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.

First Report of Curtobacterium flaccumfaciens pv. flaccumfaciens Causing Bacterial Wilt and Blight on Sunflower in Russia.

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.

First Report of Pectobacterium polaris Causing Soft Rot and Black Leg of Potato in Russia.

Blackleg and soft rot of potato (Solanum tuberosum) were monitored in the Central European part of Russia within a period of 2012- 2019. Symptoms included decay of tubers, blackening of stem vascular bundles, and partial yellowing of leaves. The disease causes serious potato yield losses in the field and storage. Pectobacterium parmentieri, P. brasiliense, P. versatile (syn. Ca. Pectobacterium maceratum), P. carotovorum, P. atrosepticum, Dickeya dianthicola, and D. solani are considered as main causal agents of soft rot and blackleg disease in Russia (Voronina et al. 2019, Ngoc Ha et al., 2019, Shirshikov et al. 2018, Kornev et al. 2012). Potato plant samples collected in commercial fields in routine plant health assay were used for bacteria isolation on crystal violet pectate agar (CVP) (Helias et al. 2012) as described previously (Voronina et al. 2019). Bacterial colonies producing pitting on CVP were re-isolated and purified on nutrient broth yeast extract medium. DNA of bacterial isolates was extracted, and polymerase chain reaction (PCR) amplifications were performed using gapA primers (Cigna et al. 2017) followed by sequencing. DNA sequence alignment showed that the isolates F099, F100, F106, F109, and F118 were identical (deposited as part of NCBI Ref.Seq. for F109 NZ_RRYS01000004.1, locus KHDHEBDM_RS06360) and grouped together with the type strain Pectobacterium polaris NIBIO1006T (CP017481), a new species described as a potato pathogen (Dees et al. 2017). These strains were negative in diagnostic PCR assays using specific primers Y45/Y46 for the detection of P. atrosepticum, Br1f and L1r for P. brasiliense (Duarte et al. 2004), and ADE1/ADE2 for Dickeya sp. (Nassar et al. 1996). To further validate the identification, strain F109 of P. polaris was selected for genome sequencing. The genome of P. polaris strain F109, (NCBI Reference Sequence NZ_RRYS00000000.1) reveals >99% sequence similarity with type strain P. polaris IPO_1606 (GenBank accession GCA_902143345.1). The strain F109 was deposited to All-Russian Collection of Microorganisms under number VKM V-3420. Thus, the characterization of five isolates provided evidence that a previously unreported pathogen was present in the surveyed fields. The isolates were uniform in genetic and physiological properties; they were gram negative, facultative anaerobes with pectinolytic activity, negative for oxidase, urease, indole production, gelatin liquefaction. All isolates were catalase positive, produced acid from lactose, rhamnose, saccharose, xylose, and trehalose, and were tolerant to 5% NaCl, unable to utilize malonate and citrate. All the isolates grew at 37°C. All isolates caused soft rot symptoms on 10 inoculated potato tubers. They produced typical black leg rot symptoms in young potato plants inoculated with 107 CFU/ml of the pathogen by stem injection and incubated at 25°C for 48 h. The bacteria were re-isolated successfully from symptomatic potato and pathogen confirmed by gapA sequencing to complete Koch's postulates. To our knowledge, this is the first report of blackleg and soft rot caused by P. polaris on potato in the Russian Federation. According to the data of commercial diagnostic laboratory "PhytoEngineering" (Moscow region), P. polaris occurred in 5% potato seed stocks harvested in 2017-2019 in the Moscow region. This finding may indicate that new Pectobacterium strains have adapted to a diverse environment, which is consistent with widespread distribution of commercial seed potatoes. The author(s) declare no conflict of interest. Funding: This work was supported by Russian Science Foundation grant #16-16-00073.