Mitrović, J.

‘Candidatus Phytoplasma cynodontis’ is widespread in bermudagrass and has only been found in monocotyledonous plants. Molecular studies carried out on strains collected in Italy, Serbia, and Albania enabled verification of molecular variability in the 16S ribosomal RNA (rRNA) gene. Based on restriction fragment length polymorphism and sequence analyses, the strains from Serbia were clearly differentiated from all others and assigned to a new ribosomal DNA (rDNA) subgroup designated as 16SrXIV-C. A system for amplification of fragments containing the ‘Ca. P. cynodontis’ groEL gene was developed to enable study of its variability in related strains belonging to different 16SrXIV subgroups. Despite the fact that the groEL gene exhibited a greater sequence variation than 16S rRNA, the phylogenetic tree based on groEL gene sequence analysis was highly congruent with the 16S rDNA-based tree. The groEL gene analyses supported differentiation of the Serbian strains and definition of the new subgroup 16SrXIV-C. Phylogenetic analyses of both genes confirmed distinct phylogenetic lineages for strains belonging to 16SrXIV subgroups. Furthermore, groEL is the only nonribosomal marker developed for characterization of ‘Ca. P. cynodontis’ thus far, and its application in molecular surveys should provide better insight into the relationships among these phytoplasmas and correlation between strain differentiation and their geographical distribution.

Corn reddening (CR) or maize redness is a severe disease of corn (Zea mays L.) associated with ‘Candidatus Phytoplasma solani’ or stolbur phytoplasma (16SrXII-A). In Serbia, CR is continually present at a low frequency, while two outbreaks occurred in the late 1950s and 1990s. Its etiology was molecularly determined in 2006 (1). The first severe outbreak in Bulgaria was observed in Kneja in 1992, and in 2010 typical CR symptoms (leaf reddening, premature drying, and shriveled grains) were observed from Byala Slatina to Pleven. Although the number of CR affected plants was highly variable in different fields, the disease incidence in most cases was 30 to 50%, with an estimated yield reduction of about 20%. Leaf samples from four symptomatic corn plants were collected from Kneja, northwestern Bulgaria, in mid-August 2013. Extraction of DNA was performed from the main leaf midrib tissues using the CTAB method. Separate PCRs were carried out for amplification of the phytoplasma 16S rDNA and tuf genes using the phytoplasma specific primers P1/P7 and TufAyf/r, respectively. DNA from asymptomatic corn plants and reactions without template DNA were employed as negative controls, while DNA from periwinkle tissue infected with ‘Ca. P. asteris’ was used as a positive control. Amplicons of the expected sizes (1.7 and 0.9 kbp, respectively) were produced with DNA from three out of four symptomatic corn samples, while no amplification was observed with DNA from one symptomatic corn sample (probably because samples were collected during a drought period) nor the DNA from asymptomatic plants and negative control. RFLP analyses performed on 16S rDNA and tuf gene amplicons using Tru1I and HpaII restriction enzymes, respectively, revealed the presence of ‘Ca. P. solani’ (16SrXII-A, tuf type b) in all three positive samples (3). Both amplicons of a selected representative sample 241/13 were directly sequenced by a commercial service and the obtained sequences were deposited in NCBI GenBank under the accession number KF907506 for the16S rDNA (1,684 bp) and KF907507 for the tuf gene (896 bp). The 16S rDNA sequence of phytoplasma detected in Bulgarian corn shared a complete sequence identity with ‘Ca. P. solani’ strain from Serbian corn (JQ730750.1) and >99.7% sequence identity with the reference strain STOL (AF248959), while the tuf gene nucleotide sequence shared complete sequence identity with several ‘Ca. P. solani’ (e.g., Serbian strain 284/09, FO393427), thus confirming, with both genes, the affiliation of phytoplasmas in Bulgarian corn to ‘Ca. P. solani’. This study adds new information on CR prevalence, previously reported in neighboring countries. Further studies will investigate the roles of Hyalesthes obsoletus and Reptalus panzeri, both polyphagous Cixiidae reported as CR vectors (2,4) in disease transmission in Bulgaria. References: (1) B. Duduk and A. Bertaccini. Plant Dis. 90:1313, 2006. (2) J. Jović et al. Eur. J. Plant Pathol. 118:85, 2007. (3) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (4) N. Mori et al. Bull. Insectol. 66:245, 2013.