Authors : D. Caplik, M. Kusek, S. Kara, A. Seyrek, Y. Celik
DOI : 10.1002/ndr2.12070
Volume : 45
Issue : 1
Year : 2022
Page No : 1
Maize is a significant crop widely cultivated in Turkey. In 2020, maize production reached 6.5 Mt from approximately 700,000 hectares. Dickeya zeae (syn. Erwinia chrysanthemi pv. zeae) is a devastating maize pathogen, causing bacterial stalk rot of maize and significant yield losses (Samson et al., 2005). Dickeya zeae also causes soft rot in many plant species, including essential crops such as potato, rice and banana (Toth et al., 2011; Pu et al., 2012; Zhang et al., 2014). In the summer of 2021, a stalk rot disease symptom, i.e. maceration of the stalk and brown discolouration of infected vein tissues with soft rot, emitting an unpleasant odour, was observed on maize in Bismil county in Diyarbakir province, Turkey (Figures 1-3). Four samples, two samples from two diseased stems, were collected and placed in 70% ethanol for one minute. The plant samples were then immersed in 1% NaOCl suspension for 30 seconds and rinsed in sterile water three times. Following surface sterilisation, samples were placed into sterile tubes containing 1 ml sterile water. The samples were ground separately and 50 μl of the suspension plated on nutrient agar and King's B media. Plates were incubated for two to three days at 27°C and two representative colonies of the bacterial isolate were selected according to their morphology. The colonies on King's B medium were white in colour, slimy, and shiny, while the colonies were grey, non-mucoid and round on nutrient agar medium. Pure cultures grown on King's B medium, were selected after twenty-four hours’ growth for a pathogenicity assay. Eight three-week-old maize seedlings were inoculated with the pure pathogen culture using a sterile toothpick and incubated in the greenhouse at 30°C, 90% humidity with a 12-hr photoperiod. Plants were inoculated with sterile water as a negative control. The soft stalk rot symptom was observed on maize seedlings ten days’ post-inoculation (Figure 4). Re-isolated bacteria were isolated from the inoculated tissue and were stored at −80°C in 30% glycerol for identification and long-term storage. Two isolates (DBM1 & DBM2) were selected for classic and molecular identification assays. Both isolates grew at 37°C, were gram-negative, and positive based on a pectolytic activity assay. For molecular diagnosis, a 420 bp pel gene region specific to the genus Dickeya was amplified using PCR with the ADE primer set (Nassar et al., 1996). In addition, the dnaxF / dnaxR primer pair (Slawiak et al., 2009) was used to identify Dickeya species. Sequencing and BLAST analysis of the resulting PCR products showed that the dnax gene of the two isolates (GenBank Accession Nos. OL873319-OL873320) had 100% identity with the D. zeae strain DZ15SB06 isolated from sweetcorn in Thailand ((KY817915.1). The sequences of the dnax gene region were used to develop a phylogenetic tree with MEGA software (version 11). The sequences of dnax gene region of maize isolates DBM1 and DBM 2 were clustered in the same clade with D. zeae strains. To our knowledge, this is the first record of Dickeya zeae causing bacterial stalk rot disease on maize in Turkey.