Сontrolled breeding of the psychrophilic strain G-034 VIZR of Trichoderma asperellum for fast crop residues’ polymers utilization and soil enhancement

Genus Trichoderma strains as the natural plant residues’ biodestructors, highly active antagonists of soil phytopathogens and phytoregulators with the widest range of optimum conditions for their development, are widely used in biologics development. Of particular importance in Russia’s northern regions, especially in winter crop cultivation, is the ability of a microorganism’s strain used in agro-technologies to maintain viability and target biological activity at low temperatures. In this connection, this work purpose is to select a psychrotolerant strain of T. asperellum for the rapid crop residues’ polymer utilization and soil enhancement at low temperature, as well as to evaluate its activity under laboratory and field conditions. In the work process, the following tasks were addressed: selecting psychrotolerant strains of T. asperellum with high cellulolytic activity; further controlled breeding of psychrophilic strains capable of rapid growth, active colonization of plant substrates and high sporulation at 4–8 °C; evaluating the target activity of the selected psychrophilic strain as a cellulolytic as well as antagonistic activity against cereal pathogens; obtaining laboratory samples of bioformulations by deep-surface cultivation on non-sterile peat and multirecycled wastes from the edible mushrooms production and assessing their efficacy in field small-plot trials. The methods for inoculum cultivation, sporulation capacity determination, modified wet chamber, estimating antagonistic activity and biologics’ quality, field small-plot trials management, quantitative estimates of biomass losses, cellulose and lignin content were used in the work. The active psychrophilic strain for the rapid crop residues’ polymer utilization and soil enhancement controlled breeding was selected during a four-step screening of 29 T. asperellum strains from All-Russian Research Institute of Plant Protection (VIZR) State Microorganisms’ Collection with high cellulolytic and antagonistic activities. In terms of linear growth rate, antagonistic and hyperparasitic activities at 4–8 °С, a high rate of wheat and maize stubble residues’ colonization, a perspective psychrophilic strain G-034 of T. asperellum was selected for developing the laboratory samples of biologics and for running field trials. In small-plot trials, the active maize crop residues’ decomposition under the T. asperellum G-034 influence was revealed, resulting in the complete loss of plant intact state in 12 months due to more than 80 % cellulose and 20 % lignin biodestruction. The maximum loss of maize crop residues biomass for 12 months was more than 70 %. The T. asperellum strain G-034 was active after field hibernation in an amount of ×104 cfu/g, resulting in a titer increase with seasonal temperature rising and the trophic base bioavailability growth.

1. Алимова Ф.К. Trichoderma/Hypocrea (Fungi, Ascomycetes, Hypocreales): таксономия и распространение. Казань, 2005. [Alimova F.K. Trichoderma/Hypocrea (Fungi, Ascomycetes, Hypocreales): Taxonomy and Distribution. Kazan, 2005. (in Russian)]

2. Алимова Ф.К., Тухбатова Р.И., Тазетдинова Д.И., Кабрера Ф.Х.А., Каримова Л.Ю. Взаимоотношения Тrichoderma, распространенной на территории Республики Татарстан, с микроорганизмами и растениями. Грибы и водоросли в биоценозах: Материалы междунар. конф., посвящ. 75-летию биологического факультета МГУ им. М.В. Ломоносова. Москва, 31 янв.–3 февр. 2006 г. М., 2006;12-13. [Alimova F.K., Toukhbatova R.I., Tazetdinova D.I., Cabrera F.J.A., Karimova L.Yu. Interactions of Тrichoderma, widespread throughout the Republic of Tatarstan, with microorganisms and plants. In: Fungi and Algae in Biocenoses: Proc. Int. Conf., dedicated to the 75th anniversary of the Department of Biology, Lomonosov Moscow State University. Moscow, January 31–February 3, 2006. Мoscow, 2006;12-13. (in Russian)]

3. Аринбасарова А.Ю., Баскунов Б.П., Меденцев А.Г. Низкомолекулярный антимикробный пептид из Trichoderma cf. aureoviride Rifai ВКМF-4268D. Микробиология. 2017;86(2):258-260. [Arinbasarova A.Yu., Baskunov B.P., Medentsev A.G. A low-molecular-mass antimicrobial peptide from Trichoderma cf. aureoviride Rifai VKMF-4268D. Mikrobiologiya = Microbiology. 2017;86(2): 258-260. (in Russian)]

