Multirecycled polyfunctional biologics based on Bacillus subtilistogether with compost in potato organic farming

Multirecycled polyfunctional biologics based on Bacillus subtilistogether with compost in potato organic farming | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Multirecycled polyfunctional biologics based on Bacillus subtilis together with compost in potato organic farming Irina Novikova, Julia Titova, Vladislav Minin, Anton Zakharov, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4317900/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Bacillus strains have long been widely and successfully used as the polyfunctional biologics’ basis in various systems for crops cultivation and protection. The research goal was to evaluate application effect from experimental prototypes of multirecycled polyfunctional biologics based on Bacillus subtilis I-5/12–23 together with compost in potato organic farming. A significant stimulation of potato plants Udacha variety growth and development up to the flowering phase was observed regardless of the growing season hydrothermal conditions. The stimulation was by the additive effect of joint biologics and compost use in proportion to its dose. The multirecycled substrate-associated and liquid polyfunctional biologics prototypes together with compost almost doubled the potato tubers biological yield compared to the control regardless the growing season conditions. In the flowering phase, the biological efficacy with respect to the potato fungal diseases incidence and development was 90% under optimal hydrothermal conditions and up to 75% under drought conditions. At the vegetation end the efficacy in the potato fungal diseases development reached 70% (compost efficiency itself more than 45%) regardless of the vegetation period conditions. Four-year scientific and producing approbation of the technological application rules for the biologics’ prototypes based on B. subtilis I5-12/23 and compost developed for the North-West region’s various weather and climatic conditions showed their high efficacy in protection the organic potato. Disease incidence on plants decreased about 80%, on tubers about 50%, their quality improved, and the marketable products yield increase at 9 t ha − 1 (3 t ha − 1 , on average). The application’s technological rules optimizing for various weather and climatic conditions is carried out by changing the consumption norms of protection means in proportion to the limiting factors. Multirecycled polyfunctional biologics prototypes combining the properties of biopesticides, biofertilizers and inoculants can be used to ensure stable organic potato production. Agricultural Engineering Agroecology Food Science & Technology Biotechnology and Bioengineering Applied & Industrial Microbiology plant protection in organic agriculture organic potato farming agro-industrial waste multibiorecycling Bacillus subtilis producer strains organic compost multirecycled substrate-associated and liquid polyfunctional biologics Figures Figure 1 Figure 2 Figure 3 Figure 4 1 Introduction 1.1 Advantages and disadvantages of conventional and organic farming Contemporary intensive crop cultivating technologies cause serious changes in the functioning of agroecosystems [ 1 , 2 ] . Repeated mechanical tillage during the vegetative season constantly disrupts the vital activity of a huge number of living organisms that ensure soil fertility, its structure and suppressivness [ 3 , 4 ] . As a result of plowing, microorganisms' habitat conditions are disturbed, the arable layer microbial composition changes, and thus the ability to natural maintain soil fertility [ 5 , 6 ] . Together increases the weathering and leaching of elements necessary for plants development [ 7 ] . As these negative effects of intensive agricultural practices accumulate, it becomes necessary to use mineral fertilizers and other chemicals to maintain high yields [ 2 , 8 ] . Disturbances in crop health and soil suppressivness due to the composition, structure and functioning of its microbiota lead to plant diseases, and thus to more frequent use of pesticides, high doses of mineral fertilizers as well as to further soil restructuring, poor antagonistic microbiota and shift towards phytopathogenic and phytotoxic species in its structure [ 9 , 10 ] . Organic (natural, biological, precision) farming cannot yet be an alternative to intensive agriculture, as the productivity of such organic production is usually 20% lower compared to conventional intensive one [ 11 – 13 ] . Organic farming excludes the use of pesticides, chemical fertilizers, various plant growth regulators, ionizing radiation, and genetically modified sowing material [ 14 ] . In such farming, the soil is not plowed, but only loosened superficially to 5–10 cm depth [ 15 ] . Intensive cultivation of agricultural crops involves medium or deep soil fertile layer plowing to 25 cm depth and more [ 16 ] . Instead of mineral fertilizers, soil fertility is maintained by mulching with the use of siderates, various composts and organic fertilizers [ 17 , 18 ] . To protect plants from diseases and pests, only biologics are used [ 11 ] . It is biological plant protection that provides opportunities for sustainable organic agriculture, as it is an alternative to the use of chemicals with the advantages of wider application and reduced environmental impact [ 9 , 19 ] . 1.2 Biological crop protection benefits Biological crop protection provides such benefits as: assists in reducing damage caused by phytopathogens and pests, enhances nutrient availability and uptake, improves overall productivity and health of the crop, assists in residue and resistance management, extends the window of application around harvest [ 2 , 20 ] . Development of technological schemes for biologicals applying and their adopting to the particular farm conditions always contribute to greater agrotechnical measures efficiency in every agricultural system [ 21 , 22 ] . The majority of biologics applying technologies initiate and produce functioning one or both ways of natural regulatory mechanisms in agroecosystems [ 9 , 23 ] . The first one uses agricultural technological methods and biofertilizers to produce conditions for natural mass reproduction of antagonists, entomophages and entomopathogens – the originally occurring microbe pool determining natural biocenotic regulation in agroecosystems and soil suppressivness [ 24 ] . Such microorganisms underpin the recycling of nutrients by decomposing organic residues, generate humus, restore fertility and recycle pesticides, as well as promote growth, development and immunity in agricultural crops [ 22 , 25 ] . The second way makes the artificial saturation of biocenosis by various polyfunctional biologics applying, the producer strains of which are the microorganisms that possess not only antagonistic, but also soil-fertilizing, phytoregulatory, and other properties due to their polyfunctionality: the ability to synthesize a wide range of secondary metabolites [ 9 , 26 ] . High ecological adaptability of such microorganisms is the result of their polyfunctionality [ 27 ] . The particular necessity lies in the technologies development for biologics producing and applying used in organic farming [ 28 ] . The most important is the various necessary properties of fertilizer, stimulant and biopesticide combining in one contemporary polyfunctional biological [ 29 , 30 ] . In addition, inoculants and biopesticides for crop production have rather strict requirements: pathogenic and toxic properties absence dangerous for humans, warm-blooded animals, beneficial insects, fish and soil microorganisms; high target activity in maximum beneficial properties manifestations (antagonism to pathogens, nitrogen fixation, phosphate mobilization, etc.); producibility in obtaining and applying in agriculture conditions [ 25 , 31 , 32 ] . Biologics should be convenient for transport, store and application. Biologics use optimizing helps the practical solution of many problems related to both environment biological purification, fertility maintenance and full quality yield obtaining [ 31 , 33 ] . In addition, such optimization allows to minimize the main risks of organic crop cultivation which are ensuring plants mineral nutrition and agroecosystems phytosanitary optimization. Organic fertilizers and various composts use reduces the first risks, and biological plant protection is a key element in ensuring a stable phytosanitary condition [ 22 , 34 ] . 1.3 Contemporary polyfunctional biologics producing technologies For the development of polyfunctional biologics, the most interesting microbes are those that are non-pathogenic for humans and beneficial organisms, capable of producing a wide range of secondary metabolites, with low demand for their cultivation conditions, high producibility and great ecological adaptability [ 22 , 25 , 35 ] . The development of such technology comprises a number of stages, the foremost of which is selection and optimization of nutrient substrates [ 25 , 36 ] . The composition of components in the substrates should not adversely affect their biological activity; they should underpin the desired polyfunctionality [ 22 , 25 ] . Since the foundations of polyfunctional biologics are both the cultures of living microorganisms and the products of their metabolism (toxins, enzymes, etc.), there exist various approaches toward cultivation of producer strains [ 37 ] . Submerged and solid phase fermentations are enough technologically to result in a highly standardized final product as polyfunctional biologic [ 38 ] . Recycling of agro-industrial wastes allows the return of useful components to the biosphere cycle, to ensure sustainability and increase the efficiency of production processes, and to preserve the biological diversity [ 39 ] . The problem is that most microorganisms are unable to provide thorough biorecycling of agro-industrial wastes because they contain hard-to-degrade lignocellulose complex [ 40 ] . Wood-destroying basidiomycetes are the only known group of organisms capable to actively degrade lignin all the way to complete mineralization [ 41 ] . Basidiomycetes are an important component of biocenoses, they have a significant role in the biodegradation processes during which the biogenic substances are regenerated [ 42 ] . Therefore, biorecycling of lignin-cellulose complexes with xylotrophic basidiomycetes is a promising feature not only for resource saving and low waste, but also as an environmentally friendly technology [ 43 ] . Agro-industrial wastes have long been utilized in the production of edible mushrooms [ 44 ] . In the process of their cultivation, annually renewable materials are recycled, which are the organomineral mixtures [ 43 , 45 ] . During the cultivation of edible mushrooms on low-value plant wastes, their enrichment with fungal protein and easily degradable carbohydrates, micronutrients, vitamins and biologically active substances, which as the result of biorecycling are also included into the mycelium enzyme complexes composition [ 41 , 43 ] . Such enriched substrate, after harvesting of fruit bodies, is a very valuable biotechnology product for subsequent stages of its biorecycling [ 46 , 47 ] . Such biorecycled wastes are the cheapest, nutrient-rich and readily available substrates for cultivation of producer strains and the development of multi-biorecycled substrate-associated polyfunctional biologics (MSAPB) based on them [ 42 , 48 ] . In such biologics, both the potential of SMS substrates as organic fertilizers and biostimulants, and polyfunctionality traits of producer strains are employed. At the same time, the development of a methodology for their production is an important step in sustainable resource management, turning wastes into useful products and making agricultural technologies eco-friendlier [ 40 , 43 , 49 ] . 