First Report of Providencia rettgeri Causing Soft Rot on Calla lily in China

Calla lily (Zantedeschia hybrida) is widely cultivated as an ornamental plant in China and commonly used as cut flower. Water soaked lesions on the tuber and petiole followed by collapse of entire plant along with foul smell were noticed in a 4-hectare calla lily plantation in the Yanqing District of Beijing, China during August 2022. It was revealed 20%-30% of the plants had these symptoms. Petiole (5 cm) and tuber (5 mm3) samples were collected from 10 plants randomly from fields. The samples were disinfected with 75% ethanol for 5 s, and then rinsed three times with distilled water (DW). Five small pieces for each collected petiole and tuber (about 0.25 cm2 in area) were ground in DW; the suspension was plated on Luria Bertani agar and incubated for 12 h at 28°C. Single colonies were picked and re-streaked three times to purify the bacterial culture. The colonies were creamy, short rod-shaped, gray translucent, and with shiny texture. Gram staining revealed red color, confirming it as Gram-negative bacteria (Werkman et al. 1932). For molecular analysis, DNA was directly extracted from single colony using FastPure Bacteria DNA Isolation Mini Kit (Vazyme Biotech Co., Ltd. Nanjing, China). Four gene-specific markers (16S rRNA, dnaJ, atpD, and ropB) were then amplified using their respective primer pairs (Monciardini et al. 2002). The sequences obtained from four strains, namely ZAPR22R, ZAPR22L, ZAPR22P and ZAPR22S, were submitted after editing to GenBank with accession numbers OP740771.1-OP740774.1 for 16S rRNA, OP759518.1-OP759521.1 for atpD, OP759514.1-OP759517.1 for dnaJ, and OP759510.1-OP759513.1 for ropB genes. All of these gene sequences have the highest BLAST hit to Providencia rettgeri with 94.8% to 99.8% sequence identity (expcept dnaJ, 88.3%) and 96% to 100% of coverage. Phylogenetic tree constructed based on sequences of all four genes using RAxML v8.2.12 (github.com/stamatak/standard-RAxML) showed that the calla lily bacterial strains were clustered with type strain of P. rettgeri). Koch's postulate was performed to confirm the pathogenicity of the bacterial strain ZAPR22R by inoculating petiole base of 20 seedlings of Zantedeschia cv. 'Jingcai Yangguang' with the classical injection procedure. Three sites 1-2cm above the petiole base of individual plant were injected with 100 µl of 108 CFU/ml bacteria, and each was spaced 1 cm apart. All samples were incubated with 16 h of light and 8 h of darkness in a culture chamber at 70-80% relative humidity and 30℃. Soft rot symptoms were developed at the inoculated site after twenty days of inoculation, while no symptoms appeared in the controls. Bacteria reisolated from the inoculated tissues had similar phenotypes and identical molecular characteristics as the original isolate. To our knowledge, this is the first report of the occurrence of P. rettgeri on calla lily in China. P. rettgeri was described as a rhizobacterium that promotes the growth of vegetable crops and lichens from Brazil (Cavalcante da Silva et al. 2020; Swamy et al. 2016). In contrast, it was recently identified as the pathogenic bacterium which cause brown slime flux on Populus tomentosa, a Chinese deciduous tree (Zhou et al. 2020). In the present study, the soft rot was identified as a major disease affecting the growth of calla lily and posing a great threat to commercial calla lily industry. It also provides insights that more studies should be performed on the epidemiology of the disease in different parts of China to establish effective disease management strategies.