APPLICATION OF SALICYLIC ACID AND MOISTURE LEVELS ON GROWTH, YIELD AND POLYPHENOL CONTENT OF Withania somnifera (ASHWAGANDHA) 

The roots of Withania somnifera (L.) Dunal (Ashwagandha) are key constituents of many herbal medicinal products due to their high levels of biologically active secondary  metabolites. Abiotic stress conditions like water stress and elicitors are known to trigger the  production of these metabolites. The current research has been conducted to determine the  impact of substrate water availability and salicylic acid (SA) foliar spray on growth, root  yield, and polyphenolic content of Ashwagandha under protected cultivation in low-country wet zone of Sri Lanka. There were three (3) polyhouse trials. In the first trial, the effects of four (4) substrate water regimes (50, 60, 80, and 100% field capacity (FC) and four (4) foliar SA applications (0, 0.1, 1, and 10 mM) on each other were studied using a split plot design with four (4) replications. This trial was done for preliminary identification of the most effective combinations as well as the existence of any significant interaction between substrate moisture and SA application before conducting the optimization of both factors individually. Each replicate contained only two Ashwagandha plant growing in a coir dust pot. The combination of 50% FC and 1 mM SA was the most effective in respect of all parameters. It resulted in increase in root dry matter content from 2.80 to 7.62 g plant⁻¹ (2.7-fold), total polyphenols content from 0.29 to 3.64    mg GAE g⁻¹ DW (12.49-fold), flavonoids content from 0.83 to 3.88 mg QE g⁻¹ DW (4.67-fold) and in increase in DPPH radical scavenging activity from 10.50 to 45.70 % (4.35  times) compared to the control (100% FC without SA application). In view of the above observations, the second experiment was conducted to study the impact of moisture in the soil alone through seven different levels of substrate moisture (50–100% FC (control), 45 50%, 40–45%, 35–40%, 30–35%, 25–30%, and 20–25% FC) under the completely randomized design having four (4) replicates each. In this experiment, each experimental unit consisted of one Ashwagandha plant grown in sand filled pots. Plants kept at 50–100% FC showed the highest shoot growth. But when compared to the well-watered control (50–100% FC), plants kept at 35–40% FC showed increased dry matter of roots (0.23 to 0.59 g plant⁻¹; (2.56 fold), total polyphenols (2.22 to 4.81 mg GAE g⁻¹ DW; 2.16 fold), flavonoids (1.88 to 2.91 mg QE g⁻¹ DW; 1.54 fold), and DPPH radical scavenging activity  (57.79 to 81.03 %; 1.40 fold). The third experiment was conducted to optimize the  effects of SA application under well watered condition (100% FC) by using eight levels of foliar applications of SA (0, 50, 75, 100, 125, 150, 175, and 200 ppm; 0-1.45 mM) in completely randomized design having four replications. Salicylic acid worked as an effective elicitor, with increase in growth, SPAD  values, root dry matter and antioxidant indices up to optimum level of 125-150 ppm and slight decrease in values above 175 ppm. As compared to untreated control plants (0 ppm  SA), 125 ppm SA application increased root dry matter from 3.30 to 6.73 g plant⁻¹ (2.03- fold), total polyphenol from 1.14 to 2.50 mg GAE g⁻¹ DW (2.19-fold), flavonoids from 1.18 to 2.70 mg QE g⁻¹ DW (2.28-fold) and DPPH radical scavenging activity from 45.91 to 73.97 % (1.60-fold). Generally speaking, a medium level of substrate moisture stress along with foliar spray of SA increased root biomass and phytochemicals related to antioxidants in Ashwagandha. In cases of moisture-stress conditions, root biomass and polyphenols increased most efficiently when treated with 50% FC and 1mM SA, but when well-watered, foliar spray of 125 ppm SA was found to be more efficient. 

Keywords: Ashwagandha, substrate moisture, field capacity, abiotic stress, secondary 
metabolites, polyphenols.