1.4 Bacillus producer strains use for multibiorecycled polyfunctional biologics developing Bacillus bacteria, one of the most widespread, diverse and commercially useful groups of microorganisms, are widely used as biologics producer strains [ 50 , 51 ] . The low pathogenicity of Bacillus species and the diversity of metabolic processes have caused the representatives of this group to be used in various industrial fields [ 52 ] . They are extremely unpretentious with respect to growth conditions and are capable of producing a great biological active substances variety of protein nature, which can induce the plant disease resistance [ 25 , 50 ] . Bacillus strains having polyfunctionality – the ability to synthesize a wide range of hydrolytic enzymes and active secondary metabolites –are capable to utilize in solid-phase fermentation the spent mushroom substrates (SMS) and their aqueous extracts in liquid-phase one, as the cheapest, nutrient-rich and available substrates for producer strains cultivation and multirecycled substrate-associated (MSAPB) and liquid polyfunctional biologics (MLPB) development based on them [ 42 , 45 ] . Such biologics utilize both the SMS potential as biofertilizers and biostimulants and the producer strains polyfunctionality as well [ 38 ] . Combining various properties in one bioproduct is the most important trend in resource saving, waste transforming into useful products and agriculture greening [ 42 , 48 ] . The research goal was to evaluate application effect from experimental prototypes of multirecycled polyfunctional biologics based on B. subtilis I-5/12–23 together with compost in potato organic farming. To achieve this goal, the tasks for production and application technological rules developing for polyfunctional biologics experimental prototypes in substrate-associated and liquid formulations and organic compost for optimal and dry growing seasons were solved and phytoregulatory activity and biological efficacy estimating as well. 2 Materials and methods The research was conducted in FSBSI VIZR Microbiocontrol Laboratory and in organic crop rotation, located at the IEEP branch Experimental Station of the FSBSI FSAC VIM. Chemicals have not been used on these lands for more than 20 years. Only biologicals and organic fertilizers are used in production multi-field crop rotation with assessment of their biological efficiency. The station is located in the south of St.-Petersburg region (northwestern Russia). Organic Experimental Station of the FSBSI FSAC VIM has sod-podzolic light loamy gley soil on residual carbonate moraine loam characterized by reaction close to neutral and increased content of organic matter. Research studies covered optimal and dry growing seasons. 2.1 Objects The research objects were the MSAPB and MLPB prototypes liquid and granulated formulations and organic compost in various doses. The MSAPB and MLPB prototypes were obtained by sequential multibiorecycling the sterilized agro-industrial waste by Lentinula edodes (Berk.) Pegler, Pleurotus ostreatus (Jacq.) P. Kumm. and further by B. subtilis I5-12/23 (Ehrenb.) Cohn. The biologics prototypes were developed in accordance to FSBSI VIZR approved regulations, specifications and producer strain’s toxicological passport. The high active producer strain B. subtilis I-5 12/23 is certified, deposited and maintained at FSBSI VIZR State Collection of Microorganisms Pathogenic for Plants and Their Pests registered on 28.01.1998 No. 760 in the World Federation for Culture Collections, World Data Center for Microorganisms (WDCM WFCC, Japan). Prototypes titer was not less than ×10 10 colony forming units (CFU) g − 1 for granulated MSAPB and was not less than ×10 11 CFU mL − 1 for MLPB ones. The biologics experimental prototypes were used to protect organic potatoes from fungal and bacterial diseases during vegetation and storage. The active units of biologics experimental prototypes were cells (spores) and B. subtilis I-5 12/23 metabolome; formulations were the suspension concentrate (SC) and granulated (G) ones. The compost characterized by nearly 40% dry matter was prepared in IEEP’s Lab for organic waste bioconversion from chicken manure using bioconvector. Depending on the season weather conditions, compost doses in terms of nitrogen were 80, 110, 160 kg N×ha − 1 [ 53 ] . 2.2 Materials The materials were semi-synthetic and natural media, such as dried nutrient medium (DNM): pancreatic sprat hydrolysate – 15 g L − 1 ; NaCl – 4.59 g L − 1 ; microbiological agar – 20 g L − 1 ; H 2 O – 1 L; pH = 7.2 (Microgen Co. Ltd., Russia); natural nutrient media based on multirecycled spent mushroom substrates (SMS) and their aqueous extracts: double spent SMS – 200 g L − 1 , H 2 O – 1 L, pH = 6.5–7.5 (FSBSI VIZR, Russia). Multirecycled SMS was obtained as sequential solid-phase fermentation the agricultural and technogenic sphere waste by macromycetes L. edodes (shiitake mushroom) and P. ostreatus (oyster mushroom). Such waste content as commercial L. edodes cultivating substrate was: oak sawdust – 88.9%, wheat bran – 10%, CaCO 3 – 0.1%, CaSO 4 × 2H 2 O – 1%. The multirecycled SMS obtaining process included a sequential vegetative and reproductive stages’ change of L. edodes 4080, then P. ostreatus HK-35 (Sylvan, Inc.). The double mushroom culture industrial waste after basidiomata gathering were classified as a multirecycled granulated SMS – another research material. This waste substrate was used for solid-phase fermentation by producer strain after its components crushing into 0.5–2.5 cm pieces and soaking in water for 20–24 h to complete moisturizing. The prepared multirecycled granulated SMS having 70–80% moisture content and stabilized pH = 7.0–7.5 were packed in 1500 g each in 1 L polypropylene bags. The bags were sealed up and sterilized for 1 h at 133°C (202.7 kPa). For the liquid-phase fermentation the double spent (multirecycled) SMS were used as preliminarily milled, and boiled in amount of 200 g L − 1 for 1 h, filtered, and restored to the previous 1 L volume. Sterilization modes for the liquid nutrient media were 30–60 min at 50.7–81.1 kPa, for the granulated SMS was 60 min at 202.7 kPa in steamer 5075ELVPV D (Tuttnauer Europe BV, Netherlands). The bedding poultry manure supplied by Leningrad Region poultry farm was used as an organic material for the compost production [ 54 ] . 2.3 Research Methods The standard micro-, myco- and phytopathological research methods were used in the work: liquid- and solid-phase fermentation for the inoculums and stock cultures, for biologics prototypes developing; SMS preparing for multirecycling; serial dilutions’ method for titers assessing and the quality biologics experimental prototypes; conducting field tests, field testing results and potato yield accounting. There used 4–5-fold replications in major trials including the field ones [ 42 , 48 , 55 – 61 ] . The MSAPB and MLPB prototypes were developed by liquid- and solid-phase fermentations in FSBSI VIZR Microbiocontrol lab. The inoculums’ and prototypes’ titers were determined by the serial dilutions’ method on DNM. The producer strain inoculum was preliminarily stored and developed in test tubes on the same nutrient medium. The liquid-phase fermentations were developed in shaking flasks with 750 mL volume, containing 100 mL of SMS-extract. Submerged producer strain inoculum was grown at 27–28°C for 3 days with aeration (180 rpm, New Brunswick™ Innova® 44 Shaker Incubator, Eppendorf, Germany). The samples were taken away every day to control the bacterial growth stage and the contamination as well. The fermentation process finished at 85–90% spores had been produced in the culture liquid (CL). To obtain the final formulation the CL was concentrated for 10 min at 3000 rpm in the centrifuge OS-6MC (Dastan Inc., Kyrgyzstan) and then 0.2% potassium sorbate was added to the spore suspension concentrates put into 1 L wide-mouth bottles, PE-LD (VITLAB GmbH, Germany). The initial CL titers were not less than ×10 10 , the finished ones were near ×10 11 CFU mL − 1 [ 42 , 48 ] . The solid-phase fermentation of the 1500 g multirecycled SMS with the help of B. subtilis I-5 12/23 was occurred in sterile 1 L polypropylene bags having after its inoculation with submerged producer strain's cultures in sterility conditions. Incubation was carried out during 5 days at 27–28°C in a thermostatic chamber PRO TC 30/120–500 (SIA Prooborudovanie Co. Ltd., Russia) using substrate shaking and stirring every other day until the last one was completely overgrown. The MSAPB prototypes titers were not less than ×10 10 CFU g − 1 [ 56 ] . The compost was produced by aerobic solid-phase fermentation of bedding poultry manure in the special biofermenter (IEEP–BRANCH OF FSAC VIM, St-Petersburg, Russia) [ 54 , 57 ] . The compost shelf life is 2 years from the production date under − 20–+30°C air temperature and 60–75% air humidity. The MSAPB and MLPB prototypes based on B. subtilis strain I-5 12/23 as well as the compost were developed for field trials of their biological efficacy. The area used for the field trials carried out in 2019–2022 was located at 59°65 N and 30°38 E (Leningrad region, Russia). The soil in the organic crop rotation is well cultivated and has up to high levels of available P and K, pH = 6.5–6.6 and 5.6% organic matter content as well. The compost was used before ridging and its subsequent embedding was made by the ridge space loosening with a chisel cultivator (IEEP–BRANCH OF FSAC VIM, St-Petersburg, Russia) up to 30 cm depth from the bottom of the furrow. Such a deep loosening between organic potatoes rows as an inter-row treatment resulted in a decrease in soil compaction both in the potato rooting zone and directly in the aisle together with hilling and destroying weeds. Super elite and elite classes of potato variety Udacha was used in the field trials. The 30–50 t ha − 1 average yield and 12–15% starch content are this variety characteristics. Tubers mature early and this variety is well adapted to various types of soil and climatic zones. Udacha variety tubers have good taste and a smooth surface [ 57 ] . It is midresistant to late blight, rhizoctonia disease, wrinkled mosaic, black leg, wet rot and common scab (State register of selection achievements approved for use in Russian Federation from FSBI “State Breeding Commission”, 2022). Potato tubers and then plants were three-fold treated with B. subtilis I-5 12/23 formulations at planting and then by foliar spray after 10 days and 20 days at the MLPB prototypes’ consumption rate 3 and 6 L ha − 1 under optimal and dry growing conditions, respectively. Spraying was carried out with the help of specially designed sprayer installed on the planter and cultivator. Second week after planting the inter-row cultivation was begun and the carried out regularly using the row-crop cultivator for deep loosing of inter-rows (IEEP–BRANCH OF FSAC VIM, St-Petersburg, Russia). Weeds were removed mechanically using small rotary harrow BRU-0.7 harrows mounted on the cultivator. The reciprocally orthogonal scheme of field trials was applied with standard placement of variants (3 series): 4 replicates in a complete randomized design [ 58 – 60 ] . Accounting plot size according to growing season conditions was 20–60 m 2 . The studied indicators were the potato plants developmental biometry and productivity, diseases incidence and their development in dynamics. Potato plant growth rate (mm/day) and productive/flowering stems number (pieces) parameters and the healthy tubers crop were used to evaluate phytoregulatory activity. To assess the damage of fungal disease complex to plants and potato tubers, the disease incidence and development on potato plants and on tubers, crop losses, biological efficacy were used. The dominant fungal diseases on potato plants during research period were leaf spots, late blight, Fusarium wilt and rhizoctoniosis. On tubers there were common and silver scab, anthracnose, late blight and rhizoctoniosis. The field trials data were gathered 5 times: 2 biometric accountings with disease symptoms appearance fixating were carried out on 3–5-week-old seedlings in the 1–3 d leaf tiers phase and on 6–7-week-old potato plants in the 9–10th leaf tiers phase; 2 phytopathological accountings were carried out at the beginning and ending of the flowering phase; 1 accounting was carried out at tubers harvesting. Every third potato plant in the 4-fold trial plot (up to 100 plants) was examined [ 58 – 61 ] . 2.4 Statistical processing methods Statistical processing was produced using the Microsoft Excel 2010 and Statistica 10.0 software packages (StatSoft, Inc., Tulsa, OK, USA). Statistics included checking the analyzed data normal distribution by Shapiro-Wilk's W -test, calculating the means ( M ) and standard errors (± SEM ) as well, analysis of variance ( ANOVA) . In options pairwise comparison the statistical differences significance was assessed by Student's t- test [ 62 , 63 ] . 3 Results All the results obtained in various research years on the peculiarities of organic potato cultivation in the conditions of northwest Russia are considering the weather conditions of vegetation seasons. 