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ISSN (E): 2349 1183

ISSN (P): 2349 9265


4(2): 192202, 2017
DOI: 10.22271/tpr.2017.v4.i2.028
Research article

Change in physico-chemical character of nutrient media and


carpet effluent in presence of six algae monocultures
and their consortia
Akash Kumar Patel1, M. R. Suseela1, Munna Singh2 and Sanjeeva Nayaka1*
1
Algology Section, Plant Biodiversity and Conservation Biology Division, CSIR-National Botanical Research
Institute, Lucknow-226001, Uttar Pradesh, India
2
Department of Botany, University of Lucknow, Lucknow-226007, Uttar Pradesh, India
*Corresponding Author: nayaka.sanjeeva@gmail.com [Accepted: 21 May 2017]

Abstract: Synthetic microalgae ecology is a promising approach to regulate the concentrations of


nutrients in wastewater reservoirs. The aim of experiments was to evaluate the role of six
microalgae monocultures and their consortia in restoring the water quality of water reservoirs
represented here in the form of media BG11+ (nitrate rich), BG11 (nitrate poor) and wastewater
from carpet effluent (CE). The algal species utilized in the present experiment were Anabaena sp.,
Chlorella sp., Oscillatoria sp., Nannochloropsis sp., Scenedesmus sp., Phormidium sp., and their
synthetic consortia. The water parameters analyzed were dissolved oxygen (DO), water
conductivity, pH and concentrations of nutrients (nitrates, phosphates, nitrites). We observed that
Chlorella sp. produced maximum biomass of 2.2170.126 g.L1 in BG11+ media followed by
Anabaena sp. 1.8970.045 for BG11 media and 1.2330.29 g.L1 for synthetic consortia of BG11
media. On other hand, biomass yields of 1.2330.017 and 0.4600.010 g.L1 were obtained
respectively for consortia in the nitrate deficient BG11 media and CE water. Consequently, with
inoculation of consortia in BG11+ and CE media nitrate level was increased by 295.27% (748.08
mg.L1) and 123.14% (1.33 mg.L1) respectively, while decreased in BG11 by 68.91% (5.1 mg.L
1
). Nitrite concentration was enhanced up to 35.52, 0.038 and 0.028 mg.L1 in BG11+, BG11 and
CE respectively. In contrast, with inoculation of the consortia, the phosphate levels were found to
be depleted by 35.35% (1.31 mg.L1) in BG11+, 95.72% (3.64 mg.L1) in BG11 and 94.97% (1.7
mg.L1) in CE respectively. The range of pH value and DO for different media was found between
8.258.87 and 6.1410.54 mg.L1 respectively, and conductivity was increased with the growth of
algae. The study suggests that beneficial algae and their consortia can be logically selected for
biomass production and for improving the water quality.
Keywords: Wastewater treatments - Microalgae consortia - Water parameters - Biomass - Carpet
effluent.

[Cite as: Patel AK, Suseela MR, Singh M & Nayaka S (2017) Change in physico-chemical character of nutrient
media and carpet effluent in presence of six algae monocultures and their consortia. Tropical Plant Research
4(2): 192202]

INTRODUCTION
The idea of employing the microalgae for wastewater treatments has been originated about six decades ago
(Oswald et al. 1957). Today microalgae are widely utilized in industrial and household wastewater treatments.
These microalgae can perform photosynthesis to generate their own energy and releases excess amount of O 2 to
the atmosphere. Microalgae wastewater treatment plants require minimal supply of energy resources that
reduces the use of sophisticated instruments such as energy-intensive electromechanical blowers (Green et al.
1995), catalytic hydrothermal gasification (Elliott 2008), anaerobic digestion (McCarty et al. 2011) and
hydrothermal liquefaction (Chakraborty et al. 2012). In conventional wastewater treatments nitrate is converted
into the N2 gas through the oscillation within nitrification or denitrification system while phosphates are
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Received: 16 February 2017 Published online: 31 May 2017
https://doi.org/10.22271/tpr.2017.v4.i2.028
Patel et al. (2017) 4(2): 192202

precipitated by metal salts. Microalgae remove nitrates and phosphates in more eco-friendly manner. Through
mixotrophic metabolism, microalgae consortia could perform better and can be used as a sustainable method for
treatment of wastewater. Mixotrophic metabolism utilizes nitrate and phosphate from wastewater to enhance
microalgae biomass (Henkanatte-Gedera et al. 2015). Microalgae consortia based wastewater treatments has
numerous advantages over the conventional methods. The consortia could be efficient in detoxification of
organic and inorganic pollutants from wastewater as compared to the individual microalgae (Subashchandrabose
et al. 2011). Natural consortia improve water quality (bioremediation of hazardous waste viz. nitrates and
phosphates), soil quality (nutrient recycling), plant health etc., and help in maintaining the major ecosystem
(Lugtenberg & Kamilova 2009, Gilbert et al. 2012, Craggs et al. 2013, Samor` et al. 2013). Engaging
microalgae consortia for the nutrient recycling in different wastewater reservoirs to produce biomass for a broad
range of value added bio-chemicals (Martins et al. 2010) has revitalized the concept of synthetic microalgae
ecology as emerging technology (Pittman et al. 2011, Craggs et al. 2013).
Microalgae require nitrate and phosphate in a specific ratio for biomass production. Therefore, removal of
nitrate depends on the levels of phosphate in wastewater and vice-versa. In household wastewater nitrate occurs
between 15 to 90 mg.L1 while levels of phosphate vary between 4 to 20 mg.L1 (Christenson & Sims 2011,
Abdelaziz et al. 2013, Cai et al. 2013). Microalgae utilize N and P in various biochemical reactions to produce a
number of primary and secondary metabolites that alternatively determine the overall biochemical composition
of biomass (Klausmeier et al. 2004, Loladze & Elser 2011). Nitrogen participates in the synthesis of protein
while phosphorus helps in the synthesis of ribosomal RNA and ribosome (French et al. 2003, Dennis et al.
2009). A number of studies have shown that lower supply of nitrate and phosphate resulted in more
accumulation of carbohydrate or lipid or both in their biomass. However, their higher levels increase the algae
biomass as well as their concentration in water or media (Xin et al. 2010, Arbib et al. 2013, Samor` et al. 2013,
Beuckels et al. 2015). Published literature showed that the concentration of any one nutrient (either nitrate or
phosphate) fluctuate at a time while other remains constant (Xin et al. 2010, Arbib et al. 2013, Samor` et al.
2013, Zhang & Hong 2014). Therefore, levels of both nutrients vary simultaneously without changing their
ratios (Aslan & Kapdan 2006, Akerstrm et al. 2014). At high levels of nitrate reduced absorption of phosphate
from water has been reported (Pittman et al. 2011, Craggs et al. 2013). High amount of nitrate content reduces
the nitrate uptake. In the presence of high amount of nitrate, some algae synthesize nitrate (Philips et al. 1992).
Nutrient availability influences the biochemical composition of microalgae biomass that alternatively provokes
the biosynthesis of some primary and secondary metabolites. Keeping their facts in view the aim of present
study is to know up to what extent the microalgae monocultures and their consortia can mitigate the levels of
nitrate, nitrite and phosphate in carpet effluent (CE), and how they respond to BG11+ and BG11 media
supplemented with and without nitrate respectively. We have also evaluated that up to what extent the water
physico-chemical parameter in media and natural CE water can affect the physiology and biomass production.

MATERIALS AND METHODS


Algae Cultures
Six different microalgae species belonging to three phyla such as Cyanophyta (Anabaena sp., Oscillatoria
sp., Phormidium sp.), Orchrophyta (Nannochloropsis sp.) and Chlorophyta (Chlorella sp., Scenedesmus sp.)
were isolated and purified from the carpet industries effluent of Bhadohi district, Uttar Pradesh, India. These
microalgae monocultures were regularly maintained on BG11+ agar plates as well as in the liquid BG11+ media.
Each algae taxon was identified by following the standard procedures and relevant literature (Desikachary 1959,
Philipose 1967, Hibberd 1981). CE media was sterilized through 0.22 m membrane filter while BG11+ and
BG11 media were steam sterilized. All the cultures were incubated at 271 C temperature with an illumination
of 92 mole.m2.S1 photons in a regime of 14:10 light-dark period in 500 mL microcosms.
Experimental design
A set of 21 experiments were executed in 200 mL of sterilized BG11+, BG11 media and CE water for six
monocultures and their synthetic consortia in three replications. The BG11+ media was supplemented with 1.5
g.L1 NaNO3 while BG11 media was maintained without nitrate supplement and CE acted as natural media.
The algae biomasses were harvested after 35 days of cultivation, and dried biomass was obtained by the
gravimetric method. At the end of experiments, different physico-chemical parameters such as changes in the
pH values, dissolved oxygen, water conductivity, nitrates, nitrites and phosphate of the cell-free supernatants of

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different media were analyzed by using DREL 2800 Complete Water Quality Lab with Meter (Hach Co. USA).
Statistics
All the data for different physico-chemical parameters yields were analysed statistically by two-way
ANOVA followed by post hoc Bonferroni analysis, significant differences among the treatments were
considered at 95% confidence level (P value < 0.05). Various graphs were prepared using the Graph Pad
PrismTM (version 5.0, GraphPad Software). All the experiments were done in three replications, and data
presented here as mean and standard deviation in graphs and in tables.

RESULTS AND DISCUSSION


Estimation of physico-chemical parameters
The pH of cultivation media was variably influenced by the growth of monocultures and their synthetic
consortia, and an interesting pattern was observed. The pH values were found to be in the range between 8.25 to
8.87 for different monocultures and consortia. The initial pH value was found to be highest in the case of both
nitrate enriched BG11+ (9.020.00) and nitrate deprived BG11 (8.690.01) media, while it was lowest in
natural CE (7.930.58) water. The pH invariably increased in CE with inoculation of monocultures and
consortia, and highest change was found in the case of consortia. The pH value was considerably decreased in
BG11+ with growth of Anabaena sp. while in BG11+ with Nannochloropsis sp. Significant changes in pH were
recorded with growth of Anabaena sp. in BG11+ (P < 0.001), CE (P < 0.001) and BG11 (P > 0.05, ns), and
with Chlorella sp. in BG11+ (P > 0.05, ns), BG11 (P < 0.01) and CE (P < 0.001). In contrast, Oscillatoria sp. in
BG11+ did not affect the pH (P > 0.05, ns) while it significantly affected in BG11 (P < 0.001) and CE (P <
0.001). On other hand, Nannochloropsis sp. affected the pH of CE (P < 0.001), Scenedesmus sp. significantly
altered the pH in BG11+ (P < 0.05), BG11 (P < 0.01) and CE (P < 0.001). Growth of Phormidium sp. also
affected the pH significantly in BG11+, BG11 (P < 0.01) and CE (P < 0.001). Consortia significantly altered
the pH in BG11+ (P < 0.05), BG11 (P < 0.01) and CE (P < 0.001) when compared with respective controls. The
means of pH and their standard deviation are given in figure 1.

Figure 1. pH of media and carpet effluent of blank (Initial) after growth of monoculture and consortia.
The monocultures have some distinct inherent traits that determine their metabolic features and
morphological appearance. However, variations in environmental conditions could highly influence their
physiology and morphology (Smith & Schindler 2009). A little variation in the environment can affect their
metabolite profile and biomass (Piersma & Drent 2003), and pH is one of the major factors that affect the
physiology including growth rates, primary and secondary metabolite production. Most of the algae can make
their own food through the process of photosynthesis which involves a number of enzymes that work optimally
at a particular pH. When we cultured these monocultures and consortia, pH of different growth media fluctuated
in a wide range. Any variation in the pH can affect the function or may denature those important enzymes which
are involved in the photosynthesis. Alternatively, it affects the overall biomass yields. Since CO2 is a major
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byproduct of respiration, microalgae utilizes CO2 and water during photosynthesis to produce the photosyntax,
carbonic acid that alternatively decreases the pH (Wurts & Durborow 1992). Therefore, pH values of the media
and CE water oscillated between 8.25 and 8.87 for optimal CO2 sequestration. As more CO2 is absorbed into the
solution, the acidity of the solution gradually increases. At the higher acidity point no more CO 2 can be
absorbed. The change in pH is responsible for the increased synthesis of biomass by carbon fixation reactions.
The pH of CE media was increased from 7.93 to 8.69 for all the organisms and consortia which indicate that it is
the range of pH at which these organisms could grow well in natural water reservoirs (Yeh et al. 2005).
Initial dissolve oxygen recorded in BG11+ was 6.790.02 mg.L1, in BG11 media 6.830.02 and in CE
water 2.640.02 mg.L1. In all the experiments Anabaena sp. and Chlorella sp. significantly increased DO
levels in media and effluent water at a range of 9.700.05 to 10.540.16 mg.L1. Monoculture namely Anabaena
sp., Chlorella sp., Oscillatoria sp., Scenedesmus sp., Phormidium sp. and consortia significantly optimized (P <
0.001) the DO levels of media and CE water. Nannochloropsis sp. could not improve the DO content of BG11+
and BG11 (P > 0.05, ns) while improved significantly in CE water (P < 0.001). Mean values and standard
deviation of DO of cell-free supernatant for different culture media are shown in figure 2. In general, if
consumption of DO is more than the production its level declines.

Figure 2. Dissolve oxygen of media and carpet effluent of blank (Initial) after growth of monoculture and consortia.
The microbial growth is directly related to the available amounts of nutrient and DO in water. Here, we
found that the DO level of un-inoculated CE media was lowest when compared to BG11 and BG11+ media.
However, after inoculation, DO level significantly increase up to 5 folds. Microalgae growth significantly
increased the DO levels by releasing the oxygen that added to the media of their growth. In the case of
Anabaena sp. and Chlorella sp. highest DO levels were observed than other microalgae in all the three media
with its maximum values in CE. Therefore, inoculation of Anabaena sp. and Chlorella sp. in CE water could
significantly enhance DO levels. In contrast, the lowest increase in DO levels was observed in all the media with
inoculation of algae consortia.
DO levels varies with temperature and altitude, cold water holds more oxygen than warm water but at the
higher altitude water holds less oxygen. DO concentrations highly depend on the photosynthetic rates of
microalgae and levels of nutrients in the water. In general, high temperature and salinity reduce oxygen levels.
The maximum amount of DO (7.95 mg.L1) occurs at 27 C (Manasrah et al. 2006). In our study at the end of
experiment DO was found to be in the range between 6.1410.54 mg.L1. However, published literature of
microalgae consortia showed the range of DO between 6.26.4 mg.L1 (Badran 2001, Renuka et al. 2013).
Initial conductivity recorded in BG11+, BG11 media and CE water were 2440.010.0, 351.67.6 and
934.08.5 respectively. Conductivity increased in all set of experiments expect for Anabaena sp. and Chlorella
sp.in CE water. In BG11+ and BG11 media, inoculation of all the monocultures and consortia significantly
increased the water conductivity. Oscillatoria sp. significantly enhanced the water conductivity in all the media.
Anabaena sp. significantly increased in BG11+ (P < 0.001) and CE water (P < 0.01) whilst in BG11 (P > 0.05,

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ns) it did not affected significantly. On other hand, significant changes in conductivity were recorded with
inoculation of Chlorella sp. (BG11+, P < 0.01), Oscillatoria sp. (BG11+ and CE, P < 0.001), Nannochloropsis
sp. (BG11+, P < 0.001), Scenedesmus sp. (BG11, P < 0.05), Phormidium sp. (BG11+, P < 0.001) respectively.
In contrary, the consortia could change the conductivity only in BG11+ (P < 0.001). The mean values of
conductivity along with their standard deviation are presented in figure 3.

Figure 3. Conductivity of media and carpet effluent of blank (Initial) after growth of monoculture and consortia.
Since water conductivity is used to measure the ability of water to carry the electricity, pure water is
considered as a poor conductor. It is the main reason that water shows significant conductivity in the presence of
dissolved salts. In general, conductivity is directly proportional to the amount of dissolved salts in the water
(Iyasele & Idiata 2015), and microalgae growth increases the water conductivity (Potapova & Charles 2003).
Microalgae growth adds a number of minerals such as iron, nitrogen and phosphorus, which significantly
involve in enhancing the water conductivity (Townsend et al. 2012). In addition, the increased water
conductivity and the algae density have a direct relationship (Potapova & Charles 2003, Anonymous 2012).
Biomass Estimation
Biomass production was found to be highest in the case of Chlorella sp. (2.2170.126 g.L1) in BG11+
media, followed by Anabaena sp. (1.8970.045 g.L1) and consortia (1.2330.029 g.L1) in BG11 media;
Phormidium sp. (1.1030.057 g.L1), Scenedesmus sp. (1.0200.026 g.L1) and consortia (0.9230.025 g.L1) in
BG11+ media. Biomasses produced by different cultures are shown in figure 4.

Figure 4. Biomass of media and carpet effluent of monoculture and consortia.


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A number of published articles are available dealing with how the different species of Chlorella sp. behaves
in the nitrogen enriched environment, and utilised for the large-scale biomass production. In general, various
species of Chlorella sp. needs nitrogen and nutrient enriched water for their healthy growth. Therefore, it grew
well in nitrogen enriched media and able to produce higher biomass in BG11+ media (Min et al. 2011). The
algae consortia produced second highest biomass in nitrate deficient BG11 media after Anabaena sp. It clarifies
that under nitrogen depleted conditions and in the presence of phosphate Anabaena sp. can efficiently fix the
nitrogen to produce biomass (Buikema & Haselkor 1991). However, when Anabaena sp. was grown in consortia
the nitrogen fixed by it got used up by other non-nitrogen fixing organisms for their healthy growth. Possibly,
some of these organisms inhibited the growth of Anabaena sp. by producing the biochemicals that have
allelopathic interactions (Hu & Hong 2008). Anabaena sp., it may be responsible for inhibiting the growth of
other organisms in the consortia. Therefore, biomass yield of consortia was found to be less than Anabaena sp.
In the CE maximum biomass yield was recorded with Phormidium sp. (0.480.010 g.L1) followed by algae
consortia (0.4600.010 g.L1). As Phormidium sp. has a strong capability to grow in nutrient enriched or
polluted water (De-Bashan & Bashan 2010), and carpet effluent is a natural source of a list of hazardous heavy
metals, therefore, due to its inherent traits to utilise all these things as supplements for healthy growth could
ultimately help it to produce the highest biomass in CE water.
Nitrate, Nitrite and Phosphate estimation
The initial value of nitrate recorded in BG11+ was 253.351.75 in BG11 7.400.05 media and in CE
1.080.10 mg.L1. From the growth of synthetic consortia in BG11+ media nitrate content was enhanced by
295.27% (748.08 mg.L1) while its cultivation in nitrate deficient BG11 media resulted in a decrease of 68.91%
(5.1 mg.L1) due to biosorption of nitrate from media (Table 1). However, in CE media 123.14% (1.33 mg.L1)
increase in the nitrate content was obtained. Overall in nitrogen rich BG11+ media, algae consortia synthesized
the nitrate and efficiently participated in the bio-addition process. In BG11+ media Nannochloropsis sp.,
Phormidium sp. and consortia synthesized nitrate while other algae used the nitrate from the media and
significantly contributed in biosorption process. Cultivation of nitrogen fixing microalgae Anabaena sp. in
BG11 media significantly enhanced the nitrate content by 1258.10% (93.1 mg.L1). In contrast, almost all the
microalgae species and consortia depleted the nitrate to the surrounding habitat in the CE water that resulted in
the valuable quantity of biomass production.
Table 1. Nitrate concentration of initial, monoculture and consortia.
Algae BG11+ BG11 Carpet effluent
(Nitrate mg.L1) (Nitrate mg.L1) (Nitrate mg.L1)
Initial (Blank) 253.351.75 7.400.05 1.080.10
Anabaena sp. 113.230.87 100.501.25 2.130.15
Chlorella sp. 85.450.56 3.580.35 3.180.07
Oscillatoria sp. 152.532.10 1.30.05 44.261.04
Nannochloropsis sp. 271.801.32 1.940.04 2.310.07
Scenedesmus sp. 191.966.04 3.750.15 2.480.07
Phormidium sp. 341.433.46 1.900.05 2.310.07
Consortia 1001.196.53 2.300.15 2.410.07

Table 2. Nitrites concentration of initial, monoculture and consortia.


Algae BG11+ BG11 Carpet effluent
(Nitrate mg.L1) (Nitrate mg.L1) (Nitrate mg.L1)
Initial (Blank) 0.00.000 0.000.000 0.0110.002
Anabaena sp. 0.3970.012 0.1540.005 0.0260.004
Chlorella sp. 1.4270.008 0.0220.004 0.0220.003
Oscillatoria sp. 4.7320.023 0.0250.005 0.2080.006
Nannochloropsis sp. 12.8070.169 0.0250.004 0.0170.004
Scenedesmus sp. 18.4200.270 0.1190.003 0.0390.004
Phormidium sp. 13.7100.135 0.0360.005 0.0180.003
Consortia 35.5200.665 0.0380.004 0.0390.004

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In comparison to nitrate the concentration of nitrites was increased in all the experiment. With inoculation of
algae consortia the maximum increase in the concentration of nitrite was recorded in the BG11+ media, followed
by BG11 and CE respectively (Table 2). Microalgae consortia and monocultures showed varying performance
towards bio-addition activity in different cultivation media. Maximum activity was recorded by consortia in the
case of BG11+ (35.52 mg.L1 from zero value) followed by BG11 (0.038 mg.L1 from zero value) and CE 254%
(0.028 mg.L1). Therefore, cultivation of these photosynthetic organisms significantly added the nitrate and
nitrite in the nitrate enriched BG11+ media.

Figure 5. Phosphate of media and carpet effluent of blank (Initial) after growth of monoculture and consortia.
Initial phosphate recorded in BG11+ was 4.070.02 mg.L1, in BG11 media 3.810.00 mg.L1 and CE
1.790.00 mg.L1. In BG11+ media Anabaena sp. and Nannochloropsis sp. added 27.10% (1.10 mg.L1) and
22.28% (0.90 mg.L1) phosphate respectively whilst other algae and consortia were actively involved in its
biosorption in figure 5. Bio-sorption in BG11+ and BG11 by algae consortia was 35.35% (1.31 mg.L1),
95.72% (3.64 mg.L1) and in CE water 94.97% (1.7 mg.L1) respectively. All monocultures and consortia
significantly affected the phosphate absorptions (P < 0.001) except Chlorella sp. in BG11+ (P > 0.05, ns) media.
The ratio of nitrogen to phosphorus available for the growth of algae is an important parameter that
determines the overall growth. The Redfield ratio of 106C:16N:1P is commonly used to indicate the required
ratio of carbon, nitrogen and phosphorus (Redfield 1958). Nitrogen is the second limiting factor in plant growth
taken up by cells in the form of ammonia, nitrate and nitrite (Evans 2001). Phosphorus is the third most
important macronutrient that is absorbed by plant cells in the form of phosphate. It can bind to other ions and
precipitate and enhance its limiting concentrations for optimal growth (Xin et al. 2010). With enhanced nitrogen
levels bio-sorption of phosphorus decreases (Grobbelaar 2004). Hence, uptake of phosphate depends on the
availability of nitrate. In BG11+ media supplementation of sodium nitrate decreased the phosphate absorption.
In BG11 media higher the phosphate absorption in the relation of BG11+ was recorded. The higher
concentrations of nitrate in media provoke the fixation of nitrogen by heterocystous filamentous and unicellular
morphological types of algae (Philips et al. 1992). However, higher nitrate content reduces the biosorption of
nitrate. In the presence of higher concentrations of nitrate, some algae synthesize nitrate (Manhart & Wong
1980, Philips et al. 1992). Nitrogen is mainly involved in the synthesis of proteins; therefore, low nitrogen
supply limits the synthesis of required amounts of the proteins (Geider & Roche 2002, Loladze & Elser 2011)
that alternatively results in a low number of ribosomes and the total amount of ribosomal RNA. Since most of
the phosphate in the cell is stored in the ribosomal RNA, reduced numbers of the ribosomes are due to the
lowering of cellular phosphate levels (Sterner & Elser 2002, Beuckels et al. 2015).
In microalgae consortia high nitrate content could not influence the higher biomass synthesis. However, low
nitrate supply enhanced the higher biomass synthesis due to the association of nitrogen fixating as well as non-
nitrogen fixating algae species. In general, at the low nitrogen supply higher biomass yields are reported
(Arumugam et al. 2013). On other hand, higher content of nitrate in media reduces the biosorption of phosphate
by microalgae. However, in CE algae growth released very little nitrate in wastewater. Therefore, with limited
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or lower supply of nitrates the rate of nitrate and phosphate bio-absorption was found to be higher (Ma et al.
2012, Zhang & Hong 2014). High nitrate concentration induced the nitrate reductase activity to form nitrite
which alternatively converted to the ammonium ion by the action of another enzyme nitrite reductase. Nitrate
reductase is inhibited by nitrite and ammonium ions, regulators of the nir operon (Jha et al. 2007). In non-
nitrogen fixing cyanobacteria, nitrate, nitrites and ammonium ions chiefly regulate the nir operon by fine-tuning
the activity of nitrate reductase (NR) enzyme. Nitrate is utilized as the chief source of inorganic nitrogen for the
majority of plants (Li et al. 2010) and is converted to ammonium (Stitt et al. 2002). Nitrate reduces to ammonia
in two consequent steps, first, the reduction of nitrate to nitrite which is catalyzed by NADH2-nitrate reductase
enzyme and secondly, the reduction of nitrite to ammonia that is catalyzed by ferredoxin-nitrite reductase. The
presence of ammonia suppresses the uptake of nitrate by inactivating the nitrate reductase system by ammonia
(Losada et al. 1970, Herrera et al. 1972, Serra et al. 1978). As a result, nitrate was synthesized with
supplementation of high nitrate levels (Hansell et al. 2004). Some algae synthesize several types of phosphate
enzymes such as alkaline phosphates, acid phosphates and protein phosphates. Phosphatase is an enzyme that
removes a single phosphate group from its substrate through hydrolysis of phosphoric monoesters into a
phosphate (Babi et al. 2013). Therefore, uptake of phosphate is dependent upon the supply of nitrate and with
low supply of nitrate biosorption of phosphate increases. Similarly, with high nitrate, biosorption of phosphate
from waste water was decreased (Beuckels et al. 2015) with inoculation of these organisms.

CONCLUSION
Microalgae monocultures and synthetic consortia are capable biosorption of phosphate in the media and CE
water, and played distinct roles in the ecosystem by altering the DO, pH and conductivity. It was observed in the
present experiment where a higher amount of phosphate was removed from BG11 and CE water in comparison
to nitrate enriched BG11+ media. Microalgae consortia added nitrogen to the media which was already
supplemented with considerable amounts of nitrogen i.e. BG11+ media. Therefore, presence of nitrogen
enhances the bio-addition of nitrogen to media. To optimize the services of these monocultures in their natural
habitats, there is a need to screen the suitable candidate algae species that might be helpful in mitigating the
nutrient levels, and that can produce affordable amounts of biomass. Consequently, by applying the modern
principles of the synthetic ecology suitable consortia of selected algae species can be developed that could be a
sustainable way of a better method for waste water treatment and biomass production at industrial scale.
Synthetic consortia of BG11 could be helpful in getting the valuable amounts of biomass by adjusting the DO,
pH, conductivity and biosorption of nitrate and phosphate. Carpet effluent is a rich source of nutrients, therefore,
it can be targeted as media for such algae species which could improve the water quality and simultaneously can
produce the biomass for various algae based value added bio-processes.

ACKNOWLEDGEMENTS
This research was financially supported by UGC, New Delhi under UGC-SRF fellowship. We are thankful
to Director, CSIR-NBRI, Lucknow for his constant support, encouragement and necessary laboratory facilities
during the course of this research work. Dr. Kiran Toppo, Technical Officer and member of algae laboratory are
thanked for her cooperation during the study.

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ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(2): 203209, 2017
DOI: 10.22271/tpr.2017.v4.i2.029
Research article

Integration of mulching and row spacing on weed management in


Green Pepper varieties
Edgar N. Ocharo1, 2*, Nicholas K. Korir1 and Joseph Gweyi-Onyango1
1
Department of Agricultural Science and Technology, Kenyatta University, P.O. Box 43844-00100,
Nairobi, Kenya
2
KEPHIS-Kenya Plant Health Inspectorate Services, P.O. Box 49592-00100, Nairobi, Kenya
*Corresponding Author: ocharoedgar@yahoo.com [Accepted: 23 May 2017]

Abstract: A field experiment was conducted at the Kenya Agricultural and Livestock Research
Organization, Alupe Research Farm, Busia County for two cropping seasons during the year 2015
to determine the impact of row spacing and mulching materials on weed management of two green
pepper varieties. The two varieties California Wonder and Yolo Wonder were sown in a
randomized complete block design in factorial arrangements, comprising three-row spacing
treatments and four mulching materials then replicated thrice. The row spacing were; 30, 40 and
50 cm whereas the mulching materials were; black plastic mulch, transparent plastic mulch, straw
mulch and bare soil which was the control. Data collected was subjected to SAS statistical
software for analysis and means separated using LSD at P0.05. Significant differences (p0.05)
were observed between the treatment combination of mulch and row spacing in the fresh and dry
weed biomass, weed species density per m2 and the weed growth vigor. The highest fresh weed
biomass (5008 g.m-2) was elicited by the control mulch treatment at the widest row spacing during
the long rains season while the lowest (188 g.m-2) was observed in the black plastic mulch at the
narrow row spacing of 30 cm. The weed species density was highest (7) in the control at 40 cm
row spacing with the lowest elicited in the black plastic mulch at the 40 cm row spacing (1.7)
during the short rain seasons. The weed growth vigor was highest in all the control mulch
treatments at 30 cm row spacing while the lowest vigor was in the black plastic mulch which was
closely followed by the transparent mulch at the various row spacings. The competitiveness of
sweet pepper with weeds can be significantly enhanced by using black plastic as mulch under
narrow row spacing with an optimum of 30 cm.
Keywords: Row spacing - Green pepper - Mulch.

[Cite as: Ocharo EN, Korir NK & Gweyi-Onyango J (2017) Integration of mulching and row spacing on weed
management in Green Pepper varieties. Tropical Plant Research 4(2): 203209]

INTRODUCTION
Green pepper (Capsicum annum L.), which belongs to the Solanaceae family, is one of the most varied and
widely used foods in the world; it originated from Mexico and Central America regions and Christopher
Columbus encountered it in 1493 (Kelley & Boyhan 2009). Its fruits are harvested and consumed at different
maturity stages; green, red and not fully ripe. A phenolic compound called capsaicin is responsible for the
pungency in peppers. Green pepper is grown as an annual crop due to its sensitivity to frost and it is actually a
herbaceous perennial that can survive and yield for several years in a tropical climate (Leja et al. 2008). It is a
high-value crop and its fruits are highly nutritious, rich in vitamins particularly pro-vitamin-A, vitamin-B,
vitamin-C and minerals such as Ca, P, K and Fe. However, weed infestation is a major bottleneck to higher
green pepper production. Weeds are omnipresent pests that compete with crops for water, nutrients, space, and
light; host pests and diseases; and release allele-chemicals into the rhizosphere (Khaliq et al. 2013a,). The
magnitude of weed-related losses, however, depends on the type and density of a particular weed species, its
time of emergence, and the duration of interference (Estorninos et al. 2005). Yield losses are most severe when

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Received: 19 February 2017 Published online: 31 May 2017
https://doi.org/10.22271/tpr.2017.v4.i2.029
Ocharo et al. (2017) 4(2): 203209

resources are limited and weeds and crops emerge simultaneously (Zimdahl 2007).
There is a significant reduction of yields due to weeds which compete for light, space, water and nutrients
that weaken the cultivated crops, while in other occurrences some weeds serve as alternate hosts for other pests,
like, insects, diseases, viruses and or nematodes (Shah et al. 2013). Weeds reduce fruit yield by 70%, depending
on stage and duration of competition (Marana et al. 1986). The first four weeks are critical in many vegetable
crops, therefore during this period weeds should be removed. Although herbicides can be effective in controlling
weeds, they are also expensive and often beyond the budget of farmers in Kenya. In addition, herbicide use
requires special equipment and expertise to ensure proper herbicide rates use and proper human health and
safety precautions (Saleem et al. 2013). Increasing use of herbicides to manage agricultural weeds is a primary
concern today in most developing countries (Khaliq et al. 2013b). The use of herbicides indiscrimately for the
last few decades has caused ecological and environmental problems, such as weed resistance, weed population
shifts, and dominance of minor weeds and therefore the widespread application should be minimized (Chauhan
& Johnson 2010, Chauhan et al. 2012). To decrease dependence on herbicides and establish more weed control
methods, integrated weed management (IWM) programs have received increasing attention (Chauhan &
Johnson 2010, Chauhan et al. 2012, Khaliq et al. 2013c). Therefore, all those approaches which can prevent
weed germination, suppress weed growth, and enhance crops competiveness are integrated to control weeds
(Rasmussen 2004). Mulching is a recent and important non-chemical weed control method used in high-value
vegetable crops. Reducing crop row spacing and delaying time of weed emergence may also provide the crops
with a competitive edge over weeds.
In modern agriculture, field crops are planted in distinct rows with various row spacings and plant densities
(Chen et al. 2008). In a study by Kaur et al. (2002) have shown that even seed distribution increases crop
establishment and per unit area and thus resulting in higher competitiveness against weeds. In another
experiment, significant effects of both crop density and spatial distribution on weed growth were observed
(Weiner et al. 2001). They further reported that high crop density and grid sowing pattern recorded 60% less
weed biomass and produced 60% higher yield as compared to lower crop densities and normal sowing in rows.
In addition, sowing crops at wider row spacing resulted in increased competition within the crop plant clusters
(i.e. the rows), resulting in decreased crop growth and yield compared to narrow row spacing (Shapiro &
Wortmann 2006).
Considering the importance of green pepper, the costs of weeds in terms of yield reduction, expenditure on
their control, and the different options available for weed control, farmers in Kenya need more information
about the effectiveness and economics of various methods for managing weeds. Therefore, the present study
was carried out to investigate the influence of using different mulching materials in varying row spacing for
controlling weeds in green pepper in the County of Busia, Western region of Kenya.

MATERIALS AND METHODS


Study site
The study was conducted during the two cropping seasons (long and short rains) of 2015 at the Kenya
Agricultural and Livestock Research Organization (KALRO), Alupe Crops Station in Busia County in western
Kenya region. It lies within latitude 030'0" N, longitude 3407'50" E with an elevation of 1157 m above sea
level. The average annual relative humidity for the period from March 2015 to March 2016 ranged between
73.6% and 78.9%. Average annual rainfall (mm) at the study area ranged from 49.6 to 215.8 mm with average
annual maximum and minimum air temperatures ranging from 29.1 to 35.9 and 16.9 to 18.3 C respectively.
Experimental design
The two varieties California Wonder and Yolo Wonder were sown in a randomized complete block design in
factorial arrangements. The row spacing treatments comprised of 30, 40 and 50 cm with a constant intra-row
spacing of 40 cm while the four mulching materials were black plastic mulch (0.25 m thick), transparent
plastic mulch (0.25 m thick), straw mulch (finger millet) and bare soil which was the control. The row spacing
and mulch treatment combinations were then replicated three times.
Cultural practices
Ploughing and harrowing were performed on the land before nursery beds were made. The seedlings of the
green pepper were transplanted when they were 30 days old on the 23 rd March, 2015 for the long rain season
and 26th September, 2015 for the short rain season to the experimental field. Uniform seedlings with between 3
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to 5 leaves were transplanted to the experimental plots measuring 3.0 m 2.0 m. The transplanted seedlings
were irrigated right after transplanting. They were also watered depending on the available soil moisture
content. All other agronomic practices, except treatments were kept constant.
Data management and analysis
Data were recorded on weed species density (m-2), fresh and dry weed biomass, and the weed growth vigor
on a scale of 1 to 3 where 1 was most vigorous and 3 least vigorous. All the parameters were subjected to
analysis technique using SAS computer software (Version 9). When significant differences were obtained
(P0.05), means were separated with Fishers LSD test.

RESULT
Weed species Density
The number of weed species per meter squared was significantly different among the treatment combinations
for both seasons. The highest number of weed species during the long rains season was observed in the control
mulch at a row spacing of 40 cm with an average of 7 weed species per square meter. At the same row spacing,
the straw mulch showed the lowest number of weeds with an average of 4.7 during the long rain season under
the California Wonder variety. During the short rain season, the highest weed species density was elicited by the
control mulch at the widest row spacing under both varieties. Yolo Wonder variety in the black plastic mulch at
40 cm row spacing elicited the lowest number of weed species with only an average of 1.7 per meter squared
(Table 1).
Table 1. The influence of mulching material and row spacing on the number of weed species per square metre during the
short rain season of September December 2015 and long rain season of March August 2015 in Busia, Kenya.
Long Rain Season Short Rain Season
Variety Mulch
3040cm 4040cm 5040cm 3040cm 4040cm 5040cm
Black Plastic 5.3b 5.3b 6.0a 3.0d 3.0d 3.3d
California

Control 6.3a 7.0a 6.0a 5.0b 5.0b 6.3a


Wonder

Straw Mulch 6.3a 4.7b 6.7a 4.3c 5.7b 5.7b


Transparent Plastic 6.7a 6.0a 4.7c 3.7c 4.3c 3.3c
Black Plastic 5.0b 5.3b 6.3a 3.0d 1.7e 4.0c
Wonder

Control 6.3a 6.7a 5.7b 5.7b 4.7c 6.3a


Straw Mulch 6.0a 5.7b 5.7b 5.7b 5.7b 4.3c
Yolo

Transparent Plastic 6.3a 5.7b 5.3b 2.7d 2.7d 3.7c


LSD (0.05) 0.759 0.812
Note: Different letters within each column refer to significant differences among the means.

Table 2. The influence of mulching material and row spacing on the weed growth vigor during the short rain season of
September December 2015 and long rain season of March August 2015 in Busia, Kenya.
Long Rain Season Short Rain Season
Variety Mulch
3040cm 4040cm 5040cm 3040cm 4040cm 5040cm
Black Plastic 3.0a 3.0a 3.0a 2.7a 2.7a 2.7a
California

Control 1.7c 1.0d 1.7c 1.3d 1.7c 1.3d


Wonder

Straw Mulch 2.0c 2.3b 1.7c 2.0b 1.7c 2.0b


Transparent Plastic 2.0c 2.3b 2.0c 2.0b 2.0b 2.3b
Black Plastic 2.7b 2.7b 3.0a 2.7a 2.3 3.0a
Wonder

Control 1.0d 1.7c 1.3d 1.0d 1.3d 2.0b


Straw Mulch 1.7c 2.0c 1.7c 1.7c 1.7c 1.7c
Yolo

Transparent Plastic 2.0c 2.0c 2.0c 2.0b 2.3b 2.0b


LSD (0.05) 0.22 0.18
Note: Different letters within each column refer to significant differences among the means.
Weed growth vigor
On a scale of 13, where 1 was the most vigorous and 3 the least vigorous, it was observed during the long
rains season that the black plastic mulch across all the row spacings showed the least weed growth vigor in both
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Ocharo et al. (2017) 4(2): 203209

varieties as shown in table 2 while the most vigorous growth vigor of weeds was observed in the control at the
row spacing of 40 cm under California Wonder and 30 cm under Yolo Wonder variety. During the short rain
season, the same trend as that of the long rain season was observed where the control mulch had the most
vigorous growth of weeds but at 30 cm and also at 50 cm row spacing treatments under California Wonder and
Yolo Wonder varieties respectively. The black plastic mulch had the lowest growth vigor for both varieties
while the transparent mulch also showed poor to intermediate growth vigor of the weeds (Table 2).
Fresh and dry weed biomass (g.m-2)

Figure 1. The interaction effect between mulching materials and row spacing on the fresh weed biomass in Busia, Kenya: A,
Long rain season of March August 2015; B, Short rain season of September December 2015.
There were significant differences between the treatment combinations of mulching materials and row
spacing on the fresh and dry weed biomass in both seasons. During the long rain season, the control mulch and
the widest row spacing (50 cm) had the highest fresh weed biomass (5008 g.m-2) while the black plastic mulch
had the lowest (188 g.m-2). During the short rain season, the highest fresh weed biomass was observed in the
control mulch at two-row spacing levels where at 30 cm the fresh weed biomass was 1083 g.m-2 which was not
significantly different from the 50 cm row spacing which had 1003 g.m-2; while the black plastic mulch had the
lowest (278 g.m-2) (Fig. 1). On the other hand, the highest dry weed biomass was recorded as 357 g.m-2 for the
control mulch with widest row spacing (50 cm) and lowest (66 g.m-2) for black plastic mulch at 30 cm row
spacing during the long rain season. During the short rain season, dry weed biomass was again recorded the
highest (419 g.m-2) for the control mulch & widest row spacing (50 cm) and the lowest (37 g.m-2) for transparect
plastic mulch at 40 cm row spacing (Fig. 2).

DISCUSSION
The control treatments had the highest weed density which may be attributed to the open soil surface and the
niches available provided a conducive environment for free and aggressive weed growth. These results are also
in accordance with those of Fathi et al. (2003), and Hassan & Ahmad (2005) who also found the highest number
of weeds m-2 in weedy check plots. Weed seedlings emerging at different times after crop emergence may have
differed in growth and productivity, depending on the conditions during the early development of the crop plants
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Ocharo et al. (2017) 4(2): 203209

(Lindstrom & Kokko 2000). Therefore, the black mulch probably discouraged the growth of weed as compared
to the bare soil while the narrow spacing enhanced the crops competitiveness with the weeds. Kaur et al. (2002)
have shown that even seed distribution increased crop establishment and dry matter production per unit area,
resulting in higher competitiveness against weeds. In another experiment, significant effects of both crop density
and spatial distribution on weed growth were observed (Weiner et al. 2001).

Figure 2. The interaction effect between mulching materials and row spacing on the dry weed biomass in Busia, Kenya: A,
Long rain season of March August 2015; B, Short rain season of September December 2015.
These results on the wider row spacing could be explained by the fact that wider green pepper rows lacked
an overlapping canopy early in growth, allowing substantial weed growth and consequent competition. It was
earlier suggested by Holt (1995) that by manipulating the crop row spacing and orientation in row cultivation,
there may be reduction in light interception by weeds. Crop shading via narrow row spacing can improve weed
control without extra costs or negative environmental impacts (Barberi 2002) whereby narrow rows provide a
competitive edge to the crop over weeds due to early and rapid canopy closure (Kristensen et al. 2008,
Mashingaidze et al. 2009, Chauhan & Johnson 2010, Khaliq et al. 2014). Significant differences (p0.05) were
recorded in the dry weed biomass for both seasons among the treatment combinations. In the long rain season,
the control mulch at 50 cm row spacing showed the highest dry weed biomass (357 g.m-2) while the lowest (67
g.m-2) being elicited by the black plastic mulch at 30 cm row spacing (Acciaresi & Chidichimo 2007, Ashraf et
al. 2014). Uscanga-Mortera et al. (2007) reported that crops benefit from narrow row spacing and higher
cropping densities in the weed-crop competition. Narrow row spacing inhibits weed germination and growth by
reducing the incoming radiant energy and altering the spectral composition of the weed seeds and seedlings.
During the short rain season, the control mulch had the highest dry weed biomass throughout the different row
spacing levels peaking at the widest with 419 g.m-2. The lowest dry weed biomass was recorded in the
transparent mulch at the narrow row spacing of 30 cm with only 41 g.m-2. The availability of resources (water,
nutrients, and space) and environmental factors like light and temperature affect the extent of the competition in
weed-crop settings (Guillemin et al. 2013). Studies with a number of crops like wheat (Kristensen et al. 2008,
Mashingaidze et al. 2009), rice (Chauhan & Johnson 2010), barley (Kolb et al. 2010), cotton (Reddy 2001),
millet (Shinggu et al. 2009), sorghum (Grichar et al. 2004), and soybean (Hock et al. 2006) found inverse
relationships between narrow crop rows and weed growth.
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Ocharo et al. (2017) 4(2): 203209

CONCLUSION
The interaction effect of mulching materials and row spacings was significant in all the weed control
parameters under study for both seasons. The black plastic mulch was the most effective at the narrow spacing
of 30 cm in controlling the weeds possibly by reducing their competitiveness. Therefore, this study highly
recommends the combination between black plastic mulch and a row spacing of 30 cm.

ACKNOWLEDGEMENTS
All the Authors are thankful to the Head of the Department, Agricultural Science and Technology, Kenyatta
University, Nairobi, Kenya and Director, KEPHIS-Kenya Plant Health Inspectorate Services, Nairobi, Kenya
for providing necessary laboratory facilities during the study.

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ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(2): 210224, 2017
DOI: 10.22271/tpr.2017.v4.i2.030
Research article

Nitrogen level affects growth and reactive oxygen scavenging of


fenugreek irrigated with wastewater
Saima Kausar1*, Shahla Faizan2 and Irfana Haneef2
1
Department of Botany, Jhunjhunwala Degree College, Faizabad, Uttar Pradesh, India
2
Environmental Physiology Laboratory, Department of Botany, Aligarh Muslim University, Aligarh, India
*Corresponding Author: kausar.saima25@gmail.com [Accepted: 23 May 2017]

Abstract: Today, due to the constraint in availability of the freshwater for irrigation, wastewater is
being used for irrigation of agriculture fields. Wastewater contain plant nutrients that favour crop
growth but leave a burden of heavy metals which can enter the food chain and is a cause of great
concern. This research work aimed at evaluating the potential utilization of wastewater as an
alternative source of water and nitrogen (N) for fenugreek. For this purpose, a pot experiment was
conducted with wastewater and four doses of nitrogen (0, 20, 40 and 60 kg.ha-1) to observe a
comparative effect on growth and biochemical characteristics using fenugreek as the test crop.
Wastewater and soil used in the experiment were analyzed for their physicochemical properties.
Results indicate that wastewater was richer in essential plant nutrients but contained some heavy
metals in amounts well below the permissible limits for its use as irrigation water. Use of
wastewater with 20 kg.N.ha-1 improved growth, proline and antioxidant enzymes of the plant as
compared to application of groundwater with no fertilizer. Lipid peroxidation increased with
wastewater but decreased with the increasing nitrogen doses, so the best combination (WWN20)
has negative impact on this parameter. These results concluded that wastewater can efficiently
substitute groundwater and also works as a nutrient source whereas application of nitrogen could
provide protection against the oxidative stress by increasing the antioxidant protective system.
Keywords: Trigonella foenum-graecum - Proline - Lipid peroxidation - Catalase - Peroxidase -
Superoxide dismutase.

