Elucidating the role of Trichoderma in the triple combination ‘Copper-Chitosan-Trichoderma’ for the management of late blight disease of potato (Solanum tuberosum L.)
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Date
2017-06
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G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand)
Abstract
A ‘triple combination’ comprising of low dose of fungicide (CuOH), biocontrol agent (Trichoderma) and plant defence activator compound (chitosan) developed at Biocontrol laboratory, G.B.P.U.A.&T., Pantnagar and field tested over years was found very effective in the management of late blight of potato. In the present study, the ‘triple combination’ was further investigated by testing its different variants in two geographically different conditions and it was found that the ‘triple combination’ in its various forms was found very effective in managing late blight in both hills as well as plains. Its use led to reduction of disease severity, increased plant growth and increased yield that was at par with the standard recommended fungicides, viz., mancozeb, CuOH used for late blight management. Present study also revealed that spraying of the novel combination at proper concentration (CuOH 500 ppm+CS651 500 ppm+ Tri. (1%)), proper time (5 sprays at 35,45,55,65 and 75 DAS) with proper volume of water (1000 L/ha in 1st spray, 1083 L/ha in 2nd spray, 1166 L/ha in 3rd spray, 1333 L/ha in 4th spray and 1416 L/ha in 5th spray) resulted into significant gains in terms of disease severity reduction, plant growth promotion and tuber yield. Results of in-vitro studies conducted to find out interaction of CuOH-tolerant Trichoderma asperellum strains and chitosan showed that PCR amplification with the primers designed specifically to detect chitosanolytic genes viz., N-acetyl-β-D-glucosaminidase (NAG), hexosaminodase (HEXO) and chitinase (CHIT) resulted in expected bands of ~200bp, ~80bp and ~1450bp in case of NAG, HEXO and CHIT genes, respectively in both CuOH-tolerant Trichoderma asperellum strains TCMS-36 and SBIT-32, which indicated presence of chitosanolytic genes in the two strains. Expression profiling of the chisanolytic genes through real time-PCR revealed that chitosan incorporation into the media led to upregulation of different chitosanolytic genes. In TCMS-36, maximum expression of NAG (6.71±1.20) and HEXO (9.53±0.51) genes was observed at chitosan concentration of 500 ppm, while in case of SBIT32, maximum expression of NAG (2.82±1.45) and HEXO (4.84±1.09) was observed at 125 ppm chitosan concentration. However, in case of CHIT gene, both TCMS-36 and SBIT-32 showed maximum expression (3.29±0.91, 4.32±0.72 respectively) at 125 ppm chitosan concentration. Expression profiling of chitosanolytic genes at protein level through SDS-PAGE revealed the presence of typical band of approximate 93 KDa when these strains were grown in PDB media amended with chitosan (125ppm & 500ppm) and the band was absent when both Trichoderma strains were grown in absence of chitosan, indicating that the production of chitosanase enzyme was substrate specific and that the enzyme corresponded to an exo-type chitosanase (exo-β-Dglucosaminidase). Results of Gas chromatography-mass spectrometry revealed that in addition to efficient chitosanolytic enzymatic battery, these CuOH-tolerant Trichoderma strains also possessed important secondary metabolites such as 1,2-Benzenedicarboxylic acid, 2H-Pyran-2-one, Tri- methylsilyl palmitate, phenolic isomers, etc. that reportedly have antimicrobial and plant growth promotion activity. It is postulated that the CuOH-tolerant Trichoderma asperellum strains used in the triple combination interact with chitosan in a synergistic manner using its chitosanolytic machinery to breakdown chitosan into smaller oligomers that have antimicrobial, plant growth promoting and defence inducing activity. This aspect need to be further addressed in order to understand the mechanism of action of the triple combination in the management of late blight of potato.
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