Rhodococcus fascians is a bacterium that causes growth abnormalities such as leafy galls, fasciation, and shoot proliferation in many plants, including ornamental plants. In February 2020, the Animal and Plant Quarantine Agency of South Korea detected 492,000 contaminated lily bulbs using an in-house PCR test based on the R. fascians fasD gene, and subsequently 1.3 million imported bulbs were destroyed. Because no pathogen isolation was associated with this diagnosis, there has been great cultivator demanded for bacterial isolation evidence of lily bulb infection with pathogenic R. fascians. To isolate the causal bacterium of the PCR tests, we sampled leaf, stem, and bulb tissues from 130 lilies with growth abnormality symptoms, collected from 24 South Korean mass production lily farms from June to August 2020. Supernatants of the homogenized samples were spread on mD2 medium (Kado and Heskett 1970) and incubated at 28°C for 10 days. Yellow to orange colonies were isolated into pure culture on mD2. Total DNA was extracted from cultures grown in yeast extract broth (YEB) at 28°C for 24 hours with Wizard DNA prep kit (Promega, Madison, WI, USA). PCR was performed to test for pathogenicity genes fas (A,D, and R) and att (A and R) (Putnam and Miller 2007). Colonies that produced at least one amplicon from these pathogenicity genes were analyzed by partial 16s rRNA gene sequencing to determine the corresponding species. Three strains that were isolated from the bulbs of fasciated lilies from Wanju (35°56´22.1˝N; 127°08´52.0˝E), Gwacheon (37°26´51.6˝N; 127°00´11.8˝E), and Yeongwol (37°18´45.8˝N; 128°11´05.6˝E), or W1, G3, and Y5 strains, yielded PCR products of the expected size for fas and att genes with the primer sets published in Serdani et al. (2013) and developed in this study (attAF: 5'–CCCGGCTACACGCATTCGC-3', attAR: 5'-CGAACGCGGTGTGCAGGT-3' and attRF: 5'-AGTGTCCCGTCGGCGAG-3', attRR: 5'-CGCGGCAGATCGAAGTCCT-3'). Sequences of the three strains were deposited in Genbank for fasA (accession MW122940-942), fasD (G3:MW122935 and 936), and fasR (MW122937-939); all shared 98.3 - 100% nucleotide identity to corresponding sequences from phytopathogenic R. fascians A25f (CP049745.1 Protein_ID fasA:QII09280.1, fasD:QII09282.1, and fasR:QII09277.1). The attA and attR products were only present in G3 (attA: MW122943 and attR: MW122944) and resulted in 100% identity to those of A25f (CP049745.1 Protein_ID attA:QII09269.1, attR:QII09267.1). Partial 16s rRNA gene sequences were obtained (MW064131-133) and clustered with phytopathogenic R. fascians strains D188, A21d2, and A25f. Thus we concluded that strains (W1, G3, and Y5) corresponded to R. fascians. To test the pathogenicity of these three strains, 10 seeds of garden peas for each strain were inoculated at 108 CFU/ml according to Nikolaeva et al. (2009), and the length of the main stem of each seedling was calculated 22 days post-inoculation. Seedlings inoculated with G3 and Y5 resulted in a stunted phenotype with up to 40% height reduction (p ≤ 0.001) compared to non-inoculated seedlings. As for the seedlings inoculated with W1, they exhibited as much as 15% height reduction (p ≤ 0.001). Colonies were recovered from the inoculated seedlings, identity was confirmed through colony PCR for fas and att genes. To our knowledge, this is the first report of phytopathogenic R. fascians in lilies cultivated in South Korea.
Nasal carriage of Staphylococcus aureus among a healthy suburban population: genotypic diversity and frequency of pathogenicity genes