Biocontrol potential of Trichoderma asperellum mutants T39 and T45 and their growth promotion of poplar seedlings

Abstract

This study investigates the biocontrol potential of Trichoderma asperellum mutants against Rhizoctonia solani, Alternaria alternata, and Fusarium oxysporum and growth promotion of Populus davidiana ×  P. alba var. pyramidalis (PdPap poplar) seedlings. A T-DNA insertion mutant library of T. asperellum was constructed using Agrobacterium tumefaciens-mediated transformation. Sixty-five positive transformants (T1–T65) were obtained. Growth rates of the mutants T39 and T45 were the same, 39.68% faster than the WT. In toxin tolerance tests, only T39 had greater tolerance to A. alternata fermentation broth than the WT, but mutant T45 had the same tolerance as the WT to all fermentation broths. Furthermore, T39 and T45 had a greater antagonistic ability than the WT strain against R. solani and A. alternata. The inhibition rate of the mutants T39 and T45 against A. alternata are 73.92% and 80.76%, respectively, and 63.51% and 63.74%, respectively. Furthermore, the three strains increased the activities of superoxide dismutases, peroxidase, catalase (CAT) and phenylalanine ammonia lyase (PAL) in PdPap seedling leaves. CAT and PAL activity in the PdPap seedling leaves was 11.25 and 5.50 times higher, respectively, in the presence of T39 than in the control group and 12 and 6.35 times higher, respectively, in the presence of T45 than in the control group. All three strains promoted seedling growth and the root and stem development, especially mutant T45. Mutants T39 and T45 reduced the incidence of pathogenic fungi in poplar and stimulated poplar seedling growth.

Keywords

Biocontrol / Trichoderma asperellum / Mutant library / Fungal disease / Poplar growth

References

[1] Brotman Y, Landau U, Cuadros-Inostroza Á, Takayuki T, Fernie AR, Chet I, Viterbo A, Willmitzer L. Trichoderma-plant root colonization: escaping EARLY plant defense responses and activation of the antioxidant machinery for saline stress tolerance. PLoS Pathog, 2013, 9: 130-139.

[2] Dong ZY, Wang ZZ. Isolation and characterization of an exopolygalacturonase from Fusarium oxysporum f.sp. cubense race 1 and race 4. BMC Biochem, 2011, 12: 51-60.

[3] Druzhinina IS, Seidl-Seiboth V, Herrera-Estrella A, Horwitz BA, Kenerley CM, Monte E, Mukherjee PK, Zeilinger S, Grigoriev IV, Kubicek CP. Trichoderma: the genomics of opportunistic success. Nat Rev Microbiol, 2011, 9: 749-759.

[4] Fu J, Liu ZH, Li ZT, Wang YF, Yang KJ. Alleviation of the effects of saline-alkaline stress on maize seedlings by regulation of active oxygen metabolism by Trichoderma asperellum. PLoS ONE, 2017, 12: e179617.

[5] Huang Y, Mijiti G, Wang ZY, Yu WJ, Fan HJ, Zhang RS, Liu ZH. Functional analysis of the class II hydrophobin gene HFB2-6 from the biocontrol agent Trichoderma asperellum ACCC30536. Microbiol Res, 2015, 171: 8-20.

[6] Jalón JGD, Gutiérrez-López MD. Development of a mutant of Trichoderma citrinoviride for enhanced production of cellulases. Bioresour Technol, 2009, 100: 1659-1662.

[7] Ji SD, Wang ZY, Fan HJ, Zhang RS, Yu ZY, Wang JJ, Liu ZH. Heterologous expression of the Hsp24 from Trichoderma asperellum improves antifungal ability of Populus transformant Pdpap-Hsp24 s to Cytospora chrysosperma and Alternaria alternata. J Plant Res, 2016, 129: 921-933.

[8] Kovács K, Megyeri L, Szakacs G, Kubicek CP, Galbe M, Zacchi G. Trichoderma atroviride mutants with enhanced production of cellulase and β-glucosidase on pretreated willow. Enzyme Microb Technol, 2008, 43: 48-55.

[9] Latifian M, Hamidiesfahani Z, Barzegar M. Evaluation of culture conditions for cellulase production by two Trichoderma reesei mutants under solid-state fermentation conditions. Bioresour Technol, 2007, 98: 3634-3637.