[Cite as: Kausar S, Faizan S & Haneef I (2017) Nitrogen level affects growth and reactive oxygen scavenging of
fenugreek irrigated with wastewater. Tropical Plant Research 4(2): 210224]

INTRODUCTION
In many parts of the world freshwater resources is scarce, and there is with gradual destruction and increased
pollution of fresh water resources, requiring alternatives to supply water for agriculture need to be considered
(Yoshida & Ishii 1993). One solution to insufficient water supplies is water reuse. Sewage water for irrigation is
a way to dispose of sewage; provide the reliability of large volumes of water for irrigation; to reduce discharge
into potable water bodies; serve as an alternative source to chemical fertilizers, provide organic matter; improve
soil infiltration capacity, permeability, cation and anion exchange capacities, water holding capacity and texture
(Schneider & Erickson 1972, FAO 1992, Avemelech 1993, Brady & Weil 1999, Van der Hoek et al. 2002).
However, there is a concern about contamination and bioaccumulation in plants of potentially toxic elements
such as chromium (Cr), lead (Pb), nickel (Ni), cadmium (Cd), copper (Cu) and zinc (Zn) from domestic and
industrial sources (Chen & Chia 2002, Kiziloglu et al. 2007).
In urban areas, especially in developing countries, raw wastewater for irrigation is used to grow vegetables
for nearby urban markets (Lucho-Constantino et al. 2005). If the wastewater is used after proper dilution for
irrigation hazards of heavy metals can be minimized (Pathak et al. 1999). The water used in the proposed
research work has already been diluted from the source (Tak et al. 2012, Akhtar et al. 2012, Iqbal et al. 2012,
2017, Chalkoo et al. 2014, Sahay et al. 2015) and the contents of all the heavy metals except Ni were well
within the permissible limits of Awasthi (2000) (Table 1). The presence of pathogenic microorganisms in
wastewater poses an additional health-hazard. In order to avoid this problem, side-on-soil-surface watering was

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Received: 05 February 2017 Published online: 31 May 2017
https://doi.org/10.22271/tpr.2017.v4.i2.030
Kausar et al. (2017) 4(2): 210224

used with watering can and practiced in pots to avoid direct contact with crop foliage. However, two weeks
before the harvest, this technique was replaced by direct watering on the foliage (Cisse 1997, Akponikpe et al.
2011). Wastewater provides variable nutrients and improves attributes of the plant compared to fresh water, but
the nutrient concentrations are not sufficient to fulfill requirements of the crop. External inputs need should be
guided by the efficiency of their use, as the use of fertilizers beyond limits, in addition to being uneconomical, is
potentially harmful to the environment by leaching through the soil beyond the root zone, eventually reaching
ground water, or through surface runoff be deposited into nearby water bodies and cause eutrophication.
Table 1. Physicochemical characteristics of ground water (GW) and 100% wastewater (100%WW). All
determinations in mg.l-1 or as specified.
Determinations GW WW FAO acceptable level
pH 7.30 8.00 6.58.4a
EC ( mhos cm-1) 700.00 1310.00 0.253.0a
TS 955.00 1550.00 2200c
TDS 560.00 1060.00 <2000a
TSS 400.00 576.00 -
BOD 16.25 105.55 <25b
COD 65.45 150.25 30-160b
NO3-N 0.83 2.75 <10.0a
NH4-N 0.11 4.45 5.0a
Na+ 20.56 59.35 -
K+ 4.94 20.44 <2.0a
Ca++ 15.45 52.35 <400a
Mg++ 27.35 118.72 <61a
Cl- 55.25 113.22 <350a
CO3-- 54.54 147.74 -
HCO3- 105.33 80.48 <610a
SO4-- 30.86 88.95 -
PO4--- 0.36 1.24 <2.0a
Cu - 0.209 0.20d
Cd - 0.009 0.01d
Ni - 0.331 0.20d
Zn - 0.153 2.0d
Cr - 0.039 0.10d
Pb - 0.028 5.0d
Note: aAyers and Westcot, (1994); bPescod, (1992); cIndian Standard Institution (1974); dAwasthi, (2000).
In India, vegetable consumption is many times more than the European countries due to the vegetarian food
habit of a large proportion of the population, and the lower cost of the common vegetables compared to meat,
fish, egg and milk products. It is also important that, compared to other crops, many vegetables can be grown
throughout the year and be marketed regularly, due to a short life-cycle.
Fenugreek (Trigonella foenum-graecum L.) is used fresh or dried, as spice (seeds) and vegetables (fresh
leaves, sprouts and microgreens). Fenugreek leaves contain calcium, phosphorus and iron. Seeds are bitter in
taste and have been known over 2500 years for their medicinal qualities (chronic dysentery, diarrhoea, chronic
cough, enlargement of liver and spleen).The seeds contain the steroidal substance diosgenin which is used as a
starting material in the synthesis of sex hormone as oral contraceptive. The study examined application of urban
wastewater for irrigation and its effect on Fenugreek supplemented with nitrogen.

MATERIALS AND METHODS


Wastewater (WW) was collected in 50 L Jerry cans for watering the pots, as and when required, from the
drain running along the Aligarh Mathura road, 6 km away from the Department. The water analyzed at the start
and end of each experiment. Tap water was used as the control (GW) pumped directly from under the earths
surface and stored in syntax water tanks. The water types were analyzed for various physicochemical
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characteristics (Table 1) following standard procedures (APHA 1998). Determination of heavy metals in the
wastewater samples was with an Atomic Absorption Spectrophotometer (SENSAA GBC Avanta var. 2.02,
Aligarh, India) according to the method of Ademoroti (1996). Microbiological examination of wastewater was
conducted in the Microbiology Laboratory, Department of Agricultural Microbiology, Aligarh Muslim
University, Aligarh, following quality guidelines of WHO (1989) and FAO (1992).
Before sowing, samples of the sandy loam soil were randomly accumulated from each pot, air-dried, mixed
well, ground in a mortar and pestle, and passed through a 2 mm sieve before analysis. Soil pH and electrical
conductivity were determined following the procedure of Jackson (1958). Organic matter was measured using
the method described by Walkley & Black (1934). Estimation of carbonates and bicarbonates was followed
Richards (1954). Nitrate nitrogen was estimated according to Ghosh et al. (1983). Phosphorus was measured
following Olsen et al. (1954); potassium was estimated following Jackson (1958). Soil cation exchange capacity
was determined following Ganguly (1951). Soil heavy metals were determined following Lindsey & Norwell
(1978).
The experiment was conducted in the November 2010 to March 2011. The experiment was a completely
randomized design replicated 3 times with main treatment of 100% urban wastewater (WW) and groundwater
(GW). Doses of NPK fertilizer (N0P0K0, N20P30K30, N40P30K30 and N60P30K30) were mixed into the soil with the
fertilizers from urea, single super phosphate and muriate of potash.
Effects of treatment were determined by measurement of plant: length, fresh and dry weights, numbers of
leaves, leaf area, leaf fresh and dry weights, proline content, malondialdehyde content and enzymatic
antioxidants (catalase, peroxidase and superoxide dismutase) at vegetative, flowering and fruiting stages at 30,
60 and 90 DAS, respectively. Plants were uprooted and lengths of the main tap root and shoot were measured.
To assess dry mass, plants were dried for about 72 hrs in a hot air oven (forced air oven) maintained at 80C and
then weighed. After separating all the leaves at the petiole and stem junction, leaf area was measured using a
leaf area meter (LA 211, Systronics, India). Proline content in fresh leaves was estimated following the
procedure of Bates et al. (1973). The level of lipid peroxidation products in leaves was determined as
malondialdehyde content by a modified version of the method described by Cakmak & Horst (1991). Catalase
activity (EC 1.11.1.6) was measured according to Aebi (1984). SOD activity (EC 1.15.1.1) was estimated by
recording the decrease in absorbance of superoxide nitroblue tetrazolium complex by the enzyme (Sen Gupta et
al. 1993). The method of Bergmeyer et al. (1974) was used to measure oxidation of pyrogallol to purpurogallin
by peroxidase.
The data were analyzed according to Panse & Sukhatme (1985) using a two-way analysis of variance after
Steel & Torrie (1962) SPSS (ver. 11.0, Chicago, IL). If interactions were significant they were used to explain
results using Duncan Multiple Range Test (Duncan 1955).

RESULTS
The physicochemical and microbiological properties of water types varied (Table 1). Electrical conductivity,
total solids, total dissolved solids, BOD, COD, nitrate nitrogen, ammonia nitrogen, sodium, calcium,
magnesium, chlorine, carbonate, sulphate and phosphate had higher values in wastewater than in groundwater
(Table 1). Contents of Cu, Cd, Zn, Cr and Pb were within permissible limits; the Ni content exceeded
permissible limits. The presence of microorganisms in wastewater from highest to lowest was faecal coliforms
(7.2102 cfu/100 mL), total heterotrophic bacteria (2.83106 cfu/100 mL), total coliforms (1.9103 cfu/100 mL)
and Salmonella-Shigella sp. (1.3102 cfu 100/mL).
Soil characteristics prior to the study and after the study varied (Table 2). The pH of the pre-sowing soil was
desirable in agricultural soil. In pots irrigated with wastewater, the pH of soil extract decreased from before
sowing to after harvesting. The organic carbon of wastewater irrigation was higher than well water and soil
irrigated with wastewater had higher organic carbon than pre-sowing soil. This indicates that wastewater
irrigation helps improve fertility after crop harvest. Wastewater provided essential nutrients to the soil and the
crop because wastewater irrigated soil had more nitrogen, phosphorus and potassium than pre-sowing soil.
Concentrations of heavy metals were within permissible limits (Awasthi 2000).
The wastewater and nitrogen combination, WWN20, was most effective as it produced the longest plants,
higher plant fresh and dry weights, more leaves, higher leaf area, and higher leaf fresh and dry weight at the 3
growth stages (Figs. 1, 2 & 3). There was an increase of 65.57% at 30 DAS, 35.06% at 60 DAS and 35.03% at
90 DAS in plant length over the GWN0. For plant fresh weight, interactions were significant only at fruiting
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stage, where WWN20 and WWN40 had similar values with increases of 35.14 and 29.13% over GWN0,
respectively. The best combination increased 54.89, 52.13 and 51.81% in plant dry weight over GWN0 at the 3
growth stages. Among interactions, WWN20 increased leaf number by 35.16, 35.58 and 35.53% over GWN0
at the successive stages. For leaf area, WWN20, produced increases of 34.95, 35.29 and 35.37% over GWN0
at the 3 growth stages, respectively. Interactions were significant at vegetative and flowering stages in leaf fresh
weight. The WWN20 had the best causing increases of 40.27 and 39.13% over GWN0 at the vegetative and
flowering stages, respectively. This optimum combination recorded an increase of 39.2, 35.95 and 37.06% in
leaf dry weight over GWN0 at the 3 growth stages, respectively. The combinations WWN20 and WWN40
were similar. The WWN0 combination was similar with GWN20, GWN40 and GWN60. The linear
regression curves worked out for plant height with leaf number and leaf area with leaf fresh weight favour the
present findings (Fig. 4).
Table 2. Physicochemical characteristics of soil collected before sowing. All determinations in mg.l -1 in 1:5 (soil-water
extract) or as specified.
After harvest
Determinations Pre-sowing soil Normal Range
(soil layer irrigated with wastewater)
Texture Sandy Loam Sandy Loam -
CEC (meq 100 g-1 soil) 3.32 3.92 -
pH 7.74 6.85 -
Organic Carbon (%) 0.419 1.15 -
EC ( mhos cm2) 242 - -
TDS 758 - -
NO3N (g kg-1 soil) 0.352 0.283 -
Phosphorus (g kg-1 soil) 0.109 0.151 -
Potassium 8.2 11.6 -
Calcium 27.14 23.89 -
Magnesium 16.49 12.94 -
Chloride 29.47 - -
Carbonate 19.34 - -
Bicarbonate 92.43 - -
Sodium 14.11 15.02 -
Sulphate 17.32 -
Cu - 1.11 135270
Cd - 0.22 36
Ni - 0.75 75150
Zn - 2.09 300600
Cr - 0.053 -
Pb - 0.72 250500

Proline content increased with increasing nitrogen dose as well as with wastewater; the WWN60 interaction
caused an increase of 24.09, 27.27 and 22.84% over GWN0 at 3 stages, respectively. Wastewater treated plants
had increased malondialdehyde content over GW irrigated plant. However, malondialdehyde content declined
with increase in nitrogen. Among combinations, WWN0 and WWN20 and WWN40 were best and similar
(Fig. 5).
Wastewater irrigation increased the activity of catalase, peroxidase and superoxide dismutase. Nitrogen dose
also positively influenced the enzyme activity with most activity under N60. The WWN60 combination
produced increases of 25.19, 20.74 and 22.58% in catalase over GWN0 at the 3 samplings, respectively.
However, this combination increased peroxidase by 24.56% at the vegetative stage, and 20.04 and 18.91% in
superoxide dismutase at vegetative and flowering stages over GWN0, respectively. The interactions were not
significant at flowering and fruiting stages for peroxidase but were significant at the flowering stage for
superoxide dismutase (Fig. 6). The proline content and antioxidant enzymes increased with the combination of
WWN60. The proline content was positively correlated with CAT (r2=0.9479), POD (r2=0.8991) and SOD
(r2=0.9056) (Fig. 7).
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Kausar et al. (2017) 4(2): 210224

Figure 1. Effect of waste water on length, fresh weight and dry weight/plant of fenugreek supplemented with different
nitrogen at 30, 60 and 90 DAS.

Figure 2. Effect of waste water on leaf number and leaf area/plant of fenugreek supplemented with nitrogen at 30, 60 and 90
DAS.

Figure 3. Effect of waste water on leaf fresh weight and leaf dry weight/plant of fenugreek supplemented with nitrogen
levels at 30, 60 and 90 DAS (at 30 DAS, leaf dry weight10).
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Kausar et al. (2017) 4(2): 210224

Figure 4. Linear regression curve of fenugreek showing correlation of plant height with leaf number and leaf area with leaf
fresh weight at 60 DAS.

Figure 5. Effect of waste water on proline and malondialdehyde contents of fenugreek supplemented with nitrogen at 30, 60
and 90 DAS.

Figure 6. Effect of waste water on catalase, peroxidase and superoxide dismutase activity of fenugreek supplemented with
nitrogen.
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Figure 7. Linear regression curve of fenugreek showing correlation of proline with catalase, peroxidase and superoxide
dismutase at 60 DAS.

DISCUSSION
The maximum values of various parameters were recorded with 100% concentration of wastewater which
was black in color. The higher value of TDS is associated with higher EC of wastewater (Bhargava et al. 2008).
The Cl-, CO3-2 and HCO3- were within the permissible limits (ISI 1974, Pescod 1992, Ayers & Westcot 1994).
High BOD, COD, NO3- and PO4-3 might be due to the presence of high oxidizable organic matter and rapid
consumption of dissolved inorganic materials (Biswas et al. 2009, Kumar & Chopra 2012). The TSS content
contributed to high salinity. Heavy metals at high levels in water bodies could cause toxicity to aquatic life
(Zyadah & Abdul-Bakky 2000, Indra & Sivaji 2006) and buildup in river sediments (Singh et al. 1997). Total
bacterial count and most probable number in wastewater are likely due to higher organic load of wastewater
(Kumar & Chopra 2012). The pH is an important parameter as many nutrients are available for plant uptake
only at a particular pH range. A pH value of 6.08.2 supports bacterial activity and is favourable for maximum
yield. A shift in pH outside that range renders nutrients less available, even though they remain in the soil.
Under acidic conditions, iron, aluminium, manganese, and heavy metals become highly soluble and may create
problems for plants (Charman & Murphy 1991). The higher concentration of Na in the soil after effluent
irrigation may be associated with the presence of high concentration of carbonate and bicarbonate in the effluent
(Thompson et al. 2001). Reduction in water infiltration can occur when irrigation water contains high sodium
relative to the calcium and magnesium content. This condition is called sodicity which reduces soil permeability
(Kelley 1951). The overall increase in nitrogen is likely due to the nitrate and ammonium ions in wastewater.
Irrigation with wastewater generally adds significant quantities of salts to the soil.
The beneficial effect on growth may be attributed to the nutrients present in wastewater (Table 1). However,
reduced growth at higher concentrations may be due to the accumulation of salt which causes increased
electrical conductivity (EC) with a link between EC and reduced plant growth (Kumar & Chopra 2012). The
source of wastewater was diluted urban sewage which appears to be beneficial for the development of fenugreek
because properly diluted wastewater can minimize the hazards of heavy metals (Pathak et al. 1999). Use of
diluted wastewater from the same source produced beneficial effects on Triticosecale Wittm. (Shah et al. 2005),
Lens culinaris Medikus (Tabassum et al. 2007a), Brassica juncea L. (Tabassum et al. 2007b), Abelmoschus
esculentus (L.) Moench (Kausar 2009, Faizan et al. 2014, Kausar & Faizan 2015), Cicer arietinum L. (Tak et al.
2010, 2012, 2013), Triticum aestivum L. (Akhtar et al. 2012), Capsicum annuum L. (Iqbal et al. 2012, Chalkoo
et al. 2014, Iqbal et al. 2015).
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Improvement in growth might be due to the presence of nitrogen. Fenugreek responded better to relatively
low nitrogen dose because it is a leguminous crop and able to fulfill nitrogen needs by symbiotic nitrogen
fixation. However, the process of symbiosis takes time to establish and became fully operative and the nitrogen
accumulated in this manner is only available to the soil when the legume plant dies. During early plant growth, a
low starter dose of nitrogen is necessary because higher doses hinder nodule formation (Mann 1968, Arrese-Igor
et al. 1997, Ram & Verma 2001, Shahroz 2009). Other reports concerning nitrogen application on the same crop
differ (Chaudhary 1999a, b, Sharma 2000, Thapa & Maity 2003, Datta et al. 2005, Tuncturk et al. 2011) where
comparatively higher rates of nitrogen were reported.
Nitrogen is an important nutrient required in large amounts by plants. It is an integral constituent of proteins,
nucleic acids, chlorophyll, co-enzymes, phytohormones and secondary metabolites. The availability of N to
roots is therefore a decisive factor for plant growth (Marschner 2012). The urban wastewater used for irrigation
contained nitrogen in the ionic forms, NH4+ and NO3- and further supplementation of nitrogen in the form of
urea yielded nitrate rapidly within a few days which is absorbed by plants possibly contributing to a cumulative
effect on growth (James 2010).
Proline is an amino acid that easily accumulates in plants under stress and is a marker of imbalance in a
metabolic pathway. It also functions as an osmolyte to alleviate damage caused by biotic or abiotic stresses
(Ashraf & Foolad 2007, Chandra et al. 2009, Myriam et al. 2009, Bedouh & Bekhouche 2012, Kumar et al.
2012a, b). Resistant plants accumulate more proline than less resistant plants (Naidu et al. 1992). Accumulation
of proline preserves the structure and activity of protein, reduces enzyme denaturation and protects
biomembranes from damage by inactivating hydroxyl radicals or other reactive chemical species (Pollard &
Wyn Jones 1979, Smirnoff & Cumbes 1989, Saradhi et al. 1995). Proline accumulates in plants exposed to
heavy metal stress (Zhao 2011, Bauddh & Singh 2012a, b). Fenugreek irrigated with wastewater exhibited
increase in the proline content. The wastewater contained heavy metals that cause stress to plants likely
increasing proline content. Heavy metal induced proline accumulation has been reported (Lalk & Dorfling 1985,
Bhattacharjree & Mukherjee 1994, De & Mukherjee 1998. Handique & Handique 2009, Kumar et al. 2015).
Proline accumulation helps the plant to withstand the heavy metal stress by protecting the key enzyme such as
glucose-6-phosphate dehydrogenase and nitrate reductase from being inactivated by toxic heavy metal ions by
forming metal-proline complex (Handique & Handique 2009). In this study, proline content increased with
increased nitrogen application. This may be because it is a nitrogen-storage compound (Ahmad & Hellebust
1988) and that synthesis and accumulation of proline are stimulated by nitrogen supply (Sanchez et al. 2002).
This result is in agreement with the findings of many workers (Naidoo & Naidoo 2001, Sanchez et al. 2001,
2002, Zhao & Liu 2009, Ahmadi et al. 2010, Wang et al. 2011).
Malondialdehyde content (MDA) or thiobarbituric acid reactive substances (TBARS) is the decomposition
product of polyunsaturated fatty acids of biomembranes obtained when reactive oxygen species (ROS) initiates
the process of lipid peroxidation, which is considered an indication of oxidative stress in plants (Gosset et al.
1994, Blokhina et al. 2003). Production of the ROS superoxide radical (O2-), hydrogen peroxide (H2O2) and
hydroxyl radical (OH-) increases as a result of abiotic stress such as can occur with heavy metal toxicity, salinity
and drought in plants. Under normal conditions, these ROS are well regulated by cell metabolism but under
abiotic stress, they may exceed the scavenging capacity of the antioxidant system. The wastewater contained
heavy metals which may enhance the level of H2O2 which can result in the formation of hydroxyl radicals that
can cause lipid peroxidation (Loggini et al. 1999). Oxidative damage to bio-membranes of plants can be
primarily attributed to the presence of Cd, a redox active metal catalyzing the generation of hydroxyl radicals
and O2- (Arora et al. 2002). The non-redox metals Zn, Ni, Cr, Cu and Pb do not produce ROS directly but
generate oxidative stress by interfering with the plant's antioxidant defence system (Garnczarska & Ratajczak
2000, Aravind & Prasad 2003, Panda & Choudhary 2005). These metals might have contributed to increased
MDA levels (Olorunfemi & Lolodi 2011). Reduction of MDA content due to nitrogen application was probably
due to increased activity of antioxidant enzymes (Shin et al. 2005, Zhang et al. 2007, Zhao & Liu 2009, Lin et
al. 2011). Xiao et al. (1998) reported that nitrogen application can improve light reaction of photosynthesis
which ultimately leads to the lower ROS production.
Biotic and abiotic stresses affect plants by causing excess accumulation of ROS, which can react with certain
biomolecules, and alter or inactivate biochemical activities (Mittler 2002, Mittler et al. 2004, Choudhary et al.
2012). Even under optimal conditions, ROS are generated as by-products of normal metabolism in subcellular
components (Asada & Takahashi 1987, Elstner 1991, Asada 1994, del Rio et al. 1998). These radicals are
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capable of initiating peroxidation reaction (Bandyopadhyay et al. 1999). The ROS also influence degeneration
of DNA, oxidation of deoxyribose sugar, the reforming and breaking of DNA strands and cause mutations
(Zhang & Kirkham 1996, Chirkova et al. 1998). To migrate and repair damage initiated by ROS, plants
developed complex antioxidant systems (del Rio et al. 2002). This defense system includes enzymatic and non-
enzymatic components. The enzymatic defense system includes the enzymes superoxide dismutase (SOD),
catalase (CAT), ascorbic peroxidase (APX), dehydroascorbate reductase (DHAR), and glutathione reductase
(GR) (Yordanova et al. 2003). SOD is an enzyme acting as a first line of defence in combating oxidative stress
in plants, which dismutates superoxide anions to H2O2 (Shalini & Dubey 2003, Mishra et al. 2006, Davies et al.
2009). CAT and POD are involved in building resistance against accumulation and toxicity of hydrogen
peroxide by converting H2O2 to water and oxygen in cells (Msttes 2000). Increases in levels of all antioxidant
enzymes occurred in fenugreek irrigated with wastewater. This is likely due to the pollutants present that can
lead to oxidative stress causing biochemical changes (Lee et al. 2001, Panda & Upadhyay 2003, Singh et al.
2003, Fatima & Ahmad 2005, Baghel 2008, Xu et al. 2010, Olorunfemi & Lolodi 2011, Jain & Srivastava 2012,
Sangeetha et al. 2012). Application of levels of nitrogen increased CAT, POD and SOD activities in fenugreek.
This may be due to the alleviation of heavy metal damage by the nitrogen increased activity of antioxidant
enzymes. Williams et al. (1967) suggested that addition of nitrogen in soil increases the formation of NO3-
which decreases soil pH. Lowering pH increases the solubility of heavy metals and decrease the efficiency of
adsorption (Moreno et al. 2000, Zhang et al. 2007, Zhao & Liu 2009, Ahmadi et al. 2010, Lin et al. 2011).

CONCLUSION
From the present study we concluded that the wastewater used for irrigation is suitable for fenugreek as the
nutritive and toxic chemical elements present in wastewater were accumulated in the soil at low level and this
accumulation did not cause any problem to the plants tested. Use of wastewater can provide economic benefits.
Substitution of ground water by wastewater at some contents increased growth, proline, malondialdehyde
content and antioxidant enzymes. Integrated application of wastewater and fertilizer increased growth and
biochemical characteristics in fenugreek. Even if oxidative stress is induced in fenugreek plants irrigated with
wastewater, application of N could provide protection against oxidative stress by increasing the antioxidant
protective system.

ACKNOWLEDGEMENTS
The Authors are thankful to the Head of Botany Department, Aligarh Muslim University, Aligarh, India for
his valuable support by providing the necessary facilities.

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ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(2): 225234, 2017
DOI: 10.22271/tpr.2017.v4.i2.031
Research article

Oxidative stress induced by lead in Vigna radiata L. seedling


attenuated by exogenous nitric oxide
Himani Singh, N. B. Singh*, Ajey Singh, Imtiyaz Hussain and Vijaya Yadav
Plant Physiology Laboratory, Department of Botany, University of Allahabad, Allahabad-211002, India
*Corresponding Author: singhnb166@gmail.com [Accepted: 27 May 2017]

Abstract: The present study deals with the effectiveness of nitric oxide (NO) on some biophysical
and biochemical variables in Vigna radiata subjected to lead (Pb) toxicity. Pb adversely affected
seedling growth and biochemical parameters of the test crop. The seedlings were grown in soil
supplemented with graded concentrations of Pb. Pb toxicity caused a marked decrease in growth.
Seedlings growth reduced maximum at the highest concentrations of Pb. The effect of Pb on
seedlings was mitigated by NO donor sodium nitroprusside (SNP). Pre-treatment of seeds with
SNP caused amelioration of detrimental effects of heavy metal stress on growth. The oxidative
stress induced by Pb elevated malondialdehyde (MDA) content in the seedlings. Nitric oxide
decreased MDA content in Pb treated seedlings. Exposure of seedlings to Pb enhanced antioxidant
enzyme activities of the seedlings, but exogenous SNP decreased the activities of superoxide
dismutase (SOD), catalase (CAT) and peroxidase (POX) in single (SNP) and SNP combined with
Pb (Pb+SNP) treatment. Pb induced oxidative stress by enhancing the reactive oxygen species.
SNP demonstrated a positive role against Pb toxicity which was evident from decreased activities
of antioxidant enzymes. The SNP treatment enhanced plant tolerance against Pb toxicity.
Keywords: Antioxidants - Heavy metal - Stress - Lead - MDA - Vigna radiata.

[Cite as: Singh H, Singh NB, Singh A, Hussain I & Yadav V (2017) Oxidative stress induced by lead in Vigna
radiata L. seedling attenuated by exogenous nitric oxide. Tropical Plant Research 4(2): 225234]

INTRODUCTION
Plants absorb most of the elements from the soil. Some of the engrossed elements are essential because they
are required by plants to complete their life cycle. Plants also absorb heavy metals like Pb (Sharma & Dubey
2005), chromium (Hayat et al. 2012), mercury (Chen & Yang 2012), cadmium (Yadav & Singh 2013, Singh et
al. 2005), and arsenic (Kumar et al. 2015), which show toxicity at very low concentrations. Heavy metals are
present in waste water and runoff in agricultural fields which affect crop metabolism and growth of plants.
Heavy metals are defined on the basis of size, density and number of atoms or position in the periodic table. Pb
is a highly toxic element that occurs in soil in low concentration. Pb is not found in native form, but it forms
complex with other elements in the soil. In soil, Pb contamination increased because of its wide application in
gasoline, paints, water pipes, fertilizers, paper making and many other industrial processes (Pallavi & Rama
2005). Pb is highly immobile and persists for a long duration in the soil. Some plants can tolerate or avoid Pb,
but others experience toxicity because it easily affects some metabolic pathways (Wierzbicka 1999). Pb has low
solubility and accessibility for plant uptake (Blaylock & Huang 2000). It dissolves in water slowly. It
precipitates as phosphates and sulphates and is usually transported to different plant parts from soil-rhizosphere
(Blaylock & Huang 2006). Pb has a detrimental effect on plant physiological processes and modifies growth
parameters, thus affects growth and productivity. Pb is known to affect the contents of photosynthetic pigments,
sugar and protein (Bhardwaj et al. 2009). Alteration in pigments influences the biosynthesis of photo-
assimilates. The detrimental expressions of Pb are in forms of chlorotic spots, senescence, necrotic lesions on
the leaf surface, and stunted growth of plants. The various enzymes are inactivated by Pb binding with their SH-
groups (Pinho et al. 2012). Sugars and proteins are energy rich compounds which provide energy for the
biosynthesis of compounds required by the plants. Contents of sugar and protein are altered in plant subjected to

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Received: 05 January 2017 Published online: 31 May 2017
https://doi.org/10.22271/tpr.2017.v4.i2.031
Singh et al. (2017) 4(2): 225234

unfavourable conditions. The plant membrane is the target site of heavy metals. Membrane lipids are oxidised to
peroxides and reactive oxygen species are produced. Thus, permeability of the membrane is altered. Reactive
oxygen species induces activities of antioxidant enzymes. Nitrate is one of the important forms of nitrogen
required in the biosynthesis of amino acids, the building block of protein. The nitrate reductase enzyme which
converts NO3- into NH3 or NH4+ is induced by the substrate. Metabolic changes influence overall plant growth
and health. Thus shoot length, pigments viz. chlorophyll and carotenoids, sugar, protein, and activities of nitrate
reductase and antioxidant enzymes were taken into consideration in the present study.
SNP issued as a source of NO which acts as a signalling molecule. SNP alleviates the drastic effects of metal
stress in various plant species. NO is a labile gaseous messenger molecule which involves in various
physiological responses, biotic and abiotic stresses like salinity, pathogen attack, drought, herbicides,
temperature and heavy metal toxicity (Wendehenne et al. 2005, Singh et al. 2017). NO has both detrimental and
positive effects on plants depending on its concentration and location in the cells (Leitner et al. 2009).
In plant cells in vivo production of NO is nitrate reductase dependent in cytosol (Gill et al. 2013). NO
production, in vivo is due to nitrate reductase enzyme, which converts nitrate into nitrite (Yamasaki & Sakihama
2000). The NO formation through nitrate reductase has been reported in several plant species (Xu & Zhao
2003). Plants have several enzymes viz. cytosolic nitrate reductase, nitric oxide synthase and xanthine
dehydrogenase involved in NO production (Gill et al. 2013).
SNP alleviates oxidative stress in Solanum (Singh et al. 2012) and other in crops (Gill et al. 2013). SNP
directs various signalling cascade pathways in plant cell (Neill et al. 2003) and initiates diverse hormone
signalling pathways (Pagnussat et al. 2004), Ca++ dependent stomata closing (Garcia-Mata et al. 2003) and
change in gene expression (Hao & Zhang 2010, Hasanuzzaman et al. 2010). Exogenous application of SNP
mitigates the toxicity of heavy metals like cadmium in rice (Hsu & Kao 2004), copper (Yu et al. 2005), and
aluminium (Zhang et al. 2008) and allelochemicals (Singh et al. 2012).
The survey of the literature reveals that there are comprehensive studies on the effect of SNP on Vigna
radiata L. seedlings stressed by Pb. The study was undertaken to observe alteration of plant biophysical
processes by lead and amelioration of toxic effects by SNP as a growth regulator.

MATERIALS AND METHODS


Seeds of Vigna radiata L. (mungbean) were procured from certified seed agency of Allahabad, Uttar
Pradesh, India. Lead in form of lead acetate [(CH3.COO)2Pb3H2O] (Molecular weight 379.33g.mol-1) was
purchased from LobaChemie and sodium nitroprusside (Na2[Fe(CN)5NO].2H2O) [sodium pentacyanonitrosyl
ferrate (II)](molecular weight 297.95g.mol-1) from Merck.
Growth and pot culture
The experiment was conducted in May, 2014 in the glasshouse in the Department of Botany, University of
Allahabad, Allahabad (24o47 and 50o 47N latitude; 81o 91 and 82o 21E longitude; 78 m above sea level).
Healthy seeds of Vigna radiata L. were selected and surface sterilized with 0.1% HgCl2 for 5 min and then
washed thoroughly with double distilled water (DDW). One set of the seeds was soaked in DDW and other set
was soaked in SNP (250 M) for 3 hours. The fire clay pots (height 7 cm and diameter 21 cm) each filled with 1
kg of garden soil, were divided into two sets. One set of pots was without Pb and other contained varying
concentrations of Pb, 0.5g (Pb1), 1g (Pb2) and 2g (Pb3) per kg of soil according to the treatments. The first set
was divided into two subsets; in one subset of pots seed soaked in distilled water were sown (C) while in other
subset seeds soaked in SNP were sown (SNP). The other set of pots with Pb was divided into two subsets. In
one subset of pots DDW soaked seeds were sown and in other subset SNP treated seeds were sown. The
experiment was conducted in triplicate in glass house condition. After 14 days first fully expanded leaves of the
seedlings were sampled for measurement of biophysical and analyses of biochemical parameters.
Determination of growth
The seedling growth was measured as shoot length of the plants. The seedlings were uprooted from the pots
and washed gently with tap water to remove soil particles. The plants were blotted with filter papers to remove
excess of water and measured the length of shoots by using a meter scale.
Photosynthetic pigments
The amount of photosynthetic pigments was quantified following the method of Lichtenthaler (1987). Fresh
leaves (100 mg) of the seedlings were homogenised in 80% (v/v) acetone. The extracts were centrifuged until
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Singh et al. (2017) 4(2): 225234

the supernatant became clear. The absorbance of the resulting solutions was recorded on 663, 645, and 470 nm,
spectrophotometrically (Shimadzu single beam UVvisible spectrophotometer-1700).
The pigments were quantified by the following formulae:

Chlorophyll a (g.ml-1) = 12.21xA663-2.81xA645

Chlorophyll b (g.ml-1) = 20.13xA645-5.03xA663

Total chlorophyll (g.ml-1) = (20.2x A645+8.02x A663)

Carotenoids (g.ml-1) = [1000xA470-3.27(Chl. a)-104(Chl. b)]/198


Where A is the observed OD
Sugar and Protein content
The quantification of total soluble sugar (TSS) was done following Hedge& Hofreiter (1962). About 50 mg
of the sample was homogenized in 3 ml 95% ethanol. After centrifugation 0.1 ml of supernatant was mixed with
4 ml anthrone reagent and heated in water bath at 95C temperature and cooled. Absorbance was recorded at
620 nm. The amount of sugar was determined by the standard curve prepared from glucose.
Protein content was determined according to the method of Lowry et al. (1951). Ten mg of first fully
expanded fresh leaves of the plants were homogenized with 1 ml 1 N NaOH for 5 minutes at 100 oC. Five ml of
alkaline copper reagent were added to it and the mixture was allowed to stand at room temperature for 10
minutes followed by addition of 0.5 ml of Folin-Ciocalteau reagent immediately in the tube. The absorbance of
the solution was measured at 650 nm after 30 minutes. The standard curve of bovine serum albumin was used to
calculate the amount of protein.
Lipid peroxidation
Lipid peroxidation was measured as the amount of malondialdehyde (MDA) content determined by
thiobarbituric acid reactive substance as described by Heath & Packer (1968). Fresh leaf (200 mg) was
homogenized in 0.1% (w/v) trichloroacetic acid and centrifuged at 10,000g for 10 min. Malondialdehyde level
was used as index of lipid peroxidation and was expressed as nmol.g-1 FW. One millilitre supernatant was
mixed with 4 ml 0.5% thiobarbituric acid. The mixture was heated at 95C for 30 min and centrifuged after
cooling. The absorbance of the supernatant was recorded at 532 nm and corrected by subtracting the non-
specific absorbance at 600 nm. The MDA concentration was calculated using the extinction co-efficient of 155
mM-1.cm-1 and expressed as n mol.g-1 FW.
Nitrate reductase activity
Nitrate reductase (NR) activity was measured following the p procedure of Jaworski (1971). Fresh leaf tissue
(250 mg) was incubated in 4.5 ml medium which contained 100 mM phosphate buffer (pH 7.5), 3% KNO 3 and
5% propanol. About 0.4 ml aliquot was treated with 0.3 ml 3% sulphanilamide in 3N HCl and 0.3 ml 0.02% N-
1-naphthyl ethylene diamine dihydrochloride. The absorbance was measured at 540 nm. NRactivity was
calculated with a standard curve prepared from NaNO2 and expressed in mol NO2g-1 FW h-1.
Extraction and assay of activities of antioxidant enzymes
Enzyme extract was prepared by homogenizing leaves with 0.1 M sodium phosphate buffer (pH 7.0)
containing polyvinyl pyrrolidone. The homogenate was centrifuged at 4oC at 15000 g for 30 min in cooling
centrifuge (Remi instruments C 24). The supernatant was used for the assay of superoxide dismutase, catalase
and peroxidase.
Superoxide dismutase assay
The activity of superoxide dismutase (SOD) was determined by the nitroblue tetrazolium (NBT)
photochemical assay following Beyer & Fridovich (1987). The reaction mixture (4 ml) consisted of 20mM
methionine, 0.15mM ethylene diamine-tetra-acetic acid, 0.12 mM NBT, 1.3 M riboflavin, 0.05 M sodium
carbonate (pH 10.2) and enzyme extract. Test tubes were exposed to fluorescent lamp for 30 min and identical
unilluminated assay mixture served as blank. The photo reduction of NBT (formation of purple formazone) was
recorded spectrophotometricallyat 560 nm and compared with blank sample having no enzyme extract. One unit
of SOD activity was defined as the amount of enzyme required to cause 50% inhibition of the reduction of NBT.

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Catalase assay
Catalase (CAT) activity was assayed following Cakmak & Marschner (1992). Assay mixture contained 25
mM potassium phosphate buffer (pH 7.0), (0.2 ml) 10 mM H2O2 and 0.5 ml enzyme extract. The rate of H2O2
decomposition for 1 min was monitored at 240 nm and calculated using extinction coefficient of 39.4 mM-1.cm-1
and expressed as enzyme unit g-1 FW. One unit of CAT was determined as the amount of enzyme required to
oxidize 1 mM H2O2 min-1.
Peroxidase assay
Peroxidase (EC 1.11.1.7) activity was assayed following McCune & Galston (1959). Fresh leaf tissue (0.2 g)
was homogenized in 0.1 M potassium phosphate buffer (pH 6.0) and centrifuged at 10,0009 g for 20 min at 4C.
The reaction mixture contained 2.0 ml enzyme extract, 2 ml potassium phosphate buffer, 1.0 ml 0.1 N
pyrogallol and 0.2 ml 0.02% H2O2 and determined spectrophotometrically at 430 nm. One unit of enzyme
activity was defined as the amount which produced an increase of 0.1 OD per minute.
Statistical analysis
Data represent the mean standard errors of results from three data were statistically analysed using analysis
of variance (ANOVA) by using SPSS software (Version 16 SPSS Inc., Chicago, IL, USA). The appropriate
standard error of means was calculated for presentation with tables and graphs. The treatment means were
analysed by Duncans multiple range test (DMRT) at level of P < 0.05.

RESULTS
Growth
One time pre-soaking of seeds was effective as it caused the significant modification in biophysical and
biochemical parameters of Vigna. Hayat et al. (2012) also recorded the effects of SNP on tomato seeds soaked
in SNP for eight hours. Pre- soaking of tomato seeds with SNP solution of 250 M concentration was effective
to cause maximum growth during preliminary experiments (Singh et al. 2012).
The growth of seedlings was measured in terms of shoot length. Shoot length decreased significantly (P<.05)
in treatments with Pb. The decrease of 6, 23 and 34% in shoot length, was recorded in the seedlings with Pb1,
Pb2 and Pb3 treatments respectively. Pre-soaking with SNP without Pb enhanced the shoot length of the
seedlings by 12.5% as compared with control. The seedlings pre-soaked with SNP and treated with Pb1, Pb2 and
Pb3 showed an alleviation of 3, 14 and 14% in shoot length respectively (Fig. 1A).
Photosynthetic pigments
The amounts of photosynthetic pigments viz. Chl a, Chl b, total Chl and carotenoids are shown in table 1.
The contents of Chl a, Chl b, total Chl and carotenoids declined with graded doses of Pb in Pb1, Pb2 and Pb3
treatments. The detrimental effect of Pb is evident in Chl a, Chl b, total Chl and carotenoids and decrease of 72,
86, 76 and 88% was recorded in the highest dose of Pb. However in the seedlings pre-soaked with SNP without
Pb photosynthetic pigments were greater than that of Pb and control. The Pb treated seedlings pre-soaked with
SNP caused significant improvement in Chl a, Chl b, total Chl and carotenoids contents but were always lower
than that of control.
Table 1. Effect of SNP on photosynthetic pigments in seedlings of Vigna radiata grown under lead stress.
Chl a (mg.g-1FW) Chl b (mg.g-1FW) Total Chl (mg.g-1FW) Carotenoids (mg.g-1FW)
Treatment
-SNP +SNP -SNP +SNP -SNP +SNP -SNP +SNP
C 7.170.34b 9.310.47a 2.530.18b 4.050.14a 9.700.51b 13.370.34a 1.840.31b 2.710.35a
Pb1 5.070.56c 7.050.47b 1.050.10d 1.420.16c 6.130.652c 8.470.61b 0.900.12d 1.570.14bc
Pb2 4.040.56e 5.440.53cd 0.560.29e 0.950.22d 4.600.27d 6.390.30c 0.610.14de 1.070.28c
Pb3 1.960.58f 3.350.69e 0.360.08f 0.460.16e 2.330.49e 3.82.53d 0.210.14f 0.700.14de
Note: Data are means standard error of three replicates. Bars followed by different letters show significant differences at P < 0.05
significance level between treatments according to the Duncans multiple range test.

Sugar and Protein


Total soluble sugar (TSS) declined by 15, 23 and 36% and protein 20, 48 and 75% in Pb 1, Pb2 and Pb3
treatments respectively as compared with control. Exposure to SNP enhanced TSS and protein contents to 13
and 28% respectively as compared with control (Fig. 1BC).

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Figure 1. Effect of NO in seedlings of Vigna radiata under lead stress: A, Shoot length; B, Sugar; C, Protein; D, Lipid
peroxidation. [Data are means standard error of three replicates. Bars followed by different letters show significant
differences at P < 0.05 significance level between treatments according to the Duncans multiple range test]
Lipid peroxidation and nitrate reductase activities
The content of MDA increased significantly in the seedlings treated with graded concentrations of Pb. The
lipid peroxidation (LP) increased by 72, 115 and 174% in the seedlings under Pb1, Pb2 and Pb3 treatments
respectively. MDA content in SNP treated seedlings decreased to 53% as compared with control. SNP caused a
decrease in the level of MDA content in the seedlings under the influence of Pb as compared with treatments
with the respective concentration of Pb but was always higher than that of control (Fig. 1D).
The nitrate reductase activity in the leaves of Vigna seedlings was affected by Pb. The activity of NR
decreased in dose-dependent manner. The maximum 45% inhibition of NR was recorded in the seedlings treated
with Pb3. Maximum 15% increase in NR activitywas recorded in the seedlings preexposed to SNP. The
activities of NR enhanced by 8.7,18 and 20% in the seedlings pre exposed to SNP in Pb1, Pb2 and Pb3 treatments
respectively (Fig. 2a).
Activities of antioxidative enzymes
The result pertaining to the activities of antioxidative enzymes viz. superoxide dismutase (SOD), catalase
(CAT) and peroxidase (POD) in leaves are presented in figure 2BD. The activities of SOD, CAT and POD
increased in treatments with Pb in concentration dependent manner. The activities of SOD increased by 25, 67
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Singh et al. (2017) 4(2): 225234

and 87% CAT by 49, 76 and 145% and POD by 17, 34 and 64%, in the seedlings exposed to Pb 1, Pb2 and Pb3
treatments respectively. The activities of antioxidant enzymes recorded at the lowest level in the seedlings, pre
treated with SNP. The activities of antioxidative enzymes exhibited a significant reduction in the seedlings
grown from the seeds pre-treated with SNP and exposed to Pb. The exposure of seedlings to SNP declined the
activities of SOD, CAT and POX by 9, 20 and 12% respectively.

Figure 2. Effect of NO in seedlings of Vigna radiata under lead stress: A, Nitrate reductase; B, Superoxide dismutase; C,
Catalase; D, Peroxidase. [Data are means standard error of three replicates. Bars followed by different letters show
significant differences at P < 0.05 significance level between treatments according to the Duncans multiple range test]

DISCUSSION
Growth is the key factor for evaluating plant responses to various biotic and abiotic environmental stresses.
The growth of the seedlings decreased significantly as the concentration of Pb increased. The growth reduction
may be due to decrease in photosynthetic pigments and increased lipid peroxidation. Singh et al. (2003) also
reported decrease in growth parameters of Vigna radiata under lead stress. The inhibition in growth parameters
by Pb stress has been observed in various plant species as a consequence of deregulation of various primary
physiological and metabolic processes (Singh et al. 1997a, b). Exogenous SNP appears to provide protection to
plant against Pb toxicity which is evident from increased plant growth. The application of exogenous SNP
significantly (P<0.05) mitigated Pb toxicity.
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The photosynthetic pigments in Pb treated seedlings decreased progressively with increased concentration of
Pb. Reduction in the content of Chl and carotenoids in the seedlings under stress induced by Pb may be due to
the inhibitory effect of Pb on the enzyme which involved in pigment biosynthesis. Pb stress, either inhibited
biosynthesis or stimulated degradation of photosynthetic pigments. The decrease in chlorophyll contents
resulted in decreased biomass production and inhibition of seedling growth. The Pb toxicity affected various
biochemicals, morphological and physiological processes which caused stunted plant growth (Wang et al.
2003). According to Pinho et al. (2012) Pb deactivates several enzymatic processes by binding with SH-groups
causing impairment of metabolic activities which may be responsible for reduced plant growth.
The present study showed that Pb decreased protein content. The decrease of protein content may be due to
inhibition of protein synthesis or degradation of protein (Palma et al. 2002). Costa & Spitz (1997) recorded
decrease in protein content in Lupinus albus under heavy metal stress. Oxidative stress caused by Pb may be
another possible reason for the decrease of protein content (Bharwana et al. 2013). Exogenous supply of SNP
increased protein concentration (Hsu & Kao 2004). The heavy metal in plants caused impairment of metabolic
activities which gradually decreased sugar contents. The decreased photosynthetic pigments inhibited
photosynthetic activity which reduced sugar content (Hussain et al. 2013). According to Stiborova et al. (1987)
heavy metals interact with ribulose biphosphate carboxylase reactive centre and show the negative impact by
inhibiting the carboxylation. Our results reveal that photosynthetic pigments, total soluble sugar and protein
content decreased in presence of Pb. These findings are in agreement with Bhardwaj et al. (2009).
Heavy metal stress elevated the level of MDA. Impaired metabolic activities caused by Pb resulted in the
generation of reactive oxygen species which enhanced oxidative stress leading to lipid peroxidation and
membrane damage. In the present study higher concentration of Pb (2g.kg-1) increased MDA content to the
maximum. Increase in MDA content was also recorded by Mroczek-Zdgrka & Wojick (2012) in Pb treated
Vicia faba seedlings. Differential reduction in MDA content is evident in treatment with SNP. The decreased
MDA content was also observed in wheat seedlings treated with SNP (Singh et al. 2008). NO is known to
inhibit ion leakage and protect plants tissue against membrane damage by lipid peroxidation (Beligini et al.
2002). This appears that SNP protects membrane from damage caused by Pb stress.
Nitrate reductase activity decreased in higher concentration of Pb. NR activity may also be inhibited by the
NADH regulated supply of NO3- at the site of enzyme synthesis (Burzynski & Grabowski 1984, Dabas 1992).
Nitrate reductase is the substrate induced enzyme. Decreasein NR activity may be due to restricted supply of
NO3-to the roots. Pb appears to interfere with absorption of NO3- from the soil. It may ensure the absorption of
NO3- to the roots and increases NR activity to the maximum.
Plants have antioxidant defence systems to maintain their growth and health. Plants face oxidative damage
when exposed to Pb and various heavy metals (Bharwana et al. 2013). Plants develop a machinery of
antioxidant system to prevent the oxidative damage. In the present study activities of antioxidative enzymes like
SOD, CAT and POD increased in the seedlings under Pb stress. Heavy metals increase the activities of SOD in
Avena sativa L. (Luna et al. 1994) and Oryza sativa L. (Verma & Dubey 2003). This evinced over production of
ROS in the seedlings under the influence of Pb stress.
The SNP is known to increase or decrease the activities of antioxidant enzymes. According to Zhang et al.
(2008) SNP enhanced the activities of antioxidants. Plants have antioxidant potential to scavenge ROS and have
greater stress tolerance (Ashraf & Akram 2009). Exogenous NO decreased the activities of antioxidant enzymes,
levels of H2O2 and MDA in the maize plants exposed to saline stress (Kaya et al. 2015). In the present study
decreased activities of antioxidant enzymes on the application of SNP showed that plant mitigated the Pb stress
to maintain plant health. Thus NO by decreasing the activities of antioxidant enzymes prevented the energy
diversion from the biosynthesis of antioxidative enzymes and thus supported plant growth. Decreased activity of
SOD may be correlated with decreased level of lipid peroxidation and enhanced level of photosynthetic
pigments. These alterations protected plants against oxidative stress in various stress conditions (Laspina et al.
2005, Hung & Kao 2003). The decreased activity of SOD reflects that the production of ROS (O2-) is low due to
the action of NO (Caro & Puntraulo 1998). Plants pre-treated with SNP in different concentrations (100 and 200
M) decreased POD activity (Ferreir et al. 2010). SNP inhibits activities of CAT and POD in Nicotiana
tabacum (Clark et al. 2000). Xiong et al. (2010) reported that rice seedlings pre-treated with NO decreased ROS
production and prevented Pb dependent negative impacts. It is evident that SNP inhibits production of ROS and
regulates the activities of antioxidant enzymes. In plants, the growing in normal environmental condition the
production of ROS corresponds to the activities of antioxidant enzymes. SNP buttresses the defence system of
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Vigna seedlings treated with Pb. Concentration dependent increase in SOD activities was recorded in Pb. POD
and CAT detoxify H2O2 produced by the activities of SOD. CAT appears to play a major role in detoxification
of H2O2 produced in response to Pb treatment of Vigna seedlings. SNP decreased the activities of antioxidant
enzymes in Pb treated seedlings. Thus, SNP prevents the energy diversion required to increase the activities of
antioxidant enzymes. The SNP appears to mitigate the toxic effect of Pb on the Vigna seedlings.

