Research On Archaeal Community Structrue And Diversity In Red Paddy Field

Limited reports demonstrated that Archaea,such as methanogenus,plays an important role in elements and energy circulation in soil ecosystem.Red soil is the most common soil type in southern China.In this study,to investigate the Archaeal community structures and diversity in red soil,the soil samples collected from Jiangxi Guangfu base?N28°37?,E116°03??in May?before rice-sowing?,July?rice-tillering stage?,August?rice-filling stage?,September?rice-maturity stage?and October?after rice-harvest?,2014,were analyzed by optimized DGGE and High-throughput sequencing.The meta-genomic DNA extracted from July,August and September soil samples were first analyzed by DGGE.2 phyla,4 classes,5 orders of Archaea were identified by 16S rRNA V4-V5 reagion analysis and bands recovery,sequencing.Methanomicrobia was the dominate class accounting for 49.15%of total Archaeal abundance at class level.Methanosarcinale,as dominate order,accounted for 39.45%of total abundance at order level.And the abundance of Nitrosotalea was about 6.07%.Highly similiarity of quantity and sequence of the bands from soil samples collected at different time was identified by DGGE gel image analysis.No significant difference was detected?P=0.534?between the Shannon indexes of rice-tillering stage and rice-filling stage calculated from DGGE images,which suggested that Archaea diversity and abundance of rice-tillering and rice-filling stage is highly similar.The Shannon index of rice-maturity stage soil samples was slightly higher than that of rice-tillering and rice-filling stage?P=0.017 and P=0.009 respectively?,which might be caused by the significant decrease of water content in soil.In order to acquire more comprehensive information of Archaeal community,Archaea 16S rRNA V3-V6 hypervariable region of May,August and October samples were further analyzed by 454 pyrosequencing platform.A total of 244,136 valid reads obtained were assigned into 10848 OTUs.All these OTUs can be classified into 3 phyla,20 classes,31 orders,37 families and 47 genera after searching against GREENGENE database.No more than 20%of obtained fragments could be identified to genus level,and most of the identified sequences belonged to methanogenus?96.6%?.Shannon indexes of Archaea in red paddy calculated from High-throughput sequencing were4.88,5.06 and 5.11 in May,August and October,respectively,no signifccant difference was detected among them.Consistent with DGGE result,Archaeal community structure remained stable through out the rice-growing period,which maybe resulted from long term of co-evolution between Archaea and human farming activities.Since methanogenus was proved to be the dominate taxa in Archaea by both DGGE and High-throughput sequencing.Soil samples of May,August and October were analyzed by RT-PCR to investigate the abundance of Methanosaeta,Methanobacterium and Methanosarcina,three representative methanogenus genera with different metabolic pathway of methane.The abundance of Methanosaeta in red soil collected in this work was around 10¹⁰ copies/gram dry soil,which was about 100 times higher than that of previous reported.Methanosaeta is a group of Archaea that specifically utilizes acetate to produce methane,the research on its ecological function in red soil should be paid much attention in future,eapcially the remediation of the acidification of red paddy soil.For Methanobacterium,specifically utilizing hydrogen and carbon dioxide to produce methane,and Methanosarcina,using both acetate and hydrogen and carbon dioxide to produce methane,though abundance of Methanobacterium and Methanosarcina is 10-100 times lower than that of Methanosaeta,significantly increased by 2.49 folds?P<0.001?and 1.62 folds?P=0.005?repectively in August.Modulating the abundance of Methanobacterium and Methanosarcina maybe one effective way of increasing rice yield,because hydrogen in the soil can considerably promote the growth of rice and enhance the tolerance of rice to aluminum,one of the main problem of red soil.Besides previous reports that methanogenus exclusively releases biogenic methane,with stronger greenhouse effect than carbone dioxide,our finding from this study further suggested that Archaea plays extensive and profound roles in ecological system of red paddy,i.e.,soil acidification,promotion of rice growth and ion tolerance of rice etc.Extensive and in-depth study on abundance,diversity and ecological functions of Archaea in soil etc.,should be conducted in future,which will notably contributed to the establishment of novel sustainable farming scheme and farmland remediation.