3.1 Weather conditions in the research years The weather conditions in late spring and summer of the research years differed to various significant extent from each other (Table 1 ). Research years 2019 and 2021 were warm and to various extent dry, meanwhile 2020 and 2022 were wet and not so warm. The hottest and driest year was 2021, and the coolest and wettest year was 2020. The month May was the coolest opposite to July that was the warmest month during every vegetation season. The weather conditions in optimal 2020 and 2022-years vegetation periods were characterized by essentially comfortable temperatures and suitable rainfall during the active potato development. Table 1 Summer weather conditions in the research years month weather indicator, hydrothermal coefficient (HTC) yearly average average for cumulative years 2019 2020 2021 2022 - May temperature °С 12.1 10.0 11.6 8.5 11.3 precipitation, mm 79.3 53.0 172.0 15.0 46.0 HTC 2.1 0.6 0.3 0.3 - June temperature °С 18.7 19.2 20.9 17.4 15.7 precipitation, mm 79.3 129.4 16.6 47.6 71.0 HTC 1.4 2.3 0.3 0.9 - July temperature °С 16.5 17.6 22.0 18.7 18.8 precipitation, mm 179.8 186.2 16.8 85.2 79.0 HTC 3.5 3.4 0.3 1.5 - August temperature °С 17.0 17.2 15.8 20.0 16.9 precipitation, mm 94.6 195.9 109.2 149.6 83.0 HTC 1.8 3.8 2.2 1.7 - The maximum precipitation for the entire growing seasons was registered in 2020. To establish the aridity conditions for a certain period in a given area one can use Selyaninov’s Hydrothermal Coefficient (HTC). It is determined by the ratio of the precipitation amount during the growing season to the sum of temperatures above 10°C, reduced 10-fold: the natural moistening conditions are considered optimal, if HTC 1–2, if less than 1 are considered dry [ 64 ] . It helped in defining the driest conditions in June and July 2021 for the active potato development. The HTC ratios allowed grouping all the data obtained into optimal and dry periods for potato plant development and yield formation. 3.2 Phytoregulatory activity as bioinoculants The phytoregulatory activity study of various compost doses, the polyfunctional MSAPB and MLPB prototypes based on B. subtilis I-5/12–23, as well as their various combinations applied in the experimental variants in every studied case revealed their positive effect on the potato plants growth and development (Fig. 1). The pronounced tendency of growth and development stimulation after various compost doses application as well as their combinations with polyfunctional MSAPB and MLPB prototypes is typical for dry growing seasons, when against the background of not very favorable development conditions organic fertilizers and biologics’ experimental batches help potato plants to grow and develop (Fig. 1b). Under optimal growing conditions, such a trend is not so obvious, and the potato plants growth rate of potato plants in various variants of compost and MSAPB and MLPB prototypes application was fixed within the measurement error (Fig. 1a). Nevertheless, in all cases it was higher than the control values without compost and MSAPB and MLPB prototypes applying (Fig. 1a). For such an important indicator in potato cultivating as the productive/flowering stems number per bush, which ultimately determines the productivity of plants, there was found a direct proportional dependence on the compost various doses application regardless of the growing season conditions (Fig. 1). Reliability of differences was recorded under optimal conditions for the development of organic potato (Fig. 1a). Although the trend was also characteristic for dry vegetative periods (Fig. 1b). Polyfunctional MSAPB and MLPB prototypes based on B. subtilis I-5 12/23 applying without compost completely replaced its application regardless of organic potatoes vegetation period weather and climatic conditions (Fig. 1). Under optimal growing conditions for organic potatoes, no significant differences in plant development were observed after MSAPB and MLPB prototypes application (pre-planting treatment of tubers or application into the soil at planting) (Fig. 1a). When cultivated in dry periods, the application of granular forms into the soil was significantly ( P < 0.05) more important: growth and development indicators of potato plants were higher (Fig. 1b). The same trends in phytoregulatory activity described by the parameters of potato plant growth rate and productive stems formation (Fig. 1) were revealed in the potatoes total yield forming under the organic compost application and under the influence of polyfunctional MSAPB and MLPB prototypes based on B. subtilis I-5 12/23 applied (Fig. 2). There was observed a significant and reliable ( P < 0.01) increase in total biological yield in every trial variant compared to the control regardless the growing season conditions (Fig. 2). The same way in all variants of the compost application recorded the dose-effect relationship: a significant and in most cases reliable ( P < 0.05) increase in the total potatoes biological yield with compost dose increasing, and regardless growing season conditions as well (Fig. 2a, b). Under the influence of polyfunctional MSAPB and MLPB prototypes based on B. subtilis I-5 12/23, a significant reliable increase in the total biological yield ( P < 0.05) of organic Udacha potatoes compared to the control was observed in 1.3 times on average by 5 t ha − 1 under both optimal and dry growing season conditions (Fig. 2). Moreover, the effect of experimental biologics prototypes was equivalent to the low and medium nitrogen doses of compost applying (Fig. 2a, b). Maximum and reliable ( P < 0.01) increase in potato yield by 1.5 times on average by 9 t ha − 1 under both optimal and dry growing season conditions was achieved by the coeffect of experimental biologics prototypes and high organic compost doses (Fig. 2a, b). Regardless the vegetation period conditions of organic potato development, no significant differences in yield forming were observed after various biologics prototypes formulations applying (pre-planting tubers treatment or application into the soil at planting) (Fig. 2a, b). In polyfunctional MSAPB and MLPB prototypes based on B. subtilis I-5 12/23 and compost coapplying a reliable ( P < 0.05) increase in the total biological yield of Udacha organic potato under every weather condition during cultivation was recorded when the compost dose was increased (Fig. 2a, b). The amount of organic potatoes disease damaged yield was regulated mainly by polyfunctional MSAPB and MLPB prototypes applying – decrease observed in 3.5 times (Fig. 2a, b). This trend was especially pronounced during suboptimal and dry periods in organic potato development (Fig. 2b). The compost dose increasing often had a positive effect on the 3–7 times reduction of the disease damaged yield (Fig. 2a, b). 3.3 Protective activity as biopesticides The protective effect of the polyfunctional MSAPB and MLPB prototypes applying sometimes together with compost was evaluated using the biological efficacy indicator (Fig. 3). To identify biological efficacy relative dynamics, it was evaluated both in the fungal disease complex incidence and development of organically cultivated potato (Fig. 3). The data presented in Fig. 3 indicate high biological efficiency of the applied polyfunctional MSAPB and MLPB prototypes – 50–95% in optimal and 50–75% in dry vegetation periods in organic potato variety Udacha development. Suboptimal vegetative conditions mostly reduced the biological efficacy in the fungal disease complex development (Fig. 3b). Biological efficiency in diseases incidence on organic potatoes was 8–30% inferior to that in their development on plants (Fig. 3a, b). The most effective (60–95%) in the protection of organic potato variety Udacha against a diseases complex were combinations of polyfunctional MSAPB and MLPB prototypes and medium compost doses (Fig. 3a, b). The intrinsic compost biological efficiency did not exceed 50%, and with increasing dosage it decreased both under optimal and suboptimal development conditions (Fig. 3a, b). Under optimal growing organic conditions for potato plants revealed the maximum field efficacy in polyfunctional MSAPB and MLPB prototypes application – 75–95% (Fig. 3a). During the organic potatoes’ development in dry periods, the polyfunctional MSAPB and MLPB prototypes various formulations applying allowed to protect (biological efficiency 50–70%) plants from diseases complex most effectively (Fig. 3b). Compost application did not always support the protective effect from the polyfunctional MSAPB and MLPB prototypes application regardless the growing season conditions (Fig. 3a, b). Biological efficacy of the polyfunctional MSAPB and MLPB prototypes application, conditioning the disease development level on plants of organically cultivated Udacha variety potatoes was 5–20% higher than that in disease incidence both in optimal and dry conditions of potato cultivation (Fig. 3a, b). Biological efficacy of applied MSAPB and MLPB prototypes protecting the organic potato yield was 15–55% in optimal and 10–17% in dry periods of Udacha variety potato organic development. Suboptimal vegetative conditions the most reduced biological efficacy in fungal disease complex development on tubers (Fig. 4b). Biological efficacy in disease incidence on tubers was 5–35% inferior to that in disease development and only under optimal growing conditions (Fig. 4a, b). The most effective (35–55%) in protecting the yield of variety Udacha organic potato were combinations of various MSAPB and MLPB prototypes formulations (Fig. 4a). The intrinsic biological efficacy of the compost did not exceed 5–18%, and with dosage increasing it fell both under optimal and suboptimal development conditions (Fig. 4a, b). Under optimal growing conditions of organic potatoes revealed the maximum biological efficiency in polyfunctional MSAPB and MLPB prototypes applying for tubers crop protection – 50% (Fig. 4a). During the organic potatoes’ development in dry periods, the use of polyfunctional MSAPB and MLPB prototypes various formulations allowed to protect the yield from the diseases complex with 10% biological efficacy (Fig. 4b). 4 Discussion 4.1 Ways to use antagonist microbes in biocontrol There are two ways to use antagonist microbes in phytopathogens microbiocontrol. One of them involves the conditions developing for natural mass reproduction of microbes through agronomic practices and fertilizers [ 6 ] . Another one is based on artificial saturation of agrobiocenosis with antagonistic microorganisms through the various biologics use, and better polyfunctional ones, as they have a direct antagonistic effect on pathogens due to the BAS complexes and increase the stress resistance in plants and their immunity to diseases due to their phytoregulatory activity [ 22 , 51 ] . These two ways formed the contemporary approaches to the production of polyfunctional biologics which should suit two concepts of phytosanitary optimization and long-term biocenotic harmonization of agroecosystems [ 25 , 65 ] . The first one is based on the use of natural resources of microorganisms possessing complex biological activity, in particular polyfunctionality: the ability to synthesize a wide range of secondary metabolites developed in a long evolutionary process of soil-dwelling microorganisms under rigid natural selection in extremely saturated habitat [ 22 , 66 ] . High ecological adaptability of microorganisms is the result of their polyfunctionality [ 67 ] . According to the second concept, polyfunctional microorganisms that produce a variety of BAS complexes as secondary metabolites, that turned out to be the most competitive as a result of evolution, are incorporated into a pool of natural enemies of detrimental organisms in agroecosystems regulating the number and harmfulness of the latter [ 25 , 68 ] . 