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. 2004), respectively. While P. psychrotolerans and P. oryzihabitans form the same clade in phylogenies, strains of P. psychrotolerans do form a unique sub-clade. Isolate ZC5 clustered more closely with the type strain of P. psychrotolerans LMG 21977 in the phylogenetic tree. Therefore, based on the concatenated sequences of three genes (gyrB, rpoB and rpoD), the isolate ZC5 was confirmed as P. psychrotolerans. Based on morphological, Biolog characteristics and phylogenetic analysis, the isolate was identified as P. psychrotolerans. The tobacco plants at fast growing stage were selected for pathogenicity tests. Pathogenicity tests were conducted by injecting 10 μL bacterial suspension (108cfu/mL) of ZC5 into tobacco leaves with a syringe. Sterile water was inoculated into the tobacco leaves in the same way as the control. Six plants were selected for pathogenicity tests each time and five leaves of each tobacco plant were inoculated, and the tests were repeated three times. To simulate disease conditions in the natural environment, the inoculated plants were moved outdoors. The average temperature was 32°C during the day and 20°C at night. To maintain humidity, the tobacco leaves were sprayed with water every two days. Symptoms appeared on the pathogen inoculated leaves seven days after inoculation, whereas the control treatment remained symptomless. The pathogens were reisolated from diseased leaves and identified as P. psychrotolerans based on morphological, molecular and phylogenetic analysis, which fulfilled Koch’s postulates. To our knowledge, this is the first report of tobacco bacterial leaf spot caused by P. psychrotolerans.