Effect of polyacrylamide soil-dressing on

Journal of Herbs, Spices & Medicinal Plants

ISSN: 1049-6475 (Print) 1540-3580 (Online) Journal homepage: http://www.tandfonline.com/loi/whsm20

Effect of polyacrylamide soil-dressing on physiological attributes, essential oil content, and composition of vetiver (Vetiveria zizanioides) Asfia Shabbir, M. Masroor A. Khan, Yawar Sadiq, Hassan Jaleel, Bilal Ahmad & Moin Uddin To cite this article: Asfia Shabbir, M. Masroor A. Khan, Yawar Sadiq, Hassan Jaleel, Bilal Ahmad & Moin Uddin (2018): Effect of polyacrylamide soil-dressing on physiological attributes, essential oil content, and composition of vetiver (Vetiveria zizanioides), Journal of Herbs, Spices & Medicinal Plants, DOI: 10.1080/10496475.2018.1440363 To link to this article: https://doi.org/10.1080/10496475.2018.1440363

Published online: 20 Feb 2018.

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JOURNAL OF HERBS, SPICES & MEDICINAL PLANTS https://doi.org/10.1080/10496475.2018.1440363

Effect of polyacrylamide soil-dressing on physiological attributes, essential oil content, and composition of vetiver (Vetiveria zizanioides) Asfia Shabbira, M. Masroor A. Khana, Yawar Sadiqa, Hassan Jaleela, Bilal Ahmada, and Moin Uddinb a

Department of Botany, Aligarh Muslim University, Aligarh, India; bBotany Section, Women’s College, Aligarh Muslim University, Aligarh, India ABSTRACT

ARTICLE HISTORY

A pot experiment was conducted on vetiver (Vetiveria zizanioides) in a completely randomized design to study the effect of soil-dressing with polyacrylamide (PAM) on the quality and quantity of vetiver essential oil (EO). Among the PAM doses tested, 120 mg kg−1 increased 17.3% and 39.6% in the EO content and yield, respectively. Gas chromatography (GC) revealed that khusimol content and yield was increased by 26.7% and 77.0% over the respective control. The study suggests that PAM increased the root biomass by improving soil environment thus increasing the growth parameters, physiological attributes, and EO production.

Received 1 May 2017 KEYWORDS

Gas chromatography; khusimol

Introduction Vetiver (Poaceae) roots, being the most valuable part of this grass, forms an intertwined network in the soil which is the storehouse of its oil, which is composed of highly complex volatile sesquiterpene and its derivatives as indicated by its characteristic balsamic note (1). Apart from its direct applications in perfumery industry, vetiver oil in its diluted form is extensively used in aftershave lotions, air freshener products, and bathing soaps; it is used to flavor syrups, ice creams, cosmetic items, and food preservatives (2). Vetiver essential oil (EO) is beneficial to human health due to its anti-inflammatory, antiseptic, analgesics, anticataleptic, rheumatism, nervine, sedative, tonic, vulnerary, and calming properties, acting as antimicrobial, antimycobacterial, and antifungal agent (3). Vetiver oil has also been reported to have antioxidant and anticancer activities (1). Soil conditioners have been in extensive use for many years to amend the soil and induce physical preconditioning. Polyacrylamide (PAM), one of the synthetic conditioners, is a water soluble polymer grouped in a class of compounds formed by the polymerization of acrylamide (4). One of the conditioning features of PAM, which contrasts it with many other soil conditioners, is that only a small quantity CONTACT Asfia Shabbir [email protected] Department of Botany, Aligarh Muslim University, Aligarh, 202002, India. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/whsm. © 2018 Taylor & Francis Group, LLC

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of PAM (in few kg ha−1) is required to obtain the desired effects compared to other types of conditioners that need to be applied in larger quantities similar to that if fertilizers (5). PAM can be used to mitigate surface sealing and crusting, reduce runoff and erosion, increase seedling emergence, and reduce the losses of fertilizers and pesticides (6). The water-soluble polymeric conditioners improved soil physical properties, thereby improving root penetration, infiltration, aeration, erosion resistance, and drainage (5–7). The use of polymeric conditioners usually resulted in the increased root biomass and plant interception of nutrients and water, which indirectly improved the plant nutrition in Ammophila arenaria (8), Triticum aestivum (9), Oryza sativa (10), Solanum lycopersicum (11), Vicia faba (12), Concarpus lancifolius (13), and Zea mays (14). This study examined the effect of PAM soil application on the root growth and overall performance of the vetiver plant, including the EO production and active constituents.

