Detection, Isolation and Characterization of Two Egyptian Isolates of Spiroplasma Citri from Citrus Sinensis Trees
Abdou Omar, Ayman Faisal (2006) Detection, Isolation and Characterization of Two Egyptian Isolates of Spiroplasma Citri from Citrus Sinensis Trees. PhD thesis, Universiti Putra Malaysia.
Citrus stubborn disease is considered one of the most destructive diseases of citrus in the Mediterranean. It caused by Spiroplasma citri (Mollicutes, Order Entomoplasmatales, Family Spiroplasmataceae). Preliminary detection of S. citri of infected plants from Kafr El-Sheikh and El Qualubia governorates, Egypt was carried out using Dienes’ stain of stem sections and electron microscope of ultrathin sections of leaf midribs. Spiroplasma citri was isolated from common orange (Citrus sinensis) trees showing typical symptoms of citrus stubborn disease and previously detected by Dienes’ stain and electron microscope, one each from Kafr El-Sheikh and El-Qualubia. They were referred to as Fewa and Qualubia isolates, respectively. The organisms were isolated from young leaves. Fried-egg shape colonies were obtained when S. citri was subcultured on C-3G solid medium. S. citri was confirmed using darkfield microscope, electron microscope and ELISA from S. citri cultures. Small helical filamentous and small round bodies were observed by dark field microscope, whereas electron microscope inspection detected helical structure of the filamentous organism and vesicular blebs on the filaments. The isolated organisms were confirmed by a positive reaction in ELISA test. Differences between the two S. citri isolates were detected by crossed immunoelectrophoresis (CIE) with intermediate gel and polyacrylamide gel electrophoresis. In the homologous reactions of Fewa isolate, eleven precipitin peaks were detected using CIE. Identical homologous reaction of Qualubia isolate was produced. One antigen (a) was specific for the Fewa isolate and one antigen (b) was specific for the Qualubia isolate when CIE with intermediate gel was used. One-dimensional electrophoresis analysis demonstrates very similar patterns of protein with two different minor protein bands between the two isolates of S. citri. Two Egyptian isolates of S. citri (Fewa and Qualubia) were studied based on nucleotide sequences of their spiralin and16S rDNA genes and 16S/23S rDNA intergenic spacer region. The 16S rDNA of the two isolates comprised 1529 nucleotides with a similarity between them at 99.80% where only three nucleotide differences were observed. Both have more than 99.67% similarity to three published strains of S. citri, the R8A2HP, Car4 and Car6. Their 16S/23S rDNA intergenic spacer region has 305 nucleotides, with a similarity of about 99.76 %, where only one nucleotide difference was observed. The 16S/23S nucleotide sequence of Fewa isolate was identical to that of strains 169, Maroc and ATCC 27556. Five nucleotide differences were observed in spiralin gene sequences of the two isolates, which have 99.30 % similarity. The two spiralins comprised 241 amino acids with only three amino acids differences were observed between them, and their amino acids similarity was about 98.75%. Our findings indicated that both Fewa and Qualubia isolates have high similarity in the nucleotide sequences of their 16S rDNA, 16S/23S rDNA intergenic spacer region and the nucleotides and amino acids of spiralin gene. However, they were not identical and could be differentiated by using restriction enzyme BfaI that digested at the recognition site CTAG within their spiralin gene sequence. Therefore, based on the above studies the two Egyptian isolates of S. citri (Fewa and Qualubia) are not identical.
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