4.2 Polyfunctional biologics final formulation produce the efficacy guaranty For the manifestation by producer strains their polyfunctionality, the final formulation obtained during the final technological stage of biologics production is very important [ 69 , 70 ] . This final step is closely related to the technologies for biologics' applying and affects their storage duration [ 71 ] . The biologic efficiency depends on the extent to which the final formulation will contribute to the producer strain potential manifestation [ 72 ] . Biologics standardizing and quality assessing are related to the final form [ 73 ] . The main requirements for such final formulations are: long-term (at least 1–2 years) preservation of microorganisms’ cells viability, biological activity during biologics storage and their application. Nowadays final biologics’ formulations are liquid (fluids, suspensions, concentrates of suspensions, pastes), dry (wetting powders, tablets), immobilizing formulations with nutrients for producer strains: dispersive solid bulk (on the basis of peat, vermiculite, chitin-chitosan carriers), granulated (based on hydrogels and porous mineral sorbents, some of them crafted as nanoscale materials) [ 8 , 74 ] . Some final formulations having unacceptable additives used in technological schemes for polyfunctional biologics producing cannot participate in organic crop production [ 11 ] . Providing organic agriculture with suitable plant protection products caused to develop new approaches to the polyfunctional biologics production through multibiorecycling of agro-industrial wastes [ 31 , 47 ] . Such approaches provide the necessary substrate–producer strain association and the latter polyfunctionality, which lead modern biologics to combine in their final formulations the properties of biofertilizers, biostimulants and biopesticides, providing a sustainable increase in crop yields with their application [ 34 , 49 , 75 ] . 5 Conclusions Multibiorecycled polyfunctional MSAPB and MLPB biologics prototypes combining the properties of biopesticides, biofertilizers and biostimulants (inoculants) can be used to ensure stable organic potato production. The substrate base of such experimental prototypes obtained by multibiorecycling technogenic and agricultural waste by edible shiitake and oyster mushrooms – the product enriched with protein, vitamins, macro- and microelements – provided enhanced development of active producer strain B. subtilis I-5 12/22 with a titer of ×10 10 CFU g − 1 for granulated MSAPB and not less than ×10 11 CFU mL − 1 for MLPB ones. Polyfunctional MSAPB and MLPB prototypes based on B. subtilis I-5 12/23 strain showed growth-stimulating and protective effects tested in field trials in organic production of the variety Udacha potato. High biological activity of B. subtilis strain I-5 12/23 in all formulations of biologics experimental batches based on it provided reliable ( P < 0.01) 1.2–2.5 times increase in biometric indices of organic potato plant development as well as 1.2–1.7 times in tuber yield under optimal and dry vegetative conditions. Four-year scientific and producing approbation of the technological application rules for the MSAPB and MLPB prototypes based on B. subtilis I5-12/23 and compost developed for the various weather and climatic conditions of northwest Russia showed their high efficacy in protection the organic Udacha variety potato. Disease incidence on vegetative potato plants decreased by 50–95%, on tubers by 25–70%, their quality improved, and the marketable products yield increase at 9 t ha − 1 (3 t ha − 1 , on average). The application's technological rules optimizing for various weather and climatic conditions is carried out by changing the consumption norms of protection means in proportion to the limiting factors. Declarations Acknowledgements: this research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4317900","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":294982381,"identity":"116f215b-595f-450a-8a06-a171cba5c384","order_by":0,"name":"Irina Novikova","email":"","orcid":"https://orcid.org/0000-0003-2816-2151","institution":"Federal State Budget Scientific Institution All-Russian Research Institute of Plant Protection” (FSBSE VIZR), St. Petersburg, Russia","correspondingAuthor":false,"prefix":"","firstName":"Irina","middleName":"","lastName":"Novikova","suffix":""},{"id":294982382,"identity":"f63b9198-e97a-401e-9835-29ad097a42c9","order_by":1,"name":"Julia Titova","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYBACxgbmhgMJFf/k+EE8HuK0MDYceHDmgLFkA7FawJoeth1I3HCAWC3MMxIbDyS23TE2vpH87MGbisMM5jMSCNgxIxHol3PP5MxupJkbzjmTxiBzgygtZczGZjcSzKR522wYJCSI0sLGnLh5Rvo3ad5/EsRqaTucuEEiB2hLAzG29DwEajmTZixx5k2Z5JxjaTwSPA/wazFsTz788UeFjRx/e/o2iTc1h+Uk2AnYYtgAYwlAVBKOGnk4i/8AQcWjYBSMglEwQgEAFo1LeLkiNmMAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-8188-1852","institution":"Federal State Budget Scientific Institution All-Russian Research Institute of Plant Protection” (FSBSE VIZR), St. Petersburg, Russia","correspondingAuthor":true,"prefix":"","firstName":"Julia","middleName":"","lastName":"Titova","suffix":""},{"id":294982383,"identity":"74a82b26-9308-44d3-8c09-24b784a2662d","order_by":2,"name":"Vladislav Minin","email":"","orcid":"https://orcid.org/0000-0002-5207-8921","institution":"Institute for Engineering and Environmental Problems in Agricultural Production (IEEP) – branch of Federal State Budgetary Scientific Institution «Federal Scientific AgroEngineering Centre of All Russia Institute for Mechanization» (IEEP FSBSI FSAC VIM), Saint Petersburg, Russia","correspondingAuthor":false,"prefix":"","firstName":"Vladislav","middleName":"","lastName":"Minin","suffix":""},{"id":294982384,"identity":"2fbae281-704b-4c77-ae0f-64803782e905","order_by":3,"name":"Anton Zakharov","email":"","orcid":"https://orcid.org/0000-0003-3501-0543","institution":"Institute for Engineering and Environmental Problems in Agricultural Production (IEEP) – branch of Federal State Budgetary Scientific Institution «Federal Scientific AgroEngineering Centre of All Russia Institute for Mechanization» (IEEP FSBSI FSAC VIM), Saint Petersburg, Russia","correspondingAuthor":false,"prefix":"","firstName":"Anton","middleName":"","lastName":"Zakharov","suffix":""},{"id":294982385,"identity":"827b667b-9b72-46bf-b8c3-c46527e80371","order_by":4,"name":"Irina Krasnobaeva","email":"","orcid":"https://orcid.org/0000-0001-9166-4475","institution":"Federal State Budget Scientific Institution All-Russian Research Institute of Plant Protection” (FSBSE VIZR), St. Petersburg, Russia","correspondingAuthor":false,"prefix":"","firstName":"Irina","middleName":"","lastName":"Krasnobaeva","suffix":""},{"id":294982386,"identity":"92e9147e-da86-47f7-98ef-96110efbd19b","order_by":5,"name":"Eugeniy Murzaev","email":"","orcid":"https://orcid.org/0000-0001-5143-7665","institution":"Institute for Engineering and Environmental Problems in Agricultural Production (IEEP) – branch of Federal State Budgetary Scientific Institution «Federal Scientific AgroEngineering Centre of All Russia Institute for Mechanization» (IEEP FSBSI FSAC VIM), Saint Petersburg, Russia","correspondingAuthor":false,"prefix":"","firstName":"Eugeniy","middleName":"","lastName":"Murzaev","suffix":""}],"badges":[],"createdAt":"2024-04-24 11:34:39","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-4317900/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4317900/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55277591,"identity":"051f322b-ebdd-42b5-b8a2-68379393a5e5","added_by":"auto","created_at":"2024-04-25 05:30:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":350112,"visible":true,"origin":"","legend":"\u003cp\u003ePhytoregulatory activity of the polyfunctional MSAPB and MLPB prototypes based on \u003cem\u003eB.\u0026nbsp;subtilis\u003c/em\u003e I-5 12/23 and compost in doses of 80, 110 and 160 kg N ha\u003csup\u003e−1\u003c/sup\u003e in organic potato growing during: a – optimal vegetative season; b – dry vegetative season.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4317900/v1/5853ff813ddeac3dd0868fff.png"},{"id":55277593,"identity":"a704a98a-4d05-4fc1-bd58-ed4d08e10cf1","added_by":"auto","created_at":"2024-04-25 05:31:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":414450,"visible":true,"origin":"","legend":"\u003cp\u003eTotal biological organic potatoes yield and damaged yield affected by polyfunctional MSAPB and MLPB prototypes based on \u003cem\u003eB.\u0026nbsp;subtilis\u003c/em\u003e I-5 12/23 and compost in doses of 80, 110 and 160 kg N ha\u003csup\u003e−1\u003c/sup\u003e during: a – optimal vegetative season; b – dry vegetative season.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4317900/v1/e22785c8b00cfc8c8380d251.png"},{"id":55277590,"identity":"8b4cdcb9-f702-4cfa-a6d7-17df6ef5f6ce","added_by":"auto","created_at":"2024-04-25 05:30:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":366215,"visible":true,"origin":"","legend":"\u003cp\u003eBiological efficacy of application the polyfunctional MSAPB and MLPB prototypes based on \u003cem\u003eB. subtilis\u003c/em\u003e I-5 12/23 and compost in doses of 80, 110 and 160 kg N ha\u003csup\u003e−1\u003c/sup\u003e in protecting the organic potato plants from the dominant fungal diseases complex during: a – optimal vegetative season; b – dry vegetative season.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4317900/v1/bcda901322678243fef12bcb.png"},{"id":55277572,"identity":"642c1299-6f8d-4c49-a9fe-82637b9cc398","added_by":"auto","created_at":"2024-04-25 05:30:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":205751,"visible":true,"origin":"","legend":"\u003cp\u003eBiological efficacy of application the polyfunctional MSAPB and MLPB prototypes based on \u003cem\u003eB. subtilis\u003c/em\u003e I-5 12/23 and compost in doses of 80, 110 and 160 kg N ha\u003csup\u003e−1\u003c/sup\u003e in protecting the organic potato yield from the dominant fungal diseases complex during: a – optimal vegetative season; b – dry vegetative season.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4317900/v1/686c71c3e079132de8ded945.png"},{"id":55277626,"identity":"84737320-759b-403a-8940-e2a3784ba9df","added_by":"auto","created_at":"2024-04-25 05:31:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1780342,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4317900/v1/69018791-7a67-4ee6-a785-d360174d43d3.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eMultirecycled polyfunctional biologics based on \u003cem\u003eBacillus subtilis\u003c/em\u003etogether with compost in potato organic farming\u003c/p\u003e","fulltext":[{"header":"1 Introduction","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003e1.1 Advantages and disadvantages of conventional and organic farming\u003c/h2\u003e \u003cp\u003eContemporary intensive crop cultivating technologies cause serious changes in the functioning of agroecosystems \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Repeated mechanical tillage during the vegetative season constantly disrupts the vital activity of a huge number of living organisms that ensure soil fertility, its structure and suppressivness \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. As a result of plowing, microorganisms' habitat conditions are disturbed, the arable layer microbial composition changes, and thus the ability to natural maintain soil fertility \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Together increases the weathering and leaching of elements necessary for plants development \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. As these negative effects of intensive agricultural practices accumulate, it becomes necessary to use mineral fertilizers and other chemicals to maintain high yields \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Disturbances in crop health and soil suppressivness due to the composition, structure and functioning of its microbiota lead to plant diseases, and thus to more frequent use of pesticides, high doses of mineral fertilizers as well as to further soil restructuring, poor antagonistic microbiota and shift towards phytopathogenic and phytotoxic species in its structure \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOrganic (natural, biological, precision) farming cannot yet be an alternative to intensive agriculture, as the productivity of such organic production is usually 20% lower compared to conventional intensive one \u003csup\u003e[\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Organic farming excludes the use of pesticides, chemical fertilizers, various plant growth regulators, ionizing radiation, and genetically modified sowing material \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. In such farming, the soil is not plowed, but only loosened superficially to 5\u0026ndash;10 cm depth \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Intensive cultivation of agricultural crops involves medium or deep soil fertile layer plowing to 25 cm depth and more \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Instead of mineral fertilizers, soil fertility is maintained by mulching with the use of siderates, various composts and organic fertilizers \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. To protect plants from diseases and pests, only biologics are used \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. It