Study On The Mechanism Of Silicon Fertilizer Induced Resistance Of Wheat To Defense Against Cyst Nematode Heterodera Filipjevi

The cereal cyst nematode is widespread in China,and the area and degree of damage in Huang-huai valley wheat areas is especially serious,which poses a serious threat to the safe production of wheat.There are two main species of Heterodera filipjevi and Heterodera avenae,which are harmful to wheat in China.Since the discovery of Heterodera filipjevi in Henan Province in 2010,its occurrence area has been expanding and the damage degree has become more serious.As an inducer,silicon plays an important role in plant disease resistance.The current reports on the induction of plant resistance to silicon mainly focus on sheath blight,rice blast,powdery mildew and other diseases.However,there are few reports on the mechanism of application of silicon fertilizer in wheat defense against Heterodera filipjevi.The research object of this study was wheat,using pot experiment,applying silicon fertilizer by mixing soil,and the nematode was Heterodera filipjevi.This study is divided into three parts.Test one:Design of 5×2 for inoculation Heterodera filipjevi and applying silicon fertilizer.Silicon level is 0,0.25,0.5,1,2 g/kg.The inoculation of Heterodera filipjevi was divided into non-inoculation(Si0,Si0.25,Si0.5,Si1,Si2)and inoculation(Si0N,Si0.25N,Si0.5N,Si1N,Si2N).We took samples two months after inoculation.By measuring the indexes of the number of cysts of each wheat plant,the fresh weight of wheat shoot and root,stem length,SPAD value,root morhology and root activity of wheat,evaluated the effect of silicon fertilizer on the effect of Heterodera filipjevi to find out the optimal concentration of silicon fertilizer against Heterodera filipjevi.Test two:The optimum concentration of silicon fertilizer selected by test one was used for the test.Complete experimental design of two factors for application of silicon fertilizer and inoculation of cyst nematode.There were four treatments,non-silicon and non-inoculation(CK),application of silicon and non-inoculation(Si),non-silicon and inoculation(N)application of silicon and inoculation(SiN).We took samples two months after inoculation.By measuring the indexes of the silicon content of wheat shoot and root,the content of nutrient substance(soluble sugar,soluble protein content,free amino acids),the content of secondary metabolites(lignin,total)and H2O2 content of wheat,we explore the physiological and biochemical mechanism of applying silicon fertilizer to wheat defense against cyst nematode.Test three:The optimum concentration of silicon fertilizer selected by test one was used for the test.We set four treatments,CK,N,Si and SiN,respectively.we sampled at 0,12,24,48,72,96 h after inoculation.The most sensitive time points of wheat reaction to inoculation were selected by comparing the dynamic changes of resistance-related enzyme activity(MDA、LOX、CAT、SOD、PPO、PAL)and resistance-related gene(PAL gene,AOS gene,Chi3 gene,Glul gene)expression in wheat treated with N and SiN.We extracted the total RNA from the wheat root in CK,Si,N,SiN of the most sensitive time points.Through RNA-seq analysis of the differential genes of Silicon group(SiNvsSi)and Control group(NvsCK)after inoculation cyst nematodes,the resistance mechanism of wheat defense against Heterodera filipjevi was further discussed from the molecular level,and the molecular mechanism of increasing wheat resistance to cyst nematode was deeply revealed,which provided a better theoretical basis for using induced resistance to control crop nematode disease.The results are as follows:(1)With the addition of different concentrations of silicon fertilizer,the amount of systs of Heterodera filipjevi decreased and the growth and development of wheat increased.A silicon fertilizer with a concentration of 0.5 g/kg promoted wheat growth most obviously and had the best resistance to Heterodera filipjevi.Compared with the control,the number of cysts of each wheat plant treated with 0.5 g/kg silicon fertilizer decreased by 67.74%;The fresh weight of wheat shoot,stem length and SPAD values increased significantly by 55.95%,38.50%and 31.58%,respectively;(2)A study on the physiological and biochemical mechanism of applying silicon fertilizer to enhance wheat defense against Heterodera filipjevi was carried out