Materials and methods Plant material and growth conditions

A pot experiment was conducted on vetiver (Vetiveria zizanioides) in a completely randomized design under natural conditions of net-house at Department of Botany, A.M.U., Aligarh, India (27º 88ʹ N latitude, 78º 08ʹ E longitude, and 187.45 m altitude). Slips of vetiver were obtained from Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow, India. PAM was purchased from Wallace Laboratories, 365 Coral Circle, El Segundo, CA, USA. Prior to transplantation, each pot was filled with 5 kg homogenous mixture of soil and organic manure (4:1). Soil samples were analyzed in the Central Laboratory for Soil and Plant Analysis, Indian Agricultural Research Institute, New Delhi. Physicochemical characteristics of the soil were: texture-sandy loam, pH: 8.07, electrical conductivity: 0.36 m mhos−1, available N, P and K: 167.4, 94.6, and 286.0 mg kg−1 of soil, respectively. A uniform recommended basal dose of N (as urea), P (as diammonium phosphate), and K (as muriate of potash) was applied before transplantation at 85.5, 102.3, and 45.0 mg kg−1 soil, respectively. There were five treatments including control (deionized water). The soil application was by mixing PAM homogenously with the soil. The treatments consisted of: i) Treatment 1 (Control (No PAM)), ii) Treatment 2 (PAM 40 mg kg−1 soil), iii) Treatment 3 (PAM 80 mg kg−1 soil), iv) Treatment 4 (PAM 120 mg kg−1 soil), and v) Treatment 5 (PAM 160 mg kg−1 soil). Each treatment was replicated three times. Each pot contained a single healthy plant. The plants were irrigated as required. Sampling for plant analysis was carried out at 300 days after transplanting (DAT).

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Determination of growth attributes

The growth attributes, namely, fresh and dry weights of plant, shoot and root lengths per plant were evaluated at 300 DAT. Three plants of each treatment were uprooted and washed with tap water, surface-dried using blotting paper and the fresh weight of shoot and root recorded. Plants were dried at 80°C for 48 h using a hot-air oven and the dry weights recorded. The aboveground plant height (shoot length) and root length were measured using a meter scale. Determination of physiological and biochemical attributes Estimation of total chlorophyll and carotenoids Concentration of chlorophyll and carotenoids was estimated in the fresh leaves (15). Fresh tissue from the interveinal leaf area was ground with 80% acetone, using a mortar and pestle. The optical density (OD) of the pigment-extract was recorded at 645 nm and 663 nm for chlorophyll content and at 480 nm and 510 nm for total carotenoids content, using a spectrophotometer (Shimadzu UV-1700, Tokyo, Japan) and expressed as mg g−1 FW. Estimation of carbonic anhydrase (CA) activity The activity of CA (EC 4.2.1.1) was measured in fresh leaves (15). Chopped leaf-pieces (0.2 g) were transferred to Petri plate, dipped in 10 mL of 0.2 M cystein hydrochloride solution for 20 min at 4◦C. To each test tube, 4 mL of 0.2 M sodium bicarbonate solution and 0.2 mL of 0.002% bromothymol blue were added. The reaction mixture was titrated against 0.05N HCl using methyl red as indicator. The enzyme activity was expressed as M CO2 kg−1 leaf FW s−1. Estimation of nitrate reductase (NR) activity The activity of NR (E.C. 1.7.1.1) was estimated by the intact tissue assay method (15). The amount of nitrite formed was determined spectrophotometrically at 540 nm. NR activity was expressed as nanomoles of nitrite produced per g fresh weight of leaf tissue per hour (nM NO2−1 g−1 FW h−1). Estimation of net photosynthetic rate (PN), internal CO2 concentration (Ci), and stomatal conductance (gs−) PN, Ci, and gs were measured on sunny days at 11:00 h on the youngest fully expanded leaves using IRGA (Infra Red Gas Analyzer, LI-COR 6400 Portable Photosynthesis System, Lincoln, NE, USA) at 300 DAT. Estimation of chlorophyll fluorescence (Fv/Fm) Fv/Fm was measured in diurnal time using a saturation-pulse fluorometer PAM-2000 (Walz, Effeltrich, Germany). All measurements were carried out

