1. Introduction {#sec1}
===============

India has 15 agro climatic zones and 17,000--18,000 species of flowering plants of which 6000--7000 species are estimated to have medicinal usage in folk and other systems of medicine like, Ayurveda, Siddha, Unani and Homoeopathy [@bib1]. Amongst them one of the important medicinal plants is *Withania somnifera* Dun. (Solanaceae), also known as '*Ashwagandha*', and has been listed in 32 prioritized medicinal plants of India [@bib2]. The estimated production of *W. somnifera* roots in India is more than 1500 tonnes and the annual requirement is about 7000 tonnes necessitating the increase in its cultivation and higher production [@bib3]. In India, the popular approach to achieve the increased yield and production is by application of inorganic fertilizers. Knowing the demerits of using these chemicals, there is a thrust for alternatives for better yield and quality, which are eco-friendly too. Such practices for cultivation are available in the literature of ancient India, the major one being *Vrikshayurveda*. '*Vrikshayurveda*', an ancient science of plant life deals with healthy growth of plants and their productivity. It is an age old agro practice which is of great relevance even today in agriculture and horticulture sectors. It deals with pest and disease management of plants and also encompasses storage of seeds, sowing, germination, plant propagation, manuring etc. [@bib4], [@bib5], [@bib6].

Though, the chemical fertilizers can enhance the yield, however it will be at the cost of the soil fertility. Meantime, the alternatives such as age old *Vrikshayurveda* practices which are organic in nature should be evaluated on medicinal plants for their acceptance and wider utility. To the best of our knowledge, there are no reports available as on date of evaluation of such practices especially on medicinal plants of high demand like *W. somnifera*. Hence, the present work was designed to evaluate the effect of two of the *Vrikshayurveda* treatments *viz*. *Kunapa jala* (*KJ*) and *Pancha gavya* (*PG*) in comparison with control, organic and inorganic fertilizer treatments on growth, physiological, biochemical, yield and quality attributes of *W. somnifera* harvested at different stages.

2. Materials and methods {#sec2}
========================

2.1. Land preparation, sowing and treatments {#sec2.1}
--------------------------------------------

The treatment groups comprised of control, *Kunapa jala* (*KJ*), *Pancha gavya* (*PG*), farmyard manure (FYM), inorganic fertilizer (NPK) and humic acid (HA). Mature seeds of *Ashwagandha* (variety: Jawahar *Ashwagandha* 20) were procured from KRC College of Horticulture, Arabhavi, India. The plant specimen has been authenticated and voucher specimen was deposited at Regional Medical Research Centre (ICMR), Belagavi, India for future reference (Voucher specimen no. RMRC-1100). Other raw materials required for preparation of *KJ*, *PG*, FYM, branded HA and NPK were obtained from local market and manufacturers.

A field experiment was conducted at Horticulture Research Station, Kanabargi, India. The soil was sandy loam with pH 7.31, Electrical Conductivity (EC) 0.098 ds m^−1^ and Organic Carbon (OC) 0.79%. The available N, P and K were 220, 18.86 and 280 kg ha^−1^ respectively. The net plot sizes were 2.0 m × 2.0 m. The seeds were sown in field at depth of 1 cm. Light irrigation was provided immediately after sowing. The crop was thinned at 30 days after sowing (DAS) to retain one seedling per hill with spacing of 30 × 15 cm. Full dose of *KJ*, *PG*, FYM and HA and inorganic fertilizer *viz*. phosphorus (P) in the form of Diammonium phosphate (DAP), potassium (K) in the form of Muriate of Potash (MOP) and 50% nitrogen (N) in the form of urea were applied to soil a week before sowing, remaining half dose of N and full dose of *KJ*, *PG*, FYM and HA were top dressed at 45 DAS to respective groups (Based on the results of pilot studies, only 10% of *KJ* and *PG* were used for full dose application).

