Studies On Stimulation Effect Of Brassica Napus From Root Exudates On Resting Spores Of Plasmodiophora Brassicae

Plasmodiophora brassicae can cause root clubroot of cruciferous crops such as Brassica napus,which can result in reduced or even no harvest.At present,the commonly used comprehensive prevention and control strategy for clubroot can prevent and reduce the occurrence of the disease to a certain extent.However,due to the lack of systematic research on the pathogenesis,it has been impossible to completely eradicate clubroot.In this study,hydroponics and solvent extraction were used to obtain crude extracts of root exudates of Brassica napus.The crude extracts were screened in groups with five solvents,and the biological activity of each group of screened materials was tested to stimulate the germination of resting spores of P.brassicae.The structural analysis was completed by HRMS,and the effective stimuli of resting spores of P.brassicae were screened by metabonomics multivariate statistical analysis.Finally,the stimulation effect of the selected substances was tested to verify its effectiveness.Taking plant root exudates as an entry point to explore the association and mechanism of action between root exudates and pathogenic bacteria is a strategy to explore the pathogenesis of clubroot from the root,and can also provide reference for studying the pathogenesis of other soil-borne diseases.The specific research contents are as follows:1.Extraction of root exudates of Brassica napus and their stimulating effect on resting spores of P.brassicae.Huayouza 62 was selected as the research object.After 28days of hydroponic cultivation,the root exudates of Brassica napus were collected in ultrapure water.After freeze-drying to remove solvents,approximately 0.7 mg of crude extract was obtained for each rapeseed plant.The crude extract was used to stimulate the germination of resting spores of P.brassicae.The concentration of the spore suspension extracted from the diseased roots of susceptible rapeseed is about 5.2×10~8/m L,the average diameter of resting spores is about 2.5μm,and the initial germination rate is 8%.The PBS buffer solution with p H 6.50 is the most conducive to the germination of resting spores.The crude extract stimulated the germination of resting spores in different degrees within five days,and the stimulation ability was the strongest on the third day,with the stimulation rate of 59%.The root exudates of Brassica napus extracted from hydroponic experiments contain active substances that can stimulate the germination of resting spores,which can be further separated and analyzed.2.Group screening and composition analysis of the root exudates in Brassica napus.Solvent screening was carried out on the crude extract of root exudates of Brassica napus,and 5 kinds of solvents were used for screening and extraction,including ultrapure water,methanol,acetone,ethyl acetate and n-hexane.The polarity of solvents is different,and the main components and the proportion of components extracted from the crude extracts are different,which leads to the different stimulating activities of the screened extracts on resting spores of P.brassicae.The stimulation rates of the screened extracts of ultrapure water and acetone were 78%and 67%,respectively,which were significantly higher than the other three groups(P<0.05).Five groups of screened extracts were tested by LC-HRMS,using metabolomics and other related analysis,according to the results of OPLS-DA and Student’s t test,three potential stimulating compounds were screened,namely hypoxanthine,thymine and L-isoleucine.The stimulating effect of these compounds in practice was further tested by the germination test in resting spores.The experiment showed that on the third day,their stimulation rates reached 25%,19%and17%,respectively,which were significantly different from the blank control(P<0.05),and were obviously different from the stimulation effects of other substances.The test also found that the stimulation rate of the compound of several substances increased to 33%.These results indicate that in the crude extract of root exudates of Brassica napus,more than one substance has a significant stimulating effect on resting spores,hypoxanthine,thymine and L-isoleucine have synergistic effects on each other,and their ratio composition is also an element factor affecting the stimulating effect.The research on the structure of the components of the stimulant and the synergistic effect between the components will provide a reference for the research on the pathogenicity mechanism of P.brassicae.Systematic and in-depth research has dual significance of basic theory and practical application.