RESEARCH ARTICLE

Effect of shade level and mulch type on

growth, yield and essential oil composition of
damask rose (Rosa damascena Mill.) under
mid hill conditions of Western Himalayas
Meenakshi Thakur ID1,2, Vinod Bhatt3, Rakesh Kumar1,2*

1 Academy of Scientific and Innovative Research, New Delhi, India, 2 Agrotechnology of Medicinal, Aromatic
and Commercially Important Plants Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur
a1111111111 (HP), India, 3 Natural Product Chemistry and Process Development Division, CSIR-Institute of Himalayan
a1111111111 Bioresource Technology, (Council of Scientific and Industrial Research), Palampur (HP), India
a1111111111
a1111111111 * rakeshkumar@ihbt.res.in
a1111111111

Abstract
A field experiment was conducted at the experimental farm of CSIR-Institute of Himalayan
OPEN ACCESS
Bioresource Technology, Palampur, India for two consecutive years (2015–16 and 2016–
Citation: Thakur M, Bhatt V, Kumar R (2019) Effect
17). The aim of the study was to test the hypothesis whether different shade level and mulch
of shade level and mulch type on growth, yield and
essential oil composition of damask rose (Rosa type would influence the growth, flower yield and essential oil profile of R. damascena. Yield
damascena Mill.) under mid hill conditions of attributes viz., numbers of flowers plant-1, fresh flower weight plant-1, flower yield, and
Western Himalayas. PLoS ONE 14(4): e0214672. essential oil yield were significantly higher under open sunny conditions as compared to
https://doi.org/10.1371/journal.pone.0214672
25% and 50% shade levels. However, plants grown under 50% shade level recorded signifi-
Editor: Manuel Joaquı́n Reigosa, University of cantly higher plant height (cm), plant spread (cm) and the lowest numbers of branches as
Vigo, SPAIN
compared to control. Among mulches, black polyethylene mulch recorded significantly
Received: November 28, 2018 higher growth, and yield attributes of damask rose as compared to other mulches. Black
Accepted: March 18, 2019 polyethylene mulch recorded 74.5 and 39.2% higher fresh flower yield as compared to with-
Published: April 4, 2019 out mulch, during 2015–16 and 2016–17, respectively. Correlation studies showed a posi-
tively significant correlation between quality and quantity traits. A total of twenty-six
Copyright: © 2019 Thakur et al. This is an open
access article distributed under the terms of the essential oil compounds were identified which accounted for a total of 88.8 to 95.3%. Plants
Creative Commons Attribution License, which grown under open sunny conditions along with the applications of black polyethylene mulch
permits unrestricted use, distribution, and produced a higher concentration of citronellol and trans-geraniol. Damask rose planted in
reproduction in any medium, provided the original
open sunny conditions and mulched with black polyethylene sheet recorded significantly
author and source are credited.
higher flower yield.
Data Availability Statement: All relevant data are
within the paper.

Funding: The source of funding that supported the

work, received from Scientific and Engineering
Research Board (SERB), department of Science
and Technology, GOI, New Delhi, India. Specific 1. Introduction
grant numbers: SB/S4/AS-134/2013 Initials of
Damask rose (Rosa damascena Mill.) is considered as one of the important aromatic crops.
authors who received each award: Rakesh Kumar.
Full names of commercial companies that funded
The plant is highly valuable as it has application in perfumery, cosmetics and flavors industries.
the study or authors: No commercial funding was Besides this, it has application in pharmacology such as anti-HIV, antibacterial, antioxidant,
received. Initials of authors who received salary or hypnotic, antidiabetic activities [1] and is used to cure depression, insomnia and for stress

PLOS ONE | https://doi.org/10.1371/journal.pone.0214672 April 4, 2019 1 / 14

 Effect of shade level and mulch type on growth, yield of damask rose (Rosa damascena)

other funding from commercial companies: No reduction [2]. Bulgaria, Turkey, Morocco, Iran, India, Egypt, China, and Russia are known to
salary was received from commercial funding. produce damask rose for producing rose oil, rose water, concrete, attar and absolute [3]. The
URLs to sponsors’ websites: http://www.dst.gov.
price of its essential oil varies from 7 to 10 lakh kg-1 [2]. The quality of essential oil of damask
in. The funders had no role in study design, data
collection and analysis, decision to publish, or rose is due to the high percentage of the monoterpene alcohols such as citronellol, trans-gera-
preparation of the manuscript. niol, linalool, phenyl ethyl alcohol and hydrocarbons such as nonadecene and nonadecane.
The growth and yield of damask rose are affected by agronomic factors viz., nutrient manage-
Competing interests: The authors have declared
that no competing interests exist. ment, varieties, harvesting stage, distillation process, diurnal variability and storage conditions
of flowers.
Light is one of the most important environmental factors for plant growth and development
[4]. Different light intensities influence plant growth, leaf gas exchange, and water use effi-
ciency. In aromatic crops, different light intensities have been demonstrated to alter essential
oil content and composition. The substantial amount of light energy is required to reduce car-
bon which combines with CO2 producing oxygen, carbohydrates, ATP and NADPH. The sug-
ars converted into amino acids, hormones, and secondary metabolites. However, plants grown
under shade level leads to reduce the stomatal conductance, photosynthesis rate, ATP synthe-
sis, carbon assimilation and plant growth [5,6]. The study of microclimate modification by
using different green shade nets found that different crops behaved differently under shade
conditions [7]. These variations in the microclimate modify the rate of CO2 assimilation and
photosynthesis, consequently, crop growth and productivity. Crop yield and productivity can
also be increased by application of mulch in the soil as it improves soil conditions for plant
growth, including soil temperature, soil moisture, weed control, reduction in leaching of fertil-
izers [8]. Mulching is also known to buffer soil temperature, increases early production and
higher yields along with better quality; enhance water and fertilizer use efficiency and decrease
the incidence of pests [9]. Therefore, different types of mulching materials are used to change
the micro environment along with the improvement of crop growth and yield.
Plant acclimation to different light intensities depends on environmental conditions and
plant genotype, and thus is species-specific [10,11]. There is a dearth of information on shade
effect and mulch type for the production of damask rose in the world. Modification of micro-
climate by shade and mulch is an alternative approach to meet the increasing demand of dam-
ask rose under such conditions. However, the effects of shade level and mulch type on R.
damascena have not been studied under Western Himalayan conditions. In view of the above
facts, an experiment was conducted to study the effect of shade level and mulch type on
growth, flower yield, essential oil content, and composition of damask rose under mid hill con-
ditions of Western Himalayas.

