Bio-elicitation of Ashwagandha (Withania Somnifera (L.) Dunal) for improved growth, yield and secondary metabolite production

Abstract

The study titled “Bio-elicitation of Ashwagandha (Withania somnifera (L.) Dunal) for improved growth, yield and secondary metabolite production” was carried out in the Department of Plantation, Spices, Medicinal and Aromatic Crops, College of Agriculture, Vellayani, during December 2019 to September 2023 with the objective to study the effect of individual application of chitosan and the root endophytic fungus, Piriformospora indica and their combined application on plant growth, yield and secondary metabolite production in Withania somnifera. The study was carried out in W. somnifera as four experiments (i) Biopriming of seeds using chitosan, (ii) Bio-elicitation by foliar application of chitosan, (iii) Bio-elicitation using P. indica, and (iv) Bio-elicitation by combined application of chitosan and P. indica. The second experiment consisted of two parts, preparation of chitosan nanoparticles and foliar application of chitosan. In the first experiment, seeds of Ashwagandha were primed with fourteen treatments, comprising of four different concentrations of chitosan (0.5 mg mL-1, 1 mg mL-1, 2.5 mg mL-1, and 5 mg mL-1) for three different durations (2 h, 4 h and overnight), along with overnight hydropriming and control (without priming). Different germination and seedling growth parameters of the seeds and seedlings were recorded. The highest germination per cent (76.00) and survival per cent (73.33) were observed in the priming treatment chitosan 5 mg mL-1 for 4 h. This was on par with the treatment chitosan 5 mg mL-1 for 2 h (69.33) and hydropriming treatment (70.67) with respect to germination per cent. Hydropriming recorded the highest germination index (1.79) and the treatment chitosan 2.5 mg mL-1 for 2h recorded the highest allometric index (0.91). Respective results were on par with the priming treatment chitosan 5 mg mL-1 for 4 h. Hydropriming recorded the lowest mean germination time (8.83 days). The highest values of seedling vigour indices I (8890.00) and II (45.15) were also recorded in the treatment chitosan 5 mg mL-1 for 4 h. The duration of priming had a significant effect on seedling growth parameters. Overnight priming treatments with chitosan were found to have an inhibitory effect on germination and seedling growth parameters with values on par with or lower than that of the control. Based on the results, seed priming with chitosan 5 mg mL-1 for 4 h, was selected as the best priming treatment for further experiments in the study. The preparation of chitosan nanoparticles was carried out by ionotrophic gelation method by dropwise addition of tripolyphoshate (TPP) solution to chitosan solution, under stirring at room temperature. Eighteen treatments formed by three levels of chitosan (1 mg mL-1, 2 mg mL-1 and 3 mg mL-1), three levels of TPP (0.5 mg mL-1, 0.75 mg mL-1 and 1 mg mL-1) and two speeds of rotation (700 rpm and 800 rpm) were tried for preparing the nanoparticles. The absorption spectra of the resultant solutions were observed at eight different wavelengths, i.e., 200 nm, 220 nm, 240 nm, 260 nm, 280 nm, 300 nm, 320 nm and 340 nm. The factor combinations with chitosan 1 mg mL-1 and TPP 0.5 mg mL-1 at 700 rpm, chitosan 2 mg mL-1 and TPP 0.75 mg mL-1 at 700 rpm, and chitosan 3 mg mL-1 and TPP 1 mg mL-1 at 700 rpm had an absorption peak at 220 nm. These were further characterized for particle size and were observed to have mean particle sizes of 222.9 nm, 333.4 nm and 366.9 nm, respectively. As the particles formed by various chitosan - TPP combinations did not fall in the nanoscale range of less than 200 nm, the further experiments involving chitosan nanoparticles were carried out using commercially available chitosan nano particles of size 80 to 100 nm. In the experiment involving foliar application of chitosan, seeds primed with the best priming treatment selected (chitosan 5 mg mL-1 for 4 h) were sown in protrays and thirty-day old seedlings were transplanted to growbags. The eight treatments in this experiment consisted of chitosan and chitosan nanoparticles spray, each at three different concentrations (1 mg mL-1, 2.5 mg mL-1 and 5 mg mL-1). The primed and non-primed seeds (absolute control) without any foliar spray application served as the control treatments. The treatments were imposed as foliar spray at transplanting, 15, and 45 days after