CONCLUSION
In the present study, SNP appears to play a significant role to improve growth and development of the Vigna
seedlings exposed to the Pb stress. It is evident that Pb decreased growth, pigments, sugar, protein and nitrate
reductase activity and adversely affected the metabolism of the seedlings. The increase in MDA content is an
index of oxidative stress caused by Pb. The activities of antioxidant enzymes SOD, CAT and POD were
stimulated to cope with the oxidative stress caused by Pb. SNP mitigated the Pb toxicity and played important
role to buttress antioxidant defence system. Exogenous application of SNP protected Vigna radiata L. seedlings
against Pb toxicity.

ACKNOWLEDGEMENTS
The authors are thankful to the University Grant Commission, New Delhi and University of Allahabad,
Allahabad, India for providing financial assistance to Himani Singh.

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ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(2): 235241, 2017
DOI: 10.22271/tpr.2017.v4.i2.032
Research article

Effect of vehicular traffic on pollen size and viability of


Apocynaceous plant species
Mandeep Kaur and Avinash Kaur Nagpal*
Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar-143005, Punjab,
India
*Corresponding Author: avnagpal@yahoo.co.in [Accepted: 04 June 2017]

Abstract: Vehicular traffic adds up different kinds of contaminants like heavy metals,
polyaromatic hydrocarbons, gaseous toxins etc. to the atmosphere. Pollen grains of different plants
have been found to be sensitive to these pollutants, which show varied effects on their viability,
and size and hence can be used as bioindicators of air pollution. Keeping this in mind, the present
work was designed to measure the pollen viability and pollen size of three common roadside
plants, namely Alstonia scholaris, Nerium oleander and Tabernaemontana divaricata belonging to
Apocynaceae family growing at seven sites of Amritsar city with varying levels of traffic stress.
Pollen viability and pollen size were studied using 2, 3, 5-triphenyl tetrazolium chloride (TTC)
staining method. The percentage of pollen viability of A. scholaris ranged from 32.45% to
65.74%, for N. oleander it ranged from 30.3% to 45.11% and for T. divaricata it ranged from
14.31% to 49.62%. Pollen size ranged from 22.86 to 23.94 m for A. scholaris, 29.58 to 32.17 m
for N. oleander and 29.83-32.43 m for T. divaricata. Pollen viability was found to be inversely
proportional to a load of traffic at a given site whereas no remarkable variation was found in pollen
size. For A. scholaris, a significant negative correlation (r= -0.762, p0.05) was observed
between pollen viability and traffic load, while for other two species correlation between pollen
viability and traffic load was not statistically significant at p0.05.
Keywords: Air pollutants - Indicator - Traffic stress - Pollen viability - Pollen size - TTC.

[Cite as: Kaur M & Nagpal AK (2017) Effect of vehicular traffic on pollen size and viability of Apocynaceous
plant species. Tropical Plant Research 4(2): 235241]

INTRODUCTION
Vehicular exhaust adds up huge amounts of soot particles, smoke, poisonous gases (SO2, NO2, CO2, VOCs
etc.), heavy metals and organic molecules on the roads all over the world. All these air pollutants are known to
produce adverse effects on the health of plants, animals and humans (Rezaei et al. 2010, Atkinson et al. 2012).
Environmental pollution may also play a significant role in increasing pollen allergenicity and hence asthma
problems in humans residing at various polluted areas (Majd et al. 2004). Plants growing outdoors are one of the
main and primary targets to all types of air pollutants (Shannigrahi et al. 2004). Plants can act as great
candidates for reduction of air pollution at a particular place by absorption, deposition, accumulation and
detoxification of harmful pollutants (Prajapati & Tripathi 2008).
Many studies have shown effects of automobile emissions on leaves, stomatal apertures, pollen viability and
growth patterns of plants (Iannotti et al. 2000, Verma et al. 2006). One of the major consequences of air
pollution is its effect on pollen grain size, shape, exine sculpturing, viability etc. (Iannotti et al. 2000, Duro et al.
2013, Kaur et al. 2016). The mature pollen grains which are released from the flower buds are dry and while
absorbing humidity from the ambient air, they also absorb some contaminants which influence their viability
hence, plants reproductive system (Kaur et al. 2016). Due to lipophilic nature of pollen and its exine surface
stiffness, gaseous/particulate and organic/non-organic pollutants generally get accumulated inside it and
resulting in a decrease in their size and different morphological deformities (Bist et al. 2004). Pollen, being
sensitive indicators of air pollution, can provide interesting and useful information about the adverse effects of
air pollution on living systems (Gottardini et al. 2004, Noori et al. 2009, Yousefi et al. 2011, Kaur et al. 2016).

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Received: 08 December 2016 Published online: 30 June 2017
https://doi.org/10.22271/tpr.2017.v4.i2.032
Kaur & Nag (2017) 4(2): 235241

Reproductive success of any plant is mainly dependent on the quantity and quality of pollen grains produced.
Contaminants like pesticides, heavy metals, fluorides, etc. can accumulate on the surface of pollen grains and
make them active and sensitive bio-indicators of atmospheric pollution (Noori et al. 2009, Yousefi et al. 2011).
The literature survey has revealed enormous reports on the impact of air pollution on pollen grains structure and
exine properties but very less work is accessible on the use of pollen grains as bioindicators of air pollution.
For assessment of pollen viability and size, different methods such as in vitro germination tests and staining
methods are being used. TTC staining method is one of the widely and fast used methods based on
dehydrogenase enzymatic reaction in mitochondria of living cells (Firmage & Dafni 2001, Soares et al. 2013).
Amritsar, a fast growing city of Punjab (India), is witnessing an exponentially increasing number of vehicles
on the roads during the last few decades. Myllyvirta & Dahiya (2015) in their recent report published by WHO
reported Amritsar to be 13th out of 20 most polluted Indian cities and it was found that vehicular exhaust adds an
annual average of 92 g.m-3 of PM2.5 (Particulate matter) into the atmosphere. Since roadside trees/shrubs are
recipients of air pollution; their pollen studies can give an estimate of the biological effects of air pollution
(Gottardini et al. 2004). Hence, the aim of the present study was to estimate the effects of traffic stress on pollen
size and viability of three species of ornamental plants belonging to family Apocynaceae has widely grown on
the roadsides of Amritsar.

MATERIALS AND METHODS


Area of study

Figure 1. Map showing location of study sites: 1, Botanical Garden, Guru Nanak Dev University (BGG); 2, Khalsa College
Campus (KCC); 3, Guru Nanak Dev University Whole Campus (GNC); 4, Kabir Park Colony (KPC); 5, Lahori Gate Road
(LGR); 6, Grand Trunk Road (GTR); 7, Ram Bagh Chowk (RBC). [Source: http://www.indmaps.com/state-map/punjab/;
https://www.google.co.in/maps/@31.6336711,74.8000795,13z?hl=en]
Table 1. Pollen sampling sites under study.
S. No. Sampling site with code Description of site
1 Botanical Garden, Guru Nanak Dev It covers whole area of Botanical Garden of Guru Nanak
University (BGG) Dev University, Amritsar.
2 Khalsa College Campus (KCC) It covers whole campus of Khalsa College
3 Guru Nanak Dev University Whole It covers whole campus of Guru Nanak Dev University
Campus (GNC) excluding Botanical Garden area
4 Kabir Park Colony (KPC) Residential colony covering road streets and park areas
5 Lahori Gate Road (LGR) Road stretch from Beri gate to Lahori Gate
6 Grand Trunk Road (GTR) Road stretch from Chheharta to Amritsar Railway Station
7 Ram Bagh Chowk (RBC) It covers area around Ram Bagh Chowk
Amritsar, one of the industrialized and rapidly growing cities, is situated at N 31.6340, E 74.8723 of the
Northwestern state (Punjab) of India. The city lies at an average elevation of 234 m above sea level and covers
almost 5075 Km2 area. Amritsar has a semiarid climate, with four seasons i.e. winter season (December to
March) with minimum temperature of 0C and maximum of about 15C; summer season or pre-monsoon (April
to June) where temperature can go up to 42C; monsoon season from July to September while post-monsoon
season is from October to November. Annual rainfall is about 681 millimetres. Its population is about 1,132,761
(Census of India 2011). In the present study, seven roadside sites of Amritsar city were selected (Fig. 1; Table
1).
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Plants under study


Three apocynaceous plant species namely, Alstonia scholaris (L.) R. Br., Nerium oleander L. and
Tabernaemontana divaricata (L.) R.Br. ex Roem. & Schult. growing along the roadsides of Amritsar city,
Punjab, India were considered. Brief description of plants under study is presented in table 2. The reason for
selection of these plants was their common occurrence at all the studied sites.
Table 2. Description of different plants under study.
Plant Name General Features observed
Botanical name- Alstonia scholaris (L.) R. Br. Grows up to100-130 ft, flowers bloom in the month of October,
Common name- Chattiyan/Indian devil tree leaves are glossy and occur in whorls of 3-10; petioles are 13 cm.
Family- Apocynaceae In Ayurveda, it is an astringent herb for treating skin disorders,
malarial fever, urticaria, chronic dysentery, diarrhea, snake bite
and milk used to treat beriberi disease.
Botanical name- Nerium oleander L. Grows up to 10-13 ft in height, leaves are 10 to 22 cm long. The
Common name- Kaner/ Oleander plant produces terminal flower heads, usually pink or white. Each
Family- Apocynaceae flower is about 5 cm in diameter and five- petalled.
The whole plant, including the milky white sap, is toxic. The one
of the most studied toxin is oleandrin.
Botanical name- Tabernaemontana divaricata (L.) Grows to a height of 5 to 6 ft, leaves are 6 inches in length and 2
R.Br. ex Roem. & Schult. inches in width, flowers are pinwheel shaped.
Common name- Chandni/Crape Jasmine Root decoction is used in treatment of ascariasis, scabies, internal
Family- Apocynaceae poisoning, kidney stones and also used as antipyretic while stems
are used to treat dental caries.
Collection of buds
Sampling was carried out once in a year, 2014. Flower buds of N. oleander and T. divaricata were collected
during the months of MayJuly while for A. scholaris sampling for flower buds was done during October
November. Fresh and young flower buds were collected during early morning hours (5.308.30 am) from each
species growing at each of the seven sites of Amritsar city. At least 10 flower buds of suitable size were
collected from each plant. For each site of the study, buds from three plants of each species were collected.
Experiments were carried out in triplicates.
Traffic load
The traffic load was calculated by counting the number of vehicles including heavy vehicles, four wheelers,
three wheelers and 2 wheelers moving at each site for 3 h at different intervals of time (89 am; 121 pm; 45
pm) with the help of a hand-held counter.
Pollen viability
Pollen viability was estimated by employing 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining method
according to the protocol of Iannotti et al. (2000) with slight modifications. Freshly prepared TTC stain kept in
dark bottles was used for making slides every time. TTC stain was prepared by taking 1% solution of TTC in
60% sucrose solution. A minimum of 10 anthers from at least 58 flower buds of suitable size of each plant
were excised with a sharp needle and placed on a slide. Further, a drop of TTC stain was added to squash
anthers on the glass slide. A cover slip was placed on the squashed material and air bubbles were removed. The
slides were sealed with dibutylphathalate xylene (DPX) and incubated for 2 hrs in daylight. Pollen grains slides
were prepared in the field or in laboratory immediately after sampling. The stained slides were then preserved in
air tight box. After one day, slides were observed under a light microscope at 400X magnification at room
temperature for viable and non-viable pollen grains. Pollen viability was calculated by counting viable and non-
viable pollen grains. The deep pink or red stained pollen grains with round shape were counted as viable while
white or yellowish pollen grains with shriveled structure were counted as non-viable. A minimum of 1000
pollen grains were studied for each plant. The experiment was carried out in triplicates.
Calculations
Pollen viability was calculated as,
( )

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Kaur & Nag (2017) 4(2): 235241

Pollen size
Light microscope fitted with an ocular micrometer was used to study the size of pollen grains under the
magnification of 400X. Ocular micrometer was calibrated by using stage micrometer. At least10 pollen grains
were observed for each plant species on average and the studies were carried out in triplicates.
Statistical analysis
Data was analysed by using the self-coded software on Microsoft Excel 2007 for one-way ANOVA test to
compare differences among the studied variables. Tukeys Honestly Significant Difference test (Tukeys HSD)
was also performed for multiple comparisons between the means. Correlations between the parameters were
also evaluated by calculating the Pearsons correlation coefficient (r).

RESULTS
In the present study, an attempt was made to assess the effect of traffic load on pollen of three plant species
growing at seven sites of Amritsar city under different levels of traffic. Results for traffic load, pollen viability
and pollen size are presented in tables 3. Table 3 depicted varying levels of traffic for each site under study. The
highest number of vehicles was observed at the site RBC whereas the lowest number was observed at site KCC.
Though vehicles are not allowed inside Botanical Garden of Guru Nanak Dev University (BGG); however, it is
adjacent to GT Road, NH 1 (Table 3).
The percentage of pollen viability for Alstonia scholaris ranged from 32.45% (GTR) to 65.74% (GNC), for
Nerium oleander 30.3% (LGR) to 45.11% (KCC) and for Tabernaemontana divaricata 14.31% (RBC) to
49.62% (KPC). Pollen viability was found to be inversely proportional to the number of vehicles at a given site.
Figure 2 shows viable and non-viable pollen grains of A. scholaris, N. oleander and T. divaricata plant species.
Pollen size of A. scholaris was observed to range from 22.86 m (GNC) to 23.94 m (GTR), for N. oleander
29.58 m (GTR) to 32.17 m (RBC) and for T. divaricata it ranged from 29.83 m (RBC) to 32.43 m (KPC).

Figure 2. Viable and non-viable pollen grains of the plant species studied.
One way ANOVA test was used to compare the differences between the parameters observed for different
plant species growing at different sites of samples collection. ANOVA for pollen viability showed the statistical
significant difference with respect to their sites of samples collection whereas no statistically significant
variation was observed in pollen size with respect to different sites studied (Table 3). Tukeys HSD test was also
used to check the differences in pollen viability and pollen size at different sites studied.
Pearson correlation coefficient (r) was calculated to find out the correlation of pollen viability with traffic
load and pollen size with traffic load at p0.05 level of significance (Table 3). In the case of A. scholaris, a
significant negative correlation between pollen viability and traffic load at different sites was observed (r= -
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Kaur & Nag (2017) 4(2): 235241

0.762, p0.05), while other two species did not show any significant correlation. Pollen size did not show any
significant correlation with traffic density for all studied species.

Table 3. Pollen viability and pollen size of Alstonia scholaris, Nerium oleander and Tabernaemontana divaricata growing at the studied sites.
Site Site No. of Pollen viability (%) (Mean S.E.) Pollen size (m) (Mean S.E.)
No. code vehicles@ A. scholaris N. oleander T. divaricata A. scholaris N. oleander T. divaricata
1. BGG 0 61.972.916a 44.36 1.503ab 42.59 2.716a 23.56 0.19 ns 31.6 0.253 ns 31.6 1.647 ns
2. KCC 1891 51.395.175a 45.11 1.241a 28.18 2.668b 23.31 0.228 ns
30.34 0.063 ns
30.72 0.228 ns
3. GNC 1989 65.743.164a 33.15 3.879ab 26.75 1.076b 22.86 0.88 ns
31.1 0.708 ns
31.79 0.522 ns
4. KPC 2071 65.512.855a 32.48 3.75ab 49.62 0.743a 23.5 0.983 ns
31.98 0.887 ns
32.43 1.082 ns
b b
5. LGR 2563 33.82.096 30.3 4.836 16.47 0.598c 23.69 0.127 ns
31.22 0.253 ns
30.84 1.513 ns
b ab
6. GTR 3923 32.452.44 37.61 2.448 27.60 1.152b 23.94 0.396 ns
29.58 0.933 ns
32.17 1.104 ns
b ab
7. RBR 4551 32.591.068 32.47 1.775 14.31 1.455c 23.37 0.396 ns
32.17 0.887 ns
29.830.190 ns
ns ns
F-value 27.00* 3.71** 56.73* 0.37 0.28 0.76ns
HSD 14.70 14.75 8.23 - - -
Pearsons correlation -0.762*** -0.529ns -0.645ns 0.213ns 0.134ns -0.356ns
coefficient (r)
Note: BGG- Botanical Garden, Guru Nanak Dev University; KCC- Khalsa College Campus; GNC- Guru Nanak Dev University Whole
Campus; KPC- Kabir Park Colony; LGR- Lahori Gate Road; GTR- Grand Trunk Road; RBR- Ram Bagh Chowk. @Total number of vehicles
counted at each site for 3 hrs at different intervals of time i.e. 89 am; 121 pm; 45 pm.
Means with same superscript showed no significant difference at p0.05 between sites using Tukeys Multiple Comparison Test.
*Data is statistically significant at p0.001; **Data is statistically significant at p0.05; ***values show significant correlation between traffic
load and pollen viability/pollen size at p0.05 level of significance; ns values are not significant at p0.05 level of significance.

DISCUSSION
Automobile generated air pollutants and different environmental stresses have played a major role in
influencing the pollen viability and pollen size of different plant species (Gottardini et al. 2004, Higashitani
2013, Rezanejad 2013, Paupire et al. 2014).
The viability of pollen grains of plants under study was determined by TTC staining method which works on
the colouring of tissue. TTC gets reduced by the dehydrogenase enzyme of live tissues and results information
of a carmine red colour compound known as formazan (Beyhan & Serdar 2008). This method is relatively
simple and gives rapid results (Soares et al. 2013). This technique has been commonly used for studying the
effects of automobile generated airborne pollutants including heavy metals on viability/fertility of pollen grains
of several plant species (Duro et al. 2013). The results obtained by this method are well comparable to those
obtained by in vitro germination tests (Huang et al. 2004). Many studies have suggested the use of TTC as a
reliable method for assessment of pollen viability (Iannotti et al. 2000, Gottardini et al. 2004, Huang et al. 2004,
Abdelgadir et al. 2012, Kaur et al. 2016). Staining techniques used for estimation of pollen viability and size of
pollen grains depend on the type of plant species. These stains/dyes can easily point out differentiation among
viable and non-viable pollen grains (Kolodziejek & Gabara 2008, Sharafi 2011).
In the present study, as a result of vehicular emissions, remarkable effects on pollen viability of three plant
species under study were observed. Viable pollen grains were found to be darkly stained and round in shape
while non-viable pollen grains were found to be transparent and have shriveled structure. Pollen viability of
plant species studied was found to be negatively correlated with the traffic load (no. of vehicles) of different
sites where these plants were growing. Effects of air pollutants on pollen viability and size have been well
documented for different plant species (Chauhan & Singh 1996, Malayeri et al. 2012, Greguskova & Micieta
2013). Dickinson (2000) and Rezanejad (2009) observed alterations in proteins involved in pollen-stigma
interactions as a consequence of automobiles generated air pollution.
Pollen size of Alstonia scholaris, Nerium oleander and Tabernaemontana divaricata at all sites was found to
be unaffected irrespective of high vehicle number. One way ANOVA test showed no significant differences
between pollen grains size from different sites of sample collection whereas pollen viability of A. scholaris
(p0.001), T. divaricata (p0.001) and N. oleander (p0.05) showed statistically significant differences with
respect to their sites of sample collection. Our results are in conformity with some of the earlier studies on
different plant species like Daucus carota L., Convolvulus sepium L. and Dactylis glomerata L. (Iannotti et al.
2000); Cassia fistula L., Cannabis sativa L. and Thevetia peruviana (Pers.) K. Schum. (Kaur et al. 2016).
Pearsons Correlation Coefficient analysis suggests that among the three species studied, Alstonia scholaris
(r= -0.762, at p0.05) was found to be most sensitive to air pollution followed by Tabernaemontana divaricata
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(r=-0.645); Nerium oleander (r=-0.529) was relatively tolerant to traffic stress (Table 3). Hence, Alstonia
scholaris and Tabernaemontana divaricata can be used as better bio-indicators of vehicular air pollution
whereas N. oleander can be the plant of choice for polluted sites. Earlier also it was reported that pollen viability
can be used as reliable bio-indicator of atmospheric pollution (Iannotti et al. 2000, Greguskova & Micieta
2013). Malayeri et al. (2012) reported the use of pollen viability and morphology of six legumes viz., Cercis
siliquastrum L., Medicago sativa L., Robinia pseudoacacia L., Melilotus officinalis (L.) lam, Trifolium repens
L., and Sophoraalo pecuroides L. for bio-monitoring of fluoride generated pollution.

CONCLUSION
The present study showed that pollen viability relatively decreases with increase in vehicle number while
pollen size did not show any significant variation. On the basis of pollen viability data, N. oleander was found to
be tolerant to air pollution while A. scholaris was found to be highly sensitive followed by Tabernaemontana
divaricata, hence, the latter two can act as better indicators of air pollution with respect to traffic stress. Present
work provides information for three species only, which is insufficient to draw a specific conclusion. The effect
on pollen viability and pollen size depends on the type of plant species and level of traffic stress. It can be
concluded that pollen grains can give important information about the biological impact of air pollutants and can
be used as good candidates as bio-indicators of the atmospheric pollution. This kind of work may be helpful in
exploration of tolerance or sensitivity level of different roadside plant species against air pollution.

ACKNOWLEDGEMENTS
Thanks are due to University Grants Commission (UGC) for financial assistance under CPEPA, UPE and
DRS programes. Mandeep Kaur is recipient of UGC-BSR fellowship. Authors also thank Prof. A. K. Thukral,
Department of Botanical & Environmental Sciences, GNDU for designing self coded software in MS Excel
2007 for statistical analysis of the data.

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4(2): 242245, 2017
DOI: 10.22271/tpr.2017.v4.i2.033
Research article

Beneficial impact of phosphate solubilizing fungi on growth of


Saraca asoca (Roxb.) de Willd. under nursery condition
Soumya Ranjan Nayak, Manas Ranjan Panigrahi and Nibha Gupta*
Plant Pathology and Microbiology Division, Regional Plant Resource Centre, Bhubaneswar -751015 Odisha
*Corresponding Author: nguc2003@yahoo.co.in [Accepted: 12 June 2017]

Abstract: Saraca asoca belonging to family Caesalpinaceae commonly known as Sita Ashok,
and assessed as endangered; and important indigenous plant of India and the state flower of
Odisha. S. asoca is a medicinal plant that claims to cure several diseases according to Ayurvedic
medicine. A study was conducted to investigate the effects of phosphate solubilizing fungi on the
growth & establishment of Saraca asoca under nursery condition. Five phosphate solubilizing
fungi were supplemented on Saraca asoca seedlings separately and their effects on morphological
and physiological growth parameters were recorded. Inoculation of fungal culture Aspergillus
kanagawaensis showed significant improvement in shoot height, root length and leaf number and
Aspergillus japonicus developed the highest number of branches, Aspergillus niger showed
highest leaf area. Overall, application of fungal culture Aspergillus kanagawaensis and Aspergillus
niger resulted in increased biomass production as compared to control experimental sets.
Keywords: Phosphate solubilization - Aspergillus - Forest - Saraca asoca - Bioinoculant.

[Cite as: Nayak SR, Panigrahi MR & Gupta N (2017) Beneficial impact of phosphate solubilizing fungi on
growth of Saraca asoca (Roxb.) de Willd. under nursery condition. Tropical Plant Research 4(2): 242245]

INTRODUCTION
Saraca asoca (Roxb.) de Willd. is an indigenous medium sized evergreen tree belonging to the family
Caesalpinaceae with several vernacular names such as, Ashoka, Sita Ashok (Hindi), Shabuqa (Arabic), Kankeli
(Sanskrit) and Ashokadamara (Kannada) (Sharma et al. 2005, Anitha et al. 2008). Saraca asoca is one of the
important indigenous medicinal plants and found throughout India (Bajpai et al. 2016, Deepa et al. 2016, Bisht
et al. 2017). Almost every part of the plant is medicinal and it has been a part of ayurvedic medicine for
centuries, known to have anti-cancer, anti-hemorrhagic, anti-oxidant, anti-oxytocic and anti-microbial activities
(Nadkarni et al. 2005, Pradhan et al. 2009). The plant is under great threat due to management practices,
increasing demand for its phytochemicals, poor seed viability and overexploitation of the plant for its bark,
flower, seeds etc.; which has resulted in the dwindling of its population throughout the globe (Pushpangadan et
al. 2004). Bioinoculation of beneficial like phosphate solubilizing fungi may play a vital role in plant growth
and development: The role of such bio-inoculants in breaking seed dormancy through plant growth promoting
substances also had been reported (Chaukiyal et al. 2000, Chopade et al. 2008). Since, S. asoca has also enlisted
in RET list, its propagation, conservation has to gain priority due to its remarkable medicinal value (Chopade et
al. 2008). So the experiment was carried out to evaluate the impact of some phosphate solubilizing fungi on
Saraca asoca in nursery condition for better establishment and growth.

MATERIALS AND METHODS


The brownish to deep brown colored seeds were decapsulated and germinated. Seedlings of 30 days were
transplanted into the poly bags containing fumigated soil. Six different phosphate solubilisng fungal isolates
(confirmed earlier through plate culture test performed on Pikovskaya medium) identified as Aspergillus
kanagawaensis, Penicillium citrinum, Aspergillus japanicus, Aspergillus niger, Fusarium oxysporum were used
for the experiment. The seven day old culture developed in Sabouraud Dextrose broth at 30C were inoculated
(@100 ml per poly bag) separately & thrice with monthly interval. Un-inoculated plants, autoclaved inoculated
cultures were considered as control and treated control, respectively. Regular watering was done to keep the

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https://doi.org/10.22271/tpr.2017.v4.i2.033
Nayak et al. (2017) 4(2): 242245

plants healthy. Finaldata was recorded on 140th day for Plant height, fresh and dry biomass, root-shoot ratio,
physiological growth parameter like relative growth rate, net assimilation rate, leaf area ratio, quality index,
were calculated Dori et al. (1990).

RESULTS AND DISCUSSION


Table 1. Growth performance of Saraca asoca seedling under different treatment.
Shoot Root Wet Biomass(g) Dry Biomass(g)
No. of Leaf Area
Treatment Height Length
leaves (mm2) Shoot Root Shoot Root
(cm) (cm)
Control 30.46.3 20.65.9 25.413.2 2645.6196.1 12.3286.5 8.0695.0 6.0622.9 3.2481.5
Aspergillus kanagawaensis 45.413.8 24.96.7 49.19.9 7955.4328.8 27.71913.1 10.5643.9 12.8325.3 5.1042.0
Aspergillus kanagawaensis TC 25.34.2 24.32.1 28.72.1 2408347.6 15.6272.0 9.7530.6 9.2212.1 5.1100.5
Penicillium citrinum 22.34.7 17.92.9 14.97.5 4414.4185.2 4.6572.7 3.8741.8 1.8471.2 1.5060.9
Penicillium citrinum TC 26.02.0 14.32.3 10.32.1 2877327.8 1.6020.8 2.7910.4 0.6060.3 0.8191.6
Aspergillus japanicus 26.37.4 15.63.5 32.914.0 4404.8356.1 11.7534.7 7.1025.1 4.8051.9 2.5221.5
Aspergillus japanicus TC 23.33.5 16.01.0 28.79.1 2190130.6 8.5362.6 6.7303.0 3.7351.6 2.7150.8
Aspergillus niger 38.36.2 19.04.3 42.39.6 9051.43050.1 20.6455.8 8.2781.9 9.6012.8 4.1201.3
Aspergillus niger TC 28.010.6 19.75.0 26.020.5 3291659.5 12.5677.1 8.9428.5 5.6033.6 2.6970.4
Fusarium oxysporum 24.35.4 18.13.7 21.811.1 5015163.6 11.4046.2 5.3572.4 4.7502.4 2.0571.0
Fusarium oxysporum TC 24.35.5 17.01.0 13.36.0 2833397.9 3.8421.3 3.1811.4 1.6890.9 1.3330.9
Note: TC= Treated Control.
A different pattern of plant growth was observed in different treatments as well as un-inoculated control and
treated control. The data of shoot height, root length of seedlings in the nursery at un-inoculated control,
inoculated heat treated control and treated seedlings are depicted the prominent and significant differences
among the treatments and it was higher in plants treated with Aspergillus kanagawaensis and Aspergillus niger
(Table 1). Aspergillus japanicus illustrated highest number of branches. Aspergillusniger treated plants showed
a significant difference in leaf area. Aspergillus kanagawaensis and Aspergillus niger exhibited growth
promoting effect over control and treated control as far as shoot height, leaf number, leaf area and plant biomass
is concerned. Differences root lengths were not reflected in all control and inoculated plants. Inoculation of
Aspergillus kanagawaensis was found to be best in enhancing shoot fresh and dry biomass over other treatments
as well as control& treated control. The inoculation of phosphate solubilizing fungi Penicillium citrinum,
Fusarium oxysporum & Aspergillus japanicus also yielded good leaf growth (leaf area) as compared to control
and treated control plants. Aspergillus kanagawaensis showed a better change in Relative growth rate (RGR)
and Net assimilation rate (NAR) was also changed due to the enhancement in dry biomass (Table 2).
Table 2. Physiological growth performance of Saracaasoca under pot culture condition.
Aspergillus Penicillium Aspergillus Aspergillus Fusarium
Prameters Control kanagawaensis citrinum japanicus niger oxysporum
LC AC LC AC LC AC LC AC LC AC
Wet RGR (d-1) 0.117 0.288 0.153 0.031 0.003 0.110 0.074 0.209 0.119 0.106 0.022
Dry RGR (d-1) 0.060 0.135 0.095 0.013 0.001 0.046 0.034 0.099 0.055 0.045 0.011
NAR (g.m-2.d-1) 60.75 853.92 73.89 35.99 1.08 127.10 19.02 735.39 90.84 152.97 13.44
LAR (m2.g-1) 0.63 0.71 0.41 2.39 3.49 1.10 0.86 1.04 0.78 1.22 1.88
QI 0.289 0.374 0.529 0.143 0.053 0.260 0.261 0.338 0.276 0.256 0.118
RSR 0.678 0.548 0.960 0.803 0.550 0.593 0.687 0.496 0.704 0.745 0.700
Note: LC= Live Culture, AC= Autoclaved Culture.
In view to evaluate the effective plant growth promoting ability of phosphate solubilizing fungion forest
trees, present experiment was set up on Saracaasoca in pot culture conditionwhich was supplemented with
liquid cultures of phosphate solubilising fungi namely: Aspergillus kanagawaensis, Penicillium citrinum,
Aspergillus japanicus, Aspergillus niger, Fusarium oxysporum. The different fungal strains used in the present
study were having phosphate solubilising potential tested in plate culture method in laboratory earlier. Odee et
al. (2002) recommended inoculation of liquid cultures to raise the healthy seedlings in the nursery conditions.
This also helps in increasing fungal population in the rhizosphere and finally mineral solubilisation. The costly
affair of chemical fertilizers, as well as limited supply of nutrient to the plants, demands an alternative like
mineral solubilising microbes. The enhancement in the growth of Saraca asoca in pot culture conditions in the
present experiment revealed the effect of fungal inoculation and their usefulness as bioinoculants for plant

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Nayak et al. (2017) 4(2): 242245

productivity. Experimental seedling of Saracaasoca supplied with different fungal cultures had exhibited good
growth in terms of plant height and biomass. Under experimental conditions Aspergillus kanagawaensis was
found to be very effective in increasing over all plant growth including shoot height, root length, number of
branches, no of leaves, leaf area and biomass. However, Aspergillus niger was also prominent in increasing leaf
area of the plants. Another fungal culture namely Aspergillus japanicus was able to increase branch numbers.
The experimental plants were surviving and growing well even after 140 days without any further addition
of chemical fertilizers indicate its role towards seedlings establishment after transfer and subsequent growth
(Maliha et al. 2004, Hossain et al. 2007, Nenwani et al. 2010) . Manyfungi like Penicillium and Aspergillus etc.
were reported as phosphate solubilisers as well as evaluated for the plant growth and productivity (Dash et al.
2013, Vibha et al. 2014). Present study also confirms these findings as inoculation of Aspergillus
kanagawaensis and Penicillium citrinum into the rhizosphere of S. asoca seedlings were found to be effective in
enhancing and improving plant growth in terms of LAR and RSR. The carbon assimilation and allocation may
be reflected in the trait of LAR as well as growth variation may be due RGR and NAR Krishnan & Satakappam
(2009). Significant differences in plants physiological parameters like relative growth rate, net assimilation rate
and leaf area ratio were observed in inoculated and uninoculated seedlings. However, data recorded through
present study will be helpful in the development of bioinoculants of Aspergillus kanagawaensis, Aspergillus
niger for the mass scale propagation of S. asoca for commercial use as thistree species is a good source of
medicinal compounds.

CONCLUSION
From the above experiment conducted to evaluate the application of phosphate solubilizing potential of
selected fungal strains in Saraca asoca was successful and the plants show tremendous growth and development
as compare to controls. This concludes the effectiveness of fungal microbes and endophytic activity with plant
root system. Further study and research has to be needed so as to confirm the fungal diversitys adaptation with
other forest trees which are more prone to be endangered.

ACKNOWLEDGMENTS
Authors acknowledge the financial assistance obtained from Forest and Environment Dept., Govt. of Odisha
through State Plan project 201516.

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4(2): 246253, 2017
DOI: 10.22271/tpr.2017.v4.i2.034
Research article

Synthesis and characterization of copper oxide nanoparticles and


its impact on germination of Vigna radiata (L.) R. Wilczek
Ajey Singh, N. B. Singh*, Imtiyaz Hussain, Himani Singh and Vijaya Yadav
Plant Physiology Laboratory, Department of Botany, University of Allahabad, Allahabad-211002, U.P, India
*Corresponding Author: singhnb166@gmail.com [Accepted: 18 June 2017]

Abstract: Nanoparticles (NPs) and its disposal via anthropogenic activities are a new concern
nowadays. The impact of copper on plant metabolism has not yet been studied in detail. In this
study, Copper oxide nanoparticles (CuO NPs) were synthesized by wet precipitation method using
copper acetate monohydrate precursor. Powder X-ray diffraction, particle size analyzer, scanning
electron and transmission electron microscope analysis revealed that synthesized CuO NPs are in
nano size range. The impact of synthesized NPs on germination and seedling growth of Vigna
radiata were tested. Germination and seedling growth of the plants were almost unaffected at
lower concentrations while significant inhibition was recorded at highest 1000 mg.l-1 concentration
of NPs as compared to control. Protein and sugar content were used as biochemical parameters to
estimate the effects of different concentration of CuO NPs on the metabolism of seedlings. The
amount of sugar and protein registered slight higher value at lower concentration and sharply
decreased with increase in concentration. By analyzing this compiled information, it is evident that
CuO NPs exhibits a toxic effect on growth and physiology of test plant while at very low
concentration effect of NPs may be little favorable also.
Keywords: Anthropogenic activities - CuO NPs - Wet precipitation - Biochemical parameters.

[Cite as: Singh A, Singh NB, Hussain I, Singh H & Yadav V (2017) Synthesis and characterization of copper
oxide nanoparticles and its impact on germination of Vigna radiata (L.) R. Wilczek. Tropical Plant Research
4(2): 246253]

INTRODUCTION
Nanoparticles (NPs) are natural or anthropogenic materials having at least two dimensions between 1100
nm. NPs possess numerous distinctive properties like high reactivity, unique optical properties and bio-
compatibility in comparison to their bulk counterparts due to their very high surface area to volume ratio
(Garima et al. 2011). In recent years, nanomaterials (NMs) have contributed in economy of many sectors
including consumer products, energy, transportation, cosmetics, pharmaceuticals, antimicrobial agents and
agriculture (Singh et al. 2015a). Nanotechnology has tremendous potential to boost agricultural production to
fulfill food, fodder and fiber requirements of humans. Use of NPs as fertilizer has a great potential to augment
crop yield and diminish environmental threats (Singh et al. 2013, Roohizadeh et al. 2015). On the other hand
extensive release of NPs in the environment causing toxicity to the flora and fauna also (Franklin et al. 2007).
The hazardous effects of metal oxide NPs can be due to the dissolved ionic metal, or there may be special
hazards from NPs themselves due to improved bioactivity. Many authors have pointed out the importance of
difference in the toxicity of NPs from heavy metal toxicity (Franklin et al. 2007, Griffitt et al. 2007, Karlsson et
al. 2008).
Among the different types of metallic NPs, Copper oxide nanoparticles (CuO NPs) have been mainly used as
preliminary material due to its natural abundance, low cost production, non-toxic nature with good electrical and
optical properties. Copper (Cu) have position in block D and period four of the periodic table and it is a
microelement required for the development of plant. Passam et al. (2007) reported that Cu in 10-14 to 10-16 M
concentrations is required for normal development of plant, below which deficiency occurs however higher
concentration than optimum exhibited toxicity. The Cu deficiency in plants is expressed as curled leaves;

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Received: 29 October 2016 Published online: 30 June 2017
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Singh et al. (2017) 4(2): 246253

petioles bent downwards and light chlorosis as well as permanent turgor loss in the young leaves while the
higher concentration of Cu leads to toxicity, growth inhibition, photosynthesis interferences, photo respiration
and increases oxidative stress (Yruela 2005, Passam et al. 2007, Manceau et al. 2008). The extensive use of
CuO NPs has increased concerns over their potential toxic impacts on ecosystem and human health due to their
discharge from different products to the environment (Chen et al. 2012). Previous reports have revealed that
exposure to CuO NPs caused toxic effects on aquatic organisms such as protozoa, crustaceans, algae, and zebra
fish (Nair & Chung 2014). The effect of Cu NPs on plant cells has not been studied sufficiently and the
available results are uncertain. It has been reported that Cu is biologically available to mung bean and wheat
germs (Lee et al. 2008). Cu exerts its toxic impact by penetrating the cell directly, apparently by causing
oxidative damage to cell structures and molecules (Garnett 2007). Cu NPs show positive effects on germination
(Shah & Belozerova 2009) but are phytotoxic at seedling growth (Lee et al. 2008).
Current progress in the field of nanotechnology has guide to the development a new perception of
synthesizing nano sized particles of preferred size and shape (Kumar et al. 2015). Synthesis of metal NPs can be
accomplished through various methods. Chemical approaches are the most popular methods for the production
of NPs. There are several schemes for the synthesis of CuO NPs like precipitation methods, decomposition
methods, plasma methods, pulsed wire explosion methods, sol gel methods, vapour deposition, electrochemical,
radiolysis methods and so on (Hussain et al. 2016).
The purpose of this study was to synthesize CuO NPs via wet precipitation method and to analyze specific
effect of CuO NPs at different concentration on the germination and growth of Vigna radiata (L.) R. Wilczek as
it is one of the most important staple food crops in India.

MATERIALS AND METHODS


Synthesis of copper oxide nanoparticles
CuO NPs were synthesized through wet precipitation method (Zhu et al. 2011). Copper acetate monohydrate
(99%), glacial acetic acid (99%) and sodium hydroxide were used without further purification as they are of
analytical grade.
In a typical procedure, about 0.2 M of Copper acetate monohydrate (Cu(CH3COO)2.H2O) was dissolved in
300 ml of water in round bottomed flask and 1 ml of glacial acetic acid (CH 3COOH) was added into that flask
and heated to boiling under continuous stirring. After 30 minutes 15 ml of 8 M sodium hydroxide (NaOH)
solution was gradually tipped as reducing agent with the help of a needle into the flask. The colour of the
solution turned black from blue immediately after addition of NaOH. The reaction was carried out under stirring
for 2 hrs, great quantity of black precipitate appeared. The mixture was cooled in room temperature and then
centrifuged, obtained wet precipitate was washed twice with DDW to eliminate the impurity. The precipitate
was then dried at room temperature for 48 hrs. Brownish black colored powder was obtained and this was
carefully collected and stored for characterization.
Characterization of synthesized copper oxide nanoparticles
First round characterization of the CuO NPs was carried out by X-Ray diffraction (XRD). The XRD pattern
of the powdered metal was recorded with the XRD equipment i.e. Rigakudmax 2200 diffracto meter with Cu-
K radiation (k = 1.5406 ). Particle size distribution of the CuO NPs was measured by computer controlled
particle size analyzer (Nanotrac wave W3372) to find out the particle size distribution. SEM studies of the
synthesized particles were done by JEOLJXA-8230. The synthesized CuO powder was mixed into DDW and
sonicated for 30 min. A tiny drop of the sample was permitted to dry on glass slide to make a thin layer of NPs
for the SEM investigation. TEM images were captured on a TECNAI 200 Kv TEM instrument. The nano
powders were diluted with DDW and dispersed by ultrasonic bath. Then, one drop was placed on a carbon-
coated copper grid and left to dry at room temperature.
Germination and seedling vigor index
Synthesized CuO NPs were suspended directly in DDW and discrete by ultra-sonic vibration for 30 min. The
solutions for seed treatments were prepared in five different concentrations: 1 mg.l-1, 10 mg.l-1, 100 mg.l-1, 500
mg.l-1 and 1000 mg.l-1 and labeled as Cu1, Cu2, Cu3, Cu4 and Cu5 respectively. Ten vigna seeds in triplicates
were soaked in 20 mL of respective concentration for 34 hrs. The seeds imbibed in DDW only were taken as
control and named as CuC. Treated vigna seeds were placed at equal distance in sterilized petriplates lined with
Whatman No 1 filter papers. 2 ml of respective concentrations was added in each petriplate. Petriplates were
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covered and kept in a dark place for seven days. Seed germination and seedlings growth were recorded at
regular interval of 24 hrs. Seedling vigor index (SVI) was calculated by the formula following Abdul-Baki &
Anderson (1973).
Seedling vigor index = Germination% (root length + shoot length)
Protein and sugar content
Quantitative analysis of protein was done following Lowry et al. (1951). The quantity of protein was
measured with reference to a typical curve of bovine serum albumin. The absorbance was calculated at 650 nm.
Total soluble sugars quantification was done by following Hedge & Hofreiter (1962). The quantity of sugar was
determined by the standard curve prepared from glucose. 0.05 mg fresh leaf tissue was homogenized in 5 ml of
95% ethanol. Following centrifugation, 1 ml of supernatant was thoroughly mixed with 4 ml anthrone reagent
and heated for 10 min in boiling water bath. Following cooling, the absorbance was recorded at 620 nm.
Statistical Analysis
Treatments were arranged in a randomized block design with three replications. Data were statistically
analyzed using ANOVA by using SPSS (Ver.10; SPSS Inc., Chicago, IL, USA). Fitting standard errors of the
means (SEM) were calculated for presentation of graphs. The treatment means were evaluated by Duncans
multiple range test (DMRT) at p < 0.05.

RESULTS AND DISCUSSION


Synthesis of copper oxide nanoparticles
The conventional and well known method for synthesis of metallic NPs is wet-chemical procedure. This
method involves growing of NPs in a liquid medium having different reactants, in particular reducing agent. The
stabilizing agent is also added to the reaction mixture to avoid the agglomeration of NPs (Singh et al. 2016).
Colour change of the reaction mixture was observed from deep blue to colour less and then to dark brown on
vigorous stirring (Fig. 1). In this process, CuO produced due to reaction between Cu++ ions from copper acetate
monohydrate and reducing agents i.e. NaOH. The synthesis process is based on the following chemical
reactions:
Cu2+ + 2NaOH = Cu(OH)2 + 2Na+ (1)
Cu(OH)2=CuO + H2O (2)

Figure 1. Picture of aqueous solution of Cu(CH3COO)2H2O before adding NaOH and after addition of NaOH.
Characterizations of copper oxide nanoparticles
X-ray diffraction (XRD) is recognized as an analytical method for detection and quantitative determination
of various crystalline forms which are also known as phases of compound there in powder and solid samples.
The XRD technique was used to determine and confirm the crystal structure of the NPs. The XRD patterns of as
synthesized CuO NPs are shown in figure 2(A). The characteristic peaks located at 2 = 30.5, 38.97 and 48.

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Singh et al. (2017) 4(2): 246253

74 are assigned to (111), (200) and (202) plane orientation of CuO (JCPDS 80-1268). XRD pattern showed
broad diffraction peaks indicating crystalline and nanoscale dimensions of particles. All diffraction peaks can be
indexed as typical monoclinic in structure and no other phases were observed.

Figure 2. Characterization of synthesized CuO NPs (A) X-ray diffraction pattern of the synthesized CuO NPs (b) Particle
size distribution image of the synthesized CuO NPs (C) SEM image of the synthesized CuO NPs (D) TEM image of the
synthesized NPs.
Dynamic light scattering (DLS) is a method used to decide the size distribution profile of minute particles in
suspension or polymers in solution. The size of the particles depends on the size of the size of surface structure,
the particle core, concentration of particle and the type of ions in the medium. Particle size analyzer study
exposes that the most of the particles in the solution are in range of 35 to 40 nm (Fig. 2B).
SEM image (Fig. 2C) of CuO NPs demonstrated the presence of some large particles, which can be
credited to aggregation or overlapping of smaller particles with sizes around 100 nm. TEM image (Fig. 2D)
showed a not well-dispersed and spherical shape for green synthesized CuO NPs with a size range of 20 to 40
nm. The larger particle size is due to the short clusters shown in the TEM image.
It is interesting to note that there is variation in the size of NPs measured with different characterization
tools. This can be attributed with the findings of Singh et al. (2016), where authors stated that XRD measures
the crystalline size which is smaller than the real particle size while diameter measured using DLS is
hydrodynamic diameter of NPs, which is bigger than the actual particle size. In DLS measurement liquid layer
moves with NPs during Brownian motion which results in the increased size of NPs than actual size.
Germination and seedling vigor index
Seed germination was recorded up to 6 days. CuO NPs did not show any toxicological effect on germination
up to 500 mg.l-1 concentration. Effect of different concentration of CuO NPs on seedling growth of Vigna
radiata (L.) R. Wilczekis shown in figure 3. However slight enhancement in seed germination was recorded in
lower concentrations of NPs i.e. 1 and 10 mg.l-1. Highest concentration i.e. 1000 mg.l-1 of NPs adversely
affected the seed germination. Maximum 27% of inhibition is found in seed germination in seeds treated with

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highest concentration (Fig. 4A). Seed germination is the beginning of the physiological process that needs water
imbibitions (Kathiravan et al. 2015). Enhanced seed germination in lower concentration of NPs may be
attributed with the findings of Khodakovskaya et al. (2012) as stated that NPs have tendency to penetrate plant
seed coats and enhance seed germination and growth. CuO NPs would have penetrated the cell wall and guides
new pores formation as a result increase in water absorption which is favorable for seed germination.
Germination of lettuce seeds was promoted by Cu NPs (14). In contrast to our result Adhikari et al. (2012)
reported that the germination of soybean and chickpea was not checked up to 2,000 ppm concentration of CuO
NPs. Seedling growth and seedling vigor index (SVI) also followed the same trend.

CuC Cu1 Cu2 Cu3 Cu4 Cu5

Figure 3. Image showing effect of CuO NPs on seed germination and seedling growth of Vigna radiata (L.) R. Wilczek
seedling. CuC = Control; Cu1 (1 mg.l-1); Cu2 (10 mgl-1); Cu3 (100 mg.l-1) Cu4 (500 mg.l-1); Cu5 (1000 mg.l-1)

Figure 4. Effect of CuO NPs on (A) seed germination, (B) seedling vigor index (SVI), (C) plumule length and (D) radicle
length of Vigna radiata (L.) R. Wilczek seedling. Data are means standard error of three independent experiments with
three replicates in each experiment. Bars followed by different letters show significant differences at p < 0.05 significance
level between treatments according to the Duncans multiple range test. CuC = Control; Cu1 (1 mg.l-1); Cu2 (10 mg.l-1); Cu3
(100 mg.l-1) Cu4 (500 mg.l-1); Cu5 (1000 mg.l-1)
Radicle (RL) and plumule length (PL) also followed the germination result. However it is interesting to note
that radicle length was found more susceptible to CuO NPs. There was no promoting effect found in RL at any
concentration of NPs however decrease RL was concentration dependent. Declining trend in RL and PL at
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concentrations higher than 1000 mg.l-1 might be due to more absorption of NPs leading to phytotoxic effects. In
this study seed germination was not significantly decreased by NPs because NPs could not pass through seed
coats. NPs could contact radicals directly after its emergence through seed coat. Since roots are the first tissue to
confront with high concentration of NPs therefore altered growth in radical has dose dependent response. NPs
clog with root openings thus nutrient uptake inhibited resulted in decreased plant growth (Adhikari et al. 2012).
It was observed that with increase in NPs concentration PL also was found to decline as PL is dependent on the
growth and health of radicle. Effect of CuO NPs seems concentration dependent and higher concentration may
be harmful to plants. Maximum inhibition of 74 and 70% was recorded in RL and PL respectively (Fig. 4).
Toxic effects of Cu-NPs in various studies reporting that concentration were higher than 200 ppm (Doshi et al.
2008, Shah & Belozerova 2009). For mung bean seedling the best growth response for radicle and plumule was
observed at lowest concentration. Ptsikk et al. (2002) and Yruela (2005) stated that elevated concentration of
Cu may injure thylakoid membranes, in that way distressing the functioning of photosystem II and the water-
oxidizing complex of chloroplasts. There is 7% stimulation was recorded in the SVI of the seeds treated with 1
mg l-1 concentration.

