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First Report of Clavibacter michiganensis subsp. sepedonicus Infecting Sugar Beet in Russia

Ignatov, A. N., Panycheva, J. S., Spechenkova, N., Taliansky, M.
Plant disease 2018 v.102 no.12 pp. 2634
Chinese cabbage, Clavibacter michiganensis subsp. sepedonicus, Pectobacterium, Pelargonium, agar, ambient temperature, antibodies, bacteria, beans, carbonates, chlorosis, cultivars, eggplants, endophytes, enzyme-linked immunosorbent assay, genes, glucose, growth chambers, kale, necrosis, pathogens, petioles, plantlets, potatoes, quantitative polymerase chain reaction, ribosomal RNA, roots, sequence analysis, spring, stems, sugar beet, summer, tap water, tobacco, tomatoes, vascular tissues, virulence, wilting, yeasts, Russia
In 2017, a new bacterial disease was observed in sugar beet plants in Krasnodar region, Russia. The disease caused severe wilt of plants, infected young petioles were curled, and whole leaves were distorted. Vascular tissues in the tap root cut longitudinally were brown to deep brown. Ten bacterial strains recovered from the petioles of wilted leaves produced white mucoid colonies on yeast dextrose carbonate agar medium (Schaad et al. 2001) after 7 days of incubation at room temperature. All tested strains were gram positive; colonies were prominent, round, and mucoid, approximately 2 to 4 mm in diameter. The strains were highly virulent on young (two to four true leaves) potato and sugar beet plants after artificial inoculation by leaf clipping and stem pinning, but they were avirulent to tomato, pepper, eggplant, tobacco, Chinese cabbage, leaf kale, beans, and pelargonium plants. Ten isolates from diseased sugar beet plants were identified as Clavibacter michiganensis subsp. sepedonicus (Cms) in taxon-specific TaqMan real-time polymerase chain reaction (PCR) (Schaad et al. 1999). Cms isolates were found on sugar beet plants at spring to early summer (May to June), and the population declined as average temperatures exceeded 22°C, substituted by secondary bacterial pathogens such as Pectobacterium spp. The isolates were deposited to the State Collection of Phytopathogenic Microorganisms at the Russian Research Institute of Phytopathology, Bolshie Vyazemy, with numbers from 2490 to 2499. All isolates had biochemical traits uniform and similar to the type strain Cms NCPPB 2137 (Schaad et al. 2001). All the isolates had positive reaction in an ELISA test with commercial antibodies (Agdia Biofords, France) for Cms. Identification of the isolates as Cms was confirmed using 16S rRNA gene universal primers fD1 and rP2 (Weisburg et al. 1991) and gyrB gene primers (Zaluga et al. 2011). DNA sequencing of 16S rRNA gene (GenBank accession nos. MH035763.1 to MH035772.1) showed a >99% relatedness to Cms type strain NCPPB 2137. GyrB gene analysis grouped the isolated strains (GenBank accession nos. MH042289.1 to MH042298.1) with Cms strains from potato (GenBank accession nos. JX890008.1, FR728141.1, and AM849034.1) with 100% similarity. Pathogenicity of the strains was tested to sugar beet and potato using suspensions of about 10⁸ cells/ml in sterile tap water. From six to eight plants with four fully developed leaves were inoculated by stem infiltration of approximately 15 µl of the bacterial suspension (Zaluga et al. 2013) and placed in a growth chamber at 20 to 25°C. Water-inoculated controls separated each test object. The potato strain of Cms was used as a pathogenic control. In sugar beet plantlets, the newly isolated Cms bacteria displayed chlorosis of leaf, wilting, or necrosis of leaf parts. The first symptoms appeared in 10 days after inoculation. In potato plants, the sugar beet and potato Cms strains caused rolling and necrosis of leaf margins, as well as mottling between veins. It was found that only 8 out of 40 tested sugar beet cultivars of Russian and German origin were susceptible to Cms inoculation, and the other 32 stayed symptomless. Each new strain was recovered from infected test plants and reidentified as Cms by TaqMan real-time PCR. The disease, caused by Cms, is considered one of the most important bacterial diseases of potato. Bugbee and Gudmestad (1988) isolated Cms as an endophyte of sugar beets and proved the presence of Cms on sugar beet seed. In our study Cms colonized roots and stems of a field-grown cultivar of sugar beet. To our knowledge, this is the first report of Cms causing bacterial disease of sugar beet in the Russian Federation.