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 ×1010 colony forming units (CFU) g− 1 for granulated MSAPB and was not less than ×1011 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; H2O – 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, H2O – 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%, CaCO3 – 0.1%, CaSO4 × 2H2O – 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 ×1010, the finished ones were near ×1011 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 ×1010 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 m2. 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 1–3d 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].