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Project titleEquipment of healthy hydroponic tubers with endophytic bacteria Bacillus subtilis as a biological method of prolonged management of yield, adaptive potential and quality of potatoes

KeywordsSolanum tuberosum L, endophytic bacteria Bacillus subtilis, healthy mini-tubers, diseases, storage, resistance, antioxidants, phytonutrients, nutritional qualities, nutritional value, functional components

Annotation
The potato (Solanum tuberosum L.) is an important crop and a valuable food product of great importance for food security throughout the planet. Tuber losses from diseases during storage reach 70% annually and cause significant damage to agriculture and the food industry. An environmentally friendly and biosafe alternative to traditional chemical fungicides/bactericides are natural resistance inducers, in particular, beneficial endophytic bacteria Bacillus subtilis, capable of inducing natural defense mechanisms in plant organisms without harming them, the environment and human health. The development of preparations based on them for use on stored fruit and vegetable products is hindered by the lack of knowledge about the mechanisms underlying the interactions in the systems “endophytic B. subtilis – host plant – phytopathogen”. Information on the effect of endophyte bacteria on the nutritional quality, nutritional value, phytonutrient composition and functional properties of potato tubers during storage is practically absent and requires detailed study. The project is aimed at studying the effect of endophyte bacteria B. subtilis on the formation of plant and tuber immunity to pathogens of the most economically harmful potato diseases (late blight, fusariosis) during the growing season and long-term storage and positively affecting the content of functionally significant phytonutrients, nutritional value and consumer qualities of stored tubers. As markers for the development of defense reactions in plants and potato tubers, it is proposed to use physiological, biochemical and molecular biological indicators of signal regulation of resistance, namely, the components of antioxidant protection (the level of lipid peroxidation, proline, etc.) B. subtilis (10-4, 26D ). In a comparative aspect, priority data will be obtained on the effect of endophyte bacteria B. subtlis on the degree of development of pathogenic micromycetes Ph. infestans and F. oxysporum, as well as on the nature of colonization of the internal tissues of plants and tubers by B. subtilis endophyte bacteria during cultivation and storage, which will make it possible to reveal the contribution of regulatory systems important in the regulation of the formation of stress resistance and to determine their significance in the implementation of the protective action. An analysis of the relationship between the immunomodulatory and protective properties of B. subtilis endophyte bacteria will be carried out in order to induce the natural protective potential of potatoes to be affected by pathogens of late blight, fusarium. For the first time, comprehensive information will be obtained on the effect of B. subtlis endophyte bacteria on the consumer properties and biological value of tubers during cultivation and storage, the content of a number of functionally significant phytonutrients (bioflavonoids, ascorbic acid, tocopherol, carotenoids, etc.), starch, sugars, dry substances, proteins, as well as table qualities (digestibility, pulp consistency, taste, smell, flouriness, wateriness, darkening of raw and boiled pulp). The implementation of this project will expand knowledge of plant-microbial interactions, will allow to determine the key mechanisms of the impact of endophytic bacteria on potato plants and tubers, to advance in understanding the regulatory mechanisms for the formation of post-vegetative immunity of potato tubers to phytopathogenic fungi Ph. infestans, F. oxysporum under the influence of endophyte bacteria, as well as to reveal their influence on consumer qualities and biological value of tubers during storage. The study of the aftereffect of treatments with endophyte bacteria will allow us to evaluate their prolonged action in subsequent field reproductions. The results obtained can be used to improve technologies for environmentally oriented storage of potatoes and will contribute to solving the problem of introducing effective biotechnologies into agriculture and the food industry.