is biological plant protection that provides opportunities for sustainable organic agriculture, as it is an alternative to the use of chemicals with the advantages of wider application and reduced environmental impact \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e1.2 Biological crop protection benefits\u003c/h2\u003e \u003cp\u003eBiological crop protection provides such benefits as: assists in reducing damage caused by phytopathogens and pests, enhances nutrient availability and uptake, improves overall productivity and health of the crop, assists in residue and resistance management, extends the window of application around harvest \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDevelopment of technological schemes for biologicals applying and their adopting to the particular farm conditions always contribute to greater agrotechnical measures efficiency in every agricultural system \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. The majority of biologics applying technologies initiate and produce functioning one or both ways of natural regulatory mechanisms in agroecosystems \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. The first one uses agricultural technological methods and biofertilizers to produce conditions for natural mass reproduction of antagonists, entomophages and entomopathogens \u0026ndash; the originally occurring microbe pool determining natural biocenotic regulation in agroecosystems and soil suppressivness \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Such microorganisms underpin the recycling of nutrients by decomposing organic residues, generate humus, restore fertility and recycle pesticides, as well as promote growth, development and immunity in agricultural crops \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. The second way makes the artificial saturation of biocenosis by various polyfunctional biologics applying, the producer strains of which are the microorganisms that possess not only antagonistic, but also soil-fertilizing, phytoregulatory, and other properties due to their polyfunctionality: the ability to synthesize a wide range of secondary metabolites \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. High ecological adaptability of such microorganisms is the result of their polyfunctionality \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. The particular necessity lies in the technologies development for biologics producing and applying used in organic farming \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. The most important is the various necessary properties of fertilizer, stimulant and biopesticide combining in one contemporary polyfunctional biological \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn addition, inoculants and biopesticides for crop production have rather strict requirements: pathogenic and toxic properties absence dangerous for humans, warm-blooded animals, beneficial insects, fish and soil microorganisms; high target activity in maximum beneficial properties manifestations (antagonism to pathogens, nitrogen fixation, phosphate mobilization, etc.); producibility in obtaining and applying in agriculture conditions \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Biologics should be convenient for transport, store and application. Biologics use optimizing helps the practical solution of many problems related to both environment biological purification, fertility maintenance and full quality yield obtaining \u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. In addition, such optimization allows to minimize the main risks of organic crop cultivation which are ensuring plants mineral nutrition and agroecosystems phytosanitary optimization. Organic fertilizers and various composts use reduces the first risks, and biological plant protection is a key element in ensuring a stable phytosanitary condition \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e1.3 Contemporary polyfunctional biologics producing technologies\u003c/h2\u003e \u003cp\u003eFor the development of polyfunctional biologics, the most interesting microbes are those that are non-pathogenic for humans and beneficial organisms, capable of producing a wide range of secondary metabolites, with low demand for their cultivation conditions, high producibility and great ecological adaptability \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. The development of such technology comprises a number of stages, the foremost of which is selection and optimization of nutrient substrates \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. The composition of components in the substrates should not adversely affect their biological activity; they should underpin the desired polyfunctionality \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSince the foundations of polyfunctional biologics are both the cultures of living microorganisms and the products of their metabolism (toxins, enzymes, etc.), there exist various approaches toward cultivation of producer strains \u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. Submerged and solid phase fermentations are enough technologically to result in a highly standardized final product as polyfunctional biologic \u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. Recycling of agro-industrial wastes allows the return of useful components to the biosphere cycle, to ensure sustainability and increase the efficiency of production processes, and to preserve the biological diversity \u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. The problem is that most microorganisms are unable to provide thorough biorecycling of agro-industrial wastes because they contain hard-to-degrade lignocellulose complex \u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. Wood-destroying basidiomycetes are the only known group of organisms capable to actively degrade lignin all the way to complete mineralization \u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. Basidiomycetes are an important component of biocenoses, they have a significant role in the biodegradation processes during which the biogenic substances are regenerated \u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. Therefore, biorecycling of lignin-cellulose complexes with xylotrophic basidiomycetes is a promising feature not only for resource saving and low waste, but also as an environmentally friendly technology \u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAgro-industrial wastes have long been utilized in the production of edible mushrooms \u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. In the process of their cultivation, annually renewable materials are recycled, which are the organomineral mixtures \u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. During the cultivation of edible mushrooms on low-value plant wastes, their enrichment with fungal protein and easily degradable carbohydrates, micronutrients, vitamins and biologically active substances, which as the result of biorecycling are also included into the mycelium enzyme complexes composition \u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. Such enriched substrate, after harvesting of fruit bodies, is a very valuable biotechnology product for subsequent stages of its biorecycling \u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. Such biorecycled wastes are the cheapest, nutrient-rich and readily available substrates for cultivation of producer strains and the development of multi-biorecycled substrate-associated polyfunctional biologics (MSAPB) based on them \u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e. In such biologics, both the potential of SMS substrates as organic fertilizers and biostimulants, and polyfunctionality traits of producer strains are employed. At the same time, the development of a methodology for their production is an important step in sustainable resource management, turning wastes into useful products and making agricultural technologies eco-friendlier \u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e1.4 \u003cem\u003eBacillus\u003c/em\u003e producer strains use for multibiorecycled polyfunctional biologics developing\u003c/h2\u003e \u003cp\u003e \u003cem\u003eBacillus\u003c/em\u003e bacteria, one of the most widespread, diverse and commercially useful groups of microorganisms, are widely used as biologics producer strains \u003csup\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. The low pathogenicity of \u003cem\u003eBacillus\u003c/em\u003e species and the diversity of metabolic processes have caused the representatives of this group to be used in various industrial fields \u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e. They are extremely unpretentious with respect to growth conditions and are capable of producing a great biological active substances variety of protein nature, which can induce the plant disease resistance \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eBacillus\u003c/em\u003e strains having polyfunctionality \u0026ndash; the ability to synthesize a wide range of hydrolytic enzymes and active secondary metabolites \u0026ndash;are capable to utilize in solid-phase fermentation the spent mushroom substrates (SMS) and their aqueous extracts in liquid-phase one, as the cheapest, nutrient-rich and available substrates for producer strains cultivation and multirecycled substrate-associated (MSAPB) and liquid polyfunctional biologics (MLPB) development based on them \u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. Such biologics utilize both the SMS potential as biofertilizers and biostimulants and the producer strains polyfunctionality as well \u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. Combining various properties in one bioproduct is the most important trend in resource saving, waste transforming into useful products and agriculture greening \u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe research goal was to evaluate application effect from experimental prototypes of multirecycled polyfunctional biologics based on \u003cem\u003eB. subtilis\u003c/em\u003e I-5/12\u0026ndash;23 together with compost in potato organic farming. To achieve this goal, the tasks for production and application technological rules developing for polyfunctional biologics experimental prototypes in substrate-associated and liquid formulations and organic compost for optimal and dry growing seasons were solved and phytoregulatory activity and biological efficacy estimating as well.\u003c/p\u003e \u003c/div\u003e"},{"header":"2 Materials and methods","content":"\u003cp\u003eThe research was conducted in FSBSI VIZR Microbiocontrol Laboratory and in organic crop rotation, located at the IEEP branch Experimental Station of the FSBSI FSAC VIM.\u003c/p\u003e \u003cp\u003eChemicals have not been used on these lands for more than 20 years. Only biologicals and organic fertilizers are used in production multi-field crop rotation with assessment of their biological efficiency.\u003c/p\u003e \u003cp\u003eThe station is located in the south of St.-Petersburg region (northwestern Russia). Organic Experimental Station of the FSBSI FSAC VIM has sod-podzolic light loamy gley soil on residual carbonate moraine loam characterized by reaction close to neutral and increased content of organic matter. Research studies covered optimal and dry growing seasons.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Objects\u003c/h2\u003e \u003cp\u003eThe research objects were the MSAPB and MLPB prototypes liquid and granulated formulations and organic compost in various doses.\u003c/p\u003e \u003cp\u003eThe MSAPB and MLPB prototypes were obtained by sequential multibiorecycling the sterilized agro-industrial waste by \u003cem\u003eLentinula edodes\u003c/em\u003e (Berk.) Pegler, \u003cem\u003ePleurotus ostreatus\u003c/em\u003e (Jacq.) P. Kumm. and further by \u003cem\u003eB. subtilis\u003c/em\u003e I5-12/23 (Ehrenb.) Cohn. The biologics prototypes were developed in accordance to FSBSI VIZR approved regulations, specifications and producer strain\u0026rsquo;s toxicological passport.