Novel solid-phase multibiorecycled biologics based on Bacillus subtilis and Trichoderma asperellum as effective potato protectants against phytophthora disease

A total of 17 biologics based on the producer strains Bacillus subtilis and Trichoderma asperellum (= T. harzianum ) are currently approved in Russia to protect potatoes from diseases. Great world experience has been gained in producing and use of traditional dry and liquid biologics. However, multirecycled industrial wastes as substrates for biologics are still not used anywhere in the world, and there is little information on effectiveness of formulations produced by industrial wastes’ multistage biorecycling. This paper reports a successful experience of the sequential use of plant wastes as substrates for mushrooms and then for microbial strains to produce granular antifungal biologics. This is a relevant approach to biotechnologies for safer utilization of wastes as resources of cheap and affordable raw materials and their transformation into useful products. Our objective was to develop brand new multirecycled biologics based on plant pathogen antagonists and to estimate their efficacy. Plant wastes were converted to substrates for B. subtilis B-10 and T. asperellum T-36 producer strains by shiitake Lentinula edodes (Berk.) Pegler and oyster mushroom Pleurotus ostreatus (Jacq.: Fr.) P. Kummer НК-35 serial cultivation. The nutritional value of the obtained double biorecycled substrate, due to decomposition of cellulose and lignin of sawdust and wheat bran mixture by shiitake and oyster mushroom, was higher as compared to that of the initial substrate used for shiitake growing or of peat, a common solid-phase fermentation substrate. In particular, the protein content was higher (9.4±0.3 % vs. 2.7±0.3 % and 4.3±0.1 %, respectively), the nitrogen level was higher (1.5±0.3 % vs. 0.4±0.1 % and 0.6±0.1 %), and the C:N ratio reduced (38.3 vs. 81.2 and 92.9). Liquid microbial inoculums were cultured in standard Czapek (Biocompas-С Ltd., Russia) and corn-molasses (Carguil Ltd., Agroresource Ltd., Russia) nutrient media. Solid-phase fermentation of the double biorecycled lignin- and cellulose-containing substrate inoculated with 0.9×109 spores/ml B. subtilis B-10 and 2.8×1010 CFU/ml T. asperellum T-36 to produce the biologics took 10 days at 25-28 °C. The obtained biologics were tested on potato cv. Elizaveta in plot trials in the Leningrad Province (Producers’ Cooperative Shushary, 2011). A reciprocally orthogonal scheme was used, and the plots were arranged in 4 replicates over 0.5 ha, with 10 m2 test plot size and 482 plants sampled in total. A single application was performed at planting on May 12, 2011. The tubers were mixed with the biologics in the bunker of the potato-planting unit at a rate of 1 kg per 1.5 ton tubers (2 kg/ha). The basic potato growing technology included i) post-planting application of Sencor® herbicide (800 g/l, Bayer Crop Science, Germany); ii) post-germination double application (with one-week interval) of Terraflex® 17/17/17 inoculant (2.8 and 1.6 kg/ha, Nu3 N.V., Belgium); iii) post-germination single application of Aquadon micro inoculant (2.0 l/ha, Orgpolymersyntes, Russia), Extrasol® microbe fertilizer agent (2.0 l/ha, BisolbyInter Ltd., Russia), Zircon inoculant (10 g/ha, ANO Nest-M, Russia), herbicides Lazurite (0.5 l/ha, AO Avgust, Russia) and Titus™ (20 g/ha, DuPont, USA); and iv) treatments with fungicides after row closure as follows: Bravo® (1.5 l/ha, Syngenta AG, Switzerland) and Ridomil gold® (1.5 l/ha, Syngenta AG, Switzerland) in 2 weeks; Revus® (250 g/ha, Syngenta AG, Switzerland) in 4 weeks, and Shirlan® (0.4 l/ha, Syngenta AG, Switzerland) in 6 weeks. The final fertilization with Terraflex® (2.8 kg/ha) combined with Shirlan® treatment (0.4 l/ha) were carried out 2 weeks before harvesting. The basic agrotechnology without biologics served as the control. Standard biometric and phytopathological indicators were used. The disease signs and biometric parameters were assessed in 3-week seedlings (1-2 leaf layer phase) and at row closure. Then two disease surveys were performed at the beginning and at the end of blooming, and final indicators for tubers were estimated at harvesting. Data processing by ANOVA and Student’s t -test for pairwise comparison revealed that the biologics caused a significant increase in plant growth rate and the leaf area growth at the beginning of vegetation. The healthy tuber yield was 240 and 690 g/m2 higher for B. subtilis B-10 and T. asperellum T-36 biologics, respectively, as compared to the control (р ≤ 0.10). Due to the biologics, the late blight intensity was 7.2 times lower and 11.6 times lower, respectively (р ≤ 0.01). The number of affected tubers, including those with signs of secondary bacterial infection, decreased almost 2 times, by 140 and 130 g/m2, respectively (р ≤ 0,01). Thus, solid plant waste multirecycling is a prospective way to produce granular environmentally safe biologics for plant protection against diseases.