Figure 5. Effect of CuO NPs on sugar and protein content of Vigna radiata (L.) R. Wilczek seedling. Data are means
standard error of three independent experiments with three replicates in each experiment. Bars followed by different letters
show significant differences at p < 0.05 significance level between treatments according to the Duncans multiple range test.
CuC = Control; Cu1 (1 mg.l-1); Cu2 (10 mg.l-1); Cu3 (100 mg.l-1) Cu4 (500 mg.l-1); Cu5 (1000 mg.l-1)
The increase in CuO NPs concentrations on vigna seedlings appeared to be negative, primarily due to
reduced sugar and protein content. A decrease in sugar and protein under unfavourable conditions allows the
conservation of energy, thereby launching the appropriate defense response and also reducing the risk of
damage. The protein and sugar content of seedling decreased significantly, reaching the minimum value in the
plant treated with 1000 mg.l-1 of CuO NPs (Fig. 5). Maximum inhibition of 44 and 46% was recorded in protein
and sugar content of the seeds treated with highest concentration respectively. Negative effects of Cu-NPs on
root (Adhikari et al. 2012), seedling growth (Shah & Belozerova 2009) and shoot growth (Stampoulis et al.
2009) on different plants have been reported. The significant reduction in protein and sugar content might be
due to the reduced biomass upon exposure to higher concentrations of CuO NPs or due to the membrane
damage under oxidative stress (Halliwell & Gutteridge 1989). It has been reported in various studies that metal
oxide NPs at their high concentration alters the physiological processes of the plants (Singh et al. 2015b,
Hussain et al. 2015).

CONCLUSION
Heavy use and release of NPs in the environment causing toxic impact on crop plants. Wet precipitation
method was found to be very efficient in producing small sized CuO NPs. The influence of synthesized NPs on
germination and seedling growth as well as protein and sugar content of Vigna radiata (L.) R. Wilczek was
investigated. The results showed that exposure to higher concentrations of CuO NPs has resulted in significant

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reduction in the seed germination and caused retardation of seedling growth in test plant. Moreover, exposure to
CuO NPs has resulted in inhibition of protein and sugar content of seedlings which affected plant metabolism.
However, it is interesting to note that lower doses of CuO NPs were found to be not even non-toxic while
favoured seed germination, seedling growth and metabolism of the test plant to some extent. Cu being a micro
element is essential for growth and metabolism of plants. Further study is required to explore the minimum and
favourable concentrations of CuO NPs which may be beneficial for plant growth and metabolism to increase the
productivity as well as safe release in the environment. Moreover these findings can guide for further
investigation of the mechanism of NPs transport within the plant and the potential implications of NPs in the
food chain.

ACKNOWLEDGMENTS
The authors are thankful to the University Grant Commission (UGC), New Delhi, India and University of
Allahabad, India for providing financial assistance to Ajey Singh. Dr. Vikas Baranwal, Nanotechnology
Application Centre, A.U, Dr. M. Dwivedi, Centre for Petrology and Mineralogy A.U, MNIT, Allahabad and
SAIF, AIIMS (New Delhi) are acknowledged for providing XRD, SEM, DLS and TEM facilities respectively.

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ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(2): 254263, 2017
DOI: 10.22271/tpr.2017.v4.i2.035
Research article

Comparative effect of arbuscular mycorrhiza, cowdung and


phosphorus on growth and yield contributing characters
of red amaranth (Amaranthus tricolor L.)
and Indian spinach (Basella alba L.)
Aporna Ghosh1, Md. Tahjib-Ul-Arif2*, S. G. Chamely1, M. R. Haque2,
Md. Mokhlesur Rahman1 and Md. Asadul Haque Bhuiyan3
1
Department of Agricultural Chemistry, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
2
Department of Biochemistry and Molecular Biology, Bangladesh Agricultural University, Mymensingh-2202,
Bangladesh
3
Soil Science Division, Bangladesh Agricultural Research Institute, Gazipur, Bangladesh
*Corresponding Author: arif1002215@gmail.com [Accepted: 22 June 2017]
Abstract: A pot experiment was conducted to study the effect of arbuscular mycorrhiza, cowdung
and phosphorus on red amaranth and Indian spinach in the net house of the Department of
Agricultural Chemistry, Bangladesh Agricultural University, Mymensingh. The experiment was
laid out in a Completely Randomized Design (CRD) with five replications and 8 treatment
combinations comprised of T1: Control, T2: Arbuscular mycorrhiza (AM), T3: Cowdung (CD), T4:
Phosphorus (P), T5: AM + CD, T6: CD + P, T7: AM + P and T8: AM + CD + P. Red amaranth
(Amaranthus tricolor L.) cv. BARI Lalshak-1 and Indian spinach (Basella alba) cv. BARI
Puishak-1 were used as test crops for the experiment. The experimental results showed that there
were significant differences in growth and yield characters such as plant height, plant diameter,
number of leaves per plant, shoot fresh and dry weights, root fresh and dry weights of red
amaranth and Indian spinach due to the application of arbuscular mycorrhiza (AM), cowdung
(CD) and phosphorus (P) independently and combinedly. When arbuscular mycorrhiza, CD and
phosphorus were applied combinedly, it exhibited a remarkably higher response in growth and
yield of these vegetables.
Keywords: Arbuscular mycorrhiza - Biofertilizer - Cowdung - Phosphorus - Growth and yield.

[Cite as: Ghosh A, Tahjib-Ul-Arif M, Chamely SG, Haque MR, Rahman MM & Bhuiyan MAH (2017)
Comparative effect of arbuscular mycorrhiza, cowdung and phosphorus on growth and yield contributing
characters of red amaranth (Amaranthus tricolor L.) and Indian spinach (Basella alba L.). Tropical Plant
Research 4(2): 254263]
INTRODUCTION
The arbuscular mycorrhiza (AM) helps most of the plant species in the uptake of moisture and nutrients from
the soil by forming symbiotic association with roots (Marschner & Dell 1994). Arbuscular mycorrhizas (AMs)
are a unique example of symbiosis between two eukaryotes, soil fungi and plants. This association induces
important physiological changes in each partner that lead to reciprocal benefits mainly in nutrient supply
(Balestrini & Lanfranco 2006). The external AM hyphae extend several centimeters from the infected root
surface and help in the exploration of greater soil volume to absorb nutrients and moisture from the soil.
Besides, they increase the rate of photosynthesis and enhance production of growth regulating substances in the
host plants (Danneberg et al. 1993). The benefit of mycorrhizae to plant nutrients is especially phosphorus.
Because of such benefits, the AM fungi can be used as bio-fertilizer for enhancing growth and yield of most
crop plants.
Field responses were often unsatisfactory; especially in high-input agricultural systems where it is concluded
by many that the mycorrhizae had little practical importance in agriculture. It is a well-documented fact that the
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Received: 17 February 2017 Published online: 30 June 2017
https://doi.org/10.22271/tpr.2017.v4.i2.035
Ghosh et al. (2017) 4(2): 254263

host plant is enormously benefited due to AM association. Certainly, agriculturists should appreciate the
distribution of mycorrhizae within their systems and understood the impact of their management decisions on
mycorrhizal functioning. With the fungi, much less fertilizer is needed, reducing the sort of over-fertilization
that leads to runoff and contamination of ground water. In Bangladesh, last few years much research has been
carried out on different crops to find out the effect vesicular-arbuscular mycorrhiza fungi. Large numbers of
VA-mycorrhizal fungi have been detected in different soil types for identification and inoculums production,
and for use in nurseries and the field as an alternative to chemical fertilizers and pesticides, and to progress
towards nature farming.
Phosphorus is one of the major limiting plant nutrients in soils world over. The presence of phosphorus in
the soil encourages plant growth because phosphorus is essential mineral macronutrients. Most of the essential
plant nutrients, including phosphorus, remain in insoluble form in soil (Abd-Alla 1994, Yadav & Verma 2007).
Most agricultural soils contain large reserves of phosphorus, a considerable part of which has accumulated as a
consequence of regular applications of P fertilizer (Richardson & Simpson 2011). A large portion of soluble
inorganic phosphate when applied on soil as chemical fertilizer is rapidly immobilized soon after application
and becomes unavailable to plants (Harrison et al. 1972, Yadav & Verma 2007). The fixation of native and
applied phosphorus either by adsorption or by chemical precipitation is generally considered the main cause of
its low availability to crops in vast majority of soils. Therefore, the release of insoluble and fixed forms of
phosphorus is an important aspect of increasing soil P availability.
Soil fertility deterioration is a major constraint for higher crop production in Bangladesh. Due to increasing
land use intensity, inadequate and unbalanced use of chemical fertilizers, little or no use of organic manure the
fertility of our soils were deteriorating which caused declining of crop production. Soil organic matter is a key
factor for sustainable soil fertility and crop productivity. Losses of soil organic matter can only be replenished in
the short term by application of organic matter such as manure (Glaser et al. 2001). Cowdung is one of the most
important organic materials and good source of organic matter. Cowdung releases nutrient slowly so that
nutrient loss is less followed by more plant uptake. It is quite promising to use of organic manure and chemical
fertilizers in integrated manner for soil fertility. The present day concern about global environmental pollution
can be reduced to a considered extent by the judicious use of chemical fertilizers and increasing the use of
organic manure.
Red amaranth (Amaranthus tricolor L.) and Indian spinach (Basella abla L.) both are the important leafy
vegetables in Bangladesh member of the family Amaranthaceae Basellaceae, respectively. Red amaranth has
been growing in Bangladesh both in winter and summer seasons. It can be harvested within a very short time.
Indian spinach is a perennial plant. In cooler areas, it can be grown as an annual during the warmer pant of the
year. It is a soft-stemmed vine. The green leaves are thick and semi-succulent. Indian spinach is a very fast
growing vine and grows in full sunlight but slowly in high pH soils. Both the crops are very tasty and nutritious.
The demand for organic vegetables is increasing notably among the health conscious people in our country
like elsewhere of the globe. Different leafy vegetables are widely grown throughout the country usually with
high doses of chemical fertilizers along with or without organic manure. Because of the beneficial effects of AM
fungi on crop plants, it was assumed that combined application of AM fungi with chemical fertilizers and
organics manure might improve growth and yield of leafy vegetables. The rate of application of chemical
fertilizers might also be reducing with the use of AM bio-fertilizer. This would help reduce the adverse effect of
high dose of chemical fertilizer on soils and the cost of production would also be reduced. Keeping the above
viewpoint, these experiments were undertaken to evaluate the response of arbuscular mycorrhiza, cowdung and
phosphorus on the growth and yield of red amaranth and Indian spinach.

MATERIALS AND METHODS


Experimental site and location
The experiment was conducted in the net house of the Department of Agricultural Chemistry, Bangladesh
Agricultural University, Mymensingh. The climate of the experimental area was under the sub-tropical climatic
zone.
Collection and preparation of soil sample
The soil used in this experiment was collected from Central Farm of Bangladesh Agricultural University,
Mymensingh. The soil was dried and the clods were broken and sieved to remove weeds, stubbles and hard
clods. Before starting the experiment, the soil was analyzed primarily for its physical and chemical properties.
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Organic carbon, soil pH, soil texture, cation exchange capacity, total nitrogen, available phosphorus,
exchangeable potassium, available sulphur were analyzed.
Table 1. Different properties of experimental soil.
pH CEC Total N Organic carbon Organic matter Available P Exchangeable K Available S
6.18 8.4% 0.10% 0.71% 1.16% 12.6 g.g-1 0.14 cmol.kg-1 10.9 g.g-1
Analysis of cowdung
Cowdung manure was analyzed for assessing the constituents of N, P, K, S, Ca, Mg and Fe following the
standard methods used for analysis. Total N (%)=1.28, P (%)=0.60, K (%)=1.65, Ca (%)=0.43, Mg (%)=0.28, S
(%)=0.16, Fe(%)=14.47.
Test crops
Red amaranth cv. BARI Lalshak-1 and Indian spinach cv. BARI Puishak-1 were used as plant materials for
the experiment. The seeds were collected from Horticulture Research Centre, Bangladesh Agricultural Research
Institute, Joydebpur, Gazipur.
Treatment combination and experimental design
There were eight treatment combinations for each test crop. The experiment was laid out in Completely
Randomized Design (CRD) with five replications and total treatment combinations were 40. T1=Control, T2 =
AM, T3=CD, T4=P, T5=AM+CD, T6=CD+P, T7=AM+P, T8=AM+CD+P.
Fertilizer dose-
For red amaranth: AM=80 g/polybag, CD= 20 g/polybag, P=0.30 g TSP/polybag
Basal dose- Urea @ 110 kg ha-1 (0.95 g/polybag), MOP @ 25 kg ha-1 (0.20 g/polybag) and Gypsum @ 4 kg ha-1
(89 mg/polybag)
For Indian spinach: AM=80 g/polybag, CD= 32 g/polybag, P=0.30 g TSP/polybag
Basal dose- Urea @ 120 kg ha-1 (1.04 g/polybag), MOP @ 50 kg ha-1 (0.40 g/polybag) and Gypsum @ 13 kg ha-
1
(290 mg/polybag)
Per polybag 8 kg soil was used. Basal doses were added from Fertilizer Recommendation Guide-2005.
Application of arbuscular mycorrhizal (AM) fungi in soil and sowing of seed
Soil based AM inoculum was used in the seed furrows of about 3 cm depth at the rate of 80 g/polybag. Then
seeds of each test crop were sown in each furrow on AM inoculums and covered them by side soil. After sowing
the seed, the soil was saturated with water.
Collection of data
Plant height, Plant diameter, Total number of leaves per plant, Shoot and root fresh weights, Root length,
Shoot and root dry weights of red amaranth were recorded after 36 DAS and of Indian spinach after 50 DAS, 80
DAS and 110 DAS.
Statistical analyses
Analysis of variance was done following the Completely Randomized Design (CRD) with the help of
computer package MSTAT developed by Russel (1984). The data were analyzed statistically by F-test (Gomez
& Gomez 1984). The mean differences of the treatments were adjusted by the least significant difference (LSD)
test.

RESULTS
Effect of arbuscular mycorrhiza, cowdung and phosphorus on growth and yield contributing characters
of red amaranth
Plant height and diameter
Application of AM, CD and P significantly (P0.01) influenced the plant height and diameter of red
amaranth (Table 2). Combined application of AM, CD and P increased plant height and diameter. The highest
plant height and diameter was recorded from T8 treatment (AM + CD + P) which was statistically identical with
T6 treatment (CD + P) in the case of plant height and T7 treatment (AM + P) in the case of diameter. The lowest
plant height and diameter was obtained in T1 (control) treatment. Results suggested that AM biofertilizer along
with phosphorus and CD application exert a positive effect on plant height and diameter of red amaranth.
Application of only AM, CD and P showed significantly higher plant height and diameter over control plant.

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Number of leaves per plant


Significant (P0.01) effect of AM, CD and P was found on number of leaves per plant in red amaranth
(Table 2). The highest number of leaves per plant was observed in T8 where AM, CD and phosphorus were
applied. The treatment T6 (CD + P), T5 (AM + CD), T4 (P), T3 (CD) and T2 (AM) all were statistically identical.
It was noted that the number of leaves per plant increased successively with the application of AM.
Fresh and dry weight per plant
Significant (P0.01) variation in fresh and dry weight of red amaranth was observed due to the effect of
AM, CD and P (Table 2). It was observed that the maximum plant fresh and dry weight was recorded in T8 (AM
+ CD + P) that was significantly higher than all other treatments. The lowest fresh and dry weight was obtained
in treatment T1 (control). Combined effect of AM and P in T7 was showed statistically identical fresh and dry
weight per plant with T2 (AM), T3 (CD), T4 (P) T5 (AM + CD) and T6 (CD + P).
Root length
The result revealed that root length and fresh weight of red amaranth varied significantly (P0.05) by the
effect of AM, CD and P (Table 2). The highest root length and fresh weight was recorded from T8 treatment
(AM + CD +P) and the lowest length was recorded in T1 (control). Root length of T5, T6 and T7 treatments were
higher than T2, T3 and T4, respectively. This indicated that application of AM + CD, CD + P or AM + P always
gave higher root length than individual application of AM, CD or P.
Table 2. Effect of arbuscular mycorrhiza (AM), cowdung (CD) and phosphorus (P) on growth and yield of red amaranth (after
36 days of sowing).
Plant Plant Number of Shoot fresh Shoot dry Root Root fresh
Treatments height diameter leaf per weight per plant weight per plant length weight per plant
(cm) (cm) plant (g) (g) (cm) (g)
T1: Control 18.5e 0.20d 8.07d 1.29c 0.17c 3.58b 0.19c
T2: AM 27.2d 0.46c 11.2c 10.8b 0.79b 4.55ab 0.68b
T3: CD 34.3bcd 0.52c 12.7bc 12.0b 0.95b 5.04ab 0.96ab
T4: P 36.3bc 0.53c 12.4bc 11.2b 0.79b 5.66a 0.97ab
T5: AM+CD 35.6bd 0.56bc 13.4abc 11.5b 1.07b 4.81ab 0.93ab
T6: CD+P 38.2ab 0.57abc 12.8bc 13.4b 1.05b 5.22a 0.98ab
T7: AM+P 39.3ab 0.69ab 15.2ab 14.2ab 1.13b 5.44a 1.20a
T8: AM+CD+P 45.7a 0.70a 16.4a 17.9a 1.80a 6.03a 1.29a
LSD (0.05) 8.67 0.13 3.12 3.87 0.39 1.56 0.45
CV (%) 3.64 11.26 5.60 7.90 9.26 9.02 9.66
Level of sig. ** ** ** ** ** * **
Note: * Significant at 5%; ** significant at 1% level of probability; In a column, the figure(s) having same letter are not
significantly different at 5% level of probability by DMRT.

Table 3. Correlation matrix among growth and yield attributes of red amaranth.
Correlation coefficient (r value)
Characters Plant Plant Shoot fresh Shoot dry Root
Number of
height diameter weight per plant weight per plant length
leaf per plant
(cm) (cm) (gm) (gm) (cm)
Plant diameter 0.702**
No. of leaf per plant 0.490** 0.745**
Shoot fresh weight 0.689** 0.789** 0.735**
Shoot dry weight 0.743** 0.759** 0.623** 0.742**
Root length 0.356* 0.396* 0.446** 0.629** 0.461**
Root fresh weight 0.762** 0.738** 0.644** 0.761** 0.686** 0.437**
Note: ** = Significant at 1% level of probability.
The treatment T7 (AM + P) achieved comparatively higher root fresh weight than T4 with phosphorus. No
significant differences in root fresh weights were observed in T3 (CD), T4 (P), T5 (AM + CD) and T6 (CD + P)
treatments. It is revealed from the results that AM application with phosphorus and CD exerted some effect on
increasing the fresh weight of root per plant.
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Correlation among growth and yield attributes of red amaranth


The correlation between different growth parameters of red amaranth are shown in table 3. In the present
experiment, shoot fresh weight (yield) was significantly positive correlated with plant height, shoot diameter,
leaves number and root length (P0.01). Also, most of the plant characters were strongly correlated among
themselves.
Effect of AM, cowdung and phosphorus on growth and yield contributing characters of Indian spinach
Plant height and diameter
The plant height and diameter of Indian spinach was positively influenced by the application of AM and CD
with P (Table 4). Significant variation was observed in Indian spinach at 50, 80 and 110 days after sowing
(DAS). The highest plant heights were recorded in T8 treatment (AM + CD + P) which were higher than all
other treatments. At 50 DAS, the second highest plant height was noted in T7 treatment (AM + P) which was
identical with T3 (CD), T5 (AM + CD) and T6 (CD + P) treatments. At 80 DAS, application of AM and cowdung
(T5 treatment) produced the second highest plant height that was statistically identical with T3 (CD), T6 (CD + P)
and T7 (AM + P). But at 110 DAS, the same treatment (T5) gave the second highest plant height which was
identical with T6 (CD + P) and T8 (AM + CD + P). The lowest plant heights were found in control treatment.
At 50 DAS, the shoot diameters were found in T8 (AM + CD +P) and in T7 (AM + P) treatments which were
comparatively higher than all other treatments but statistically identical with all the treatments except control
treatment. At 110 DAS, the treatment T8 (AM + CD + P) produced highest shoot diameter and T7 (AM + P) and
T5 (AM + CD) gave same shoot diameter. The treatments T8, T7, T6, T5 and T2 gave statistically similar results.
At 80 DAS, the maximum shoot diameter was obtained where plants were treated with AM, CD and phosphorus
that was statistically identical with all the treatment except T4 with phosphorus and control treatment.
Application of AM with CD achieved shoot diameter which was higher than T3 without AM at 80 DAS but
statistically similar. When AM was used in T2, it gave better response to control treatment.
Number of leaves per plant
The number of leaves per plant was statistically significant (P0.01) due to application of AM, CD and P at
three stage of harvesting (Table 4). The maximum number of leaves per plant were obtained with application of
AM, CD and P combinedly which was superior to all other treatments. At 110 DAS, combined application of
CD and phosphorus in T6 treatment achieved the second highest number of leaves per plant which was identical
with T7 (AM + P) but different from all other treatments. In three stages of harvesting, control gave the lowest
number of leaves per plant than the treatments treated with AM biofertilizer. Application of arbuscular
mycorrhiza with CD in 50 and 80 DAS obtained higher response than the treatment treated without arbuscular
mycorrhiza but found statistically identical result at 110 DAS.
At 50 DAS, T6 (CD+ P) produced number of leaves per plant which was higher than T3 (CD). At 80 DAS,
application of CD with phosphorus achieved numbers of leaves per plant which was higher than the results
found in T4 with phosphorus but statistically identical with T3 where CD was used without phosphorus.
Application of CD with arbuscular mycorrhiza in T5 produced higher value than the treatment treated with AM
but statistically similar with T3 (CD) at 110 DAS.
Shoot fresh weight
Shoot fresh weight of Indian spinach was influenced significantly (P0.05) by the application of AM,
phosphorus and CD (Table 4). The maximum shoot fresh weight was recorded in T8 (AM + CD + P) plants at 50
DAS, 80 DAS and 110 DAS which was identical to T7 (AM + P). At each stage of harvesting, the shoot fresh
weights of Indian spinach gave better response with the application of AM and P in T7 than the plants was
treated without AM in T4, respectively. Application of AM in T2 produced shoot fresh weights which were
much higher than control treatment. Application of CD was significantly influenced the shoot fresh weight of
Indian spinach.
Shoot dry weight
Shoot dry weight of Indian spinach was significantly (P0.01) influenced by arbuscular mycorrhiza, CD and
phosphatic fertilizer (Table 4). Maximum dry weight was recorded in T8 treatment (AM + CD + P) at 50, 80 and
110 DAS. At 50, 80 and 110 DAS, the shoot dry weights were found in T7 (AM + P) which were statistically
higher than T4 (P). Application of arbuscular mycorrhiza with CD produced the shoot dry weights in T5 which
were higher than the value found in T3 (CD) at 50, 80 and 110 DAS, respectively. Application of AM in T2
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produced dry weight which was statistically higher than control treatment at 50 DAS. Same indication was
observed at 80 and 110 DAS.
Root length at 110 DAS
The result revealed that root length of Indian spinach did not vary significantly (P0.01) by the effect of
AM, CD and P (Table 5). With respect to root length, the highest length was recorded from T8 treatment (AM +
CD + P) and the lowest length was recorded in T1 (control). Statistically there were no differences among root
lengths of T5 (AM + CD), T6 (CD + P) and T7 (AM + P) treatments.
Table 5. Effect of arbuscular mycorrhiza (AM), cowdung (CD) and phosphorus (P) on root length, fresh and dry
weights of Indian spinach at 110 DAS.
Treatments Root length Root fresh weight Root dry weight
(cm) (gm) (gm)
T1: Control 10.8 3.87e 1.32d
T2: AM 13.0 14.4c 5.57b
T3: CD 13.1 10.7d 4.46c
T4: P 11.9 14.5c 5.74b
T5: AM+CD 13.5 16.8b 6.91a
T6: CD+P 13.5 15.1c 6.07b
T7: AM+P 13.4 19.0a 7.59a
T8: AM+CD+P 14.3 19.3a 7.68a
LSD (0.05) 3.58 1.34 0.79
CV (%) 5.07 6.15 10.01
Level of sig. NS ** **
Note: ** Significant at 1% level of probability; NS = Not significant; In a column, the figure(s) having same letter
are not significantly different at 5% level of probability by DMRT.
Root fresh and dry weight at 110 DAS
The result showed that root fresh and dry weight of Indian spinach varied significantly (P0.01) by the
application of AM, CD and P (Table 5). The highest root fresh and dry weight was recorded in T8 treatment
where AM, CD and P were applied which was statistically identical with the result found in T7 (AM + P) and T5
in case of dry weight only. Control treatment produced the lowest root fresh and dry weight. Application of AM
in T2 achieved comparatively higher root fresh and dry weight per plant than control. Use of AM with CD in T 5
achieved higher fresh weight value than the plants was treated without arbuscular mycorrhiza in T3. Application
of CD with phosphorus in T6 produced root fresh and dry weight which was statistically similar with the root
fresh weight found in T4 without CD.
Correlation among growth and yield attributes of Indian spinach
The correlation between different growth parameters at 110 DAS are shown in Table-6. In the experiment
shoot fresh weight (yield) was significantly positive correlated with plant height, shoot diameter, leaves number,
root length and root fresh weight. Also among the growth parameters there were found positive correlations.
Table 6. Correlation matrix among growth and yield attributes of Indian spinach at 110 DAS.
Correlation coefficient (r value)
Characters Plant Shoot Number of Shoot fresh Shoot Root Root fresh
height Diameter leaf per plant weight dry weight length Weight
Shoot diameter 0.408**
Number of leaf par plant 0.748** 0.316*
Shoot fresh weight 0.860** 0.379* 0.757**
Shoot dry weight 0.688** 0.401** 0.739** 0.837**
Root length 0.342* 0.102 NS 0.410** 0.345* 0.275NS
Root fresh weight 0.842** 0.479** 0.696** 0.906** 0.826** 0.327*
Root dry weight 0.849** 0.382* 0.734** 0.894** 0.817** 0.366* 0.928**
Note: * = Significant at 5% level of probability; ** = Significant at 1% level of probability; NS= Non Significant.

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DISCUSSION
Different growth parameters such as plant height, plant diameter and number of leaves per plant are
positively correlated with the yield of red amaranth and Indian spinach. The present study revealed that AM
inoculants had significant effect on plant growth of red amaranth and Indian spinach. Similar finding was
reported by (Karagiannidis et al. 2002) in tomato and egg plants, (Ergin & Glser 2016) in lettuce and (Caglar
& Akgun 2006) in three Pistacia species, who found that mycorrhiza treatment increased plant growth compared
to control. Results indicated that application of cowdung with phosphorus fertilizer increased the plant height,
diameter and number of leaves. This result was supported by Wang et al. (2006) and Jin et al. (1996) where they
reported that application of cattle manure resulted in an increase of plant height. Bargali (2006) observed the
effect of AM inoculation on growth performance of Indigofera heterantha at seedling stage. He found that
arbuscular mycorrhiza fungi inoculation resulted in 5.9 cm increment in shoot length and with increasing time
the positive effect of AM fungi inoculation was also increased. From the results stated above suggested that AM
biofertilizer along with phosphorus and cowdung application exert positive effect on plant height of red
amaranth and Indian spinach.
Mycorrhizal inoculation improved higher shoot diameter in AM inoculated seedlings than in non-inoculated
seedlings (Giri et al. 2005). The shoot diameter of Indian spinach was always higher in the treatments where
cowdung was applied with phosphorus or AM than individual application of CD or P or AM. Similar result was
reported by Subhan (1988) that application of organic manure increased the head diameter of cabbage. The
result showed that the plant diameter of red amaranth and Indian spinach was significantly influenced by the
effect of AM biofertilizer, cowdung and phosphorus (Guo et al. 2006).
It is revealed from the results that AM along with phosphorus and cowdung application exerted significant
effect on increasing the number of leaves per plant. Similar result was found by (Lins et al. 2006) in Leucaena
leucocephala (Lam.) and (Samanhudi et al. 2014) in ginger.
Shoot fresh weight and dry weight of red amaranth and Indian spinach was also influenced significantly by
the application of AM, phosphorus and CD. Shoot fresh and dry weight was increased by AM colonization
(Tawaraya et al. 2006). The shoot dry weight was increased with AM inoculation (Andersen & Andersen 2006)
and the increasing rate of manure (Asghari et al. 2015). Under nursery conditions mycorrhizal inoculation
improved growth of Glomus fasciculatum, G. macrocarpum and Cassia siamea seedlings. Shoot dry weight
was higher in mycorrhizal than non-mycorrhizal plants (Giri et al. 2005).
Arbuscular mycorrhiza fungi inoculation resulted in 1.9 cm increment in root length and with increasing
time the positive effect of AM fungi inoculation also increased in Indigofera heterantha at seedling stage
(Bargali 2006) Similar result was found in the present study that AM application increased root length of red
amaranth and Indian spinach significantly. It is also revealed from the results that AM application with
phosphorus and cowdung exerted some effect on increasing the fresh weight of root per plant. Similarly,
(Akkopru & Demir 2005) observed that arbuscular mycorrhizal fungus (AMF) Glomus intraradices increased
morphological parameters (fresh root weight). The application of organic manure increased the yield of the
vegetables compared to vegetables applied with chemical fertilizers (Yan & An-cheng 2004).
In present study, most of the plant characters were strongly correlated among themselves. Similarly, Sarkar
et al. (2002) reported that correlation coefficient studies showed high correlation between yield and root weight,
shoot length and shoot fresh weight. Also (Solaiman 1999) found positive correlation among mungbean growth,
N uptake and yield parameters. Similar correlations were observed by Begum (2004).

CONCLUSION
From the above findings, it may be concluded that application of arbuscular mycorrhiza biofertilizer along
with cowdung and phosphorus observed better performance on growth, yield. The study ventilated that AM use
as a biofertilizer and cowdung as organic manure for production of red amaranth and Indian spinach reduced
phosphatic fertilizer uses which ultimately reduced our cost.

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ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(2): 264273, 2017
DOI: 10.22271/tpr.2017.v4.i2.036
Research article

Regional scale investigation of net primary productivity associated


to dominant land cover classes of Indian Himalayan region
Sandeep Soni*, Sandipan Mukherjee and Kireet Kumar
Watershed Processes and Management Group, G.B. Pant Institute of Himalayan Environment and Development,
Kosi-Katarmal, Almora, 263643, Uttarakhand, India
*Corresponding Author: sandeepsoni80@gmail.com [Accepted: 26 June 2017]

Abstract: This study is an attempt to understand regional variability of net primary productivity
(NPP) of four major land cover classes (LCC), namely evergreen broadleaf forest (EBL), mixed
forest (MF), grassland (GL) and crop land (CL), over the Indian Himalayan Region (IHR)
including Nepal and Bhutan. The study period is chosen to be the 11 years of 2001-2011.
Therefore, the spatial distribution along with inter-annual and total NPP is analysed using
Moderate Resolution Imaging Spectroradiometer (MODIS) LCC and NPP products having a
spatial resolution of 0.1 degree. MODIS NPP is based on algorithm developed by applying the
radiation conversion efficiency logic to the prediction of daily Gross Primary Productivity (GPP),
fractional absorbed photosynthetically active radiation, photosynthetically active radiation and
other surface meteorological fields. Irrespective of the spatial distribution, the monthly maximum
NPP for EBL, MF, GL and CL were found to be 6.4, 6.3, 4.5 and 4.6 gC.m-2.day-1, respectively.
The annual total NPP for EBL, GL and CL over the entire IHR were found to be decreasing with
the rate of 370.3, 185.1 and 822.5 gC.m-2.day-1 whereas, the annual total NPP value for the MF
category was found to have a small rate of annual enhancement of 16.0 gC.m-2.day-1. Overall for
the period of 20012011, the annual reduction in the NPP values of all the four major LCC was
found to be 1.3 KgC.m-2.day-1 indicating a significant carbon loss in terms of biomass of IHR.
Keywords: Net Primary Productivity - Land Cover Changes - MODIS - Himalayan Region.

[Cite as: Soni S, Mukherjee S & Kumar K (2017) Regional scale investigation of net primary productivity
associated to dominant land cover classes of Indian Himalayan region. Tropical Plant Research 4(2): 264273]
INTRODUCTION
Net primary productivity (NPP) is an important component in biosphere functioning and carbon balancing at
regional and global scale as it provides a measure of the amount of CO2 removed from the atmosphere through
photosynthesis and respiration (Liu et al. 1999). NPP is also an essential step in providing means of evaluating
spatial patterns in productivity as well as inter-annual variation and long-term trends in biosphere behaviour.
NPP represents the net carbon input from the atmosphere to the terrestrial vegetation (Melillo et al. 1993) and is
one of the important biophysical variables presenting vegetation activities. Therefore, NPP is one of the most
important components of the biogeochemical carbon cycle (Tan et al. 2007). However, studies on the impact
analysis and spatiotemporal dynamics of NPP are a new research topic and have attracted much attention
recently. Such research activities are spurred by global issues, such as global change, indirect sustainable
management and conservation of biodiversity (Jinguo et al. 2006). Since NPP is the fundamental ecological
variable, not only because it measures the CO2 assimilation, but it also indicates type of the land surface area
(e.g., crops, forests) and status of a wide range of ecological processes (Roy et al. 2001), continuous monitoring
of NPP, would therefore, be important to gather useful information for estimation of amount of CO2 removed
from the atmosphere by biosphere (Bonan 2008, Grosso et al. 2008), to understand the management of natural
resources (Liu et al. 1997) and the global carbon cycle.
The Himalayan region is a hotspot of natural biodiversity and is extremely vulnerable to intensive
anthropogenic activities and natural calamities. This highly fragile region having rugged terrain and unique
topography is sensitive to regional and global-scale changes in climate (Palni et al. 1998). Furthermore,

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https://doi.org/10.22271/tpr.2017.v4.i2.036
Soni et al. (2017) 4(2): 264273

changing landscape pattern, changing ecology and forest degradation are amongst the major environmental
issues of Himalaya (Singh et al. 1984, Ives & Messerli 1990). Due to the heterogeneity and complexity of
Himalayan ecosystems, a better understanding of the relationship between climate change and net primary
productivity distribution is important to predict future carbon dynamics (Zhang et al. 2012). Although,
information regarding the changes in land cover classes (LCC) (Soni 2017) and NPP for the few selected
regions of Indian subcontinent (Kumar et al. 2011) are available for large scale analyses for the entire country
(Ramakrishnan 1988, Chhabra & Dadhwal 2004, Singh et al. 2011, Bala et al. 2013) and for some Himalayan
states of India (Agrawal & Goyal 1987, Sharma et al. 1998, Dhaulakhandi et al. 2010, Joshi & Pant 2012,
Shrestha et al. 2012), no detailed regional scale Himalaya specific analyses of changes in NPP associated with
changes in LCC are available. Therefore, this study aims at exploring the temporal variation of regional scale
NPP values over the Himalayan region using significant LCC. Within this overarching aim, particular objectives
of this study include: (i) identification of trends of spatial changes in the major LCC, (ii) investigation of mean
month wise NPP value for significant LCC (iii) variability estimation of annual sum of NPP over the Himalayan
region for selected LCC and (vi) assessment of correlation between major LCC specific annual NPP and
precipitation.

METHODOLOGY
Study area and data used

Figure 1. Location map of the study area.


In order to address the above objectives, regional scale land cover data from MODIS (Moderate Resolution
Imaging Spectroradiometer, www.neo.sci.gsfc.nasa.gov, spatial resolution 0.1 degree) were used for the Indian
Himalayan region (IHR) that include Nepal and Bhutan Himalaya for the period 2001 to 2011. The study area is
extended from 21o5630 N to 37o530 N latitude and 72o3150 E to 97o2450 E longitude (Fig. 1). The
entire domain has approximate size of 9.16 lac Km2. There were 17 different land cover classes for the entire

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domain. However, we have restricted our analyses for four major classes namely, evergreen broadleaf forest
(EBL, accounts for 12.2% of the total land cover), mixed forest (MF, accounts for 24.8% of the total land
cover), grass land (GL, accounts for 13.7% of the total land cover) and cropping land (CL, accounts for 11.1%
of the total land cover) which altogether accounts for 61.8% of the total area of the IHR (Table 1). However, the
four major LCC do not include barren or sparsely vegetated land cover as, presumable, they are responsible for
minute variation in the NPP values. Algorithm for the global scale production of annual land cover supplied by
MODIS can be found in Strahler (1999). In short, the MODIS land cover data were produced by compositing
eight different land cover parameters from different MODIS channels with inputs from Earth Observatory
Satellite on monthly basis. The final land cover data from MODIS were then validated with several ground
observations for data improvement.
Table 1. Land cover classes and area of land cover classes for the Indian Himalayan region.
Average area % of the total area
S/N Land cover classes
covered (km2) of IHR
1 Water 3120.6 0.3
2 Evergreen Needleleaf Forests 14345.7 1.6
3 Evergreen Broadleaf Forests 112060.6 12.2
4 Deciduous Needleleaf Forests 0.0 0.0
5 Deciduous Broadleaf Forests 0.0 0.0
6 Mixed Forests 227488.7 24.8
7 Closed Shrublands 67.4 0.0
8 Open Shrublands 4153.3 0.5
9 Woody Savannas 66250.8 7.2
10 Savannas 78.6 0.0
11 Grasslands 125205.2 13.7
12 Permanent Wetlands 639.8 0.1
13 Croplands 101856.9 11.1
14 Urban and Built-Up Lands 696.0 0.1
15 Cropland/Natural Vegetation 46202.7 5.0
16 Permanent Snow/Ice 56788.0 6.2
17 Barren/Sparsely Vegetated Land 156994.8 17.1
The NPP values for the four dominant LCC of our study were obtained from the MODIS monthly global
data set of NPP (www.neo.sci.gsfc.nasa.gov) for the time period of 2001 to 2011 having a spatial resolution of
0.1 degree. Details of the algorithm used to derive the MODIS NPP values can be found in Running et al.
(1999). However, a brief description of the algorithm is provided here. The fundamental notion of linear
relationship between the absorbed photosynthetically active radiation (APAR) and the NPP (Monteith 1972,
1977) of a well-watered crop plant was used to produce this data. Although, such simplistic algorithm is
insufficient to derive NPP values for the entire earth as many other meteorological and biological factors can
modulate NPP of a plant (see Running et al. 1999 for detail). Therefore, the present data were developed by
applying the radiation conversion efficiency logic to the predictions of daily Gross Primary Productivity (GPP),
using satellite-derived fractional absorbed photosynthetically active radiation (fPAR) and independent estimates
of photosynthetically active radiation (PAR) and other surface meteorological fields. Subsequently, the NPP
values were computed by subtracting the maintenance and growth respiration terms from GPP. The maintenance
respiration and growth respiration components were derived from allometric relationships linking daily biomass
and annual growth of plant tissues to satellite-derived estimates of leaf area index. To address fourth objective,
TRMM (Tropical Rainfall Measuring Mission) precipitation data having spatial resolution of 0.25 degree (see
TRMM 2011 for detail) was downloaded from the website http://disc.gsfc.nasa.gov/datacollection/TRMM_3
B43_V7.shtml for above mentioned time period and further analysed with ArcGIS software.
In order to address the first objective of this study, at first, the annual global land cover values were
extracted for the Himalayan region over the period 2001 to 2011 using the ArcGIS version 10.0 software. Next,
the four dominant LCC (EBL, MF, GL and CL) were identified and number of pixels within the study domain
for individual LCC was counted. The spatial resolution of remote sensing data was 0.1 degree. Therefore, total
area covered by individual LCC for each year of study was computed. Finally, trend of LCC-wise spatial
distribution was computed using simple linear regression. To address the second and third objective of this
study, using the similar method of LCC extraction, NPP values were extracted for each LCC. Then, arithmetic

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Soni et al. (2017) 4(2): 264273

mean of NPP values of each month was estimated and their spatial distributions were studied. The average
monthly maxima of NPP () over the 11 years period was computed following:
My 11
1
NPP m
max
Y

My 1
m
NPPmax (1)

m
Where, NPPmax = maximum NPP value of each month for 2001 to 2011 over the entire domain, Y = number of
years and My = summation of months (e.g. Jan. to Jan.) for 2001 to 2011.
Similarly, the average total NPP value for a particular month and summed over the 11 years of data period
() was computed following:
1 My 11
NPP NPPtotm
m
tot (2)
Y My 1
m
Where NPPtot = total NPP value of each month for 2001 to 2011, Y = number of years.

Finally, total annual sum of NPP for a single year for the four dominant classes were estimated
following:
DecY
NPPtotAnn NPP
JanY
tot (3)

Where, = total NPP value of each year, Y = year (2001 to 2011).


RESULTS AND DISCUSSION
The four major LCC for this study are EBL (Class 1), MF (Class 2), GL (Class 3) and CL (Class 4). The
annual land cover distribution of these classes for the entire IHR over the period of 20012011 shown in figure
2. A quick look on the LCC throughout 20012011 reveals that the maximum forest (EBL and MF) distribution
of the IHR is concentrated in the eastern Himalaya. When the spatial distribution of each of this classes were
analysed for the entire Himalayan region, it was found that the distribution of EBL was restricted within the
eastern Himalayan region with exception of the Brahmaputra river valley area, which was mostly dominated by
croplands, presumably rice paddies. The westernmost significant distribution of EBL was observed near the
border of the state of West Bengal of India and Nepal. The MF was found to be ubiquitously distributed over the
eastern and central Himalayan region, whereas over the north-western IHR, distribution of MF was found to be
sparse. As expected Most of the CL areas were observed near the lower altitudes and, GLs were observed in the
higher altitudes. Two significant concentrations of GL were observed in the upper region of the Indian state of
Kashmir and over the high altitudes of Central Nepal.
In order to estimate the rates of changing area distribution of the selected LCC, trend lines were produced for
an area of each LCC using linear regression method (Fig. 3). It was observed that except for MF, land cover
areas of all the other three classes were decreasing. The rate of land cover area enhancement of MF was found to
be 177.2 Km2.year-1 (r2 = 0.13) for the study period. Although, rates of land cover area decrement for the EBL,
CL and GL were found to be 43.5 (r2 = 0.03), 30.9 (r2 = 0.03) and 81.0 Km2.year-1 (r2 = 0.22). Reduction of the
EBL forests of the eastern Himalayan region can be attributed to the decreasing trend of rainfall and moisture
availability of the region for which a significant proportion of the EBL forest type is replaced by MF category
(Chakraborty 2009, Reddy et al. 2013). Similarly, reduction of high altitude GL of IHR can be attributed to
overgrazing and decrement of rainfall resulting replacement of GL by barren and sparsely vegetated land (Pandit
et al. 2007, Farooq & Rashid 2010, Reddy et al. 2013).
In order to investigate the average month-wise variation in the NPP values of selected classes, first we have
explored the spatial variation. It is obvious that the spatial distribution of NPP (Fig. 4) of a LCC would only
correspond to the spatial distribution of that particular LCC, however, the month-wise maximum and minimum
of NPP would be different. To start with the spatial distribution of monthly averages of NPP of EBL, it was
observed that the maxima of NPP (> 5 gC.m-2.day-1) for EBL forests were mostly concentrated over the
Manipur, Mizoram states of the north-east Himalayan region. Similarly, the maxima of NPP for MF (> 5 gC.m-
2
.day-1) were mostly observed for those forests situated in the foothills of Himalaya. The maxima of NPP for GL
(> 3 gC.m-2.day-1) were mostly observed for those regions which were adjacent to the MF category.
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Figure 2. Annual land cover distribution for the period 20012011: Class 1- Evergreen broadleaf forest (EBL); Class 2-
Mixed forest (MF); Class 3- Grass land (GL); Class 4- crop land (CL).
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Figure 3. Trends in the changing area distribution of selected LCC. The horizontal lines are produced using linear regression
method. [Class 1- Evergreen broadleaf forest (EBL); Class 2- Mixed forest (MF); Class 3- Grass land (GL); Class 4- crop
land (CL)]
The variation in the monthly maximum NPP values, estimated following equation (1), of each class for 11
years (20012011) is represented in figure (5A). The values for EBL, MF, GL and CL were found to be
6.4, 6.3, 4.5 and 4.6 gC.m-2.day-1, respectively. The for EBL and MF were observed for the month of
April and May, whereas the same for GL and CL were observed for the month of June and July. The peak
values of GL and CL for JuneJuly can be attributed the highest growth of the respective vegetation due to
monsoon rainfall. The NPP values for both EBL and MF category were found to have two maxima on April and
October, whereas the GL was found to have a single peak. This variation in the NPP values can be easily
attributed to the peak of leaf initiation and leaf senescence.
When the month-wise average of total NPP were investigate for our selected LCC (following equation (2)),
variations were found to be mostly matching the trend of values, except for the MF category, for which
the highest value of were observed for the month of October (5895.8 gC.m2.day-1) (Fig. 5B).
Finally, when the total NPP values of each year for the selected classes were estimated following equation (3), a
significant reduction was observed for the MF category during 2004 and 2005 (Pandit et al. 2007, Chakraborty
2009, Farooq & Rashid 2010, FSI 19992011). However, no such significant reduction in other LCC were
observed for that period. The overall trend in the values for the EBL, GL and CL were found to be
decreasing with the rate of 307.3 (r2 = 0.1), 185.1 (r2 = 0.5) and 822.5 (r2 = 0.7) gC.m-2.day-1. Although,
values for the MF category was found to have a small rate of annual enhancement during 20012011,
16.0 (r2=0.0) gC.m-2.day-1 (Fig. 6). The annual reduction in the NPP values of all the four major LCC was found
to be 1.3 KgC.m-2.day-1 (r2 = 0.24).
To justify above mentioned reduction in trend of major LCC and , trend was assessed between four
major LCC specific annual NPP and precipitation (Fig. 7). It was observed that precipitation was decreasing
with a rate of 6 mm.hr-1 (r2 = 0.01). However, precipitation was correlated with NPP values of all the four major
LCC to identify significant values. The observed value (r2) for of EBL was -0.5 (p-value = <0.001),
MF was 0.2 (p-value = <0.04), GL was 0.8 (p-value = < 0.001) and CL was 0.3 (p-value = <0.001) (Fig. 8).
The detailed analysis has yielded a few surprising results about LCC and NPP. In corroboration to the results
published in Reddy et al. 2013. It was found that LCC and NPP of EBL were decreasing over the Himalayan
region in the last decade (Gupta et al. 2006), while LCC and NPP of MF showed increasing trend over the study
area. However, such a significant decreasing trend was also observed for the remaining two classes i.e. GL and
CL and, the total area covered by these last two classes were comparatively smaller than EBL and MF. In the
case of GL, the primary productivity during December to February remains minimal due to very low
temperature in the Alpine region where grasslands are mostly found. Similarly, pre-monsoon months of April to
May remain non-productive for CL due to dry summer. Although, the variation in NPP associated to CL for the
summer months was found to be area specific as the Brahmaputra river basin area in the North-eastern region

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Soni et al. (2017) 4(2): 264273

showed a high variation in productivity whereas the same for the lower part of Central and Western Himalayan
region showed little variation.

Figure 4. Spatial distribution of average month wise NPP values over the entire Himalayan region for four major classes.
[Class 1- Evergreen broadleaf forest (EBL); Class 2- Mixed forest (MF); Class 3- Grass land (GL); Class 4- crop land (CL)]

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Soni et al. (2017) 4(2): 264273

Figure 5. A, Month wise maximum NPP values observed over the entire Himalayan region; B, Month wise average of total
NPP values for the entire Himalayan region for four major LCC. [Class 1- Evergreen broadleaf forest (EBL); Class 2- Mixed
forest (MF); Class 3- Grass land (GL); Class 4- crop land (CL)]

Figure 6. Year wise total NPP values for the entire Himalayan region for four major LCC. The year wise NPP values are
obtained by adding NPP values from January to December. [Class 1- Evergreen broadleaf forest (EBL); Class 2- Mixed
forest (MF); Class 3- Grass land (GL); Class 4- crop land (CL)]

Figure 7. Trend between precipitation and annual total NPP.

