First report of Curtobacterium flaccumfaciens pv. flaccumfaciens causing bacterial tan spot of soybean in Russia.

Curtobacterium flaccumfaciens pv. flaccumfaciens (H.) Collins & Jones is known as a pathogen of different legume crops, including soybean (Glycine max (L.) Merr.) (Hedges 1922; Dunleavy 1983). OEPP/EPPO (2011) considers C. flaccumfaciens pv. flaccumfaciens as present in Russia based on reports of the disease on common beans in two regions of Russia (North Caucasus and Far East) made without proper pathogen identification. During the summer of 2020 and the spring of 2021, soybean plants with tan spot disease (10-40% of plants) were reported during routine assays of several fields in Stavropol Krai (44.72°N, 43.29°E). After harvest in 2021, we inspected 48 soybean seed lots collected in different regions of Russia for the presence of C. flaccumfaciens pv. flaccumfaciens. Seed testing was performed using the OEPP/EPPO (2011) protocol. For bacteria isolation, seed extracts were spread on MSCFF agar plates (Maringoni et al. 2006). After 5 days of incubation at 28°C potential, C. flaccumfaciens pv. flaccumfaciens colonies were used for further tests on NSA and SSM agar (Tegli et al. 2017, Maringoni et al. 2016). Six seed lots produced in Stavropol, Ryazan (53.95°N, 40.62°E), Orel (52.39°N, 37.69°E) and Amur (51.31°N, 128.22°E) regions were suspect. Ten isolates (SB1 to SB4 from Stavropol, F-125-1 to F-125-3 from Ryazan, and F-30-1 to F-30-3 from Amur) were selected, and further identified by morphological, physiological, and biochemical properties, MALDI TOF MS, 16S rRNA sequences, and specific primers CffFOR2 and CffREV4 (Tegli et al. 2017). Isolates consistently formed yellow, circular, smooth colonies on agar, and were identical to C. flaccumfaciens pv. flaccumfaciens type strain DSM 20129T in diagnostic physiological properties (Tegli et al. 2017). DNA was isolated from the bacteria by the CytoSorb Kit (Sintol, Moscow). All tested strains were positive in the PCR assay (Fig. 1). 16S rRNA fragments were amplified using primers 27F/1492R (Marchesi et al. 1998) and PCR products were sequenced (Evrogen, Moscow, Russia). The obtained 16S rRNA sequences (1473 bp, Accession No. OL539808.1-OL539817.1) were 100% identical to DSM 20129T (AM410688.1) according to a BLAST NCBI search. A pathogenicity test was done by leaf-cutting with scissors wetted with inoculum (for soybeans) or by injecting 5 microliters of the bacterial suspension (108 CFU/ml) into the stem (for common beans). All ten isolates for the inoculum were grown on nutrient agar for 72 h at 28°C. Soybean cv. Kasatka plants (stage V1) were used for inoculation, and common bean (cv. Purpurnaya) plants were inoculated as well to confirm multi-host virulence. Sterile water served as a control. Ten plantlets were used as replicates for each treatment. The plants were incubated at 24°C, 80% RH, and a 14 hour light/10 hour dark cycle. Tan spots (soybean) and wilt (beans) have developed 7-21 d.p.i (Fig. 2.1-2.6). Control plants remained asymptomatic. Seed inoculation by soaking them in the same bacterial suspension repeatedly produced twisted primary root (Fig. 2.7-2.8), but typical disease symptoms on leaves developed in 4-5 weeks only. The pathogen was successfully reisolated from all infected plants and not from the controls, thus fulfilling Koch's postulates. The identity of the reisolated strains was confirmed using morphological and physiological characteristics and the DNA sequence data for the 16S rRNA. These results indicated that a causal agent of the tan spot is present on soybean in three important agricultural areas of Russia (South, Central, and the Far East). To the best of our knowledge, this is the first report of C. flaccumfaciens pv. flaccumfaciens causing a bacterial tan spot of soybean in Russia.