that. The treatment with chitosan 2.5 mg mL-1 was found to be the best in terms of parameters like plant height, number of leaves and leaf area, recording the values 123.67 cm, 178.83 leaves and 119.99 cm2 respectively, at harvest. The treatment with a higher concentration of chitosan nanoparticles (5 mg mL-1) recorded the lowest number of days to flowering from transplanting (30.67 days), and also a lesser number of days to fruitset from flowering (9.17 days). The highest number of branches (9.97) and flowering branches (8.15) at harvest were observed in the treatment, chitosan 1 mg mL-1. The treatment with the lowest concentration of chitosan nanoparticles (1 mg mL-1) recorded the lowest values in various growth parameters, viz., plant height, number of branches, number of flowering branches, leaf area and collar girth. With respect to biochemical parameters, the highest content of total proteins (7.12 mg g-1) and carbohydrates (22.41 mg 100 g-1) were observed in the treatment with chitosan nanoparticles 2.5 mg mL-1 at harvest. The treatment with chitosan 1 mg mL-1 recorded the highest peroxidase (32.35 U mg-1 protein), catalase (17.57 U mg-1 protein) and superoxide dismutase (1.68 U mg-1 protein) activities. Similar to growth parameters, chitosan 2.5 mg mL-1 recorded the highest leaf area index (25.75) and leaf area duration (1020.92 days). All the treatments involving chitosan as well as chitosan nanoparticles were observed to have significantly higher photosynthetic rate at harvest. The highest stomatal conductance (162.97 mmoles m-2 s-1) was observed in the treatment chitosan 1 mg mL-1, the highest proline content (44.03 μM g-1 tissue) was observed in the treatment chitosan nanoparticles 2.5 mg mL-1, and the highest cell membrane integrity (91.80 per cent) was observed in chitosan nanoparticles 5 mg mL-1 at harvest. This treatment also had the highest total chlorophyll content at harvest. The shoot weight was observed to be significantly higher in chitosan treatments over chitosan nanoparticles as well as the control treatments. However, with respect to the dry weight of shoot, the lowest concentration of chitosan nanoparticles (1 mg mL-1) was observed to be on par with chitosan foliar spray treatments. The treatments with chitosan nanoparticles were observed to give significantly higher berry weight and seed yield per plant over chitosan and control treatments. The root parameters, viz., root length, root diameter, root volume and root yield (fresh and dry) were also observed to be significantly higher in treatments with chitosan nanoparticles over that of chitosan and control treatments. Root being the officinal part of the plant, the secondary metabolite, withanolide content in it was recorded. The highest withanolide content was observed in the treatment chitosan 2.5 mg mL-1 (0.45 per cent), and was on par with the treatment chitosan 5 mg mL-1 (0.43 per cent). Withanolide yield per plant was the highest in the treatment chitosan nanoparticles 2.5 mg mL-1 (5.46 mg plant-1) which had 4.4 times increase in withanolide yield over control. This was on par with the treatment chitosan 5 mg mL-1 (4.99 mg plant-1) which had 3.9 times increase in withanolide yield over control. Among the foliar spray treatments, chitosan 5 mg mL-1 and chitosan nanoparticles 2.5 mg mL-1 were selected as the best two treatments in terms of higher withanolide yield, for proceeding to the combination trials. In the experiment involving application of P. indica, P. indica mycelium, culture filtrate and cell wall extract were applied to the plants raised from primed and non-primed seeds. The seedlings raised from the primed and non-primed seeds were transplanted at 30 days after sowing to growbags. P. indica mycelium was applied at transplanting; and P. indica culture filtrate and cell wall extract were applied as foliar spray at transplanting, 15 and 45 days after transplanting. With respect to growth parameters, the highest plant height (120.19 cm), number of flowering branches (7.25), leaf area (111.84 cm2) and collar girth (2.67 cm) were observed in the plants derived from primed seeds treated with P. indica mycelium. Significantly higher protein content and defense enzymes were observed in the plants raised from non-primed seeds treated with P. indica cell wall extract. Physiological parameters like leaf area index and leaf area duration were found to be the highest in the plants raised from primed seeds treated with P. indica