Differentiation of bacteria of the genus Pseudomonas pathogenic to coffee by serological techniques

ABSTRACT: Some bacterial diseases have been described causing problems in coffee, whose causal agents are Pseudomonas cichorii, P. syringae pv. garcae, P. s. pv. tabaci and Burkholderia andropogonis, all of them also occurring in Brazil. Attempts to differentiate these bacteria by double diffusion agar (dda) technique with antisera produced against whole cells of P. s. pv. garcae showed cross-reactions, especially among P. s. pv. garcae and P. s. pv. tabaci. Thus, antisera were produced against P. s. pv. garcae (pathotype strain IBSBF-248 - Phytobacteria Culture Collection of Instituto Biológico - IBSBF), using rabbit immunizations with antigens of the protein complex of membranes. These antisera were tested against different kind of antigens (autoclaved cells, cells treated with formaldehyde, capsule polysaccharides, glycoproteins, bacterial membrane proteins and bacterial suspension in 0.85% NaCl) extracted from P. cichorii, P. s. pv. garcae and P. s. pv. tabaci. The results showed that depending on the kind of antigen and the medium used in the double diffusion test (with or without MgCl2 and/or trypan blue), the antiserum reacted only with P. s. pv. garcae. Thus, these antigens may be used as an auxiliary tool in the rapid diagnosis of bacterial halo blight of coffee in the double diffusion test.

摘要：已有报道称多种细菌性病害对咖啡造成危害，其致病菌分别为菊苣假单胞菌（Pseudomonas cichorii）、丁香假单胞菌格氏致病变种（P. syringae pv. garcae）、丁香假单胞菌烟草原致病变种（P. s. pv. tabaci）以及甘蔗伯克霍尔德菌（Burkholderia andropogonis），上述病原菌在巴西均有分布。此前尝试以针对丁香假单胞菌格氏致病变种全细胞制备的抗血清，通过双向扩散琼脂（double diffusion agar, DDA）技术区分上述细菌时，发现存在交叉反应，尤其是在丁香假单胞菌格氏致病变种与丁香假单胞菌烟草原致病变种之间。为此，本研究以丁香假单胞菌格氏致病变种的膜蛋白复合物抗原免疫家兔，制备了对应的抗血清（菌株采用模式株IBSBF-248——保藏于巴西生物研究所植物细菌培养物保藏中心（Instituto Biológico - IBSBF））。随后使用该抗血清，对从菊苣假单胞菌、丁香假单胞菌格氏致病变种及丁香假单胞菌烟草原致病变种中提取的多种抗原（包括高压灭菌菌体、甲醛处理菌体、荚膜多糖、糖蛋白、细菌膜蛋白以及0.85%氯化钠生理盐水悬浮的细菌菌体）进行检测。结果显示，根据抗原类型及双向扩散试验所用培养基（添加或不添加氯化镁和/或锥虫蓝）的不同，该抗血清仅可与丁香假单胞菌格氏致病变种发生反应。因此，在双向扩散琼脂试验中，上述抗原可作为辅助工具，用于咖啡细菌性晕斑病的快速诊断。