Abstract

Background
Phytoplasma Associated with Witches' broom disease (WB) is a very serious disease of citrus with a major limiting factor for lime production. Symptoms of Witches' broom were observed on limes in AL-Qalyubia governorate, Egypt. This study identified, characterized and classified the isolated Egyptian strain of the WB causing phytoplasma.
Methods
The Phytoplasma was biologically characterized by its transmission from symptomatic Lemon (Citrus limon) to periwinkle Catharanthusroseus and to healthy Lemon (Vollka marina) plants by dodder (Cuscuta reflexa Roxb) and grafting. Cytopathological detection referred to Diene's stain was used to differentiate the phloem tissues of leaf sections from infected trees. Transmission Electron Microscopy (TEM) indicated the presence of phytoplasmas in the sieve tubes and parenchyma cells of leaf midribs. Phytoplasma was molecularly detected in symptomatic samples by the specific amplification of their 16S-23S rRNA gene.
Results
Polymerase chain reaction (PCR), utilizing the universal phytoplasma-specific primers (P1/P7 followed by R16F2n/R16R2) consistently amplified a product of expected lengths when DNA extracted from symptomatic samples. Infection with the phytoplasma associated witches' Broom was confirmed through the direct PCR amplification of the intergenic spacer region (SR) using a witches' broom-specific PCR primers (SR1 and SR2). Molecular characterization of the isolated phytoplasma strain was performed through the successful cloning and sequencing of the PCR fragment that amplified from 16S rRNA gene. The nucleotide sequences were published to the GenBank as Lemon Witches' broom-Egyptian strain (LWB-Eg strain). Phylogenetic analysis showed a high similarity (99%) with Oman isolate.
Conclusion
The phytoplasma strain of LWB-Eg Candidatus Phytoplasma that reported as a natural causal agent from Lime WB strain in Egypt was classified as 16SrII subgroup.

Key words: Witches' broom, Phytoplasma, Electron microscopy, PCR, spacer region (SR), universal primers.