\u003c/p\u003e \u003cp\u003eThe high active producer strain \u003cem\u003eB. subtilis\u003c/em\u003e I-5 12/23 is certified, deposited and maintained at FSBSI VIZR State Collection of Microorganisms Pathogenic for Plants and Their Pests registered on 28.01.1998 No. 760 in the World Federation for Culture Collections, World Data Center for Microorganisms (WDCM WFCC, Japan). Prototypes titer was not less than \u0026times;10\u003csup\u003e10\u003c/sup\u003e colony forming units (CFU) g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for granulated MSAPB and was not less than \u0026times;10\u003csup\u003e11\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for MLPB ones. The biologics experimental prototypes were used to protect organic potatoes from fungal and bacterial diseases during vegetation and storage. The active units of biologics experimental prototypes were cells (spores) and \u003cem\u003eB. subtilis\u003c/em\u003e I-5 12/23 metabolome; formulations were the suspension concentrate (SC) and granulated (G) ones.\u003c/p\u003e \u003cp\u003eThe compost characterized by nearly 40% dry matter was prepared in IEEP\u0026rsquo;s Lab for organic waste bioconversion from chicken manure using bioconvector. Depending on the season weather conditions, compost doses in terms of nitrogen were 80, 110, 160 kg N\u0026times;ha\u003csup\u003e\u0026minus;\u0026thinsp;1 [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Materials\u003c/h2\u003e \u003cp\u003eThe materials were semi-synthetic and natural media, such as dried nutrient medium (DNM): pancreatic sprat hydrolysate \u0026ndash; 15 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; NaCl \u0026ndash; 4.59 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; microbiological agar \u0026ndash; 20 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; H\u003csub\u003e2\u003c/sub\u003eO \u0026ndash; 1 L; pH\u0026thinsp;=\u0026thinsp;7.2 (Microgen Co. Ltd., Russia); natural nutrient media based on multirecycled spent mushroom substrates (SMS) and their aqueous extracts: double spent SMS \u0026ndash; 200 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, H\u003csub\u003e2\u003c/sub\u003eO \u0026ndash; 1 L, pH\u0026thinsp;=\u0026thinsp;6.5\u0026ndash;7.5 (FSBSI VIZR, Russia).\u003c/p\u003e \u003cp\u003eMultirecycled SMS was obtained as sequential solid-phase fermentation the agricultural and technogenic sphere waste by macromycetes \u003cem\u003eL. edodes\u003c/em\u003e (shiitake mushroom) and \u003cem\u003eP. ostreatus\u003c/em\u003e (oyster mushroom). Such waste content as commercial \u003cem\u003eL. edodes\u003c/em\u003e cultivating substrate was: oak sawdust \u0026ndash; 88.9%, wheat bran \u0026ndash; 10%, CaCO\u003csub\u003e3\u003c/sub\u003e \u0026ndash; 0.1%, CaSO\u003csub\u003e4\u003c/sub\u003e \u0026times; 2H\u003csub\u003e2\u003c/sub\u003eO \u0026ndash; 1%. The multirecycled SMS obtaining process included a sequential vegetative and reproductive stages\u0026rsquo; change of \u003cem\u003eL. edodes\u003c/em\u003e 4080, then \u003cem\u003eP. ostreatus\u003c/em\u003e HK-35 (Sylvan, Inc.). The double mushroom culture industrial waste after basidiomata gathering were classified as a multirecycled granulated SMS \u0026ndash; another research material. This waste substrate was used for solid-phase fermentation by producer strain after its components crushing into 0.5\u0026ndash;2.5 cm pieces and soaking in water for 20\u0026ndash;24 h to complete moisturizing. The prepared multirecycled granulated SMS having 70\u0026ndash;80% moisture content and stabilized pH\u0026thinsp;=\u0026thinsp;7.0\u0026ndash;7.5 were packed in 1500 g each in 1 L polypropylene bags. The bags were sealed up and sterilized for 1 h at 133\u0026deg;C (202.7 kPa).\u003c/p\u003e \u003cp\u003eFor the liquid-phase fermentation the double spent (multirecycled) SMS were used as preliminarily milled, and boiled in amount of 200 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for 1 h, filtered, and restored to the previous 1 L volume. Sterilization modes for the liquid nutrient media were 30\u0026ndash;60 min at 50.7\u0026ndash;81.1 kPa, for the granulated SMS was 60 min at 202.7 kPa in steamer 5075ELVPV D (Tuttnauer Europe BV, Netherlands).\u003c/p\u003e \u003cp\u003eThe bedding poultry manure supplied by Leningrad Region poultry farm was used as an organic material for the compost production \u003csup\u003e[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Research Methods\u003c/h2\u003e \u003cp\u003eThe standard micro-, myco- and phytopathological research methods were used in the work: liquid- and solid-phase fermentation for the inoculums and stock cultures, for biologics prototypes developing; SMS preparing for multirecycling; serial dilutions\u0026rsquo; method for titers assessing and the quality biologics experimental prototypes; conducting field tests, field testing results and potato yield accounting. There used 4\u0026ndash;5-fold replications in major trials including the field ones \u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan additionalcitationids=\"CR56 CR57 CR58 CR59 CR60\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe MSAPB and MLPB prototypes were developed by liquid- and solid-phase fermentations in FSBSI VIZR Microbiocontrol lab. The inoculums\u0026rsquo; and prototypes\u0026rsquo; titers were determined by the serial dilutions\u0026rsquo; method on DNM. The producer strain inoculum was preliminarily stored and developed in test tubes on the same nutrient medium.\u003c/p\u003e \u003cp\u003eThe liquid-phase fermentations were developed in shaking flasks with 750 mL volume, containing 100 mL of SMS-extract. Submerged producer strain inoculum was grown at 27\u0026ndash;28\u0026deg;C for 3 days with aeration (180 rpm, New Brunswick\u0026trade; Innova\u0026reg; 44 Shaker Incubator, Eppendorf, Germany). The samples were taken away every day to control the bacterial growth stage and the contamination as well. The fermentation process finished at 85\u0026ndash;90% spores had been produced in the culture liquid (CL). To obtain the final formulation the CL was concentrated for 10 min at 3000 rpm in the centrifuge OS-6MC (Dastan Inc., Kyrgyzstan) and then 0.2% potassium sorbate was added to the spore suspension concentrates put into 1 L wide-mouth bottles, PE-LD (VITLAB GmbH, Germany). The initial CL titers were not less than \u0026times;10\u003csup\u003e10\u003c/sup\u003e, the finished ones were near \u0026times;10\u003csup\u003e11\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1 [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe solid-phase fermentation of the 1500 g multirecycled SMS with the help of \u003cem\u003eB. subtilis\u003c/em\u003e I-5 12/23 was occurred in sterile 1 L polypropylene bags having after its inoculation with submerged producer strain's cultures in sterility conditions. Incubation was carried out during 5 days at 27\u0026ndash;28\u0026deg;C in a thermostatic chamber PRO TC 30/120\u0026ndash;500 (SIA Prooborudovanie Co. Ltd., Russia) using substrate shaking and stirring every other day until the last one was completely overgrown. The MSAPB prototypes titers were not less than \u0026times;10\u003csup\u003e10\u003c/sup\u003e CFU g\u003csup\u003e\u0026minus;\u0026thinsp;1 [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe compost was produced by aerobic solid-phase fermentation of bedding poultry manure in the special biofermenter (IEEP\u0026ndash;BRANCH OF FSAC VIM, St-Petersburg, Russia) \u003csup\u003e[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/sup\u003e. The compost shelf life is 2 years from the production date under \u0026minus;\u0026thinsp;20\u0026ndash;+30\u0026deg;C air temperature and 60\u0026ndash;75% air humidity.\u003c/p\u003e \u003cp\u003eThe MSAPB and MLPB prototypes based on \u003cem\u003eB. subtilis\u003c/em\u003e strain I-5 12/23 as well as the compost were developed for field trials of their biological efficacy.\u003c/p\u003e \u003cp\u003eThe area used for the field trials carried out in 2019\u0026ndash;2022 was located at 59\u0026deg;65 N and 30\u0026deg;38 E (Leningrad region, Russia). The soil in the organic crop rotation is well cultivated and has up to high levels of available P and K, pH\u0026thinsp;=\u0026thinsp;6.5\u0026ndash;6.6 and 5.6% organic matter content as well.\u003c/p\u003e \u003cp\u003eThe compost was used before ridging and its subsequent embedding was made by the ridge space loosening with a chisel cultivator (IEEP\u0026ndash;BRANCH OF FSAC VIM, St-Petersburg, Russia) up to 30 cm depth from the bottom of the furrow. Such a deep loosening between organic potatoes rows as an inter-row treatment resulted in a decrease in soil compaction both in the potato rooting zone and directly in the aisle together with hilling and destroying weeds.\u003c/p\u003e \u003cp\u003eSuper elite and elite classes of potato variety Udacha was used in the field trials. The 30\u0026ndash;50 t ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e average yield and 12\u0026ndash;15% starch content are this variety characteristics. Tubers mature early and this variety is well adapted to various types of soil and climatic zones. Udacha variety tubers have good taste and a smooth surface \u003csup\u003e[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/sup\u003e. It is midresistant to late blight, rhizoctonia disease, wrinkled mosaic, black leg, wet rot and common scab (State register of selection achievements approved for use in Russian Federation from FSBI \u0026ldquo;State Breeding Commission\u0026rdquo;, 2022).\u003c/p\u003e \u003cp\u003ePotato tubers and then plants were three-fold treated with \u003cem\u003eB. subtilis\u003c/em\u003e I-5 12/23 formulations at planting and then by foliar spray after 10 days and 20 days at the MLPB prototypes\u0026rsquo; consumption rate 3 and 6 L ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e under optimal and dry growing conditions, respectively. Spraying was carried out with the help of specially designed sprayer installed on the planter and cultivator. Second week after planting the inter-row cultivation was begun and the carried out regularly using the row-crop cultivator for deep loosing of inter-rows (IEEP\u0026ndash;BRANCH OF FSAC VIM, St-Petersburg, Russia). Weeds were removed mechanically using small rotary harrow BRU-0.7 harrows mounted on the cultivator.\u003c/p\u003e \u003cp\u003eThe reciprocally orthogonal scheme of field trials was applied with standard placement of variants (3 series): 4 replicates in a complete randomized design \u003csup\u003e[\u003cspan additionalcitationids=\"CR59\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]\u003c/sup\u003e. Accounting plot size according to growing season conditions was 20\u0026ndash;60 m\u003csup\u003e2\u003c/sup\u003e. The studied indicators were the potato plants developmental biometry and productivity, diseases incidence and their development in dynamics. Potato plant growth rate (mm/day) and productive/flowering stems number (pieces) parameters and the healthy tubers crop were used to evaluate phytoregulatory activity. To assess the damage of fungal disease complex to plants and potato tubers, the disease incidence and development on potato plants and on tubers, crop losses, biological efficacy were used. The dominant fungal diseases on potato plants during research period were leaf spots, late blight, \u003cem\u003eFusarium\u003c/em\u003e wilt and rhizoctoniosis. On tubers there were common and silver scab, anthracnose, late blight and rhizoctoniosis.\u003c/p\u003e \u003cp\u003eThe field trials data were gathered 5 times: 2 biometric accountings with disease symptoms appearance fixating were carried out on 3\u0026ndash;5-week-old seedlings in the 1\u0026ndash;3\u003csup\u003ed\u003c/sup\u003e leaf tiers phase and on 6\u0026ndash;7-week-old potato plants in the 9\u0026ndash;10th leaf tiers phase; 2 phytopathological accountings were carried out at the beginning and ending of the flowering phase; 1 accounting was carried out at tubers harvesting. Every third potato plant in the 4-fold trial plot (up to 100 plants) was examined \u003csup\u003e[\u003cspan additionalcitationids=\"CR59 CR60\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Statistical processing methods\u003c/h2\u003e \u003cp\u003eStatistical processing was produced using the Microsoft Excel 2010 and Statistica 10.0 software packages (StatSoft, Inc., Tulsa, OK, USA). Statistics included checking the analyzed data normal distribution by Shapiro-Wilk's \u003cem\u003eW\u003c/em\u003e-test, calculating the means (\u003cem\u003eM\u003c/em\u003e) and standard errors (\u0026plusmn;\u0026thinsp;\u003cem\u003eSEM\u003c/em\u003e) as well, analysis of variance (\u003cem\u003eANOVA)\u003c/em\u003e. In options pairwise comparison the statistical differences significance was assessed by Student's \u003cem\u003et-\u003c/em\u003etest \u003csup\u003e[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cp\u003eAll the results obtained in various research years on the peculiarities of organic potato cultivation in the conditions of northwest Russia are considering the weather conditions of vegetation seasons.