First Report of Soft Rot of Aconitum carmichaelii Caused by Pectobacterium brasiliense in China.

Abstract

Aconitum carmichaelii Debx. is a Chinese traditional medicine herb, and is widely planted in China. The processed lateral roots of A. carmichaelii is known as Fuzi, and is used for the treatment of pain and inflammation in the joints (Zhou et al., 2015). In July 2019, a high incidence (approximately 50-100%) of soft rot of A. carmichaelii was observed in several commercial fields in Jiangyou County of Szechuan Province of China. Soft rot brownish lesions developed on infected stems, leading to collapse and wilting of entire plants. From symptomatic plants, the margins between the diseased and healthy areas were cut into pieces (5 × 5 mm), which were surface sterilized using 75% ethanol for 30 s and 2% NaOCl for 1 min, followed by three rinses with sterile water. The sterilized sections were macerated in drops of sterile water, and the extract was streaked onto King's B (KB) agar medium and incubated for 48 h at 30°C. Single colonies that are round, convex and creamy on the plates after 2 days were streaked on KB agar plates. Ten bacterial strains were isolated, and the strain Fuzi915 was chosen for further analyses. The 16S rDNA gene sequence (GenBank accession MZ881946) amplified by primer pair 27F/1492R (Monciardini et al., 2002) showed 99.85% identity to the sequence of Pectobacterium brasiliense (syn. Pectobacterium carotovorum subsp. brasiliense, Pcb) strain HNP201736 (MN393938.1) and P. carotovorum subsp. carotovorum strain PJP201706 (MN394020.1), respectively, and also showed 99.78% identity to P. brasiliense strain SX309 (CP020350.1). To further identify the Fuzi915 strain, the PCR assay was carried out using primer pairs Y1/Y2, EXPCCF/EXPCCR and BR1f/L1r (De Boer and Ward, 1995; kang et al., 2003; Duarte et al., 2004), specific to P. carotovorum, P. carotovorum subsp. carotovorum and P. carotovorum subsp. brasiliense (Pcb), respectively. Specific fragments of 434 bp and 322 bp were amplified by the Y1/Y2 and BR1f/L1r primer sets, receptively, but there was no amplification by the EXPCCF/EXPCCR primer set, indicating that the Fuzi915 strain belongs to Pcb (Onkendi and Moleleki, 2014). Additional phylogenetic trees based on two housekeeping genes mdh (MZ892962) and gapA (MZ892963) were constructed using Maximum-likelihood method with 1000 bootstraps. The Fuzi915 strain clustered with all P. brasiliense strains including type strain P. brasiliense BC1. Further, a pathogenicity test was conducted on healthy A. carmichaelii roots and seedlings maintained in a growth chamber at 25°C and 95% humidity. Root inoculation was followed by drenching 107 CFU/ml of the cell suspension of Fuzi915 strain in soil surrounding the A. carmichaelii roots. Ten roots were inoculated with cell suspension while 10 roots were drenching inoculated with sterile water as negative control. Stem inoculation was followed by injecting 103 CFU/ml of the cell suspension in the stem of 10 A. carmichaelii seedlings, while 10 were injected with sterile water as negative control. After 5 days, Pcb-inoculated roots became brown and soft, and Pcb-inoculated seedlings became wilted and water soaked and started to collapse, similar to symptoms observed in the field. No symptoms were observed on the control plants inoculated with sterile water. The strain was re-isolated successfully from symptomatic A. carmichaelii and was identified as P. brasiliense by using PCR with the same primers to complete Koch's postulates. To our knowledge, this is the first report of the soft rot of A. carmichaelii caused by P. brasiliense in China.