4. Бисько Н.А., Дудка И.А. Биология и культивирование съедобных грибов рода вешенка. Киев, 1987. [Bisko N.A., Dudka I.A. Biology and Cultivation of Edible Mushrooms of the Genus Pleurotus. Kiev, 1987. (in Russian)]

5. Бисько Н.А., Фомина В.И., Володина Е.П., Билай В.Т. Изменение химического состава субстрата при культивировании Pleurotus ostreatus (Jacq.: Fr.) Kumm. Микология и фитопатология. 1986; 20(5):392-395. [Bisko N.A., Fomina V.I., Volodina E.P., Bilay V.T. Change in the chemical composition of the substrate during Pleurotus ostreatus (Jacq.: Fr.) Kumm. cultivation. Mikologiya i Fitopatologiya = Mycology and Phytopathology. 1986;20(5):392-395. (in Russian)]

6. Богданов А.И., Титова Ю.А. Антагонистическая активность штаммов Trichoderma asperellum – продуцентов мультиконверсионных биопрепаратов. Вестн. защиты растений. 2014;1:48-52. [Bogdanov A.I., Titova J.A. Antagonistic activity of Trichoderma asperellum strains, multirecycling bioformulation producers. Vestnik Zashchity Rastenii = Plant Protection News. 2014;1:48-52. (in Russian)]

7. Доспехов Б.А. Методика полевого опыта. М., 1979. [Dospekhov B.A. Methodology of Field Experience. Moscow, 1979. (in Russian)]

8. Егоров Н.С. Основы учения об антибиотиках. М., 2004. [Egorov N.S. Fundamentals of the Antibiotics Doctrine. Moscow, 2004. (in Russian)]

9. Зиганшин Д.Д., Лукьянцев М.А., Егоршина А.А., Сироткин А.С. Оценка способности консорциума микроорганизмов к утилизации стерни. Вестн. Казан. технол. ун-та. 2016;19(16):103-107. [Ziganshin D.D., Lukyantsev M.A., Egorshina A.A., Sirotkin A.S. Assessment of the ability of a microbial consortium to utilize stubbles. Vestnik Kazanskogo Tekhnologicheskogo Universiteta = Bull. Kazan Technol. Univ. 2016;19(16):103-107. (in Russian)]

10. Зиганшин Д.Д., Сироткин А.С. Особенности глубинного и поверхностного культивирования грибов Trichoderma для получения биопрепаратов на основе клеток гриба. Вестн. Казан. технол. ун-та. 2017;20(10):155-158. [Ziganshin D.D., Sirotkin A.S. Features of Trichoderma fungi deep and surface cultivation for obtaining bioformulations based on fungal cells. Vestnik Kazanskogo Tekhnologicheskogo Universiteta = Bull. Kazan Technol. Univ. 2017;20(10):155-158. (in Russian)]

11. Коломбет Л.В., Жиглецова С.К., Дербышев В.В., Ежов Д.В., Косарева Н.И., Быстрова Е.В. Микофунгицид – препарат на основе Trichoderma viride для борьбы с болезнями растений. Прикл. биохимия и микробиология. 2001;37(1):110-114. [Kolombet L.V., Zhigletsova S.K., Derbyshev V.V., Ezhov D.V., Kosareva N.I., Bystrova E.V. Mycofungicide is a biologic based on Trichoderma viride to control plant diseases. Prikladnaya Biokhimiya i Mikrobiologiya = Applied Biochemistry and Microbiology. 2001;37(1):110-114. (in Russian)]

12. Лабынцев А.В., Целуйко О.А. Способ разложения растительных остатков. Пат. РФ RU 2485752 C1. 2013. [Labyntsev A.V., Tseluyko O.A. A method of plant residues destruction. Patent RU 2485752 C1. 2013. (in Russian)]

13. Лилли В., Барнетт Г. Физиология грибов. М., 1953. [Lilly V., Barnett G. Physiology of the Fungi. New York: McGrawHill Publ., 1951. (Russ. ed.: Lilly V., Barnett G. Physiology of the Fungi. Moscow, 1953. (in Russian))]

14. Методы экспериментальной микологии: Справочник. Киев, 1982. [Experimental Mycology Methods: Handbook. Kiev, 1982. (in Russian)]

