In the summer of 2018, wilt and leaf spots were observed on sunflower (Helianthus annuus L.) plants in fields near Kursk (51.74°N, 36.02°E) in Russia. In the following years, incidence of this disease was 5 to 20% in the inspected fields. Marginal chlorosis on seedling leaves developed into wilt and necrosis about one week later (Fig. 1). Mature plants had leaves with blight and reduced height compared to symptomless plants. Pathogen isolation from seeds was done by the method of Tegli et al. (2002) with modifications. Bacteria from diseased plants were isolated by streaking inoculum from symptomatic tissues on nutrient dextrose agar (NDA) (Schaad et al. 1988). The plates were incubated at 30°C for 7 to 10 days. Isolates consistently formed slow-growing, yellow, circular, smooth colonies without soluble pigment. The isolated bacteria were aerobic, gram-positive, and rod-shaped. Eight strains, CF-20 to CF-26 from plants, and Curt1 and Curt3 from seeds, were identified by MALDI TOF MS analysis as Curtobacterium flaccumfaciens pv. flaccumfaciens or C. flaccumfaciens pv. poinsettiae. All strains had GENIII MicroPlate (BIOLOG) test results identical to C. flaccumfaciens pv. flaccumfaciens strain DSM20129T. Further analysis was done by specific PCR (Tegli et al. 2002) and 16S rDNA, gyrB, and atpD gene sequencing. For PCR amplification, DNA was extracted by the CitoSorb Kit (Syntol Co., Moscow). Primers 27F/1492R (16S rRNA) (Marchesi et al. 1998), 2F/6R (gyrB) (Richert et al. 2005), and aptD2F/aptD2R (Jacques et al. 2012) were used to amplify the target gene sequences. The PCR products were sequenced by Evrogen (Moscow). The 16S rRNA sequences (OL584192.1 to OL584199.1) were identical to that of C. flaccumfaciens pv. flaccumfaciens strain DSM20129T (AM410688.1; 1,477/1,477 bp). The phylogenetic tree of concatenated gyrB (560 bp) and atpD (716 bp) sequences (OL548915.1 to OL548922.1 and OL548923.1 to OL548930.1, respectively) clustered the strains from sunflower among C. flaccumfaciens pv. flaccumfaciens, C. flaccumfaciens pv. betae, and C. flaccumfaciens pv. oortii (Fig. 2) with high genetic similarity to other C. flaccumfaciens strains: 96.3 to 100% for atpD and 95 to 100% for gyrB. A pathogenicity test for each of the strains was performed by injecting 5 μl of a bacterial suspension (108 CFU/ml) grown for 72 h on NDA into the stems of five plantlets (four true leaf stage) of the sunflower cv. Tunka (Limagrain, France) and soybean cv. Kasatka (VIM, Russia). Strain DSM20129T was a positive control, while sterile water was a negative control. The plants were incubated at 24°C, 80% relative humidity, and 14-h light/day. Wilting and blight on sunflower (Fig. 3) and tan spots on soybean were observed in 15 to 20 days after inoculation for all sunflower strains and strain DSM20129T. The negative control plants were asymptomatic. The bacteria re-isolated from the inoculated plants exhibited the same morphological characteristics and 16S rDNA sequence as the original culture, thus fulfilling Koch's postulates. The presence of C. flaccumfaciens pv. flaccumfaciens in sunflower seeds indicated that the bacterium was transmitted via seed. Sunflower has been previously reported as a host for the pathogen (Harveson et al. 2015). The presence of C. flaccumfaciens pv. flaccumfaciens on beans in Russia was suggested from the disease symptoms (Nikitina and Korsakov 1978), but, to our knowledge, this is the first report of the pathogen affecting sunflower in Russia. Phytosanitary categorization placed C. flaccumfaciens pv. flaccumfaciens in the EPPO A2 list (EPPO 2011). Thus, sunflower seeds should be tested to protect pathogen-free areas from introduction of this pathogen.
Keywords: Curtobacterium flaccumfaciens; MALDI TOF; bacterial wilt; sunflower.