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on the first pair of unifoliolate, fully expanded leaves. The upper surface of leaf was clipped to measure the Fv/Fm. Estimation of N, p, and k contents in leaves Leaf samples from each treatment were digested for the estimation of leaf—N, P, and K contents. The leaves were dried for 24 h in a hot-air oven maintained at 100°C. The dried leaves were ground using mortar-pestle and the leaf-powder was sieved using a 72 mesh and used for the estimation of N, P, and K contents. 100 mg of the oven-dried leaf-powder was transferred into a digestion tube, to which 2 mL of AR (analytical reagent) grade concentrated sulfuric acid was added subsequently. The content was heated on a temperature controlled Kjeldahl assembly at 100°C for ~ 2 h, and cooled for ~ 15 min at room temperature. To the cooled content, 0.5 mL of 30% hydrogen peroxide (H2O2) was added. The addition of H2O2 was followed by gentle heating (at 50ºC) of the content, followed by its cooling at room temperature. This step was repeated until the content of the tube turned colorless. The aliquot (peroxide-digested leaf-material), thus prepared, was used to estimate the per cent N, P and K contents in the leaves on DW basis. Estimation of nitrogen content Leaf-N content was estimated as reported (15). The dried leaf-powder samples were digested with H2SO4 in the digestion tubes using temperature controlled digestion assembly. A 10 mL aliquot (peroxide-digested leaf material) was transferred into a 50 mL volumetric flask, 2 mL of 2.5 N sodium hydroxide solution, and 1 mL of 10% sodium silicate were added to neutralize the excess acid and prevent turbidity, respectively. A 5 mL aliquot of the peroxidedigested leaf material was transferred into a 10 mL graduated test tube, followed by addition of 0.5 mL of Nessler’s reagent. The OD of the solution was recorded at 525 nm using the spectrophotometer. Estimation of phosphorus content Previously described method (15) was used to estimate the leaf-P content in the peroxide-digested leaf material. A 5 mL aliquot was poured into a 10 mL graduated test tube. To it, 1 mL of molybdic acid (2.5%) was added, followed by addition of 0.4 mL of 1-amino-2-naphthol-4-sulfonic acid. When the color of the content turned blue, the volume of the test tube was made up to 10 mL using double distilled water and its OD recorded at 620 nm using the spectrophotometer. Estimation of potassium content Leaf-K content was determined in the peroxide-digested material by a flame-photometer (Model, C150, AIMIL, India) as per the emission spectra, using specific filter (15). In the flame photometer, the solution (peroxide-digested


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leaf material) was discharged into a chamber through an atomizer in the form of a fine mist, where it was drawn into a flame. Combustion of the elements produced the light of a particular wavelength [λmax for K = 767 nm (violet)]. The light, thus produced, was passed through an appropriate filter to impinge upon a photoelectric cell that subsequently activated a galvanometer. Determination of yield and quality attributes of EO

The yield and quality attributes were determined estimating the EO yield per plant and khusimol yield per plant. Isolation and compositional analysis of EO Estimation of EO content. The EO was extracted through hydrodistillation method using Clevenger’s apparatus (Borosil, India) and then quantified gravimetrically (16). The fresh roots (200 g) were chopped into small pieces. EO was extracted by distillation of roots for 10 h. The extracted oil was dried over anhydrous sodium sulphate and subsequently preserved in sealed glass vials at 4ºC for the gas chromatography (GC) analysis of the EO. The amount of EO obtained from the plant material (roots) was calculated as: EO contentð%v=wÞ ¼ ½observed volume of oil ðmLÞ=weight of sampleðgÞ 100

