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

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.