2. Materials and methods

2.1 Experimental site
The field experiment was conducted at the experimental farm of CSIR-Institute of Himalayan
Bioresource Technology, Palampur, India, located at 32˚06’05”N latitude, 76˚34’10”E longi-
tude and an altitude of 1390 m above mean sea level during 2015–16 and 2016–17. Maximum
temperature (14.3 to 30.0˚C and 11.6 to 31.9˚C), minimum temperature (1.8 to 16.0˚C and 1.7
to 19.0˚C), relative humidity (22 to 76% and 31 to 68%), and total rainfall 254.8 mm and 373.2
mm) were recorded during both the seasons (Fig 1).

2.2 Experimental details

Damask rose variety Jwala was used to study the effect of microclimate modification during
2015–16 to 2016–17. The soil of the experimental field was silty clay in texture (10.76% sand,
55.48% silt and 33.76% clay), acidic in reaction (pH 5.25), low in organic carbon (0.2%) and

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 Effect of shade level and mulch type on growth, yield of damask rose (Rosa damascena)

Fig 1. Meteorological standard weeks (A) during growing season of damask rose (2015–2016) (B) during growing season of damask rose (2016–2017).
https://doi.org/10.1371/journal.pone.0214672.g001

exhibited a low availability of nitrogen (49.6 mg/kg of soil), low phosphorous (3.1 mg/kg of
soil) and high potassium (182.2 mg/kg soil). Application of nutrients NPK was applied in the
ratio of 120:60:40 kg/ha through fertilizer urea (46% N), single super phosphate (16% P2O5)
and muriate of potash (60% K2O), respectively. The experiment was laid out in a split plot
design (SPD) with four replications. The agro-shade net houses (shade level) were organized
in main plots, while the different mulches were subjected to sub-plot. Shade houses (3 m height
x 12 m width x 22 m length) were constructed on a 0.2 ha section of an open field at the
Chandpur farm of the institute. Green polypropylene shade fabrics of different mesh gauges
were erected on frames to simulate shade treatments: 25 and 50%. Frames without shade fabric
served as control (i.e., 0% shade). The shade houses were placed on ground cover in the open
field, 2 m a part to prevent treatment overlap.
The experimental area was divided into three blocks to account for any shading effect from
adjacent sides. After pruning of damask rose bushes in the month of December, nine treat-
ments consisting of three shade net houses viz., control (without shade), 25% shade and 50%
shade level were made. Three mulch types viz., control (without mulch), organic mulch at the
rate of 5 t/ha (poplar leaf mulch) and black polyethylene mulch (25/30 micron size) in sub
plots were organized.

2.3 Growth and yield attributes

Biometric observations were recorded at the time of flowering. Growth information on plant
height (cm), numbers of branches plant-1, plant spread (cm) and leaf area index (LAI) was
recorded. Five plants plot–1 were selected for recording plant height, plant spread, number of
branches plant–1, and leaf area. Plant height was measured from the ground level to tip of the
top leaf. Plant spread was recorded in North-South and East-West directions. Yield parameters
viz., numbers of flowers plant-1, fresh flower weight plant-1 (g) flower yield (kg ha-1), essential
oil yield (kg ha-1), essential oil content (%) and essential oil components were recorded. Plant
height and leaf area index (LAI) was measured with the help of measuring scale and digital
plant canopy imager CI-110/120, respectively. Photosynthetically Active Radiations (PAR)
under each shade and mulch treatment was measured during the flowering season with a LI-

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 Effect of shade level and mulch type on growth, yield of damask rose (Rosa damascena)

190 quantum sensor (LI-COR, Inc., USA). The observations were taken around 8:00 AM to
4:00 PM after each interval of two hours as the sun’s angle to normal (zenith angle) is less than
2˚C on most days of observations. Each reading was expressed as μmol-1m-2s-1.

2.4 Extraction of essential oil

Damask rose flowers were collected separately from each treatment during April and mid of
May months of the crop season. In each treatment, 1000 g flowers were used for distillation.
Damask rose flowers were harvested early in the morning (5:00–7:00 AM) by manual plucking
and were immediately hydro-distilled in the Clevenger type apparatus within 10 minutes after
harvest to prevent the loss of volatile compounds from the flowers. The ratio of flower and
water was 1:2 (w/v) and the essential oil content of damask rose was measured as mL and %
ratio (v/w) on the fresh weight basis. Essential oil yield (kg ha-1) was calculated by multiplying
fresh flower yield, essential oil content and specific gravity (0.85) of rose essential oil. The
extracted essential oil samples were dried over anhydrous sodium sulphate and were stored at
4˚C before analysis. Three replicates were used to identify the essential oil composition
through GC and GC-MS.

