Slimy potato (Solanum tuberosum) tubers with a foul-smelling odor were obtained from a potato field during harvest in Huize county (25.94°N; 103.40°E), Yunnan province, China in August 2021. The incidence of symptomatic potato tubers was approximately 5% while no symptoms were observed on potato stems. To isolate the causal agent, potato tubers were surface-disinfected and infected tissues were mashed in a 15 ml sterile centrifuge tube with 2 ml sterile distilled water. After a series of dilutions, a volume of 200 μl bacterial suspension from each dilution was spread on nutrient agar (NA) medium and incubated at 28 °C for 48 h. Single colonies randomly selected from NA plates were then picked and separately subcultured in nutrient broth (NB) medium. Pure cultures were acquired by successive streaking on NA medium. The colonies with irregular shapes were white and opaque (Fig. S1A). Among all the isolates, strain Q1 exhibited pectinolytic activity on inoculated potato tubers using a pin prick method with an inoculum concentration of 108 CFU/ml while no symptoms appeared for the control group using sterile water (Fig. S1B). However, no obvious pits were observed for strain Q1 when tested on crystal violet pectin (CVP) medium. To further identify the strain Q1, total DNA was extracted using the TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit. PCR amplification of 16S rDNA of strain Q1 was performed using the universal PCR primer pair 27F/1492R and followed by Sanger sequencing. The BLASTn analysis of the sequence (NCBI accession number ON631256) based on NCBI rRNA/ITS databases revealed 100% query coverage and 99.78% identity to the 16S rDNA sequences of type strain Pseudomonas tolaasii ATCC 33618 and Pseudomonas palleroniana CFBP 4389, respectively. Whole-genome sequencing of strain Q1 was then performed using the Illumina and Nanopore sequencing platform. A single contig (NCBI accession number CP092411) with a length of approx. 6.2 MB was obtained by de novo assembly using Raven (v1.5.1) (Vaser and Šikić 2021) and Pilon (v1.24) (Walker et al. 2014). The completeness and redundancy of this assembly was evaluated by BUSCO (v5.2.2) (Manni et al. 2021), and a score of 100% completeness indicated a high quality of the assembly. The genome sequence of strain Q1 was uploaded to the Type Strain Genome Server (TYGS) for a whole genome-based taxonomic analysis (Meier-Kolthoff and Göker 2019). The distance-based phylogeny revealed that strain Q1 is well clustered together with Pseudomonas palleroniana (Fig. S1C). Furthermore, a digital DNA-DNA hybridization (dDDH) value (86.9%) between strain Q1 and P. palleroniana was above the species boundary (≥70%), indicating that strain Q1 should be classified as P. palleroniana. In addition, P. palleroniana was re-isolated from potato tubers inoculated using a pin prick method with an inoculum concentration of 108 CFU/ml and was identified by morphological similarities and 16s rDNA sequencing described above, thus fulfilling Koch's postulates. Furthermore, aerial stem rot symptoms were observed after 3-5 days post inoculation of strain Q1 with a concentration of 108 CFU/ml on stems of young potato plants using a pin prick method (Fig. S1D). Notably, a couple of Pseudomonas spp. have been reported to cause soft rot in carrot (Godfrey and Marshall 2002) and melon (Zhang et al. 2016). To our knowledge, this is the first report of P. palleroniana causing soft rot on potato tuber in China.
Keywords: Causal Agent; Crop Type; Field crops; Pathogen detection; Prokaryotes; Subject Areas.