Effects Of Penicillium Purpureus On GmC4H And GmCAD Under Soybean Cyst Nematode Stress And Their Resistance Mechanism

Soybean cyst nematode,as one of the important pathogens that harm economic crops,has caused irreversible losses to soybean production worldwide.In recent years,biological control technology with the advantages of being environmentally friendly,non-toxic and harmless to human body and high control effect has become a hot research hotspot.Studies have shown that plants can resist external stress by regulating cell wall resistance,inhibiting cell wall degradation or lignification,thereby preventing the transport of nutrients to fight external stress.Penicillium janthinellum Snef1650 is a biocontrol fungus with high control effect against soybean cyst nematode screened in our laboratory.In this study,starting from lignin,the technology of coating seeds with fungal fermentation broth combined with the expression changes of key enzymes cinnamic acid-4-hydroxylase(C4H)and cinnamyl alcohol dehydrogenase(CAD)in lignin synthesis under nematode stress,To explore the specific mechanism of the anticystic nematode in soybean induced by Penicillium purpureus Snef1650.The results of the study are as follows:1.All concentrations of the fermentation broth of Penicillium purpureus Snef1650 showed nematicidal effects on soybean cyst nematodes,and the corrected mortality rate of the fermentation broth at 48 h could reach about 95%.Pot experiments found that Snef1650 could inhibit the development of nematodes in soybean roots.It was found that at 3 dpi(days post inoculation),the number of J2 in soybean roots treated with Snef1650 was reduced by about 35% compared with the control group,and the control group appeared at 9 dpi J4 appeared at 15 dpi in Snef1650-treated group.In addition,the number of J3,J4 and adult females in Snef1650-treated soybean roots was significantly lower than that in control group at 20 dpi.The induction effect of Snef1650 on soybean nematode resistance was explored by root splitting system,and it was found that the treatment group responded to root nematode infection by about 53%.2.In order to study the specific action mechanism of Penicillium purpureus Snef1650 inducing soybean resistance to soybean cyst nematode,using q PCR technology,the lignin synthesis-related genes Gm C4 H,Gm CAD1 and Gm CAD2 in the roots of susceptible cultivar Williams 82 and disease-resistant cultivar D.The expression situation under insect stress was analyzed.In the process of Snef1650-induced nematode stress in susceptible varieties,Gm C4 H was significantly upregulated at 1 dpi and 9 dpi,but there was no significant difference in resistant varieties,indicating that C4 H gene may be induced to participate in the colonization and development of soybean resistance to nematodes process.Gm CAD1 and Gm CAD2 responded strongly at 9 dpi and 15 dpi during Snef1650-induced nematode resistance in susceptible cultivars,and were temporally consistent with their expression in resistant cultivars.It indicated that Snef1650 induced CAD gene and played a role in the development and reproduction of soybean resistance to nematodes.The above results show that C4 H only plays a role in susceptible varieties and is not sensitive to resistant varieties.C4 H may be involved in other pathways against cyst nematodes,while CAD1 and CAD2 both show positive responses in resistant varieties.As the main research goal,to further verify the important mechanism of Penicillium micropurulenti Snef1650-induced lignin synthesis to resist soybean cyst nematode stress.3.The KEGG enrichment results showed that the CAD gene family was a key enzyme in the regulation of phenylpropane lignin.The relevant bioinformatics analysis software analysis showed that the CDS sequences of Gm CAD1 and Gm CAD2 genes were both 1074 bp in size,and the corresponding physicochemical properties,secondary structure and phosphorylation site prediction of the protein were obtained.In addition,tissue-specific expression showed that Gm CAD1 and Gm CAD2 were highly expressed in roots,and the gene responses in roots were more sensitive to pathogen infection.Phloroglucinol lignin staining indicated that Snef1650 may induce lignin to participate in soybean cyst nematode stress and play a similar role to the disease-resistant varieties.4.Using PCR technology,clone the CDS region sequences of the target genes Gm CAD1 and Gm CAD2,add Nco I and Spe I enzyme cleavage sites at both ends of the sequences,transfer them into a plant vector and connect to obtain an overexpression vector p CAMBIA1302-GFP-Gm CAD1 and p CAMBIA1302-GFP-Gm CAD2 were injected and transformed into tobacco leaves in vitro.Subcellular localization showed that Gm CAD1 and Gm CAD2 were mainly distributed in the cell membrane.It was speculated that the regulation of cell wall thickness may be involved in the process of resistance to soybean cyst nematode through cell deposition.