CONCLUSIONS
This study is aimed at measuring the satellite-based NPP values to quantify the spatial and interannual
variations over the IHR for a period of 20012011. Out of seventeen different land cover classes of the IHR,
four dominant LCC were identified. It was observed that the land cover area and NPP of MF are maximum
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Figure 8. Significant curves between precipitation and NPP of four major LCC: A, Evergreen broadleaf forest (EBL); B,
Mixed forest (MF); C, Grass land (GL); D, crop land (CL).
among all other LCC of IHR having an increasing trend for the period of 2001 to 2011. It was also observed that
EBL is dominant in the North-eastern Himalaya having comparatively lesser spatial area than MF. The total
annual NPP of EBL was also found to be decreasing. The total annual NPPs of GL and CL were also observed
as decreasing the rates of 185.1 and 822.5 gC.m-2.day-1. The NPP products available from the website
(www.neo.sci.gsfc.nasa.gov) were used for this study which had a spatial resolution of 0.1 Degree. No extra
algorithm was used in this work to improve the regional scale estimate of NPP. Therefore, the NPP estimation
presented in this work could be further improved if a finer resolution NPP and LCC product is used in the
future. This work can further be extended in the context of climate change if long term climate and NPP
products are available.
ACKNOWLEDGEMENTS
Authors are grateful to Director, GB Pant Institute of Himalayan Environment and Development
(GBPIHED), Kosi-Katarmal, Almora, India for providing the necessary computational facilities and a research
grant to carry out this work.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(2): 274285, 2017
DOI: 10.22271/tpr.2017.v4.i2.037
Research article

Assessment of allometric models for leaf area index estimation


of Tectona grandis
R. K. Chaturvedi1*, Shivam Singh2, Hema Singh2 and A. S. Raghubanshi3
1
Community Ecology and Conservation Group, Xishuangbanna Tropical Botanical Garden,
Chinese Academy of Sciences, Menglun, Mengla, Yunnan 666303, China
2
Ecosystems Analysis Laboratory, Department of Botany, Banaras Hindu University, Varanasi 221005, India
3
Institute of Environment and Sustainable Development, Banaras Hindu University, Varanasi 221005, India
*Corresponding Author: ravikantchaturvedi10@gmail.com [Accepted: 17 July 2017]

Abstract: We developed allometric models for accurate estimation of Tectona grandis (teak) leaf
area index (LAI), for different stem diameter-classes (D-classes). In this study, we harvested teak
trees in the tropical dry region of India in the ten stem diameter classes and measured LAI, and
developed regression models for the non-destructive estimation of LAI with the help of wood
density (), diameter at breast height (D) and plant height (H). Models used for the prediction of
biomass of tree components were of the form, linear, logistic, Gompertz and Chapman. Among the
four models, non-linear models were more efficient compared to the linear model. We observed
more than 60% variability in the LAI, explained by non-linear regression models. The models
including and D had greater R2 and lower standard error of estimate. Our study detected logistic
models more appropriate for broad diameter range and Gompertz models for small D-classes. The
regression models developed in our study can be applied separately for the ten D-classes, and this
could minimize the error occurring during indirect estimation of teak LAI.
Keywords: Tectona grandis - Leaf area index - Allometric models - Wood density - D-classes -
Tropical dry region.

[Cite as: Chaturvedi RK, Singh S, Singh H & Raghubanshi AS (2017) Assessment of allometric models for leaf
area index estimation of Tectona grandis. Tropical Plant Research 4(2): 274285]
INTRODUCTION
Exchange of mass and energy of resources through plant canopies is functionally related to leaf area index
(LAI) (Law et al. 2001, Moser et al. 2007, Woodgate et al. 2015). In forest ecosystems, LAI is an essential
variable which controls solar energy, carbon flux and water. Usually, LAI is defined as the total one-sided leaf
tissue area per unit of ground surface area (Watson 1947). It is a dimensionless quantity and an important
characteristic feature of the canopy of an ecosystem. It is the most important parameter for modeling forest
ecosystem processes, such as canopy photosynthesis, respiration and evapotranspiration.
Mostly, the studies for LAI have been carried out in temperate forests (Dufrne & Brda 1995, Sellin 2000,
Temesgen & Weiskittel 2006, Weiskittel & Maguire 2006, Urban et al. 2009), and very few for tropical
ecosystems (Maass et al. 1995, Nascimento et al. 2007, Vyas et al. 2010). The quantification of LAI is
unfortunately, very difficult, due to its spatial and temporal variability (Brda 2003). LAI has been observed to
be sensitive to site differences and tree density (Gspaltl & Sterba 2011). It can be determined by harvesting,
litter trap methods, optical sensors and laser scanning (LiDAR). For indirect measurements, optical methods and
LiDAR are commonly used, which are based on Beer-Lambert law. Optical measurements are generally
performed with using canopy analysers, hemispherical photography, and remote sensing (Brda 2003,
Jonckheere et al. 2004, Zhang et al. 2005, Dovey & Toit 2006, Arias et al. 2007, Nascimento et al. 2007, Urban
et al. 2009, Huang & Pretzsch 2010). LiDAR applies threshold-based separation of laser reflections from the
canopy and from the ground (Hosoi & Omasa 2006, Bland et al. 2011, Stark et al. 2015, Lin & West 2016).
However, due to some functional limitations, these indirect methods, fail to measure LAI accurately in tropics,
where the vegetation is dense and multi-layered (Moser et al. 2007). Inaccuracy in measurements has also been
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Received: 22 April 2017 Published online: 31 July 2017
https://doi.org/10.22271/tpr.2017.v4.i2.037
Chaturvedi et al. (2017) 4(2): 274285

observed with non-random leaf area distribution and shading caused by plant parts (e.g. branches, stems), other
than leaves (Pokorn & Marek 2000, Huang & Pretzsch 2010).
In the direct estimation of LAI, leaf areas of total leaves of the canopy have to be summed and divided by
the canopy projected area. However, this process is time-consuming and impossible for the whole stand.
Therefore, generally a limited number of trees are sampled, allometric equations are parameterized, and used for
the other trees in the stand (e.g. Ledermann & Neumann 2006, Hietz et al. 2010, Laubhann et al. 2010, Gspaltl
& Sterba 2011). Across the environmental gradients, LAI estimated by direct methods have been observed to be
more accurate compared to the indirect methods (optical measurements) (Khan et al. 2005). A significant
underestimation of LAI by indirect methods compared to the direct methods have also been observed, when the
estimates were cross-validated (Brda 2003). Therefore, for the accurate non-destructive estimation of LAI,
allometric models are needed.
In the tropical dry region, plants shed their leaves during the dry period, therefore, for non-destructive
estimation of LAI, leaf litter is collected in litter traps plotted below the canopy during the dry period (Marshall
& Waring 1986, Brda 2003). By using specific leaf area (SLA), allometric equations relating littermass and
DBH (diameter at breast height) have been developed for the estimation of LAI (Gower et al. 1999). Several
studies have also developed allometric models for LAI estimation relating foliage biomass, littermass, DBH, and
ground area covered by the canopy (Li et al. 2005, Pretzsch & Mette 2008, Vyas et al. 2010).
Since, LAI is utilized to understand community, ecosystem, and global processes, there is great demand of
better temporal and spatial data sets of LAI (Cornwell 2015). For accurate estimation of LAI, multi-
species/multi-site allometric equation could not be applied over a large area. In different stem diameter-classes
(D-classes), there are architectural changes in trees, therefore, allometric models are required also for different
D-classes, separately (Chaturvedi & Raghubanshi 2015a). Common allometric models for LAI estimation have
foliage biomass or littermass as the estimator, however, foliage allometric equations are supposed to be affected
by tree size, species and edaphic conditions (Law et al. 2001). Errors in the measurement of LAI by littermass
have also been observed due to environmental effects on litter SLA, particularly those concerning canopy
structure and soil conditions (Bouriaud et al. 2003). Therefore, to minimize the error in LAI estimate, we chose
wood specific gravity or wood density () as an estimator in the allometric model for LAI estimation. is linked
to stem strength, stiffness and safety (i.e. measured as dry wood mass per green wood volume), and ranges from
0.1 to 1.5 g.cm-3 among tropical trees (Chave et al. 2009, Zanne et al. 2009, Bastin et al. 2015). It is often used
as a proxy for understanding the size and functioning of the tropical tree species (Swenson & Enquist 2007,
Chave et al. 2009, Reich 2014). In several studies has been used for developing biomass allometric models
(Chave et al. 2005, Chaturvedi et al. 2010, Chaturvedi et al. 2012a, Chaturvedi & Raghubanshi 2013, Chave et
al. 2014, Chaturvedi & Raghubanshi 2015a, Chaturvedi et al. 2017a).
Teak (Tectona grandis L.f.) is the most important timber-yielding, deciduous plant of the tropical belt
(Sousa et al. 2012). This broadleaved tree species is native to India, Myanmar (Burma), Thailand and Laos, but
nowadays grows in the whole intertropical region. Worldwide, teak plantations cover approximately 6.0 mha
area (Bhat & Ma Hwan 2004) in which around 94% area is in tropical Asia, with a major part in India (44%)
and Indonesia (31%) (Prez 2005). In India, about 29% of the total forest area is dominated by teak (FSI 1998).
Mostly teak is naturally found in western and southern India (Champion & Seth 1968). Its northern limit is
western Aravallis and eastward through central India. Teakwood is moderately hard, easily worked and
extremely durable, therefore, it has a high demand for yatching, building and furniture industries (Troup 1921,
Tewari 1992). Due to high market demands, the establishment of plantations of teak is continuously increasing
in several tropical countries beyond its native origin (Prez & Kanninen 2003). In this study, we measured LAI
of teak in ten D-classes, occurring in the tropical dry region of India, by harvest method and developed
regression models for the non-destructive estimation of LAI with the help of , D and tree height (H). Further,
we observed the strength of similarity between the actual harvested LAI and those estimated by the regression
models.

MATERIALS AND METHODS


Study area
The study was executed in the Vindhyan highlands situated in Sonebhadra District of Uttar Pradesh, India
(21 2925 11 N and 78 1584 15 E). The area experiences tropical monsoon climate with three seasons in
a year, viz, summer (Aprilmid June), rainy (mid JuneSeptember) and winter (NovemberFebruary). The
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months of March and October constitute transition periods, respectively between winter and summer, and
between rainy and winter seasons. The maximum monthly temperature varies from 20 C in January to 46 C in
June, and the mean minimum monthly temperature reaches 12 C in January and 31 C in May. According to the
data collected from the meteorological stations of the state forest department for 19802010, the mean annual
rainfall ranges from 865 to 1196 mm (Chaturvedi et al. 2011a). About 85 % of the annual rainfall occurs during
the monsoon (rainy) season from the south-west monsoon, and the remaining from the few showers in
December and in MayJune. There is an extended dry period of about 9 months (Octobermid June) in the
annual cycle (Jha & Singh 1990). The monthly rainfall varies from 6 mm in April to 334 mm in August
(Chaturvedi et al. 2012b). Soils of the study area are residual, ultisols, sandy-loam in texture, reddish to dark
gray in colour and extremely poor in nutrients (Chaturvedi & Raghubanshi 2011). Recently estimated physico-
chemical properties of soils of the study region have been described in Chaturvedi & Raghubanshi (2015b). The
forest region exhibit patchiness in the species composition due to small variations in the environmental variables
(Chaturvedi et al. 2011b). Species composition, distribution and diversity (Chaturvedi & Raghubanshi 2014,
Chaturvedi et al. 2017b), and seasonal growth (Chaturvedi et al. 2011c, 2013, 2014) of woody species is highly
influenced by soil moisture content. The density-DBH distributions in the forest region exhibited low DBH-
biased structure, where the average density of seedlings, saplings and adults were 9,2611,511, 799154 and
29762, respectively (Chaturvedi & Raghubanshi 2014).
Leaf area index (LAI) estimation
The data was collected from the private teak plantation in the forest region of Vindhyan Highlands. The field
sampling was done in September, when teak trees were fully-leaved. We selected ten teak trees in each of the
ten stem diameter (D, cm) classes (viz. 3.2 to < 6.4; 6.4 to < 9.6; 9.6 to < 12.7; 12.7 to < 15.9; 15.9
to < 19.1; 19.1 to < 22.3; 22.3 to < 25.5; 25.5 to < 28.7; 28.7 to < 31.8; 31.8). For the individuals <
3 m height, D was measured below the lowest branch and away from stem irregularities, and for others having
3 m height, D was measured at 1.37 m above ground. Projected crown area for each selected individual was
estimated by the line-intercept method, which is expected to provide most accurate canopy cover estimate (Fiala
et al. 2006). In this method, horizontal distances covered by the live crown was recorded along a line transect
radiating out from the main stem for each tree, in 6-8 directions depending upon the crown irregularities. For
each tree, along with each transect line, the distance of the first point of interception of the crown to the last
point of interception was recorded (to the nearest dm), with the help of a clinometer to verify the interception of
the crown directly overhead. After canopy cover estimation, all selected individuals were harvested, and their
height (H) and D were recorded. The foliage of trees having < 9.6 cm D was separated and dry biomass of
leaves for each individual tree was estimated by drying in an oven at 80 C to constant weight. For the trees
having 9.6 cm D, it was impractical to pick off all leaves of the large tree, therefore, initially, twigs with
leaves were cut and weighed in bulk for each tree separately on a hanging scale (0.1 kg precision, 200 kg
capacity). Around 10 to 20% of the bulk was defoliated (foliage was sampled proportionally to the position of
foliage along the branch) and leaves and twigs were weighed separately to calculate the leaf-to-twig mass
fraction for that tree. This fraction was used to detect the foliage green weight for each individual tree. The
defoliated leaves were dried at 80 C to constant weight, separately for each tree to know the moisture content in
the leaves. By subtracting the moisture content from the foliage green weight, foliage dry weight for each
individual tree was calculated. Before drying, 50 leaves were randomly taken from each individual tree for the
estimation of specific leaf area (SLA) following Cornelissen et al. (2003). After SLA estimation, the dry weight
of the leaves was pooled with the foliage dry weight of the tree from where the leaves were taken. Wood
samples for the estimation of were collected by using a stem borer (Turner & Cole1973). After removing the
bark, wood core was taken from each sapling at the height where D was measured and the samples were sealed
in polythene bags and were taken to the laboratory. Volumes of fresh wood samples were measured by water
displacement method and the samples were dried in a well-ventilated oven at 101105 C till constant weight
(Williamson & Wiemann 2010). The values were expressed as g.cm-3.
Statistical analysis
To estimate the one-sided leaf tissue area for each tree, their foliage dry weights were multiplied with their
SLA. Thus, we determined LAI of each tree by harvest method as the ratio of total one-sided leaf area and the
canopy cover. Standard deviation, skewness and kurtosis of the LAI of each D-class were calculated with the
help of Microsoft Excel 2007. Data of all the ten D-classes were used to develop regression models for the
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estimation of LAI on the basis of , D and H. The regression models used for the prediction of LAI were of the
form, linear, logistic, Gompertz and Chapman. The best model for each D-class was selected by assessing the
goodness of fit based on the coefficients of determination, standard errors of the estimate, P values, and average
deviation (%) from the observed data. We applied t test for comparison of LAI estimated by harvest method and
that by the regression model. Similarly, we developed regression models for the estimation of LAI for all the ten
D-classes, and selected most appropriate models. All the statistical analyses were done using SPSS (ver. 16)
package. To check for the strength of the relationship between LAI estimated by the two methods, we regressed
the harvest data of each D-class against the estimates obtained through the newly developed model by using
SigmaPlot (ver.11).

RESULTS
Table 1. Range of wood specific gravity (), stem diameter (D), height (H), specific leaf area (SLA) and canopy
cover (CC) in the 10 stem diameter classes of teak.
S.No. D-class (cm) (g.cm-3) D (cm) H (m) SLA(cm2.g-1) CC (m2)
1. 3.2 < 6.4 0.523-0.592 3.25-6.15 3.8-8.2 70.1-73.2 0.95-1.77
2. 6.4 < 9.6 0.611-0.647 6.50-9.36 8.4-11.9 68.4-70.6 2.01-3.80
3. 9.6 < 12.7 0.639-0.661 9.83-12.6 12.3-15.2 67.3-70.2 2.83-6.60
4. 12.7 < 15.9 0.654-0.693 13.2-15.5 15.4-17.8 66.2-69.1 4.91-10.2
5. 15.9 < 19.1 0.688-0.726 16.2-18.6 17.8-19.8 65.2-67.9 10.2-13.2
6. 19.1 < 22.3 0.718-0.747 19.4-22.1 19.1-21.2 64.3-67.8 11.3-16.6
7. 22.3 < 25.5 0.735-0.749 22.6-25.4 20.4-23.3 63.8-66.8 15.9-27.3
8. 25.5 < 28.7 0.726-0.755 25.6-28.5 22.8-25.8 62.9-65.8 26.4-30.2
9. 28.7 < 31.8 0.751-0.768 28.9-31.6 24.3-26.4 61.9-64.6 31.2-39.6
10. 31.8 0.769-0.774 32.0-39.8 24.8-33.3 60.4-62.8 39.6-44.2

Figure 1. Relationships between the log transformed values of D2 and the log transformed values of LAI estimated by
harvest method for teak trees. For regression models, see Table 2. , wood specific gravity (g cm-3); D, stem diameter (cm);
H, height (m). n = 100.
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( )
Table 2. Regression models [linear, logistic, Gompertz, ; Chapman,
( )
( ) ] for estimating leaf area index (LAI) of teak. , wood specific gravity (g cm-3); D, stem diameter at
breast height (cm); H, tree height (m).
S.No. Model R2 value Standard error Average P value
of estimate deviation
(%)
1. 0.381 0.167 4.708 <0.001
2. 0.685 0.115 4.018 <0.001

( )

3. ( ) 0.651 0.126 4.165 <0.001

4. ( ) 0.643 0.139 4.193 <0.001

5. 0.399 0.158 4.518 <0.001


6. 0.708 0.103 3.592 <0.001

( )

7. ( ) 0.670 0.124 4.036 <0.001

8. ( ) 0.651 0.132 4.101 <0.001

9. 0.353 0.171 4.945 <0.001


10. 0.672 0.116 4.057 <0.001

( )

11. ( ) 0.645 0.127 4.168 <0.001

12. ( 0.644 0.136 4.248 <0.001


)
13. 0.361 0.169 4.825 <0.001
14. 0.678 0.119 4.016 <0.001

( )

15. ( ) 0.646 0.127 4.166 <0.001

16. ( 0.647 0.132 4.232 <0.001


)

For all the ten D-classes, ranges of , D, H, SLA and canopy cover (CC) are shown in table 1. Descriptive
statistics (i.e. maximum and minimum values, mean, standard deviation, skewness and kurtosis) of the datasets
of LAI are shown in Appendix I. For the estimation of LAI, the developed regression models are reported in
table 2. These models are constructed by combining all individuals in the ten D-classes (n = 100). The
difference of variability explained between the worst and the best model was 36 % (Table 2). We observed more
than 60% variability in the LAI, explained by non-linear regression models. The models including and D had
greater R2, and lower standard error of estimate and average deviation. Among the four types of regression
models developed for predicting LAI, greater R2, and lower standard error of estimate and average deviation
were observed for the logistic model (Table 2). For the estimation of LAI, irrespective of the effect of stem
diameter, we selected model 6 from Table 2 (Fig. 1). The regression models for the estimation of LAI for all the
ten D-classes are shown in Table 3. While developing these models, we could not get significant results (at 95%
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level) for LAI for trees having stem diameter < 12.7 cm. For the D-classes having 12.7 cm stem diameter,
Gompertz model produced most significant results (Table 3).
Table 3. Regression models for the 10 stem diameter (D, cm) classes of teak. Models are of the form, linear (
( )
), and/or Gompertz ( ), where, Y = leaf area index, X = D2, Y0, a and b are constants, = wood
specific gravity (g cm-3). n = 10. Here, only linear regression models and most significant non-linear models are shown.
Model D-class (cm) Model R2 value Standard Average P value
No. error of deviation
estimate (%)
1. 3.2 to < 6.4 0.0031 0.212 0.148 >0.050
2. 6.4 to < 9.6 0.0006 0.140 0.261 >0.050
3. 9.6 to < 12.7 0.0002 0.229 0.357 >0.050
4. 12.7 to < 15.9 0.6067 0.100 1.368 <0.010
( ) 0.8579 0.069 0.792 <0.010
6. 15.9 to < 19.1 0.6448 0.068 1.178 <0.010
( ) 0.7795 0.059 0.835 <0.010
8. 19.1 to < 22.3 0.4826 0.077 1.582 <0.050
( ) 0.7141 0.066 0.736 <0.050
10. 22.3 to < 25.5 0.8449 0.046 0.937 <0.001
( ) 0.9165 0.039 0.426 <0.001
12. 25.5 to < 28.7 0.5996 0.040 1.026 <0.010
( ) 0.6719 0.039 0.728 <0.010
14. 28.7 to < 31.8 0.7819 0.014 0.992 <0.001
( ) 0.8685 0.013 0.506 <0.001
16. 31.8 0.5268 0.029 1.215 <0.050
( ) 0.6936 0.026 0.695 <0.050

Figure 2. Average LAI (1 S.E.) across the 10 diameter classes of teak estimated by the two methods.
Harvested LAI exhibited strong relationships with the predicted values used in our models, especially in
higher D-classes (Fig. 1). Average LAI for the ten D-classes estimated by harvest method and that from
regression models are shown in figure 2. For the smallest D-class (i.e. 3.2 < 6.4 cm), average LAI estimated
by harvest method and allometric method were 3.2 and 3.1, respectively, and for the highest D-class (i.e. 31.8
cm), the average LAI estimated by harvest method and allometric method was almost same, i.e. 5.2.
Statistically, the two estimates of LAI were different, only for the smaller three D-classes, i.e. 3.2 < 6.4 cm
(t test0.05 = 0.763, P = 0.049), 6.4 < 9.6 cm (t test0.05 = 0.857, P = 0.035), and 9.6 < 12.7 cm (t test0.05 =
0.716, P = 0.042), whereas for other D-classes, the two estimates were not different (Fig. 2). The linear
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regression models for LAI for all the ten D-classes were validated against the harvested LAI, and we observed
significant results only for the higher seven D-classes (Fig. 3). Here we observed strong relationships for all the
seven D-classes, with the greatest value of R2 = 0.85 for the D-class, 22.3 to < 25.5 (Fig. 3).

Figure 3. Relationships between the LAI of teak in the seven stem diameter (D) classes estimated by regression model (see
Table 3) and by harvest method. [Note: For the lower three D-classes, the relationships were not significant]
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DISCUSSION
We observed that the model containing wood specific gravity () as the estimator, better predicted LAI of
teak tree. This exhibits that the is somehow linked with LAI. Tree canopy has a determining influence on
wood quality, since the physiological processes originating in foliage regulate biological processes of wood
formation (Larson 1969). Therefore, is also influenced by the tree canopy conditions. Wood specific gravity is
also observed to be influenced by tree diameter, height and growth rate (Woodcock & Shier 2003). The range of
observed in our study (0.520.77 g.cm-3) is comparable to the average values of (0.5570 g.cm-3) found in
Costa Rica (Prez 2005) and other reportings for plantation grown teak (Bhat 1995, Brennan & Radomiljac
1998, Bhat 2000). We detected the logarithmic equation most appropriate for the estimation of LAI. According
to Kerkhoff & Enquist (2009), several allometric characteristics of organisms are multiplicative in nature,
therefore, allometric models constructed by log-transformed data is most acceptable.
Tree growth follows sigmoid/non-linear pattern (Zeide 1993) so linear regression models may not produce
significant results for a wide range of explanatory variates. It has also been reported that the linear regression
models become less effective as the number of explanatory variables and the complexity of data increases
(Death & Fabricius 2001). When we attempted with linear as well as non-linear functions for the LAI
estimations, for all D-classes combined together, we detected non-linear models as the better predictors of LAI
estimates. Also, when we analysed the four models separately in the ten D-classes, the non-linear model (i.e.
Gompertz) was more efficient compared to the linear model. The R2 values of the non-linear models listed in
Table 2 are quite good (R2 > 0.64), and are very similar. We also observed the difference of < 7% variability
explained between the worst and the best non-linear model. If the models including are compared with the
models without but with the same variables (e.g. model 2 vs 6 and model 10 vs 14), we observed similar slope
for the models including compared to the models without . Moreover, on the basis of R2, SEE and average
deviation, all the non-linear models are very similar and equally good for the estimation of LAI.
Our allometric models predicted unsatisfactory LAI for trees with < 12.7 cm D (Table 3), however, the
ability of allometric models for predicting LAI of large trees were stronger. According to Bailey & Harjanto
(2005), teak trees at younger age (< 15 years) usually experience severe foliar damage due to cattle grazing and
harvesting by humans. Cole & Ewel (2006) also emphasized the influence of weather, herbivores, and inter-
plant competition on the foliage biomass. These might be the reasons for unpredictability in the estimation of
LAI for trees with < 12.7 cm D. In a similar study, Cole & Ewel (2006) reported unsatisfactory prediction of
leaf biomass by the allometric models applied for Cordia trees in 05 cm D-class. Similar to our observations,
Cole & Ewel (2006) also observed stronger relations for larger trees with > 5 cm DBH.
Brda (2003) compared the direct and indirect methods for LAI estimation and insisted that the
underestimation of LAI by indirect methods could vary from 25% to 50% in forest stands. Due to canopy
heterogeneity, calibration is necessary while estimating LAI through indirect methods (Brda 2003). Therefore,
for the estimation of LAI of teak we should choose the allometric equations which have fewer chances of error
and estimates accurate value. Our allometric models are easy to apply and very practical, so it could be applied
also at the locations outside where it is developed. Chave et al. (2004) also emphasized that the majority of
systematic errors may result if the allometric models are applied for trees with greater D than those used to
construct the allometry. The regression models developed in our study can be applied separately for the ten D-
classes, and this could minimize the error occurring during non-destructive estimation of LAI of teak in different
D-classes.

CONCLUSIONS
Previous studies have mostly adopted harvesting, litter trap methods and optical sensors for the estimation of
LAI for teak stands. However, still there is scarcity of the method to determine LAI of individual teak tree.
Moreover, the allometric models which are available are applied for a broad range of D-classes, due to the
scarcity of models for a range of D-classes. Therefore, for accurate estimation of teak LAI, it is necessary to
develop allometric models for different D-classes. Our study detected logistic models more appropriate for
broad diameter range and Gompertz models for small D-classes. We observed that the linear models are not
efficient in estimating teak LAI, compared to the non-linear models. Even among the three non-linear models,
there is very less difference. Our allometric model could accurately estimate the spatial variability of teak LAI.
Therefore, while choosing any appropriate technique, worker should keep in mind his own situation and the
physiological process of interest.
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ACKNOWLEDGEMENTS
R.K.C. thanks Council of Scientific and Industrial Research, India (award no. 09/13(452)/2012-EMR-I) and
Chinese Academy of Science, China for financial support.

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Appendix I. Descriptive statistics of the leaf area index of teak used for developing regression models.
Model No. D-class (cm) Min.Max. Mean Standard deviation Skewness Kurtosis
1. 3.2 < 6.4 2.6424.975 3.807 0.749 0.168 -0.715
2. 6.4 < 9.6 2.6103.873 3.382 0.434 -0.318 -0.938
3. 9.6 < 12.7 2.4134.211 3.259 0.699 0.190 -1.503
4. 12.7 < 15.9 2.5533.981 3.435 0.488 -0.753 -0.671
5. 15.9 < 19.1 2.9054.165 3.593 0.380 -0.176 -0.221
6. 19.1 < 22.3 3.3164.696 4.055 0.401 -0.275 0.043
7. 22.3 < 25.5 3.4464.740 4.168 0.452 -0.365 -1.483
8. 25.5 < 28.7 4.5155.396 4.897 0.293 0.436 -0.976
9. 28.7 < 31.8 4.7695.258 5.053 0.143 -0.757 0.512
10. 31.8 4.9605.590 5.224 0.207 0.384 -0.860

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ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(2): 286296, 2017
DOI: 10.22271/tpr.2017.v4.i2.038
Research article

Plants struggling to receive proper identity at Bhadaure Tamagi


of Kaski district, Nepal
Bimal Bahadur Kunwar*
Research, Community Development and Conservation Center (C3DR), Pokhara-25, Hemja, Nepal
*Corresponding Author: bimalnature@gmail.com [Accepted: 19 July 2017]

Abstract: Neglected and underutilized plant species have a potential to contribute to food security
and poverty alleviation. Most plant species consumed as food across the world are neglected and
underutilized. These includes plants with edible fruits, grains, leaves, nuts, oils, roots and tubers,
fibers, medicines, spices, stimulants and their derived products. Until now, there was very limited
information available on the utility of plants for different purposes in the study area. Thus, to
minimize the gaps, the research was carried out at Bhadaure village of Kaski District, Nepal. Data
were collected from the different sites through the application of Participatory Research Appraisal
methods i.e. direct observation, household survey, individual interviews and key informant
interview. This was followed by the field visit with the help of community members where
collection of the plant samples and capturing of the photograph was done. Then each species was
evaluated for nine parameters (extent of the production, extent of consumption, degree of
consumption, perceived nutritional value, cultural importance, medicinal properties, market use,
market value and contribution to household income). From the community interaction and field
visit a list of 66 plants were extracted as underutilized plants in the study area. They were from
various habitats. About 55% of the species were collected from natural or semi-natural vegetation,
particularly forest. 33% of the plants were used for vegetable. The collection or harvesting period
of the recorded plant were varies on the case of species. The availability of wild plants has also
declined drastically. Young generation was not so much interested in these plants. Some of these
plants were observed on market places being sold at competitive prices. Specific household
members consume these plants. Commercialization of these fruits is mainly undertaken by the low
resource base farmers.
Keywords: Food security - Neglected and underutilized plant - Participatory research appraisal -
Farmer.

[Cite as: Kunwar BB (2017) Plants struggling to receive proper identity at Bhadaure Tamagi of Kaski district,
Nepal. Tropical Plant Research 4(2): 286296]
INTRODUCTION
There are 350,000 plant species recorded in the world, out of them 80,000 species are edible for humans.
The cultivated plant species are represented from 55 families (Fuleky 2009). For both the poorest and the riches
socio-economic groups of the people have received important sources of protein, fats, vitamins, and minerals
from many thousands of wild plant species (Akhtar 2001, ICIMOD 2010, Aryal 2010). In many parts of the
world, the use of wild plants as a vegetable and fruits is very common (Bussmann et al. 2006, Cavender 2006,
Pieroni et al. 2007, Dutta et al. 2016). Most plant species that are used for food across the globe are neglected
and underutilized. Search for the alternative plants that are edible for humans as food or fruits havebeen done
through the process of trials and errors by various tribes, in turn domesticating the useful plants (Prescott &
Prescott 1990, Scherrer et al. 2005, Bajpai et al. 2016, Dutta et al. 2016). Current estimation of more than 7,000
plant species are cultivated or harvested from the wild for food in global scale.
In Nepal, estimation of plants between 6,500 species of flowering plants (WCMC 1994) and 5,800 (Hara et
al. 1978, 1979, 1982) have been done, out of which 1,500 are considered beneficial (Manandhar 2002). Out of
these, 651 species are economically useful including 440 species of wild food plants and about 200 plant species

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Received: 31 March 2017 Published online: 31 July 2017
https://doi.org/10.22271/tpr.2017.v4.i2.038
Kunwar (2017) 4(2): 286296

are consumed as vegetables (Manandhar 1982), most of them, however, are regarded underutilized or neglected.
The history of the utilization of the plant resources in Nepal dates back to the work of Banerji (1955). After his
work, some workers continued the ethnobotanical study in the 60's and 70's (Pandey 1964, Dobremez 1976).
Neglected and Underutilized plant species (NUS) are those species that have a potential to contribute to food
security and poverty alleviation and which has not been fully exploited. Increased public awareness about
underutilized species was prompted by the Convention on Biological Diversity and the Global Plan of Action
for the Conservation and Sustainable Utilization of Plant Genetic Resources for Food and Agriculture (FAO
1996), The Consultative Group on International Agricultural Research (CGIAR 2004) had initiated to include
underutilized species as a majorresearch agenda since 2004.
The plant struggling to receive proper identification but have benefitted to the people or NUS have received
negligible attention from research and development, and there is little scientific information about them and they
are described as neglected (Eyzaguirre et al. 1999). These plant species have value addition aspects to
diversify the use and shelf-live. Some are highly important for food security; others may prove to have greater
potential for income generation, for environmental services such as soil fertility maintenance, scenic and
biological pest control. The production and consumption of neglected and underutilized species prop up higher
incomes and in good health nutrition and preserves the cookery and cultural traditions of indigenous
communities. In many instances, these species are the only crops that can cope with harsh environments unfit
for other crops. The coverage of these plants is wide, including plants that provide edible fruits, grains, leaves,
nuts, oils, roots and tubers, fibers, medicines, spices, stimulants and their derived products. Therefore, this
demands their promotion which should be done in a sustainable manner. Food security and poverty alleviation
in rural communities can be strategically improved by diversifying the existing farming systems.
Commercialization of these plants is mainly undertaken by the low resource base farmers. The NUS have been
overlooked by scientific research and development workers despite the important role they can play in terms of
food provision and food culture of the rural poor and livelihoods of many rural and urban communities but their
use in terms of diversification is low and subsequently classified as underutilized.
Lack of attention of NUS by stakeholders has resulted in genetic erosion and ultimately their vanishing,
which are further restricting development options for the poor. Many underutilized species are particularly
beneficial as food and medicine, especially in marginal areas, where they have been selected to withstand stress
conditions and contribute to sustainable production with low-cost inputs. Unfortunately, these species have been
neglected by researchers in the past and, consequently, the information available regarding their agronomy,
yield improvement potential and quality is insufficient.
Historically, a large number 100,000 plant species have been used regularly for food, fiber, industrial,
cultural and medicinal purposes and is recognized as being underutilized. However, as a result of modernization
of agricultural practices, many of these plant species have been neglected due to their being held in low prize
and some to the level that genetic erosion of their gene pools has become extremely severe. At least 7,000
cultivated plant species are currently in use around the world (FAO 2007). By doing this study, the awareness
level of the community people regarding in NUS and its importance had been raised to a certain extent. It is
recommended that follow-up interventions need to be carried out through on-farm farmer managed trials and
value addition technologies on these plants. Priority should be given to exploration, collection, conservation and
promotion of both production and value addition technology of these species in this region. There is a wide
range of under-utilized plant species which can be cultivated and/or improved and processed. However, owing
to the lack of adequate knowledge and other technologies, full potential has not been exploited yet. Addressing
these views will be highly relevant to the development of an improved NUS agricultural portfolio. Despite their
importance for subsistence, income generation, and culture, the use of traditional vegetables is declining at an
alarming rate in all areas of Nepal, combined with genetic and cultural erosion. This occurs particularly in easily
accessible regions, where commercialization of the production is possible. The nutritional value of the wild
plant species needs to be analyzed and recognized. In addition, their utilization should be promoted to improve
livelihoods in rural and urban Nepal (Joshi et al. 2007).
There was limited information on the status of the NUS in the study area and that limits their promotion
along with their product value chain in terms of exploiting production potential, processing, effective and
efficient marketing. The purpose of this study was, therefore, to identify the underutilized plants in the studied
area, the constraints, and assess the potential, production and utilization of these plants.

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MATERIAL AND METHODOLOGY


Data were collected during expeditions from different sites through the application of Participatory Research
Appraisal tools and techniques such as direct observation, household survey, individual interviews and field
visits. The research was carried out at Annapurna Gaupalika ward no. 4 (formerly known Bhadaure Tamagi
VDC ward no. 1 and 2). It is in the West of Pokhara city (Headquarter of Western Development region of
country Nepal or Province number 4) at about 30 km and is connecting with rural gravel road with regular
access to public transportation and GPS location is Latitude 2814'4818"2814'4827", Longitude 8351'3125"
8351'3128" and altitude 14051959 masl (Fig. 1). There are ca. 243 households in these two wards (193 in
Badaure village and 50 in Talibrang).

Figure 1. Map of the research area: Bhadaure Tamagi Village.


Fifteen randomly selected individuals were interviewed using semi-structural questionnaire. In addition, a
checklist was prepared using focus group and key informant interviews. Through discussion, some key
information was recorded on each of the species identified. These are: local vernacular names, type of plant, part
used and period of availability of the part used. Each species was evaluated for nine different parameters (extent
of production, the extent of consumption, the degree of consumption, perceived nutritional value, cultural
importance, medicinal properties, market use, market value and contribution to household income). Then it was
followed by the field visit with the help of community members. Informants (local Farmers) were asked to list
the plants they considered as underutilized based on their potential and actual utilization including marketing
opportunities. Scientific names were determined using the standard book of Flora namely Flowers of the
Himalayas (Polunin & Stainton 1984), Flowers of the Himalaya (Stainton 1988), Dictionary of Nepalese Plant
Names (Shrestha 1998) and with consulting local healer. Surfing the internet for latest findings and the search
for the publications housed on the library (across national research and development institutions) were also
conducted for better documentation of the NUS recorded.

RESULT AND DISCUSSION


From the community interaction and field visit, a list of 66 numbers of plants was extracted as underutilized
plants in the field survey (Table 1). Plants belonging to herb were 22 plants, shrub 18 plants and trees 14 plants.
Climbers (13 plants) were also recorded from the present study. Collections of these plants were made from
garden (7 plants), cultivated field (32 plants) and wild habitat (63 plants). Most plants were perennial (43
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plants), followed by annual (20 plants) and biannual (2 plants).

Figure 2. Habitat of the neglected and underutilized plant species.


Special consideration was done about the habitat of the plants, i.e., wetland, farmyard, trail side, core forest,
forest, grassland, agricultural land etc. (Fig. 2). About 55% of the species were collected from natural or semi-
natural vegetation, particularly forest, whereas nearly out of 27% for total plants were found from farmyard
area. Only 11 plants were found to be cultivated.

Figure 3. Diversity of plant parts used.


Out of a total of 66 traditional, neglected plants species, 10 species were mainly used for their leaves part,
tender shoots or underground stem of 6 plant, flowers of 1 plant, fruits of 28 plant, roots and tubers of 6 plants,
whole parts were of 19 plants and so on (Fig. 3). Similarly, 33% of the plants were used for vegetable, 25 plants
for fruit or row food, 10 plants were for a medicinal purpose and so on (Fig. 4). Some plants like Pyrus pashia
Buch.-Ham. ex D.Don, Elaeagnus parvifolia Wall. ex Royle, Rhamnaceae Plant, Viburnum mullaha Buch.-
Ham. ex D. Don, Pyrularia edulis (Wall. ex Floxb.) A. DC., Castanopsis indica (Roxb. ex Lindl.) A.DC.,
Zanthoxylum armatum DC., Dioscorea deltoidea Wall. ex Griseb., Dioscorea pentaphylla L., Choerospondias
axillaris (Roxb.) B.L. Brutt & A.W. Hill, Artemisia absinthium L. and others were collected for the fruit
purposes and they are directly eaten as raw or used for medicinal purposes whereas plants like Polygonum molle
D. Don, Pouzolzia zeylanica (L.) Benn., Dendrocalamus hamiltonii Nees & Am. ex Munro, Colocasia sp. and
different types of Niuro and Mushroom are used as vegetable.
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Figure 4. Different uses of the neglected and underutilized plants.


The collection or harvesting period of the recorded plant varied with species. Some of them (20 plants) can
be collected every season, while months between June to August i.e. 12% was found to be the most appropriate
period of harvesting (Fig. 5).

Figure 5. Collecting or harvesting season for neglected and underutilized plants.


According to the respondents, the availability of wild plants has also declined drastically, e.g., because of
land-use and habitat change, excessive collection from natural habitats, climate change causing more frequent
droughts and fires, and deforestation. In the research area, species such as Dioscorea bulbifera L. and
Asparagus racemosus Willd. were endangered because they have a high demand at markets, but are mostly (and
often excessively) gathered from their natural habitats. Similarly, plants like Viburnum mullaha, Catunaregam
spinosa (Thunb.) Tirveng., Ziziphus sp., Toddalia asiatica (L.) Lam., Dioscorea deltoidea, Citrullus colocynthis (L.)
Schrad., Elaeagnus parvifolia, Coccinia grandis (L.) Viogt, Dioscorea pentaphylla, Agaricus campestris L.,
Oreocnide frutescens (Thunb.) Miq., Cyathea spinulosa Wall. ex Hook., Dioscorea bulbifera, Allium wallichii Kunth,
Baman, Pyrularia edulis or many wild species are declining day by day and turning towards rare condition due to
their low regeneration power and the lack of proper awareness about the importance of the plants.
Out of the population of the farmers interviewed 40% were males and 60% were females. The average age
of the respondents was about 38 years with the oldest being 78 years while the youngest being 25 years. The
respondent information showed that the collection or harvesting of the plants was done by female mostly and
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that was especially for their domestic purposes. In one Nepalese site, women above 35 years of age could
describe the uses of 65 percent of all edible species, while young men could only describe 23 percent (Shrestha
& Dhillon 2006). Beside this, the male respondent of the study area was comparatively more familiar towards
such plant than female. Owing to the advancement of the technology young generation seem not to be interested
in utilizing these plants.
Despite their importance for subsistence, income generation, and culture, the use of the plant is declining at
an alarming rate in the study area. This occurs particularly in easily accessible regions, where commercialization
of the production is easily possible. Only few plants such as Zanthoxylum armatum, Ficus sarmentosa Buch.-
Ham. ex Sm., Cinnamomum verum J.Presl, Persea odoratissima (Nees) Kosterm., Ficus lacor Buch.-Ham.,
Asparagus racemosus and Choerospondias axillaris were still cultivated at field-scale in the field surveyed
(Table 1) due to their known economic values. Cultivation of exotic plants for subsistence and sale increases
more and more at the expense of traditional ones, partly promoted by development programs. These studies
mostly did not consider the disadvantages of exotic plants, e.g., the high need of external inputs for successful
cultivation or the often rather low nutritional value.
The specific niches where they were growing included the inside core forest (Community as well as
National), trail side, along with the fences or farm boundaries while others were mainly growing in streamside
and uncultivated areas. However, these plants received minimum or no agronomic attention and most often get
waste at the time of peak production.
Some of these plants were observed on market places being sold at competitive prices. Specific household
members consume these plants. Some market data was recorded e.g., powder of Rhamnaceae Plant (Sutpi)
plant's fruit was sold US$1.5/Kg by a house. From Brahmin community selling of NIURO/Circinate fern
occasionally is practiced. There was no trend of good agricultural practices; they did that only in season for
paddy and millet, not for vegetable and fruit. So, their collection from wild habitat was for themselves only.
Further, the people of the study area have followed Gurung culture and per them, selling of growing plants to
generate money are against their religion. Social prestige is often worth more than money in the Gurung
societies. So, there is the absence of marketing trend. They collect the plant product for their own utilization or
use as gift items to visit their relatives. In a perfectly competitive market, no species would be considered
underutilized: its use would reflect its low value, and limitation of its collection or cultivation to specific areas
would be justified. As argued above, plant species are underutilized because of market imperfections. These
days by the commercialization and easy accessibility of market as well as other facilities, the community began
to realize the importance of money by selling their product.
The lack of economic information and the lack of product knowledge can negatively contribute to the failure
in the market. The lack of knowledge can be a market constraint, resulting in a lower demand than what it would
be under full information. Although the study is not carried out sound economic analysis but data clearly shows
that the neglected species are an important source of household incomes and can contribute to poverty reduction.
Such species can be introduced to the study area by marketing development. The market can be developed by
increasing market demand through the promotion of their value with better scientific knowledge. These plants
can be conserved and utilized in home stay which is flourishing from the study area.
Lack of attention of NUS by stakeholders in the industry meant that their potential value is under-estimated
and under-exploited. It also places them in danger of continued genetic erosion and disappearance which would
further restrict development options for the poor. To avoid or at least minimize the impending genetic and
cultural erosion concerning traditional and neglected plants, their germplasm should intensively be collected and
conserved on-farm as well as in gene banks. The related indigenous understanding urgently needs to be
documented for serving future generations. The cultivation methods of these plants should be studied and
improved. Their nutritional value needs to be analyzed and recognized. In addition, their utilization should be
promoted to improve livelihoods. Encouraging farmers to continue growing these traditional crops is also a
challenge. Recognizing the role played by farmers as custodians of local diversity is an influential way to
reinforce the self-esteem of community members and thus to contribute towards a self-sustainable and righteous
circle for on-farm conservation.
Some strategies aiming at conserving these plant species focus on the promotion of their use and
conservation including the strengthening of the market system. The starting point is, therefore, collection and
synthesis of knowledge of the obtainable situation within the authorization region through a survey. The survey
therefore aimed at i) identifying underutilized fruit species ii) identify the gaps in the product value chain where
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there was underutilization and iii) identify the major cultivation constraints. To provide policy makers with
more detailed, robust and convincing economic data, it is recommended that a scientifically well-designed
socio-economic study be conducted on the identified species.

CONCLUSION
Owing to no prior documentation of the plants from the studied area, this research focused in the
enumeration of plants may help in developing a conceptual framework whereby so called useless plants could
be employed for the benefit of the mankind. This study revealed that these plants endow several beneficial
aspects and could be highly beneficial if these resources could be tapped properly. Also, creating the awareness
about the utilization of the beneficial aspects of these plants may help uplift the socio-economic condition of the
local inhabitants. In addition, alternative beneficial aspects and the rigorous scientific discourse could also be
carried out utilizing these plants.

ACKNOWLEDGEMENT
I am highly acknowledgment the PEACE Programme of USC Canada/MDO and its employees for their
contribution in field study. The gratitude is extending towards my friend Bikash Baral, Ph.D. scholar for his
incessant encouragement to carry out this research. I cannot remain quite without giving sincere thanks to the
villager of the study area, especially Prem Gurung, Sri Prasad Gurung and Hiralal Gurung.

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ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(2): 297306, 2017
DOI: 10.22271/tpr.2017.v4.i2.039
Research article

Carbon sequestration and CO2 mitigation in a burned ecosystem


of Pinus roxburghii forest in Langtang National Park, Nepal
Biva Aryal1*, Bishnu Prasad Bhattarai2, Mohan Pandey1 and Anjana Giri3
1
Society of Natural Resources Conservation and Development, Dillibazzar-33, Kathmandu, Nepal
2
Birendra Multiple Campus, Tribhuvan University, Chitwan, Nepal
3
Nepal Academy of Science and Technology, Khumaltar, Nepal
*Corresponding Author: aryalbiva@yahoo.com [Accepted: 21 July 2017]

Abstract: Carbon (C) sequestration plays a significant role in mitigation and adaptation to the
impacts of the climate change. By contrast, fire is one of the factors that can alter the carbon
cycles. Extensive fire destroyed the whole forest severely and affects C sequestration. However,
the impact of the surface fire has been least studied. To test the impact of surface fire on C
sequestration of Pinus roxburghii forest, we measured total carbon stock of unburned (CON) and
burned sites of different intensities namely: high frequency and high intensity (HFHI), high
frequency and moderate intensity (HFMI), high frequency and low intensity (HFLI), in Langtang
National Park, Nepal. Total tree carbon stock (above ground + below ground) was analyzed by
calculating the biomass. Coverage of ground vegetation (%) was analyzed by visual estimation.
The total carbon stock between the species was highly significant (P=0.00) and the highest carbon
stock value was recorded in P. roxburghii species in all sites. Similarly, total CO2 mitigation in all
four sites according to higher to lower values were 3346.27, 3345.16, 2484.14 and 2037.95 t.ha-1 in
HFMI, HFHI, HFLI and CON, respectively. The high amounts of CO2 mitigate by HFHI and
HFMI sites strongly support that forest fire limited to the surface may have a positive impact. High
ground vegetation in HFMI site also suggested that the fire of medium intensity mitigate CO2 and
maintains the diversity of ground vegetation as well.
Keywords: Carbon stock - Climate change - Fire intensity - Ground vegetation - Surface fire.