with the optimal concentration of silicon fertilizer 0.5 g/kg.According to the results of the study,the silicon content of wheat shoot and root treated with 0.5 g/kg of silicon was 57.41%and 57.49%higher than that without silicon treatment.Silicon deposition in wheat epidermal cells to form a cuticle-bisilicon layer structure,improve the mechanical resistance of wheat.The contents of soluble sugar,soluble protein and total phenol increased significantly by 44.60%,72.66%and 35.55%,respectively,compared with the non-silicon treatment under the condition of non-inoculation.It means silicon can increase the content of composition soluble sugar,soluble protein and total phenol.The difference between the content of soluble sugar,free amino acid,lignin and total phenol increased with the application of silicon fertilizer,that is,the content of induced soluble sugar,free amino acid,lignin and total phenol could be increased by applying silicon fertilizer;The difference between the contents of soluble protein and reactive oxygen species decreased with the application of silicon fertilizer.Therefore,the application of silicon fertilizer increased the resistance of wheat to cyst nematode from three aspects:mechanical resistance,constitutive resistance and inducible resistance through the effects on wheat silicon content,nutrients,secondary metabolites and reactive oxygen species,and preliminarily clarified the physiological and biochemical mechanism of silicon fertilizer on wheat defense against Heterodera filipjevi.(3)We tested the changes of the resistance-related enzyme activity and resistance-related gene expression in wheat using dynamic sampling tests.The results showed that in the early stage of wheat inoculation with Heterodera filipjevi,silicon application could increase lipoxygenase(LOX)activity and malondialdehyde(MDA)content at 12 h and then decrease by participating in wheat metabolism;Catalase(CAT)activity decreased first and then increased.;The activity of superoxide dismutase(SOD)and phenolic related enzyme(PPO and PAL)all showed the tendency of first increasing and then decreasing,and the turning point was at 48 h;The expression of PAL genes treated with silicon was significantly up-regulated after 12 h;The expression of AOS gene and Glul gene was up-regulated after 48 h;The expression of Chi3 gene reached the maximum at 12 h under the condition of silicon application and the maximum at 48 h without silicon application.Therefore,Si application enhanced wheat resistance to cyst nematodes by inducing and regulating resistance-related enzymes and resistance-related genes in wheat,and at 48 h,Si application had the most significant effect on wheat resistance-related substance changes.(4)We performed transcriptome sequencing analysis of wheat root samples at 48 h,the most sensitive time point of the above reaction.The results showed that the differentially upregulated genes before and after inoculation KEGG annotation analysis pathway including Protein processing in endoplasmic reticulum,Ether lipid metabolism,Glycerophospholipid metabolism,Glycolysis/Gluconeogenesis and Flavonoid biosynthesis.The pathway of specific up-regulated differential genes significantly enriched in KEGG annotation analysis under Si application include Phenylpropanoid biosynthesis,Plant-pathogen interaction,Plant hormone signal transduction,Porphyrin and chlorophyll metabolism,Arginine and proline metabolism,Ubiquinone and other terpenoid-quinone biosynthesis.Application of silicon fertilizer increased wheat resistance to Heterodera filipjevi by affecting gene expression of these metabolic pathways and causing corresponding resistance substance changes,revealing the molecular mechanism of silicon application to enhance wheat resistance to Heterodera filipjevi.Therefore,in the process of interaction between Heterodera filipjevi and wheat,silicon not only works through the mechanism of physical barrier,but also causes a series of physiological and biochemical reactions by participating in the regulation of gene expression,causing a series of physiological and biochemical reactions,thus playing the role of defense against Heterodera filipjevi.In this study,the physiological and biochemical resistance mechanism and molecular resistance mechanism of wheat defense against Heterodera filipjevi were determined by using silicon fertilizer.