First Report of Bacterial Rot Caused by Pantoea endophytica on Tobacco in Liuyang, China

Tobacco, Nicotiana tabacum L., is produced largely in China (~1/3 of the global market). In the monsoon summer of 2020, tobacco plant petioles, where axillary buds were removed, became black-rotten, and thick ooze appeared, when squeezed. Lesions encompassed more than half of petiole circumference. Ten tobacco fields (100 plant/field) were investigated in Liuyang, China and 5% disease severity founded in each infected field (Fig. 1A, B, C). Six infected stalks leave of different tobacco were sampled from severe field in Liuyang (N28°21', E113°52') and were surface sterilized (1% sodium hypochlorite for 3 min.), rinsed thrice in sterile distilled water, grounded, and streaked on Luria Bertani agar (LBA). After 24 hours at 28oC, circular and convex colonies appeared. Hundred colony from ten plates were picked, amplified, and sequenced with the primer 16S-27F/16S-1492R by colony PCR (Lane et al. 1991). 16S rRNA sequence from 100 colony were assembled and fell into two sequences, either similar to Leclercia sp. (86%), or Pantoea sp. (14%). Identification and homology search was done by BLASTn analysis against NCBI and the EzBio Cloud database (Yoon et al. 2017). The Pantoea isolate HN-23 (1,408 bp, MW405831) and the other 16S sequence of 13 Pantoea showed 99.57% identity to the type strain P. endophyitca 596T (PJRT0100022) based on the EzBio Cloud database to identify novel bacteria. Colonies of HN-23 were smooth, translucent, convex with entire margin on LBA, and 1mm and 3mm (diameter), white to yellow, after 24h and 48h (Fig.1 H, I), respectively but white (Fig.1 J, K) on Nutrient Agar (NA). Phenotype of HN-23 (S-1) was performed using API 20E and API ZYM system (bioMerieux, France) and found identical to P. endophytica 596T (Gao et al. 2019). Draft genome of HN-23 (size 4.96Mbp, total Scaffold 79, Scaf N50 218,098bp and Scaf N90 61,041bp) was studied by Illumina sequencing (JAFLWX000000000) and was found to have 98.24% nucleotide identity with the genome of P. endophytica type strain 596. Average nucleotide identity (ANI) values were calculated using Ortho ANIu algorithm (Yoon et al 2017a). HN-23 had 83.89% and 83.65% ANI with P. rodasii LMG26273T and P. dispersa CCUG25232T, respectively (S-2). Six tobacco seedlings (cultivar K326, 30cm height plants grown at greenhouse at 28℃ and 70-80% humidity) were injected by 20μl of culture (109 CFU/ml) of HN-23 and three with dominant species Leclercia sp. HN-7, and reisolated from infected tissues. Pathogenic tissue extract and sterile water were also used as positive and negative control, respectively and experiments were performed in triplicate. After 20h, symptoms of water-soaked decay appeared in the injected leaf axils (Fig. 1D). After 2 days, a severe rot is developed (Fig.1 E). Though, the controls were symptomless (Fig.1 F, G). The bacterium was then isolated from the rotten tissues and identity was confirmed by 16S rDNA sequencing, thus fulfilling Koch's postulates. This species was also reported as endophytes to be isolated from root, stem and leaf of maize planted in diverse parts of China and identified as P. endophytica. To our knowledge, this is the first report of P. endophytica as a plant pathogen, which was firstly isolated from Tobacco planted in southern China.