GC analysis. The active constituent (khusimol content) of the EO was determined using GC apparatus [(Agilent) USA, 7890B] equipped with a capillary column HP5 (coated with polyimide and fused silica) of the size 30 m × 0.320 mm, flame ionization detector, and an injector. Nitrogen was used as the carrier gas; detector temperature, 300°C; oven temperature, 250°C; injector temperature, 250°C; and sample size 0.2 μL. The initial temperature was 100°C with a hold time of 20 min, increased to 270°C at 4°C per min. Identification of the active constituent (khusimol) was based on retention time. It was quantified as the per cent content comparing their peaks with the peaks obtained from the reference standard reported in the literature (17). Statistical analysis. Each pot was treated as one replicate and each treatment contained three replicates. The data (mean±S.E.) were analyzed using SPSS22 statistical software (SPSS Inc., Chicago, IL, USA) and means compared by Duncan’s Multiple Range Test (DMRT, ≤ 0.05%). Results The results showed that among the doses, PAM 120 mg kg−1 increased maximally the value of most of the studied parameters (Table 1–3).

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Table 1. Effect of Polyacrylamide (PAM) on Growth Attributes of Vetiver at 300 Days after Transplanting (DAT). Values Represent the Mean of Three Replicates ± Standard Error. Parameters Shoot fresh weight (g) Shoot dry weight (g) Root fresh weight (g) Root dry weight (g) Shoot length (cm) Root length (cm)

389.0 105.0 196.0 57.50 169.0 31.00

0 ± ± ± ± ± ±

4.9d 2.0d 2.5d 0.80d 3.0d 0.85c

PAM concentrations (mg kg−1) 40 80 120 407.2 ± 4.7c 428.9 ± 5.1b 456.5 ± 5.2a 108.9 ± 1.9cd 112.4 ± 2.0bc 119.6 ± 2.1a 206.0 ± 2.9c 216.5 ± 2.6b 234.0 ± 2.7a 60.80 ± 0.95c 63.95 ± 1.04b 68.96 ± 1.03a 178.3 ± 2.8c 187.0 ± 2.6bc 197.6 ± 3.0a 33.20 ± 0.81bc 34.90 ± 0.67b 37.70 ± 0.69a

438.0 116.9 224.0 65.62 188.6 36.20

160 ± 5.1b ± 1.9ab ± 2.2b ± 0.90b ± 2.3b ± 0.76ab

Mean separation by DMRT (p ≤ 0.05). *Values of means within a row followed by the same letter(s) are not different.

PAM improves growth attributes including fresh and dry weights of vetiver

Compared to control, the treatment 4 (PAM 120 mg kg−1) increased the fresh and dry shoot weights per plant by 17.3 and 13.9%, respectively; the fresh and dry root weights per plant by 19.4 and 19.9%, respectively; and the shoot and root lengths by 17.0 and 21.6%, respectively (Table 1).

Effect of PAM on physiological and biochemical attributes Content of total chlorophyll, NPR, Ci, gs, and fv/fm in vetiver PAM at 120 mg kg–1 increased the total chlorophyll (14.4%) and carotenoids (8.2%) at 300 DAT; photosynthetic rate, Ci and gs by 15.9, 16.8 and 16.7% respectively; and the Fv/Fm by 13.9% (Table 2). PAM enhances the enzyme activities of vetiver Maximal value of CA and NR activity were recorded on plants receiving PAM at 120 mg kg–1 with 16.0% higher CA activity and 17.2% higher NR activity. (Table 2). PAM enriches the nutrient uptake of vetiver PAM 120 mg kg–1 produced the greatest increase in the leaf—N, – P, and – K content by 14.9, 16.9, and 15.3%, respectively over their respective control (Table 2).

