Screening Of Biocontrol Bacteria Against Rice False Smut And Preliminary Study On Its Mechanism

In recent years,the occurrence area and severity of rice false smut(U.virens)have been increasing in China.The disease has risen from a secondary disease of rice to a major disease of steady-state epidemic.Relying on the extensive use of chemical fungicides to control rice false smut is not in line with the development direction of green agriculture.Biological control is one of the effective measures to replace chemical agents due to its advantages such as being environmentally friendly,harmless to human beings,non drug resistant,and it has a promising application.Therefore,screening out biocontrol strains with good inhibitory effect on rice false smut and exploring its inhibitory mechanism is of great significance for the development of new strategies for rice false smut control.In this study,the rice endophytes were isolated and comprehensively screened,and the fermentation conditions of the screened biocontrol bacteria were optimized,and preliminarily explore their biocontrol mechanisms,The main findings obtained are as follows:Screening and identification of endophytic bacteria in rice: A total of 190 endophytic bacteria of rice were collected from 10 regions(Liaoning,Jilin and Heilongjiang provinces,China),Three biocontrol strains with better inhibitory effect were obtained by confrontation culture method,spore germination method and mycelial growth rate method,Their inhibition zone diameters were 32.33 mm,32.25 mm,and 32.57 mm,respectively,The mycelial growth inhibition rates of 10%fermentation broth were 28.61%,40.82% and 30.20%,and the inhibition rates of spore germination were 68.10%、76.26%和 65.56%,respectively.In the field efficacy test,the protective effect of the three endophytic strains was better than that of the treatment,and their protective effect was higher than that of the control agent,fumonisin.All three biocontrol bacteria had broad-spectrum antibacterial activity.Through morphological,physiological,biochemical and molecular identification,SDL75-4 was identified as Bacillus velezensis,and SDL9-1 and SDL78-2 were identified as Bacillus altitudinis.Optimization of fermentation conditions for biocontrol bacteria: The fermentation base conditions of the three biocontrol strains were optimized in a single factor,Through the Plackett-Burman test design,the steepest climbing path test design,and the Box-Behnken test design,the optimal fermentation conditions for the three strains to antagonize U.virens were determined.The optimal fermentation conditions of the biocontrol strain SDL9-1 were to add beef extract with a concentration of 1 g/100 m L and 0.5 g/100 m L of copper sulfate to the basal medium,and the fermentation time was 48 h;The optimal fermentation conditions of the biocontrol strain SDL75-4 were adding beef extract with a concentration of0.3 g/100 m L and copper sulfate with a concentration of 0.4 g/100 m L to the basal medium,and the fermentation time was 48 h;The optimal fermentation conditions of the biocontrol strain SDL78-2 were to add beef extract with a concentration of 1.2g/100 m L and 0.4 g/100 m L of copper sulfate to the basal medium,and the fermentation time was 48 h.After optimization,the growth inhibition rates of strains SDL9-1,SDL75-4,and SDL78-2 against U.virens reached 90.34%,81.64%,and62.55%,respectively,which were 61.73%,40.82%,and 62.55% higher than those before optimization,respectively.Investigation of the mechanisms of biocontrol of three strains: All three strains can produce cellulase,protease,indoleacetic acid,can form biofilm,can not produce ferritin and chitinase.The fermentation broth of strain SDL75-4 showed good stability to visible light,temperature,ultraviolet light and p H;Strain SDL9-1 and SDL78-2 showed good stability to visible light and temperature.Strain SDL75-4 has the ability to dissolve potassium,and strains SDL9-1 and SDL78-2 have the ability to dissolve phosphorus.The activities of catalase(CAT),polyphenol oxidase(PPO),peroxidase(POD),phenylalanine ammonia lyase(PAL),superoxide dismutase(SOD),chitinase and superoxide anion free radical in rice treated with fermentation broth all showed a trend of increasing first and then decreasing.The content of malondialdehyde showed a trend of first decreasing and then increasing.The expression levels of five rice defense-related genes Os PR1 a,Os PR1 b,Os AOS2,Os PR10 a and Os BIMK2 all showed a trend of increasing first and then decreasing under the treatment of three endophytic biocontrol strains.It shows that after inoculation with biocontrol strains,the expression of rice-related defense genes will be promoted,the results showed that the three biocontrol strains had the characteristics of inducing disease resistance.