Effect Of Different Fertilization Treatment On Genetic Diversity Of Cultural Microorganism Associated To Rice Rhizosphere

In order to explore the effect of biogas manure on microorganisms at rice and wheat rotation soil in Chengdu plain, six treatments, including three usage amounts of biogas manure alone (T1, T2, T3), mixture use of biogas manure and chemical fertilizer (T4), fertilizer use alone(T5), and fertilizer free (T6) were designed. The rice rhizospheric soil samples were collected at tillering stage, jointing stage, and harvest stage, and microbial amount (include bacterial, fungi, and actinomycetes), soil biochemical properties (soil total N, available nitrogen, available K, available phosphorus, organic matter content, and pH value) were analyzed, and the genetic diversity of microorganism was performed based on BOXAIR-PCR, RAPD analysis, and sequence analysis. The results showed as the follow:(1) Along with the rice growing, the amount of rhizosphere bacteria and fungi increased at the joint stage, and then decreased at the harvest stage. The amount of rhizosphere bacteria, actinomycetes in T1 to T3 was lower than that of T5 at the tillering stage and joint stage; however, the quantity of rhizosphere bacteria in T1-T3 increased 10%-60% than that of T5, the quantity of rhizosphere actinomycetes in T1, T2 and T4 increased 13%-39% than that of T5 at harvest stage. But the amount of rhizosphere fungi in T1-T3 had no significant difference with T5.(2) A total of 154 bacteria isolated from all soil samples, including 48 strains at the tillering stage,56 strains at the jointing stage, and 50 strains at the harvest stage were isolated from rice rhizosphere.16S rDNA sequencing analysis distributed these strains into 9 genuses, and strains belonged to genus Bacillus was the dominant one, including 8 species such as Bacillus aryabhatti, Bacillus cereus, Bacillus lichenformis and so on, and then strains belonged to genus Ensifer. The diversity of rice rhizosphere bacteria decreased with rice growth period, but the bacillus diversity increased.(3) A total of 74 fungi isolated from all soil samples, including 20 strains at the tillering stage,26 strains at the jointing stage, and 28 strains at the harvest stage. ITS sequencing analysis showed that there were five genuses of fungal strains include Rhizopus, penicillium and Cylindrocarpon were isolated from tillering stage, jointing stage and harvest stage. While, fungal strains belonged to genus Trichoderma was the predominant at harvest stage. The fungi associated rice rhizosphere kept stable during the whole growth period.(4) There were 87 actinomycetes, including 20 strains at tillering stage,35 strains at jointing stage, and 32 strains at harvest stage were isolated from the soil samples.16S rDNA sequencing analysis revealed that all the isolates belonged to Streptomyces, but distributed into 9 species. Of them, Streptomyces deccanensis could be isolated from each growth period. The diversity of actinomycetes in T1, T2 and T4 harvest stage was higher than that of T5, which suggested that application of biogas manure properly was helpful to increase rhizosphere actinomycetes diversity.(5) Analysis of physical and chemical properties showed that, compared with chemical fertilizer application, the soil organic matter content increased 4%-37% by application of biogas slurry, but the soil total nitrogen amount was similar. Though the rice yields was not significant different between T2, T4 and T5, T2 and T4 could save 15.28%-29.13% of urea per hm2, and could increase the thousand seed weight of rice, which suggested that application of biogas slurry had good benefits and environmental effects.