15. Новикова И.И. Полифункциональные биопрепараты на основе микробов-антагонистов – основа экологически безопасной системы защиты растений от болезней. Информ. бюл. ВПРС МОББ. 2007;38:173-175. [Novikova I.I. Polyfunctional bioformulations based on antagonist microbes are the basis of an environmentally safe system of plant protection against diseases. Informatsionnyy Byulleten VPRS MOBB = IOBC EPRS Informational Bull. 2007;38:173-175. (in Russian)]

16. Новикова И.И. Биологическое разнообразие микроорганизмов – основа для создания новых полифункциональных биопрепаратов для фитосанитарной оптимизации агроэкосистем. Вестн. защиты растений. 2016;83(3):120-122. [Novikova I.I. The biological diversity of microorganisms is the basis for new multifunctional bioformulation development for phytosanitary optimization of agroecosystems. Vestnik Zashchity Rastenii = Plant Protection News. 2016;83(3):120-122. (in Russian)]

17. Оболенская А.В., Щеголев В.П., Аким Г.А. Практические работы по химии древесины и целлюлозы. М., 1955. [Obolenskaya A.V., Shchegolev V.P., Akim G.A. Practical Work on the Chemistry of Wood and Cellulose. Moscow, 1955. (in Russian)]

18. Рудаков О.Л. Микофильные грибы, их биология и практическое значение. М., 1981. [Rudakov O.L. Mycophilic Fungi: Biology and Practical Importance. Moscow, 1981. (in Russian)]

19. Рудаков О.Л. Проблемы и перспективы использования гиперпаразитов и антагонистов в защите растений от инфекционных заболеваний: Микробиологические средства защиты растений. Новосибирск, 1986;139-143. [Rudakov O.L. Problems and Prospects of Using Hyperparasites and Antagonists in Plant Protection against Infectious Diseases: Microbiological Means for Plant Protection. Novosibirsk, 1986;139-143. (in Russian)]

20. Рязанова Т.В., Чупрова Н.А., Лунева Т.А. Воздействие гриба рода Trichoderma на лигнин коры хвойных пород. Катализ в промышленности. 2014;6:64-70. [Ryazanova T.V., Chuprova N.A., Luneva T.A. Effect of Trichoderma fungi on softwood bark lignin. Kataliz v Promyshlennosti = Catalysis in Industry. 2014;6:64-70. (in Russian)]

21. Садыкова В.С., Лихачев А.Н., Бондарь П.Н. Ограничение развития комплекса возбудителей корневых гнилей ячменя антагонистами рода Trichoderma. Микология и фитопатология. 2010;44(6): 556-562. [Sadykova V.S., Likhachev A.N., Bondar P.N. Restriction of barley root rot development by antagonistic fungi of the genus Trichoderma. Mikologiya i Fitopatologiya = Mycology and Phytopathology. 2010;44(6):556-562. (in Russian)]

22. Садыкова В.С., Третьякова И.Н., Носкова Н.Е., Бондарь П.Н. Антагонистическая и ростостимулирующая активность штаммов рода Trichoderma и перспективы их использования в биоконтроле. Иммунопатология, аллергология, инфектология. 2009;2: 71-72. [Sadykova V.S., Tretyakova I.N., Noskova N.E., Bondar P.N. Antagonistic and growth-stimulating activity of the genus Trichoderma strains and prospects of their use in biocontrol. Immunopatologiya, Allergologiya, Infektologiya = Immunopathology, Allergology, Infectology. 2009;2:71-72. (in Russian)]

23. Титова Ю.А. Методология получения мультиконверсионных биопрепаратов для защиты растений. Фитосанитарная оптимизация агроэкосистем: Сб. науч. трудов III Всерос. съезда по защите растений. СПб., 2013;2:396-400. Titova J.A. Methodology for obtaining multirecycled bioformulations for plant protection. In: Phytosanitary Optimization of Agroecosystems: Proc. III All-Russian Plant Protection Congr. St. Petersburg, 2013;2:396-400. (in Russian)]

24. Титова Ю.А., Бойкова И.В., Бочкова В.Б. Биологическая активность мультиконверсионных биопестицидов на основе актиномицетов. Современная микология в России: IV cъезд микологов России. М., 2017а;7:284-286. [Titova J.A., Boykova I.V., Bochkova V.B. Biological activity of multirecycled biopesticides based on actinomycetes. In: Modern Mycology in Russia: Proc. IV Congr. Russian Mycologists. Moscow, 2017а;7:284-286. (in Russian)]