cell wall extract (1.40 and 21.65 days) at harvest. The highest stomatal conductance (129.30 mmoles m-2 s-1), photosynthetic rate (26.60 μCO2 moles m-2 s-1), proline content (45.37 μM g-1 tissue) cell membrane integrity (70.06 per cent) and total chlorophyll content (2.44 mg g-1 fresh weight) were recorded in the plants raised from primed seeds treated with P. indica mycelium. Shoot weight per plant was found to be significantly higher in plants raised from both primed and non-primed seeds treated with P. indica mycelium. Number of berries per plant, berry weight and seed yield per plant were found to be significantly higher in plants raised from primed seeds treated with P. indica mycelium and culture filtrate. All the root parameters, root length, root diameter, root volume, root yield per plant (fresh and dry) were observed to be the highest in plants raised from primed seeds treated with P. indica mycelium. The withanolide content was observed to be significantly higher in the roots of plants raised from primed seeds treated with P. indica mycelium (0.35 per cent) and culture filtrate (0.37 per cent). The same treatments showed significantly higher withanolide yield of 4.05 mg (3.82 times increase over control) and 3.60 mg per plant (3.28 times increase over control), respectively. Thus, the plants raised from primed seeds treated with P. indica mycelium and culture filtrate were selected as the best two treatments in terms of higher withanolide yield, for proceeding to the combination trials. For the combined application of chitosan and P. indica, the best two treatments selected from the individual application of chitosan and P. indica were used for studying their combined effect on growth, yield and secondary metabolite production in W. somnifera. The highest plant height (91.86 cm), number of leaves (143.70), leaf length (17.01 cm), leaf breadth (9.48 cm), and leaf area (113.28 cm2) were observed in the combination treatment of chitosan 5 mg mL-1 and P. indica mycelium. The highest number of branches (8.25), flowering branches (7.60) and collar girth (2.42 cm) were observed in the combination treatment involving chitosan 5 mg mL-1 and P. indica culture filtrate. The same treatment recorded the highest protein content (8.45 mg g-1). The highest peroxidase activity (34.74 U mg-1 protein), catalase activity (14.67 U mg-1 protein) and superoxide dismutase activity (1.48 U mg-1) were observed in the treatment involving chitosan of 5 mg mL-1 and P. indica mycelium. The same treatment recorded the highest value with respect to physiological parameters, viz., leaf area index (19.53), leaf area duration (803.07 days), stomatal conductance (137.11 mmoles m-2 s-1), photosynthetic rate (33.73 μCO2 moles m-2 s-1) and total chlorophyll content (3.76 mg g-1 fresh weight). The highest proline content was recorded in the treatment involving chitosan nanoparticles 2.5 mg mL-1 and P. indica mycelium (34.22 μM g-1 tissue) and highest cell membrane integrity in the combination treatment of chitosan nanoparticles 2.5 mg mL-1 and P. indica culture filtrate (83.69 per cent). The shoot weight and all the root growth parameters, viz., root length, root diameter, root volume and root yield were found to be the highest in the combination treatment of chitosan 5 mg mL-1 and P. indica mycelium. The combination treatments involving chitosan nanoparticles 2.5 mg mL-1 and P. indica mycelium/culture filtrate had the highest number of berries, berry weight and seed yield per plant. Significantly higher withanolide content was observed in roots of the plants treated with chitosan 5 mg mL-1 and P. indica culture filtrate. Withanolide yield per plant was the highest in the treatment comprising chitosan nanoparticles 2.5 mg mL-1 and P. indica culture filtrate and was on par with the combination treatment of chitosan 5 mg mL-1 and P. indica mycelium/culture filtrate. In the study, it was observed that all the combination treatments gave better performance than the control in terms of root yield, withanolide content and withanolide yield per plant. The concentration of chitosan and exposure time of priming had a profound influence on seed germination and seedling growth parameters in W. somnifera. The bio-elicitors, chitosan/chitosan nanoparticles and P. indica when applied individually and in combination to chitosan primed seeds have been observed to enhance growth, yield and metabolite production in a dose dependent manner in W. somnifera.