Identification Of Biocontrol Microorganism Of Cereal Cyst Nematode And The Influence Of Strain 08F04 Of Beauveria Bassiana On Soil Microbial Diversity

Cereal cyst nematode(CCN) has caused serious damage in our country. However, the resistance of the main varieties in agricultural production was poor. There was no chemical nematicide with low toxicity and high efficiency. It was impracticable to implement fallow or crop rotation. So it was urgent to find an economic and effective method. Biological control is a new method to control plant parasitic nematode through natural enemy which wouldn't break the ecological balance. The control effect of biocontrol agents depends on eco-environmental conditions and nematode species. So it was rational to screen biocontrol agents adapted to local environment and trace the colonization of the agents in soil. Also, it was necessary to survey the influence of biocontrol agents on microbial diversity in soil and conduct risk assessment, which was meaningful for developing and use bioagents rationally. Given this, this paper researches:(1)Four biocontrol agents were identified which were screened in our research group earlier. They were characterised as Beauveria bassiana(08F04), Bacillus cereus(09B18), Achromobacter xylosoxidans(09X01) and Streptomyces anulatus(S07) respectively by morphological, physiological and biocheical criteria and 16 S r RNA analysis. All of them were reported as biocontrol agents of CCN firstly.To determine the stability of the four strains and the influence on wheat plant, the trial was conducted in Xuchang and Zhengzhou diseased field for two consecutive years. Both of the four strains had good control effect against Heterodera filipjevi in the greenhouse conditions. But the stability of strain 08F04 and S07 was better than09B18 and 09X01 in the field. Also, the treatments innoculated with 08F04 or S07 strain could promote the increasing of fresh weight and yeild. It was indicated that both 08F04 and S07 strain have good prospects for farther exploitation and application. As the fungi have the advantage of easy cultivation and fast growth,strain 08F04 of Beauveria bassiana was selected for further study.(2)Real-time PCR and dilution method of plate count were conducted to study the colonization of strain 08F04 of Beauveria bassiana transformant tagged by sgfp and hph gene. Plasmid p CAM-gfp was transferred into strain 08F04 of Beauveria bassiana by Agrobacterium-mediated method. PCR and southern blot were used to validate the sgfp gene had been integrated into the genome of strain 08F04.Transformant G10 was obtained which was the same as wild-type strain in morphology, growth rate and parasitic ability.Determination system by real-time PCR was set up to investigate the copy numbers of sgfp gene per gram of soil. The colony-forming unit(CFU) was surveyed by dilution method of plate count with homomycin. The result showed that the trend of gene copy numbers and CFU in plate was same and the obtained linear fitting equation was y=0.398+0.91 x, R2=0.7995. The biomass of strain 08F04 in soil increased firstly and then decreased. The biomass reached maximum value three weeks after innoculation(3.8×105 copies/g). So we can reapply the biocontrol agents three weeks after innoculation to control CCN effectively, which was crucial time for control CCN.(3) Next generation high-throughput DNA sequencing techniques was used to research the influence of strain 08F04 of Beauveria bassiana on fungal(ITS1 region)and bacterial(16S V4 region) community in the soil of Zhengzhou and Xuchang.The result showed that the innoculation of 08F04 strain had no significant influence on the number of species of fungi population. Although strain 08F04 of Beauveria bassiana restrained the diversityof fungi in earlier stage, its diversity returned to normal level in later period. Also, the innoculation of 08F04 strain had no significant influence on the number of species of bacteria and their diversity in soil. PCA analysis showed that the influence of sampling period on bacterial and fungi community was greater than biocontrol agent and the influence of strain 08F04 of Beauveria bassiana in earlier stage was greater than later period.Analysis of sequencing indicated that the abundance of Ceratobasidium and Alternaria was decreased significantly after inoculation of 08F04 both in Zhengzhou and Xuchang samples. However, Ceratobasidium was the pathogen caused wheat sharp eyespot and Alternaria was the pathogen caused wheat black embryo and wheat leaf blotch. So it is indicated that 08F04 could control CCN and some other fungal disease in wheat. In addition, the inoculation of 08F04 strain increased the abundance of Clonostachys, which was another biocontrol agent for parasitic nematode. It may inhibit the occurrence and damage of CCN accompany with strain08F04 of Beauveria bassiana.The results above indicated that strain 08F04 of Beauveria bassiana were safe to soil ecology environment and won't destroy ecological balance when applied into the natural environment. This strain has great value of development and utilization for the control to cereal cyst nematode of wheat.