First report of citrus huanglongbing disease on orange in Iran

Huanglongbing (HLB), also known as greening, is a destructive disease of citrus that may limit production of this crop in many parts of Asia, Africa and South America. HLB is caused by a non-culturable phloem-limited bacterium of the genus ‘Candidatus Liberibacter’. Three Candidatus species of Liberibacter, namely, ‘Ca. L. asiaticus’, ‘Ca. L. africanus’ and ‘Ca. L. americanus’, have been identified (Bové, 2006). The psyllid Diaphorina citri, vector of ‘Ca. L. asiaticus’ was first detected in December 1997 in Iran in an area close to the border with Pakistan (Bové et al., 2000). Since then, high populations of D. citri have been found in citrus plantations of Hormozgan and Kerman provinces in southern Iran (Bové, 2006). Citrus trees in this region exhibit typical HLB symptoms including mottling of leaves and yellowing of shoots. In the present study leaf samples from 20 symptomatic and 20 symptomless Valencia sweet orange trees (Citrus sinensis) and over 50 psyllid samples were collected from various locations in Sistan-Baluchistan and Hormozgan provinces and total DNA was extracted using the CTAB (cetyltrimethylammonium bromide) method. DNA samples were tested for presence of ‘Ca. L. asiaticus’ by nested PCR using primer pairs F1 (5’-TGAATTCTTCGAGGTTGGTGAGC-3’)/R1(5’-GAATTCGACTTAATCCCCACCT-3’) as the first set and F2(5’ GCGTTCATGTAGAAGTTGTG-3’) / R2(5’-CCTACAGGTGGCTGACTCAT-3) as the second set. Both primer pairs were designed based on the published beta-operon of ribosomal protein encoding sequence of ‘Ca. L. asiaticus’ (Villechanoux et al. 1993). Nested PCR resulted in amplification of a 400 bp product from five out of 20 leaf samples and 26 out of 50 psyllid samples. No amplicons were obtained from symptomless sweet orange trees and psyllid samples reared on healthy sweet orange seedlings. The amplified fragment from a psyllid sample was cloned and sequenced (GenBank Accession No. FJ172759). BLAST search showed 100% identity with corresponding sequences of ‘Ca. L. asiaticus’ (M9439, AY34200 and EU078703). This is the first report on the occurrence of HLB disease in Iran. The disease appears to be widely distributed in citrus growing regions of southern Iran and may be a component of citrus decline in this area.