2.2. Preparation of *Kunapa jala* and *Pancha gavya* {#sec2.2}
----------------------------------------------------

*Vrikshayurveda* texts explains various methods for preparation of *KJ* based on ingredients involved. In the present study, *KJ* was prepared as per *Sharangadhara* [@bib7] and Sadhale [@bib4], with minor modifications. The ingredients, 1.5 kg each of sheep/goat meat, chicken meat and 1 kg of Indian mackerel fish (*Rastrelliger kanagurta*) were boiled in 16 L of water till properly cooked and transferred to an earthen pot. Each 500 g powders of black gram (*Vigna mungo* L.) and sesame (*Sesamum indicum* L.) were added along with locally available Indian breed cow\'s milk (1 L), ghee (250 g) and honey (500 g). The pot was closed with lid and kept in warm place (the room temperature was maintained at 35 °C) for 30 days with stirring clockwise and anticlockwise direction manually at regular intervals for an hour. The content of the pot was filtered on 31st day and the resultant filtrate is *KJ*.

The *PG* was prepared referring to Sarkar et al. [@bib8] and John De et al. [@bib9]. All the ingredients were collected from native Indian breed cow. Twenty kg of dung, 10 L of urine and 10 L of tap water were added in an earthen pot and kept for 15 days with stirring every day manually for an hour in clockwise and anticlockwise direction. On 16th day 5 kg of ghee was added, thoroughly mixed by stirring every day and kept for 5 more days. On 21st day, 10 L each of milk and curd were added and stirred every day till 30th day. On 31st day the content of the pot was filtered.

2.3. Growth and physiological attributes {#sec2.3}
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Plants were harvested at different growth stages (60, 90, 120 and 150 DAS). Height of the plant (cm), number of leaves and number of branches per plant were measured. The fresh leaves were detached and total leaf area (cm^2^) was measured using Leaf area meter (Biovis). Plants were dried at ambient temperature to constant weight. Physiological characteristics *viz.* leaf area index (LAI), leaf area ratio (LAR), specific leaf area (SLA), specific leaf weight (SLW), crop growth rate (CGR), leaf area duration (LAD), relative growth rate (RGR) and net assimilation rate (NAR) were studied referring to methods explained by Azarpour et al. [@bib10] and Prakash et al. [@bib11].

2.4. Biochemical attributes {#sec2.4}
---------------------------

Leaf pigments *viz*. chlorophyll a, b and the carotenoids (xanthophylls and carotenes) were estimated at different growth stages as mentioned by Lichenthaler et al. [@bib12] using Thermo Scientific multiskan Go 1510 spectrophotometer. The results were expressed in terms of mg g^−1^ fresh weight.

2.5. Yield and quality attributes {#sec2.5}
---------------------------------

Yield was calculated as dry weight of the plant (g) and dry weight of the root (g). Quality of the yield was calculated on the basis of root length (cm) and root diameter (cm).

2.6. Statistical analysis {#sec2.6}
-------------------------

Various observations collected during study were subjected to statistical analysis using Fisher\'s method of analysis of variance (ANOVA) [@bib13]. Significance of mean (n = 4) treatment squares and replication mean squares were compared by error mean squares referring to F table value at 5% probability.

3. Results and discussions {#sec3}
==========================

Results of growth attributes ([Table 1](#tbl1){ref-type="table"}.) indicated, height of the plant was maximum in *KJ* group (44.54 cm) at 150 DAS, number of leaves were found to be highest in NPK group (94.25) at 120 DAS, number of branches were found to be highest in *KJ* (8) and NPK (8) groups at 120 DAS and total leaf area was found to be highest in *KJ* group (1707.89 cm^2^) at 120 DAS, which was on par with *PG* group (1682.07 cm^2^). As the leaf area is an indicator of photosynthetic efficiency of the plants and plants grown under favourable conditions will tend to have good vegetative growth, increased number of branches, number of leaves and leaf area which was observed in *KJ* and *PG* treated groups, this observation is in lines with report of Al-Doghachi et al. [@bib14].Table 1Growth attributes.Table 1Treatment n = 4Height of the plant (cm)No. of leaves per plantNo. of branches per plantTotal leaf area (cm^2^)6090120150609012015060901201506090120150Contr15.7531.5041.4042.1521.2555.0085.0065.251.255.007.006.25450.661112.941580.571188.06*KJ*16.7538.0043.7944.5421.5064.7591.7567.751.256.008.007.25516.511254.241707.891349.36*PG*17.1335.5044.1043.2121.7566.5092.5067.751.506.507.757.00506.961215.631682.071305.75FYM15.5032.2542.1042.0618.2555.2593.5070.251.005.507.256.50464.181138.961593.841217.08NPK17.2534.0043.5643.6122.0065.5094.2569.001.506.758.007.25494.901189.221664.171279.34HA15.7531.2542.0442.0320.2565.2591.5066.751.256.257.256.50477.691067.801615.141152.87SEm ±0.591.831.191.151.473.982.522.770.220.730.610.6115.6223.528.7923.52CD at 5%1.785.513.593.464.4312.017.608.360.652.201.841.8447.0770.9126.4870.91CV%7.2010.845.565.3514.0912.845.528.1833.4024.2816.2218.026.444.051.073.77[^1]

