Biological and molecular characterization of different egyptian isolates of spiroplasma citri

Citrus Stubborn Disease (CSD) is a se serious disease of citrus caused by Spiroplasma citri, which is a helical motile, wallless and cultivable microorganism belongs to class mollicutes. Spiroplasma citri was isolated from samples collected from lime trees representing symptoms of CSD from 8 Egyptian lime, lemon and volkamere trees. The presence of S. citri was checked by examination of cross sections stained with Dienes’ stain as blue stained phloem, indicated the presence of Spiroplasma in infected phloem tissues. Culturing on C3-G specific liquid and solid media was also achieved. The isolated Spiroplasma was identified as S.citri by DAS-ELISA using specific polyclonal antibodies. By transmission electron microscopy the identity and morphology of S.citri was confirmed. S.citri units were wall-less, spiral filamentous and/or branched long helices in liquid cultures and rounded segments forming the Fried-egg shape colonies on solid culture. Examination of ultrathin sections prepared in infected plants revealed the presence of elementary helices inside the phloem sieve elements. Necrotizaton and clumping of the cytoplasm of phloem parenchyma in infected volkamer lemon leaf samples was obvious. Damaging of nucleoli in companion cells was also an effect of S.citri infection. A rep-PCR DNA fingerprinting study with BOX-A1R oligonucleotide primer matching REP sequences S.citri showed that among 8 S.citri isolates, only three isolates (EL Lux, EL1 and EL2) were shown to be in one group. Real-time PCR amplification from 8 S.citri liquid cultures showed differential specificity using Php-orf1 primer pair. Total DNA was isolated successfully from five fresh samples and used as a template for conventional PCR using the Spiralin, P58 and P89 primers pairs. The results of BLASTn analysis confirmed that the PCR product of isolate (EL K) represented the Spiralin protein coding gen and the results of the nucleotide sequence were uploaded in the GenBank with accession number “MN599050” as Spiroplasma citri EL K isolate. Furthermore, using DNAMAN sequence analysis software, the generated phylogenetic tree provided an indication that the ELK isolate was evolutionarily more related Egyptian isolates; Few (Acc. No. AM157770.1) and Qualubia isolate (Acc. No. AM157771.1) via 99.4%.