2.5 Compounds identification by GC-MS and GC

Gas chromatography—Mass spectrometry (GC-MS) analysis were carried out on a Shimadzu
QP 2010 GC-MS system equipped with AOC-5000 auto-injector and DB-5 capillary column
(30 m × 0.25 mm, 0.25 mm film thickness) from SGE, Australia; in which helium at the rate of
1.28 mL min-1, sample injection of 2 μL with a split ratio of 10; ionization energy of 70 eV was
used. Ion source temperature of 200˚C, injector temperature of 250˚C was maintained. The
oven temperature used at the initial stage was 40 oC for 4 Min, at rising 4˚C min-1 to 220˚C
and then held isothermal at 220˚C for 4 min.
The composition of rose oil samples was evaluated by a Shimadzu GC-2010 gas chromato-
graph (Shimadzu, Tokyo, Japan) equipped with flame ionization detector (FID) and DB-5
(J&W Scientific, Folsom, USA) fused silica capillary column (30 m × 0.25 mm, i.d.;0.25 μm
film thickness). Nitrogen was used as carrier gas at the column with the velocity of 1.24 mL
min-1 flow rate. The oven temperature was programmed at 40˚C for 4 min and then increased
at the rate of 4˚C min-1 from 40–220˚C. The temperature of injector and detector was main-
tained at 250˚C and samples were injected in split mode. The compounds were identified
based on retention indices and peak area percentage of the chromatogram. Retention indices
(RI) were calculated by using homologous series of n-alkanes (C8–C24), and then compounds
were identified by comparing R.I values, mass spectra, and library databases of NIST-MS
(National Institute of Standards and Technology-mass spectral) database [12,13].

2.6 Statistical analysis

Statistical analysis of data was done according to the standard analysis of variance (ANOVA)
by using the software SYSTAT-12 (SYSTAT Software Inc., Chicago, IL, USA). Variations
among treatments were assessed by LSD values at 5% probability (p� 0.05) level were pro-
cessed by multiplying standard error of the difference. Correlation studies were carried out to
investigate the relationship between growth and yield parameters. While, the standard devia-
tion method was used to prepare essential oil compounds using excel stats software. Multivari-
ate principal component analysis (software PAST3) was used to evaluate the effect of
treatment combinations of shade level and mulch type on essential oil compounds.

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 Effect of shade level and mulch type on growth, yield of damask rose (Rosa damascena)

Fig 2. Photosynthetic Active Radiation (PAR) at different time intervals during flowering season of damask rose.
https://doi.org/10.1371/journal.pone.0214672.g002

3. Results and discussion

3.1 Photosynthetic active radiation
Plant tolerance to high or low PAR condition is regulated by the microclimate modification.
Increase in shade level reduces PAR and alters light intensity, affecting the photosynthetic
rate, and crop yield production. Higher PAR was recorded at mid day under control (1500 μ
mol-1m-2s-1) followed by 25% (800 μ mol-1m-2s-1) and 50% shade level (500 μ mol-1m-2s-1) (Fig
2). The results were similar to the studies of clary sage (Salvia sclarea), and behavior of rose
cultivar in different shade levels in which decreased PAR under increased shaded conditions
was reported [4,5].

3.2 Effect of shade level on growth parameters

Microclimate modification significantly influenced crop growth and yield of damask rose. The
plants grown under shade conditions were taller than those grown under open sunny condi-
tions. The growth parameters were significantly affected by different shade levels during both
the years (Table 1). Plant height was significantly higher under 50% shade (137.4 and 144.7
cm), as compared to 25% shade (125.2 and 127.4 cm), and control (100.3 and 119.7 cm), dur-
ing both the years. This may be due to the fact that plant grown in shade resembled etiolated
plants, which have an unwanted tall appearance and are more apical dominant. It also pro-
duces a phototropism response to modify plant growth and development to receive enough
light [14]. Results were similar to the studies of rose scented geranium (Pelargonium graveolens
family; Geraniaceae) and bottle gourd (Lagenaria vulgaris) in which growth parameters were
higher in shaded conditions [15,16]. Numbers of branches were 31.4 and 22.8% higher in open
sunny conditions as compared to 50% shade level during 2015–2016 and 2016–2017, respec-
tively. This may be due to the absorption of lesser PAR under shade conditions that have
reduced the photosynthetic and respiration rate that usually inhibits plant growth and

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 Effect of shade level and mulch type on growth, yield of damask rose (Rosa damascena)