\u003c/p\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Weather conditions in the research years\u003c/h2\u003e\n \u003cp\u003eThe weather conditions in late spring and summer of the research years differed to various significant extent from each other (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Research years 2019 and 2021 were warm and to various extent dry, meanwhile 2020 and 2022 were wet and not so warm. The hottest and driest year was 2021, and the coolest and wettest year was 2020. The month May was the coolest opposite to July that was the warmest month during every vegetation season. The weather conditions in optimal 2020 and 2022-years vegetation periods were characterized by essentially comfortable temperatures and suitable rainfall during the active potato development.\u0026nbsp;\u003c/p\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003e\u0026emsp;Summer weather conditions in the research years\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003emonth\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eweather indicator, hydrothermal coefficient (HTC)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eyearly average\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eaverage for cumulative years\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2019\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eMay\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etemperature \u0026deg;С\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eprecipitation, mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e79.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e172.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eJune\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etemperature \u0026deg;С\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eprecipitation, mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e79.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e129.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e71.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eJuly\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etemperature \u0026deg;С\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eprecipitation, mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e179.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e186.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e85.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e79.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eAugust\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etemperature \u0026deg;С\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eprecipitation, mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e94.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e195.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e109.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e149.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e83.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eThe maximum precipitation for the entire growing seasons was registered in 2020. To establish the aridity conditions for a certain period in a given area one can use Selyaninov\u0026rsquo;s Hydrothermal Coefficient (HTC). It is determined by the ratio of the precipitation amount during the growing season to the sum of temperatures above 10\u0026deg;C, reduced 10-fold: the natural moistening conditions are considered optimal, if HTC 1\u0026ndash;2, if less than 1 are considered dry \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e]\u003c/sup\u003e. It helped in defining the driest conditions in June and July 2021 for the active potato development. The HTC ratios allowed grouping all the data obtained into optimal and dry periods for potato plant development and yield formation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Phytoregulatory activity as bioinoculants\u003c/h2\u003e\n \u003cp\u003eThe phytoregulatory activity study of various compost doses, the polyfunctional MSAPB and MLPB prototypes based on \u003cem\u003eB. subtilis\u003c/em\u003e I-5/12\u0026ndash;23, as well as their various combinations applied in the experimental variants in every studied case revealed their positive effect on the potato plants growth and development (Fig. 1). The pronounced tendency of growth and development stimulation after various compost doses application as well as their combinations with polyfunctional MSAPB and MLPB prototypes is typical for dry growing seasons, when against the background of not very favorable development conditions organic fertilizers and biologics\u0026rsquo; experimental batches help potato plants to grow and develop (Fig. 1b).\u003c/p\u003e\n \u003cp\u003eUnder optimal growing conditions, such a trend is not so obvious, and the potato plants growth rate of potato plants in various variants of compost and MSAPB and MLPB prototypes application was fixed within the measurement error (Fig.\u0026nbsp;1a). Nevertheless, in all cases it was higher than the control values without compost and MSAPB and MLPB prototypes applying (Fig.\u0026nbsp;1a). For such an important indicator in potato cultivating as the productive/flowering stems number per bush, which ultimately determines the productivity of plants, there was found a direct proportional dependence on the compost various doses application regardless of the growing season conditions (Fig.\u0026nbsp;1). Reliability of differences was recorded under optimal conditions for the development of organic potato (Fig.\u0026nbsp;1a). Although the trend was also characteristic for dry vegetative periods (Fig.\u0026nbsp;1b).\u003c/p\u003e\n \u003cp\u003ePolyfunctional MSAPB and MLPB prototypes based on \u003cem\u003eB. subtilis\u003c/em\u003e I-5 12/23 applying without compost completely replaced its application regardless of organic potatoes vegetation period weather and climatic conditions (Fig. 1). Under optimal growing conditions for organic potatoes, no significant differences in plant development were observed after MSAPB and MLPB prototypes application (pre-planting treatment of tubers or application into the soil at planting) (Fig. 1a). When cultivated in dry periods, the application of granular forms into the soil was significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) more important: growth and development indicators of potato plants were higher (Fig.\u0026nbsp;1b).\u003c/p\u003e\n \u003cp\u003eThe same trends in phytoregulatory activity described by the parameters of potato plant growth rate and productive stems formation (Fig. 1) were revealed in the potatoes total yield forming under the organic compost application and under the influence of polyfunctional MSAPB and MLPB prototypes based on \u003cem\u003eB. subtilis\u003c/em\u003e I-5 12/23 applied (Fig. 2). There was observed a significant and reliable (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) increase in total biological yield in every trial variant compared to the control regardless the growing season conditions (Fig.\u0026nbsp;2). The same way in all variants of the compost application recorded the dose-effect relationship: a significant and in most cases reliable (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increase in the total potatoes biological yield with compost dose increasing, and regardless growing season conditions as well (Fig. 2a, b). Under the influence of polyfunctional MSAPB and MLPB prototypes based on \u003cem\u003eB. subtilis\u003c/em\u003e I-5 12/23, a significant reliable increase in the total biological yield (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) of organic Udacha potatoes compared to the control was observed in 1.3 times on average by 5 t ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e under both optimal and dry growing season conditions (Fig. 2). Moreover, the effect of experimental biologics prototypes was equivalent to the low and medium nitrogen doses of compost applying (Fig. 2a, b). Maximum and reliable (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) increase in potato yield by 1.5 times on average by 9 t ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e under both optimal and dry growing season conditions was achieved by the coeffect of experimental biologics prototypes and high organic compost doses (Fig. 2a, b).\u003c/p\u003e\n \u003cp\u003eRegardless the vegetation period conditions of organic potato development, no significant differences in yield forming were observed after various biologics prototypes formulations applying (pre-planting tubers treatment or application into the soil at planting) (Fig. 2a, b). In polyfunctional MSAPB and MLPB prototypes based on \u003cem\u003eB. subtilis\u003c/em\u003e I-5 12/23 and compost coapplying a reliable (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increase in the total biological yield of Udacha organic potato under every weather condition during cultivation was recorded when the compost dose was increased (Fig.\u0026nbsp;2a, b).\u003c/p\u003e\n \u003cp\u003eThe amount of organic potatoes disease damaged yield was regulated mainly by polyfunctional MSAPB and MLPB prototypes applying \u0026ndash; decrease observed in 3.5 times (Fig.\u0026nbsp;2a, b). This trend was especially pronounced during suboptimal and dry periods in organic potato development (Fig.\u0026nbsp;2b). The compost dose increasing often had a positive effect on the 3\u0026ndash;7 times reduction of the disease damaged yield (Fig.\u0026nbsp;2a, b).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Protective activity as biopesticides\u003c/h2\u003e\n \u003cp\u003eThe protective effect of the polyfunctional MSAPB and MLPB prototypes applying sometimes together with compost was evaluated using the biological efficacy indicator (Fig.\u0026nbsp;3). To identify biological efficacy relative dynamics, it was evaluated both in the fungal disease complex incidence and development of organically cultivated potato (Fig.\u0026nbsp;3).\u003c/p\u003e\n \u003cp\u003eThe data presented in Fig. 3 indicate high biological efficiency of the applied polyfunctional MSAPB and MLPB prototypes \u0026ndash; 50\u0026ndash;95% in optimal and 50\u0026ndash;75% in dry vegetation periods in organic potato variety Udacha development. Suboptimal vegetative conditions mostly reduced the biological efficacy in the fungal disease complex development (Fig. 3b). Biological efficiency in diseases incidence on organic potatoes was 8\u0026ndash;30% inferior to that in their development on plants (Fig. 3a, b). The most effective (60\u0026ndash;95%) in the protection of organic potato variety Udacha against a diseases complex were combinations of polyfunctional MSAPB and MLPB prototypes and medium compost doses (Fig. 3a, b). The intrinsic compost biological efficiency did not exceed 50%, and with increasing dosage it decreased both under optimal and suboptimal development conditions (Fig. 3a, b). Under optimal growing organic conditions for potato plants revealed the maximum field efficacy in polyfunctional MSAPB and MLPB prototypes application \u0026ndash; 75\u0026ndash;95% (Fig. 3a). During the organic potatoes\u0026rsquo; development in dry periods, the polyfunctional MSAPB and MLPB prototypes various formulations applying allowed to protect (biological efficiency 50\u0026ndash;70%) plants from diseases complex most effectively (Fig. 3b).\u003c/p\u003e\n \u003cp\u003eCompost application did not always support the protective effect from the polyfunctional MSAPB and MLPB prototypes application regardless the growing season conditions (Fig.\u0026nbsp;3a, b). Biological efficacy of the polyfunctional MSAPB and MLPB prototypes application, conditioning the disease development level on plants of organically cultivated Udacha variety potatoes was 5\u0026ndash;20% higher than that in disease incidence both in optimal and dry conditions of potato cultivation (Fig.\u0026nbsp;3a, b).\u003c/p\u003e\n \u003cp\u003eBiological efficacy of applied MSAPB and MLPB prototypes protecting the organic potato yield was 15\u0026ndash;55% in optimal and 10\u0026ndash;17% in dry periods of Udacha variety potato organic development. Suboptimal vegetative conditions the most reduced biological efficacy in fungal disease complex development on tubers (Fig. 4b).\u003c/p\u003e\n \u003cp\u003eBiological efficacy in disease incidence on tubers was 5\u0026ndash;35% inferior to that in disease development and only under optimal growing conditions (Fig.