3种植物花螺原体的分离及其基本特性

Isolation and biological characteristics of spiroplasmas from flower surface

【目的】调查我国植物花上的螺原体的存在,搜集我国的螺原体资源,并研究它们的基本生物学特性。【方法】常规螺原体分离、培养方法,应用暗视野显微镜和透射电子显微镜观察螺原体形态,根据16S rDNA和ITS序列构建系统发育树研究螺原体分离菌株可能的分类地位。【结果】分别从油菜(Brassica napus)、杜鹃(Rhododendron simsii)、红花酢浆草(Oxalis corymbosa)3种植物花表分离到4株螺原体CNR-1和CNR-2、CNA-1、CRW-1,对其形态、部分生理生化特性及分子生物学特性进行了初步研究。这4株螺原体在R-2液体培养基中生长良好,都能通过孔径为0.22μm的微孔滤膜;在R-2固体培养基上呈圆形或颗粒状菌落,菌落直径约50～600μm;在生长的某个阶段可呈典型的螺旋状,菌体直径为37.04～370.40nm,长度约0.89～11.88μm;它们都能利用葡萄糖作为碳源,不能利用尿素;在不含胎牛血清的R-2培养基中,它们都不能生长;菌株CNR-1、CNA-1能强烈代谢精氨酸,而CNR-2和CRW-1不能代谢精氨酸;在氨苄青霉素钠浓度高达2000U/mL的R-2培养基中,分离菌株生长良好。根据16S rDNA序列构建的系统发育树显示,分离菌株CNR-1和CNR-2、CNA-1与蜜蜂螺原体Spiroplasma melliferum聚类较近,而CRW-1与S.clarkii聚类较近;根据ITS序列构建的系统发育树显示,CRW-1形成一个单独的分枝,其它3个菌株仍与S.melliferum聚类。【结论】以上结果初步表明,分离菌株CNR-1和CNR-2、CNA-1极有可能是Spiroplasma melliferum,而CRW-1可能是一个新的螺原体种,但还需要血清学试验进一步验证。

[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-1 and 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 μm 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.