doi: 10.15389/agrobiology.2019.5.1002eng

UDC: 635.21:632.4:632.937

This work was carried out as part of the Complex Research Program “Development of potato breeding and seed production”, Section 9 “Development of effective potato protection technologies”.



J.A. Titova, I.I. Novikova, I.V. Boykova, V.A. Pavlyushin, I.L. Krasnobaeva

All-Russian Research Institute of Plant Protection, 3, sh. Podbel’skogo, St. Petersburg, 196608 Russia, e-mail (✉ corresponding author), (✉ corresponding author),,,

Titova J.A
Pavlyushin V.A.
Novikova I.I.
Krasnobaeva I.L.
Boykova I.V.

Received June 29, 2019


A total of 17 biologics based on the producer strains Bacillus subtilisand 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.) Peglerand 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-СLtd., 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 plotswere arranged in 4 replicates over 0.5 ha, with 10 m2 test plot sizeand 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 tontubers (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/mhigher 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. In the developed three-step technology, the wastes from edible mushroom double cultivation on sawdust mixed with wheat bran possess high nutritional value as a substrate for solid microbial cultures.

Keywords: multirecycled biologics, efficacy, potato, diseases, protection, microbial antagonists, multirecycling, Bacillus subtilisTrichoderma asperellum.



  1. Zeiruk V.N., Kuz'michev A.A., Glez V.M., Derevyagina M.K., Vasil'eva S.V., Abashkin O.V. Fitosanitarnoe sostoyanie i meropriyatiya po bor'be s osnovnymi boleznyami i vreditelyami v period vegetatsii i khraneniya kartofelya [Phytosanitary condition and measures to combat the main diseases and pests during the growing season and storage of potatoes]. Moscow, 2014 (in Russ.). 
  2. Kubicek C.P., Komon-Zelazowska M., Druzhinina I.S. Fungal genus Hypocrea/Trichoderma: from barcodes to biodiversity. Zhejiang University Science B, 2008, 9(10): 753-763 CrossRef
  3. Reino L.R., Raul F., Hernandez-Galan G.R., Collado I.G. Secondary metabolites from species of the biocontrol agent Trichoderma. Phytochemistry Reviews, 2008, 7: 89-123 CrossRef
  4. Moradi H., Bahramnejad B., Amini J., Siosemardeh A., Haji-Allahverdipoor K. Suppression of chickpea (Cicer arietinum L.) Fusariums wilt by Bacillus subtilis and Trichoderma asperellum. Plant Omics Journal, 2012: 68-74.
  5. Ru Zh., Di W. Trichoderma spp. from rhizosphere soil and their antagonism against Fusarium sambucinum. African Journal of Biotechnology, 2012, 11(18): 4180-4186 CrossRef
  6. Kubicek C.P., Mach R.L., Peterbauer C.K., LoritoM. Trichoderma: from genes to biocontrol. Journal of Plant Pathology, 2001, 83: 11-23.
  7. Benítez T., Rincon F.M., Limon M.C., Codon A.C. Biocontrol mechanisms of Trichoderma strains. International Microbiology, 2004, 7(4): 249-260 CrossRef
  8. Aktuganov G.E., Galimzyanova N.F., Melent'ev A.I., Kuz'mina L.Yu. Mikrobiologiya, 2007, 76: 471-479 CrossRef (in Russ.). 
  9. Chebotar' B.K., Makarova N.M., Shaposhnikov A.I., Kravchenko L.V. Prikladnaya biokhimiya i mikrobiologiya, 2009, 45(4): 465-471 CrossRef (in Russ.). 
  10. Arinbasarova A.Yu., Baskunov B.P., Medentsev A.G. Mikrobiologiya, 2017, 86(2): 258-260 CrossRef (in Russ.). 
  11. Kolomiets E.I., Bus'ko I.I., Anan'eva I.N., Abakshonok B.C. Kartofelevodstvo, 2013, 21(1): 220-227 (in Russ.). 