[Table 2](#tbl2){ref-type="table"}, [Table 3](#tbl3){ref-type="table"} show the effect of treatments at different stages of *W. somnifera* on physiological attributes. The LAI increased with advancement of crop age up to 120 DAS and thereafter declination was observed due to senescence and shedding of leaves, the results are in accordance with Nadukeri [@bib15] in Coleus. In the present study, highest LAI was observed in *KJ* treated group (3.795) which was on par with *PG* group (3.738) at 120 DAS. A study conducted by Vaidya et al. [@bib16] on *W. somnifera* revealed highest LAI as 2.42, whereas in the present study better results were observed at given conditions. A field experiment conducted by Kubsad et al. [@bib17] on *Ashwagandha* observed highest LAI as 3.918 when fertilizer was applied at the level of 24 kg N/ha and 48 kg P/ha, which are closer to the results of *KJ* treated group from our study. It was observed from all treatment groups, the value of LAR was highest at early growth stages and later it was reduced, similar observations were made by Azarpour et al. [@bib10] and Parmar et al. [@bib18].Table 2Physiological attributes with respect to LAI, LAR, SLA and SLW.Table 2Treatment n = 4LAILAR (cm^2^ g^−1^)SLA (cm^2^ g^−1^)SLW (mg cm^−2^)6090120150609012015060901201506090120150Contr1.0012.4733.5122.64035.22631.72123.39524.1930.06140.08560.07090.0768162.843117.705141.177130.658*KJ*1.1482.7873.7952.99937.58728.26522.04222.8100.06900.07770.07170.0748145.455130.159139.516135.289*PG*1.1272.7013.7382.90237.93728.70422.60823.7480.06870.07560.07290.0789146.687134.249137.453128.304FYM1.0322.5313.5422.70535.31629.46322.59723.3360.06220.07800.07120.0758161.141129.244140.735133.267NPK1.1002.6433.6982.84335.91628.89222.74423.1390.06410.07860.07240.0771157.148128.004138.416131.072HA1.0622.3733.5892.56236.18427.74922.39821.6570.06450.07180.07100.0718155.051139.637141.052139.603SEm ±0.0350.0520.0200.0521.4600.7590.2080.4440.00230.00210.00090.00174.8863.6231.8513.008CD at 5%0.1050.1580.0590.1584.4022.2870.6271.3390.00700.00620.00280.005214.72710.9205.5799.066CV%6.4384.0451.0713.7688.0335.2091.8383.8377.15745.32322.60474.53336.3165.5812.6494.522[^2]Table 3Physiological attributes with respect to CGR, LAD, RGR and NAR.Table 3Treatment n = 4CGR (g m^−2^ day^−1^)LADRGR (g g^−1^ day^−1^)NAR (g m^−2^ day^−1^)60--9090--120120--15060--9090--120120--15060--9090--120120--15060--9090--120120--150Contr0.1850.271−0.15452.12089.78492.2880.01460.0095−0.00460.04400.0354−0.0194*KJ*0.2560.276−0.15259.02598.738101.9090.01700.0081−0.00390.05370.0327−0.0174*PG*0.2430.266−0.16157.42096.59099.5940.01670.0081−0.00440.05250.0323−0.0189FYM0.2120.266−0.15353.43891.09393.6970.01560.0087−0.00440.04930.0343−0.0190NPK0.2280.266−0.14856.13795.11398.1170.01580.0083−0.00400.05040.0329−0.0176HA0.2110.280−0.15751.51789.43292.2670.01550.0091−0.00440.05010.0367−0.0199SEm ±0.0050.007−0.9240.9590.9900.9900.00030.00030.00010.00180.00100.0006CD at 5%0.0160.0220.0132.8892.9852.9850.00100.00090.00040.00550.00310.0019CV%4.8935.457−5.7063.4892.1202.0574.376.56−5.65207.266.1323−6.7610[^3]