[Cite as: Aryal B, Bhattarai BP, Pandey M & Giri A (2017) Carbon sequestration and CO2 mitigation in a
burned ecosystem of Pinus roxburghii forest in Langtang National Park, Nepal. Tropical Plant Research 4(2):
297306]
INTRODUCTION
Human activities, especially the burning of fossil fuel, deforestation, have caused a substantial increase in
the concentration of carbon dioxide (CO2) in the atmosphere and leading to global warming. Green plants
capture CO2 from the atmosphere through the natural process of photosynthesis and store carbon for a long time
called C sequestration (Watson et al. 2000). The CO2 is naturally stored in above ground biomass (trees, shrubs,
herbs, etc.), below ground parts (root, micro-organisms, etc.) and also in soil (Adam 2001). This is one of the
effective techniques for mitigating the atmospheric CO2 levels (Jina et al. 2009). Simultaneously, green plants
also release CO2 through the process of respiration and microorganism by decomposition to the atmosphere.
These carbon gain and loss processes are continuing in nature.
Forest covers more than one-third of the worlds land area and constitute the major terrestrial carbon pool
(Roberntz & Sune 1999). Tree growth serves as an important means to capture and store atmospheric CO2 in
vegetation and biomass products; about half of its standing biomass is carbon itself (Ravindranath 1997, Petsri
et al. 2007, Terakunpisut et al. 2007, Adhikari 2011). It is estimated that the worlds forests store 283 Gt of
carbon in their biomass alone (Anon 2005). In growing season, plants uptake large amount of CO2 from the
atmosphere and converted into biomass (Losi et al. 2003, Samalca 2007, Deo 2008, Adhikari 2011), which in
turn mitigates the level of CO2 in the atmosphere. Atmospheric CO2, contributed from different medium
including biological sources, is a primary Green House Gas (GHG) and its concentration in the atmosphere has

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Received: 20 March 2017 Published online: 31 July 2017
https://doi.org/10.22271/tpr.2017.v4.i2.039
Aryal et al. (2017) 4(2): 297306

been increasing steadily since 1958 (Keeling et al. 1989, Kumar et al. 2013), due to fossil fuel combustion,
cement manufacture, land use, deforestation, etc. The level of CO2 in todays atmosphere is 31% higher than it
was at the start of the Industrial Revolution about 250 years ago (IPCC 2007). Similarly, atmospheric levels of
CO2 have risen from 280 ppm at the pre-industrial era (1750) to the present level of 385 ppm (WMO 2008).
Because of the increase in the concentration of CO2, mean earths temperature has already increased by 0.60.2
C, and is projected to increase by 24 C towards the end of the 21st century (IPCC 2007). These increases
global warming will result in climate changes like sea level rise, increased frequency and intensity of wildfires,
floods, drought, unusual rain, snow, etc.
Nepal's contribution to the global annual GHG emission is 0.025% (MoPE 2004). The total GHG emission
from Nepal is estimated at 39,265 Gega gram (Gg) and per capita emission is 1,977 kg (GoN 2008a). Over the
last twentyfive years, the temperature in Nepal has also been increasing at the rate of 0.06C per year (GoN
2008b). In high altitudes, it increased by 0.6C over the last thirty years (Liau & Rasul 2007). In Nepal, CO2
emissions are those stemming from the burning of fossil fuels and the manufacture of cement
(http://data.opennepal.net-/content/carbon-dioxide-emissions-nepal).
In Nepal, the forest is frequently under fire and considered as one of the dominant disturbances generally
repeats every year during dry seasons (visual observation). Additionally, the frequency and intensity of fire in
protected and non-protected areas differ significantly (local communications and visual observation). In
protected areas usually controlled fire has been practiced by the authorities, however, in non-protected areas and
sometimes in protected areas it happens accidentally or intentionally. It is well-known fact that forest fire causes
a change in the vegetation composition (Romanya et al. 2001, Mackenzie & DeLuca 2006), damages flora,
fauna, human livelihoods, local climate change every year and also change the carbon sequestration.
Carbon sequestration and reduction of emission are the major mitigative options to reduce the consequences
of climate change (IPCC 1995). It was observed that the maximum carbon is stored in the order of
conifers>deciduous>evergreen>bamboos (Negi et al. 2003). Among conifers Pinus roxburghii Sarg. forests
perform maximum C sequestration (Negi et al. 2003, Sheikh & Kumar 2010, Panthi 2011, Afzal & Akhtar
2013). In contrast, high concentration of resin in its tree trunk and needles make the plant more sensitive to fire
(Paudyal 2008, Kumar 2015), however, the quantity of destruction mainly depends on the intensity of the fire. It
is also observed that P. roxburghii forests are still surviving after surface fire (visual observation, local
communication). In this background, we hypothesized that surface fire has a low impact on forest structure and
C sequestration. Therefore, the present study focused on determining the status of tree C stock of various surface
fire intensities in a burned affected ecosystem to provide total CO2 mitigation in burned ecosystem of P.
roxburghii forest.

MATERIALS AND METHODS


Study area

Figure 1. Study area in Langtang National Park showing four sites in black dots (Dhunche and Syapru VDCs of Rasuwa).
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The study was carried out in Langtang National Park, Rasuwa district, Central Nepal. The district lies in
between 27 55' to 28 25' N latitude and 85 00' to 85 50' E longitudes (Fig. 1). The altitude ranges from 617
m to 7,227 m within 1,512 km2. It encompasses three distinct geographical zones: the Himalaya, the mid hill
and basin. Mid hill and basin covers less area but the productive land lies in this region. According to the
interview with park authorities, local people and also the visual observation of forest fired conditions, the Pinus
roxburghii forest of Dhunche (headquarter of Rasuwa District) and Syapru VDCs (Village Development
committee) of Rasuwa District were selected for the study. Sites 2 and 3 are located in Dhunche VDC (286'42"
N, 8517'52" E, 2,030 m) while sites 1 and 4 are selected from Syapru VDC (2816'80.47" N, 8532'90.87" E,
1,352 m) (Fig. 1).
Vegetation
The vegetation of the P. roxburghii forest in both selected VDCs was dominated by tree species like P.
roxburghii, Rhododendron arboreum Sm., Lyonia ovalifolia (Wall.) Drude, etc. Similarly, understory was
occupied by saplings of Pinus roxburghii, Rhododendron arboreum, Lyonia ovalifolia; shrubs like Indigofera
cassioides DC., Inula cappa (Buch.-Ham. ex D.Don) DC., Desmodium sp., Salix sp., Berberis aristata DC. and
Rubus ellipticus Sm., etc. Ground vegetation are dominated by Ageratina adenophora (Spreng.) R.M.King &
H.Rob., Anaphalis sp., Artemesia sp., Aconogonum sp., Anaphalis sp., Arisaema sp., Bidens bipinnata L.,
Calicarpa sp., Capillipedium sp., Carex sp., Cyprus sp., Dryopteris sp., Hypericum sp., Melastoma sp.,
Persicaria capitate (Buch.-Ham. ex D.Don) H.Gross, Trifolium repens L., Selaginella sp., etc.
Climatic conditions
The climate of Nepal falls within the monsoon system of the Indian subcontinents with dry periods in the
winter and wet periods in the summer. More than 80% of the rainfall occurs during the four summer months
(June to September). Mean maximum temperature of the study area was 24.52C during the June and mean
minimum air temperature was 2.26C in January. Similarly mean maximum precipitation was 593.45 mm in
July, minimum in December (6.6 mm). These values are mean from 1999 to 2013 AD (Department of
Hydrology and Metrology, Nepal Government).
Site selection criterion
Key informants who have knowledge about the issues and an idea about the sites were requested to prepare a
community map including different intensity burned affected forest ecosystem. With the help of community
map, forest sites were surveyed with the local people. After visiting the surface burned affected forest, three
sites were classified on the basis of frequency (fire event within a time period) and intensity (fire severity) of
surface fire and one site as a control. The sites were categorized as high frequency and high intensity site
(HFHI), high frequency and moderate intensity site (HFMI), high frequency and low intensity site (HFLI), and a
control (unburned) site, after collecting the basic data of height of ground vegetation (Armour et al. 1984),
charcoal accumulation on ground (Turcios et al. 2016), needle accumulations, fired level on tree trunks of Pine
and other species (Fire Science Brief 2009), presence of olive trees (James et al. 1985), coverage of bare land,
number of burned species etc. The criterion has been summarized as below:
a) High frequency and high intensity (HFHI) site: Characterized by low canopy coverage (22%) of ground
vegetation (Fig. 2), total ground coverage lower than in control, site affected by annual surface fire events
during dry season (according to park authority, local communication, visual observation) and remains of
burned needles and bark on the ground, burned stumps and even trunk of pine tree up to 4 meters.

Figure 2. Canopy coverage (%) of ground vegetation, litter, bare land and crown coverage of all sites.
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b) High frequency and moderate intensity (HFMI) site: The site was occupied by higher canopy of understory
(131%) than high-intensity site. Although, the impact of fire has been observed on the stem of ground
vegetation as well as on the trunk of P. roxburghii, regenerating young leaves on ground vegetation
suggested the low severity of fire (Fig. 2).
c) High frequency and low intensity (HFLI) site: Characterized by the vigorous growth of understory species
and burned have been observed at the base of mature trees. The site has almost similar features with HFMI
site except for the height of the understory, which was up to 2m and the value of shrub coverage (87%) and
litter accumulation (74%) was higher with negligible damage (Fig. 2).
d) Control (CON) site: The control (unburned) site has been supported by high coverage of species, the
absence of bare land (Fig. 2), almost no charcoal accumulation, and luxuriant growth of epiphytes (data not
shown).
Sampling
Random sampling method was used for sampling the above ground (AG) vegetation. Five replicates of the
sampling plots were surveyed in three burned sites and four replicates in control site. The sampling was done in
August to September 2015 with the help of 3 nested circular plots of radii 10 m, 5 m and 2.5 m for trees,
saplings, shrubs, respectively (FRA 2011). Similarly, for the ground vegetation 1m 1m plots were laid down
within the main plots (Fig. 3). For carbon (C) analysis, plant species having DBH >10cm and height >1m was
considered as a tree. To calculate the coverage (%) of understory vegetation, plant species having height of <1m
was considered as shrubs (Shrestha & Singh 2008) and DBH <10cm was considered as sapling.

Figure 3. Showing plot design - CT - Central tree, 10 m, 5 m and 2.5 m circular plot for trees, sapling and shrub respectively
and 1 m 1 m for grass and herb (FRA 2011).
Tree diameter at breast height and tree height
Diameter at breast height (DBH) of each tree within each plot was measured using DBH tape, and height of
each tree was calculated using Sunto Clinometer. Plants were identified by consulting literature and
photographs.
Above ground biomass (AGB) estimation
The AGB of the tree includes the wood, branches, leaves, barks, etc., were measured by following methods:
a. Volume: Volume of the trees were calculated by r2h.
b. Biomass of wood: The biomass of wood was calculated following the method given by Chaturvedi &
Khanna (1982). Biomass of branch and foliage were calculated by MPFSN (1988).
Below ground biomass (BGB) estimation
The BGB includes all biomass of live roots. The below ground biomass has been calculated following Oli &
Shrestha (2009).
Carbon analysis
Above ground carbon (AGC) like wood, branch and foliage carbon were analyzed according to Negi et al.
(2003). The below ground carbon (BGC) has been calculated by Oli & Shrestha (2009).
CO2 mitigation (t.ha-1)
CO2 mitigation was calculated by the method adopted by Bhattarai et al. (2012).
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Statistical data Analysis


The significance of differences in AGC and BGC stock between sites was evaluated by one way ANOVA
and Duncans multiple range tests by using SPSS software (SPSS Inc., Chicago, IL, USA).

RESULTS
Ground Coverage
Highest canopy coverage (%) of ground vegetation (grass, herb, shrub, etc) was observed in HFMI site while
lowest in HFHI site. HFLI site was characterized by the vigorous growth of shrub coverage (87%) was highest
than other sites. Similarly, highest coverage (35%) of grass species was recorded in CON site as well as HFMI
(34.5%). Lowest grass coverage (12%) was recorded in HFHI site (Fig. 2).
Above ground carbon (AGC) stock
Among the 4 sites, the maximum mean value of AGC for P. roxburghii is observed in HFMI
(704.78160.13 t.ha-1) and the minimum mean value was for CON (445.72198.83 t.ha-1). The minimum mean
value of AGC was observed in the associated species like R. arboreum (2.971.76 t.ha-1) in HFMI, L. ovalifolia
(6.382.66 t.ha-1) in HFHI. The AGC stock between the species was highly significant (P=0.00) while within
the species was insignificant at all sites (Fig. 4).

Figure 4. Above ground carbon stock (t.ha-1) of all investigated species in all sites. The box plots show the median, and 25
to 75 percentile. Whiskers indicate maximum and minimum values. Different letter denotes the highly significant difference
between the species tested by Duncan multiple range tests at (P<0.05).
The mean value of total AGC stock of P. roxburghii forest was maximum in HFMI (705.96160.43 t.ha-1),
followed by HFHI (703.3797.80 t.ha-1) and HFLI (523.1767.11 t.ha-1). The lowest value was recorded in
CON (454.35198.59 t.ha-1) (Fig. 5). There was the insignificant difference (P=0.472) between the total above
ground carbon stock and fired sites tested by one way ANOVA.
Below ground carbon (BGC) stock
The mean value of BGC for P. roxburghii was maximum in HFMI (206.3046.44 t.ha-1) and minimum in
CON (131.7056.98 t.ha-1). The minimum mean value was also observed in other associated species like L.
ovalifolia (1.660.84 t.ha-1) in HFHI, and R. arboreum (2.210.81 t.ha-1) in HFLI. Likewise, the AGC stock and
BGC stock also showed the highly significant difference in mean values between the P. roxburghii and
associated species (Fig. 4 & 6).

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Figure 5. Total Carbon stock of AGC and BGC from different sites. The Same letter indicates insignificant difference tested
by Duncan multiple range tests. Error bar showed SE (N=7-11).
The mean maximum value of total BGC stock of P. roxburghii forest was recorded in HFHI (208.9427.55
t.ha-1) followed by HFMI (206.6546.53 t.ha-1) and HFLI (154.3119.47 t.ha-1). The lowest value was recorded
in CON (133.8157.10 t.ha-1) (Fig. 5). One way ANOVA analysis showed there was the insignificant difference
(P=0.450) of total BGC stock between the sites.

Figure 6. BGC stock (t.ha-1) of all investigated species in all sites. The box plots show the median, and 25 to 75 percentile.
Whiskers indicate maximum and minimum values. Different letter denotes the highly significant difference between the
species tested by Duncan multiple range tests (P<0.05).
CO2 mitigation (t.ha-1)
Our measurement showed that the CO2 mitigation value by vegetation (above ground + below ground) in P.
roxburghii forest followed similar trend with mean total carbon stock. It was higher in both HFMI (3346.27 t.ha-1)
and HFHI (3345.16 t.ha-1). The minimum in CON (2037.95 t.ha-1), the intermediate value was in HFLI (2484.14
t.ha-1) (Fig. 7).

DISCUSSION
Fire is one of the factors that have negative impacts on C sequestration, although, it depends on the intensity
of the fire. We have selected two VDCs of Langtang National park, where 90% of the pure Pinus roxburghii
forest set on fire during dry season (visual observation). Although, local climatic conditions affect the annual
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fire behavior in P. roxburghii forest of Langtang National Park, various climatic agents like temperature,
relative humidity, and precipitation affect fuel moisture on the forest surface. The long, hot and dry summers
that last from February until May convert the Pine understory into a continuous sheet of dry and highly
inflammable fuel load (IFFN 2005). The rainfall during the dry season is very low and range between 44.48 to
103.69 mm. During the dry season, the maximum temperature fluctuates from 15.25C to 23.69C (Data not
shown) increasing the risk of ignition to high levels. Similarly, sometimes wind and its direction are also
dominant factors for fire intensity.

Figure 7. Mean total CO2 mitigation (AGC+BGC) from different sites.


In fired P. roxburghii forest of the present study, the highly significant difference between the species in
terms of C sequestration has been observed. The maximum mean value was observed in P. roxburghii among all
three fired sites. Minimum mean value observed in associated species like Rhododendron arboreum and Lyonia
ovalifolia. The study carried out by previous authors (Panthi 2011, Afzal & Akhtar 2013) in unfired mixed
Pinus roxburghii forest reported maximum value for P. roxburghii similar to present study; however, there is a
significant difference between the values of other associated species. In our study, the numbers of associated
species were less compared to unburned forests. Therefore, the differences may be due to frequent annual fire on
forest surface in the study area which allows only the growth of fire prone species like P. roxburghii but affects
associated species. The study carried out by James et al. (1985) suggested that low intensity fires on lodgepole
pine stands have no serious damage. Similarly, P. roxburghii has several fire-adaptive traits that enable mature
trees to survive on low-intensity surface fires (Tiwari 1994, Semwal & Mehta 1996, Sangye 2005). The survival
of P. roxburghii after burned of outer bark up to 14 m height in low-high intensity burned sites observed in the
present study further justified its fire resistant nature. Among the three burned sites the total C Stock showed no
significant difference. However, the highest values were observed in HFMI and HFHI sites and lowest in CON
site. Highest value in HFMI, HFHI could be because of the highest number and age (girth) of P. roxburghii in
these sites than CON i.e. with maximum young trees.
The intensity of fire determines the growth of ground vegetation. In high-intensity sites, there is maximum
loss of ground vegetation (Armour et al. 1984), however luxuriant growth of shrubs occurs in medium and low-
intensity sites (Fire Science brief 2009). Among the three burned sites of the present study similar results has
been observed. It is possible because herbaceous plants propagate through seeds are highly affected by forest
surface fire than the plants propagate through rootstocks or rhizome (underground). However, the situation is
different for shrubs and saplings; only fire prone species can grow in such conditions. The reason of the
luxuriant growth of shrubs (Indigofera cassioides, Inula cappa) in medium and low intensity burned sites may
be due to its capacity to grow even after burning. The overall results of ground vegetation suggested that burned
of any intensity can change the diversity of the species.
The increased amount of CO2 in the atmosphere is a global concern and considered as one of the reasons for
changed climatic conditions due to an increase of temperature. Mitigation of CO2 by green plants using the
machinery of photosynthesis is the easiest and inexpensive natural pathway. However, the rate of photosynthesis
i.e. capacity to mitigate CO2 may vary among the different species (Devi et al. 2013). Of them, conifers are
regarded as one of the efficient plants to mitigate CO2 (Negi et al. 2003). However, disturbances such as
deforestation as well as fire are recognized as a driver of climate change. In our study, total CO2 mitigation was
found highest in HFMI, HFHI and lowest in CON site. Highest CO2 mitigation in HFMI and then HFHI
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suggested that in the case of P. roxburghii forest the fire of high to medium intensity limited to the forest surface
has a positive impact on CO2 mitigation.

CONCLUSIONS
Forest surface fire only encourages burned up to a certain height of the tree trunk without damaging Pinus
roxburghii trees, sequestrate more C and sink as a long term C pool which increased the rate of CO2 mitigation
ultimately reduces CO2 from the environment. Therefore, we concluded that the controlled forest surface fire is
beneficial to the global carbon mitigation in case of P. roxburghii forest. The study conducted on three different
fire frequency and intensity sites suggested that in terms of CO2 mitigation, both HFHI and HFMI fire condition
are best. However, HFHI site with low ground vegetation with few species suggested that high-intensity surface
fire reduces the overall diversity because only fire prone species can grow in such areas. Therefore, the intensity
of the fire in P. roxburghii forest can minimize either by removing the fuel load (litter) from the forest surface or
by increasing frequency of induced forest surface fire in control manner.
Finally, the fire of high to medium intensity showed positive impacts on CO2 mitigation by maintaining P.
roxburghii forest and ultimately essential for conservation and management of these forests. Therefore, regular
monitoring of this forest is necessary to induce control fire in manage form to increase CO2 mitigation by
increasing sequestration and maintaining the diversity of the area as well.

ACKNOWLEDGEMENTS
This paper is based on research financed under the Climate Change Research Grants Program implemented
by the Nepal Academy of Science and Technology. The Program is part of the Mainstreaming Climate Change
Risk Management in Development project. This project is a component of Nepals Pilot Program for Climate
Resilience and is executed by the Ministry of Population and Environment (Nepal), financed by the Climate
Investment Funds, administered by the Asian Development Bank with technical assistance from ICEM,
METCON and APTEC. The authors are thankful to the DNPWC and Park authorities for providing permission
for field work. Authors are also thankful to the members of Society for Natural Resources Conservation and
Development, Mr. Gyanendra Neupane (Journalist, Rasuwa), Mrs. Sushma Devkota, Mr. Ram Ranabhat to
support this study.

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ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(2): 307313, 2017
DOI: 10.22271/tpr.2017.v4.i2.040
Research article

Ethnomedicinal aspects of climbing plants of Palpa district, Nepal


Anant Gopal Singh1 and Akhilesh Kumar2*
1
Department of Botany, Tribhuvan University, Butwal Multiple Campus, Butwal, Nepal
2
Department of Botany, Dayalbagh Educational Institute (Deemed University), Agra, Uttar Pradesh, India
*Corresponding Author: akhilpbh@rediffmail.com [Accepted: 23 July 2017]

Abstract: Climbers are those plants which germinate on land and grow by adhering to other plants
to attain great stature. Climbers are different in their mechanical characters, well adapted to climb
on support like large trees, hedgerows or rocks by twining their stems, some climbers climb on
support by the way of adventitious clinging roots, with twining petioles or by using tendrils. A
total of 51 climbing plants species belonging to 40 genera and 22 families used in folk medicine
have been documented from different VDCs of Palpa district.
Keywords: Climber - Clinging roots - Tendrils - Ethnomedicine - Liane.

[Cite as: Singh AG & Kumar A (2017) Ethnomedicinal aspects of climbing plants of Palpa district, Nepal.
Tropical Plant Research 4(2): 307313]
INTRODUCTION
Climbing plants are one of the most interesting group but a much-neglected group of plants. A climbing
habit has evolved independently in several plant families, using many different climbing methods because of
their weak stem. Climbers are different in their mechanical characters (Isnard et al. 2009), well adapted to climb
on support like large trees, hedgerows or rocks. Some climbers climb by twining their stems around a support
another climb by way of adventitious clinging roots, with twining petioles or using tendrils which can be
specialized shoots, leaves or even inflorescence.
Climbers are the plants that germinate on the floor and grow for part of their life by winding ground,
anchoring or adhering to other plants (Jongkind & Hawthrone 2005), to attain great stature (Swaine et al. 2005).
They comprise 7% and 20% of regional flora in temperate and tropical forest respectively (Gentry 1991) and
thus become an important constituent of an area. Climbers are not only the important components of plant
diversity but, are also valuable for their medicinal uses, nutrient cycling etc. (Schnitzer & Bongers 2002). The
climbers are rooted in the ground but need support for their weak stems (Richards 1952). In nature, climbers
generally use plants growing nearby as external support media to ascend (den Dubbelden & Oosterbeek 1995).
Schimper (1903) classified climbers into scrambles, root climbers, twiners and tendril climbers. The term vine
and liane are generally used as synonymous for climber and woody climbers respectively (Kelly 1985).
Today the climbers are an important constituent of non-timber forest products (NTFPs) According to the
best of our knowledge there is no comprehensive study assessing the role of climbers in Nepalese forests for
healthcare management and economic subsistence of local people. Keeping above view in mind present study
was proposed to document the ethnomedicinal uses of climbers for the first time in the Palpa district of Western
Nepal.

MATERIALS AND METHODS


Study Area
Palpa district is a part of Lumbini Zone, is one of the seventy-five districts of Nepal, landlocked country of
South Asia, is located at 2734" to 2754" N and 8315" to 8422" E with an altitude ranging from 152 m to
1936 m above the sea level. It has unique physiography due to Churia and Mahabharat ranges. The district, with
Tansen as its headquarters, covers an area of 1,373 Km2 and has a population of 2, 61,180 of which male and
female proportions are 1, 15,840 and 1, 45,340 respectively (CBS 2011). Palpa district is divided into two
municipalities i.e. Tansen and Rampur and 61 VDCs. It is bounded by Gulmi and Arghakhanchi districts from
West; Gulmi, Syangja, and Tanhun district from North; Nawalparasi and Tanhun from East and Rupandehi and

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Received: 14 November 2016 Published online: 31 July 2017
https://doi.org/10.22271/tpr.2017.v4.i2.040
Singh & Kumar (2017) 4(2): 307313

Nawalparasi from South (Fig. 1). Palpa district is located in hilly region of Nepal. The average temperature of
the district is maximum 32C and minimum 4C (DDC 2009) with dry winter and wet summer. The average
annual rainfall is around 2006 mm, with a fluctuating pattern for total of 29 years between 1985 and 2013 A.D.,
(DCEP 2016). The monsoon starts from June and pronounce rainfall occurs during June to mid-September.
Palpa district of Western Nepal is inhabited by many ethnic communities of which 50% or majority is
comprised by Magars and rest by Brahmin, Newar, Chhetri, Gurung, Kumal, Sarki, etc.
Geographically Palpa district is divided into mid-mountain hill region (82%) and Chure hill region (18%).
Total forest in Palpa district is 67607 ha, the land area used for agriculture is 44,332 ha, shrubs area is 23,736 ha
with 538 ha area of water bodies and 70 ha of barren land. The vegetation of the Palpa district is dominated on
the southern side by lower tropical Sal (Shorea robusta Gaertn.) and mixed broad leaves forest. Terminalia
alata Heyne ex Roth, T. chebula Retz., T. bellirica (Gaertn.) Roxb., Dalbergia sissoo DC., Ficus semicordata
Buch.-Ham. ex Sm., Castanopsis indica (Roxb. ex Lindl.) A.DC., Berberis asiatica Roxb. ex DC., Zanthoxylum
armatum DC., Albizia species, Schima wallichii (DC.) Korth., and Pinus roxburghii Sarg. etc. are also found
dominated in the upper and mid belt of the district.
Palpa district of Western Nepal is selected for research activities because this district is potential in terms of
ethnomedicine. Little works have been carried out by some workers (Shrestha 1985, Mahato 1998, Mahato &
Chaudhary 2003, Singh et al. 2011) but no any work has been done on climbing plants.

Figure 1. Location map of study site: Palpa district.


Data Collection
A preliminary survey was done in different VDCs like Kachal, Dobhan, Koldanda, Gothadi, Rahabas, and
Jyamire to get information about the study area along with local persons and knowledgeable peoples. The area
was visited three times in different seasons (summer, monsoon, and winter) to avail most of the plant resources
in their flowering condition for this purpose. The study was conducted from October 2014 to August 2015. The
methodology was based on interviews using checklist and questionnaire of information.
Plants collected from study area were identified by using previous literature (Hooker 18721897, Bailey
1949, DMP 1969, 1970, 1984, 1986, Polunin & Stainton 1984, Mc Crackers & Shrestha 1992, Stainton 1997,
Chaudhary 1998, Manandhar 2002). Photographs were taken of the plants habit in the flowering conditions,
abundant plants were taken to prepare herbarium sheets. Herbarium specimens and photographs were identified
by taxonomists and finally deposited in the Herbarium of the Department of Botany, Butwal Multiple Campus,
T.U., Butwal, Nepal for future use. The questionnaire was prepared in terms of local language following
Martins (1995) manual. The Scientific name of the collected climbing plants were provided by Hara et al.
(1978, 1979, 1982) and Press et al. (2000) have been adopted.

RESULTS AND DISCUSSION


Survey of palpa district revealed that 51 climbing plants species belonging to 40 genera and 22 families are
used in folk medicine by the inhabitants of different VDCs like Kachal, Dobhan, Koldanda, Gothadi, Rahabas,
and Jyamire of Palpa district. Out of these climbing plants 38 plants belong to Dicots, 12 belong to Monocots
and 1 belongs to Fern. The highest number of climbing plants belong to family Cucurbitaceae (12 species)
followed by Fabaceae (8 species), Convolvulaceae, Dioscoreaceae, Piperaceae (3 species in each), Vitaceae,
Menispermiaceae, Asclepiadaceae, Liliaceae, Smilaceae (2 species in each) etc. Singh (2016) described
ethnobotany of 18 climbers of Paras district of Nepal. Out of these, 15 namely climbers Abrus precatorius,
Acacia rugata, Aristolochia indica, Bauhinia vahlii, Cissampelos pareira, Cuscuta reflexa, Dioscorea bulbifera,

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Figure 2. Some ethnomedicinally important climbers: A, Abrus precatorius L.; B, Ampelocissus barbata (Wall.) Planch; C,
Argyreia nervosa (Burm.f.) Bojer; D, Asparagus racemopsus Willd; E, Basella alba L.; F, Benincasa hispida (Thumb.)
Cogn.; G, Bauhinia vahlii Wight & Arn; H, Boerhaavia difusa L.; I, Capparis zeylanica L.; J, Cissampelos pariera L.; K,
Clitorea ternatea L.; L, Coccinia grandis (L.) Voigt.

Figure 3. Some ethnomedicinally important climbers: A, Cucumis sativus L.; B, Cucurbita maxima Duch. ex Lam.; C,
Cucurbita pepo L.; D, Cuscuta reflexa Roxb.; E, Dioscorea deltoidea Wall. ex Griseb.; F, Gloriosa superba L.; G, Ipomea
aquatic Forssk.; H, Lagenaria sineraria (Molina) Standl.; I, Lygodium japonicum (Thunb.) Sw.; J, Mucuna pruriens (L.)
DC.; L, Pothos scandens L.; M, Tinospora cordifolia (Willd.) Miers.
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D. deltoidea, Gymnema sylveste, Lygodium japonicum, Mucuna pruriens, Passiflora edulis, Quisqualis indica,
smilax aspera and Tinospora cordifolia were recorded during the present study. Climbing plants used in folk
medicine are enumerated in table 1 arranged in alphabetical order by their botanical name along with their
family, local name and ethnomedicinal uses. The photographs of some of these plants have also been provided
(Fig. 2 & 3). These plants with high medicinal values are suggested here for the biological screening to develop
the valuable pharmacological products.
Table 1. Ethnomedicinal usages of climbing plants of Palpa district, Nepal.
Botanical name and Family Local name Ethnomedicinal uses
1. Abrus precatorius L. Ratti Gedi Bronchitis, diuretic, tonic, abortifacient, boils, eczema,
Fabaceae leucoderma, jaundice, back bone pain and gonorrhoea.
2. Accacia rugata (Lam.) Sikakai Fruit used as detergent, young shoot cooked as vegetable.
Voigt
Fabaceae
3. Ampelocissus barbata Jarilo Lahara Bone fracture, skin diseases.
(Wall.) Planch
Vitaceae
4. Asparagus racemosus Kurilo Aphrodisiac, galactagogue, urinary trouble, nervous
Willd. debility, bronchitis, dysentery & throat infection.
Liliaceae
5. Argyreia nervosa Samundra phal Aphrodisiac, dyspepsia, colic, anaemia, anti-diabetic,
(Burm.f.) Bojer cardiac debility, nervous debility, weakness & syphilis.
Convolvulaceae
6. Aristolochia indica L. Ishaharmool Diuretic, stimulant, anti-arthritic, anti-diabetic, blood
Aristolochiaceae pressure, emetic, abortifacient & emmenogogue
7. Basella alba L. Poi Sag Demulcent, insomnia, dermatitis, diuretic, & laxative
Basellaceae
8. Bauhinia vahlii Wight & Bhorla Aphrodisiac, diarrhoea, malarial fever, tonic, boils,
Arn. pimples, blisters & blood dysentery.
Fabaceae
9. Benincasa hispida Kubindo Cooling, styptic (stop bleeding), laxative, diuretic,
(Thumb.) Cogn. aphrodisiac, & anthelmintic.
Cucurbitaceae
10. Boerhavia diffusa L. Punarnawa Pimples, scabies, cuts, wounds, stomachache, cardiac
Nyctaginaceae disorders, jaundice, anemia, constipation, cough,
inflammations and bronchitis.
11. Calamus erectus Roxb. Anti-diabetic, dyspepsia, stomach problems, eczema &
Arecaceae wounds
12. Capparis zeylanica L. Kukurkande Diuretic, resolvent, antipyretic, anti-arthritic, toothache,
Capparaceae jaundice and spleen problems.
13. Cissampelos pariera L. Batulpate Diuretic, diarrhoea, dysentery, dyspepsia, malarial fever,
Menispermiaceae pulmonary disease, piles, cough, antipyretic, cut, wounds,
burns & ring worm.
14. Cissus quadriangularis L. Hathzode Boils, ulcers, rheumatism.
Vitaceae
15. Clinopodium umbrosum Suparnasa/ Birajor Cuts, burns, wounds
(M. Bieb) C. Koch.
Lamiaceae
16. Clitoria ternatea L. Aparajita Leucoderma, liver disease, nervous tonic, abdominal
Fabaceae pain.
17. Coccinia grandis (L.) Golkankri/ Cataract, anti-diabetic, carminative, antipyretic, hepato-
Voigt. Kunaroo protective
Cucurbitaceae
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18. Cucumis sativus L. Kankro Galactagogue, refrigerant, diuretic.


Cucurbitaceae
19. Cucurbita maxima Duch. Pharsi Refrigerant, diuretic, neuralgia.
ex Lam.
Cucurbitaceae
20. Cucurbita pepo L. Pharsi Anthelmintic, anti-inflammatory, kidney stones, urinary
Cucurbitaceae tract infection.
21. Cuscuta reflexa Roxb. Akashbeli/ Jaundice, constipation, bronchitis, fever,
Cuscutaceae Amarbel antihypertensive, cardio-tonic, emetic, antiviral &
antibacterial.
22. Dioscorea alata L. Ghar tarul Wounds, leprosy, gonorrhoea, blood pressure & skin
Dioscoreaceae diseases.
23. Dioscorea bulbifera L. Githa/ Ban tarul Piles, leprosy, asthma, cough, cold, tuberculosis,
Dioscoreaceae contraceptive, constipation, indigestion, dysentery, syphilis,
cardiac debility, aphrodisiac, refrigerant, pulmonary
disease and ulcers.
24. Dioscorea deltoidea Kukur tarul/ Stomachache, kill body lice, wash clothes.
Wall. ex Griseb. Bhyakur
Dioscoreaceae
25. Dolichus lablab L. Rajsimi Emmenagogue, febrifuge, stomachic, anti-spasmodic,
Fabaceae aphrodisiac.
26. Entada phaseoloides (L.) Pangra Epilepsy, constipation, anthelmintic
Merr.
Fabaceae
27. Gloriosa superba L. Karihari Stomachic, anthelmintic, dermatitis & abortifacient.
Liliaceae
28. Gymnema sylvestris Gudmar/ Anti-diabetic, malarial fever, cough, cold, jaundice.
(Retz.) R.Br. Madhunasini
Asclepiadaceae
29. Ipomea aquatic Forssk. Laharo pani sag/ Emetic, purgative, gastric troubles, nervous and general
Convolvulaceae Kerunga sag debility. Tender shoot used as green vegetable.
30. Ipomea batatas (L.) Lam. Sakharakand Refrigerant, laxative, aphrodisiac, diuretic & tonic.
Convolvulaceae
31. Lagenaria sineraria Lauka Anti-inflammatory, refrigerant, expectorant, purgative,
(Molina) Standl. hepato-protective, emetic & diuretic
Cucurbitaceae
32. Luffa acutangula (L.) Pate Ghiroula/ Skin diseases, demulcent, diuretic, tonic, nutritive,
Roxb. Taroi purgative, emetic & expectorant.
Cucurbitaceae
33. Luffa cylindrica (L.) Ghiu Taroi/ Diuretic, emollient, laxative, expectorant, tonic,
Roem. Ghrimala anthelmintic, galactagogue, fever & bronchitis.
Cucurbitaceae
34. Lygodium japonicum Janai Lahara Expectorant, diuretic, cough, cold, fever, anti-arthritic.
(Thunb.) Sw.
Lygodiaceae
35. Marsdenia roylei Wight. Baahuni Lahara Stomachache, purgative, gonorrhoea.
Asclepiadaceae
36. Momordica charantia L. Tite Karela Peptic ulcer, anti-diabetic, rheumatism. Anthelmintic,
Cucurbitaceae antipyretic, purgative & carminative.
37. Momordica dioica Roxb. Bankarela Rheumatism, anti-diabetic
ex Willd.
Cucurbitaceae

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38. Mucuna monosperma Baldhengra Expectorant, cough, asthma


DC.
Fabaceae
39. Mucuna pruriens (L.) Kauso Anthelmintic, aphrodisiac, kidney problems, uterine
DC. Fabaceae. trouble & general debility.
40. Paederia foetida L. Bari Lahara/ Anti-arthritic, colic, flatulence, tonic, astringent, nervous
Rubiaceae Gandha Prasarini tonic, gastro protective, antioxidant & anti-inflammatory.
41. Passiflora foetida L. Sanojhar Anti-ulcer, antioxidant, diuretic, asthma & skin diseases.
Passifloraceae
42. Piper bettle L. Pan Mouth refreshment; improve digestion, diuretic,
Piperaceae analgesic, cough & cold.
43. Piper longum L. Pipli Cough, digestion, bronchitis, fever
Piperaceae
44. Piper nigrum L. Kalo marich Gastric trouble, cough, fever.
Piperaceae
45. Pothos scandens L. Money plant Antioxidant, antipyretic, epilepsy, rheumatism.
Araceae
46. Quisqualis indica L. Madhumalati Purgative, diarrhoea, fever, rheumatism & fever.
Combrataceae / Baja phul
47. Smilax aspera L. Kukurdaino Paralysis, syphilis, diuretic, skin diseases, blood purifier.
Smilaceae
48. Smilax ovalifolia Roxb. Ramdatun Venereal diseases, rheumatism, abdominal pain,
ex D. Don dysentery,
Smilaceae
49. Tinospora cordifloia Gurjo/ Giloy Anti-diabetic, malarial fever, hepato-protective,
(Willd.) Miers. rheumatism, stomach troubles, diuretic, diarrhoea,
Menispermiaceae dysentery.
50. Trichosanthes dioica Parwal Febrifuge, laxative, refrigerant.
Roxb.
Cucurbitaceae
51. Trichosanthes anguina L. Chichinda Purgative, anthelmintic, emetic, refrigerant, syphilis &
Cucurbitaceae verminopsis.

ACKNOWLEDGEMENT
Authors are thankful to the peoples of Palpa district who actively participated and shared valuable
information in the field; without their active participation, this research would not have been completed. The
first author was also thankful to the National Herbarium and Plant Laboratories, Godavari (KATH), Nepal and
Herbarium of Tribhuvan University, Butwal Multiple Campus, Butwal for allowing me to consult herbaria and
for other facilities.

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DOI: 10.22271/tpr.2017.v4.i2.041
Research article

Comparative studies on some biochemical parameters of Cajanus


scarabaeoides (L.) Thouars and Cajanus cajan (L.) Millsp.
Joyeeta Dey*, Puja Rani Saha, Priyanki Debnath and R. K. Sinha
Plant Physiology and Biochemistry Lab., Department of Botany, Tripura University, Tripura, India
*Corresponding Author: joyeeta.dey10@gmail.com [Accepted: 18 August 2017]

Abstract: Studies of some biochemical parameters in wild Cajanus scarabaeoides was carried out
and compared with Cajanus cajan as its cultivated relative. C. scarabaeoides is considered to have
a potent source of genetic variation carrying genes for resistance to various biotic and abiotic
stresses and other morphological traits. Comparative analysis of biochemical parameters revealed
distinct genomic diversity between the two species. Electrophoretic study of leaf isoperoxidases in
two species of Cajanus indicated the presence of species specific variability in terms of relative
mobility values of isozymes. Higher values of total amino acids, sugar and protein content in C.
scarabaeoides also indicates ethnobotanical significance of the species as compared to widely
cultivated C. cajan.
Keywords: Cajanus species - Biochemical attributes - Isoperoxidase - Genomic diversity.

[Cite as: Dey J, Saha PR, Debnath P & Sinha RK (2017) Comparative studies on some biochemical parameters
of Cajanus scarabaeoides (L.) Thouars and Cajanus cajan (L.) Millsp. Tropical Plant Research 4(2): 314318]
INTRODUCTION
Cajanus scarabaeoides (L.) Thouars belongs to the family Fabaceae, locally known as Bonkalai. This
species is the closest wild relative to Cajanus cajan (L.) Millsp. [Pigeon pea]. C. scarabaeoides is reported to be
used as ethnomedicine by the tribal healers around different states of India. Tribal people of Madhya Pradesh
use the plant decoction as a tonic after delivery; the fresh leaf paste is applied on swellings of leg. The pods are
also eaten for this purpose. In Bihar tribal people use the root paste and use it with coconut oil to check falling
hairs to cure baldness (Sharma & Kumar 2013). Native people of Andhra Pradesh use this plant to cure piles
(Murty & Rao 2010), skin diseases (Rao et al. 2006). This plant is also used as fodder and effective in reducing
diarrhea in cattle. Besides, the species is an important weed legume with various ethnomedicinal properties. It
also possesses wound healing, anti-diabetic, anti-inflammatory, hepatoprotective, anti-diarrheal, anti-bacterial
activities (Pattanayak et al. 2009, 2011). Significance of C. scarabaeoides as a source of ethnobotanicals in
terms of carotenoid content was recorded (Dey & Sinha 2015). C. scarabaeoides was also reported to have
higher levels of draught tolerance, and resistance to insect pests compared to cultivated types (Tikka et al.
1997). The genetic traits of drought tolerance and the higher levels of resistance to insect pests of Cajanus
scarabaeoides can be utilized to improve the crops productivity of Cajanus cajan (Sharma et al. 1987,
Upadhyaya et al. 2011). In view of the above context, the present study is aimed to study certain biochemical
attributes of wild C. scarabaeoides and its comparision with cultivated C. cajan.

MATERIALS AND METHODS


The two species, Cajanus cajan (L.) Millsp. and Cajanus scarabaeoides (L.) Thouars were collected in an
around Suryamaninagar area (Fig. 1) of West Tripura district; having a geographical location of N 234540.6
and E 0911604.3. The healthy plant materials were collected during the month of March, 2016. Herbarium
specimen of respected species were prepared and identified with the help of floristic literature (Deb 1983,
Hooker 1885). The herbarium specimen with accession number TU/BOT/471 and 472 were assigned for C.
cajan and C. scarabaeoides respectively and submitted to the herbarium of the Department of Botany, Tripura
University for reference (Fig. 2).
Healthy fresh leaves were used as a source of ethnobotanicals purposes and thus biochemical investigation

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Dey et al. (2017) 4(2): 314318

of respective species were performed to estimate the total soluble sugar with Anthrone Reagent by the method
of Yemm & Willis (1954). In this, 500 mg freshly harvested leaves of plants were homogenized in 10 ml of
50% aqueous ethanol with a pinch of activated charcoal. The slurry was centrifuged at 5000 rpm for 10 min and
free amino acids were extracted in the form of a clear supernatant. The volume of supernatant was raised to 10
ml with aqueous 50% ethanol. To 1 ml of the supernatant 2 ml of 2% freshly prepared anthrone reagent was
added. The absorbance of the green coloured complex was taken at 620nm. Glucose was used as standard.

Figure 1. Map showing the study area (Suryamaninagar) of the West Tripura district, Tripura.

Figure 2. A, Herbarium specimen of Cajanus cajan; B, Herbarium specimen of Cajanus scarabaeoides.


The amount of total free amino acid was estimated following the method of Yemm & Cocking (1955).
Freshly harvested leaves (500 mg) of plants were homogenized in 10 ml of 50% aqueous ethanol with a pinch of
activated charcoal. The slurry was centrifuged at 5000 rpm for 10 min and free amino acids were extracted in
the form of a clear supernatant. The volume of supernatant was raised to 10 ml with aqueous 50% ethanol. To 1
ml of the supernatant 2 ml of 2% Ninhydrin (w/v in dehydrated alcohol) was added. The mixture was kept on
water bath at 752 C for 10 minutes and after cooling, aqueous alcohol (1:1) was added to make up the volume
to 3 ml. The absorbance of the violet complex was measured at 570 nm on a spectrophotometer. The amount of
total free amino-acids was calculated with the help of a standard curve prepared from glycine and was expressed
as mg of amino acids per gram fresh weight of the sample.
The phosphate buffer soluble protein was estimated following the procedure of Lowry et al. (1951). Fresh
leaves (500 mg) were crushed in 5 ml cold potassium phosphate buffer solution (pH 7.5; 0.1 M) on ice-bath
using pestle and mortar. The homogenate was filtered through cheese cloth and the filtrate was centrifuged at
12000 rpm for 25 min in a refrigerated centrifuge. The supernatant was taken in a 10 ml measuring cylinder and
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its volume was raised to 10 ml with same buffer. The pellet was used for insoluble protein. This supernatant
contains soluble protein. In 1 ml of supernatant 1 ml of tri-chloro acetic acid (TCA) solution (10%, w/v) was
added in a centrifuge tube. Immediately a whitish or cream coloured precipitate appeared. The precipitate was
centrifuged at 5000 rpm for 5 min and after centrifugation the supernatant was discarded and the pellet
contained precipitate of soluble protein. To this pellet 2 ml of ethyl alcohol (95%) was added and stirred the
precipitate so as to remove the TCA sticking on the surface of the protein as well as to remove pigments, if any.
The tube containing the precipitate was re-centrifuged (1000 rpm for 5 min) and the supernatant was discarded.
The pellet was used for estimation of soluble protein using reagents prescribed in the Lowry method and the
absorbance of blue colour complex was read at 690 nm using Spectrophotomter. BSA was used as standard
protein.
The amount of total soluble phenol were determined by the following the methods Swain & Hillis (1959).
For this 500 mg fresh leaves were taken and crushed with 80% chilled ethanol. Then the slurry was centrifuged
at 5000 rpm for 20 mins. The supernatant was collected and evaporated to dryness. After drying the pellet was
dissolved in water and this was used as aliquot. From this aliquot 0.05ml was taken in a test tube and 0.5 ml
Folin reagent, 2.5 ml distilled water and 2 ml saturated sodium carbonate solution were gradually added. The
OD of the blue coloured complex was taken at 560 nm . Tannic acid was used as a standard.
Assay of in vivo Nitrate Reductase was measured in fresh leaves by the method of Hageman & Hucklesby
(1971). Fresh 500 mg leaves were cut into thin strips (2 mm 3 mm size) from respective plant species and
incubated in the assay mixture containing 4 ml of potassium-phosphate buffer (0.1 M; pH 7.4), 0.5 ml KNO 3
(100 mM) and 0.5 ml of 5% aqueous propanol. After 30 mins of incubation period at 322 C 1 ml of incubated
assay was transferred to a test tube contain 1 ml of sulphanilamide and 1 ml of NED mixture. The pink coloured
complex was used for determination of nitrite spectrophotometrically at 540 nm. Sodium nitrate was used as
standard. Antioxidant activity in the leaves was measured by following the method of Patel & Patel (2011).
For isozyme study slab vertical polyacrylamide (without SDS) gel electrophoresis (Laemmli 1970) was
adopted. In this process, 500 mg freshly harvested leaves were taken and homogenized in 3 ml extraction buffer
in cold; and the homogenate was centrifuged at 12,000 rpm for 45 mins at 4C for 78 times. The supernatant
was collected and used as the material source for isozyme study. For the isozyme study, the vertical PAGE was
performed without using SDS. Respective mean values of the biochemical attributes were compared following
the simple studentss t test between the means.

RESULTS AND DISCUSSION


Total free amino acid and soluble sugar was found to be higher in the leaves of C. scarabeoides (Table 1)
whereas the amount of total soluble phenol was significantly higher in C. cajan. Efficiency of nitrate reductase
activity was much higher and significant in C. cajan though soluble protein content was almost similar in both
the species. The antioxidant activity measured in the leaves of C. scarabaeoides was 45.54% inhibition and
much less against the reported data of C. cajan (Mahitha et al. 2015).
Table 1. Comparison of some biochemical parameters of Cajanus cajan and Cajanus scarabaeoides.
Parameter Cajanus cajan Cajanus scarabaeoides t-value
Total free amino acid 5.59 0.50 7.18 0.95 3.31*
(mg.g-1 fresh wt.)
Soluble sugar 6.67 1.20 8.34 1.44 1.99(NS)
(mg.g-1 fresh wt.)
Soluble phenol 1.290.12 0.970.08 4.96*
(mg.g-1 fresh wt.)
Soluble protein 28.211.74 28.261.22 0.06(NS)
(mg.g-1 fresh wt.)
Antioxidant activity 52.12% (inhibition) 45.54% (inhibition) -
Nitrate reductase activity 3.4 0.29 1.590.05 7.45*
( moles NO2 produced h-1g-1leaf fr. wt.)
Note: *Significant at 5% level; NS= Not Significant; **Mean of five replicates (source: Mahitha et al. 2015).
Cajanus scarabaeoides is having high ethnomedicinal values as it is used in tonic after delivery, fresh leaf
paste or pod used to cure swelling of leg, root paste also used in night fever, dropsy, anaemia, burns and wounds
by the indigenous people of Tripura (Majumdar & Datta 2013). Since leaves are also used as vegetables by

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certain communities (Choudhury et al. 2015) , the present study highlights significant differences in terms of
high soluble sugar and free amino acid content as compared to C. cajan. The antioxidant property and phenolic
contents of the leaves are however superior in C. cajan. Soluble protein content in the leaves is very close
between the two taxa in spite of high Nitrate Reductase activity recorded in C. cajan. Iso-enzyme pattern reveal
remarked genetic differences between the two taxa. As many as seven isozymes were recorded in C. cajan with
different relative mobility (Rm) values ranging from 0.08 - 0.61 compared to five isozymes ranging from 0.08
0.59 in C. scarabaeoides (Fig. 3). Only three isozymes were found common in both the species. In spite of
differences in many biochemical traits and isozyme patterns between the two taxa, C. scarabaeoides is reported
to be compatible with C. cajan. Successful F1 hybrid production between the two taxa was already reported by
other workers (Mishra et al. 2012). This result also suggests that genetic distance between cultivated C. cajan
and its wild relative is not related to their hybridization barrier as also reported by Mudaraddi et al. (2013).