Table 1. Effect of shade level and mulch type on growth parameters of damask rose.
Treatment Plant height (cm) Numbers of branches Plant spread (cm) Leaf area index (LAI)
plant-1
N-S E-W
2015–2016 2016–2017 2015–2016 2016–2017 2015–2016 2016–2017 2015–2016 2016–2017 2015–2016 2016–2017
Shade level
Control (0% Shade) 100.3c 119.7c 22.2a 25.3a 93.5c 99.7c 111.2c 116.3 4.0bc 4.8c
b b b ab b ab b ab
25% Shade 125.2 127.4 18.1 23.8 106.9 112.3 127.9 132.6 5.0 6.3ab
a a bc c a a a a
50% Shade 137.4 144.7 16.9 20.6 122.6 118.9 140.4 141.6 5.6 6.6a
SEm± 2.7 1.1 0.5 0.7 0.9 2.8 1.6 3.5 0.3 0.3
LSD (p � 0.05) 9.6 3.7 1.7 2.4 3.2 9.5 5.5 NS 1.1 0.9
Mulch type
Without Mulch 109.0c 121.4c 17.9 21.4bc 104.2b 96.8c 122.2bc 112.2c 4.8 5.5
ab b ab a b a
Organic Mulch 123.1 131.4 19.3 23.7 117.2 113.2 133.7 131.6b 4.9 5.8
Black polyethylene Mulch 129.9a 139.1a 20.0 24.6a 101.5bc 120.9a 123.5b 146.7a 4.8 6.4
SEm± 3.0 1.5 0.9 0.8 2.6 3.2 2.8 10.0 0.2 0.2
LSD (p � 0.05) 8.9 4.5 NS 2.5 7.6 9.4 8.2 11.2 NS NS

Note: N-S: North South; E-W: East West; NS: Not significant; LSD: Least Significant Difference; SEm: Standard Error of Mean;LAI: Leaf area index; Mean values with
same letter in same columns do not differ significantly (p�0.05)

https://doi.org/10.1371/journal.pone.0214672.t001

productivity by affecting the gas exchange as compared to full sunlight [17]. During both the
years of studies, plant spread (N-S) was significantly higher in 50% shade level as compared to
open sunny conditions (control). Significantly, lowest plant spread was recorded under open
sunny conditions which might be due to higher absorption of PAR, that had enhanced the
photosynthetic rate compared to shaded conditions (Fig 2). The presented data revealed signif-
icantly higher LAI under 50% shade level followed by 25% shade and control, respectively, for
both years of studies. Similar results were observed by modifying the microclimate in S. offici-
nalis to different shade levels resulted in maximum leaf areas when planted in 50% shade
[5,18,19].

3.3 Effect of different mulch types on growth parameters

Growth parameters were significantly affected by different mulch (Table 1). During both the
years, plant height was significantly higher under black polyethylene mulch (129.9and 139.1
cm), as compared to control. Results were in accordance with the studies of sweet-scented
geranium and stevia in which plant height increased in mulched conditions as compared to
control [20,21]. This is due to increased temperature under black polyethylene mulch that has
enhanced the microbial activity of nitrifying bacteria that access the nitrification and nitrate to
plant roots under soil [22,23]. Significantly higher numbers of branches were recorded under
black polyethylene mulch followed by organic and without mulch (control) during both the
years. During the first year, organic mulch recorded significantly higher plant spread (N-S and
E-W direction) followed by black polyethylene mulch and control. However, during the sec-
ond year, plant spread in both directions was significantly higher in black polyethylene mulch
followed by organic mulch and control. Similar results were reported in the studies of African
marigold in which maximum plant spread was recorded in plastic mulch [24]. Plant growth is
influenced by the use of mulch, such as moisture content, reduced competition for light and
availability of nutrients, absence of weeds, pest reduction and increased soil temperature [24].
LAI was not affected significantly by different mulch during both the years.

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 Effect of shade level and mulch type on growth, yield of damask rose (Rosa damascena)

Table 2. Effects of shade level and mulch type on yield attributes and yield of damask rose.
Treatment Number of flowers plant-1 Fresh flower weight plant-1 (g) Flower yield (kg ha-1) Essential oil yield (kg ha-1)
2015–2016 2016–2017 2015–2016 2016–2017 2015–2016 2016–2017 2015–2016 2016–2017
Shade level
Control (0% Shade) 186.7a 131.8a 511.3a 381.6a 4544.7a 3392.4a 2.0a 1.5a
b b b ab b ab b
25% Shade 79.2 111.5 230.7 334.2 2050.3 2970.3 0.9 1.4ab
c c c c c c c
50% Shade 34.2 78.7 105.3 207.2 936.1 1841.4 0.4 0.9c
SEm± 5.4 7.2 19.6 44.1 174.1 392.4 0.01 0.08
LSD (p � 0.05) 6.2 8.3 22.6 50.9 200.9 452.7 0.05 0.3
Mulch type
Without Mulch 72.8c 90.1c 203.1c 256.3c 1805.0c 2278.1c 0.8c 1.0c
b b b b b b ab
Organic Mulch 102.1 109.0 289.9 309.7 2576.7 2753.2 1.2 1.3b
a a a a a a a
Black polyethylene Mulch 125.2 122.9 354.3 356.9 3149.4 3172.7 1.4 1.5a
SEm± 11.2 6.5 30.9 44.1 274.3 147.0 0.3 0.2
LSD (p � 0.05) 11.0 6.5 30.6 16.4 271.5 145.5 0.1 0.1
LSD: Interaction (S X M) 19.1 11.2 52.9 28.4 470.3 252.0 0.2 NS