At 90 DAS highest LAR was recorded in the control group (31.721 m^2^ g^−1^) whereas *KJ* group shown lowest (28.265 m^2^ g^−1^). SLA indicates the ratio of photosynthetic tissues surface to the weight of photosynthetic tissues. Higher SLA is indicative of more leaf area and lesser dry weight, hence the reduction of SLA increases photosynthetic efficiency of leaves. Though, amongst 4 growth stages 60 DAS shown lowest SLA in all treatment groups may be because of lowest leaf area and dry weight. However in subsequent stages 90 DAS shown highest SLA followed by 150 and 120 DAS. Amongst the treatment groups at 90 DAS, lowest SLA was recorded in HA (0.0718 cm^2^ g^−1^). Parmar et al. [@bib18] explains high rate of photosynthesis requires a large amount of enzymes and light harvesting complexes per unit leaf area and possibly an extra layer of palisade parenchyma, all of which decrease SLA and thus LAR. Often SLW is related to photosynthetic efficiency of the crop plants, higher values indicate increased photosynthetic efficiency. HA group shows 139.637 mg cm^−2^ SLW, indicating respective photosynthetic activity.

The CGR is the rate of dry matter accumulation per unit ground per unit time and it is increased with the advancement of crop age. The highest CGR was recorded in HA group (0.280 g m^2^ day^−1^) at 90--120 DAS ([Table 3](#tbl3){ref-type="table"}.). However highest CGR was obtained at 60--90 DAS in *KJ* group (0.256 g m^2^ day^−1^), which was on par with *PG* group (0.243 g m^2^ day^−1^). The increase in the CGR at 90--120 DAS might be due to the maximum plant height, number of branches and leaves per plant resulting in maximum dry matter accumulation in plants, this is in accordance with Venkatesha et al. [@bib19]. It is also observed that negative values of CGR at 120--150 DAS suggesting defoliation or senescence, this is in compliance with Pirzad et al. [@bib20] and Azarpour et al. [@bib10]. The LAD signifies the duration of the functional activity of the leaves, it denotes the ability of the plant with reference to the photosynthetic duration. The results indicate ([Table 3](#tbl3){ref-type="table"}.), highest LAD was achieved in *KJ* treated group (101.909) at 120--150 DAS, which was on par with *PG* group (99.594). The influence of treatments on the LAD was similar to LAI because it was derived from the LAI data, which is justified by Irshad et al. [@bib21]. The RGR denotes the rate of increase in dry matter per unit dry matter present. The RGR decreased with advancement of crop growth. The results show ([Table 3](#tbl3){ref-type="table"}), higher RGR was recorded at 60--90 DAS, which indicates grand growth period. In this period highest RGR was recorded in *KJ* treated group (0.0170 g g day^−1^), which was on par with *PG* group (0.0167 g g day^−1^). These results are in accordance with Pirzad et al. in *Pimpenella anisum* [@bib20] and Nadukeri et al. in coleus [@bib15]. The highest NAR was recorded at 60--90 DAS and it decreased thereafter till harvest due to lower LAI. The NAR is essentially an estimation of canopy photosynthesis achieved per unit leaf area which can measure photosynthetic efficiency. The results indicate ([Table 3](#tbl3){ref-type="table"}), that the highest NAR was recorded in *KJ* group (0.0537 g m^2^ day^−1^) which was on par with *PG* (0.0525 g m^2^ day^−1^), NPK (0.0504 g m^2^ day^−1^), HA (0.0501 g m^2^ day^−1^) and FYM (0.0493 g m^2^ day^−1^) at 60--90 DAS. Maximum NAR in all the treatments was observed at the beginning stage (60--90 DAS) and when dry matter accumulation reached to its maximum, NAR declined to zero and thereafter to negative values. Similar changes in NAR curve were reported by Singh et al. [@bib22] and Yang et al. [@bib23]. As stated by Azarpour et al. [@bib10] and also evident from our study, the reason of declination of CGR, RGR and NAR curves pattern to negative levels may be due to acceleration in leaf production and early closure of canopy. This condition enables absorption of less solar radiation by the leaves resulting into declination of values.

