RNAi-MEDIATED EFFECT OF BIOSTIMULANT REGOPLANT IN PROTECTION OF COMMON HORSE CHESTNUT OF Aesculus L. GENUS AGAINST THE DAMAGING ACTION OF HORSE CHESTNUT LEAF MINER Cameraria ohridella DESCHKA & DIMIC

Horse chestnut leaf miner (HCLM), Cameraria ohridella Deschka & Dimic (Lepidoptera: Gracillariidae) is severe pest of common horse chestnut of Aesculus L. genus. Due to the climate change and lack of natural enemies, the pest has been spreading rapidly in the countries of the world in last ten years. Using of chemical insecticides (pyretroids, neinicotinoids) and insect growth regulators is the predominating method to control НСLM. However it is not economically feasible and environmentally friendly. Non- chemical control of the pest using natural biostimulants may be promising alternative to chemical method. The bioprotective properties of biostimulant Regoplant against the damaging effect of НСLM on various species of common horse chestnut is studied. The treatment of plants with biostimulant Regoplant provides the improvement of physiological parameters of common horse chesnut, i.e. water content is increased - up to 4.19%, photosynthesis rate is increased - up to 2.80 mg CO2·dm-2·h-1 as well as water deficit is decreased – up to 5.19% and transpiration intensity is decreased – up to 6.79 mg m -2·s-1 as compared to control. The significant reducing of the degree of leaf surface damage - up 50.24% is observed while using biostimulant Regoplant as compared to control. The positive effect of biostimulant on physiological parameters of common horse chestnut against HCLM damage is slightly lower then in case of conventional insecticide Thiamethoxam application. In the molecular-genetic experiments the decrease of degree of homology between cytoplasmic mRNA and si/miRNA populations, isolated from control and experimental treated by biostimulant Regoplant various species of common horse chestnut of Aesculus L. genus is found. It is shown that index of hybridization between mRNA and si/miRNA is decreased on 28-37% (at resistant to HCLM species) and on 21-27% (at nonresistant to HCLM species) as compared with the same index obtained in control №1 common horse chestnut. On the contrary the increase of degree of homology between si/miRNA, isolated from treated by biostimulant Regoplant resistant and nonresistant to HCLM common horse chestnut species, and mRNA, isolated from the most resistant to HCLM - yellow buckeye of Aesculus octаndra Marsh genus, is observed. It is shown that index of hybridization between mRNA and si/miRNA is decreased on 8-16% (at resistant to HCLM species) and on 10-19% (at nonresistant to HCLM species) and became more similar to the same index obtained in control №2 yellow buckeye. Similarly the increase of degree of homology between mRNA, isolated from species that usually are not damaged by HCLM - sweet chestnut of Castanea sativa Mill. genus, and si/miRNA, isolated from the both resistant and nonresistant to HCLM species of common horse chestnut of Aesculus L. genus, is shown. It is set that index of hybridization between mRNA and si/miRNA is decreased on 21-31% (at resistant to HCLM species) and on 27-32% (at nonresistant to HCLM species) and became more similar to the same index obtained in control №3 sweet chestnut. The increase of silencing (inhibiting translation of mRNA) activity of si/miRNA, isolated from both resistant to HCLM (up to 75-87%) and nonresistant to HCLM (up to 56-64%) species of experimental common horse chestnut infected by HCLM and treated by biostimulant Regoplant, as compared to control is observed in the wheat-germ cell-free protein synthesis system. These results testify that protective action of biostimulant Regoplant occurs through stimulation of RNAi-mediated resistance to HCLM of common horse chestnut Aesculus L. genus.