First report of a 16SrII-D phytoplasma associated with Albizia lebbeck witches' broom disease in Iran

The lebbek tree, Albizia lebbeck, family Fabaceae is a deciduous, perennial tree mainly distributed in tropical and subtropical areas of the world. Lebbek is widely used as a shade tree on the margin of agricultural fields and urban parks in the south of Iran.

In the spring of 2018, two A. lebbeck trees showing shoot proliferation, short internodes, witches' broom and twig dieback symptoms (Figs. 1-2) were observed in Jiroft, Kerman province, Iran. Total genomic DNA was extracted from leaf midribs of the symptomatic and two symptomless lebbek trees according to a CTAB-based method (Zhang et al., 1998). The DNA was used for nested PCR testing with P1/P7 (Schneider et al., 1995) followed by R16F2n/R16R2 universal primer pairs (Gundersen & Lee, 1996) for phytoplasma detection as previously described (Azadvar et al., 2011). The amplified PCR products were directly sequenced and the nucleotide sequences obtained were analysed by BLAST and iPhyClassifier (Zhao et al., 2008) for phytoplasma identification and group/subgroup classification, and using the MEGA 6.0 programme (Tamura et al., 2013) for phylogenetic analysis.

Results indicated that 1.25 kb nested PCR amplicons corresponding to the phytoplasma 16S rRNA gene were amplified from each symptomatic but not from the symptomless lebbek trees. BLAST analyses of the consensus sequences showed that the phytoplasma associated with lebbek witches' broom disease in Iran (GenBank Accession No. MN121115) was identical to that of the 16SrII phytoplasma 'Candidatus Phytoplasma aurantifolia' (MH547068).

Virtual restriction fragment length polymorphism (RFLP) analyses of the 16S rDNA F2nR2 sequences by AluI, BamHI, BfaI, BstUI (ThaI), DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, Sau3AI (MboI), MseI, RsaI, I and TaqI restriction enzymes using iPhyClassifier (Zhao et al., 2009) confirmed that the RFLP profile of the phytoplasma associated with lebbek witches' broom disease in Iran was identical (similarity coefficient 1.00) to that of the 16Sr group II, subgroup D (Y10097). Moreover, the phylogenetic tree (Fig 3) constructed based on the partial 16S rDNA sequences confirmed that the phytoplasma associated with A. lebbeck witches' broom disease is clustered with phytoplasmas belonging to the 16SrII group, including the reference strain of subgroup D.

To our knowledge, this is the first report of A. lebbeck witches' broom disease and its association with a 16SrII-D phytoplasma in Iran. Phytoplasma-infected A. lebbeck trees may pose an epidemiological threat to other crops or nearby plant species. It will be important to identify the natural hosts of 16SrII-D phytoplasma and to determine the insect vectors of the phytoplasma.