Apple Replant Disease Key Factor In Old Orchards And Mechanism Of Rootstock Response To Its Infection

Apple replant disease(ARD)has become the limiting factor of the sustainable development of apple industry in China.It is of theoretical and practical significance to find out the key pathogens of apple replant disease and the mechanism of apple rootstock response to infection.The physicochemical properties and fungal community structure of rhizosphere soil in 57 perennial tree soils(over 25 years)were investigated.The correlation between soil main factors and the degree of ARD was analyzed.And we identified the key factors of ARD.Effects of F.solani on physiology and molecular mechanism of apple rootstock‘M9T337'were studied.At the same time,a gene of MdWRKY74 was screened and cloned for the first time from the roots of‘M9T337',which responded positively to the changes of F.solani inection,and its function was explored.Results were as follows:1.It was analyzed that Fusarium is the key factor of ARD.The results showed that the degree of ARD varied in different orchards.The fungal communities and physicochemical properties of soil in different orchards were also different.A linear regression and Pearson correlation indicated that the abundance of Mortierella,the contents of phloridin in the soil and SOM were significantly negatively correlated with the inhibition rate of plant dry weight,and the abundance of Fusarium significantly positively correlated.There were no correlation between HN,AP,AK,p H and the rate of inhibition rate of plant dry weight.Among them,the Pearson correlation coefficient of Fusarium was 0.695(P<0.01)and the highest.It indicated that the relative abundance of Fusarium had a strongly correlation with the degree of ARD,and Fusarium is the key factor causing ARD.2.It was clarified that the root physiological mechanism of apple rootstock response to F.solani infection.The results showed that F.oxysporum,F.proliferatum,F.solani and F.moniliforme all inhibit the growth of apple rootstock‘M9T337'.Among them,the inhibitory effect of F.solani was stronger.At the early stage of infection with F.solani,the defense system of apple roots was activated,there were a rapid burst of ROS,antioxidant enzyme activities and the abundance of PR protein transcription was increased.And the apple root was normal and had small amount brown areas.With the extension in infection time,the plant defense system was destroyed,the activities of antioxidant enzymes and the abundance of PR protein transcription decreased gradually after reaching their peak.And the apple root tissue had massive brown,root tip was necrosis.This showed that the antioxidant system play an active role in the early stage of apple rootstock resistance to F.solani infection.With the extension in infection time,the coordination of ROS and antioxidant system was destroyed in apple roots,root structure was damaged and plant growth was inhibited.3.The inhibitory effects were investigated on photosynthesis of apple rootstock by F.solani infection.The result showed that apple rootstock‘M9T337'was infected by F.solani after 20 days,the content of chlorophyll and gas exchange parameter were significantly reduced.When F.solani infected apple rootstock,the OJIP curve shape changed obviously,and the J point value was decreased.This suggested that the performance degradation of the PSII reaction center was exacerbated by F.solani infection.In addition,the chlorophyll fluorescence parameter was decreased,and the non-photochemical quenching(NPQ)was increased by the F.solani infected apple rootstock.F.solani infection could reduce the contant of chlorophyll,decresse the photosynthetic parameter,inhibit the activity of PS?in apple rootstock.And F.solani infection reduced the energy distribution of leaf light system and inhibited photosynthesis of apple rootstock.4.Slightly acid electrolyzed oxidizing water could reduce apple replant disease.Compared with control,slightly acid electrolyzed oxidizing water(physical and chemical criterion:p H 6.0±0.05,ORP 1050±20m V,ACC 70±5 mg·L^-1)had a strong inhibitory effect on the mycelial growth of F.oxysporum,F.fusobacterium,F.moniliforme and F.solani,the inhibition rates were as follows 43.35%,48.19%,27.55%and 40.71%.In pot experiments,slightly acid electrolyzed oxidizing water increased the length of roots,surface area of roots,total roots volume and root activity by 0.47,1.41,1.01,0.37 times,respectively.Slightly acid electrolyzed oxidizing water decreased the numbers of fungi and bacteria,by 48.1%and21.4%.Based on the T-RFLP,slightly acid electrolyzed oxidizing water on the replanted soil formed independent community structure.Based on the q PCR analysis,slightly acid electrolyzed oxidizing water reduced the copy number of F.oxysporum,F.fusobacterium,F.solani and F.moniliforme by 57.16%,39.59%,49.05%and 43.52%,respectively.Therefore,slightly acid electrolyzed oxidizing water can alleviate apple replant disease by changing the soil environment.5.Apple rootstock‘M9T337'in response to F.solani infection was analyzed by RNA-seq.The result showed that 667 differentially expressed genes was identified in F.solani infected rootstocks,of which 543 were up-regulated and 124 were down-regulated.GO enrichment analysis showed that F.solani infection had a significant impact on the oxidation-reduction process,response to biotic stimulus,chitin catabolic process and other biological processes of apple roots.KEGG enrichment analysis showed that F.solani infection caused significant changes in genes related to terpenoid biosynthesis,pentose glucuronate interconversions,phenylpropanoid biosynthesis and other signaling pathways in apple roots.In addition,the analysis of transcription factors,19 differentially expressed transcription factors were enriched.Among them,ERF and MYB transcription factor families have the most members responding to F.solani infection,as well as transcription factor family genes such as b HLH,WRKY.This showed that apple rootstocks‘M9T337'respond to F.solani infection by producing and transporting terpeniods,sugars,phenylpropanoids and other metabolites.6.The function of MdWRKY74,in response to F.solani infection in apple rootstock‘M9T337',was investigated.The results showed that the expression of MdWRKY74 induced by F.solani infection apple rootstock‘M9T337'.MdWRKY74 belongs to WRKY IId group according to the WRKY domain and zinc finger motif characteristics.Subcellular localization assays showed that MdWRKY74 was targeted to nucleus.The overexpression of MdWRKY74gene in apple‘Orin'callus,the mycelium growth of F.solani was decreased.This showed that the overexpression of MdWRKY74 apple callus can significantly improve the resistance to F.solani.By the F.solani infection,the relative expression levels of endonuclease gene(Md ECHT)in the OE-MdWRKY74 calli were higher than those in the control calli.MdWRKY74 could bind Md ECHT promoter by yeast one-hybrid and electrophoretic mobility transfer analysis.As per the luciferase reporter analysis,MdWRKY74 significantly increased the activity of Md ECHT promoter.MdWRKY74 is a positive regulator of apple disease resistance,which promotes its expression by binding to the promoter of endostatinase gene,thus improving the resistance of apple to F.solani.