  12. Dzhalilov F.S. Kartofel' i ovoshchi, 2018, 8: 2-4 CrossRef (in Russ.). 
  13. Kolombet L.V., Zhigletsova S.K., Derbyshev V.V., Ezhov D.V., Kosareva N.I., Bystrova E.V. Prikladnaya biokhimiya i mikrobiologiya, 2001, 37(1): 110-114 (in Russ.). 
  14. Zaika N.A., Gromovykh T.I., Ushanova V.M. V sbornike: Lesnoi i khimicheskii kompleksy — problemy i resheniya (ekologicheskie aspekty) [In: Forest and chemical complexes — problems and solutions (environmental aspects)]. Krasnoyarsk, 2004, 3: 34-37 (in Russ.). 
  15. Sánchez C. Modern aspects of mushroom culture technology. Applied Microbiology and Biotechnology, 2004, 64(6): 756-762 CrossRef
  16. Sánchez C. Cultivation of Pleurotus ostreatus and other edible mushrooms. Applied Microbiology and Biotechnology, 2010, 85(7): 1321-1337 CrossRef
  17. Chitamba J., Dube F., Chiota W.M., Handiseni M. Evaluation of substrate productivity and market quality of oyster mushroom (Pleurotus ostreatus) grown on different substrates. International Journal of Agricultural Research, 2012, 7(2): 100-106 CrossRef
  18. Li C., Chen C., Wu X., Tsang C.-W., Mou J., Yan J., Liu Y., Lin C.S.K. Recent advancement in lignin biorefinery: with special focus on enzymatic degradation and valorization. Bioresource Technology, 2019, 291: 121898 CrossRef
  19. Djarwanto, Tachibana S. Screening of fungi capable of degrading lignocellulose from plantation forests. Pakistan Journal of Biological Sciences, 2009, 12: 669-675 CrossRef
  20. Chukwurah N.F., Eze S.C., Chiejina N.V., Onyeonagu C.C., Ugwuoke K.I., Ugwu F.S.O., Nkwonta C.G., Akobueze E.U., Aruah C.B., Onwuelughasi C.U. Performance of oyster mushroom (Pleurotus ostreatus) in different local agricultural waste materials. African Journal of Biotechnology, 2012, 11(37): 8979-8985 CrossRef
  21. Čvančarová M., Křesinová Z., Filipová A., Covino S., Cajthaml T. Biodegradation of PCBs by ligninolytic fungi and characterization of the degradation products. Chemosphere, 2012, 88(11): 1317-1323 CrossRef
  22. Jafarpour M., Eghbalsaeed S. High protein complementation with high fiber substrates for oyster mushroom cultures. African Journal of Biotechnology, 2012, 11(14): 3284-3289 CrossRef
  23. Sales-Campos C., Pires D.A., Barbosa S.R.L., Abreu R.L.S., Andrade M.C.N. In vitro cultivation of Pleurotus ostreatus and Lentinula edodes in lignocellulosic residues from Amazon. African Journal of Biotechnology, 2013, 12(46): 6526-6531 CrossRef
  24. Titova Yu.A., Khlopunova L.B., Fedorova R.A., Zykov I.O. V sbornike: Sovremennaya mikologiya v Rossii [In: Modern mycology in Russia]. Moscow, 2017, tom 7: 389-391 (in Russ.). 
  25. Titova Yu.A., Dolgikh V.V., Bogdanov A.I. Vestnik zashchity rastenii, 2014, 3: 46-49 (in Russ.). 
  26. Novikova I.I. Vestnik zashchity rastenii, 2016; 87(3): 120-122 (in Russ.). 
  27. Titova Yu.A. Materialy III Vserossiiskogo s"ezda po zashchite rastenii [Proc. III All-Russian Congress on Plant Protection]. St. Petersburg, 2013, 2: 396-400 (in Russ.). 
  28. Segarra G., Aviles M., Casanova E., Borrero C., Trillas I. Effectiveness of biological control of Phytophthora capsici in pepper by Trichoderma asperellum strain T34. Phytopathologia Mediterranea, 2013, 52(1): 77-83 CrossRef
  29. Fedorenko V.F., Mishurov N.P., Konovalenko L.Yu. Sovremennye tekhnologii proizvodstva pestitsidov i agrokhimikatov biologicheskogo proiskhozhdeniya [Modern technologies for production of pesticides and biologicals]. Moscow, 2018 (in Russ.). 
