| Walnut blight is a bacterial disease caused by Xanthomonas arbicola pv.Juglandis(Xaj)and the serious walnut disease on leaves and nuts all over the world.The pathogen attacks various organs of walnut including catkins,female flowers,fruit,green shoots,buds and leaves,which reduces the productivity of walnut orchards and negatively influences the quality of the nuts.Current control measures are based on the use of copper-based biocides.However,a chief concern moving forward is increased resistance to copper-based biocide in natural populations of Xaj,and an immediate and longterm concern is the viability of the continued registration of these compounds,due to their toxicity and their impact on the environment and on natural ecosystems.A genetic solution to this problem using traditional breeding approaches requires a good evaluation of existing genetic variation in susceptibility to blight among walnut cultivars.Moreover,study on the mechanism of walnut response to Xaj can further guiding breeding.Due to the lack of walnut disease resistance materials and their genetic information,there are few studies on the mechanism of interaction between walnut and Xaj,especially the researches on the pathogenicity of pathogens and the immune system of plant disease resistance,which greatly increased the difficulty of breeding resistant varieties.To establish a method for evaluating the disease resistance of walnuts,and to study the response of the physiological and biochemical indexes of walnuts with different sensitivity to walnut blight,and then analyze the expression of disease-related genes during the immune process,is helpful to analyze the immune mechanism of disease resistance and tap its key disease resistance genes,which has important guiding significance for cultivating walnut resistant varieties.In this study,field investigation and artificial inoculation(in vitro leaf inoculation and in vivo leaf inoculation)were used to evaluate the resistance of 18 walnut varieties or genotypes to walnut blight,and by comparing the results of field investigation and artificial inoculation evaluation,a good walnut blight evaluation system was established.In addition,by selecting 10 varieties with different sensitivity to walnut blight,the response patterns of various physiological and biochemical indexes during the infection of Xaj were analyzed.And then using RNA sequencing,analyze the changes of gene expression levels in healthy and diseased leaves of susceptible variety and resistant genotypes,screen the main disease-related genes by comparing differentially expressed genes between variety and genotype,which providing candidate gene resources for resistance breeding of walnut blight and has important guidance and practical significance for revealing the resistance mechanism of walnut blight.The results are as follows:1.Through field investigation,the sensitivity of 18 different varieties or genotypes to walnut blight were determined,and the disease resistance of each variety or genotype was: JS 91(JS 92,JS 86,Qingxiang,JS 71,JS 64)>JS 199(JS 200,Shuo Xing)>Panhe No.1(Yanyuanzao)>Chuanzao No.1(Chuanzao No.2,Liaohe,JS 100)>Shujiang No.1(JS 65,Xiangling).Then the sensitivity of these 18 walnut varieties or genotypes was evaluated artificially by in vitro leaf inoculation and in vivo leaf inoculation,and the results were compared with the field investigation.The results showed that the result of in vivo leaf inoculation evaluation was the most correlated with the field survey results(r = 0.789),indicating that in vivo leaf inoculation evaluation was more reliable than the in vitro leaf inoculation.Therefore,in vivo leaf inoculation evaluation method in this study can be the supplement of field investigation,and provides a basis for the screening of resistant materials to walnut blight.2.Ten walnut cultivars or genotypes with different sensitivity to walnut blight were selected and in vivo leaf inoculation method was used to observe the changes of redox-related enzyme activity,phenolic compound content and their related enzyme activity and membrane lipid peroxidation level in the leaves at 0 d,3 d,7 d,14 d and 28 d after inoculation(DAI)by Xaj.The results showed that SOD activity and MDA content were induced after inoculated but the change had no cotrrelation with disease resistace;the change of CAT activity had no regulation after inoculation;POD activity,total phenolic content,PPO and PAL activity changed significantly after infection,and were significantly correlated with disease resistance(P ≤ 0.01)at early stages.The total phenolic content and their related enzyme activity were induced after inoculation and the change was positively correlated with disease resistance,indicating that phenolic metabolism plays an important role in the early defense of walnut blight,which can be used as a reference biomarker for walnut blight evaluation.3.Using walnut blight resistance genotype JS 91 and susceptible variety Xiangling as materials,the transcriptome sequencing of healthy leaves and diseased leaves was carried out.Then the transcriptome expression profile of walnut response to Xaj was constructed through annotation of gene and metabolic pathways and comparison of differential gene expression in healthy leaves and diseased leaves.The response of walnuts to pathogens and the differences in response of disease related genes were analyzed.In this study,eight walnut samples were sequenced by transcriptome,and a total of 77,325 annotated Unigenes were obtained.The annotation results provide an important reference for gene annotation of other related studies in walnut.By analyzing the differential expression of disease related genes,the following results were obtained: For the recognition of pathogen,the induction of FLS2 and XA21 was specific found in resistance genotype,indicating that the resistance genotype may establish PTI to defense by recognize pathogens.Induction of the genes involved in the FLS2 and XA21 pathways(RING/U-box and E3 ubiquitin ligase;ubiquitin ligase,plasma membrane ATPase,and protein phosphatase 2C)were found in susceptible variety,which means this defense mechanism may be inhibited.Besides,in cell wall reconstruction,the XTH-related genes involved in cell wall hydrolysis were both found in resistant genotypes and susceptible varieties,but in the resistant genotypes,pectin esterase,transcription factor MYB,S-adenosylmethionine decarboxylase enzymes,callus synthase and a large number of bHLH and wall-associated kinases are involved in cell wall remodeling,which hinders the continued spread of pathogens.While in the susceptible varieties,no induction of genes related to cell wall reconstitution was found,and XET and cellulase were induced to promote cell wall degradation as well as the specific induction of MLO-related genes,indicating that the constitutive defense in susceptible variety was overcome by the pathogen,and its MYB transcription factor accumulated after the infection without the accumulation of bHLH,indicating that its flavonoid synthesis pathway may be inhibited.Meanwhile,in addition to the significant up regulation of POD-related genes in resistant genotype and SOD related genes in susceptible varieties,the other ROS scavenge enzymes(such as GST,CAT,etc.)related genes were down regulated or no significant change.In addition,expression of PPO was induced and expression of PAL was down-regulated in susceptible variety,which indicating that the phenylpropanoid pathway was inhibited as well.Furthermore,resistant genotypes and susceptible variety appear to be involved in different signaling pathways.In the resistant genotype,a large number of calcium-related genes were induced,while only c40744_g1,c92619_g1 and c94745_g1 were induced in the susceptible variety,and a large number of related genes were significantly down-regulated or not significantly changed.Moreover,the genes related to ethylene signal transduction were only induced or inhibited in susceptible varieties.The difference between resistance genotypes and susceptible varieties in signaling pathways may related to the difference in their sensitivity to walnut blight.In this study,the sensitivity to walnut blight of 18 varieties or genotypes were evaluated by field investigation and artificial inoculation,and then the method of artificial inoculation evaluation was clarified.The determination of physiological and biochemical indicators found that total phenolic content and enzymes(POD,PPO and PAL)related to phenols activities were induced by pathogen infection,and the changes at early stages were related to the sensitivity to walnut blight,which could be used as candidate biomarker for disease resistant evaluation.In addition,the results of RNA sequencing are helpful to understand the defense mechanism to walnut blight.The difference in disease resistance genotype and susceptible variety may reveal differences in the sensitivity to walnut blight,the related genes provide a certain direction for the subsequent research on the walnut blight defense mechanism and disease resistance breeding. |
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