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Phytoplasma associated with Hibiscus Witches’-Broom in Egypt

Phytoplasma is considered as one of the most important plant pathogens reducing the productivity of several economic crops including hibiscus. Sensitive detection and characterization as well as the diagnosis of phytoplasmas are of paramount importance for effective prevention strategies, particularly because phytoplasmas may have a very long latency period. Molecular diagnosis confirmed the infection of hibiscus plants with Phytoplasma Associated with Witches’-Broom based on the symptoms associated with phytoplasma diseases. Symptoms of hibiscus witches' broom disease caused by phytoplasma were observed on hibiscus ornamental plant growing in El-Giza, Alexandria, Qlubia, El-Fayom, and El-Mansoura governorates. The infected plants were characterized with the following symptoms, excessive axillary branching, leaf yellowing, short internodes, proliferation of shoots, abnormally small leaves, deformed flowers, and in some cases, premature flower dropping. Highly infected plants may die due to severe infection. The previously stained hand sections of hibiscus leaves midrib were examined using light microscope. Shows that,phloem was colored bright turquoise blue indicating the presence of phytoplasma in midrib sections prepared from infected hibiscus sample. The phloem of sections prepared from healthy samples remained unstained. Samples showed positive results for the nested PCR, using the primer pairs R16F2n/R16R2, were analyzed for hibiscus witches’ broom. The direct PCR for detection of the hibiscus witches’ broom was performed using the specific primer pair SR1/SR2 and yielded a strong band of approximately 325 bp for DNA extracted from hibiscus samples showed witches'-broom symptoms. PCR amplifications were obtained by direct, as well as nested PCR for the DNA extracted from Hibiscus tissue. The nested PCR using the universal phytoplasma- specific primers P1/P7 and R16F2/R16R2 gave amplification of the expected fragment at1.2 kb, while the direct PCR using the witches' broom-specific PCR primers (SR1/SR2) showed a clear band at ~ 325 bp corresponding to the 5’-end of the 23S rDNA and the partial 16S rRNA gene. The first round of the nested PCR using the P1/P7 primers didn’t show a band due to the low phytoplasmatic DNA concentrations as compared to plant DNA. That agrees with the fact that, the detection of phytoplasmatic DNA was achieved only with a nested-PCR. In the second PCR reaction, conditions are optimized and the sensitivity of the technique is increased facilitating visualization of the amplicons that refers to the use of the PCR product of the first Round PCR as a DNA template for the second one. DNA amplified from template DNA isolated from any of the healthy, non-symptomatic, plant samples were found to be negative for phytoplasma presence in both direct and nested PCR. Those PCR results clearly demonstrated the natural infection of hibiscus with phytoplasma associated with Witches’-Broom. Phylogenetic analysis and homology with the sequence of the 16S ribosomal RNA (rRNA) gene, the 16-23S rRNA spacer region, and the 5`-end of the 23S rRNA gene identified the phytoplasma as belonging to the 16Sr II group (97-99% homology). Sequence obtained from the1246 bp PCR product associated with infected Hibiscus rosa-sinensis was submitted to BLAST analysis which showed a 100 % similarity with reference strain of hibiscus witches' broom from Brazil, belonging to 16SrII-group. DNA sequencing and phylogenetic analysis indicated that this phytoplasma clustered in the 16SrII group. A 1,246 bp sequence of the 16S rRNA gene from the hibiscus witches’ broom phytoplasma showed 100% homology with the 16S rRNA gene, hibiscus witches' broom in Brazil (HQ230579-SiWB-B) and peanut witches'-broom (EU099547-YN02). Nucleotide sequence determined in this study was submitted to the gene bank and accessioned by Gen Bank accession number “KF716175” as the first report of phytoplasma infection affecting hibiscus witches' broom in Egypt.