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BIOCHEMICAL AND MOLECULAR ASPECTS OF INDUCED RESISTANCE BY TRICHODERMA IN GROUNDNUT (Arachis hypogaea L.) AGAINST COLLAR ROT (Aspergillus niger Van Tieghem)

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Title BIOCHEMICAL AND MOLECULAR ASPECTS OF INDUCED RESISTANCE BY TRICHODERMA IN GROUNDNUT (Arachis hypogaea L.) AGAINST COLLAR ROT (Aspergillus niger Van Tieghem)
 
Creator Gajera Harsukhray Popatbhai
 
Contributor Vakharia D.N.
 
Subject GROUNDNUT
BIOCHEMISTRY
 
Description Experiment was conducted in three parts. (1) In vitro antagonistic effect of various isolates of Trichoderma strains as bio-agents against disease causing A. niger. (2) Molecular characterization of various isolates of Trichoderma strains and disease causing pathogen A. niger. (3) Biochemical and molecular aspects on host-pathogen interaction during collar rot disease in groundnut seedlings and induced resistance by Trichoderma. Among the 12 isolates of 3 Trichoderma strains, T. viride 60 exhibited highest (86.2%) in vitro per cent growth inhibition of fungal pathogen A. niger followed by T. harzianum 2J (80.4%) on PDA media. This per cent inhibition was positively correlated with specific activities of lytic enzymes: chitinase, -1,3 glucanase and protease, and negatively correlated with cellulase and poly galacturonase (PG). T. viride 60 was found suitable strain to be used in biological control of plant pathogen A. niger.
The RAPD profiles of genomic DNA of Trichoderma isolates and A. niger demonstrated presence of high level of polymorphism between them. The calculated PIC values for RAPD markers ranged from 0.172 to 0.401 and RAPD primer index (RPI) differed from 0.99 to 6.01. RPI revealed that RFu C-5 gave best results among the primer used. RAPD markers showed 10 marker loci for diagnosis of T. viride 60 and T. harzianum 2J (first two highest inhibitory acting antagonist). Dendrogram constructed on the basis of Jaccard's similarity coefficient which illustrated two distinct clusters of 12 isolates of Trichoderma and A. niger pathogen, and shared 19% similarity.
However, the in vitro highest growth (A. niger) inhibitory Trichoderma isolates - T. viride 60 and T. harzianum 2J were in same out group and shared 63% similarity. In pot culture study, per cent collar rot disease incidence were found higher in susceptible (GG-20; 67.4%) variety, moderate in GAUG-10, GG-13; (46%) and minimum in tolerant varieties (J-11, GG-2; 30%) in A. niger infected pot at 15 DAS. The rate of oxalic acid deposition was higher in susceptible variety as compared to the tolerant in infested pot culture, and further it was declined at higher rate (27.8 and 36.9 %) due to Trichoderma treatment. Oxalic acid is an indicator for pathogenicity of collar rot. The malondialdehyde (MDA) content as product of lipid peroxidation increased upto 9 DAS in tolerant varieties and upto 12 DAS in GG-13 and GG-20 varieties under A. niger infection. This result is correlated with high lipoxygenase activity in tolerant varieties. Ascorbic acid was higher in tolerant varieties as compared to the susceptible and the rate of increases was also higher in tolerant varieties. After collar rot infection, free amino acids content increased in GG-20 (@ 422%) upto 9 DAS, while same was accumulated by 55% in J-11 and GG-2. Total phenol content increased in GG-20 at a greater rate (193%) upto 9 DAS than tolerant GG-2, where it accumulated 146% during 0 DAS to 3 DAS stage. The content was higher upto S1 stage followed by declined during subsequent stages in all groundnut varieties of Trichoderma treated seedlings. Phenolic acids profiling indicated that gallic, hydroquinone, chlorogenic, ferulic, salicylic and cinnamic acids were increased during disease developmental stages in Trichoderma treated groundnut seeds sown in A. niger infected soil.
The specific activities of ROS scavenging enzymes: superoxide dismutase (SOD), peroxidase (Px) and ascorbate peroxidase (APx) increased upto 12 DAS in tolerant varieties in sick soil and sick + Trichoderma treatment. Overall, Trichoderma treated seedling had 1.25 to 3.25 fold higher activity than sick treatment. The lipoxygenase (LOX) activity was found to be increased in tolerant varieties (3.5 fold) during 0 to 12 DAS in sick soil and sick + Trichoderma treatment, while a reverse trend was observed in susceptible variety. LOX activity was induced by Trichoderma bio-control
agents in tolerant varieties, and hence it may be a marker for collar rot disease resistance. Among phenol metabolism enzymes, Trichoderma treated seedling had 1.5 fold higher and early induction (6 DAS) of phenylalanine ammonia lyase (PAL) specific activity than sick treatment (9 DAS). The polyphenol oxidase (PPO) activity induced by 1.0 fold in bio-control agents T. viride 60 treated groundnut seeds and challenged with A. niger compared to sick treatment upto 3 DAS. The level of PAL and PPO was higher in tolerant varieties and it maintained through out the disease developmental stages. Among the elicitor releasing enzymes, the chitinase activity increased about 2.3 to 2.8 fold in tolerant varieties and it was about 2.6 to 3.2 fold in moderately susceptible varieties. The minimum enzyme induction observed in susceptible GG-20 (2.0 fold) upon Trichoderma treatment upto 9 DAS stage. The level of ß-1,3 glucanase was higher in tolerant varieties and it maintained through out the disease development stages. Overall, Trichoderma treated seedling had 0.75 fold higher ß-1,3 glucanase activity than sick treatment. The densitometric analysis for Native PAGE revealed that some of the new bands (102, 33.4 and 22.6 KDa) observed in Trichoderma treated groundnut seedlings which might be considered as positive marker for induced resistance against collar rot disease. The RAPD and ISSR were utilized for the characterization of five groundnut varieties. RAPD and ISSR markers showed 5 and 7 markers loci respectively in tolerant varieties against collar rot. SSR primer PM-50 was the best primer for identifying the collar rot disease resistance reaction in groundnut varieties. RAPD and ISSR data analyzed through Jaccard's similarity coefficient which showed two distinct clusters and out grouped either on tolerant or susceptible varieties of groundnut. These results suggested that chlorogenic acid (phenolic), native protein banding pattern, LOX, chitinase and ß-1,3 glucanase enzymes can work as biochemical markers, and RAPD and ISSR as a molecular markers may be used for large scale screening for disease reaction traits against collar rot in groundnut.
 
Date 2016-09-21T09:04:17Z
2016-09-21T09:04:17Z
2009-05
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/77861
 
Language en
 
Format application/pdf