枣疯病和泡桐丛枝病原植原体分离物的组织培养保藏和嫁接传染研究

Propagation and Long-Term Preservation of Several Isolates of Jujube Witches' Broom and Paulownia Witches' Broom-Phytoplasmas in in vitro Cultured Plantlets and Grafting Transmission of the Pathogens from the Diseased to Healthy Plantlets

分别从河北唐县赞皇大枣、辽宁凌源梨枣和河南濮阳扁核酸3个品种的枣疯病和来自山东、江西和北京的不同无性系的泡桐丛枝病树上采集丛枝枝条作为组织培养材料,获得的枣疯病和泡桐丛枝病组培苗皆表现典型的丛枝症状。其中感染植原体的赞皇大枣组培苗(Ft)和扁核酸组培苗(HPD)在未加任何激素的MS培养基和其它培养基交替继代培养1a以上仍能维持丛枝苗生长;而发病梨枣(LD)除产生丛枝外,还出现叶片黄化和植株矮化、枯梢等衰退症状。泡桐丛枝病植原体可在不同无性系组培苗上通过各种培养基交替和单纯的MS培养基继代培养,并已在实验室内连续保藏达10a,其引致丛枝症状的能力无明显的改变。用枣树Ft染病材料作接穗,以健康冬枣(DJ)和抗病婆枣(W14)砧木进行组培苗间嫁接传病试验,可使部分DJ和W14发病;而嫁接未发病的砧木多数像健苗一样正常生长。用感染山东泡桐丛枝病植原体ZD株系丛枝组培苗为接穗,嫁接健康泡桐无性系组培苗致使无性系MB33、TY2T和C125发病。用植原体16SrDNA通用引物进行PCR,确证了泡桐和枣树发病苗和嫁接发病组培苗体内存在植原体。用DAPI荧光显微镜技术比较组培苗韧皮部筛管中的植原体浓度,结果显示,Ft和嫁接发病冬枣(DJ Ft)筛管中植原体浓度相对较高,但仍低于各泡桐无性系染病丛枝组培苗;

The diseased shoots of three jujube (Ziziphus zizyphus) cultivars,Pozao from Tang County,Hebei Province,Lizao from Lingyuan County,Liaoning Province and Bianhesuanzao from Puyang,Henan Province,respectively infected with different isolates of jujube witches'broom (JWB)-phytoplasmas as well as paulownia (Paulownia spp.) witches'broom(PWB)-phytoplasma from Shandong,Jiangxi Provinces and Beijing were collected and cultured in vitro on various media.The in vitro cultured and infected plantlets displayed typical witches' broom symptoms,except for the hypertrophy and whiting of axillary and top buds often associated with some paulownia plantlets with PWB-phytoplasma.Plantlets with JWB-phytoplasma Pozao (jujube) isolate (Ft) and Bianhesuanzao (jujube) isolate (HPD) were subcultured on MS medium without addition of any hormone and grew well all along with witches' broom symptom for more than one year,while the Lizao (jujube) isolate (LD) showed obvious decline symptoms such as leaf yellows,dwarf and dieback besides ordinary witches'broom. PWB-phytoplasma isolates had been maintained in the laboratory in variety of paulownia tissue-cultured plantlets for over 10 years and no pathogenic mutation associated with these phytoplasma isolates was detected.When Ft scions with JWB-phytoplsams were grafted on the healthy jujube stocks,certain percentages of stocks of Dongzao (jujube) (DJ) and Pozao (W14) cultivars became infected and appeared typical witches' broom symptoms,while there was no typical symptom on unsuccessful-grafting stocks.The ZD scion with PWB-phytoplasma C85-028D isolate was also used to graft-transmit the phytoplasma into three paulownia clones,C125,TY2T and MB33,consequently causing typical witches'broom symptoms.The existence of both JWB- and PWB-phytoplasmas in diseased plantlets and graft-transmitted stocks was certified by polymerase chain reaction (PCR) using phytoplasmal 16SrDNA universe primer pairs,R16mF2/R2.The relative concentrations of both phytoplasmas in the phloems of JWB and PWB plantlets were also evaluated by DAPI staining fluorescence microscopy.The results showed that Ft and graft-infected DJ had relative more strong JWB-phytoplasma fluorescence emitted from the phloem than that of HPD and LD,but still rather lower than that of paulownia plantlets infected with PWB-phytoplasma; whereas less phytoplasmas and less number of phloem with JWB-phytoplasmas were detected in W14 stock already with witches'broom symptom which coincided with its relatively high resistance to JWB-phytoplasma.There were no phytoplasma fluorescence observed in the healthy plantlet as well as the stock grafted unsuccessfully.