Etiology and Transmission of Cowpea Phyllody Associated Phytoplasma in East Azarbaijan Province of Iran

Phytoplasmas were first discovered in 1967 and named mycoplasma-like organisms or MLOs. They are wall-less prokaryotes of the class Mollicutes causing diseases in many economically important plant species. Phytoplasmas are transmitted by phloem-feeding leafhoppers and planthoppers. Plants infected by phytoplasmas exhibit a variety of symptoms suggesting profound disturbances to the normal plant physiology. Restriction fragment length polymorphism (RFLP) and sequence analyses of rDNA have been used for the taxonomic characterization and phylogenetic analysis of phytoplasmas. Because of the high economic losses, in particular those of cultivated crops, annuals and high-value vegetables, detection of phytoplasmas is very important. Cowpea (Vigna unguiculata) is an important vegetable crop cultivated in Iran. Progressive growth abnormalities and phyllody symptoms with high economic loss have been observed in cowpea fields of East Azarbaijan province. Causal agent of the disease is, however, unclear. This study was, therefore, carried out to identify pathogen associated with cowpea phyllody in East Azarbaijan province of Iran.
Infected cowpea plants were collected from fields in East Azarbaijan province of Iran in summer of 2016. The disease agent was transmitted by dodder to periwinkle plants under controlled greenhouse conditions. Leaf midribs from healthy control and naturally-infected hosts were used for DNA extraction. Total DNA was extracted from midrib of six symptomatic cowpeas, inoculated periwinkle plants using CTAB buffer as previously described by Zhang et al., 1998. The universal primer pair P1/P7 was employed in first round PCR to amplify an 1830 bp fragment of 16S rDNA. A 30-fold dilution of the first round PCR product was then used as template for nested PCR using primer pair R16F2n/R16R2 which amplified an internal fragment of 1245 bp from the 16S rDNA. PCR products were ligated into pTZ57R/T using InsT/AcloneTM PCR Product Cloning Kit and transformed to Escherichia coli DH5a cells. The recombinant plasmids were sequenced. The sequences were assembled using DNA baser V. 3 program. About 100 ng of DNA from R16F2n/R16R2 primed PCR product from each province was separately digested with 2.5 μL of AluI, HaeIII, HpaII, HinfI, RsaI and TaqI restriction enzymes according to the manufacturer’s instructions. Restricted DNA fragments were analyzed by electrophoresis through a 2% agarose gel. Phylogenetic tree was generated using neighbour-joining method with MEGA7 software. Acholeplasma laidlawii, a culturable mollicute that is phylogenetically related to the phytoplasmas, was designated as the out-group to root the tree. The reliability of the tree was assessed by bootstrap analysis with 1000 times replication. Further, the sequences of the 16S rRNA gene were subjected to virtual restriction fragment length polymorphism (RFLP) using iPhyClassifier software (http:// plantpathology. ba. ars. usda. gov/ cgi- bin/ resource/ iphyclassifier. cgi) to determine ribosomal group affiliation.
Cowpea plants collected from East Azarbaijan province showed disease symptoms similar to those caused by phytoplasmas. In infected cowpea plants, symptoms of little and thickened leaves, phyllody, proliferation of shoot, wrinkled and malformed leaves, stem fasciation and stunting were observed. Inoculated periwinkles showed little leaf, internode shortening, yellowing, virescence, phyllody, stem and floral proliferation and witches’ broom. Amplification of 1830 and 1245 bp fragments in PCR and nested-PCR, respectively, and Blast analysis of the fragment sequences confirmed phytoplasma infection of cowpea plant associated with phyllody symptoms. No band was found at healthy control. Consensus sequences of 1,830 bp of three isolates (Tabriz, Nazarlou, Shabestar) were submitted in the NCBI GenBank under accession numbers KC633092 to KC633094. Phylogenetic analyses and restriction fragment length polymorphism (RFLP) of 1245 bp fragment with 17 restriction endonucleases using iPhyClassifier software indicated that phytoplasma associated with cowpea phyllody belong to Clover proliferation group or 16SrVI A subgroup (similarity coefficient 1.00).
Tomato (Solanum lycopersicum), potato (Solanum tuberosum), cucumber (Cucumis sativus), cabbage (Brassica oleracea) and periwinkle (Catharanthus roseous) were reported as hosts for 16SrVI phytoplasma group in Iran. Recently, maize (Zea mays) has also been reported as a natural host of the 16SrVI group. In addition, different phytoplasma groups and subgroups were reported infecting cowpea crops from different countries over the globe. A 16SrXII-B strain of 'Ca. Phytoplasma australiense' associated with witches’ broom and small leaves of V. unguiculata var. sesquipedalis in Australia. A Vigna little leaf phytoplasma belonging to group 16SrV has also been reported from Australia. In India, different phytoplasmas associated with cowpea from 16S rDNA groups 16SrI-B, 16SrXIV-A and 16SrII-D, have been characterized. Identification of the causal agent of phyllody of cowpea in Iran should facilitate studies concerned with epidemic aspects of the disease, and could contribute to knowledge of the genetic diversity of phytoplasmas in Iran. To the best of the present study authors’ knowledge, it is the first report of the Vigna unguiculata infection with phytoplasmas related to the “Clover Proliferation Group” from East Azarbaijan province of Iran.