Screening Of Sweet Orange Mutants Resistant To Xanthomonas Citri Subsp. Citri

Xanthomonas citri subsp. citri (Xcc) is the causal agent of citrus canker, a quarantine disease worldwide. Citrus canker is difficult to control completely, and causes seriously damages to the citrus industry.’Bingtang’sweet orange is the one of the most important citrus cultivars in Hunan province, but the leaves, sticks and the fruits are highly susceptible, and the disease spot on the surface of the fruits can seriously reduce their commercial value. On the production, chemical prevention was the main method to control citrus canker, but never completely eradicate it. Also, the chemical pesticides can cause environmental pollution and excessive residue in fruit may cause fruit safety problem. Breeding disease-resistant varieties become the only way to control the canker disease.Therefore, this thesis is, by T-DNA insertion mutagenesis, radiation mutagenesis and chemical mutagenesis, to induce mutation of sweet orange, and through in vitro and in vivo inoculation to select mutants resistant to the canker disease. It could provide the important materials to study the resistant mechanism to citrus canker disease; and better understanding the relationship between’Bingtang’sweet orange or citron C-05 with the pathogen Xcc. The main results are as follows:1.’Bingtang’ sweet orange embryonic callus suspension and ’Dahong’ sweet orange were transformed with the activation tagging PSKI074-GUS. The transformants were detected by GUS,38 clones were GUS-positive. After detected by RT-PCR,5 transgenic clones were obtained. But they all had typical canker symptoms after being inoculated with Xcc in vitro assay.2. Different doses of gamma rays were used to treat bud sticks of ’Bingtang’sweet orange, then grafted on ponkan trees. The detached leaves of each budded shoots were inoculated with 108 CFU/ml of Xcc. Eight out of 7463 irradated shoots did not show typical symptom of citrus canker. By in vivo assay of the 8 shoots, three (1-3-5,1-3-5,199) of them had a certain resistant to citrus canker.1-3-5 appeared necrosis spot in the area of injection, the expression of CERK1 was higher than CK after being inoculated with Xcc.2-6-8 had lighter symptom, the expression of CERK1 was decline and the expression of LYP2 was higher.199 appeared symptom later, the expression of CERK1 and LYP2 were all increased.3. EMS treated seeds of’Dahong’ sweet orange germinated later and the rate of seed germination was lower. The growth of seedlings were declined as the increase of the EMS concentration. After inoculating,3 (1-9-7、2-8-4、3-24-7) out of 743 mutants did not appear symptom of citrus canker. By in vivo inoculation, they were all susceptible to canker disease.4. When the callus of ’Bingtang’ sweet orange inoculated with Xcc, the expression of CERK1 and LYP2 genes were lower than the control, and the Xcc grew quickly around the callus. While the callus of citron C-05 had higher expression of CERK1 and LYP2 gene than the control treatment under the same conditions. The Xcc did not grow around the callus, but the callus changed brown and hypersensitive death. Therefore, the Xcc was not suitable as selection stress for screening of resistant mutants from sweet orange somaclones.5. The callus of ’Bingtang’ sweet orange or citron C-05 induced Xcc to grow quickly around the callus of ’Bingtang’ sweet orange, but not around the callus of citron C-05. The callus and leaves of ’Bingtang’ sweet orange could cause the Xcc proliferation, the callus of liquid suspension promoted the formation of biofilm, and the extract of leaves enhanced the compound of exopolysaccharides (EPS). The substance promoting the growth of Xcc was of diffusion and thermal instability. Some substance in the callus and leaves of citron C-05 might control the growth of Xcc.6. The supernatant of Xcc cultures was concentrated 10 times to prepare the Xcc-crude extract. When the leaves of ’Bingtang’ sweet orangethe was inoculated with Xcc-crude extract, the symptom was similar as the typical symptom of citrus canker. When the new shoots of citron C-05,’Bingtang’ sweet orange and kumquat were cultured in 10% Xcc-crude extract solution, the leaves of ’Bingtang’ sweet orange and kumquat became yellow and then dropped, and the stems rotted to died; while the shoots of citron C-05 grew normally for 7 days. And the callus suspension died completely when 10% Xcc-crude extract was added in the suspension for 3 weeks. So, the Xcc-crude extract could be the best selection pressure to obtain resistant somaclones of ’Bingtang’ sweet orange.7. EMS mutagen had lethal effect to ’Bingtang’ sweet orange somatic callus. The half lethal concentration was 1.5% EMS treated for 1h. After selected in the Xcc-crude extract for 3 weeks,271 resistant were obtained. After cultured in the Xcc-crude extract solution, only 19 of them survived after 2 months. Through in vitro and in vivo assays, just one somaclones showed steady resistantce to canker disease.