Note: S: Shade; M: Mulch; LSD: Least Significant Difference; SEm: Standard Error of Mean; Mean values with same letter in same columns do not differ significantly
(p�0.05)

https://doi.org/10.1371/journal.pone.0214672.t002

3.4 Effect of shade levels on yield attributes

Yield attributes of damask rose were significantly affected by shade level and mulch types
(Table 2). Significantly higher numbers of flowers were recorded under open sunny conditions
as compared to 25 and 50% shade level during both the years. Light affected chlorophyll con-
tent, photosynthetic enzyme activity, stomatal opening, and the distribution of carbohydrates in
plants [25,26,27]. A perusal of data presented in Table 2 revealed that fresh flower weight plant-1
was significantly affected by shade level and mulch type. At 50% shade level, fresh flower weight
plant-1 was found significantly lowest (105.3 and 207.2 g), however, control recorded signifi-
cantly higher fresh flower weight plant-1 (511.3 and 381.6 g), respectively, during both the years.
During the second year of study, fresh flower weight per plant was found at par with control
and 25% shade level. It may be because of the lesser weed growth, more numbers of branches
and numbers of flowers resulted in higher fresh flower weight under open sunny conditions
and 25% shade level. Similar results were observed in clary sage in which fresh weight biomass
was reduced with increase in shade level [28]. Reduction in light intensity reduces the rate of
photosynthesis and rate of the growth. While, more vegetative growth, lesser numbers of flowers
resulted in significantly lesser fresh flower weight plant-1 under 50% shade conditions [29,30].
Plants grown under open sunny conditions produced significantly higher flower yield dur-
ing both the years (Table 2). During the first year, open sunny conditions recorded signifi-
cantly higher flower yield (4544.7 kg ha-1) than 25% (2050.3 kg ha-1) and 50% shade level
(936.1 kg ha-1), respectively. During the second year, plants under open sunny conditions reg-
istered significantly higher flower yield as compared to 50% shade level but remained at par
with 25% shade level. It may be attributed to the favorable environment for increased numbers
of branches consequently increased the numbers of flowers that resulted into higher flower
yield. The essential oil obtained by hydro distillation in Clevenger-type apparatus was influ-
enced significantly by different shade levels and mulch types (Table 2). Significantly higher
essential oil yield was recorded in open sunny conditions as compared to 25% and 50% shade
level during the first year of study. Similarly, during the second year, essential oil yield was sig-
nificantly higher in control as compared to 50% shade level but remained at par with 25%

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 Effect of shade level and mulch type on growth, yield of damask rose (Rosa damascena)

shade level. This may be due to the higher flower yield and essential oil content under control
and 25% shaded conditions as compared to 50% shade level (Table 2). The production and
quality of essential oil of medicinal and aromatic species responds differently to shade condi-
tions due to genetic makeup and environmental conditions. Essential oil yield of Thymus vul-
garis and Matricaria chamomilla increased under open sunny conditions, however, Ocimum
gratissimum, Anethum graveolens and Pothomorphe umbellate recorded significantly higher
essential oil yield when cultivated under shade [31].

3.5 Effect of mulch type on yield attributes

Among mulch types, black polyethylene mulch recorded significantly higher number of flow-
ers as compared to organic mulch and control during both the years. Black polyethylene
mulch, registered 72.0 and 36.4% higher number of flowers as compared to control, during
2015–16 and 2016–17, respectively. It might be due to favorable soil temperature and a mois-
ture condition that has enhanced the microbial activity in the soil resulting in enhanced nutri-
ent availability and physiological activities of plant which ultimately resulted in the higher
number of flowers [24,32]. Black polyethylene mulch produced a significantly higher fresh
flower weight plant-1 as compared to organic mulch and control. Black polyethylene mulch,
enhanced the fresh flower weight plant-1 by 74.4 and 39.3% as compared to control during
both the years. Similar results were reported under black polyethylene mulch in rose [8]. Black
polyethylene mulch, retained higher soil moisture content with efficient use of nutrients,
which might have maximized the number of flowers, hence, fresh flower weight.
Plants mulched with black polyethylene, produced significantly higher flower yield (3149.2
and 3172.7 kg ha-1) as compared to organic mulch (2576.7 and 2753.2 kg ha-1) and without
mulch (1805.0 and 2278.1 kg ha-1), respectively, during both the years. Black polyethylene
mulch, recorded an increase of 74.5 and 39.3% as compared to without mulch, respectively.
Mulch types, could be attributed to water use efficiency, alter the soil temperature and soil
moisture content, proper utilization of nutrients which might favor the environmental condi-
tions for the plant. The results obtained were consistent with the studies of Pogostemon cablin
and Java citronella [32,33]. Among mulches, black polyethylene mulch recorded significantly
higher essential oil yield as compared to organic and control during both the years.
3.5.1 Interaction effect on yield attributes and yield. Interaction effect of shade level and
mulch type on numbers of flowers plant-1 and fresh flower weight plant-1 were found significant
during both the years (Table 2). Plants grown under open sunny conditions recorded significantly
higher numbers of flowers plant-1 as compared to 25 and 50% shade level at each mulch type.
Black polyethylene mulch recorded significantly higher number of flowers plant-1 and fresh flower
weight plant-1 as compared to organic mulch and control at each shade level. In all treatment
combinations, black polyethylene mulch under control produced significantly higher number of
flowers plant-1 and fresh flower weight plant-1. Interaction effect of shade and mulch factors on
fresh flower yield of damask rose was found significant during both the years. Open sunny condi-
tions produced significantly higher flower yield, however, shaded condition of 50% level produced
significantly lower flower yield. Plants under open sunny conditions produced significantly higher
flower yield with black polyethylene mulch. The different mulch produced significant difference
with different light intensities. Black polyethylene mulch, produced a significantly higher flower
yield followed by organic and without mulch, respectively in open sunny conditions.