  30. Novikova I.I., Boikova I.V., Pavlyushin V.A., Zeiruk V.N., Vasil'eva S.V., Derevyagina M.K. Vestnik zashchity rastenii, 2015, 86(4): 12-19 (in Russ.). 
  31. Zeiruk V.N., Vasil'eva S.V., Novikova I.I., Boikova I.V. Zashchita kartofelya, 2018, 1: 23-28 (in Russ.). 
  32. Novikova I.I., Titova Yu.A., Boikova I.V., Zeiruk V.N., Krasnobaeva I.L. Biotekhnologiya, 2017, 33(6): 68-76 CrossRef (in Russ.). 
  33. Dospekhov B.A. Metodika polevogo opyta [Methods of field trials]. Moscow, 1979 (in Russ.). 
  34. Metody eksperimental'noi mikologii: Spravochnik /Pod redaktsiei V.N. Bilai [Methods of experimental mycology: a handbook]. Kiev, 1982 (in Russ.). 
  35. Metodicheskie ukazaniya po registratsionnym ispytaniyam fungitsidov v sel'skom khozyaistve [Guidelines for registration testing fungicides during registration]. St. Petersburg, 2009 (in Russ.). 
  36. Ivanov A.I., Koryagin Yu.V., Anokhin R.V. XXI vek: itogi proshlogo i problemy nastoyashchego plyus, 2015, 27(5): 120-128 (in Russ.). 
  37. Fomin I.V., Kshnikatkin S.A. Kontsept, 2016, 11: 2791-2795 (in Russ.). 
  38. Pol'skikh S.V., Mel'kumova E.A., Fedyukina Yu.A., Odilovna Kh.K. Vestnik Michurinskogo GAU, 2015, 2: 31-36 (in Russ.). 
  39. Segarra G., Casanova E., Bellido D., Odena M.A., Oliveira E., Trillas I. Proteome, salicylic acid, and jasmonic acid changes in cucumber plants inoculated with Trichoderma asperellum strain T34. Proteomics, 2007, 7(21): 3943-3952 CrossRef
  40. Yoshioka Y., Ichikawa H., Naznin H.A., Kogure A., Hyakumachi M. Systemic resistance induced in Arabidopsis thaliana by Trichoderma asperellum SKT-1, a microbial pesticide of seedborne diseases of rice. Pest Management Science, 2012, 68(1): 60-66 CrossRef
  41. Singh V., Upadhyay R.S., Sarma B.K., Singh H.B. Trichoderma asperellum spore dose depended modulation of plant growth in vegetable crops. Microbiological Research, 2016, 193: 74-86 CrossRef
  42. Zhang F., Ruan X., Wang X., Liu Z., Hu L., Li C. Overexpression of a chitinase gene from Trichoderma asperellum increases disease Resistance in transgenic soybean. Applied Biochemistry and Biotechnology, 2016, 180(8): 1542-1558 CrossRef
  43. Su S., Zeng X., Bai L., Williams P.N., Wang Y., Zhang L., Wu C. Inoculating chlamydospores of Trichoderma asperellum SM-12F1 changes arsenic availability and enzyme activity in soils and improves water spinach growth. Chemosphere, 2017, 175: 497-504 CrossRef
  44. Asad S.A., Ali N., Hameed A., Khan S.A., Ahmad R., Bilal M., Shahzad M., Tabassum A. Biocontrol efficacy of different isolates of Trichoderma against soil borne pathogen Rhizoctonia solani. Polish Journal of Microbiology, 2014, 63(1): 95-103.
  45. Mahmoud H.E.K., Amgad A.S., Anas E., Younes Y.M. Characterization of novel Trichoderma asperellum isolates to select effective biocontrol agents against tomato Fusarium wilt. The Plant Pathology Journal, 2015, 31(1): 50-60 CrossRef
  46. Li Y., Sun R., Yu J., Saravanakumar K., Chen J. Antagonistic and biocontrol potential of Trichoderma asperellum ZJSX5003 against the maize stalk rot pathogen Fusarium graminearum. Indian Journal of Microbiology, 2016, 56(3): 318-327 CrossRef
  47. Qiong W., Ruiyan S., Mi N., Jia Y., Yaqian L., Chuanjin Y., Kai D., Jianhong R., Jie C. Identification of a novel fungus, Trichoderma asperellum GDFS1009, and comprehensive evaluation of its biocontrol efficacy. PLoS ONE, 12(6): e0179957 CrossRef







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