Effect of PAM on EO yield attributes

PAM 120 mg kg−1 also increased the EO content (17.3%) and the EO yield (39.6%) per plant (Table 3). Khusimol (Figure 1) is one of the main components of vetiver EO which was increased by the PAM at 120 mg kg−1 by 26.7% (Figure 2 a and b and Figure 3) (Table 3).

1.670 0.540 330.0 233.6 0.666 13.1 269.3 0.215 1.626 0.0625 1.229

0 ± ± ± ± ± ± ± ± ± ± ± 0.005e 0.005d 4.3d 2.7e 0.007d 0.13d 2.18e 0.002d 0.028d 0.002d 0.052a 1.760 0.553 344.2 243.9 0.697 13.7 280.6 0.228 1.710 0.0659 1.257

40 ± 0.006d ± 0.007cd ± 4.5c ± 2.4d ± 0.008c ± 0.16c ± 2.40d ± 0.003c ± 0.025c ± 0.002cd ± 0.034a

Mean separation by DMRT (p ≤ 0.05). *Values of means within a row followed by the same letter(s) are not different.

Parameters Total chlorophyll content (mg g−1 FW) Total carotenoids content (mg g−1 FW) N R activity (nM NO2 g−1 FW h−1) C A activity (µM CO2 kg−1 leaf FW S−1) Chlorophyll fluorescence (Fv/Fm) Net photosynthetic rate (µmol CO2 m−2 s−1) Internal CO2 concentration (µmol(CO2) kg−1 FW s−1) Stomatal conductance (µmol CO2 m−2 s−1) Leaf nitrogen content (%) Leaf phosphorous content (%) Leaf potassium content (%)

PAM concentrations (mg kg−1) 80 1.850 ± 0.006c 0.562 ± 0.003bc 363.7 ± 3.8b 254.7 ± 2.2c 0.730 ± 0.005b 14.3 ± 0.14b 295.0 ± 2.25c 0.236 ± 0.002b 1.790 ± 0.021b 0.0681 ± 0.001b 1.296 ± 0.070a

120 1.910 ± 0.008a 0.584 ± 0.006a 386.9 ± 4.1a 271.1 ± 2.7a 0.762 ± 0.004a 15.2 ± 0.16a 314.6 ± 2.23a 0.251 ± 0.003a 1.870 ± 0.021a 0.0731 ± 0.001a 1.319 ± 0.081a

1.880 0.574 371.5 262.7 0.740 14.7 303.0 0.242 1.810 0.0702 1.304

160 ± 0.007b ± 0.005ab ± 4.2b ± 2.1b ± 0.006b ± 0.17b ± 2.05b ± 0.002b ± 0.015ab ± 0.001ab ± 0.062a

Table 2. Effect of Polyacrylamide (PAM) on Physiological and Biochemical Parameters of Vetiver at 300 Days after Transplanting (DAT). Values Represent the Mean of Three Replicates ± Standard Error.

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Table 3. Effect of Polyacrylamide (PAM) on Yield Attributes of Vetiver at 300 Days after Transplanting (DAT). Values Represent the Mean of Three Replicates ± Standard Error. Parameters Essential oil content (%) Essential oil yield (mL) Khusimol content (%) Khusimol oil yield (mL)

0 0.710 ± 0.006e 1.390 ± 0.043d 8.78 ± 0.026e 0.122 ± 0.002e

PAM concentrations (mg kg−1) 40 80 120 0.748 ± 0.004d 1.540 ± 0.040c 9.67 ± 0.036d 0.149 ± 0.001d

0.780 1.680 10.29 0.173

± ± ± ±

0.006c 0.039b 0.025c 0.001c

0.833 1.940 11.12 0.216

± ± ± ±

0.009a 0.045a 0.036a 0.002a

160 0.800 1.740 10.59 0.184

± ± ± ±

0.006b 0.040b 0.021b 0.002b

Mean separation by DMRT (p ≤ 0.05). *Values of means within a row followed by the same letter(s) are not different

Figure 1. Chemical structure of khusimol: An important odour-influencing active constituent of the essential oil obtained from Vetiveria zizanioides L. Nash.

