First Report of Bacterial Leaf Spot on Tobacco Caused by Pseudomonas psychrotolerans in China

Tobacco (Nicotiana tabacum) is an economically important crop, and its productivity is challenged due to pathogen infection. In 2020 and 2021, a previously uncharacterized disease was observed on field grown tobacco (Variety NC102) in Zhucheng City, Shandong Province, China (119°7'14" E, 36°0'58" N), where tobacco has been grown for decades. The disease can be found throughout the growth period of tobacco and mainly occurred from fast growing period (about 13-16 leaves) to leaf maturity stage. In severely diseased areas, the incidence rate can reach 100%. The symptoms first began as chlorotic water stain like small spots, then the spots merged into larger irregular necrotic maculae around the chlorotic halos. Small pieces of symptomatic leaves from 10 different infected plants were collected for pathogen isolation. The small pieces of discolored leaves were surface sterilized with 75% ethanol for 40s and washed with sterile water for three times. The sterilized leaves were ground with a glass rod with 1mL sterile water, and 100 μL suspensions were spread on nutrient agar medium then incubated at 28oC for 48 hours. Yellow round colonies with undulating edges were showed up on nutrient agar medium 48 hours later. Three isolates were randomly picked up from each of the 10 plates for subsequent analysis. After purification and culture on nutrient agar plate, the 16S rRNA gene of the 30 isolates were amplified with primers 27F and 1492R and the amplicons were sequenced and analyzed by sequence alignment. The sequence alignment results showed that the 16S rRNA nucleotide identity of the 30 isolates were 100%. One typical isolate named ZC5 was selected for subsequent analysis, and the resulting 16S rRNA sequence was deposited at GenBank, NCBI under accession OK092624. The 16S rRNA sequence identity with those of P. psychrotolerans strain K3-2 (KY882083) and M3-1 (KY882120) were 100%, respectively. The phenotypic analysis by Biolog Gen Ⅲ indicated that the bacterial isolate (ZC5) showed highest similarity (98.3%) with strain Pseudomonas oryzihabitans. P. oryzihabitans and P. psychrotolerans have a high degree of homology in the phylogenetic relationship based on the phylogenetic analysis of three concatenated sequences of gyrB, rpoB and rpoD genes (Mulet et al. 2010). The gyrB (ON462356), rpoB (ON462355), rpoD (ON462357) gene of isolate ZC5were also amplified and sequenced by using primers gyrB-For/gyrB-Rev, rpoB-For/rpoB-Rev and rpoD-For/rpoD-Rev (Hauser et al. 2