As the chlorophylls and carotenoids absorb light energy and transfer it into the photosynthetic apparatus of leaves, determination of leaf pigments will provide a valuable tool to integrate and understand the physiological and biochemical function of leaves [@bib24]. Results of biochemical estimation ([Table 4](#tbl4){ref-type="table"}, [Table 5](#tbl5){ref-type="table"}, [Table 6](#tbl6){ref-type="table"}) suggest that chlorophyll a, b and carotenoids contents were found to be highest in *KJ* treated group (1.877, 0.745 and 1318.14 mg g^−1^ respectively) at 90 DAS. Kannan et al. [@bib25] and Lichenthaler et al. [@bib12] reported, lower level of leaf pigments due to stressed conditions; similar observations were made in the present study treatment groups with lower level of leaf pigments. Sarkar et al. [@bib8] reported, chilli, tomato and cow pea plants showed higher chlorophyll contents in *PG* and *KJ* combination treatment group which is supportive to the present study.Table 4Estimation of Chlorophyll A (mg g^−1^).Table 4Treatment n = 46090120150Contr0.9091.0780.9960.650*KJ*1.3381.8771.6171.096*PG*1.0231.3791.2260.870FYM0.8531.1180.9980.733NPK1.1941.4201.1500.926HA1.2211.3281.1600.878SEm ±0.0730.0910.0630.049CD 5%0.2210.2750.1900.148CV%13.46213.34810.56211.416[^4]Table 5Estimation of Chlorophyll B (mg g^−1^).Table 5Treatment n = 46090120150Contr0.2920.3760.3850.035KJ0.4980.7450.5170.223PG0.4420.5710.4780.242FYM0.4120.4570.4190.237NPK0.4170.5340.5710.172HA0.3770.5350.5340.154SEm ±0.0090.0290.0300.015CD 5%0.0270.0890.0900.044CV%4.36810.97012.28916.553[^5]Table 6Estimation of Carotenoids (mg g^−1^).Table 6Treatment n = 46090120150Contr779.11900.57815.72451.58*KJ*1112.241318.141113.25979.03*PG*907.531052.01994.55931.10FYM780.51936.02832.80645.42NPK976.661075.521038.95836.45HA935.541066.52981.51869.73SEm ±47.2944.7748.6158.85CD 5%142.54134.95146.51177.40CV%10.338.4610.1014.98[^6]

Results of yield and quality parameters at harvest ([Table 7](#tbl7){ref-type="table"}.) indicated that the highest dry root yield was recorded in *KJ* group (118.63 g plot^−1^ or 5.93 quintal hectare^−1^), which was on par with *PG*, NPK and HA groups. Wankhade et al. [@bib26] observed 5.47 quintal hectare^−1^ root yield at maturity stage. Vajantha et al. [@bib27] observed, the dry root yield was significantly influenced by different treatments of *Panchakavya* on *Ashwagandha*, which is in compliance with present study results of *Vrikshayurveda* treatment groups. The quality of yield was assessed on the basis of root length and diameter. Root length was found to be highest in *KJ* group (15.66 cm) which was on par with *PG*, FYM and HA and root diameter was found to be highest in *PG* group (1.36 cm) which was on par with NPK, *KJ* and HA. According to Hasanuzzaman et al. [@bib28] an increased trend of plant weight was observed with the increase of cow dung amount due to the beneficial effect of organic matter in soil properties and plant growth. Similar trends were observed in *KJ* and *PG* groups.Table 7Quality and Yield attributes at harvest (150 DAS).Table 7Treatment n = 4Dry root yieldRoot length (cm)Root diameter (cm)g plot^−1^q hect^−1^Contr92.404.6213.981.17*KJ*118.635.9315.661.26*PG*116.415.8215.561.36FYM95.444.7714.981.20NPK108.655.4314.311.28HA107.885.3914.851.19SEm ±4.130.210.530.05CD at 5%12.440.621.590.16CV%7.747.747.108.57[^7]

