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Biological and Molecular Characterizations for Identification of a Phytoplasma Associated With Lemon Witches’-Broom in Egypt

Biological and Molecular Characterizations for Identification of a Phytoplasma Associated With Lemon Witches’-Broom in Egypt

Rasha M. Mahrous1,4, Ahmed K. El-Attar2, Ahlam A. AlWatban1, Sohair I. EL-Afifi3,
Nagwa M. Aref1,3

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 and classified the isolated WB
phytoplasma.
Method: Phytoplasma was isolated from lime trees exhibited Witches' broom Symptoms in ALQalyubia
governorate, Egypt by grafting on indicator (Vollka marina ) plants. The Phytoplasma was
transmitted from naturally infected Lemon (Citrus limon) to healthy periwinkle (Catharanthus roseus)
by dodder (Cuscuta reflexa Roxb) and Lemon (Vollka marina) plants by eye bud grafting.
Cytopathological detection referred to Diene's stain was used to differentiate the phloem tissues of leaf
petiole sections from infected trees. The phytoplasma was detected in the sieve tubes and parenchyma
cells of leaf midribs by Transmission Electron Microscopy (TEM). Phytoplasma was molecularly
detected in symptomatic samples using the specific primers of their 16S-23S rRNA gene by PCR.
Results: The Phytoplasma was isolated on indicator (Vollka marina) plants. It was transmitted on
Lemon (Citrus limon) by grafting and on periwinkle by dodder. It was detected in the sieve tubes and
parenchyma cells of leaf midribs tissues using Diene's stain and TEM. Infection of Lemon trees with
Phytoplasma associated witches' Broom was detected by PCR amplification of the intergenic spacer
region (SR) using a witches' broom-specific PCR primers (SR1 and SR2). Molecular characterization
of the isolated Phytoplasma 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) with accession number
“KJ948653”. Phylogenetic analysis showed a high similarity (99%) with Oman isolate.
Conclusion: 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.
 

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Journal of Virological Sciences

July

Vol. 8, Pages 1-27

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