First Report of Clavibacter michiganensis subsp. michiganensis Infecting Potato in Russia

Abstract

Yellow-pigmented clavibacteria were isolated from diseased potato plants and tubers from five regions of Russia between 2011 and 2017. Severe outbreaks of the disease were recorded in the central and northwest parts of the Russian Federation. Plants exhibited yellowing and necrosis of the leaf and wilting of whole leaves and plants, and brown veins around potato tuber eyes were visible on cross sections. Vascular tissue was removed from discolored nodes, crushed in sterile 10 mM phosphate buffer, and dilution plated on yeast extract dextrose calcium carbonate agar. Creamy yellow colonies developed from the extracts after 7 days incubation at 26°C. Isolates from each diseased potato plant (35 isolates deposited to the State Collection of Phytopathogenic Microorganisms at Russian Research Institute of Phytopathology, Bolshie Vyazemy) were identified as Clavibacter michiganensis subsp. michiganensis (Cmm) using taxon-specific polymerase chain reaction (PCR) with primers CMM5/CMM6 that gave expected product of 614 bp (Dreier et al. 1995). Additionally, isolates were positive for Cmm and negative for C. michiganensis subsp. sepedonicus using a commercial ELISA test (Agdia Biofords, France). Bacteria were gram positive aerobic rods with biochemical traits uniform and similar to the type strain Cmm NCPPB2979T (Schaad et al. 2001). All isolates were positive for esculin hydrolysis, gelatin liquefaction, H₂S production from peptone, utilization of citrate and succinate, and acid from d(+)mannose, and negative for starch hydrolysis, casein hydrolysis, methyl red reaction, acid from inulin, mannitol, d(+)melezitose and d(–)sorbitol, and utilization of acetate, formate, lactate, propionate, and ribose. Sequencing of the 16S rRNA (Weisburg et al. 1991) and gyrB gene (Zaluga et al. 2011) was performed for all 35 isolates. 16S rRNA sequencing (GenBank accession nos. MH035728.1 to MH035762.1) showed a >99% relatedness to Cmm type strain NCPPB2979ᵀ. gyrB gene analysis grouped the new potato-isolated strains (GenBank accession nos. MH042254 to MH042288) with Cmm strains from tomato obtained from the same regions. Pathogenicity of four typical isolates was tested on tomato (cv. Dubok) and potato (cv. Lion Heart) plants using suspensions of about 10⁸ cells/ml in sterile tap water. Four to six plantlets with two to four fully developed leaves were inoculated by stem infiltration (Zaluga et al. 2013) with 10 µl of bacterial suspension and were placed in a growth chamber at 20 to 25°C. The type strain of Cmm from tomato was used as a pathogenic control. The first symptoms appeared 14 days after inoculation and progressed slowly. In both tomato and potato plantlets, the four Cmm isolates displayed chlorosis of leaf margins, mottling and yellowing between veins, wilting or necrosis of individual leaf parts, and finally wilting of whole leaves. Each isolate was recovered from infected test plants and reidentified as Cmm by PCR with primers CMM5/CMM6. No symptoms were observed on negative control plants inoculated with sterile water and on potato inoculated with the Cmm type strain. 16S rRNA gene (GenBank accession nos. MH035728.1 to MH035762.1) showed a >99% relatedness to Cmm type strain NCPPB2979ᵀ. This is the first report of Cmm affecting potato in Russia.