Isolation and Characterization of a Non-Helical Strain of Spiroplasma citri

Abstract

A non-helical strain of Spiroplasma citri from little-leaf diseased oranges was subcultured over 40 times in different media without producing helical cells. It was non-motile and produced colonies of a characteristic morphology on soft agar plates. Serology, DNA hybridization and toxin production confirmed that it was a strain of S. citri but its membrane protein pattern after polyacrylamide gel electrophoresis differed from those of other S. citri strains in that one band was absent. The organism caused symptoms in plants identical to those produced by a pathogenic helical strain of S . citri. I N T R O D U C T I O NSpiroplasmas differ from other Mollicutes in shape and motility. All those isolated are typically helical during at least part of their growth phase and show rotary or 'screw'-like motility as well as flexing movements. The helical shape is apparently maintained without the assistance of a rigid cell wall or axial filaments (Cole et al., 1973).Spiroplasma citri was isolated and characterized by Saglio et al. (1973) and is the leafhopper-transmitted agent of little-leaf disease of citrus (Markham et al., I 974). We have isolated a pathogenic strain of S. citri, which lacks the characteristic helical morphology, from diseased citrus material. M E T H O D SIsolation and culture. A non-helical spiroplasma and six normal strains of S. citri were isolated from 'lop-sided' fruits of little-leaf diseased sweet orange trees by the method of Daniels et al. (1973). Isolates were subcultured twice in complete sorbitol medium (SMC; Saglio et al., 1973) without arginine and tryptone, before 0.1 ml samples were plated on SMC solidified with I % (w/v) agar (Oxoid no. I). After incubation for 7 days at 32 "C, colonies showing unusual morphology were picked off into SMC medium. One of these isolates was cloned three times and designated ASP-I. All cultures were maintained in SMC at 32 "C. Characterized strains of S. citri ~8 -~2 (type strain) and c189 were kindly supplied by Dr P. Saglio, and the pathogenic strain SP-A was isolated in this laboratory (Daniels et al., 1973).Media and incubation conditions. The colony and cell morphologies of strains SP-A and ASP-I were compared under different conditions. Plates of SMC solidified with 0.75 % and I % (wlv) agar were spread with 0-1 ml of diluted spiroplasma culture and incubated at 32 "C for 7 days in air or in N2/C02 (95 : 5, v/v). Cultures in SMC were incubated at 25, 28, 2 M I C 100

Nucleotide sequence of the Spiroplasma citri fibril protein gene

“…Although the gradient does concentrate the fibrils, SDS-polyacrylamide gel electrophoresis (PAGE) revealed that other spiroplasmal proteins copurify with them. Spiralin, the major membrane protein of both S. citri (32) and S. melliferum (6) was reported by Townsend et al (27) to copurify with the fibrils.…”

“…Individual fibrils are 4 nm wide. These fibrils are made from one single protein with a molecular mass of 55 kDa (27). The protein pattern of the fibril preparation is shown in Fig.…”

“…The following procedures for solubilization of the cells and purification of the fibrils were based on those reported by Townsend et al. (27). Each Polyacrylamide gel electrophoresis of the fibril suspension was performed by the method of Laemmli (15) on 12.5% polyacrylamide separating-5% polyacrylamide stacking gels.…”

“…Individual fibrils are 4 nm in diameter and possess a 9-nm periodicity along their length (31). Polyacrylamide gel electrophoresis of density gradient-purified fibrils showed them to be composed of a protein with an approximate molecular mass of 55 kDa (27). The early history of the fibrillar protein has been recently reviewed (1,2).…”

Deoxyribonucleic Acid Relationship Between Spiroplasma citri and the Corn Stunt Spiroplasma

“…Saglio et al (25) and Townsend et al (32) also found a high level of relatedness among Spiroplasma citri isolates. The degree of DNA homology between the CSS and Spiroplasma citri isolates Cl89 and Cir3B is between 63 and 74% (average, 68%); in the reciprocal experiment a similar value (64 to 75% [average, 68%]) was obtained.…”