Screening And Performance Evaluation Of Promoting-growth And Antagonistic Bacteria In Rice Roots

Fungal disease of rice becomes increasingly serious with the expansion of rice production scale.Effectively preventing and controlling fungal disease of rice were scientific problems to be solved in the process of improving quality and increasing yield of rice.The long-term use of chemical pesticides to control rice pests and diseases is prone to lead to problems such as drug resistance and environmental pollution.Nevertheless,the development and application of biological pesticide can protect the green production of rice.Rice root endogenous bacteria with promoting-growth and bio-control role are ideal materials for development of biological pesticides.However,the investigation of rice root endogenous bacterial resources in saline-alkali soil is rarely reported.Based on it,the rice roots in Daqing saline-alkali soil were used to be material,the endogenous bacteria with promoting-growth and bio-controling plant disease were selected by traditional cultivable methods,effect of promoting-growth and bio-controlling plant pathogen were illuminated,the antibacterial mechanism of rice root endogenous bacteria was preliminarily discussed.The study can provide materials for the development of biological pesticide used to cultivate rice in saline-alkali soil and lay the foundation of the development and application of biological pesticide.The results in the study are as follows.1.Traditional cultivable methods were used to isolate rice root endogenous bacteria in Daqing saline-alkali field,the result shows that the number of cultivable bacteria in rice root at booting stage was 1.6×10⁴ CFU/g fresh weight,the number of endogenous culturable bacteria in rice roots at filling stage was 1.1×10⁴ CFU/g fresh weight,a total of 74 strains of bacteria were purified from rice root systems at two stages,including 48 strains at booting stage,26 strains at filling stage,plate confrontation and improved Salkowski colorimetry method were used to screen antagonistic strains and determine the ability of producing IAA among 74 strains rice root endogenous bacteria,It was found that 14 strains at booting stage could be antagonistic to one more the tested plant pathogens,22 strains at filling stage could do that,36 strains at booting stage and 21 strains at filling stage could secrete IAA of more than 1μg/mL.2.A root endogenous strain SM13 was identified and selected from obtained rice root endogenous bacteria according to secreting IAA（5.56 g/mL）and inhibiting the growth of plant pathogens.The bacteriostasis rates for Curvularia crescent,Sclerotinia Sclerotinia（Lib.）de Bary,Magnapporthe grisea,and Fusarium graminearum Schw of SM13 strain were 59.38%,78.13%,53.12%and 37.50%respectively.The morphological characteristics,physiological and biochemical characteristics and the sequence analysis of 16S rRNA gene and functional genes were used to identify strain SM13,which was identified as a subspecies of bacillus subtilis.3.The application of propagated SM13 bacterial solution to potted rice(3.4×10¹³ CFU/667m²)showed that,SM13 bacterial solution could improve the activity of soil Sucrase at full heading stage of rice（p<0.05）,the tiller number of rice and chlorophyll content was decreased,plant height was increased,The spike dry weight of rice was significantly increased at full heading stage,but had no effect on dry weight of the sheath,stem and leaf.The results showed that SM13 bacterial solution could increase rice yield by 12.96%.Compared with the control group,SM13 solution could increase the grain number and grain weight of secondary branches（p<0.01）,and have no effect on rice quality.4.High throughput sequencing results of bacterial and fungal groups in potted paddy soil treated with SM13 bacterial solution showed that the application of SM13 strain increased the abundance of beneficial bacteria and fungi,including mycorrhizal fungi（Dentiscutata,Gyrophanopsis）which could promote the growth of crop and inhibit plant disease fungi,other beneficial bacteria and fungi strains with degrading lignin（Podospora）,cellulose（Koribacter,Roseiflexus）and hemicellulose（Roseiflexus）,resisting pathogenic bacteria and pathogenic fungi（Ochrolechia）,using methyl groups（Methylomicrobium）.The abundance of plant pathogens（Pyrenophora）in soil was decreased.5.The crude protein and crude polysaccharide of the fermentation broth of SM13 strain were extracted by saturated ammonium sulfate precipitation method,Sevag method combined with alcohol precipitation method.The antibacterial effects of crude protein,crude polysaccharide,fermentation broth and bacterial liquid against Magnapporthe grisea and Rhizoctonia solani were compared by Oxford-cup tests.The results showed that cude polysaccharide had not effect on Magnapporthe grisea and Rhizoctonia solani,crude protein,fermentation broth and bacterial liquid had the inhibiting effect on Magnapporthe grisea and Rhizoctonia solani.Clear bands of crude protein were obtained by SDS-PAGE separation.Comparative analysis of ten bands identified by mass spectrometry showed that the crude protein in SM13 fermentation broth had high identity to Minor extracellular protease and Peptidase S8which inhibits pathogenic fungi.The obtained proteins should be verified after purification.In summary,a Bacillus with promoting-growth and antagonistic ability was isolated from Daqing saline-alkali paddy soil.Rice yield applied with the strain were increased by 12.96%under potting condition.The strains could increase the beneficial bacteria and fungi groups in soil and decrease the potential plant pathogenic bacteria groups in soil.Bio-control functional proteins of the strain were primarily speculated and were close to minor extracellular protease and Peptidase S8.The function of SM13 strain will be further verified in the field.