Figure 3. Zymogram pattern of leaf isoperoxidases: A, Cajanus cajan; B, Cajanus scarabaeoides.

CONCLUSION
The present study highlights the genetic diversity of wild C. scarabaeoides in terms of certain biochemical
attributes and its relative significant difference in free amino acids, phenol contents and nitrate reductase
activities between the two species. Relatively high value of soluble sugar and protein contents is also attributing
characteristic of nutritional value of C. scarabaeoides leaves.

ACKNOWLEDGEMENT
Authors are thankful to the University Grants Commission, (UGC) New Delhi for providing financial support
to the Department of Botany, Tripura University, Suryamaninagar - 799022, India.

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4(2): 319329, 2017
DOI: 10.22271/tpr.2017.v4.i2.042
Research article

Bioprospecting fungal diversity from crude oil infiltrate soil of


Assam, Indias Northeast
N. F. Islam*
Department of Botany, N.N. Saikia College, Titabar-785630, Jorhat, Assam, India
*Corresponding Author: nazimforidislam@yahoo.co.in [Accepted: 22 August 2017]

Abstract: The Screening of native fungi from crude oil contaminated soil of Assam is conducted.
Total crude oil concentrations varied from 39000 mg.Kg-1 to 51500 mg.Kg-1. The soil fungal
population varied from 14.6106 CFU.g-1 to 19.2106 CFU.g-1 soil. Twenty fungal strains were
isolated and out of them seven were selected on the basis of their ability to grow in PDA medium
where crude oil is used as sole carbon source. The growth rate and tolerance of each strain to crude
oil were examined and were found quite satisfactory. The radial extension rate varied from 0.03
cm.d1 to 0.50 cm.d1 and exhibited significant differences (P< 0.01) among the selected strains.
The degradation percentage of crude oil was in the range of 71% to 89% in mineral salt medium
(MSL) after the incubation for 28 days showing outstanding potential of isolates.
Keywords: Crude oil contamination - Polycyclic aromatic hydrocarbons - Indigenous fungi -
Biodegradation.

[Cite as: Islam NF (2017) Bioprospecting fungal diversity from crude oil infiltrate soil of Assam, Indias
Northeast. Tropical Plant Research 4(2): 319329]
INTRODUCTION
Crude oil is the most important and predominant energy resource for humans and the raw material of various
petroleum products which are essential for daily life. However, during crude oil exploration, a vast amount of
drilling mud/fluid is generated (Sarma et al. 2017). Further, oil spills, leaks, and other releases of petroleum
products occur frequently during its transportation and result in the contamination of cultivated soil and
groundwater, especially when associated with accidental spills (Prasad & Katiyar 2012). This huge amount of
toxic and persistent pollutants like PAHs, HMs, oil, grease, phenols, drilling fluid and mud affect organisms in
the biosphere from genetic through molecular levels (Vagi et al. 2005).
Crude oil is a complex mixture of hydrocarbons consisting of saturated hydrocarbons, aromatic
hydrocarbons and polar organic compounds. The exploration of crude oil has been by no means without some
visible side effects in the ecosystem. The transport of crude oil from point source to various components of the
ecosystem is one of the major factors that jeopardize the wellbeing of the environment (Sarma et al. 2016). Oil
spillages from drilling sites and oil storage facilities or loading stations are the chief source of contamination of
soil or aquatic areas. The term total petroleum hydrocarbons (TPH) is generally used to describe the
measurable amount of petroleum-based hydrocarbons in the environment. The polycyclic aromatic
hydrocarbons (PAHs) present in the crude oil may cause damage to vital organs like lungs, liver, kidneys,
intestines and other internal organs and are the major carcinogenic pollutants (Mandal et al. 2012). The
biodegradation of these stable compounds are very difficult particularly in case of high molecular weight PAHs
compounds. It has been found that the remediation of persistent contaminants such as PAHs from soils is too
expensive and time consuming process. Moreover the disposal of these contaminants is posing a serious
problem. The most promising and eco-friendly approach for remediation of petroleum hydrocarbon
contaminated soil is through biological methods (Mandal et al. 2012).
The biodegradation of crude oil (Uzoamaka et al. 2009, AI-Jawhari 2014) in the environment has been
extensively studied and documented in the literature and a large diversity of microorganisms, including bacteria
and fungi, are capable of oxidizing or mineralizing a range of PAH present in the crude oil. Of the
microorganisms identified to have the capability to degrade PAHs in the environment, fungi have been shown to

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Islam (2017) 4(2): 319329

be relatively more successful in breaking down the higher molecular weight compounds (Potin et al. 2004) than
bacteria, principally because of the production of non-specific extracellular enzymes such as laccase, lignin
peroxidase and manganese dependent peroxidase (MnP). They also function well in non-aqueous environments
where hydrophobic PAHs accumulate; a majority of other microbial degradation occurs in aqueous phase.
The oil industry is one of the major industries in Assam. The main activity of this industry is drilling for
exploration and production of oil. The Oil India Limited (OIL) and Oil and Natural Gas Commission (ONGC)
are the two leading oil producing companies which are at present actively engaged in crude oil exploration in
various sites of Assam. Most of the drilling sites are based at the periphery of the human settlement including
agricultural belt causing tremendous health hazards to plants and animals by releasing carcinogenic
polyaromatic hydrocarbon of various molecular weight. There is an evidence of heavily contaminated agro
ecosystem neighbouring the oil fields of Assam (mainly Digboi, Naharkatia, Rudrasagar, Lakua, Geleki and
Borhola, oil fields of upper Assam. Therefore, for effective biodegradation using indigenous organisms are
always preferred and research in this area is found to be very scanty in India particularly in the northeastern
region. Hence, the present work is based on screening of a set of suitable fungi from crude oil contaminated sites
that can be potentially exploited in bioremediation processes.

MATERIALS AND METHODS


Site location and description

Figure 1. Location map of the study area showing sampling sites.

The study area is located in Assam one of the northeastern states of India (Fig. 1). The area geographically
forms part of the Himalayan range. Four districts of Assam comprising of 6 sampling sites viz., Digboi,
Naharkatia, Rudrasagar, Lakuwa, Gelakey and Borholla were selected for the study. These are crude oil
exploration sites adjacent to which rice fields or tea plantations is predominant, experiencing high level of PAHs
contamination. This region is characterized by a humid tropical climate with a mean maximum and minimum
temperature of 33.0C and 9C respectively. The average annual rainfall of Assam is 2332 mm. The soils of the
study area are of old alluvial type and sandy loam in texture (Table 1).
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Table 1. Physical characteristics of soil.


Characteristics Coarse sand (%) Fine sand (%) Silt (%) Clay (%) Textural class
Soil test values 5.39 60.24 21.22 14.73 Sandy loam
Collection of soil samples
Soil samples from the six different contaminated areas were collected aseptically. The soil samples were
designated as HC-1, HC-2, HC-3, HC-4, HC-5 and HC-6 for Digboi, Naharkatia, Rudrasagar, Lakuwa, Galeki
and Borholla respectively (Table 2). These are the oil exploration and drilling sites of Oil India Limited (OIL)
and Oil and Natural Gas Corporation (ONGC) that suffers from moderate to high level of crude oil
contaminations at regular intervals due to seepage and other faulty practices (Sarma et al. 2016). The
subsurface soil samples (115 cm) contaminated by crude oil were collected randomly from each sites, in sterile
dark coloured bottles and poly bags to reduce photo-oxidation and carried to the laboratory. A similar set of soil
samples were collected from non-contaminated site as control for comparison. Analysis was commenced
immediately upon arrival in the laboratory and samples were refrigerated at 4C until further use.
Table 2. Description of the sampling location of the study area.
S.N. Site ID Sampling site Geographical Elevation Category
Coordinates (m asl)
1. HC-1 Digboi 2723'35"N 157 Urban and Industrial
9537'6"E
2. HC-2 Naharkatia 2716'55"N 117 Semi urban/Agricultural
9520'27"E
3. HC-3 Rudrasagar 26 58' 38"N 92 Semi urban/Agricultural
94 34' 52"E
4. HC-4 Lakua 26 59' 36"N 101 Semi urban/Agricultural
94 49' 09"E
5. HC-5 Galakey 26 47'24"N 105 Semi urban/Agricultural and green belt
94 41'42"E
6. HC-6 Borholla 261'50"N 107 Rural/Agricultural and green belt
9352'36"E
Analysis of soil chemical properties
Soil pH, organic carbon (%), available phosphorous (mg.Kg-1), total nitrogen (%), potassium (ppm), organic
matter (%) and water holding capacity were analysed using standard protocol (Kalra & Maynard 1991).
Extraction of crude oil from soil
The extraction of crude oil from soil was carried out using method of AI-Jawhari (2004) with slight
modification. Five gram soil sample from each sampling sites was mixed with 1:1 ratio of methylene
chloride/acetone solvent and centrifuged at 10,000 rpm for 10 min. The solvent-oil layer was separated from the
soil extract and placed in rotary evaporator at 40C. After complete evaporation of solvent the amount of oil was
measured for gravimetric value.
Isolation of indigenous fungal strains
Cultivable native fungi were isolated from crude oil contaminated soil. Soil from each site were
homogeneously mixed and carefully sorted to remove stones and other unwanted soil debris. 10 g of soil
samples (wet weight) from each site were added to 90 mL saline solution [085%(w/v) NaCl] in a sterile 250
mL Erlenmeyer flask, and the solution was suspended by shaking for 5 min at 150 rev min-1 .Further dilution
upto to 10-6 were done in saline solution and plated on Czapek-Dox Agar (CDA) medium containing
streptomycin (25 g.ml-1). The plates were incubated at 2830oC for 5 to 7 days. Results were recorded as CFU
per gram of soil. The grown cultures were carefully and aseptically sub-cultured onto fresh CDA plates to obtain
pure cultures for identification. Altogether, 20 different morphological fungal isolates including one unidentified
type, maintained on SDA slant were obtained and identified. The fungal strains were identified on the basis of
colonial appearance and microscopic examination of hyphae and spore morphology (Raper & Thom 1968,
Barnett & Hunter 1972). The abundance of the fungi with respect to different genera was also determined.
Screening of potential isolates based on growth and tolerance to crude oil
Screening of potential fungal isolates was carried out based on their growth and tolerance on medium

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containing crude oil by surface plate assay. To evaluate growth rate of each isolated fungal strain and to test
their tolerance to crude oil, discs of agar plug from actively growing fungal culture plates were used. 5-mm disc
from colonised Czapek-Dox Agar (CDA) plates was taken from the edge of a fungal colony and placed in the
middle of the Potato Dextrose Agar (PDA) medium. Sterile crude oil (1 % w/v) was added to serve as the
carbon source through vapour-phase transfer (Raymond et al. 1976). Prior to inoculation, 90-mm diameter discs
of sterile cellophane membrane were placed aseptically on the surface of the agar in each Petri dish. The plates
without crude oil and inoculated with each of the fungal strains were taken as controls. All treatments were
carried out in triplicate and the plates were incubated at 27C for two weeks. The colony radius from the edge of
the CDA piece was measured on daily basis in order to access the growth rate of each fungal species.
Biodegradation Studies
Biodegradation studies were carried out using, 2 mL of Digboi crude oil (as a sole source of carbon and
energy) in 98 mL mineral salts (MS) medium in 250 ml flasks following the procedure of Uzoamaka et al.
(2009) and AI-Jawhari (2014) with minor modification. Mineral salts medium (MSM) was composed of (g.L-1
water) 3 NH4NO3, 0.5 KH2PO4, 0.5 K2HPO43H2O, 0.008 MgSO47H2O, 0.002 CuSO45H2O, 0.002
MnSO4H2O, 0.002 FeSO47H2O and 0.002 CaCl22H2O (Jia et al. 2008). The pH was adjusted to 7.5 with
either HCl or NaOH solutions. 5mm disk from each of the seven potential strains (Aspergillus terreus,
Cunninghamella elegans, Aspergillus flavus, A. versicolor, A. fumigatus, A. niger and Trichoderma harzianum)
of 7 days old pure culture were inoculated separately in 250 mL flasks containing MS medium and incubated at
27C for a period of 28 days. The control flasks without organisms were prepared accordingly. All treatments
were carried out in triplicate. 5mL of culture broth from each flask were collected at a regular interval of 7 days
and subjected to centrifugation (10,000 rpm) to obtain cell free supernatant. The estimation of crude oil
degradation was determined by gravimetric method. For separation of residual crude oil in the culture
supernatant, 5 mL of n-hexane was used in a separating funnel. The solvent extraction was carried out twice for
complete recovery of oil. Hexane was evaporated in a hot air oven (50C) and the residual oil obtained was
weighed. The percentage degradation of the crude oil was determined as described by the
equation, , where a is the initial weight of undegraded crude oil (control), b is
the weight of the remaining crude oil after degradation.
Statistical data analysis
All the data were examined for significant differences between treatments using analysis of variance
(ANOVA). To establish if differences between individual treatments were significant (P < 0.05) the test for least
significant differences and least information criterion was used (SPSS version 24).Correlation between various
components were analyzed. P values < 0.01 and < 0.05 were considered as significant. Variance components
like coefficient of variations were also calculated. Means were separated with Duncan Multiple Range Test
(DMRT) in respective tables.

RESULTS
Soil chemical properties
Table 3. Chemical properties contaminated soil.
Soil parameters HC-1 HC-2 HC-3 HC-4 HC-5 HC-6
pH 6.21 6.13 5.03 6.15 5.98 6.01
Soil organic carbon (%) 3.16 3.24 2.56 1.65 3.96 3.82
Available phosphorus (mg.kg-1) 3.6 1.5 5.5 5.8 4.09 3.9
Total nitrogen (%) 0.03 0.17 0.68 0.55 0.04 0.07
Potassium (ppm) 145 132 162 146 180 132
Organic matter (%) 4.33 5.11 4.30 3.25 6.04 6.22
Water holding capacity 56.36 60.18 64.12 52.28 58.60 55.18
Note: Value are mean of standard deviation of (n=5).
The crude oil degradation processes is reported to be affected by multifarious factors. Some of the important
ones are biological factors, nutrients availability, soil pH and degree of contaminations by petroleum
hydrocarbons. The results of the soil chemical parameters are shown in the Table 3. The result ranged as
follows: pH, 5.036.21; soil organic carbon, 1.65 to 3.96%; Available phosphorus 1.5 to 5.8 mg.kg-1; Total
nitrogen 0.03 to 0.68%; Potassium 132 to180 ppm; Organic matter 3.25 to 6.22%.

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Extraction of crude oil from soil


Extraction of crude oil from all the six contaminated soils showed significant variations. The extractable
crude oil ranged from 39000 mg.Kg-1 to 51500 mg.Kg-1of contaminated soil (Fig. 2). The highest concentration
(51500 mg.Kg-1) was recorded from site HC-6. The spatial variations of crude oil across the sampling sites
showed significant variations [F (2.256), P < 0.05] based on linear independent pair wise comparison. The least
information criteria of distribution of petroleum hydrocarbons according to the Akaikes information criterion
and Hurvich and Tsais criterion was [(721.813) (R2=0.84)].

60000
Mean concentrations of Crude oil

50000

40000
(mg.Kg-1)

30000

20000

10000

0
HC-1 HC-2 HC-3 HC-4 HC-5 HC-6
(20.20) (07.40) (12.01) (08.10) (06.13) (10.11)
Sampling sites
Figure 2. Concentrations of Crude oil in the sampling sites. (Values in the parentheses denote Standard deviation across the
mean).
Isolation of indigenous fungal strains
30
25
Relative abundance (%)

20
15
10 y = 14.939e-0.117x
R = 0.3231
5
0

Figure 3. Relative abundance of fungal genera.

A total of 20 fungal isolates including one unidentified were obtained from crude oil-contaminated soils. The
morphological identification of the fungal strains showed that they belong to 9 genus with typical structures of
Aspergillus (5 species), Cladosporium (2 species), Cunninghamella (1 species), Fusarium (3 species),
Gliocladium (1 species), Mucor (1 species), Penicillium (4 species), Rhizopus (1 species) and Trichoderma (1
species). The relative abundance of fungal genera is shown in the figure 3. The most dominant genus is
Aspergillus (25%) followed by Penicillium (15.3%) and Fusarium (15%). The fungal population was highest in
the soil sample collected from HC-5 (19.2106 CFU.g-1 soil) and HC-6 (17.7106 CFU.g-1 soil), and lowest in
HC-1 (14.6106 CFU.g-1 soil). In the other three sampling sites the count is almost similar varying from
15.2106 CFU.g-1 soil to 16.6106 CFU.g-1 soil. The dominant species among the fungal population across all
the sites were Aspergillus fumigates (11.6106 CFU.g-1 soil) and Aspergillus terreus (10.76) (Table 4).
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Table 4. Fungal population (CFU g-1 x 106) isolated from different crude oil contaminated sites.
Fungal species Strain ID HC-1 HC-2 HC-3 HC-4 HC-5 HC-6
Aspergillus flavus NF1 2.6 2.7 0.7 2.9 1.7 1.0
A. fumigatus NF2 - 0.02 1.6 1.8 - 2.7
A. niger NF3 0.09 0.03 1.5 1.2 0.7 0.1
A. versicolor NF4 0.06 0.7 1.8 0.6 0.08 1.7
A. Terreus NF5 1.1 1.6 0.06 2.7 2.8 2.5
Cladosporium cladosporioides NF6 0.2 1.5 - 0.9 3.0 2.0
C. resinae NF7 - 0.8 - 1.7 2.6 2.5
Cunninghamella elegans NF8 1.4 2.3 2.4 0.13 0.02 -
Fusarium equiseti NF9 2.5 - - 0.6 1.7 1.2
F. oxysporum NF10 - 0.3 1.2 0.1 - -
F. solani NF11 - 0.7 0.4 2.0 2.6
Gliocladium viride NF12 0.01 0.07 1.2 0.09 - 0.5
Mucor racemosus NF13 2.0 1.3 1.8 1.0 - 1.5
Penicillium citrinum NF14 0.09 - 1.4 0.03 2.0 0.06
P. commune NF15 - 0.5 1.2 0.01 - -
P. funiculosum NF16 - 1.2 - - - 0.08
P. glabrum NF17 0.9 0.04 - - - 0.03
Rhizopus stolonifer NF18 0.8 - 1.0 0.01 0.08 -
Trichoderma harzianum NF19 1.8 2.1 0.3 0.4 1.0 1.1
Unidentified UI 1.0 0.3 0.04 - 0.9 0.6
Screening of potential isolates based on growth and tolerance to crude oil
The growth rates of different fungal isolates in the presence of crude oil in surface plate assays are shown in
table 5. Mycelial extension rates exhibited significant differences between the strains with respect to their
growth ability in the presence or absence of 1 % (w/v) crude oil for two weeks. Species of Aspergillus,
Cunninghamella and Trichoderma did not show any significant inhibitory effect. Species of Penicillium were
the most sensitive isolates (50.499.9% sensitivity) as the rate of growth notably decreased in the presence of
the crude oil. The most tolerant species to crude oil was Aspergillus terreus showing 0.50 cm.d-1 of radial
growth compared to control (0.44 cm.d-1) (P < 0.05). This is followed by Cunninghamella elegans (0.41 cm.d-1),
Aspergillus flavus (0.40 cm.d-1), A. versicolor (0.39 cm.d-1), A. fumigatus (0.38 cm.d-1), A. niger (0.30 cm.d-1)
and Trichoderma harzianum (0.29 cm.d-1) on the surface of crude oil. The slowest growth rate was exhibited by
Penicillium commune (0.03 cm.d-1). The significant (R2 = 0.90, P< 0.05) increase in the growth of Aspergillus
terreus and Cunninghamella elegans in the presence of crude oil suggests that they can better utilize it as a
growth substrate.
Table 5. Radial extension rate (cm.d1) of fungal isolates in presence of crude oil.
Isolate Strain ID Control Crude oil treatment
Aspergillus flavus NF1 0.409 (0.09) 0.401 (0.09)
A. fumigatus NF2 0.404(0.02) 0.388(0.004)
A. niger NF3 0.330(0.01) 0.300(0.03)
A. versicolor NF4 0.385(0.08) 0.398((0.02)
A. Terreus NF5 0.446(0.009) 0.500(0.01)
Cladosporium cladosporioides NF6 0.191(0.02) 0.105(0.01)
C. resinae NF7 0.132(0.01) 0.102(0.001)
Cunninghamella elegans NF8 0.408(0.008) 0.418(0.04)
Fusarium equiseti NF9 0.211(0.03) 0.108(0.01)
F. oxysporum NF10 0.115(0.01) 0.098(0.01)
F. solani NF11 0.203(0.04) 0.109(0.01)
Gliocladium viride NF12 0.205(0.03) 0.170(0.01)
Mucor racemosus NF13 0.100(0.01) 0.067(0.03
Penicillium citrinum NF14 0.222( 0.02) 0.110(0.007)
P. commune NF15 0.113(0.03) 0.030((0.001)
P. funiculosum NF16 0.150(0.06) 0.076(0.005)
P. glabrum NF17 0.145(0.05) 0.047(0.004)
Rhizopus stolonifer NF18 0.196(0.06) 0.101(0.01)
Trichoderma harzianum NF19 0.298(0.05) 0.291(0.03)
Unidentified UI 0.156(0.02) 0.089(0.002)
Note: Values in the parentheses shows standard deviation from mean, n=3.
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Biodegradation Studies

Control Aspergillus flavus A. fumigatus


A. niger A. versicolor A. terreus
90
Cunninghamella elegans Trichoderma harzianum
80
Biodegradation of crude oil (%)

70
60
50
40
30
20
10
0
7d 14d 21d 28d
Incubation time (Days)
Figure 4. Biodegradation of crude oil by selected fungi.
Out of the 20 fungal isolates only seven of them showed their ability to degrade crude oil contaminant (Fig.
4). The potential fungal strains were tested based on their ability to degrade crude oil when grown in mineral
salt medium for 28 days. The highest degradation of crude oil after 28 days of incubation was observed with
Aspergillus terreus (89%) and Cunninghamella elegans (87%) followed by Aspergillus flavus (85%), A.
versicolor (82%), A. fumigatus (79%), A. niger (76%) and Trichoderma harzianum (71%) respectively. A.
flavus, A. fumigatus, A. versicolor, A. terreus and Cunninghamella elegans showed more than 60% degradation
within 7 days of incubation. There was positive correlation between the crude oil degrading ability of the fungi
(Fig. 5) and the growth rate of the fungi.
100
90 6 5
4
80 3 1
2
70
Degradation (%)

7
60
50 y = 8.4x + 49.45
40 R = 0.859
30
20
1. Aspergillus flavus, 2. A. fumigatus, 3. A. niger, 4. A. versicolor, 5. A. terreus,
10 6. Cunninghamella elegans, 7. Trichoderma harzianum
0
-0.1 6E-16 0.1 0.2 0.3 0.4 0.5 0.6
Growth rate (cm.d-1)
Figure 5. Correlation between crude oil degrading ability and growth rate of seven different fungal isolates.

DISCUSSION
In the present study soil parameters like pH, Soil organic carbon, phosphorus, nitrogen, potassium, organic
matter and water holding capacity, from contaminated sites were assessed to determine its effect on fungal
population to biodegrade petroleum hydrocarbons of crude oil (Fierer & Jackson 2006). The high soil pH in the
present study may be due to disparity in leaching of essential salts due to presence of petroleum hydrocarbons.
Hydrocarbon degradation has been reported to be favoured in slightly alkaline soils (Maeir et al.2000). In the
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present study four out of six sampling sites showed pH above 6 and pH range between 6 to 8 has been suggested
conducive for bioremediation. The organic carbon content and total nitrogen of the polluted soils was in the
range of 1.65 to 3.96% and 0.03 to 0.68%. This may be attributed to the steady input of petroleum
hydrocarbons. The soil phosphorus was in the range of 1.5 to 5.8 mg.Kg-1. The optimum C:N:P ratio in soil is
critical for biodegradation of petroleum hydrocarbon as microorganisms utilize these elements for incorporation
into biomass and the synthesis of cellular components (Atlas & Bartha 1992, Stroud et al. 2007). The water
holding capacity or the presence of moisture level is one of the essential parameters for the biodegradation of
crude oil at the optimum level. Low soil moisture may impede the microbial activity by affecting the petroleum
hydrocarbon transport and bioavailability (Sarma et al. 2016). The water holding capacity in the present study is
between 52 to 64% which was at par to the earlier finding (Vadali 2001).
The amount of crude oil in soil is largely dependent on soil physical, chemical or biological processes The
removal of crude oil from the soil depends on soil type, soil organic matter content, soil minerals, the physico-
chemical properties of the individual PAH, soil temperature and moisture, redox potential, nutrients availability,
presence and activity of degrading microorganisms and bioavailability of the PAH to degrading microorganisms
(Meijer et al. 2002, Nam et al. 2008, Okere & Semple 2012). The variations in the amount of crude oil may be
due to leaching phenomenon, volatilization or photo-oxidation (Wilcke et al. 1999). These variations were also
dependent on the distance from the source of pollution and the prevailing climatic conditions (Wild & Jones
1995). The amount of crude oil in the soil can be used to estimate the sources of pollution. The levels of crude
oil in the spilled soils are similar to levels reported earlier (Cheraghi et al. 2015).
A total of 20 fungal isolates were initially obtained from CDA medium. Of these, 7 potential isolates were
screened based on ability to degrade crude oil. The potentials fungal strains were identified as Aspergillus
terreus, Cunninghamella elegans, A. versicolor, A. flavus, A. fumigatus, A.niger and Trichoderma harzianum.
Some of these fungi have been reported earlier as hydrocarbon bio-degraders (Uzoamaka et al. 2009). The
fungal population in the contaminated sites varied from 17.710 6 CFU.g-1 soil to 19.2106 CFU.g-1 soil. Low
CFU count in some of the sites may be due to low moisture contents which might be responsible for the
inhibition of fungal growth. The result is in consistence with the previous findings (Sims & Overcash 1983,
Yuan et. al. 2001).
An increasing trend in the fungal population with the increase in crude oil contamination has been observed
in some of the experimental sites. The results clearly demonstrated the positive relationship between the crude
oil contents and the fungal population in the contaminated soils. The present results contradict the findings of
Sims & Overcash (1983) who demonstrated that the higher concentration of PAH in the soil retards the
depletion of PAHs by microbes due to high level of PAHs toxicity. Increased fungal population may be due to
high pH and moisture and their adaptability to the prevailing environmental conditions (Rahman et al.
2002).The isolated fungal population might have the potentiality to assimilate petroleum hydrocarbon in the
contaminated soil. The growing trend in the fungal population with the increase in petroleum hydrocarbon
compounds has been also discussed by different researchers (Shi et al. 2002). Similarly, in some of the earlier
studies, large population of bacteria, actinomycetes and fungi were recorded from oil-spilled environment
(Venkateswaran et al. 1993, Wrenn & Venosa 1996, Cabello 1997).
In the present study screening of potential fungal isolates was based on their ability to grow on crude oil by
surface plate assay. The method proved to be advantageous over other traditional methods used to screen
hydrocarbon degrading strains, as the degradation is directly correlated to growth and metabolism of the fungi
growing on hydrocarbon substrates using it as the source of carbon and energy. The increment and reduction in
growth among different fungal isolates was due to varying degree of tolerance to crude oil. The isolated crude
oil degraders belong to the genera Aspergillus, Cladosporium, Cunninghamella, Fusarium, Gliocladium, Mucor,
Penicillium, Rhizopus and Trichoderma. The most tolerant species to crude oil as noted in the present study was
Aspergillus terreus.
Biodegradation of crude oil is a crucial process involving different soil microbial population. The role of
some of these microorganisms have been extensively reviewed (Shuttleworth & Cerniglia 1995, Macleod et al.
2001, Macleod & Semple 2002, Zhang et al. 2006, Peng et al. 2008). Most of the works were confined to the
degrading ability of bacteria and as such development of suitable mycoremediation strategies using potential
fungal strains is a real challenge. The growth and distribution of crude oil degrading microbial populations
depends on environmental conditions (Margesin & Schinner 2001), soil properties (Manilal & Alexander 1991),
bioavailability of the PAH (Ogram et al. 1985) and presence or absence of compounds toxic to microbes (Sarma
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et al. 2017). Thus lack of tolerance of microbial population to these factors may lead to persistence of petroleum
hydrocarbons in contaminated soils. In the present study, 20 fungal isolates were obtained from different crude
oil-contaminated soils however only seven of them were found to be as potential candidates to degrade crude oil
contamination. This is exemplified by their capacity to grow over crude oil layer viz., Aspergillus terreus,
Cunninghamella elegans, Aspergillus versicolor, A. flavus, A. fumigatus, A. niger and Trichoderma harzianum.
In the present study, species of Aspergillus showed maximum degrading ability, which may be due to their
better adaptability to crude oil contamination sites. The observations are in consonance with the previous
findings (Uzoamaka et al. 2009). The role of some of these fungi in degradation of different hydrocarbons has
been described elsewhere by different workers (Massaccesi et al. 2002); however the data is insufficient on their
growth rate and range of tolerance to crude oil. The variability of fungal isolates in the present findings may be
due to different degree of crude oil contamination, their range of tolerance with respect to sites and seasonal
impact. Moreover, variations in soil parameter like pH, moisture and organic matter have profound effect on
their distribution. Thus the types of exposure largely determine the ability of indigenous fungi to degrade crude
oil contaminated soils. As most of the researches were focused on the degradation of crude oil by various
biological, chemical and physiological processes, the ability of fungi needs attention as because fungi have more
tolerance to crude oil. The use of highly tolerant fungal isolates could boost up the degradation process.

CONCLUSION
This work demonstrated that the seven out of twenty isolated fungi from the crude oil contaminated
environment exhibited exceptional ability in degrading crude oil. The preliminary findings indicated that these
fungi have higher tolerance potential than other tested fungal strain which makes them more attractive potential
candidates for further investigations regarding their ability to remove crude oil from contaminated sites.

ACKNOWLEDGMENT
The author is grateful to University Grant Commission, Governmentof India for financial support (Grant No.
F.5-53/2014-15/MRP/NERO/1683 dated 05th Feb, 2015) and to Dr. Hemen Sarma, N.N. Saikia College for
technical support.

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bacteria by a most probable number procedure. Canadian Journal of Microbiology 42: 252258.
Yuan SY, Wei SH & Chang BV (2001) Biodegradation of polycyclic aromatic hydrocarbons by a mixed
culture. Chemosphere 41: 1463.
Zhang XX, Cheng SP, Zhu CJ & Sun SL (2006) Microbial PAH-degradation in soil: degradation pathways and
contributing factors. Pedosphere 16: 555565.

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DOI: 10.22271/tpr.2017.v4.i2.043
Short communication

A new distributional record for Gujarat state:


Dipcadi saxorum Blatter
Rupesh R. Maurya1, Umerfaruq M. Qureshimatva1*, Sandip B. Gamit1,
Rajdeo Singh2 and Hitesh A. Solanki3
1
Department of Botany, USSC, Gujarat University, Ahmedabad, Gujarat, India
2
St. Xaviers College, Mumbai University, Maharashtra, India
3
Department of Botany, USSC, Gujarat University, Ahmedabad, Gujarat, India
*Corresponding Author: ufmqureshi@yahoo.in [Accepted: 24 August 2017]

[Cite as: Maurya RR, Qureshimatva YM, Gamit SB, Singh R & Solanki HA (2017) A new distributional record
for Gujarat state: Dipcadi saxorum Blatter. Tropical Plant Research 4(2): 330331]

The Shoolpaneshwar Wildlife Sanctuary is an important protected area of Gujarat State, India, established in
1989, encompassing an area of 607 km2. along the north-eastern border of Bharuch district. The area lies
between 21 03 N to 21 59 N latitude and 73 05 E to 74 10 E longitude at an altitude of 800 to 900 msl.
Dense forest of the sanctuary constitutes catchment of two major irrigation dams viz. Sardar Sarovar and Karjan
dams. Hills of the sanctuary are part of the Satpudas, where they merge with the Vindhayan. Dipcadi Medik. is
represented by 41 species in the world, India as one of the center of diversity having 10 species with 4 varieties
(Prabhugaonkar et al. 2009). Initially this genus was placed in Liliaceae, later in Hyacinthaceae and recent
phylogenetic synthesis of Angiosperms available as APweb (Stevens 2001) and The Plant List (2013) places it
now in Asparagaceae (sensu lato). With the publication of Red Data Book of Indian Plants six species of
Dipcadi were assessed as threatened in India and thus prioritized for conservation (Dasgupta & Deb 1981).
Dipcadi saxorum Blatter is one of the six threatened plants in India which has been reported as endemic to
Maharashtra earlier (Gaikwad et al. 2014). The specimens have been collected from Shoolpaneshwar forests
area, Narmada District. Therefore, it forms a new distributional report to the Gujarat after Maharashtra (Shah
1978, Raghavan et al. 1981, Almeida 2009, Meena 2012). Voucher specimens RM-2036, 2037 are deposited in
Herbarium, Department of Botany, USS, Gujarat University, Ahmedabad.

ENUMERATION
Depcadi saxorum Blatter J. Bombay Nat. Hist. Soc. 32: 736 1928. (Fig. 1)
Depcadi maharashtrensis Deb. & Dasgupta J. Bombay Nat. Hist. Soc. 72: 822-3, t. 1, 1975.
Ornithogalum saxorum (Blatt.) Manning & Goldblatt Edinburgh J. Bot. 60: 552 2003.
Bulb tunicate. Scape about 50 cm. long, terete, smooth. Racemes up to 20 cm long, rather lax, about 20
flowered. Bracts broadly ovate, long-acuminate, scarious, slightly finged near the base about 7 mm long.
Pedicels stout, 1/2 longer than the bracts. Perianth 1214 mm long, tubular, slightly trigonous, the 3 outer lobes
connate for 1/3 this length, linear-oblong, obtuse, recurved from about the middle with a thick, glandular tip, the
3 inner connate for almost 2/3 their length, 3 mm broad ovate-obtuse, with a recurved and slightly thickened tip,
all lobes whitish with an olive coloured central band. Filaments about 1 mm long, slightly flattened; anthers 3
mm long, versatile, yellow. Pistil 10 mm long. Ovary 5 mm long, trigonous, clavate, shortly but distinctly
stipitate; style very stout, 5 mm. long minutely grandular upwards; stigma slightly thicker than style, 3-lobed.
Capsule broader than long, deeply 3-sulcate, loculicidally 3-valved, membranous, stipitate, stalk stout, about 2.5
m long; 5 seeds slightly elliptical to nearly orbicular in outline, reaching 5 mm in diameter, compressed, with a
raised margin, the lowest and top most in each cell plano-convex.
Flowering: JulyAugust.
Fruiting: JulyAugust.

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Maurya et al. (2017) 4(2): 330331

Figure 1. Photographs of Depcadi saxorum Blatter: A, Habit; B, Inflorescence; C, Flowers.

REFERENCES
Almeida MR (2009) Flora of Maharashtra. Orient press, Mumbai, Vol. 5 (A-B): pp. 177.
Dasgupta S & Deb DB (1981) Liliaceae: Tribe Scilleae. Fascicles of Flora of India 7: 123.
Gaikwad S, Ramchandra G, Krushnadeoray G, Sampatrao G (2014) Endemic flowering plants of northern
western Ghats (Sahyadri ranges) of India: check list. Check List 10(3): 461472.
Meena SL (2012) A checklist of vascular plants of Banaskantha district, Gujarat, India. Nelumbo 54: 3991.
Prabhugaonkar A, Yadav US & Janarthanam MK (2009) Dipcadi goaense (Hyacinthaceae), a new species from
the foothills of the Western Ghats, India. Kew Bulleten 64: 743746.
Raghavan RS, Wadhwa BM & Ansari MY (1981) A checklist of the plants of Gujarat. Botanical Survey of
India 21(2): 51.
Shah GL (1978) The flora of Gujarat State. Vol-I and II, Sardar Patel University Press, Vidhyanagar, Anand,
India.
Stevens PF (2001) Angiosperm Phylogeny Website. Version 12, July 2012. Available from:
http://www.mobot.org/MOBOT/research/APweb/ (accessed on 3 August 2013).
The Plant List 2013. Version 1.1. Published on the Internet; http://www.theplantlist.org/ (accessed 21 May
2014).

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DOI: 10.22271/tpr.2017.v4.i2.044
Short communication

Recollection of Didymoplexis micradenia (Rchb. f.) Hemsl.


(Orchidaceae) from Western Javan populations
Wendy A. Mustaqim*1, Dimas H. Pradana2, Reza Saputra1 and Margi Hartanto1
1
Organisasi Mahasiswa Pecinta Tumbuhan (OMPT) Canopy, Department of Biology, Faculty of Mathematics
and Natural Sciences, Universitas Indonesia, Depok, West Java
2
Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Indonesia, Depok, West
Java
*Corresponding Author: wendyachmmadm@gmail.com [Accepted: 26 August 2017]

[Cite as: Mustaqim WA, Pradana DH, Saputra R & Hartanto M (2017) Recollection of Didymoplexis
micradenia (Rchb. f.) Hemsl. (Orchidaceae) from Western Javan populations. Tropical Plant Research 4(2):
332334]

Didymoplexis Griff. is a genus of mycoheterotrophic orchids which consists of about 20 species. The genus
is widely distributed in the Old World tropics (Cribb & Whistler 2011). Didymoplexis micradenia (Rchb. f.)
Hemsl. is one of the species which occur in Java.
The first record of this species in Java was made by Smith (1900) under the new species discovery, namely
Didymoplexis minor J.J. Sm. He mentioned the Buitenzorg (now Bogor) as the locality. This information also
has been used by him in his further treatment of the orchids of Java, Die Orchideen von Java (Smith 1905). A
year before the time Comber (1990) published the latest comprehensive account of the orchids in Java,
Didymoplexis minor has been listed as a synonym of Didimoplexis micradenia by Lewis & Cribb (1989). The
latest state has been used by many recent authors, including Hsu & Chung (2007), Cribb & Whistler (2011) and
last by Phueakkhlai et al. (2014).
The first discovery of Didymoplexis micradenia in Universitas Indonesia has been made around the year of
2012 to 2013. But, no good specimens were available, especially the flowers. Around the middle of October
2016, around 50 individual have been found and adequate materials have been collected in the form of spirit
materials. The recently collected specimens were deposited in the herbarium of Bogor Botanical Garden (KRB).
Didymoplexis micradenia (Reichb. f.) Hemsl., J. Linn. Soc. Bot. 20: 311, 1883; Cribb & Whistler,
Lankesteriana 11 (2): 135. 2011; Lewis & Cribb, Orch. Vanuatu 1989: 24; Hsu & Chung, Taiwania 52 (4): 360,
fig. 2 & 3. 2007; Phueakkhlai, Bunpha & Tetsana, Thai. For. Bull. (Bot.) 42: 71. 2014. Type: Ovalau, Seemann
610 (holotype W n.v., isotype K n.v.).
Didymoplexis minor J.J.Sm., Bull. Inst. Bot. Buit. 7: 1. 1900; Die Orchideen von Java - Figuren Atlas, t. LII.
1908; Comber, Orchid Java 1990: 83. (Fig. 1)
Terrestrial herbs, achlorophyllous, holomycotrophic, leafless. Rhizome just or below the ground, fusiform or
moniliform, 1.01.5 1.05 cm, pale brown. Roots few, 4 or more, white or flushed with bright yellow,
especially at the apex, bearing nodules or not. Peduncle pale brown or white, 8.259.00 cm long, 1.22.0 mm
diam., glabrous, with 45 scales, up to 3 mm long, obtuse. Racemes 1.63.6 cm long, flowers more or less 78,
bracts ovate-triangular, acuminate, about 1 mm long, reflexed at fruiting. Flowers white, not opening widely,
pedicels and ovary ca. 7 mm long. Sepals and petals connate at the base into floral tube. Dorsal sepals 7.5 mm
long, adnate to petals for 3/4 of its length, forming a hood over the column, totally to ca. 3 mm wide, apex
shallowly 3-lobed, apex of the central lobe rounded. Lateral sepals connate, free portion 2-lobed, obtuse or
rounded, 6.25 1.50 mm. Lip cuneiform, 5.75 ca. 2.5 mm, callus of 3 ridged brown papillae, apex rounded in
outline, apical margin denticulate, rounded in outline, lateral margins erect, slightly incurved. Column white, 5.5
mm long, 2-winged at apex on both sides, foot ca. 0.5 mm long. Capsules cylindrical, 1.852.15 0.55 cm
wide, peduncle lenghtening to 17.520.0 cm or longer at maturity, ca. 2 mm diam., fleshy.

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Mustaqim et al. (2017) 4(2): 332334

Figure 1. Didymoplexis micradenia, urban forest of Universitas Indonesia, Depok, West Java. A, Flowering individual; B,
Plant showing the tuber; C, Close up of flower; D, Plant in fruiting stage (Photograph: Wendy A. Mustaqim).

Specimens Examined: INDONESIA, West Java Province, Depok, urban forest of Universitas Indonesia (Hutan
Kota UI), Depok, West Java, 5070 m, 1013 October 2016, W.A. Mustaqim & Reza Saputra 1915 (KRB!).
Distribution: Indochina, Taiwan, Samoa, Vanuatu, New Caledonia, Fiji, Tonga, Niue, Palau, Vietnam,
Thailand, Java (Depok (formerly part of Buitenzorg)).
Habitat: Shaded urban forest, once found in a forest margin, usually found in areas where herbs are absent or
few. Several plant species exist around the localities, identified in the field, are listed in table 1.

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Table 1. Plants grow in the localities of Didymoplexis micradenia in the urban forest of Universitas Indonesia, Depok,
West Java.
Family Species Life form
Wild Native Species
Araceae Amorphophallus variabilis Bl. Herbs
Araceae Alocasia cf. flemingiana A. Hay Herbs
Dilleniaceae Tetracera scandens (L.) Merr. Woody climbers
Dilleniaceae Tetracera indica (Christm. & Panz.) Merr. Woody climbers
Dioscoreaceae Dioscorea hispida Dennst. Climbers
Euphorbiaceae Macaranga tanarius (L.) Mull. Arg. Trees
Ferns Cyclosorus sp. Herbs
Ferns Pteris sp. Herbs
Ferns Nephrolepis sp. Herbs
Moraceae Ficus hirta Vahl Shrubs
Orchidaceae Zeuxine clandestina Bl. Herbs
Poaceae Cyrtococcum patens (L.) A. Camus Herbs (grass)
Primulaceae Ardisia humilis Vahl Shrubs
Rubiaceae Psychotria viridiflora Reinw. ex Blume Shrubs
Taccaceae Tacca palmata Bl. Herbs
Tiliaceae Microcos tomentosa Sm. Shrubs to trees
Verbenaceae Clerodendrum villosum Bl. Shrubs
Non-Native (Introduced) and Cultivated Native Species
Araceae Caladium bicolor (Aiton) Vent. Herbs
Cecropiaceae Cecropia peltata L. Trees
Fabaceae Bauhinia cf. purpurea L. Trees
Fabaceae Gliricedia sepium (Jacq.) Walp. Trees
Meliaceae Swietenia macrophylla King Trees
Notes: The presence of this species show the significantly important role of this urban forest for the existence of
this species in Java since this species has been recorded only from Depok (W), as mentioned in the previous
literature (Smith 1905, Backer & Bakhuizen van den Brink 1968, Comber 1990). In fact, the locality which is an
urban forest shows that the species also can be found in a human affected ecosystem. There is a doubt that this
species probably is a relict of cultivation, but no previous record about the cultivation of this species are
available.

ACKNOWLEDGMENTS
The first author thanks to Unit Pelaksana Teknis (UPT) K3L Universitas Indonesia, who gave permission to
conduct research during part of the study. Also to Mr. Yasman who give support during the field study. Thanks
are also due to anonymous reviewers for the comments on the early manuscript.

REFERENCES
Backer CA & Bakhuizen van den Brink RC Jr (1968) Flora of Java 3. N.V.P. Nordhoff. Groningen.
Comber J (1990) Orchids of Java. Royal Botanic Gardens, Kew.
Cribb PJ & Whistler (2011) The orchids of Tonga, Niue, and the Cook Islands. Lankesteriana 11(2): 9677.
Hsu T-C & Chung S-W (2007) Didymoplexis micradenia: A newly recorded orchid (Orchidceae) in Taiwan.
Taiwania 52(4): 360364.
Lewis B & Cribb P (1989) Orchids of Vanuatu. Royal Botanic Gardens, Kew.
Phueakkhlai O, Bunpha K & Tetsana N (2014) Didymoplexis micradenia and Gastrodia theana (Orchidaceae),
new records for Thailand. Thai Forest Bulletin (Botany) 42: 7174.
Smith JJ (1900) Kurze beschreibungen neuer malaiischer orchideen. Bulletin de lInstitut Botanique de
Buitenzorg 7: 15.
Smith JJ (1905) Die orchideen von Java. E.J. Brill., Leiden. [doi: http://dx.doi.org/10.5962/bhl.title.86938]

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DOI: 10.22271/tpr.2017.v4.i2.045
Research article

Floristic composition and plant community analysis of vegetation


in Ilu Gelan district, West Shewa Zone of Oromia region,
Central Ethiopia
Zerihun Tadesse1, 2*, Ensermu Kelbessa1 and Tamrat Bekele1
1
Department of Plant Biology and Biodiversity Management, the National Herbarium, Addis Ababa University,
P.O.Box: 3434, Addis Ababa, Ethiopia
2
Wollega University, Faculty of Agriculture, Department of Plant Science, Shambu, Ethiopia
*Corresponding Author: zerihunbio@gmail.com [Accepted: 26 August 2017]

Abstract: This study was conducted on Dirki and Jato sites vegetation in Ilu Gelan district, west
Shewa zone of Oromia region, 195 km west of Addis Ababa, to identify floristic composition and
plant community types the vegetation. Systematic sampling method was used to collect vegetation
data from 54 (20 m 20 m) plots. To collect data for herbaceous plants, five 1 m 1 m subplots
were laid in each of the main plot, where four were at the corners and one at the center.
Cover/abundance values were visually estimated to use for cluster analysis. Shannon - Wiener
Diversity Index was used to calculate species diversity, richness and evenness whereas Soresens
Similarity ratio was used to compare the vegetation with other related vegetation. Two hundred
and thirteen species were recorded from the sample plots, where Fabaceae, Asteraceae and
Poaceae were the most dominant families with 23, 22 and 12 species respectively. Eleven endemic
and two near endemic species were recorded from the study area. Based on IUCN Red Data List,
nine species were least concern; three were near threatened while one was vulnerable. Three plant
community types were recognized from the study area.
Keywords: Plant community - Diversity - Endemic species - Near endemic species - Abundance.

