Molecular comparison of phytoplasmal agent of lime witches'-broom disease in Kerman, Hormozgan and Sistan-Baluchestan provinces

Lime witches′-broom (LWB) is caused by Candidatus Phytoplasma aurantifolia and is a destructive disease of lime in southern provinces of Iran. Restriction fragment length polymorphism (RFLP) and sequence analyses were used to compare phytoplasmal LWB agents in Kerman, Hormozgan and Sistan- Bluchestan Provinces. Total genomic DNA of infected samples from three provinces was extracted and a 300-bp fragment of 16S–23S rDNA spacer region (SR) was amplified by PCR using P3/P7 primer pair. Amplified products were cloned and sequenced in both directions. Sequence analysis of SR regions showed that phytoplasmal agent of LWB disease in three infected Provinces of Iran, Oman and the United Arab Emarates are Identical. Furthermore, an 1800-bp fragment of ribosomal operon consisting of the 16S rRNA gene, 16–23S SR and 5′-end of 23S rRNA gene was amplified by PCR using P1/P7 primer pair. Amplified P1/P7 fragments were subjected to restriction fragment length polymorphism (RFLP) analysis after separate digestion by Alu I, Rsa I, Taq I, Hinf I, Hpa I and Hha I restriction enzymes. Comparison of RFLP patterns indicated that the phytoplasmal agent of LWB in Kerman, Hormozgan and Sistan- Bluchestan are indistinguishable. Therefore, on the basis of sequence and PCR-RFLP analyses, phytoplasmal agent of LWB disease in Oman, the United Arab Emarates and southern provinces of Iran are identical and probably originated from a common source. To determine distribution of the disease in Kerman province, samples were collected and checked by PCR test during 2004-2005. The results showed that lime is severely infected in lime growing areas of Kerman Province including Kahnooj, Jiroft and Manoojan.