25. Титова Ю.А., Долгих В.В., Богданов А.И. Особенности биоконверсии компонентов растительных субстратов штаммами-продуцентами биопрепаратов. Вестн. защиты растений. 2014;3:46-49. [Titova J.A., Dolguikh V.V., Bogdanov A.I. Biorecycling features of plant substrate components by bioformulation producer strains. Vestnik Zashchity Rasteniy = Plant Protection News. 2014,3:46-49. (in Russian)]

26. Титова Ю.А., Сокорнова С.В., Полякова О.Ю. Особенности роста энтомопатогенных видов Beauveria при мультибиоконверсии отходов производства съедобных грибов. Современная микология в России: IV cъезд микологов России. М., 2017б;6: 405-406. [Titova J.A., Sokornova S.V., Polyakova O.Yu. Growth features of Beauveria entomopathogenic species in the multibiorecycling of edible fungi production wastes. In: Modern Mycology in Russia: Proc. IV Congr. Russian Mycologists. Moscow, 2017b;6:405-406. (in Russian)]

27. Титова Ю.А., Хлопунова Л.Б., Федорова Р.А., Зыков И.О. Производство вешенки путем мультибиоконверсии отходов производства шии-таке. Современная микология в России: IV cъезд микологов России. М., 2017в;7:389-391. [Titova J.A., Khlopunova L.B., Fedorova R.A., Zykov I.O. Oyster mushroom production by shiitake waste multibiorecycling. In: Modern Mycology in Russia: Proc. IV Congr. Russian Mycologists. Moscow, 2017c;7:389-391. (in Russian)]

28. ТУ 9291-005-59147141-2006. Технические условия. Триходермин нова. 2006. [ТS 9291-005-59147141-2006. Technical specifications. Trichodermin nova. 2006. (in Russian)]

29. Alamri S., Hashem M., Mostafa Y.S. In vitro and in vivo biocontrol of soil-borne phytopathogenic fungi by certain bioagents and their mode of action. Biocontrol Sci. 2012;17(4):155-167.

30. Benítez T., Rincon F.M., Limon M.C., Codon A.C. Biocontrol mechanisms of Trichoderma strains. Int. Microbiol. 2004;7(4):249-260.

31. Bheemaraya P.M.B., Ramesh N.M.K., Kalyanrao. Effect of substrates on growth and shelf life of Trichoderma species. J. Mycol. Plant Pathol. 2011;41(4):618-621.

32. Boureghda H., Renane R. In vitro study of antagonistic activity of some isolates of Trichoderma spp. against Fusarium spp. isolates the causal agent of wheat head scab. Arab. J. Plant Protection. 2011;29: 51-59.

33. Devi P., Prabhakaran N., Kamil D., Pandey P., Borah J.L. Characterization of Indian native isolates of Trichoderma spp. and assessment of their biocontrol efficiency against plant pathogens. Afr. J. Biotech. 2012;11(85):15150-15160. DOI 10.5897/AJB12.2007.

34. Haran S., Schickler H., Chet I. Molecular mechanisms of lytic enzymes involved in the biocontrol activity of Trichoderma harzianum. Microbiology. 1996;142:2321-2331.

35. Heidi I.G., Abo-Elnaga. Biological control of damping off and root rot of wheat and sugar beet with Trichoderma harzianum. Plant Pathol. J. 2012;11(1):25-31. DOI 10.3923/ppj2012.25.31.

36. Kubicek C.P., Mach R.L., Peterbauer C.K., Lorito M. Trichoderma: from genes to biocontrol. J. Plant Pathol. 2001;83:11-23.

37. Parra T.J., Maniscalco P.D. Effect of Trichoderma spp. on Rhizoctonia solani and some physiological attributes on Zea mays L. under greenhouse conditions. Fitopatol. Venez. 2012;25:10-15.

38. Schwanninger M., Hinterstoisser B. Klason lignin: Modification to improve the precision of the standardized determination. Holzforschung. 2002;56:161-166.

39. Whipps J.M., Lumsden R.D. Commercial use of fungi as plant disease biological control agents: Status and prospects. In: Butt T.M., Jackson C., Magan N. (Eds.). Fungi as Biocontrol Agents: Progress, Problems and Potential. Wallingford, UK: CABI Publ., 2001;9-22.