Development Of Seed Coating Agent For Inducing Resistance To Cyst Nematode In Soybean And Preliminary Study On Its Biocontrol Mechanism

Soybean cyst nematode,as a common disease in soybean production,seriously affects the yield and quality of soybean.At present,the compatibility to ecological environment and low toxicity caused by biological control have become the focus of the development of nematode control agents.In this paper,Penicillium flavum(Penicillium janthinellum)Snef1216 strain obtained from(Penicillium chrysogenum)Snef1650;and(Bacillus megaterium)Sneb207 strain were compounded according to a certain proportion and mixed with seed coating auxiliaries to prepare soybean cyst nematode biological seed coating agents SN105 and SN106.Through indoor pot experiment,field experiment,expression of resistance gene and determination of hormone in fermentation broth of biocontrol bacteria,the induced resistance effect and mechanism of biological seed coating agent were studied systematically.1.New biocontrol seed coating agents SN105 and SN106 were developed for the control of soybean cyst nematode: in this study,soybean seeds were coated with metabolites of microbial fermentation broth to regulate and affect the soybean plant itself and improve the resistance of soybean roots to soybean cyst nematode.The high efficient strains Snef1216 and Snef1650 screened in the laboratory were mixed with Sneb207 respectively.The best proportion of the compound was selected by seed germination experiment and toxicity test to soybean cyst nematode,and new biocontrol seed coating agents SN105 and SN106 were formed.The indoor pot experiment further showed that the fermentation broth under the optimum ratio had a certain growth-promoting effect on the plant,and could significantly improve the resistance of the roots of susceptible soybean varieties to race 3 of soybean cyst nematode.2.The field control effects of new biological seed coating agents SN105 and SN106 on soybean cyst nematode were determined.The best proportion of metabolites of biocontrol bacteria obtained by indoor screening was mixed with appropriate amount of seed coating auxiliaries to make biological seed coating agents SN105 and SN106.Field experiments were conducted in Kangping county and Shenhe district of Shenyang City in 2021.The results of field investigation showed that the number of cysts on soybean roots and the number of cysts in root soil treated with biological seed coating agents SN105 and SN106 decreased significantly at seedling stage.Soybean seeds coated with biological seed coating agents SN105 and SN106 could significantly improve the resistance of susceptible soybean varieties to physiological races 1 and 3 of soybean cyst nematode,and the number of soybean cyst nematode in roots decreased significantly.In the mature stage of soybean,the treatment of biological seed coating agent could promote the plant height and the number of seeds per plant,but there was no significant change in the number of pods per plant and 100-seed weight after treatment.3.New biological seed coating agents SN105 and SN106 could significantly up-regulate soybean-related disease course protein genes: real-time fluorescence quantitative PCR was used to detect the expression of soybean-related disease course protein genes(PR1,PR2,PR3 A,PR3B,PR5,PR9)in soybean roots at different stages of soybean cyst nematode infection(6 d,12 d,18 d).It was found that the genes of PR1,PR3 A,PR3B,PR5 and PR9;in soybean roots coated with biological seed coating agent SN105 were significantly upregulated compared with the control,and the up-regulation time of PR2,PR3 A,PR3B,PR5 and PR9 genes were slightly different.The results suggest that biological seed coating agents SN105 and SN106 may activate the immunity of soybean through the expression of different resistance genes at different stages so as to achieve the purpose of soybean resistance to soybean cyst nematode infection.4.It was confirmed that the fermentation broth of high efficiency strains snef1216,Snef1650 and Sneb207 all contained a certain concentration of salicylate hormone,and that soybean seeds treated with higher concentration of salicylic acid solution could significantly improve the resistance of soybean to soybean cyst nematode.Salicylic acid in the metabolites of three high efficiency strains was detected by LC-MS.The results showed that all the metabolites contained salicylic acid,and the content was slightly different.Soybean seeds were treated with salicylic acid with different concentration gradients.The root cracking test showed that soybean could be induced to develop systemic resistance to soybean cyst nematode by coating with 1000 ng/ml salicylic acid.The content of salicylic acid detected by the metabolic producing species of the three biocontrol strains was not enough to cause a strong defense response of the soybean plant itself.Under the coating treatment of 1000 ng/ml salicylic acid,the number of nematodes in soybean roots was significantly reduced in 12 dpi after inoculation.The results showed that genes related to salicylic acid pathway,jasmonic acid pathway and defense enzyme activity played an immune role in stimulating soybean resistance to soybean cyst nematode infection at all stages of nematode infection under exogenous salicylic acid treatment.