Identification Of Fusarium Spp.causal Agent Of Peanut Root Rot And Enzymatic Properties Of Cell Wall Degradation Of The Pathogen

Peanut is an important oil crop,and root rot is one of the most serious diseases in peanut production,which can be infected in the whole growth period of peanut.The pathogens that cause peanut root rot are mainly Fusarium.At present,there is a lack of systematic research on the species and pathogenic mechanisms of Fusarium spp.causal agent of peanut root rot,especially the enzymatic properties of the fungal cell wall degrading enzymes.In this study,the species of root rot fungus in the main peanut producing areas of Shandong were identified,and the enzymatic properties of cellulase and xylanase produced by the fungus were analyzed.The main research results are as follows:1.Species identification and biological characteristics of peanut root rot fungusPeanut root rot samples were collected from different areas in Shandong Province,and60 isolates were isolated.The pathogenicity test showed that different strains were pathogenic to peanut seedlings,but there were significant differences in pathogenicity.Morphological observation and EF-1αsequence analysis showed that 51 of the 60 isolates were Fusarium oxysporum,and the other fungi were Fusarium proliferatum（3 isolates）and F.solani（4isolates）,F.acuminatum（1 isolate）and F.moniliforme（1 isolate）.Eight representative strains were selected,and the effects of pH and temperature on the growth rate of pathogenic fungi mycelium were determined.The results showed that the growth temperature range of each strain was wide,all of them could grow at 15-33℃,and the optimum temperature was 25-28℃;the optimum pH of different strains was different,and most of them grew at pH 5-7.2.Determination of enzymatic activity of cell wall degrading enzymes of Peanut root rotTwo strains of F.oxysporum（Foxy4 and Foxyh）with strong virulence,Foxy4 and Foxyh,and the less virulent strain of F.solani（Foxy2）were selected to compare the cell wall-degrading enzymes produced by each strain.The results showed that the activities of cellulase and xylanase produced by Foxy4 were the highest and Foxy2 had the lowest activity.Enzymatic determination found that the optimum reaction temperature and optimum pH of cellulase of strains Foxy4 and Foxyh were basically the same,which were 60℃and pH 4,respectively;the optimum reaction temperature and optimum pH of xylanase of the two strains were 50℃and pH 4.Microcrystalline cellulose,sodium carboxymethyl cellulose,xylan and soybean meal were used as induction medium to determine the cellulase and xylanase activities of the three strains.The results showed that different induction media had different effects on inducing cellulase and xylanase.Among them,the sodium carboxymethyl cellulose medium had the best effect of inducing cellulase,and the xylan medium had the best effect of inducing xylanase.3.Determination of the enzymatic properties of F.oxysporum Foxy4 xylanase Focel10The enzymatic properties of the recombinant xylanase Focel10 were analyzed by heterologous expression.Using xylan as the substrate,the optimal reaction temperature and optimal reaction pH of xylanase Focel10 were determined,and it was found that the optimal reaction temperature was 50℃,and the optimal reaction pH was 4;the xylanase Focel10 was treated at 50℃for 1 h,and still had high activity;the substrate specificity assay showed that the xylanase Focel10 had higher activity on sodium carboxymethyl cellulose,amylopectin,arabinogalactan and mannan,glucan and phosphoric acid swollen cellulose have no obvious hydrolysis;metal ions Co²⁺,Mg²⁺,Mn²⁺,Cu²⁺,Fe³⁺,Ca²⁺can reduce the activity of xylanase Focel10;Tween-20,Triton X-100,Methanol,Ethanol,Isopropand,EDTA,SDS can reduce the activity of xylanase Focel10.The hydrolyzate of xylan degraded by xylanase Focel10 was analyzed by thin-layer chromatography,and it was found that most of the hydrolysis products were oligosaccharides with low degree of polymerization,and the content of oligosaccharides increased with the increase of time.