Discussion The results are in accordance with the findings conducted on Ammophila arenaria (8), Triticum aestivum (9), Oryza sativa (10), Solanum lycopersicum (11), Vicia faba (12), Concarpus lancifolius (13), and Zea mays (14). Reports argued that enhanced plant growth, following the application of hydrophilic polymers, could result from increased water supply (18). Liu et al. (19) revealed that superabsorbent polymer (a hydrophilic polymer), when added with fertilizer, increased the plant dry matter as well as seedling emergence time. Other reported benefits of PAM application included improved soil parameters such as hydraulic properties, infiltration rate, aeration, root penetration and aggregate stability, and boosted plant establishment and growth rate (5). Improvement in the values of these parameters may be due to readily available soil nutrients facilitated by the application of this polymer, which thereby, increased the growth by cell division, expansion, and elongation (20). These effects may be ascribed to the increased growth attributes of vetiver in the present study (Table 1). The photosynthetic parameters, viz. leaf-content of photosynthetic pigments (chlorophylls and carotenoids), PN, Ci, gs, and Fv/Fm, were improved by the application of PAM at 120 mg kg−1. The result could be corroborated with others (21) who reported a polymer-mediated increase in the photosynthetic pigments


a

b

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Khusimol 8.78%

Khusimol 11.12%

Figure 2. a) GC chromatogram of essential oil obtained from vetiver control plants (deionised water). b) GC chromatogram of essential oil obtained from vetiver treated with polyacrylamide (PAM) 120 mg kg−1.

that resulted in an overall improvement in photosynthesis. Photosynthesis involves chlorophylls, enzymes, and coenzymes, which heavily depend upon nutrient availability (22). The retention of moisture or water in the soil is the fundamental process upon which all plants depend and that addition of organic materials can help in water retention and increased nutrient availability (12). It reflects clearly that PAM works by assuring improved soil environment with high water retention capacity, maximizing nutrient availability, and improving photosynthetic parameters. An integrated part this may be increased CA and NR activities which might have ultimately resulted in an increase in the photosynthetic rate and the related attributes.

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Plastid

Cytosol

Glyceraldehyde-3-P

Acetyl CoA

Methyl-D-erythrytol 4-phosphate (MEP)

Mevalonic Acid (MVA)

HMBPP

IPP

DAMPP

IPP

DMAPP

GPP

FPP Synthase

FPP

Zizaene Synthase Zizanal Oxidation Zizaene

Khusimol

Oxidation

Zizanoic acid

Figure 3. An elucidation to biosynthetic pathway of khusimol (dashed arrow means more than one step) (Laule et al., 2003; Schalk and Deguerry, 2013). DMAPP, dimethylallyl pyrophosphate; FPP, farnesyl pyrophosphate; GPP, geranyl pyrophosphate; HMBPP, 1-Hydroxy-2-methyl-(E)-butenyl 4-diphosphate; IPP, Iso pentenyl pyrophosphate; MEP, Methyl-D-erythrytol 4-phosphate; MVA, Mevalonic acid.

An important aspect of plant growth is its nitrate reducing power. This reduction process, however, mainly depends on metabolic sensors and/or signal transducers (23). The progressive increase in the NR activity might be ascribed to the PAM-amended enhanced nutrient content (Table 2), specifically nitrogen content that might have increased the nitrate concentration in leaves to be acted upon by NR.