3.6 Correlation analysis

The correlation study for phenotypic level, growth and yield attributes are given in Table 3.
Numbers of flowers plant-1 and fresh flower weight plant-1 produced significantly (p � 0.01)

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 Effect of shade level and mulch type on growth, yield of damask rose (Rosa damascena)

Table 3. Estimate of correlation coefficients at the phenotypic levels for quantitative and quality traits during crop season (Pooled data of two years).
Parameters Fresh flower weightplant-1 Plant height Number of N-S plant E-W plant Leaf area Essential oil Flower yield
(g) (cm) branches plant-1 spread (cm) spread (cm) index content (v/w%) (kg ha-1)
(LAI)
Numbers of flowers 0.99�� -0.69 0.44 -0.59 -0.53 -0.77 0.77� 0.99��
plant-1
Fresh flower weight plant-1 (g) -0.67 0.45 -0.59 -0.51 -0.76 0.77� 0.99��
�� �� ��
Plant height (cm) 0.14 0.88 0.82 0.89 -0.24 -0.67
Numbers of branches plant-1 0.24 0.32 0.20 0.71� 0.45
N-S Plant spread (cm) 0.94�� 0.78� -0.08 -0.59
E-W plant spread (cm) 0.78� -0.10 -0.51
Leaf area index (LAI) -0.33 -0.76
Essential oil content (v/w%) 0.77�

Note
� ��
, , Significant correlation with p values � 0.05, � 0.01, respectively.

https://doi.org/10.1371/journal.pone.0214672.t003

higher positive correlation with flower yield (0.99), essential oil content (0.77), while, showed
positive correlation with numbers of branches plant-1 (0.44). Similar results were observed in
correlation matrix of damask rose in which data revealed that flower yield was significantly
and positively correlated with the number of flower [34]. Plant height (cm) had highly positive
significant correlation for LAI (0.89), N-S plant spread (0.88) and E-W plant spread (0.82),
however, had positive correlation for number of branches plant-1. Numbers of branches plant-
1
produced significant positive correlation for essential oil content (0.71), while, showed posi-
tive correlation for growth and yield attributes. N-S plant spread showed highly significant
positive correlation for E-W plant spread (0.94) and showed significant and positive correla-
tion for LAI (0.78). Likewise, E-W plant spread showed positive correlation for LAI (0.78).
Essential oil content produced significantly higher positive correlation for flower yield (0.77).
Similar results were observed in Tanacetum parthenium, in which positive correlation was
observed between essential oil % and flower yield [35].

3.7 Essential oil yield and composition

Essential oil yield ranged from 0.051 to 0.055% in different treatments (Table 4). Damask rose
essential oil yield was not significantly affected by different shade level and mulch types. The
analysis of essential oil of R. damascena led to the identification of twenty six compounds. The
total area percentages of damask rose components were 88.8 to 95.3%. The components were
grouped into oxygenated monoterpene (53.2 to 68.0%), oxygenated sesquiterpene (3.6 to
6.3%), sesquiterpene hydrocarbon (3.0 to 4.9%) and aliphatic hydrocarbon (16.1 to 31.8%)
(Table 4). The analyzed component of damask rose essential oils mainly contains oxygenated
monoterpenes and aliphatic hydrocarbon as a major fraction. The major components were cit-
ronellol (35.5 to 49.2%), trans-geraniol (12.6 to 18.4%), nonadecane (8.9 to 15.1%), heneico-
sane (3.8 to 8.7%) and nonadecene (1.6 to 3.6%).
The percentage of oxygenated monoterpene and sesquiterpene components like citronellol,
geranyl acetate, linalool and trans-rose oxide were higher in open sunny conditions as com-
pared to 25% shade and 50% shade level (Table 4). Results were similar to the study of S.
sclarea and T. minuta in which oxygenated monoterpenes and sesquiterpene components
decreased with increase in shade levels [5,17]. Light played an important role in essential oils
production that depends upon the metabolic processes, as well as the physiology of the whole
plant. Black polyethylene mulch produced higher percentage of citronellol and trans-geraniol

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 Effect of shade level and mulch type on growth, yield of damask rose (Rosa damascena)