[10] Lü XX, Zhang WX, Chen GJ, Liu WF. Trichoderma reesei Sch9 and Yak1 regulate vegetative growth, conidiation, and stress response and induced cellulase production. J Microbiol, 2015, 53: 236-242.

[11] MarkSchubert SiegfriedFink, Schwarze FMR. Field experiments to evaluate the application of Trichoderma strain (T-15603.1) for biological control of wood decay fungi in trees. Arbor Assoc J, 2008, 31: 249-268.

[12] Mastouri F, Björkman T, Harman GE. Seed treatment with Trichoderma harzianum alleviates biotic, abiotic, and physiological stresses in germinating seeds and seedlings. Phytopathology, 2010, 100: 1213-1221.

[13] Mathiyazhagan S, Kavitha K, Chandrasekar G, Nakkeeran S, Manian K, Krishnamoorthy AS, Sankaralingam A, Fernando WGD. Toxin production by Corynespora cassiicola in Phyllanthus amarus, the stem blight pathogen. Acta Phytopathol Hung, 2005, 40: 55-65.

[14] Michielse CB, Hooykaas PJJ, van den Hondel CAMJ, Ram AFJ. Agrobacterium-mediated transformation as a tool for functional genomics in fungi. Curr Genet, 2005, 48: 1-17.

[15] Mirzaee M, Moieni A, Ghanati F. Effects of drought stress on the lipid peroxidation and antioxidant enzyme activities in two canola (Brassica napus L.) cultivars. J Agric Sci Technol Iran, 2013, 15: 593-602.

[16] Mohamed HAA, Wafaa Haggag M, Roche L, Edin M, Mathieu V, Laurens F. Biocontrol potential of salinity tolerant mutants of Trichoderma harzianum against wilt of tomato. Braz J Microbiol, 2006, 37: 181-191.

[17] Mullins ED, Chen X, Romaine P, Raina R, Geiser DM, Kang S. Agrobacterium-mediated transformation of Fusarium oxysporum: an efficient tool for insertional mutagenesis and gene transfer. Phytopathology, 2001, 91: 173-180.

[18] Noël A, Levasseur C, Le VQ, Séguin A. Enhanced resistance to fungal pathogens in forest trees by genetic transformation of black spruce and hybrid poplar with a Trichoderma harzianum endochitinase gene. Physiol Mol Plant, 2005, 67: 92-99.

[19] Rawat L, Singh Y, Shukla N, Kumar J. Seed biopriming with salinity tolerant isolates of Trichoderma harzianum alleviates salt stress in rice: growth, physiological and biochemical characteristics. J Plant Pathol, 2012, 94: 353-365.

[20] Sun WL, Liu LX, Hu XL, Tang J, Liu P, Chen J, Chen YP. Generation and identification of DNA sequence flanking T-DNA integration site of Trichoderma atroviride mutants with high dichlorvos-degrading capacity. Bioresour Technol, 2009, 100: 5941-5946.

[21] Tsuji G, Fujii S, Fujihara N, Hirose C, Tsuge S, Shiraishi T, Kubo Y. Agrobacterium tumefaciens-mediated transformation for random insertional mutagenesis in Colletotrichum lagenarium. J Gen Plant Pathol, 2003, 69: 230-239.

[22] Vidhyasekaran P, Ponmalar TR, Samiyappan R, Velazhahan R, Vimala R, Ramanathan A, Paranidharan V, Muthukrishnan S. Host-specific toxin production by Rhizoctonia solani, the rice sheath blight pathogen. Phytopathology, 1997, 87: 1258-1263.

[23] Wang B, Liu LX, Gao YD, Chen J. Improved phytoremediation of oilseed rape (Brassica napus) by Trichoderma mutant constructed by restriction enzyme-mediated integration (REMI) in cadmium polluted soil. Chemosphere, 2009, 74: 1400-1403.

[24] Wang WB, Kim YH, Lee HS, Kim KY, Deng XP, Kwak SS. Analysis of antioxidant enzyme activity during germination of alfalfa under salt and drought stresses. Plant Physiol Biochem, 2009, 47: 570-577.

[25] Yi HW, Chi YJ. Biocontrol of Cytospora canker of poplar in north-east China with Trichoderma longibrachiatum. Forest Pathol, 2011, 41: 299-307.

[26] Zhou XY, Xu SF, Liu LX, Chen J. Degradation of cyanide by Trichoderma mutants constructed by restriction enzyme mediated integration (REMI). Bioresour Technol, 2007, 98: 2958-2962.