(“ASSESSMENT OF INDUCED RESISTANCE BY Pseudomonas fluorescens AGAINST COLLAR ROT (Aspergillus niger Van Tieghem) DISEASE IN GROUNDNUT (Arachis hypogea L.) USING BIOCHEMICAL AND MOLECULAR MARKERS’’)
The experiment was designed to study biochemical and molecular aspects related to host-pathogen and biocontrol agent interaction to find out most effective Pseudomonas fluorescens (Pf) isolate as a biocontrol agent against collar rot. The present experiment was conducted in two parts. (1) In vitro antagonistic effect of various isolates of Pseudomonas fluorescens as biocontrol agent against disease causing fungi Aspergilllus niger (2) Biochemical and molecular characterization of groundnut seedling infected with Aspergillus niger Van Tieghem and their interaction with biocontrol agent Pseudomonas fluorescens, in pot. Among the ten Pseudomonas fluorescens isolates, Pf-3 exhibited highest in vitro per cent growth inhibition (78.88%) against the fungal pathogen A. niger followed by Pf-9 (64.52%) and Pf-5 (62.30%). The culture media containing Pf-3 strain had higher activities of pathogenesis related enzymes chitinase (21.32 U.mg-1protein), β-1,3-glucanase (77.12 U.mg-1 protein) and reduced activities of cell wall degrading enzymes cellulase (12.33 U.mg-1 protein) and PG (18.50 U.mg-1 protein). Five different groundnut varieties grown in pots were treated with normal seed (T1), P.fluorescens (T2), A. niger (T3) and A. niger + P.fluorescens (T4), studied at three different disease developmental stages i.e. S1 pre-diseased (6 DAS), S2 diseased (12 DAS) and S3 post-diseased (18 DAS). The per cent collar rot disease incidence in groundnut was higher in semi-spreading (GG-20; 53% and GG-21; 46%) varieties while minimum in bunch (GG-2 and GG-5) varieties. Variety GG-20 had the highest free amino acid and phenol content in S2 stage. The amount of these metabolites increased upto diseased stage followed by reduction. The highest free amino acid content was found in T3 whereas T4 recorded highest phenol content. Phenolics profile was carried out using HPLC at post-disease stage which resulted in presence of higher gallic acid in both T3 and T4 whereas the treatment T4 contained higher chlorogenic acid only. The specific activities of ROS scavenging enzymes peroxidase and superoxide dismutase increased from stages S1 to S2 stages followed by decrease in activity at S3 stage. Superoxide dismutase showed higher activity in T1 whereas T4 recorded the lowest activity. Semi-spreading varieties showed maximum peroxidase activity as compared to bunch type during the disease developmental stages. Amongst all the treatments the treatment T3 recorded the highest peroxidase activity. Lipoxygenase showed an increase in activity with the progression of stages. Biocontrol treatment resulted in highest enzyme activity. Polyphenol oxidase showed a decline in activity from stage S1 to S2 followed by a slight increase upto S3. Higher polyphenol oxidase activity was found in T4 as compared to T3 at stages S1 and S2. Both chitinase and ß-1,3 glucanase activity was maximum in treatment T4 among all the treatments. The Native-PAGE of protein gave ten, six and eight bands at 6, 12 and 18 days. Treatment T3 gave more number of bands. In S3 stage two unique bands were found to be present in T4 with Rm value 0.23 and 0.15 which may be involved in defence. Dendrogram of Native-PAGE revealed two major clusters of which cluster A comprised of treatment T1 and T2 whereas cluster B contained treatment T2, T3 and T4. Out of four isozymes studied viz. peroxidase, esterase, superoxide dismutase and polyphenol oxidase, peroxidase at S3 stage showed the presence of novel isoforms in treatment T3 and T4. Esterase and superoxide dismutase banding pattern gave differences in intensity (quantitative) as well as in presence/absence (qualitative). Pooled dendrogram of Native-PAGE and all isozyme revealed similar clustering as seen in Native-PAGE. The ISSR profiles for five groundnut varieties gave total 26 bands with polymorphism ranging from 28 to 100%. On the basis of biochemical and molecular results, the isolate Pf-3 has potential to inhibit the pathogen A. niger. This was due to its ability to produce higher amount of mycolytic enzymes and inhibiting the cell wall degrading enzymes produced by pathogen. Seed treatment with Pf-3 also induced ROS scavenging enzymes and enzymes involved in phenol metabolism. The enzymes related to pathogenesis like chitinase and ß-1,3 glucanase were also triggered in response to Pf-3 treatment. Thus, the isolate Pf-3 effectively reduced the collar rot in groundnut by inducing resistance mechanism in plant.