Relativity Study Between Endophytic Bacteria Flora Of Different Orange Varieties And Resistance Or Susceptibility Of Host To Citrus Canker

Different citrus varieties have obvious differences in resistance to canker.Sweet orange is highly susceptible,while mandarin orange is resistant.Endophytic bacteria are a group of microbial that grow in plants without causing any symptoms.A large number of studies have shown that endophytic bacteria have many beneficial biological effects on plants.So,are there any differences of endophytic bacterial flora among different citrus varieties and whether the resistance of citrus to canker is affected by the endophytic bacteria?For this reason,this study takes Satsuma Mandarin（SM,resistant variety）and Newhall Navel Orange（NNO,susceptible variety）as materials.By enriching endophytic bacteria in citrus,culturing aseptic seedlings and using Illumina sequencing,we study and analyze the endophytic bacterial communities in citrus with different resistance to canker,and preliminarily explore whether endophytic bacteria affect the production of defensins in citrus.The results are as follows.（1）Screening of enrichment methods of endophytic bacterial flora from citrus.When the sample tissues were pretreated with solution A（1.5%macerozyme R-10+1.5%cellulase R-10+8.55%sucrose）,solution B（1.5%macerozyme R-10+1.5%cellulase R-10+12.8%mannitol+0.12%MES+0.36%CaCl₂·2H₂O+0.011%NaH₂PO₄）and solution C（8.55%sucrose）,respectively,the proportion of chloroplast DNA in metagenomic DNA decreased,and the lowest chloroplast DNA proportion appeared in the sample tissues treated with solution A or B.For NNO leaves and SM peel,solution A resulted in the lowest proportion of 7.34%and 74.89%,respectively,while for NNO peel and SM leaves,Solution B resulted in the lowest proportion of36.07%and 9.65%,respectively.Compared to the control,both the number of OTU taxa and the amount of Proteobacteria and Firmicutes in the samples treated with three solutions increased,and more types of endophytic bacteria were identified.Based on the result,it can be inferred that by using appropriate methods the interference of chloroplast DNA to endophytic bacteria can be effectively reduced,and the endophytic bacteria can be easily enriched.（2）Difference analysis of endophytic bacteria flora between resistant and susceptible citrus varieties.The endophytic bacteria in each sample were mainly distributed in the Proteobacteria and Firmicutes,and a small amount of bacteria were distributed in the Actinobacteria and the Bacteroidetes.OTU quantitative analysis and beta diversity analysis revealed that the structure of the endophytic bacteria flora in two citrus varieties is different and the endophytic bacterial flora of the two citrus changed with the season.Compared with NNO,SM contain more antagonistic bacteria of Firmicutes in three seasons,and the content of Firmicutes decreased sharply in summer.It was found that the predominant bacteria were different between the two citrus varieties.There were more antagonistic endophytic bacteria in SM,while the antagonistic bacteria and synergetic bacteria were both found in the NNO.（3）The relationship between the citrus cultivable endophytic bacteria from citrus and canker resistance to canker disease.10 strains of main cultivable endophytic bacteria（the colony forming unit is 7.19×10⁴ in 1 gram fresh tissue）were isolated from SM,of which the colony number of the strongest antagonistic strain WZ131 accounted for 47%of the total colonies,and its diameter of antibacterial circle was 2.50cm.While 18 strains of main cultivable endophytic bacteria（the colony forming unit is1.29×10⁴ in 1 gram fresh tissue）were isolated from NNO,of which the colony number of the strongest antagonistic strain GN231 accounted for 46%of the total colonies,and its diameter of antibacterial circle was 0.90cm.Through inoculation experiment of detached leaves of NNO,it was found that all antibacterial strains obtained had inhibitory effects on canker,obviously reducing the incidence.Therefore,there may be a correlation between cultural endophytic bacteria in orange and its host resistance to canker,that is,endophytic bacteria in resistant varieties are fewer species but more numerous,as well as had better antibacterial effects than in susceptible varieties.In addition,the experiment also found that the existence and difference variation of antagonistic endophytic bacterial flora may affect the ability of citrus to resist canker.（4）Culture of aseptic seedlings of citrus.The optimum disinfection method of SM explants was the treatment with 70%alcohol and 0.1%mercury chloride for 6minutes and disinfect with 2 times;The optimum disinfection method of NNO explants was the treatment with 70%alcohol and 15%NaClO solution for 2 times.The addition of 1.0 mg/L 6-benzylaminopurine（6-BA）and 0.1 mg/L naphthylacetic acid（NAA）in MS solid medium was beneficial to the germination of axillary buds in citrus explant.The optimum disinfection method of rootstock seeds was that removing their inner and outer coat and disinfect them in 70%alcohol and 1.5%NaClO solution.Grafting of microbuds was carried out using inverted"T"grafting method,and the grafted citrus seedlings were successfully obtained.（5）Preliminary study of expression of defensin gene PDF1.2.The antagonistic endophytic bacteria were able to inhibit the occurrence of citrus canker significantly when they were inoculated to the aseptic seedlings.The inoculated seedlings were detected by PCR for the expression of PDF1.2 gene,but the expected band was absent.The possible reasons are that the structure of citrus defensins may be more complex,the primer matching is not good,and the endophytic bacteria in citrus may inhibit the occurrence of canker by other means or other substances.In conclusion,there are a certain differences of the endophytic bacterial flora between citrus canker resistant varieties and susceptible varieties.It is preliminarily considered that this difference is related to the host’s ability to resist canker,that is,there are more antagonistic endophytic bacteria in the resistant varieties,while there are both antagonistic bacteria and susceptible synergistic bacteria in susceptible varieties.The difference of endophytic bacteria affects the ability of host to resist canker disease.In addition,antagonistic effect of endophytic bacteria on citrus canker may be achieved through non-induced resistance.