Biocontrol agents based on strains of microorganisms that participate in mutualistic relationships with host plants, including those based on strains of endophytic bacteria, can be an alternative to chemical pesticides. There are endophytic B. thuringiensis strains that produce insect-toxic proteins and induce systemic resistance of plants to pathogens. An important issue is the possibility of regulation of the relationship of endophytic bacterial strains with the host-plant and their ability to induce plant defense reactions against pathogens by signaling molecules, including salicylic acid (SA). The aim of this work was to study the effect of SA and the endophytic bacterial strain B. thuringiensis B-5351 on the activity of potato plants reactions associated with signaling and biosynthesis of SA, as well as the activity of a potato trypsin inhibitor upon infection with the late blight pathogen. We used sterile test tube potato plants (Solanum tuberosum L.) of the Early Rose cultivar. 20-days-old potato plants were inoculated with a bacterial suspension of B. thuringiensis B-5351 (1*108 cells / ml) in distilled water, or in 1 μM SA solution by applying 5 μl of bacterial suspension to 4 upper leaves (20 μl / plant). Control plants were treated with distilled water, and some plants were treated with 1 μM SA solution in the same volume. On the 7th day after inoculation with B. thuringiensis B-5351, the plants were infected with oomycete P. infestans spores (5 μl of 1*105 spore/ ml suspension per leaf). To assay the damaged area, leaves were photographed; the images were analyzed using the ImageJ software. 24 h after infection with late blight pathogen spores, plants were fixed to assess the transcriptional activity of PR6, PR1 and PAL genes (See Table 1) (using CFX Connect Real-Time PCR Detection System Bio-Rad (USA)), and after 6, 24 and 48 h - to analyze the activity of plant proteinase inhibitors and the content of hydrogen peroxide. We estimated the number of colonyforming units (CFU) of microorganisms in plant tissues after their surface sterilization on the 7th day after inoculation of plants with B. thuringiensis B-5351 bacteria. The significance of the differences between the samples was accessed using Student’s t-test (p<0.05). Tables and Figures show data as the mean of the replicates and their standard deviations (M±SD). All experiments were conducted in three-five biological and three analytical replicates. In this work, we showed that SA presence increased the content of living cells of B. thuringiensis B-5351 bacteria in the internal tissues of plants (2,3±1,5×106 CFU/g) as compared to individual treatment with the bacterial strain under investigation (7,3±1,6×105 CFU/g) (See Table 2). Using the method of RAPD analysis, we established the identity of bacteria isolated from internal tissues of potato plants and the initial strain of B. thuringiensis B-5351 (See Fig. 1). Apparently, such titer of bacterial population regulated by plant signaling molecules in plant tissues is an important factor in the formation of plant resistance. Thus, we did not observe a decrease in the area of late blight symptoms on the leaves of potato plants treated only with B. thuringiensis B-5351, while under the individual action of SA this parameter decreased by half, and upon combined treatment of plants with SA and bacteria B. thuringiensis B-5351 - almost 4 times as compared to water-treated plants (See Fig. 2). Treatment with bacteria B. thuringiensis B-5351 increased the activity of proteinase inhibitors in potato plants only 6 h after infection with the late blight pathogen; in plants treated with bacteria cells of B. thuringiensis B-5351 together with SA, there was an almost a twofold increase in the activity of proteinase inhibitors after 6 and 24 h after infection with oomycete P. infestans. Under the influence of SA, a 15-20% increase in the content of H2O2 in intact plants was observed in all variants. In infected plants treated with SA, the H2O2 content exceeded the control values by more than 40%. In plants treated with a suspension of bacteria B. thuringiensis B-5351 and SA plants, the presence of the late blight pathogen caused a threefold increase in the H2O2 content 6 h after infection (See Fig. 3). In the variant with combined treatment with SA and bacteria B. thuringiensis B-5351 of noninfected plants, the content of PR6 gene transcripts exceeded the control level by 25%, as in infected plants treated with SA. Treatment of plants with SA together with bacteria B. thuringiensis B-5351 increased the number of PR6 gene transcripts by more than 50% relative to water-treated non-infected plants (See Fig. 4). In this work, we found that the strain of endophytic bacteria under investigation promoted the accumulation of transcripts of genes encoding PR1 and PAL proteins to the same extent as SA, both individually and when used together. In the presence of the late blight pathogen, more than a twofold (relative to this level in water-treated intact plants) increase in the content of PAL gene mRNA in potato plants was observed in cases of both individual and combined application of SA and B. thuringiensis B-5351 cells. Thus, treatment with B. thuringiensis B-5351 bacteria together with SA induces salicylate-dependent defense reactions (PR1, PAL), as well as an increase in the transcriptional activity of the PR6 gene, which, according to the literature, is a jasmonate-dependent gene. Our results showed the effectiveness of the composition based on live bacteria B. thuringiensis B-5351 and SA in protecting potato plants from late blight by inducing systemic defense responses in plants. Taking into account the insecticidal activity of the strain under investigation against Russian wheat aphids and Colorado potato beetle, which we previously described, the possibility of increasing plant resistance to late blight by biocontrol agent combining B. thuringiensis B-5351 with SA can open one of the approaches to the development of multifunctional protection of plants from biotic influences.
Decrosslinking enables visualization of RNA-guided endonuclease–in situ labeling signals for DNA sequences in plant tissues