Study On Microbial Diversity In Vineyard Soil From Regions Of Guizhou

Highly complex microorganisms involve in vineyard soils,which play an important role in the vineyard ecological system.In this study,we performed PCR-denaturing gradient gel electrophoresis,quantitative PCR and Illumina Hiseq sequencing to compare microbial compositions in four different soil depths(0-5cm,5-10 cm,10-20 cm,20-40 cm)from a vineyard in Tongren,Guizhou and analyzed their vertical distribution characteristics,in the rhizosphere and non-rhizosphere soils from a vineyard in Zhenyuan,Guizhou,repectively.We also investigated the diversity of yeasts in vineyard soil,grape peel,and spontaneous fermentation process of the grapes,by combining plate isolation,5.8S-ITS-RFLP classification and 26 S rDNA D1/D2 sequencing.The results were as follows:The organic matter and available phosphorus had significant differences in different soil depths,and the activities of urease,catalase and phosphatase significantly decreased with the increasing soil depth in Tongren,Guizhou.Quantitative PCR analysis indicated that the bacterial levels ranged from 9.10 to 10.03 Log copies g-1,which was significantly correlated with soil moisture,organic matter,total phosphorus,and the activity of amylase,and significantly higher in bacterial quantities were displayed at 0-5 cm depth soil than 20-40 cm depth soil at each site.In contrast,the fungal biomass vary from 5.97 to 6.91 Log copies g-1 at different depths and significantly correlated with soil moisture,organic matter,total nitrogen,and the activity of saccharase.The PCR-DGGE and Illumina HiSeq sequencing results revealed that bacterial community was dominated by Proteobacteria,Acidobacteria and Actinobacteria,and the fungal community was dominated by Ascomycota and Basidiomycota in all soil samples.We also found that Pseudomonas and Arthrobacter were the predominant bacterial genera,and Mortierella and Aspergillus were major fungal genera.ANOSIM and MRPP tests showed obviously differences in bacterial and fungal communities at 0-5 cm depth compared to 10-20 cm and 20-40 cm depths.Pearson correlation analysis and Redundancy analysis suggested that potential relations existed in soil physicochemical properties,enzyme activities and soil microbial community structure.The water content,organic matter,total N,total P in the rhizosphere soils were higher than that in the non-rhizosphere soil;the activities of amylase in the rhizosphere soils were higher than that in the non-rhizosphere soil,while the activities of urease,catalase,and phosphatase in the rhizosphere soils were lower than that in the non-rhizosphere soil from Zhenyuan,Guizhou.Quantitative PCR results indicated that the bacterial 16 S rRNA and fungal 18 S rRNA gene copy numbers in the rhizosphere soils were higher than that in the non-rhizosphere soil.PCR-DGGE and Illumina Hiseq sequencing analysis showed that bacterial communities was dominated by Proteobacteria and Actinobacteria,and the fungal community was dominated by Ascomycota and Basidiomycota in the rhizosphere and non-rhizosphere soils.Alpha diversity analysis showed that the bacterial richnesses in the rhizosphere soils were lower than that in the non-hizosphere soil,while the fungal richnesses in the rhizosphere soils were higher than that in the non-hizosphere soil.Compared with the non-rhizosphere soil,the rhizosphere soils contained 33 special bacterial genera and 208 special fungal genera.A total of 257 yeast strains were isolated from vineyard soils,grape peel,and the spontaneous fermentation process of the grapes by combining plate isolation,5.8S-ITS-RFLP classification and 26 S rDNA D1/D2 sequencing.First,the yeast strains were preliminarily morphological identified by WL medium.Second,the yeast strains were divided into 32 categories using 5.8S-ITS-RFLP classification.Vineyard soils from Tongren,Guizhou were most rich in yeast species,there were 14 categories;vineyard soils from Zhenyuan,Guizhou were followed,which were 12 categories;10 categories of yeasts were isolated from grape peel;and only 4 categories of yeast species were isolated during spontaneously fermented process of the grapes.Finally,32 categories of yeast strains were further identified as 2 phyla and 32 genera by 26 S rDNA D1/D2 sequencing analysis.Overall,Wickerhamomyces pijper and Cyberlindnera saturnus were the most abundant species in vineyard soils;Hanseniaspora lachance was the most abundant species in grape peel;and during the spontaneous fermentation process,Hanseniaspora lachancei and Issatchenkia terricola dominated the initial stages,Hanseniaspora lachancei,Issatchenkia terricola and Pichia occidentalis dominated the middle stages,Pichia manshurica dominated the last stages.