Isolation,Identification And Biocontrol Functional Study Of Pantoea Ananatis LY01

Recent studies have shown that Pantoea is widely distributed in paddy fields.Isolation and sequencing analysis showed that Pantoea ananatis was the most abundant and dominant bacterium in rice growing environment.On the one hand,P.ananatis as pathogenic bacteria cause diseases on many crops in our country,such as rice white leaf blight,corn leaf blight,strawberry stem rot,strawberry bacterial leaf blight and peach soft rot disease;On the other hand,P.ananatis can also antagonize the infestation of cinerea or inhibit the growth of other pathogens by producing antifungal compounds.In mid-July 2021,a suspected bacterial stripe disease occurred in the rice fields of Yongyou 2640 and Chunyou 584 in Linyi City,Shandong Province.This research through the isolation and identification of pathogenic bacteria,bacteriostatic test indoor medicament,plate inhibition test of P.ananatis fermentation supernatant,inoculation experiment of pathogenic bacteria,reactive oxygen species burst was detected by NBT staining,detection of callose deposition by aniline blue staining,mass spectrometry screening of antibacterial active substances and prokaryotic expression in P.ananatis fermentation supernatant and other methods were carried out.The main results were as follows:(1)From susceptible samples suspected to be symptomatic of bacterial leaf streak in rice,followed by 16S r DNA sequencing,molecular identification,sequence alignment and pathogenicity test of P.ananatis housekeeping genes leu S and rpo B.The newly discovered rice disease on indica-japonica hybrid rice combination in Linyi rice field of Shandong province was identified as bacterial stripe disease caused by P.ananatis LY01.The pathogen strain was named P.ananatis LY01.(2)In order to prevent and control a new type of rice stripe disease caused by P.ananatis,tetramycin,xinjunan and Zhongshengmycin,which are conventional and effective in preventing rice bacterial diseases,were selected for bacteriostatic test indoor medicament.It was found that tetramycin water had the best inhibition effect on P.ananatis in pineapple.(3)It has been reported that P.ananatis can produce antifungal compounds to inhibit the tomato Botrytis cinerea.In order to investigate whether P.ananatis has inhibitory effect on B.cinerea,plate bacteriostatic test was conducted.The results showed that LY01 and other strains inhibited the growth of B.cinerea.The effect of P.ananatis officinalis on Rhizoctonia solani Kühn YWK196,pathogenic bacteria of bacterial leaf streak Xanthomonas oryzae pv.oryzicola RS105 and white leaf blight pathogen X.oryzae pv.oryzae PXO99~A.The plate inhibition test showed that LY01 and other strains also showed inhibitory effects on the growth of YWK196,RS105 and PXO99~A.(4)P.ananatis showed inhibitory effects on multiple pathogenic bacteria on the plate.In order to explore whether P.ananatis secreted antibacterial active substances,the fermentation supernatant of P.ananatis affected the pathogenicity and growth of RS105.The inoculation experiment showed that the fermentation supernatant of P.ananatis reduced the pathogenicity of RS105 in rice leaves,and the plate inhibition experiment showed that the fermentation supernatant of P.ananatis inhibited the growth of RS105.(5)In order to further explore whether the fermentation supernatant of P.ananatis activated plant immune response,NBT staining and aniline blue staining experiments showed that P.ananatis could stimulate the outbreak of reactive oxygen species and callose deposition in rice.(6)The supernatant was treated with chloroform,ethanol and high temperature,and the antibacterial activity of the supernatant disappeared after treatment,indicates that the main bacteriostatic substances may be protein.In order to further explore the bacteriostatic effect of proteins,mass spectrometry was performed on them,and Omp A,Lol B and Pal,which contained exogenic signal peptides,were selected for functional verification.At present,Lol B-p MAL-c2X-MBP has been successfully constructed and induced to express Lol B protein.NBT staining was used to detect superoxide,and it was found that Lol B protein supernatant could stimulate superoxide accumulation in rice leaves.In summary,P.ananatis was identified as a new discovery of bacterial leaf streak in Shandong Province,and named P.ananatis LY01 strain.Through the laboratory experiment,it was found that the tetramycin agent had the best inhibitory effect on P.ananatis,which provided some reference value for the future prevention and control of field bacterial diseases caused by P.ananatis.The study also found that LY01 showed inhibitory effects on various fungi and bacteria of rice on the plate,and its fermentation supernatant could reduce the incidence of RS105 in rice,and stimulate the outbreak of reactive oxygen species and callose deposition in rice.This study selected contains secrete signal peptide Omp A,Lol B and Pal as bio-control proteins for exploratory research,and will provide new green rice disease control and prevention immune precursor protein.