Expected results
The implementation of this project will expand knowledge of plant-microbial interactions and will advance in understanding the regulatory mechanisms of the formation of immunity, including post-vegetation immunity, of potato tubers to phytopathogenic fungi, induced by B. subtilis endophyte bacteria and to identify their effect on the consumer qualities and biological value of tubers when cultivation and storage. Priority data will be presented on the impact of endophytic bacteria B. subtilis on the species composition of the pathomicrobiome, on the degree of development of diseases detected during the growing season of plants and in the post-harvest period of storage of potato tubers, as well as the quality and culinary properties of tubers. It is assumed that the introduction of endophytic bacteria B. subtilis into agrocenoses can be an effective and safe way to protect potato crops from adverse environmental factors and extend the shelf life while maintaining product quality. the previous year, and it was also revealed whether the protective properties of the treatment are preserved in subsequent generations of plants. The obtained complex picture of the formation of resistance of plants and potato tubers to diseases under the influence of endophytic B. subtilis in combination with data on the content of functionally significant phytonutrients will allow us to identify the contribution of these systems important in the regulation of the formation of stress resistance and to determine their significance in the implementation of the protective effect of B. subtilis on tubers during infection with pathogens, which, ultimately, will help to reveal the significance of changes in these parameters caused by B. subtilis in the implementation of the physiological effect of endophytic B. subtilis on potato tubers during cultivation and storage. The aftereffect of tuber inoculation with B. subtilis endophytic bacteria on disease resistance in subsequent field generations will be studied. The expediency of using improved planting material of minitubers inoculated with endophytes to increase the shelf life, nutritional value, commercial quality, as well as prolonged action in subsequent reproductions in the field will be substantiated. The approach used in the project can be recommended for evaluating new preparations, which makes it possible to screen out inactive compositions from tubers inducing post-harvest immunity, to determine the effectiveness of their use against pathogens, and to select the most effective for implementation as preparations-compositions that modulate the natural protective potential of tubers and positively affect the biological value and functional properties of stored products. The results obtained can be used to improve technologies for environmentally oriented potato production and will contribute to solving the problem of introducing effective biotechnologies into agriculture and the food industry.