\u0026nbsp;4a, b). The most effective (35\u0026ndash;55%) in protecting the yield of variety Udacha organic potato were combinations of various MSAPB and MLPB prototypes formulations (Fig.\u0026nbsp;4a). The intrinsic biological efficacy of the compost did not exceed 5\u0026ndash;18%, and with dosage increasing it fell both under optimal and suboptimal development conditions (Fig.\u0026nbsp;4a, b).\u003c/p\u003e\n \u003cp\u003eUnder optimal growing conditions of organic potatoes revealed the maximum biological efficiency in polyfunctional MSAPB and MLPB prototypes applying for tubers crop protection \u0026ndash; 50% (Fig.\u0026nbsp;4a). During the organic potatoes\u0026rsquo; development in dry periods, the use of polyfunctional MSAPB and MLPB prototypes various formulations allowed to protect the yield from the diseases complex with 10% biological efficacy (Fig.\u0026nbsp;4b).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Ways to use antagonist microbes in biocontrol\u003c/h2\u003e \u003cp\u003eThere are two ways to use antagonist microbes in phytopathogens microbiocontrol. One of them involves the conditions developing for natural mass reproduction of microbes through agronomic practices and fertilizers \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Another one is based on artificial saturation of agrobiocenosis with antagonistic microorganisms through the various biologics use, and better polyfunctional ones, as they have a direct antagonistic effect on pathogens due to the BAS complexes and increase the stress resistance in plants and their immunity to diseases due to their phytoregulatory activity \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. These two ways formed the contemporary approaches to the production of polyfunctional biologics which should suit two concepts of phytosanitary optimization and long-term biocenotic harmonization of agroecosystems \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]\u003c/sup\u003e. The first one is based on the use of natural resources of microorganisms possessing complex biological activity, in particular polyfunctionality: the ability to synthesize a wide range of secondary metabolites developed in a long evolutionary process of soil-dwelling microorganisms under rigid natural selection in extremely saturated habitat \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]\u003c/sup\u003e. High ecological adaptability of microorganisms is the result of their polyfunctionality \u003csup\u003e[\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]\u003c/sup\u003e. According to the second concept, polyfunctional microorganisms that produce a variety of BAS complexes as secondary metabolites, that turned out to be the most competitive as a result of evolution, are incorporated into a pool of natural enemies of detrimental organisms in agroecosystems regulating the number and harmfulness of the latter \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Polyfunctional biologics final formulation produce the efficacy guaranty\u003c/h2\u003e \u003cp\u003eFor the manifestation by producer strains their polyfunctionality, the final formulation obtained during the final technological stage of biologics production is very important \u003csup\u003e[\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]\u003c/sup\u003e. This final step is closely related to the technologies for biologics' applying and affects their storage duration \u003csup\u003e[\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]\u003c/sup\u003e. The biologic efficiency depends on the extent to which the final formulation will contribute to the producer strain potential manifestation \u003csup\u003e[\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]\u003c/sup\u003e. Biologics standardizing and quality assessing are related to the final form \u003csup\u003e[\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]\u003c/sup\u003e. The main requirements for such final formulations are: long-term (at least 1\u0026ndash;2 years) preservation of microorganisms\u0026rsquo; cells viability, biological activity during biologics storage and their application. Nowadays final biologics\u0026rsquo; formulations are liquid (fluids, suspensions, concentrates of suspensions, pastes), dry (wetting powders, tablets), immobilizing formulations with nutrients for producer strains: dispersive solid bulk (on the basis of peat, vermiculite, chitin-chitosan carriers), granulated (based on hydrogels and porous mineral sorbents, some of them crafted as nanoscale materials) \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]\u003c/sup\u003e. Some final formulations having unacceptable additives used in technological schemes for polyfunctional biologics producing cannot participate in organic crop production \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Providing organic agriculture with suitable plant protection products caused to develop new approaches to the polyfunctional biologics production through multibiorecycling of agro-industrial wastes \u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. Such approaches provide the necessary substrate\u0026ndash;producer strain association and the latter polyfunctionality, which lead modern biologics to combine in their final formulations the properties of biofertilizers, biostimulants and biopesticides, providing a sustainable increase in crop yields with their application \u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003eMultibiorecycled polyfunctional MSAPB and MLPB biologics prototypes combining the properties of biopesticides, biofertilizers and biostimulants (inoculants) can be used to ensure stable organic potato production. The substrate base of such experimental prototypes obtained by multibiorecycling technogenic and agricultural waste by edible shiitake and oyster mushrooms \u0026ndash; the product enriched with protein, vitamins, macro- and microelements \u0026ndash; provided enhanced development of active producer strain \u003cem\u003eB. subtilis\u003c/em\u003e I-5 12/22 with a titer of \u0026times;10\u003csup\u003e10\u003c/sup\u003e CFU g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for granulated MSAPB and not less than \u0026times;10\u003csup\u003e11\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for MLPB ones. Polyfunctional MSAPB and MLPB prototypes based on \u003cem\u003eB. subtilis\u003c/em\u003e I-5 12/23 strain showed growth-stimulating and protective effects tested in field trials in organic production of the variety Udacha potato.\u003c/p\u003e \u003cp\u003eHigh biological activity of \u003cem\u003eB. subtilis\u003c/em\u003e strain I-5 12/23 in all formulations of biologics experimental batches based on it provided reliable (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) 1.2\u0026ndash;2.5 times increase in biometric indices of organic potato plant development as well as 1.2\u0026ndash;1.7 times in tuber yield under optimal and dry vegetative conditions.\u003c/p\u003e \u003cp\u003eFour-year scientific and producing approbation of the technological application rules for the MSAPB and MLPB prototypes based on \u003cem\u003eB. subtilis\u003c/em\u003e I5-12/23 and compost developed for the various weather and climatic conditions of northwest Russia showed their high efficacy in protection the organic Udacha variety potato. Disease incidence on vegetative potato plants decreased by 50\u0026ndash;95%, on tubers by 25\u0026ndash;70%, their quality improved, and the marketable products yield increase at 9 t ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (3 t ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, on average). The application's technological rules optimizing for various weather and climatic conditions is carried out by changing the consumption norms of protection means in proportion to the limiting factors.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003ethis research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. The authors very much appreciate the timely assistance of their personal sponsors and editors Valeria and Christopher Robert Hearsey.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with ethics guidelines:\u003c/strong\u003e the authors declare that they have no conflict of interest or financial conflicts to disclose. All applicable institutional and national guidelines for the care and use of animals were followed.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGiller KE, Delaune T, Silva JV, Descheemaeker K, van de Ven G, Schut AG, van Wijk M, Hammond J, Hochman Z, Taulya G (2021) The future of farming: Who will produce our food? 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Microbiol Res 257:126978. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.micres.2022.126978\u003c/span\u003e\u003cspan address=\"10.1016/j.micres.2022.126978\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"plant protection in organic agriculture, organic potato farming, agro-industrial waste multibiorecycling, Bacillus subtilis producer strains, organic compost, multirecycled substrate-associated and liquid polyfunctional biologics","lastPublishedDoi":"10.21203/rs.3.rs-4317900/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4317900/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eBacillus\u003c/em\u003e strains have long been widely and successfully used as the polyfunctional biologics\u0026rsquo; basis in various systems for crops cultivation and protection. The research goal was to evaluate application effect from experimental prototypes of multirecycled polyfunctional biologics based on \u003cem\u003eBacillus subtilis\u003c/em\u003e I-5/12\u0026ndash;23 together with compost in potato organic farming. A significant stimulation of potato plants Udacha variety growth and development up to the flowering phase was observed regardless of the growing season hydrothermal conditions. The stimulation was by the additive effect of joint biologics and compost use in proportion to its dose. The multirecycled substrate-associated and liquid polyfunctional biologics prototypes together with compost almost doubled the potato tubers biological yield compared to the control regardless the growing season conditions. In the flowering phase, the biological efficacy with respect to the potato fungal diseases incidence and development was 90% under optimal hydrothermal conditions and up to 75% under drought conditions. At the vegetation end the efficacy in the potato fungal diseases development reached 70% (compost efficiency itself more than 45%) regardless of the vegetation period conditions. Four-year scientific and producing approbation of the technological application rules for the biologics\u0026rsquo; prototypes based on \u003cem\u003eB. subtilis\u003c/em\u003e I5-12/23 and compost developed for the North-West region\u0026rsquo;s various weather and climatic conditions showed their high efficacy in protection the organic potato. Disease incidence on plants decreased about 80%, on tubers about 50%, their quality improved, and the marketable products yield increase at 9 t ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (3 t ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, on average). The application\u0026rsquo;s technological rules optimizing for various weather and climatic conditions is carried out by changing the consumption norms of protection means in proportion to the limiting factors. Multirecycled polyfunctional biologics prototypes combining the properties of biopesticides, biofertilizers and inoculants can be used to ensure stable organic potato production.\u003c/p\u003e","manuscriptTitle":"Multirecycled polyfunctional biologics based on Bacillus subtilistogether with compost in potato organic farming","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-25 05:30:23","doi":"10.21203/rs.3.rs-4317900/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aeb18527-722e-4f93-9162-9efbb365ca2c","owner":[],"postedDate":"April 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":31100278,"name":"Agricultural Engineering"},{"id":31100279,"name":"Agroecology"},{"id":31100280,"name":"Food Science \u0026 Technology"},{"id":31100281,"name":"Biotechnology and Bioengineering"},{"id":31100282,"name":"Applied \u0026 Industrial Microbiology"}],"tags":[],"updatedAt":"2024-04-25T05:30:23+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-25 05:30:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4317900","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4317900","identity":"rs-4317900","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}