Objective: To obtain the spiroplasma resources and investigate the characteristics of the spiroplasmas on the surface of flowers in China.
Methods: The spiroplasma morphology was examined by dark-field microscope and transmission electron microscopy. The biological characteristics of the spiroplasmas were investigated by using conventional culture-dependent method and phylogenetic analysis based on 16S rRNA gene and ITS (16-23S rDNA intergenic space) sequence comparisons. Phylogenetic analysss was performed using the software package MEGA4.0 after multiple alignment of sequence data by CLUSTAL X.
Results: Four spiroplasma isolates were obtained from three kinds of flower surface. Isolates CNR-1 and CNR-2 were from Brassica napus; CNA-land CRW-1 from Rhododendron simsii and Oxalis corymbosa respectively. All isolates grew well in R-2 liquid medium and exhibited contractive movements. The colonies of all isolates were circinal and grain-like in solid medium. Through electron microscopy, all isolates exhibited helicity during their growth phase. All isolates could pass through a 0.22 microm filtrate membrane and resist to penicillin (2000 U/mL). They must grow in medium with serum. Glucose could be used as their carbon source instead of sucrose. The ability to hydrolyze arginine varied from different spiroplasmas but urea could not be hydrolyzed. The phylogenetic relationships based on 16S rDNA supported CRW-1 was close to S. clarkii, and others were close to S. melliferum. The phylogenetic relationship based on ITS sequence supported CRW-1 formed a separate clade, and others were also close to Spiroplasma melliferum.
Conclusion: The result indicated that spiroplasma isolate CRW-1 might be a new species and other three isolates were S. melliferum, but this need further support of serological test.