Annotation of the results obtained in 2023
In the first year of the project implementation, a healthy (disease-free) hydroponic mini-tubers of potato (Solanum tuberosum L., Bashkir variety) were obtained, and experiments (two parallel) were conducted in the field and one preliminary vegetation experiment under controlled conditions. Priority data on the complex effect of endophytes B. subtilis (strains 10-4 and 26D) have been obtained using pre-planting inoculation of seed tubers of reproduction of super elite and healthy potato mini-tubers on the intensity of growth processes and the nature of colonization of internal plant tissues by them, photosynthetic activity, oxidative and osmotic status of cells, resistance to diseases, changes in the species composition of the phytopathogenic complex of plants and tubers, as well as the structure and the quality of the harvest. It was revealed that the pre-planting treatment of seed reproductive tubers of super elite and mini-tubers accelerated the phenology of plants, led to earlier emergence of seedlings, the formation of buds and flowers in potatoes, as well as the death of the tops and the onset of physiological maturity of tubers. Plants inoculated with both strains (10-4 and 26 D) were characterized by an increased content of photosynthetic pigments (chlorophyll a, b and carotenoids) and a significantly larger total leaf area during the growing season. It was shown that pretreatment of reproductive tubers super elite with endophytic B. subtilis contributed to an increase in the proline content in the leaves in the second half of the growing season by 36.3 (strain 26D) and 47.3% (strain 10-4) compared with the control, whereas inoculation of mini-tubers with B. subtilis 10-4 ensured the accumulation of proline throughout the growing season: from 3.8 micrograms/g (+52%) in the germination phase is up to 2.5 micrograms/g (+31.5%) in the end-flowering phase. B. subtilis 10-4 reduced the level of MDA accumulation in leaves by 23.4 and 24.5%, respectively, during the germination and budding phase compared with the control (55.5 nMg/g), which indicated a decrease in oxidative stress in plants, and the protective effect was more pronounced in the experiment with mini-tubers. Endophytes (10-4 and 26D) increased the activity of the soil enzyme catalase in the potato rhizosphere in variants with both reproductive super elite and mini-tubers by 2 times (flowering phase) and 1.5 times (harvest ripening) compared with the control. This indicates an intensification of redox processes and an increase in plant resistance. It was revealed that the studied strains 10-4 and 26D successfully colonize the internal tissues of plants, however, the nature of their penetration/colonization (titer changes) depends on the characteristics of the strain itself: before the budding phase, the cells of the root system (strain 10-4) and leaves (strain 26D) were a favorable organ for the symbiotic existence of endophytic bacteria, in particular during the flowering phase, the largest number of bacterial cells of strain 10-4 was noted in the stems, strain 26D – in the leaves of plants. The data obtained by RAPD-PCR analysis confirmed the identity of bacteria isolated from the internal tissues of plants with the used initial strains of B. subtilis 10-4 and 26D. It was found that inoculation of reproductive tubers with B. subtilis 10-4 reduced the prevalence of alternariosis on plants during the budding phase and the end of flowering by 1.5 and 1.9 times, respectively, in the experiment with mini-tubers during the budding phase, no signs of damage were found, by the end of flowering, the lesion was 2.5 times lower compared with the control. The analysis of the microbiota of potato leaves carried out by high-performance sequencing showed that Verticillium dahlia and Cladosporium were found in the phytopathogenic complex of micromycetes, in addition to the predominant Alternaria alternata. Phytopathological analysis of potato tubers conducted after harvesting revealed a 3.4-fold decrease in the incidence of silver scab and dry rot (fusarium) by 4.8 times in endophyte-inoculated variants. Metagenomic analysis of tubers showed that in the phytopathogenic complex of micromycetes, in addition to the predominant causative agents of fusarium (Fusarium solani and Fusarium oxysporum), Alternaria alternata, Alternaria oregonensis, Verticillium dahlia and Helminthosporium solani were found. The species composition of the microbiota of mini-tubers without treatment (control) was represented by the species Fusarium oxysporum, Alternaria alternata and Alternaria oregonensis, in the variant of pre-planting treatment of tubers with B. subtilis 10-4, only Alternaria alternata was identified. In variant B. subtilis 26D – Fusarium solani, Fusarium oxysporum, Alternaria alternata. Thus, pre-planting treatment of reproductive tubers of the superelite with endophytes contributed to a decrease in the prevalence of plant and tuber diseases, and inoculation of healthy mini-tubers ensured a decrease in the species diversity of the pathogenic complex of micromycetes. In B. subtilis inoculated variants, an increase in productivity was revealed due to the weight and number of tubers per bush: by 16.6% and 35.4% in the experiment with reproductive tubers and by 35.5% and 68.5%, respectively, in the experiment with mini-tubers. With double treatment of B. subtilis 10-4, the productivity level remained at the level of a single application, and in the case of 26D, it increased by 12.3% compared with the control. It was found that pre-planting inoculation with strains 10-4 and 26D in all variants contributed to an increase in the content of ascorbic acid, anthocyanins, which indicates not only higher antioxidant properties and nutritional value of the products, but also the potential ability of these tubers to resist infection, both during the growing season and during storage. In all variants, the use of strains 10-4 and 26D had practically no effect on the taste, smell, consistency of the pulp, mealyness and watery of the freshly harvested tubers. However, it contributed to the inhibition (deceleration) of the browning rate of raw and boiled pulp, which is probably due to the revealed increased content of ascorbic acid and anthocyanins with antioxidant properties. The totality of the data obtained indicates that the pre-planting treatment of seed tubers of super elite reproduction and hydroponically growth healthy mini-tubers with endophytic B. subtilis 10-4 and 26D improves the yield and quality indicators of harvest tubers in the field. Moreover, the method of pre-planting enrichment of healthy seed mini-tubers grown on hydroponics with endophytic B. subtilis has an advantage over the pre-planting treatment of super elite reproductive seeds, as an inexpensive, environmentally friendly approach to the production of high-yielding and nutrient-rich potatoes.

Publications

1. Liudmila Pusenkova, Oksana Lastochkina, Sezai Erci sli The Potential of Hydroponic Seed Minituber Enrichment with the Endophyte Bacillus subtilis for Improving the Yield Components and Quality of Potato (Solanum tuberosum L.) Open Access Journal MDPI Agriculture, Basel, Switzerland, Volume 13, Issue 8, P.1626. (year - 2023) https://doi.org/10.3390/agriculture13081626

2. Pusenkova L., Garipova S., Lastochkina O., Shpirnaya I., Valieva V. ВЛИЯНИЕ ЭНДОФИТНЫХ БАКТЕРИЙ BACILLUS SUBTILIS НА АКТИВНОСТЬ МЕТАБОЛИЧЕСКИХ ПРОЦЕССОВ, ПРОТЕКАЮЩИХ В КЛУБНЯХ КАРТОФЕЛЯ ПРИ ХРАНЕНИИ Известия вузов. Прикладная химия и биотехнология, - (year - 2023)