Detection, Isolation and Characterization of Two Egyptian Isolates of Spiroplasma Citri from Citrus Sinensis Trees

Field: Agricultural and Biological Sciences (Life Sciences)

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.