Study on Genetic diversity of Phytoplasmas associated with Lime Witches-broom in Iran using PCR-RFLP and analysis of ribosomal protein and 16S rDNA genes

Lime witchs' Broom (LWB) is one of the debilitating diseases of the lime trees in southern Iran and it is caused by Candidatus Phytoplasma aurantifolia. The symptoms included yellowing, little leaves, short internodes and shoot proliferation. In this project, we have done a comparative analysis on this phytoplasma isolated from different areas and worked on its detection in weeds and other citrus plants including oranges and mandarins. A survey was carried out in 2007-2008 by collecting 125 samples from the following provinces in Iran: Hormozgan, Sistan-Baluchestan and Kerman. A PCR assay was carried out using P1/P7 as outside primers and R16F2/R16R2 working as internal (nested) primers. Nested PCR gave a product of 1250 bp which was cut with the restriction enzymes HaeIII, EcoRI and MspI and analyzed by agarose gel electrophoresis. The RFLP profiles from selected isolates were similarindicating lack of heterogeneity among the isolates collected from various provinces. Since16S_23S rRNA spacer region gives a more precise genetic variability in phytoplasmas; we targeted this region by using the primer pair fP3/M23sr which gives a PCR product of 340 bp. We used isolates LWB12, LWB25, LWB44, LWB97, LWB119 which were collected from distant geographical regions. In further analysis, we designed the primer pair rTuf1/fTuf1 from the elongation factor gene which is less conserved as compared to 16S rRNA gene. However, since the product from this pair was weak, we looked to replace it with the pair pTBB14F/pTBB14R on the DNA polymerase beta III chain gene which gave a product of 650 bp. In further analysis the following crop samples growing within lime orchards were tested for phytoplasmas: Medicago Sativa, Trifolium pratense, Sesamum indicum, Alhagi camelorum, Solanum nigrum, Plantago major and Convolvulus arvensis. Only a phytoplasma in Sesamum indicum with phyllody symptoms was detected. The 16S rRNA sequence of the phytoplasma in sesame showed 99% similarity to that of the lime isolates of LWBP. It is worth noting that sesame phyllody is an endemic pathogen in Iran.