| Jujube witches' broom is a kind of destructive disease that is caused by phytoplasma. It is widely distributed, causing great economic loss in jujube fruit production, and has become a major obstruction to jujube development in China. There is no safe and effective cortrol method at present. The author in this experiment laid the foundation of the biological control of jujube witches' broom, by studying on low temperature preservation and cryopreservation of phytoplasma, living dynamics and seasonal change of phytoplasma in Ziziphus jujuba vat. po zao (po zao), the colonization and movement of plant endophytic bacteria in jujube seedlings and screening of antagonistic microbes and antibiotics against jujube witches' broom. The main results are as follows: 1 Cryopreservation of jujube and Catharanthus roseus materialsBy using encapsulation-vitrfication, vitrification, simple vitrification and common preservation, the author studied preservation methods of the stem apex and stem with axillary buds of tissue culture seedling with symptom of Jujube witches' broom, po zao, C. roseus and callus of Jujube witches' broom seedlings under -20℃, -76℃,-196℃. The result showed that the stem apex of tissue culture seedling of C roseus was successfully recovered in liquid nitrogen; the other preservation methods of C roseus under -20℃and -76℃and cryopreservation of jujube materials did not success. 2 Seasonal change and living dynamics of phytoplasma in a jujube tree with light symptom of witches' broomThe diseased branches of po zao with symptom of witches' broom were studied from two aspects. First, through symptom observation of the diseased branches which were collected respectively in late Autumn, Winter, and early Spring and cultured in hydroponic system, and detection by PCR technique, the specific band of phytoplasma with the size of 1.5 Kb fragment was all amplified from the diseased branches' phloem and the bud. The results indicated that phytoplasma can survive safely in the regions with the lowest temperature at -10℃in the winter. Secondly, no phytoplasma was detected in the root, trunk and branches without visible symptom of infected tree through the PCR except for diseased branches. Based on these results, it can be inferred that jujube phytoplasma only exists in diseased branch in newly infected jujube trees. 3 Isolatation, detection and the colonization of plant endophytic bacteria in jujube seedlingsIn this study, the Streptomyces strains CNI20 and CNI21 were isolated respectively from the root of the healthy po zao and the fruit of healthy Ziziphus jujuba var. fu ping da zao (fu ping da zao). The experiments were also carried out by studying the presence of unculturable endophytic bacteria in the tissue culture seedlings with symptom of jujube witches' broom, tetracycline-treated tissue culture seedlings with symptom of jujube witches' broom, tissue culture seedlings of healthy po zao and tissue culture seedlings germinated from seed of healthy fu ping da zao, using microscope and polymerase chain reaction. Bacteria with different shape and size were detected under microscope with magnification of 1000 times and the specific band of bacteria with the size of 1.5 Kb fragment was all amplified from the different materials. The colonization and movement of endophytic bacterias and Streptomyces isolated from different plant materials were studied as well. The CN015r, CN017r and CN122 strains were inoculated into po zao and detected by dilution plating methods on the 7~th day, 15~th day and 30~th day after inoculation. The results showed that CN015r, CN017r and CN122 strains can move into tissue culture seedlings of jujube, and the number of endophytic bacteria increased with time in the same part and decreased from stem base to the middle part of stem. CN017r was inoculated into tetracycline-treated tissue culture seedlings of healthy po zao and detected on the 30~th day. In the base of stem, the number of endophytic bacteria is 2.14×10~9 CFU/g in the healthy tetracycline-treated tissue culture seedlings and 1.70×10~7 CFU/g in the healthy tissue culture seedlings of po zao, respectively. It can be concluded that tetracycline can kill or suppress parts of unculturable bacteria and the number of culturable endophytic bacteria greatly increased in tetracycline-treated tissue culture seedlings of healthy jujube. 4 Screening of antagonistic microbes and antibiotics against jujube witches' broomAntagonism against phytoplasma of Jujube witches' broom was performed, using 9 bacteria strains CN015r, ('N017r, CN068, CN078, CHA0r, JM218, WCS358r, WCS358::phl, WCS417r, 6 Streptomyces species CN120, CN121, S. rimosus, S. aureofaciens, S. ahygroscopicus var. wuyiensis, Men-myco-93-63 and their metabolites and 6 kinds of antibiotics 2, 4-diacetylphloroglucinol(DAPG), chloramphenicol, rifampin, streptomycin sulphate, tylosin tartrate and tilmicosin by setting tetracycline and oxytetracycline as effective control. It can be concluded that Jujube witches' broom seedling treated by S. rimosus did not show visiable symptom change on the 15~th day; leaves at the top of affected seedlings became wider, the stems elongated and symptom began to ease on the 30~th day, most of the leaves changed to normal and symptom disappearred on the 45~th day. the metabolite of S. rimosus can ease the symptom of tissue culture seedlings with Jujube witches' broom. Bacterial strains, other Streptomyces species and their metabolite and antibiotics except for tetracycline and oxytetracycline can not suppress phytoplasma, and the affected seedlings didn not show any visiable change. |
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