Development of a multiplex real-time PCR method for the detection of Pseudomonas savastanoi pv. glycinea and Curtobacterium flaccumfaciens pv. flaccumfaciens in soybean seeds.

Multiplex real-time PCR with TaqMan® probes has been developed for the simultaneous detection of soybean pathogens Pseudomonas savastanoi pv. glycinea and Curtobacterium flaccumfaciens pv. flaccumfaciens. The method specificity has been confirmed using 25 strains of target bacteria and 18 strains of other bacteria common to soybean seeds as endophytes. The multiplex real-time PCR developed has been shown to have high sensitivity - a positive result was achieved at 0.01 ng/µl of DNA for both target organisms, and at 100 CFU/ml of bacteria in soybean seed homogenate. The robustness of the multiplex real-time PCR developed has been verified by the detection of the pathogens in 25 commercial seed stocks, in comparison with previously published PCR protocols. In all tests, three seed stocks were positive and 22 were negative. The multiplex real-time PCR can be applied in diagnostic practice for the simultaneous detection of two important pathogens of leguminous plants.

Genetic and phenotypical diversity of Pseudomonas syringae population in the Russian Federation.

Proteobacteria comprising species of Pseudomonas syringae group cause diseases of many plants around the world. The phytopathogen has a complex taxonomic structure, which is constantly being revised due to the emergence of new molecular and biochemical diagnostic methods. Here for the first time, we describe the genetic and phenotypic diversity of 57 strains of Pseudomonas syringae isolated from affected soybeans, cereals, sunflowers, and other plants in the Russian Federation from 1950 to 2019. Genetic diversity was assessed by Multi Locus Sequence Analysis (MLSA) using fragments of the genes of glyceraldehyde-3-phosphate dehydrogenase (gapdh), the DNA-directed RNA polymerase subunit D (rpoD), gyrase (topoisomerase) B subunit (gyrB), and citrate synthase I (gltA). The synthesis of syringomycin and coronatine by bacteria was assessed by the reaction of susceptible yeast culture, seedlings of barley, tomato, and sunflower, and by presence of toxin genes confirmed by PCR test. The pathogenicity of the strains was confirmed on seedlings of dicotyledonous and monocotyledonous plants of peas, soybean, sunflowers, barley and wheat, as the most affected crops. The sensitivity of bacteria to 10 antibiotics of the main mechanisms of activity and two bactericidal commercial products was tested by standard disc method. The obtained results showed a high genetic homogeneity of the Russian population of P. syringae, which infects various agricultural crops, and an increase in the proportion of antibiotic-resistant strains over the years.

[A virulence gene from Xanthomonas campestris pv. campestris homologous to the avrBs2 locus is recognized in race-specific reaction by two different resistance genes in Brassica plant species]. / Gen avirulentnosti Xanthomonas campestris pv. campestris, gomologichnyi lokusu avrBs2, uznaetsia pri rasovo-spetsifichnoi reaktsii dvumia genami ustoichivosti rastenii roda Brassica.

Race-specific interaction between the Brassica plants and Xanthomonas compestris pv. campestris bacteria follows the "gene-for-gene" rule. Expression of the avirulence genes recognized by two dominant resistance genes of Brassica, Rxc1 in plants with the BB genome, and Rxc3 in the CC plants, was lost after bacterial mutation in planta. The mutation was distinguished by the elongation of CGCGC pentanucleotide repeat in the gene, which was designated as avrRxc1/3. This gene displayed strong structural similarity to the avrBs2 locus from the related species X. vesicatoria. Thus, it is the first description of the avrRxc1/3 avirulence gene conferring race-specific interaction between X. campestris pv. campestris and Brassica plants. Structural homologues of the avrBs2 are found in many Xanthomonas species, but in all cases except X. vesicatoria, their function remains unknown.