First Report of Stolbur Phytoplasma Associated with Maize Redness Disease of Maize in Bosnia and Herzegovina.

Maize redness (MR), caused by stolbur phytoplasma (16SrXII-A, 'Candidatus phytoplasma solani') and vectored by the cixiid planthopper Reptalus panzeri (Löw), is a severe and emerging disease of maize in southeastern Europe (2). Symptoms of MR include midrib, leaf, and stalk reddening, followed by desiccation of the entire plant, abnormal ear development, and incomplete kernel set. MR may cause significant economic losses (2). During 2010, 2011, and 2012, the presence of MR-like symptoms on maize accompanied by significant yield losses were frequently observed in maize fields in the Semberija region of northeastern Bosnia and Herzegovina. From mid-June to early July, potential vectors were collected using mouth-aspirators from maize plants in fields at three locations in the Semberija region where MR-like symptoms were previously observed. At the end of July, symptomatic maize plants were collected from six fields in the same region for phytoplasma identification. In addition, we sampled asymptomatic johnsongrass (Sorghum halepense L.), bindweed (Convolvulus arvensis L.), and volunteer wheat (Triticum aestivum L.) in areas adjacent to maize fields with MR-like symptoms, as potential phytoplasma reservoirs (2,3). A total of 49 plants (38 maize, 6 johnsongrass, 3 bindweed, and 2 wheat) and 43 R. panzeri were tested for the presence of stolbur phytoplasma. Leaves of four maize seedlings, grown in insect-proof greenhouse conditions, were used as controls. Total DNA was extracted from roots of each plant and R. panzeri using the CTAB methods (2). Initial phytoplasma detection was conducted on 16S rRNA gene using nested PCR assay with phytoplasma universal primers P1/P7 and F2n/R2 (4). Subsequently, all phytoplasma positive samples were retested employing stolbur-specific Stol11 protocol with the f2r/f3r2 primer set (1). Molecular characterization of identified phytoplasmas was performed by PCR-RFLP analysis of the tuf gene (3) and by sequence analyses of the 16S rRNA nested PCR products (GenBank Accession No. KC852868). All samples that tested positive on 16S rRNA gene using phytoplasma generic primers gave positive reaction in assays with stolbur-specific primers. Stolbur phytoplasma was identified in 36 of 49 plant samples (34 of 38 symptomatic maize plants and in 2 of 6 johnsongrass) and in 2 of 43 R. panzeri individuals. None of the control plants, bindweed, or wheat samples were positive for the presence of any phytoplasma. Tuf gene RFLP analyses enabled affiliation of all isolates to the stolbur type tuf-b. Comparison of the 16S rRNA sequence using BLAST analyses further confirmed identification of the phytoplasmas as being 'Candidatus phytoplasma solani.' The obtained sequence showed 100% identity with 'Candidatus phytoplasma solani' from corn in Serbia (JQ730750). These data clearly demonstrated association of stolbur phytoplasma with MR symptoms on maize in Semberija, which represents the first report of the MR disease and stolbur phytoplasma in maize, R. panzeri, and johnsongrass in Bosnia and Herzegovina. In the Semberija region, maize-wheat crop rotation is a traditional practice, which is a key factor for MR occurrence and persistence (2). References: (1) D. Clair et al. Vitis 42:151, 2003. (2) J. Jović et al. Phytopathology 99:1053, 2009. (3) M. Langer and M. Maixner. Vitis 43, 191, 2004. (4) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.