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CA, a zinc metalloprotein is known to catalyze reversible inter-conversion of HCO3− and CO2. It is therefore, known to maintain the plant growth and development by controlling the synthesis of carbon compounds as it is directly related to photosynthesis in higher plants, which is evident by its presence in all photosynthesizing tissues. It catalyzes the reversible hydration of CO2 to carbonic acid, thereby increasing the availability of CO2 to rubisco in photosynthesis (24). An increase in gs might facilitate the diffusion of additional CO2 through the stomata to be acted upon by CA, resulting in the enhanced CA activity (25). The increase in the fresh and dry weights of the plant observed could be linked to the plant’s enhanced CA activity as a result of PAM application which might have caused the increased CO2 fixation. Thus, the observed effect of PAM on CA activity may be due to the combined effect of a number of factors associated with PAM-mediated stimulation of physiological activities. There are explicit reports confirming the role of essential elements in plant structure, metabolic function, and osmoregulation of plant cells. N, P, and K are integral nutrients that are known to control most intricate functions of plant. In the present study, application of PAM resulted in an increase in the leaf – N, – P, – K levels (Table 2). Similar results have been presented earlier in ryegrass (Lolium perenne) (18) and in Pinus pinaster (19); soil flocculation was increased with PAM-treatment even at very small application-amounts as PAM is considered to stabilize surface structure (5). El-Rehim (12) reported that polymeric substances, when added to soil, increased its water retention capacity and, hence, maximized the nutrient availability. Therefore, enhanced levels of leaf – N, – P, – K contents (Table 2) may be due to the elevated absorption rate and higher water retention capacity of soil which might allow the plant to soak up maximum amount of nutrients from it which is then successfully transported and assimilated. The synchronous increase in the fresh and dry weights of plant (Table 1) may be due to increased nutrient level in the present study. Evidently, application of PAM improved the overall growth of plant with a subsequent demand of higher uptake of these nutrients from the soil, which ultimately accumulated in the leaves. The content and yield of EO was increased by the application of various doses of PAM (Table 3). This is similar to earlier reports (5) of improved soil physical properties, viz. root penetration, infiltration rate, aeration, erosion resistance, and drainage (5). This, in turn, could lead to enhanced rooting volume and plant interception of nutrients and water, indirectly improving plant nutrition. Therefore, enhanced growth and yield of roots and greater yield of oil and oil active constituents may be credited to the influence of PAM on soil as well as on the roots which store the oil. Srivastava and Lal (26) conducted 14C labelling study to illustrate a positive association between photosynthetic rate and root biomass and demonstrated a positive association between oil content in root, root biomass, and yield. Further

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they demonstrated that the incorporation of 14C labelled carbon in the leaf assimilated metabolites which were ultimately transported to roots for their biosynthetic use. Hence, the increase in root biomass may have increased the translocation of higher portion of carbon assimilates toward increased plant secondary metabolism and related enzyme activities responsible for the complex sesquiterpene biosynthesis that might have subsequently increased the content and yield of vetiver EO. Previous studies reported soil application of fertilizers increasing the oil yields without changing the composition of oil (27). The GC chromatograms depicted the active constituents of vetiver oil in which khusimol increased by the PAM treatment (Figure 2 a and b). Vetiver oil is a complex sesquiterpene resulting from a cytosolic mevalonate dependent pathway, which is triggered by association of two acetyl coenzyme A units. The precursors in this pathway are isopentenyl diphosphate (IPP) and its allylic isomer dimethylallyl diphosphate (DMAPP). A concomitant secondary metabolic pathway takes place in the plastids commonly known as MEP pathway. Although this subcellular compartmentation allows both pathways to operate independently in plants, there is evidence that they cooperate in the biosynthesis of certain metabolites (28,29). According to Schalk and Deguerry (30), the khusimol results from oxidation of zizaene which is synthesized from FPP by the activity of zizaene synthase. As per the results of yield attributes shown in the GC chromatograms (Figure 2), it can be conjectured that PAM may have some stimulatory role in the biosynthetic pathway mainly at two steps, one at conversion of zizaene from FPP by zizaene synthase and the other at oxidation of zizaene into khusimol by cytochrome P450 reductase. This investigation suggested possibilities where PAM can be efficiently integrated as an agronomical practice in raising the production of such highvalue EO. The low cost of PAM is an additional advantage that can contribute to its economical application. Acknowledgments We are thankful to UGC, New Delhi, India for providing research fellowship to the first author. We also thank CIMAP, Lucknow, India for providing authentic planting material to carry out experimental studies.

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