4. Conclusion {#sec4}
=============

*Vrikshayurveda* treatments were effective in terms of growth, physiological, biochemical, yield and quality attributes. Further studies are needed to evaluate the effect of *Vrikshayurveda* treatments on quality and quantity of biomedically important phytocompounds. Consequently, to reduce the pressure on agriculture for higher production, the economical and ecofriendly *Vrikhayurveda* practices can be initiated for sustainable agriculture and can be considered as an alternative to conventional agricultural practices.

Sources of funding {#sec5}
==================

Indian Council of Medical Research (ICMR) intramural funds of Regional Medical Research Centre, Belagavi.

Conflict of interest {#sec6}
====================

None.

Authors wish to convey gratitude to Mr. Venkatesh A. Millanatti and Mr. Bhoopal Talwar (lab attendants), Mr. Shrikant Suryavanshi (field attendant) and Mr. Lagamanna Jindrali for their assistance during the study.

Peer review under responsibility of Transdisciplinary University, Bangalore.

[^1]: 60; 90; 120; 150: days after sowing; Contr: Control; *KJ*: *Kunapa jala*; *PG*: *Pancha gavya*; Fym: Farmyard manure; FYM: Inorganic fertilizer; HA: Humic acid; SEm: Standard error of mean; CD: Critical difference; CV: Coefficient of variation.

[^2]: 60; 90; 120; 150: days after sowing; Contr: Control; *KJ*: *Kunapa jala*; *PG*: *Pancha gavya*; Fym: Farmyard manure; FYM: Inorganic fertilizer; HA: Humic acid; LAI: Leaf area index; LAR: Leaf area ratio; SLA: Specific leaf area; SLW: Specific leaf weight; SEm: Standard error of mean; CD: Critical difference; CV: Coefficient of variation.

[^3]: 60--90; 90--120; 120--150: days after sowing; Contr: Control; *KJ*: *Kunapa jala*; *PG*: *Pancha gavya*; Fym: Farmyard manure; FYM: Inorganic fertilizer; HA: Humic acid; CGR: Crop growth rate, LAD: Leaf area duration, RGR: Relative growth rate, NAR: Net assimilation rate; SEm: Standard error of mean; CD: Critical difference; CV: Coefficient of variation.

[^4]: 60; 90; 120; 150: days after sowing; Contr: Control; *KJ*: *Kunapa jala*; *PG*: *Pancha gavya*; Fym: Farmyard manure; FYM: Inorganic fertilizer; HA: Humic acid; SEm: Standard error of mean; CD: Critical difference; CV: Coefficient of variation.

[^5]: 60; 90; 120; 150: days after sowing; Contr: Control; *KJ*: *Kunapa jala*; *PG*: *Pancha gavya*; Fym: Farmyard manure; FYM: Inorganic fertilizer; HA: Humic acid; SEm: Standard error of mean; CD: Critical difference; CV: Coefficient of variation.

[^6]: 60; 90; 120; 150: days after sowing; Contr: Control; *KJ*: *Kunapa jala*; *PG*: *Pancha gavya*; Fym: Farmyard manure; FYM: Inorganic fertilizer; HA: Humic acid; SEm: Standard error of mean; CD: Critical difference; CV: Coefficient of variation.

[^7]: Contr: Control; *KJ*: *Kunapa jala*; *PG*: *Pancha gavya*; Fym: Farmyard manure; NPK: Inorganic fertilizer; HA: Humic acid; g: gram; q: quintal; hect: hectare; SEm: Standard error of mean; CD: Critical difference; CV: Coefficient of variation.
