IDEA #3GT3RM Increase of Plant Resistance to Diseases, Pests and Stresses

In the greenhouse experiments the antipathogenic activity of new polycomponent biostimulant Regoplant, Stimpo and Radostim had been investigated at cultivation of different varieties of winter wheat, soybean and corn plants on infectious backgrounds. The best biological efficiency against phytopathogens was obtained at presowing seed treatment and spraying of crops in vegetation period by Regoplant (up to 98 %) and Stimpo (up to 89 %), less bioprotective efficiency was showed by Radostim (up to 74 %) according to control (without treatment with biostimulants). At 2nd generation of the wheat and chickpea plants (infected by pathogenic micromycetes of Fusarium L. genus without treatment with biostimulants) the increased resistance to pathogenic micromycetes of Fusarium L. genus was also found. Using DOT-blot hybridization method the considerable difference between mRNA isolated from control winter wheat and chickpea seedlings and small regulatory si/miRNA isolated from experimental seedlings (obtained from seeds of the 1st generation of plants infected by pathogenic micromycetes of Fusarium L. genus and treated with biostimulants) was found. It is proposed that indicated difference is connected with partial reprogramming of plant cell genome under the impact of biostimulants - inducers of si/miRNA synthesis with antipathogenic properties. Development of economically feasible and environmentally friendly agricultural technologies able to provide stability of agricultural ecosystems, to promote wide use of biocontrol, and to guarantee improvement of quality, is one of the challenges of modern agriculture. Pests (insects, mites, and nematodes), diseases (bacteria, viruses, fungi, nematodes), and weeds cause a significant yield reduction in agricultural production worldwide. According to the Food Agricultural Organization (FAO), the global annual crop losses due to pests, diseases, and weeds is reached up to 20-25 % [Stevens and May, 2009]. European corn borer, cutworms, wireworms, grabs cereal flies, aphids, root-knot and leaf weevils, soybean pod borer, spider mites, trips, rape beetles, flea beetles, stink, bugs, and white butterflies belong to the most widespread and dangerous pests that cause significant reduction of yield of important agricultural crops such as corn, wheat, barley, soybean, and rape. The problem of plant protection against widespread fungi (e.g., Fusarium spp., Fusarium graminearum, Fusarium oxysporum Ciceriae, Cercospora spp., Ascohyta spp., Perronospora spp., Blumeria spp., Pucinia spp., Sclerotinia spp., and Verticillim spp.); bacterial (Pseudomonas spp.), viral (Potyvirus spp.) pathogens and phytoparasitic nematodes (such as Heterodera schachtii, Meloidogyne incognita, Globodera rostochiensis, Ditilenchus dipcasi, Rotylenchulus reniformis, Tylenchulus semipenetrans) and other diseases is also economically important [Stevens and May, 2009]. Non-chemical crop protection is important component of sustainable crop production. Development of such compounds was based on achievements in modern microbiology, mycology, biotechnology, soil science, and plant protection. Long-term research and wide practical application of Stimpo, Radostim, Regoplant and Biolan - the new polycomponent biostimulants developed by the National Enterprise Interdepartmental Science & Technology Center «Agrobiotech», Natl. Acad. of Sci. and Min. of Ed. and Sci. of Ukraine, showed that these biostimulants match with economical and environmental demands of modern agriculture. These biostimulants have bioprotective and regulatory effects that are achieved by synergistic action of metabolism products (mixtures of amino acids, carbohydrates, fatty acids, polysaccharides, and microelements) of root fungus-endophyte products of ginseng Panax Ginsed M. as well as of soil streptomycete Streptomyces avermitilis metabolites [Ponomarenko et al., 2010] with phytostimulating, antiparasitic and antipathogenic effect. In our molecular-genetic experiments, we have showed that positive effects of the above-mentioned biostimulants were revealed in quantitative and qualitative changes in gene expression as a consequence of plant cell genome reprogramming with biostimulants [Tsygankova et al., 2010; Tsygankova et al., 2011]. We have found also [Tsygankova, Ponomarenko et al., 2012; Tsygankova, Stefanovska et al., 2012] that these biostimulants significantly enhanced plant resistance to different pathogens due to stimulation of the synthesis of endogenous small regulatory si/miRNA that participate in RNAi (RNA interference) process. This process called posttranscriptional gene silencing (PTGS) was found in plants, animals, and fungi [Filipowicz et al., 2005; Bakhetia et al, 2005; Katiyar-Agarwal, 2006]. Small regulatory si/miRNA with 22-24 nt size plays a leading role in silencing: together with site-specific endo- and exonucleases of RNA-induced silencing complex (RISC), it blocks (silences) translation of variable cell mRNA with complementary structure to mRNA of pathogens and parasites, or enzymatically cleaves these target mRNA molecules causing its degradation [Vaucheret, 2006; Voinnet, 2008; Zhang, 2007]. The purpose of