First Report of Bacterial Wilt Caused by Ralstonia solanacearum on Ageratum conyzoides in China.

Ageratum conyzoides L. is believed to act as reservoir host for many plant diseases. In June 2011, a 30% incidence of bacterial wilt on A. conyzoides was observed in a field of Rhizoma kaempferiae in Yangchun city of Guangdong province. The initial symptoms were wilting of the apical leaves during the day, which recovered at night. After 4 to 6 days, the leaves became totally necrotic. The basal stems of the diseased plants were blackened and the vascular tissue turned brown. To investigate the disease etiology before understanding the disease link between A. conyzoides and R. kaempferiae, 10 plants with typical wilting symptoms were collected from the field. A total of 10 bacterial isolates were isolated from the vascular tissue of each diseased plant on tripheny tetrazolium chloride (TZC) medium. After incubation at 30°C for 2 days, the plates had large, irregular round, fluidal, white colonies with a pink center. Thirty healthy A. conyzoides plants at the four- to six-leaf growth stage were inoculated by injuring the roots and soaking them in a bacterial suspension (1 × 10⁸ cfu/ml) for 20 min with the 10 bacterial isolates separately, and planted in 10-cm pots with sterile gardening soil in a glasshouse (28 to 35°C). Sterile water was used as a negative control. Five days after inoculation, a few leaves of the inoculated plants began to exhibit wilting. The inoculated plants eventually showed the same symptoms as those in the field. The same bacterium was reisolated from inoculated plants. The 30 negative control plants did not have wilt symptoms. With the same inoculation procedure, the bacterium also caused wilting on tomato (25 of 30), pepper (10 of 30), eggplant (2 of 30), ginger (11 of 15), and R. kaempferiae (8 of 15). Using the universal bacterial 16S rDNA primer set 27f/1541R (3), approximately 1,400 bp-fragments were amplified from the 10 isolates, respectively. The sequences for the 10 fragments (GenBank Accession Nos. JX294065 to JX294074) were identical and had 100% sequence identity with 16S rDNA of R. solanacearum GMI1000 (AL646052). The 10 isolates were able to oxidize disaccharides (lactose, maltose, and cellobiose) and hexose alcohols (mannitol, dulcitol, and sorbitol). According to Hayward's classification, all isolates were biovar 3 (2). Based on the pathogenicity tests, carbohydrate utilization, and near full-length 16S rDNA sequences, the bacterial isolates from the diseased A. conyzoides belonged to race 4 and biovar 3 of R. solanacearum. Furthermore, the specific 280-bp and 140-bp fragments were respectively amplified from all 10 isolates by using the multiplex PCR (1). In addition, specific 165-bp fragments were amplified from all the isolates using the specific primers AKIF/AKIR (3), which indicates the bacterium belongs to R. solanacearum Phylotype I. To our knowledge, this is the first report of a disease caused by R. solanacearum on A. conyzoides in China. References: (1) M. Fegan and P. Prior. Page 449 in: Bacterial Wilt Disease and the Ralstonia solanacearum Species Complex. C. Allen et al., eds. The American Phytopathological Society. St. Paul, MN, 2005. (2) A. C. Hayward. J. Appl. Bacteriol. 27:265, 1964. (3) M. Horita et al. J. Gen. Plant Pathol. 70:278, 2004.