Table 4. Effect of shade level and mulch type on essential oil compounds of damask rose (Pooled data of two years).
Area (%)
Treatment Control 25% Shade 50% Shade
Sr. No. Compounds Lit. R.I Exp.R.I WM OM PM WM OM PM WM OM PM
Oxygenated Monoterpene
1 Linalool 1098 1100 1.1±0.5 1.1±0.4 1.5±0.1 0.9±0.0 0.8±0.1 1.3±0.2 1.3±0.3 1.1±0.5 1.2±0.1
2 trans-rose oxide 1106 1115 0.6±0.1 0.6±0.2 0.8±0.1 0.6±0.0 0.5±0.2 0.6±0.1 0.6±0.1 0.5±0.4 0.7±0.0
3 Phenyl ethyl alcohol 1110 1116 0.1±0.1 0.1±0.1 0.1±0.0 0.2±0.1 0.1±0.1 0.1±0.1 0.1±0.0 0.3±0.4 0.2±0.1
4 cis-rose oxide 1111 1118 0.2±0.3 0.1±0.1 0.1±0.1 0.1±0.0 0.3±0.4 0.3±0.4 1.1±0.0 0.1±0.1 0.1±0.1
5 Terpineol 1189 1191 1.1±0.0 1.0±0.3 1.2±0.2 1.0±0.0 0.7±0.3 0.9±0.2 0.7±0.5 1.0±0.1 0.9±0.0
6 Citronellol 1228 1238 40.0±0.0 45.5±8.5 49.2±1.8 40.8±2.0 35.5±9.1 46.4±2.6 43.3±0.6 44.2±1.0 46.5±3.3
7 trans-geraniol 1255 1271 12.6±0.4 13.0±8.6 14.4±4.1 15.1±4.0 15.3±10.3 18.4±1.2 12.8±0.1 15.6±0.3 15.6±3.0
Sub Total 55.7±0.2 61.4±2.6 67.3±0.9 58.7±0.9 53.2±0.4 68.0±0.7 59.9±0.2 62.8±0.4 65.2±0.9
Oxygenated Sesquiterpene
8 Citronellyl acetate 1354 1354 0.8±0.2 0.8±0.1 0.7±0.3 0.5±0.1 0.5±0.1 0.5±0.1 0.6±0.1 0.5±0.0 0.5±0.0
9 Eugenol 1356 1367 0.4±0.1 0.3±0.1 0.3±0.1 0.1±0.0 0.2±0.1 0.2±0.1 0.2±0.0 0.2±0.0 0.2±0.1
10 Geranyl acetate 1383 1383 2.8±0.6 2.4±1.0 2.1±0.6 1.2±0.1 1.2±0.3 1.8±0.8 2.1±1.1 1.0±0.5 1.4±0.6
11 Methyl Eugenol 1401 1401 0.9±0.3 1.0±0.2 0.8±0.1 0.8±0.1 0.7±0.1 0.6±0.0 0.9±0.1 0.8±0.2 0.8±0.1
12 Farnesol 1722 1717 1.4±0.5 1.1±0.0 0.9±0.1 1.3±0.2 1.2±0.5 0.9±0.1 1.2±0.3 1.1±0.5 1.0±0.2
Sub Total 6.3±0.3 5.6±0.3 4.8±0.3 3.9±0.1 3.8±0.2 4.0±0.2 5.0±0.2 3.6±0.2 3.9±0.1
Sesquiterpene Hydrocarbon
13 Elemene 1391 1396 0.9±0.1 0.8±0.2 0.9±0.4 0.6±0.1 0.6±0.1 0.6±0.2 1.4±0.6 0.6±0.0 0.5±0.0
14 Caryophyllene 1404 1420 0.7±0.1 0.9±0.1 0.5±0.1 0.6±0.0 0.6±0.1 0.6±0.0 0.8±0.2 0.6±0.1 0.6±0.0
15 Alpha-guiaene 1439 1435 0.1±0.1 0.3±0.2 0.1±0.0 0.1±0.0 0.0±0.0 0.2±0.1 0.1±0.1 0.3±0.0 0.1±0.1
16 Alpha-humulene 1454 1457 0.9±0.2 1.0±0.1 0.1±0.0 0.8±0.0 0.7±0.0 0.8±0.1 0.9±0.1 0.9±0.1 0.7±0.1
17 Germacrene-D 1480 1482 0.6±0.2 0.6±0.1 0.6±0.1 0.6±0.0 0.6±0.1 0.5±0.0 0.7±0.0 0.6±0.1 0.6±0.0
18 Pentadecane 1500 1500 0.6±0.1 0.7±0.1 0.6±0.1 0.6±0.1 0.7±0.0 0.6±0.1 0.7±0.1 0.5±0.2 0.5±0.0
19 Farnesene 1508 1510 0.2±0.0 0.2±0.0 0.2±0.0 0.2±0.0 0.3±0.0 0.3±0.0 0.3±0.1 0.2±0.0 0.2±0.1
Sub Total 4.0±0.1 4.5±0.1 3.0±0.1 3.5±0.03 3.5±0.04 3.6±0.07 4.9±0.02 3.7±0.07 3.2±0.04
Aliphatic Hydrocarbon
20 Heptadecane 1700 1697 0.1±0.0 0.1±0.0 0.1±0.1 0.1±0.0 0.2±0.1 0.1±0.0 0.1±0.0 0.1±0.0 0.2±0.1
21 Octadecane 1800 1796 0.2±0.1 0.2±0.0 0.1±0.0 0.2±0.0 0.2±0.1 0.1±0.0 0.1±0.1 0.2±0.1 0.1±0.0
22 Nonadecene 1870 1870 3.0±1.0 2.5±0.2 1.6±0.1 3.4±0.2 3.6±2.2 2.1±0.2 2.7±0.5 2.7±0.3 2.5±0.3
23 Nonadecane 1900 1895 14.5±5.8 12.6±0.2 8.9±0.1 14.2±1.9 15.1±7.5 9.2±0.8 10.3±3.3 10.9±4.0 9.5±1.1
24 Eicosane 2000 2002 1.4±0.6 1.4±0.1 0.9±0.1 1.5±0.0 1.7±1.0 1.0±0.2 1.0±0.3 1.1±0.2 1.0±0.0
25 Heneicosane 2100 2096 6.6±3.2 6.1±0.2 3.8±0.4 7.1±0.4 8.7±5.2 4.8±1.1 4.6±1.6 5.5±1.0 4.9±0.1
26 Tricosane 2300 2298 1.3±0.8 1.3±0.2 0.7±0.0 1.7±0.2 2.3±1.4 1.1±0.3 0.8±0.5 1.3±0.2 1.1±0.1
Sub Total 27.1±1.6 24.2±0.1 16.1±0.1 28.2±0.4 31.8±2.5 18.4±0.4 19.6±0.9 21.8±0.8 19.3±0.2
Total 92.8±3.9 95.3±0.6 91.4±0.1 94.0±0.9 91.9±2.0 93.6±2.1 88.8±3.5 91.8±2.2 91.1±0.3

