil in P700,both exhibiting a consistent exponential increasing trend with prolonged rice cultivation duration.However,the diversity indices characterized by Shannon index,Chaol and ACE for both archaeal and methanogenic communities increased greatly in the rice soils over P0 but peaked generally in P100.Moreover,there was a fast shift in archaeal and methanogenic community composition shortly after 50 years of rice cultivation from the initial tidal marsh,though inconsistent with the ages.The methanogenic community structure reached steady in shorter time than their abundance as the salt marsh soil was shifted for rice cultivation despite of dominance by the members of the order Methanosarcinales across all the soils.Prominent archaeal community succession occurred in the initial stage of rice soil development where significant morphological and mineralogical development discerned in pedological studies.This fast biological change could be probably promoted with the fast accumulation of soil organic carbon available shortly after rice cultivation within 100 years.4.Effects of prolonged rice cultivation on soil microbial communities in PSFsHighest diversity and abundance of bacteria and fungi were found at the largest particle size fraction(coarse sand)and for bacteria also in the smallest fraction(clay).Bacterial and fungal gene abundance increased in response to the conversion to agricultural land,as indicated in the 50-year samples and then kept relatively stable with prolonged cultivation of rice for more than 300 years.This responsiveness was seen in the coarse sand and clay fractions,but not so much in fine sand and silt.Differences in bacterial and fungal community structures were also recorded between samples and between particle size fractions.Fungal community was more susceptible than bacterial under soil development.For archaea and methanogens,high abundances were found in both coarse sand and clay sized fractions.Both gene abundance and community diversity exerted profound changes with prolonged rice cultivation for coarse sand and clay sized fractions but for fine sand and silt sized fractions.Revealed by sequencing analysis of selected archaeal 16S rRNA genes,Methanocellales were the predominant group of methanogens in all size fractions of soils across the chronosequence,though Methanosarcinaceae and Methanosaetaceae were less widespread in the clay fractions across the soils.A high correlation was found between SOC and total N and microbial biomass(DNA content)in sand and silt fractions,indicating that SOC and total N might be major driving factor for microbial biomass.5.Functional and structural responses of bacterial and fungal communities to 700 years of cultivation in different depths of paddy soilThree soil depths(surface:0-5 cm;middle layer:5-10 cm;deep layer:10-20 cm)in different years of cultivation were assayed.Bacterial and fungal abundances and acid phosphatase,invertase and urease activities significantly correlated with years of rice cultivation,especially in the upper soil layer(0-10 cm).Furthermore,soil depth had significant influences on fungal abundance and acid phosphatase activity.Our data also suggested that the interaction between the years of cultivation and depth has strong effects on fungal abundance and acid phosphatase activity by repeated measures ANOVA.In addition,we found that bacterial community composition is not sensitive to soil depth,but fungal community composition changed significantly with soil development and depth.Regardless of soil depth,bacterial and fungal community diversity increased at initial stages of soil formation(50-100 years)and then reached a relatively steady state.Molecular ecological analysis of the indigenous microorganisms in combination with profile studies on soil enzymatic activities will be able to further improve our understanding of microbial composition and function in surface and subsurface soil over time scales of many hundreds of years.Using a soil chronosequence formed on salt marsh,changes in biotic and abiotic properties of soil were observed not parallel with prolonged rice cultivation.Notably in bulk soil,a general development of bacteria and archaea was characterized by a fast build-up of their community with a stable diversity shortly after rice cultivation within 100 years though the abundance increased continuously over centuries.While significant morphological and mineralogical development were seen visible only over centuries of rice cultivation,fast biological development and functioning could be seen with the ever accumulating soil organic carbon and/or improvement of soil abiotic conditions in the early stage of rice soil development.Two stages of bacterial and archaeal succession were identified,a short stage during the initial several decades when their diversity increased significantly and community structure shifted rapidly,and a long stage that lasted for centuries when bacterial and archaeal community changed slowly.In soil microhabitat,microbial population(bacteria,fungi and archaea)differed in their spatial distributions under prolonged rice cultivation.Bacterial and fungal populations had been established and formed to a certain scale within 50 years of paddy soil development,but archaeal and methanogenic populations consistently increased with 700 years rice cultivation.However,there were consistent changes in bacterial,fungal and archaeal diversities across the soil chronosequence.Variations in most soil microbial variables both in bulk soil and PSFs during the period of 700 years were significantly related to that of soil organic carbon,suggesting that soil organic carbon might be a key factor indicating and regulating the overall development of microbial communities.However,the mechanism of the biological development and the potential link to biogeochemical cycling needs further investigation. |
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