our work was verifying the possibility of increasing of the plant resistance to pathogens and parasites by above mentioned biostimulants and determination of genetic mechanisms of these biostimulants’ action and epigenetic mechanisms of these biostimulants’ post-action (i.e. effect of inheritance of increased with biostimulants plant resistance to pathogenic and parasitic organisms) [Calarco et al., 2012; Tsygankova, 2012; Ponomarenko et al., 2012]. RESULTS AND DISCUSSION. We tested bioprotective properties of new biostimulants at cultivation of winter wheat, soybean, and corn on the infectious backgrounds. The results were compared with the effect of modern pesticides produced by leading agrochemical companies such as Alpha-Cypermethrin insecticide, Yunta Quadro insecto-fungicide, Lamardor, Selest Top, Imidacloprid, Terios and Microplant micronutrients. Use of biostimulants in combination with chemical pesticides caused increase of plant resistance against different diseases caused by microbial pathogens. Biostimulants reduced phytotoxicity of chemical protectants and stimulated immune reactions of plants. As a result, commercial grain yield and seed material quality was improved. The bioprotective effect of biostimulants was found sufficiently high at their use on crops infected by nematodes, ground beetle, turnip moth, and chloropid flies hat (Table 1). biostimulants effect did not exceed an effect achieved at the use of insecto-fungicides, e.g., Yunta Quadro and Selest Top. However, the level of efficiency shown by Regoplant against wheat nematode, ground beetle, and turnip moth, and by all the biostimulants studied against chloropid flies was just set as high taking into account their economic effect and environmental safety. Regoplant and Stimpo showed also antipathogenic activity against causative agents of winter wheat (Odeska semidwarf cultivar) rot and mildew (Table 2). However, we do not consider the use of these biostimulants as alternative to chemical pesticides reliable, especially on the highly infectious background. On the low infectious backgrounds, the biostimulants application is quite possible, taking into account the level of their potential efficiency. We studied also Regoplant and Stimpo bioprotective effect on soybean and corn plants infected by dangerous pathogens of soybean and causative agents of rot and mildew of corn (Table 2 and Table 3). We found that biostimulants has a positive effect on growth and development of soybean and corn seeds. They reduced the infection impact on seed development and commodity grain quality. Our laboratory experiments demonstrated the inheritance of wheat and chickpea plants resistance to pathogenic organisms. We found that plants of the 2nd generation which were not treated with biostimulants, maintain high viability which is similar to the results obtained on the 1st generation of plants treated with biostimulants on infected background [Tsygankova, 2012]. The molecular-genetic analysis conducted by the DOT-blot method hybridization between si/miRNA with mRNA populations [Tsygankova et al., 2010; Maniatis, 1982] showed high level of homology between immuno-protective small regulatory si/miRNA and mRNA isolated from experimental plants and lower level of homology between si/miRNA isolated from experimental plants and mRNA isolated from control plants (Table 4). We called this effect "quasi-heterosis". It was found that Regoplant, Stimpo and Biolan strongly increased growth rate as well as resistance of plant to pathogenic organisms. We concluded that principal mechanism of action of these biostimulants includes almost twofold increasing of the synthesis of small regulatory si/miRNA having antipathogenic properties in the cells of the 2nd generation of the wheat and chickpea plants that were infected by pathogenic micromycetes of Fusarium L. genus and were not treated with biostimulants. CONCLUSIONS. In the greenhouse experiments the bioprotective activity of new PGRs: Regoplant, Stimpo and Radostim had been investigated at cultivation of different varieties of winter wheat, soya and corn plants on infectious backgrounds. It was found that presowing treatment of plants and spraying of crops in vegetation period with biostimulants Regoplant, Stimpo and Radostim increased plant viability and resistance to different phytopathogens. In the laboratory experiments was shown that the seedlings of the 2nd generation of winter wheat and chickpea plants (that were grown on infectious background without treatment with biostimulants), obtained from the seed of the first generation of plants (that were grown on infectious background and were treated with biostimulants: Regoplant, Stimpo and Biolan), showed high viability and resistance to pathogenic micromycetes of Fusarium L. genus due to integral indexes of germination and growth comparatively with control seedlings. The molecular-genetic indexes - % homology between si/miRNA of experimental plants and mRNA of control plants testify that at embryogenesis during formation of plant seeds there is observed reprogramming genome of plant seed embryos towards the way of “switching on” genes encoding antipathogenic si/miRNA which synthesis is induced under biostimulants’ action.