Note: WM: Without Mulch; OM: Organic Mulch; PM: Black polyethylene Mulch; ± values represents standard deviation of three replicates.

https://doi.org/10.1371/journal.pone.0214672.t004

followed by organic and without mulch. This may be because of biosynthetic activation of
pathways of essential oils due to light which depend on the carbon chain obtained by photo-
synthetic light [10]. The remaining compounds did not showed any consistent trend under
shade levels and mulch types. The variation in the compounds of damask rose essential oil
may be related both to the availability of light and favorable soil temperature and soil moisture,
since the quality of essential oil is determined by favorable environmental conditions in the
productivity of secondary metabolites of essential oil crops [36].

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 Effect of shade level and mulch type on growth, yield of damask rose (Rosa damascena)

Fig 3. Loading plot of principal component analysis (A) loading plot PC-1 (B) loading plot PC-2.
https://doi.org/10.1371/journal.pone.0214672.g003

3.8 Principal components analysis

Eighteen components of damask rose essential oil from different nine treatments were submit-
ted to principal component analysis (PCA) for analyzing compositional and different treatments
variation. PC-1 and PC-2 contributed to 45.4 and 31.5% of variance which jointly explained
76.9% of total variance (Fig 3). PC-1 clearly distinguishes the farnesol, nonadecane, nonadecene,
eicosane, heneicosane and tricosane samples having positive loading, however, linalool, rose-
oxide, terpineol and citronellol samples are negatively loaded (Fig 3A). PC-2 is mainly separated
by positive loading of linalool, rose-oxide, citronellyl acetate, geranyal acetate and methyl euge-
nol rich samples. Negative loading is due to trans-geraniol and tricosane (Fig 3B).
Four distinct clusters were observed in score plot (Fig 4). Cluster I (0% PM) explained
higher concentration of citronellol in open sunny condition with the applications of black
polyethylene mulch. In Cluster II (25% PM, 50% OM and 50% PM) concentration of citronel-
lol, trans-geraniol and geranyl acetate was found higher in 25% shade level with the application
of black polyethylene followed by 50% black polyethylene and organic mulch. Cluster III (25%
WM and 25% OM) showed that under mulching conditions, higher concentration of trans-
geraniol, geranyl acetate, nonadecene, nonadecane and heneicosane were observed. In cluster
IV (0% WM, 0% OM and 50% WM) plants grown under open sunny conditions along with
the application of mulch, produced the higher concentration of citronellol and trans-geraniol.
However, percentage of these major compounds decreased under 50% shade level (Table 5).
The analyzed data revealed that the components hydrocarbon viz., nonadecene, nonadecane,
eicosane, heneicosane and tricosane are positively correlated with each others. Hence it was
found that monoterpenes and hydrocarbons formed inverse relationship among themselves.

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 Effect of shade level and mulch type on growth, yield of damask rose (Rosa damascena)

Fig 4. Principal component analysis of essential oil compounds data. Principal component 1 and Principal component 2 jointly explained 76.9% of the
total variation. 0%, 25% and 50% are the shade levels, respectively, while WM, OM, PM are the mulch types viz., without mulch, organic mulch and black
polyethylene mulch, respectively.
https://doi.org/10.1371/journal.pone.0214672.g004

4. Conclusions
Applications of ‘‘different shade levels and mulch types” influenced the growth, yield attributes
and quality of essential oil of damask rose under the mid hill conditions of Western Himalayas.
The open sunny conditions and black polyethylene mulch promotes the production of major
secondary metabolites of damask rose. Since the adaptation of plants to shaded conditions
have reduced the yield attributes, hence from the studies we concluded that plants grown
under open sunny condition with black polyethylene mulch produced higher yield and good
quality of damask rose.

Table 5. Clusters variability in major essential oil constituents (%) of damask rose as affected by shade level and mulch type.
Sr. No. Components Cluster 1 Cluster II Cluster III Cluster IV
1 Citronellol 49.2 44.2–47.1 35.5–40.8 40.0–45.5
2 Trans-Geraniol 14.4 15.6–18.1 15.1–15.3 12.6–13.0
3 Geranyl acetate 2.1 1.0–1.6 1.2 1.7–2.8
4 Nonadecene 1.6 2.0–2.7 3.4–3.6 2.5–3.0
5 Nonadecane 8.9 9.0–10.9 14.2–15.1 11.3–14.5
6 Heneicosane 3.8 4.5–5.5 7.1–8.7 5.0–6.6
https://doi.org/10.1371/journal.pone.0214672.t005

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 Effect of shade level and mulch type on growth, yield of damask rose (Rosa damascena)

Acknowledgments
The authors are grateful to the Director, CSIR-IHBT, Palampur, (HP), India for providing nec-
essary facilities during the course of study. We also thank Mr. Kuldeep Singh, Mr. Ramdeen
Prasad and Mr. Shiv Kumar for their technical support. This is IHBT publication number
4323.

Author Contributions
Data curation: Vinod Bhatt.
Formal analysis: Meenakshi Thakur.
Supervision: Rakesh Kumar.
Validation: Vinod Bhatt, Rakesh Kumar.
Writing – original draft: Meenakshi Thakur.
Writing – review & editing: Rakesh Kumar.

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