Diversity And Activity Of Nitrogenase NifH Gene From Endophytic Diazotrophs In Rice And Wheat

Rice and wheat are the main food crops in China,and they play an important role in food prodution and people's lives.Nitrogen fertilizer is one of the main factors that affect the growth of crops.In this paper,rice and wheat grown in the fields of Heilongjiang,Hubei and Hebei provinces in China are used as materials.Real-time quantitative PCR,gradient gel electrophoresis,and reverse transcription PCR and other technologies have systematically explored the effects of application of microbial fertilizers,different crop varieties,and different growth stages on the number of nifH genes,dominant bacterial taxas,and nifH gene activity of rice and wheat endophytic nitrogen-fixing bacteria.The main findings are as follows:(1)The application of bio-fertilizer has significant effects on the number of nifH genes of endophytic nitrogen-fixing bacteria and the diversity of nitrogen-fixing bacteria taxas in rice and wheat.The number of nifH genes in rice roots,stems,and leaves are(1.51-17.80)×108 copy/g,(1.01-2.76)×107 copy/g and(2.04-19.50)×106 copy/g.Compared with the control treatment without bio-fertilizer application,the number of nifH genes in rice roots remained flat or decreased,but the number of nifH genes in stems and leaves increased significantly,and the endophytic nitrogen-fixing bacteria taxas and dominant taxas of rice have changed significantly,and the results of wheat experiment and rice have the same trend.In general,the dominant taxas are Azospirillum,Pseudomonas,Sinorhizobium,Methylocystis,Sphingomonas,Rheinheimera,Azoarcus and so on.(2)The number of endophytic nitrogen-fixing bacteria nifH genes among rice varieties was not significant,but the diversity of nitrogen-fixing bacteria taxas diversity was significant.Common taxas include Azospirillum,Ideonella,Sinorhizobium,Azoarcus,Pseudacidovorax,Methylocystis,Sphingomonas,Bradyrhizobium,Xanthobacter,Rheinheimera and so on.(3)The number of endophytic nitrogen-fixing bacteria nifH genes at different growth stages was significantly different in rice root.The number of nifH genes were(1.90-10.60)×10~6 copy/g,(1.23-2.47)×109 copy/g at seedling stage and flowering stage,and diversity was significant changes.The dominant taxas are Rubrivivax,Sideroxydans,Dechloromonas,Sinorhizobium,Pseudacidovorax,Anaeromyxobacter and so on at flowering stage.(4)cDNA analysis of the nifH gene revealed that Bradyrhizobium,Kosakonia,Sphingomonas,Ideonella,and Azohydromonas are the active endophytic nitrogen-fixing bacteria taxas of rice at seedling stage,and Bradyrhizobium,Pelomonas,Sphingomonas,and Dechloromonas are the active endophytic nitrogen-fixing bacteria taxas of rice at flowering stage;Bradyrhiburia,Pradyrhizobium are the active endophytic nitrogen-fixing bacteria taxas of wheat at greening stage.