Function Analysis Of The Cuticle On The Leaves Surface Against Infection Of Xanthomonas Citri Subsp Citri In Citron C-05

Citrus is the world’s largest fruit and the most important fruit tree in the southern region of China.Citrus canker is one of the most serious diseases,which has been affecting the development of the citrus industry,with wide distribution and serious damage.The main causative agent of citrus canker disease is the citrus subspecies of Xanthomonas citri subsp.citri(Xcc),which is mainly infected through stomata and local wounds,can harm most commercial citrus cultivars,causing fruits,leaves or branches to produce typical symptoms such as raised spots,and eventually necrosis falls.At present,Citrus canker disease is widely prevented and controlled by chemical agents such as copper preparations,but there are shortcomings,such as high cost,environmental pollution and difficulty in complete control.Therefore,at present,the selection and breeding of disease-resistant germplasm resources is the best way to control it.In addition,the leaves of Citron C-05 and‘Bingtang’Sweet Orange were sprayed with 10~8 CFU/m L of e GFP-Xcc,and it was found that the Xcc colonization on the lower epidermal surface of Citron C-05 was significantly less than that of‘Bingtang’Sweet Orange after inoculation,suggesting that the difference in leaf surface structure or composition may be an key reason for the difference in Xcc colonization between the two.Therefore,based on theoretical research and experimental basis,this paper systematically explored the mechanism of the cuticle of Citron C-05 leaf surface in the resistance to ulcerative bacteria from three aspects of cuticle structure,composition and gene expression,using Citron C-05 germplasm with high resistance and‘Bingtang’Sweet Orange germplasm with high sensitivity as test materials.The main results are as follows:(1)Citron C-05 leaves are significantly more resistant to Xcc than‘Bingtang’Sweet OrangeThe two germplasm leaves of different leaf ages(old leaves,mature leaves and young leaves)were soaked and inoculated with Xcc.It was found that the leaf maturity was positively correlated with disease resistance,and the old leaves showed no obvious disease resistance,but the disease resistance of mature leaves and young leaves of Citron C-05 was significantly stronger than that of sweet orange.Observation by scanning electron microscopy revealed that on the 10th day of inoculation Xcc,the epidermis of leaf in‘Bingtang’Sweet Orange was attached to high-density“stick-like”canker bacteria and the epidermis bulge ruptured,while only fewer canker bacteria were found on the leaf surface of Citron C-05;(2)The leaves of Citron C-05 have high resistance cuticle structureBy scanning and transmission electron microscopy to observe the two germplasm leaves under normal conditions,the results showed that the wax crystal structure of the epidermis of Citron C-05 was more than that of‘Bingtang’Sweet Orange.The wax crystal structure of the old leaves was mostly columnar,while the mature leaves were lamellar,and many stomata were covered by crystals.Compared with the smooth cuticle on the upper epidermis of Sweet orange,Citron C-05 was continuous and undulating.The stomatal density of mature leaves of Citron C-05 was significantly lower than that of‘Bingtang’Sweet Orange,and the chlorophyll retention rate was significantly higher than that of Sweet orange from 30 min,but there was no significant difference between the old leaves and young leaves;(3)The leaf surface of Citron C-05 is enriched with typical antibacterial keratin componentsThrough GC-MS analysis,the results showed that the total wax components of Citron C-05 and‘Bingtang’Sweet Orange leaves were primary alcohol,dominatedby 1-hexadecanol,and its content in‘Bingtang Sweet’Orange was 2.6 times that of Citron C-05;followed by alkanes,which were unique components of Citron C-05;the main component in keratin content was epoxy octadecanic acid,followed by 10-hydroxy-hexadecanoic acid,both of which were significantly higher in Citron C-05 than‘Bingtang’Sweet Orange.The results of the total wax composition plate experiment showed that there was no obvious bacteriostatic ring,but after the exoplasm of the wax was removed by living body,the leaves of Citron C-05 showed more severe symptoms of susceptibility than the unwatted phenotype,indicating that the exwaxing of Citron C-05 can limit Xcc proliferation to a certain extent in vitro;(4)Cm MYB30 and Cm MYB17 may be a candidate regulator for the response of Citron C-05 to XccCombined with transcriptomics and bioinformation analysis,KCS family genes(β-ketoacyl-co A synthase)KCS4 and KCS11 were identified as potential Xcc responsive genes.The upstream 2000 bp sequences of both KCS4 and KCS11 had binding sites for MYB transcription factors.Only Cm KCS11 and Cs KCS11 showed significant differences in expression trends in response to Xcc.In addition,111 MYB transcription factors were screened out through the genome database of cirtrus medica,and 6 potential R2R3-MYB transcription factors were identified with positive expression patterns in response to Xcc.Through real-time quantitative PCR analysis,only Cm MYB30 and Cm MYB17 expression levels were consistent with the phenotype of Cm MYB30、Cm MYB17 and Cm KCS11 form Xcc positive response module to participate in the specific resistance against Xcc infection of Citron C-05 leaf surface.