[Cite as: Tadesse Z, Kelbessa E & Bekele T (2017) Floristic composition and plant community analysis of
vegetation in Ilu Gelan district, West Shewa Zone of Oromia region, Central Ethiopia. Tropical Plant Research
4(2): 335350]
INTRODUCTION
Ethiopia is a country found in the horn of Africa between the geographical coordinates of 3 o 24 and 14o53
North and 32o 42 and 48o 12 East. According to MOA (2000), the total area of the land of the country is 1.12
million km2. The country has different topographic land features such as mountains, deep gorges, low lands,
valleys and flattened plateaus. These different topographic features assisted different types of flora and fauna
that have been well adapted to their own geographical features and climatic conditions. According to Fayera
Senbeta et al. (2007), the climate and topography of Ethiopia vary considerably and appear to have effects on
the distribution of biological diversities. Thus, Ethiopia has been considered a country having high biodiversity
in Horn of Africa (NBSAP 2005).
A large part of Ethiopia was believed to have been covered by forests and woodland vegetations in the past
(Friis 1992). However, due to continuous massive deforestation made on it, the vegetation cover has been
reduced through time to what it looks like at the present (Moges et al. 2010). Different researchers have studied
the vegetation of Ethiopia at different times (White 1983, Friis 1992, Teketay 1992, Bekele 1994, Ayalew 2003,
Awas et al. 2001, Senbeta 2006, Senbeta et. al. 2007, Didita 2007, Adamu et al. 2012, Dibaba et al. 2014). The
results of these studies could broadly categorize the vegetations of Ethiopia into nine major types. These
include: Afroalpine and Subafroalpine Vegetation, Dry Evergreen Montane Vegetation, Moist Evergreen
Montane Forest, Evergreen Scrub, Combretum-Terminalia (broad-leaved deciduous) woodland, Acacia-
Commmiphora (Small-leaved deciduous) woodland, Wetlands, Lowland Dry forest, and Desert and semi-Desert

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scrub. Out of the nine vegetation types, four of them occur in the dryland regions. These include,
1) Combretum-Terminalia (Broud-leaved deciduous woodland),
2) Acacia-Commiphora (Small-leaved deciduous woodland),
3) Desert and semi-desert scrub land, and
4) Dry Evergreen Montane Vegetation.
The coverage of each of the vegetation category has been declining rapidly due to the anthropogenic impacts
such as demand of land use for expansion of agriculture by local farmers, overgrazing, illegal exploitation of
forests and forest products (Friis 1992, Senbeta & Tefera 2001). Extensive agricultural investment and
expansion of road construction through vegetation are also becoming other causes of deforestation. Currently,
increasing rate of drought, desertification and shortage of food for both humans and animals are becoming
serious problems that need attentions (Moges et al. 2010). These problems are directly related to the pressures
exerted on vegetation by human beings, and thus need immediate solutions. Therefore, it is very important to
study the current status of our vegetation to identify the problems and threats associated with them and make a
useful recommendation that is helpful for planning their future conservation and sustainable management.
The aim of this study was to generate basic scientific information by identifying and documenting the
floristic composition and plant community structures of the vegetation in the study area. In addition to this, it
aimed to assess and point out the environmental factors influencing the structures of the plant communities.

MATERIALS AND METHODS


Description of the study area
Ilu Gelan District is found in West Shewa Zone of Oromia Regional State, central Ethiopia (Fig. 1). The
District is located on the Addis Ababa-Nekemte main road about 200 km from Addis Ababa to the west. IJaji is
the central town of the District and is located on geographical coordinates of 08o 5951" N and 37o 1949" E with
the altitude of 1812 m a.s.l.

Figure 1. Map of Ethiopia showing the Regional States and the study area.
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This study was conducted in the District on two nearby sites known as Dirki and Jato which are found on the
south of the main road when driving from Gedo, the central town of Cheliya District, to Ijaji at about 1o km
from Gedo to the west. The vegetation of Dirki lies on a steep mountain between 08 o 5916.108o 5950.8 N
and 37o 2245.5037o2315.8 E while that of Jato is found between the latitudes 08 o 5841.508o 5910.8 N
and longitudes 37o 2159.737o 2250.6 E.
Climate
The climate of Ilu Gelan District is considered to belong to the Weina Dega and Kolla agro-ecological zones
of Ethiopia. As most parts of the District are found in the low land, the mean annual temperature of the area is
relatively high (Amenu 2007). Meteorological data obtained from National Meteorology Service Agency
(NMSA 2015), indicates that Ilu Gelan area obtains high rainfall between May and September and low rainfall
from December to February (Fig. 2). The climadiagram figure shows that the study area is typical of forest
vegetation rainfall distribution. This indicates that the woodland vegetation was resulted from cutting effects
exerted on the original forest in the past. According to the data, the highest mean annual rainfall of the study
area recorded for twenty years (19952014) was 1351 mm and recorded in July whereas the lowest mean annual
rainfall was 11.2 mm and recorded in February. The mean maximum temperature over the twenty years was
28.1C while the mean minimum temperature was 13.8C. The highest temperature, 31.7C, was recorded in
February whereas the lowest temperature, 11.2C, was recorded in November.

Figure 2. Climadiagram showing rainfall distribution and temperature variation from 19952014 around Ijaji Town.
[Source: National Meteorological Service Agency (NMSA 2015)]
Sampling design
Systematic transect sampling following Kent & Coker (1992) was used for the study. After the highest
altitude was recorded, one 20 m 20 m quadrat was first taken at the peak of Dirki and radiating transects were
laid down from the top to the base of the mountain in four (N, W, S and E) directions. However, in the case of
Jato, vegetation cover is found only in the north and north-west facing aspects of extending escarpment. Thus,
three transect lines were laid down from the top to the base of the escarpment on the north and north-west facing
aspects, where the distance between the three consecutive transects was measured to be 300 m.
Along the transect lines of each study site, 20 m 20 m (400 m2) quadrats were laid down at every 25 m
altitudinal drop to analyze species turnover. Each transect contains different numbers of plots depending on the
length of each transect. In addition, five 1 m 1 m subplots, one at each of the four corners and one at the center
of the 20 m 20 m main plot were also laid to sample herbaceous plants. A total of fifty four 20 m 20 m
quadrats were laid for vegetation data collection, where 32 were from Dirki and the rest 22 from Jato site.
Data collection
a. Environmental data collection:
In each of the quadrat, altitude and geographical coordinates were measured using Garmin 72 GPS
(Geographical Position System) and aspect was determined using Suunnto Compass. Codes were given to
aspects following Woldu et al. (1989) as:
North= 0; East= 2; South= 4; West= 2.5 and NW= 1.3
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Ecological disturbances such as grazing and impacts of human beings (cutting, collecting firewood,
producing charcoal and trampling in the vegetation) were noticed and recorded as present or absent in the
sampled plots. Grazing intensity was estimated following Woldu & Backeus (1991) and Tekle et al. (1997) as:
0= nil; 1= slight; 2= moderate and 3= heavy
The state of human interference was estimated following Hadera (2000), Yeshitela & Bekele (2002), Kidane
et al. (2010) and codified using a 03 subjective scale to record the degree of the impacts (from cutting, fuel
wood collection, charcoal production and sign of trampling) as:
0= nil; 1= low; 2= moderate and 3= heavy
b. Vegetation data collection:
Data collection was conducted from November 06 to 20, 2014. A complete list of trees, shrubs, lianas, and
herbs was made from the systematically selected plots laid down along each transect. Plant species that occur
outside the sample plots, but inside the study area were recorded as present in the floristic composition to
produce a complete list of the plants in the vegetation area. Estimation of cover/abundance values for woody
species was made and recorded in the field.
The local name of each species, if present, was recorded during the field work. Specimens of all encountered
woody and herbaceous plants were collected, pressed, dried and brought to the National Herbarium (ETH),
Addis Ababa University, for taxonomic identification. The specimens were identified by comparing with
authenticated specimens housed at ETH and by referring to the Flora of Ethiopia and Eritrea. Voucher
specimens were kept at ETH.
Cover abundance data defined here as the proportion of area in a quadrat covered by every species recorded
and gathered from each quadrat were converted to the 19 Braun-Blanquet scale, which was later modified by
van der Maarel (1979).
Data analysis
a. Vegetation classification:
The computer program R software for windows 3.0.2 version was used to analyze the vegetation data
through Agglomerative Hierarchical Classification technique. Euclidean distance and Wards method were used
for clustering the vegetation data using R software for windows 3.0.2 version. Three plant community types
were obtained from the hierarchical clustering analysis and named using two characteristic species having the
highest mean cover abundance values in their community.
b. Diversity analysis:
Shannon-Wiener Diversity Index was used to analyze the species diversity, species richness and evenness of
the vegetation as:
H= - Pi ln Pi
Where, H: Shannon-Wiener Index; Pi: proportion of individual species; ln: log base n.
The equitability or evenness of the species in each quadrat was computed using the formula:
Equitability J = =
Where, S: the number of species; Pi: the proportion of individuals of the ith species or the abundance of the ith
species expressed as a proportion of total cover; ln: log base.
c. Phytogeographical similarity:
Sorensons Similarity ratio was used to evaluate the similarity between the three plant community types of
the vegetation in the study area and as well as the similarity between the Vegetation and four other previously
studied vegetationon the basis of their species composition.

Where, Ss: Sorensens similarity coefficient; a: number of species common to both samples /communities/ study
areas; b:number of species in sample 1; c: number of species in sample 2.

RESULTS
Floristic composition
The study showed that Dirki and Jato vegetation (Fig. 3 & Fig. 4) had high species richness in plants of
different growth forms. Plant species of different growth forms (trees, shrubs, lianas and herbs) were recorded
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from the study area (Fig. 5). Out of the total plant species recorded from the study area, trees and herbs each
comprised 32.71% while shrubs and lianas constituted 27.57% and 7.01% respectively.

Figure 3. Vegetation of Dirki site.

Figure 4. Vegetation of Jato site.


A total of 69 families with 167 genera and 214 species were recorded from the study area (Appendix 1). Of
all the families, Fabaceae, Asteraceae and Poaceae were the three most dominant families represented by 18, 15
and 11 genera, and 23, 22 and 12 species respectively. These three dominant families together constituted 57
(26.64%) of the total species richness in Dirki and Jato Vegetation. The next dominant families, Euphorbiaceae,
Acanthaceae, Rubiaceae, Lamiaceae and Combretaceae were represented by 11, 9, 9, 9 and 7 species
respectively and constituted 45 (21.03%) of the total species. Other four families which accounted 23 (10.75%)
of the total species were Malvaceae, Moraceae and Rhamnaceae and constituted six species each, while
Solanaceae was represented by five 5 species.
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Figure 5. Some of the collected plants from the study area: A, Mimusops kummel A. DC.; B, Combretum paniculatum Vent.;
C, Rothmannia urcelliformis (Hiem) Robyns; D, Acanthus polystachius Delile.
Each of the families Oleaceae, Ranunculaceae and Rosaceae were represented by four species while
Boraginaceae, Celastraceae, Loganiaceae, Sapindaceae and Verbenaceae were represented by three species
each. These eight families constituted 27 (12.62%) species while other 13 families represented by 2 species each
constituted 26 (12.15%) of the total species. The rest 36 families that contributed 16.82% of the total species
were represented by one species each. Out of the total 214 species identified from the study area, one hundred
and six, which were collected from the 54 quadrats were used in the floristic analysis. The rest 108 plant species
were collected from outside of the quadrats but inside the woodland vegetation, and included in the floristic list
to make the description of the vegetation more reliable.
Endemic plant species
Table 1. List of endemic species with their IUCN threat categories.
Species Family Habit IUCN category
Acanthus sennii Chiov. Acanthaceae S NT
Bidens ghedoensis Mesfin Asteraceae H LC
Bidens pachyloma (Oliv. & Hiern) Cufod. Asteraceae H LC
Cirsium schimperi (Valke) C. Jeffrey ex Cufod. Asteraceae H LC
Clematis longicauda Steud.ex A. Rich. Ranunculaceae L LC
Crotalaria rosenii (Pax) Milne-Redh.ex Polhill Fabaceae H NT
Echinops longisetus A. Rich. Asteraceae H LC
Lippia adoensis Hochst. ex Walp Verbenaceae S LC
Millettia ferruginea (Hochst.) Bak. Fabaceae T NT
Phyllanthus mooneyi M. Gilbert Euphorbiaceae S VU
Pycnostachys abyssinica Fresen. Lamiaceae H LC
Solanum marginatum L.f. Solanaceae S LC
Vernonia leopoldi (Sch. Bip. ex Walp.) Vatke Asteraceae S LC
Note: T= Tree, S= Shrub, L= Liana, H= Herb; NT= Near Threatened, LC= Least Concern, VU= Vulnerable.
Out of the total plant species identified from the study area, eleven species were identified as endemic to
Ethiopia while two were near endemic i.e., confined to Ethiopia and Eritrea (Table 1). Based on the IUCN
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Criteria of level of threat, nine species were least concern (LC), three species were assessed as near threatened
(NT) while one species was vulnerable (VU).
Vegetation classification
a. Plant community types:
Three plant community types were identified from the hierarchical cluster analysis using the computer
software program R for windows version 3.0.2. The computer program for determining the optimal number of
clusters was used to decide the number of plant community types. Wards method and Euclidean distance were
used to draw the dendrogram showing dissimilarity among the three clusters (Fig. 6). The vegetation
classification was done by using the cover abundance value estimate of each species included in the analysis.
Distribution of the three plant community types (C1= Community Type 1, C2= Community Type 2, and C3=
Community Type 3) along with their altitudinal range was given in table 2. The plant community types were
named by two characteristic species confied to only one of the three plant community types (Table 3).

Figure 6. Dendrogram showing the plant community types of the study area. [Agglomerativ Hierarchical Clustering Using
2
Euclidean Distance equation: ]

Table 2. Distribution of plots of the three plant communities with their altitudinal ranges.
Community Altitude (m a.s.l.) Total plots List of plots
C1 19202136 13 3, 39, 40, 41, 42, 43, 46, 47, 48, 49, 50, 51 and 52
C2 19502078 19 1, 2, 4, 5, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 33,
34 and 35
C3 18031953 22 6, 7, 8, 9, 10, 11, 12, 25, 26, 27, 28, 29, 30, 31, 32, 36, 37,
38, 44, 45, 53 and 54
Descriptions of the three plant community types
a. Acacia etbaica - Lantana trifolia community type
This community type is distributed between the altitudinal range of 19202136 m a.s.l. It is represented by
13 plots comprising 62 plant species; out of which 50 are commonly shared with community two and 51 with
community three. Acacia etbaica and Lantana trifolia are the characteristic species of this community. Acacia
abyssinica is a dominant tree species in this community type while other common tree and shrub species
associated with the community include: Maesa lanceolata, Syzygium guineense, Combretum molle, Premna
schimperi, Hypericum quartinianum, Rosa abyssinica, Stereospermum kunthianum and Nuxia congesta. In this
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community, common herbaceous species covering the ground surface are Bidens biternata, Bidens ghedoensis,
Cynodon dactylon and Oplismenus hirtellus.
b. Buddleja polystachya - Teclea nobilis community type
This community is distributed between the altitudinal range of 19502078 m a.s.l. It is represented by 19
plots consisting of 73 species; out of which 50 are shared with community one while 66 species are shared with
community three. Buddleja polystachya and Teclea nobilis are the characteristic species in the community.
Calpurnia aurea is a dominant species whereas common tree and shrub plant species found in this community
include Osyris quadripartita, Albizia schimperiana, Rhus natalensis, Euclea divinorum, Olinia rochetiana and
Schrebera alata. Whereas, Helinus mystacinun and Dioscorea shimperiana are common lianas recorded in this
community type. On the other hand, Hypoestes aristata, Panicum monticola, Justicia ladanoides and Cyperus
sesquiflorus are the dominant species at herbaceous level.
c. Combretum paniculatum - Rothmannia urcelliformis community type
This community type is distributed between the altitudinal range of 18032080 m a.s.l. It is represented by
22 plots consisting of 95 species. Combretum paniculatum and Rothmannia urcelliformis are characteristic
species of the community. Other dominant woody species of this community include: Clausena anisata,
Maytenus arbutifolia, Grewia ferruginea, Calpurnia aurea, Carissa spinarum, Croton macrostachyus, Millettia
ferruginea, Acacia abyssinica, Bersama abyssinica, Vangueria apiculata and Rytigynia neglecta. On the Other
hand, Clematis longicauda is a characteristic liana of this community whereas the characteristic herbaceous
species covering the ground surface are Achyranthes aspera, Cynodon nlemfuensis, Pycnostachys abyssinica
and Setaria megaphylla.
Table 3. Mean cover abundance estimates of species of the three communities.
Community type
Species One Two Three
Acacia abyssinica 4.15 2.21 3.59
Acacia etbaica 0.08 0.00 0.00
Albizia schimperiana 2.69 6.00 2.55
Bersama abyssinica 1.08 5.26 3.55
Buddleja polystachya 0.00 0.11 0.00
Calpurnia aurea 4.46 7.37 5.27
Carissa spinarum 2.92 6.00 4.5
Clausena anisata 2.08 7.05 6.59
Combretum molle 3.62 2.26 1.91
Combretum paniculatum 0.00 0.00 1.09
Croton macrostachyus 4.23 5.00 4.41
Euclea divinorum 0.08 5.16 0.36
Ficus sycomorus 0.00 0.00 0.23
Ficus vasta 0.00 0.00 0.09
Grewia ferruginea 3.92 4.47 5.59
Lantana trifolia 0.08 0.00 0.00
Mimusops kummel 0.00 0.00 0.23
Olea capensis 0.00 0.00 0.31
Olinia rochetiana 1.85 5.26 0.05
Osyris quadripartita 6.23 3.53 0.55
Premna schimperi 6.62 6.79 5.27
Rothmannia urcelliformis 0.00 0.00 0.32
Schrebera alata 0.38 3.94 0.05
Syzygium guineense 7.38 3.16 1.68
Teclea nobilis 0.00 0.26 0.00
Species richness, evenness and diversity of the three communities
Shannon-Wiener diversity index was computed for the three plant community types of the woodland
vegetation of Ilu Gelan District (Table 4). Community three had the highest diversity followed by community
two while community one showed the least diversity. Community type three had the highest species richness
whereas the least species rich community is community type one. However, equitability (evenness) which
measures the relative abundance of different species present in each community showed relatively the highest
value for community two followed by community three and the lowest for community one.

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Table 4. Shannon-Wiener diversity index for woodland vegetation of Ilu Gelan District.
Community Diversity Species H max Equitability Average altitude
type index (H) richness (S) (J) (m a.s.l.)
One 3.847 62 4.128 0.932 2045.50
Two 4.068 73 4.291 0.948 2014.00
Three 4.316 95 4.553 0.947 1941.50
Similarity between the three plant community types
Sorensens Coefficient Index was used to calculate the degree of similarity among the three plant community
types. The highest similarity was observed between communities one and three while the least similarity was
observed between communities two and three. The result from the analysis showed communities one and two,
community one and three, and communities two and three shared about 74.63%, 64.97% and 78.57% similarity
in species composition respectively.

DISCUSSION
Dominance of Fabaceae and Asteraceae has been reported from different floristic studies done by different
researchers at different times (Yineger et al. 2008, Tadesse et al. 2008, Abdena 2010, Kebede 2010, Didita et al.
2010, Alemu 2011, Adamu et al. 2012, Dibaba et al. 2014). Similarly, the results from analyzed data showed
that Fabaceae and Asteraceae are the most dominant species in Dirki and Jato Woodland Vegetation. Fabaceae
and Asteraceae are represented by 23 and 22 species respectively, while Poaceae followed by 12 species. The
dominance of these families is also in line with the assessment results that show their dominance positions in the
Flora of Ethiopia and Eritrea. Fabaceae and Asteraceae might have got the top dominant position probably due
to having efficient pollination and successful seed dispersal mechanisms that might have adapted them to a wide
range of ecological conditions in the past (Kelbessa & Soromessa 2008). However, the variation in topography
and environmental conditions like the amount of rainfall and temperature could be the causes of variation in
dominance positions of plants taxa. Ethiopia is considered one of the countries of east Africa known by plant
endemism (Vivero et al. 2005). The diverse topographic land features having various climatic conditions
assisted Ethiopia to be a country of high biodiversity (Gebrehiwot & Hundera 2014).
Results from data analysis showed that the woodland vegetation of Dirki and Jato consists of 13 endemic
species of the flora of Ethiopia and Eritrea. From these, 11 species are endemic to Ethiopia while two are near
endemic (Lippia adoensis and Solanum marginatum are confined to Ethiopia and Eritrea). Based on the IUCN
Criteria of the level of threat, nine species are least concern (LC); three species are near threatened (NT) while
one species has been categorized under vulnerable (VU).
Plant community types
The results from cluster analysis showed that there are differences among the three plant communities in
species composition. The differences could be attributed to variations in environmental gradients that can limit
the ecological distributions of plant species (Lulekal 2014). As described in Bekele (1993), this variation in
species composition could also be related to the effects of environmental factors such as altitude, aspect, soil
contents and moisture, human impacts and grazing intensity. Lower altitudinal ranges, aspects more exposed to
sun light directions, soils with sufficient moisture contents and part of vegetation less exposed to disturbance
accesses can support relatively more biodiversity than the reverse of each component.

CONCLUSION
The results from this study showed that plant species of different life forms (trees, shrubs, lianas and herbs)
were identified from the vegetation of Dirki and Jato in Ilu Gelan District. Out of the total plant species recorded
from the vegetation, most proportions (65.42%) were trees and herbs while the rest 27.57% and 7.01% were
shrubs and lianas respectively.
Plants recorded from the study area belong to 69 families, 167 genera and 214 species; whereas Fabaceae,
Asteraceae and Poaceae were the most dominant families with 23, 22 and 12 species respectively. These three
dominant families constituted 26.64% of the total species richness of the study area. Euphorbiaceae and
Acanthaceae were the fourth and fifth dominating families while Rubiaceae, Lamiaceae and Combretaceae were
the next three consecutive dominant families in the vegetation. These five species are represented by 11, 9, 9, 9
and 7 species respectively, and constitute 45 (21.03%) of the total species recorded from the study area.
Families Malvaceae, Moraceae, Rhamnaceae and Solanaceae constitute 23 (10.75%) of the total species and the
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first three families were represented by six species each while Solanaceae contributed five species. Families
Oleaceae, Ranunculaceae and Rosaceae were represented by four species each while families represented by
three species each in the vegetation were Boraginaceae, Celastraceae, Loganiaceae, Sapindaceae and
Verbenaceae. These eight families together contribute 27 (12.62%) of the total species while other 13 families
represented by two species each constituted 26 (12.15%) of the total species. The rest 36 (16.82%) families were
represented by only one species each.
Of the total 214 species identified from the study area, 11 are endemic to Ethiopia while two species are near
endemic (Lippia adoensis and Solanum marginatum are found in Ethiopia and Eritrea only). According to the
IUCN Red Data List, nine of these species were included in least concern (LC), two were considered near
threatened (NT) whereas one species has been put under vulnerable (VU) category.
Based on the cover abundance values of the identified species, three plant community types were recognized
from the study area and named by two characteristic species having highest mean cover abundance estimate in
each community. The three communities are Acacia etbaica - Lantana trifolia Community Type, Buddleja
polystachya - Teclea nobilis Community Type and Combretum paniculatum - Rothmannia urcelliformis
Community Type. Community three had the highest diversity and species richness than the two communities.
Communities one and two were more similar in species composition than communities one and three or two and
three.

ACKNOWLEDGEMENTS
The authors express their grateful acknowledge to Cheliya and Ilu Gelan districts for facilitating the
necessary conditions needed for the study. They also express their thanks to Addis Ababa University for
providing financial supports. Finally, they acknowledge the National Meteorology Service Agency (NMSA) and
Central Statistical Agency (CSA) for providing secondary data.

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Appendix 1. List of plant species collected from Dirki and Jato sites in Ilu Gelan District.
S.N. Species name Family Local name Ha C. code Housed at
1 Abutilon longicuspe Hoehst. ex A. Rich. Malvaceae Hincinnii S Z192 ETH
2 Acacia abyssinica Hochst. ex Benth. Fabaceae Laaftoo T Z023 ETH
3 Acacia etbaica Schweinf. Fabaceae Doddota T Z197 ETH
4 Acacia persiciflora Pax Fabaceae Laaftoo T Z204 ETH
5 Acanthus polystachius Delile Acanthaceae Sokorruu adii S Z176 ETH
6 Acanthus sennii Chiov. Acanthaceae Sokorruu S Z013 ETH
7 Achyranthes aspera L. Amaranthaceae Maxxannee H Z118 ETH
8 Acmella caulirhiza Del. Asteraceae H Z182 ETH
9 Adiantum poiretii Wikstr. Adiantaceae H Z027 ETH
10 Albizia schimperiana Oliv. Fabaceae Imalaa T Z005 ETH
11 Allophylus macrobotrys Gilg Sapindaceae Sarara T Z125 ETH
12 Allophylus africanus P. Beauv. Sapindaceae Qarxammee T Z162 ETH
13 Aloe macrocarpa Tod. Aloaceae Hargisa S Z178 ETH
14 Andropogon abyssinicus Fresen. Poaceae Baallammii H Z179 ETH
15 Apodytes dimidiata E. Mey. ex Am. Icacinaceae Qumbaala T Z157 ETH
16 Argyrolobium fischeri Taub. Fabaceae H Z016 ETH
17 Aspilia mossambicensis (Oliv.) Wild Asteraceae Keelloo S Z165 ETH
18 Asystasia mysorensis (Roth) T. Anders. Acanthaceae H Z166 ETH
19 Bersama abyssinica Fresen. Melianthaceae Lolchiisaa T Z008 ETH
20 Bidens biternata (Lour.) Merr. & Sherfft. Asteraceae Keelloo H Z183 ETH
21 Bidens ghedoensis Mesfin Asteraceae Keelloo H Z120 ETH
22 Bidens pachyloma (Oliv. & Hiern) Asteraceae Keelloo H Z153 ETH
Cufod.
23 Bridelia micrantha (Hochst.) Baill. Euphorbiaceae Agiraabaa T Z094 ETH
24 Brucea antidysenterica J.F.Mill. Simaroubaceae Qomonyoo S Z185 ETH
25 Buddelja davidii Franch. Loganiaceae Qawwwisa S Z151 ETH
26 Buddleja polystachya Fresen. Loganiaceae Qawwisa T Z075 ETH
27 Caesalpinia decapetala (Roth) Alston Fabaceae Arangamaa L Z149 ETH
28 Calpurnia aurea (Ait.) Benth. Fabaceae Ceekaa S Z001 ETH
29 Capparis tomentosa Lam. Capparidaceae Arangamaa S Z142 ETH
30 Carissa spinarum L. Apocynaceae Agamsa S Z051 ETH
31 Celtis africana Burm.f. Ulmaceae Cayii T Z108 ETH
32 Chionanthus mildbraedii (Gilg & Oleaceae Karra waayyuu T Z199 ETH
Schellenb.) Stearn
33 Cirsium schimper (Valke) C. Jeffrey ex Asteraceae H Z152 ETH
Cufod.
34 Cissampelos pareira L. Menispermaceae Hidda kalaalaa L Z018 ETH
35 Clausena anisata (Willd). Benth. Rutaceae Ulmaayii S Z003 ETH
36 Clematis hirsuta Perr. & Guill. Ranunculaceae L Z144 ETH
37 Clematis longicauda Steud.ex A. Rich. Ranunculaceae Hidda fiitii L Z063 ETH
38 Clematis simensis Fresen. Ranunuclaceae Hidda fiitii L Z020 ETH
39 Clerodendrum myricoides (Hochst.) Lamiaceae S Z131 ETH
Vatke
40 Clutia abyssinica Jaub. &- Spach. Euphorbiaceae S Z085 ETH
41 Combretum adenogonium Steud. ex A. Combretaceae Rukeessa T Z212 ETH
Rich.
42 Combretum collinum Fresen. Combretaceae T Z080 ETH
43 Combretum molle R. Br. ex G.Don Combertaceae Rukeessa T Z041 ETH
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44 Combretum nigrican Lepr. ex Guill. & Combretaceae T Z055 ETH


Perr.
45 Combretum paniculatum Vent. Combretaceae Hidda baggii L Z137 ETH
46 Commelina benghalensis L. Commelinaceae Gororaa H Z168 ETH
47 Cordia africana L. Boraginaceae Waddeessa T Z114 ETH
48 Crassocephalum macropappum Asteraceae H Z170 ETH
(Sch.Bip.ex A. Rich) S. Moore
49 Crassocephalum x picridifolium ( DC) S. Asteraceae H Z169 ETH
Moore
50 Crassula alata (Viv.) Berger Crassulaceae H Z145 ETH
51 Crepis rueppelli Sch. Bip. Asteraceae H Z136 ETH
52 Crotalaria pallida Ait. Fabaceae H Z200 ETH
53 Crotalaria quartiniana A. Rich. Fabaceae H Z132 ETH
54 Crotalaria rosenii (Pax) Milne-Redh.ex Fabaceae S Z123 ETH
Polhill
55 Croton macrostachyus Del. Euphorbiaceae Bakkanniissa T Z030 ETH
56 Cucumis dipsaceus Ehrenb. ex Spach Cucurbitaceae H Z113 ETH
57 Cyathula polycephala Bale. Amaranthaceae H Z135 ETH
58 Cymbopogon commutatus (Steud.) Stapf Poaceae Jajjaba H Z101 ETH
59 Cynodon dactylon (L.) Pers. Poaceae Coqorsa H Z103 ETH
60 Cynodon nlemfuensis Vanderyst Poaceae Waratii H Z207 ETH
61 Cyperus sesquiflorus (Torr.) Mattf. & Cyperaceae Qeexamaa H Z052 ETH
KUk.
62 Dalbergia lactea Vatke Fabaceae Sarxee T Z107 ETH
63 Desmodium repandum (Vahl) DC. Fabaceae H Z213 ETH
64 Diaphananthe candida Cribb Orchidaceae Digaluu H Z062 ETH
65 Dicranopteris linearis (Burm.f.) Underw. Gleicheniaceae Fern H Z034 ETH
66 Dioscorea schimperiana Hochst. ex Dioscoreaceae H Z047 ETH
Kunth
67 Diospyros abyssinica (Hiern) F. White Ebenaceae Ilkee T Z089 ETH
68 Dodonaea angustifolia L. f. Sapindaceae Ittacha S Z088 ETH
69 Dombeya torrida (G.F. Gmel.) P. Bamps Sterculiaceae Daannisa T Z116 ETH
70 Dovyalis abyssinica (A. Rich.) Warb. Flacourtiaceae Koshommii T Z045 ETH
71 Dracaena steudneri Engl. Dracaenaceae Meerqoo S Z150 ETH
72 Drimia altissima (L.f.) Ker-Gawl. Hycinthaceae Qullubbii H Z209 ETH
waraabessaa
73 Echinops longisetus A. Rich. Asteraceae Qoraattii harree S Z058 ETH
74 Ehretia cymosa Thonn. Boraginaceae Ulaagaa T Z021 ETH
75 Ekebergia capensis Sparrm. Meliaceae Somboo T Z095 ETH
76 Englerina woodfordioides (Schweinf.)M. Loranthaceae Digaluu S Z119 ETH
Gilbert
77 Entada abyssinica Steud. ex A. Rich. Fabaceae Ambaltaa T Z196 ETH
78 Erythrococca abyssinica Pax Euphorbiaceae Geelloo S Z067 ETH
79 Eucalyptus camaldulensis Dehnh. Myrtaceae Baargamoo T Z068 ETH
diimaa
80 Euclea divinorum Hiern Ebenaceae Mi'eessaa T Z038 ETH
81 Euphorbia schimperiana Scheele Euphorbiaceae S Z011 ETH
82 Ficus mucuso Ficalho. Moraceae Qilinxoo T Z141 ETH
83 Ficus salicifolia A. Rich. Moraceae Qilinxoo T Z167 ETH
84 Ficus sur Forssk. Moraceae Harbuu T Z164 ETH
85 Ficus sycomorus L. Moraceae Odaa T Z130 ETH
86 Ficus thonningii Blume Moraceae Dambii T Z128 ETH

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87 Ficus vasta Forssk. Moraceae Qilxuu T Z122 ETH


88 Flacourtia indica (Burm.f.) Merr Flacourtaceae Akuukkuu T Z066 ETH
89 Galiniera saxifraga (Hochst.) Bridson Rubiaceae T Z040 ETH
90 Gardenia ternifolia Schumach. &Thonn. Rubiaceae Gambeela T Z033 ETH
91 Geranium arabicum Forssk. Geraniaceae H Z070 ETH
92 Girardinia diversifolia (Link) Friis Urticaceae Doobbii H Z124 ETH
93 Glycine wightii (Wight& Am.) Verdc. Fabaceae H Z115 ETH
94 Gnidia glauca (Fresen.) Gilg Thymelaeaceae Qaqaroo S Z175 ETH
95 Gouania longispicata Engl. Rhamnaceae L Z195 ETH
96 Grewia ferruginea Hochst.ex A. Rich. Tiliaceae Dhoqonuu T Z056 ETH
97 Guizotia schimperi Sch. Bip. ex Walp. Asteraceae Tuufoo H Z069 ETH
98 Helinus mystacinus (Ait.) E. Mey. ex Rhamnaceae Hidda L Z050 ETH
Steud. hoomachoo
99 Heliotropium zeylanicum (Burm f.) Lam. Boraginaceae Maxxannee H Z029 ETH
100 Hygrophila schulli (Hamilt.) M.R. & Acanthaceae Qoraatii saree H Z096 ETH
S.M Almeida
101 Hymenodictyon floribundum (Hochst. & Rubiaceae Gaarrii T Z154 ETH
Steud.) Robinson
102 Hyparrhenia anthistirioides (Hochst. ex Poaceae Sanbaleeta H Z090 ETH
A. Rich) Stapf
103 Hypericum quartinianum A. Rich. Guttiferae Hinnee T Z181 ETH
104 Hypoestes aristata (Vahl) Nees Acanthaceae Darguu H Z092 ETH
105 Ilex mitis (L.) Radlk. Aquifoliaceae T Z206 ETH
106 Ipomoea plebeia Meeuse Convolvulaceae H Z081 ETH
107 Justicia ladanoides Lam. Acanthaceae H Z083 ETH
108 Justicia schimperiana (Hochst. ex Nees) Acanthaceae Dhummuugaa S Z171 ETH
T. Anders.
109 Kalanchoe marmorata Bak. Crassulaceae Bosoqqee H Z048 ETH
110 Laggera crispata (Vahl) Hepper & Wood Asteraceae H Z189 ETH
111 Landolphia buchananii (Hall.f.) Stapf Apocynaceae Hidda geeboo L Z208 ETH
112 Lantana trifolia L. Verbenaceae S Z198 ETH
113 Leonotis ocymifolia (Burm. f.) Iwarsson Lamiaceae S Z211 ETH
114 Lippia abyssinica (Otto & Dietr.) Verbenaceae S Z078 ETH
115 Lippia adoensis Hochst. ex Walp Verbenaceae Kusaayee S Z006 ETH
116 Loudetia flavida (Stapf) C. E. Hubb. Poaceae H Z043 ETH
117 Maesa lanceolata Forssk. Myrsinaceae Abbayyii T Z059 ETH
118 Malva verticillata L. Malvaceae Hincinnii H Z044 ETH
119 Maytenus arbutifolia (A.Rich.) Wilczek Celastraceae Kombolcha S Z007 ETH
120 Maytenus gracilipes (Welw. ex Oliv.) Celastraceae Acaacii S Z100 ETH
Exell
121 Maytenus obscura (A. Rich.) Cuf. Celastraceae Kombolcha S Z073 ETH
122 Medicago polymorpha L. Fabaceae Siddisa H Z074 ETH
123 Microglossa pyrifolia (Lam.) 0. Kuntze Asteraceae S Z110 ETH
124 Mikaniopsis clematoides (Sch. Bip. ex A. Asteraceae H Z091 ETH
Rich.) Milne-Redh.
125 Millettia ferruginea (Hochst.) Bak. Fabaceae Sootalloo T Z117 ETH
126 Mimosa pigra L. Fabaceae Arangamaa S Z214 ETH
127 Mimusops kummel A. DC. Sapotaceae Qolaatii T Z140 ETH
128 Monechma debile (Forssk.) Nees Acanthaceae H Z028 ETH
129 Myrsine africana L. Myrsinaceae Qacama S Z065 ETH
130 Nuxia congesta R.Br. ex Fresen. Loganiaceae Qawwisa T Z194 ETH

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131 Ocimum lamiifolium Hochst. ex. Benth. Lamiaceae Ancabbii diimaa S Z014 ETH
132 Ocimum urticifolium Roth. Lamiaceae Ancabbii adii S Z084 ETH
133 Olea capensis L. subsp. macrocarpa Oleaceae Gagamaa T Z147 ETH
(C.H. Wright) Verdc.
134 Olea europaea L. subsp. cuspidata Oleaceae Ejersa T Z097 ETH
(Wall.ex G.Don) Cif.
135 Olinia rochetiana A.Juss. Oliniaceae Daalachoo T Z025 ETH
136 Ophrestia radicosa (A. Rich.) Verde. Fabaceae Hidda bofaa H Z017 ETH
137 Oplismenus hirtellus (L.) P. Beauv. Poaceae Ashuffee H Z053 ETH
138 Oreosyce africana Hook.f. Cucurbitaceae H Z186 ETH
139 Osyris quadripartita Decne Santalaceae Waatoo T Z026 ETH
140 Panicum monticola Hook.f. Poaceae Marga gogorrii H Z105 ETH
141 Pavetta abyssicica Fresen. Rubiaceae S Z024 ETH
142 Pellaea calomelanos (Sw.) Link Sinopteridaceae H Z071 ETH
143 Pennisetum thunbergii Kunth Poaceae Migira saree H Z102 ETH
144 Periploca llnearlfolia Quart.-Dill. & A. Asclepiadaceae Hidda aannannoo L Z160 ETH
Rich.
145 Phaulopsis imbricata (Forssk.) Sweet Acanthaceae H Z049 ETH
146 Phoenix reclinata Jacq. Arecaceae Meexxii T Z129 ETH
147 Phyllanthus mooneyi M. Gilbert Euphorbiaceae S Z187 ETH
148 Phyllanthus ovalifolius Forssk. Euphorbiaceae Qacamoo T Z104 ETH
149 Phymatosorus scolopendria (Burn.f.) Polypodiaceae H Z184 ETH
Pic. Serm
150 Phytolacca dodecandra L'Herit. Phytolaccaceae Andoodee S Z148 ETH
151 Pittosporum viridiflorum Sims Pittosporaceae Soolee adii T Z210 ETH
152 Plectranthus punctatus (L.f.) L'H'er. Lamiaceae H Z205 ETH
153 Pliostigma thonningii (Schumach.) Fabaceae T Z127 ETH
Milne-Redh
154 Podocarpus falcatus (Thunb.) R.B. ex. Podocarpaceae Birbirsa T Z098 ETH
Mirb.
155 Polypogon schimperianus (Hochst. ex Poaceae Daggala H Z042 ETH
Steud.) Cope
156 Premna schimperi Engl. Lamiaceae Urgeessaa S Z009 ETH
157 Prunus africana (Hook.f.) Kalkm. Rosaceae Hoomii T Z203 ETH
158 Pseudognaphalium luteo-album (L.) Asteraceae H Z022 ETH
Hilliard & Burtt
159 Psychotria orophila Petit Rubiaceae S Z099 ETH
160 Pterolobium stellantum (Forssk.) Brenan Fabaceae Arangamaa L Z138 ETH
161 Pycnostachys abyssinica Fresen. Lamiaceae Bokkolluu H Z121 ETH
162 Rhamnus prinoides LHerit. Rhamnaceae Geeshoo S Z087 ETH
163 Rhamnus staddo A.Rich. Rhamnaceae Qadiidaa T Z093 ETH
164 Rhoicissus revoilii Planch. Rhamnaceae Indirifaa L Z061 ETH
165 Rhus natalensis Krauss Anacardiaceae Xaaxessaa T Z019 ETH
166 Rhus vulgaris Meikle Anacardiaceae Xaaxessaa T Z002 ETH
167 Ricinus communis L. Euphorbiaceae Qobboo S Z202 ETH
168 Rosa abyssinica Lindley Rosaceae Qaqawwii S Z060 ETH
169 Rothmannia urcelliformis (Hiem) Rubiaceae Qola-gurraa T Z146 ETH
Robyns
170 Rubia cordifolia L. Rubiaceae Maxxannee H Z036 ETH
171 Rubus apetalus Poir. Rosaceae Goraa L Z201 ETH
172 Rubus steudneri Schweinf. Rosaceae Goraa L Z188 ETH
173 Rumex nepalensis Spreng. Polygonaceae Timijjii H Z191 ETH

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174 Rytigynia neglecta (Hiern) Robyns Rubiaceae Mixoo S Z079 ETH


175 Salix mucronata Thunb. (S. subserrata Salicaceae Alaltuu T Z193 ETH
Willd)
176 Sapium ellipticum (Krauss) Pax. Euphorbiaceae Bosoqa T Z139 ETH
177 Satureja abyssinica (Benth.) Briq. Lamiaceae H Z174 ETH
178 Satureja punctata (Benth.) Briq. Lamiaceae S Z106 ETH
179 Schefflera abyssinica (Hochst. ex A. Araliaceae Gatamaa T Z086 ETH
Rich.) Harms
180 Schrebera alata (Hochst.) Welw. Oleaceae Qana'ee T Z004 ETH
181 Scutia myrtina (Burm. f.) Kurz Rhamnaceae Kombolcha adii S Z111 ETH
182 Senna petersiana (Bolle) Lock Fabaceae Gaafatoo T Z072 ETH
183 Senna septemtrionalis (Viv.) Irwin & Fabaceae S Z190 ETH
Bameby
184 Setaria megapbylla (Steud.) Th. Dur. & Poaceae Jajjaba H Z054 ETH
Schinz
185 Sida ternata L.f. Malvaceae Hincinnii H Z082 ETH
186 Sida rhombifolia L. Malvaceae Karabaa S Z156 ETH
187 Sida schimperiana Hochst. ex A. Rich. Malvaceae Cirfiggii S Z012 ETH
188 Sida urens L. Malvaceae Hincinnii S Z035 ETH
189 Solanum aculeatissimum Jacq. Solanaceae Hiddii S Z037 ETH
waraabessaa
190 Solanum anguivi Lam. Solanaceae Hiddii saree S Z133 ETH
191 Solanum giganteum Jacq. Solanaceae S Z046 ETH
192 Solanum macracanthum A. Rich. Solanaceae Hiddii S Z015 ETH
193 Solanum marginatum L.f. Solanaceae Hiddii hongorcaa S Z155 ETH
194 Sphaerantuhs suaveolens (Forssk) DC. Asteraceae Bokkolluu H Z112 ETH
195 Sporobolus africanus (Poir.) Robyns & Poaceae Murii H Z010 ETH
Tourny
196 Stereospermum kunthianum Cham. Bignoniaceae Botoroo T Z077 ETH
197 Syzygium guineense (Willd.) DC. Myrtaceae Baddeessaa T Z076 ETH
198 Tagetes minuta L. Asteraceae H Z032 ETH
199 Tapinanthus heteromorphus (A. Rich.] Loranthaceae Digaluu S Z057 ETH
Danser
200 Teclea nobilis Del. Rutaceae Hadheessa T Z159 ETH
201 Teramnus labialis (L. f.) Spreng. Fabaceae H Z158 ETH
202 Terminalia macroptera Guill & Perr. Combretaceae Dabaqqaa T Z161 ETH
203 Terminalia schimperiana Hochst. Combretaceae Gaarrii T Z126 ETH
204 Thalictrum rhynchocarpum Dill. & Ranunculaceae Sire bizuu H Z163 ETH
A.Rich.
205 Thunbergia alata Boj. ex Sims Convolvulaceae H Z143 ETH
206 Tragia ashiae M.Gilbert Euphorbiaceae Gurgubbee H Z039 ETH
207 Tragia brevipes Pax Euphorbiaceae Gurgubbee H Z031 ETH
208 Urera hypselodendron (A.Rich,) Wedd. Urticaceae Laanqisaa L Z172 ETH
209 Vangueria apiculata K. Schum. Rubiaceae Buruurii S Z064 ETH
210 Vernonia amygdalina Del. Asteraceae Eebicha T Z177 ETH
211 Vernonia hochstetteri Sch.Bip. ex Walp. Asteraceae Sooyyoma S Z173 ETH
212 Vernonia hymenolepis A. Rich. Asteraceae Sooyyoma S Z180 ETH
213 Vernonia leopoldi (Sch. Bip. ex Walp.) Asteraceae S Z109 ETH
Vatke
214 Vernonia myriantha Hook.f. Asteraceae Reejjii T Z134 ETH
Note: T= Tree; S= Shrub; L= Liana; H= Herb; C. code= Collection code; Ha= Habit; ETH= National Herbarium of Ethiopia.

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ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(2): 351357, 2017
DOI: 10.22271/tpr.2017.v4.i2.046
Research article

Evaluation of antimicrobial activity of


Phellinus linteus (Berk. & M.A. Curtis.) with their wild collections
from Western Ghats of India
Jayashree K. Kodiyalmath* and Krishnappa M.
Department of P.G. Studies and research in Applied Botany, Kuvempu University, Shankaraghatta-577451,
Shimoga, Karnataka, India
*Corresponding Author: jk.kodiyalmath@gmail.com [Accepted: 27 August 2017]

Abstract: A survey was conducted in Sagar taluk Shivamogaa district, in the month of June to
August 2015 to collect the Phellinus linteus from the wood logs of Jack fruit. The samples were
dried and powdered, subjected to extraction using petroleum ether, chloroform and methanol
successively by soxhlet method. Extracts were tested for secondary metabolites and showed the
presence of alkaloids, phenols, sterols and flavonoids. The sporocarp extract was screened for their
antimicrobial activity against different human pathogens like Klebsiella pneumonia, Escherichia
coli, Staphylococcus aureus, Candida albicans, Chrysosporium keratinophilum, Pencillium
chrysogenum, Aspergillus niger and plant pathogens like Xanthomonas campestris and
Agrobacterium tumefaciens using agar well diffusion method. The methanol extracts showed
maximum activity followed by chloroform, petroleum ether. The inhibition effects on fungi were
low compared to bacteria. GCMS analysis showed the presence of ergosterol. The study reveals
that Phellinus linteus showed good antimicrobial activity against Candida albicans and
Staphylococcus aureus.
Keywords: Phellinus linteus - Sporocarp - Extract - Pathogens - GCMS - Antimicrobial.

[Cite as: Kodiyalmath JK & Krishnappa M (2017) Evaluation of antimicrobial activity of Phellinus
linteus (Berk. & M.A Curtis.) with their wild collections from Western Ghats of India. Tropical Plant Research
4(2): 351357]
INTRODUCTION
Macrofungi are well known for their valuable food and traditional medicines around the world since times.
Mushrooms are used as antitumor, antiviral, anti-allergants (Wisitrassameewong et al. 2012) and anti-
inflammatory (Mourao et al. 2011). Elements from sporocarp and mycelia both contain antimicrobial
compounds which can be isolated for the welfare of human beings (Yamac & Bilgili 2006). Antimicrobial
includes antibacterial, antifungal and antiviral (Guler et al. 2009, David et al. 2012). Phellinus Qul. is a potent
mushroom as Ganoderma. The secondary metabolites of Phellinus were used to cure gonorrhoea, abdominal
pain, stomach ailments and diarrhoea (Sonawane et al. 2012). Few species of Phellinus are tested and proved to
be good antimicrobial nature. Phellinus baumii Pilt showed good hypoglycaemic effects against ob/ob mice
(Cho et al. 2007). Phellinus are wood inhibiting fungi and may be used as antioxidant and anticancerous
(Khatun 2012). Phellinus hartigii (Allesch. & Schnabl) Pat., P. swieteniae (Murrill) S. Herrera & Bondartseva,
P. merrillii (Murrill) Ryvarden are antimicrobial nature (Altuner & Akata 2010, Belsare et al. 2010). P.
igniarius (L.) Qul. proved good antiviral agent against influenza virus (Song et al. 2014). P. durissimus
(Lloyd) A. Roy and P. linteus (Berk. & M.A. Curtis) shows antioxidant activity (Liang et al. 2009, Lahiri et al.
2010). Out of these all, Phellinus linteus is a medicinal mushroom with a history of about 2000 years of being
used for the treatment of haemorrhage, haemostasis and menstruation in China and Korea (Chen et al. 2016).
Thus the present work is an attempt to find out the antimicrobial activity of Phellinus linteus (Berk. & M.A
Curtis.) which found in Sagara on the jack fruit (Artocarpus heterophyllus Lam.) tree which prove good
antibiotic for the welfare of the human beings.

www.tropicalplantresearch.com 351
Received: 05 June 2017 Published online: 31 August 2017
https://doi.org/10.22271/tpr.2017.v4.i2.046
Kodiyalmath & Krishnappa (2017) 4(2): 351357

MATERIALS AND METHODS


Sample collection and preparation
The fruiting bodies of Phellinus linteus (Berk. & M.A Curtis.) are collected from the Sagara taluk,
Shivamogga district, during June to August 2015. The major field characters of Phellinus linteus were recorded.
The sporocarps were brought to the laboratory and morphological characters were recorded. The samples were
oven dried at 4550C. The fruiting bodies were ground to a coarse powder using a mixer. Three hundred grams
of the same material was subjected to Soxhlet extraction for 24 hrs each, using 1000 mL of three solvents,
petroleum ether, chloroform and methanol respectively. Secondary metabolites were extracted from sporocarp
of Phellinus linteus extracts were dried to powder and kept at 4C (Dulger et al. 2004, Sridhar et al. 2011).
These three extracts were screened against different pathogenic fungal and bacterial species by the well
diffusion method. The test organisms were collected from the Microbial Type Culture Collection (MTCC), The
Institute of Microbial Technology. Chandigarh, India.
Bacteria used to check antibacterial activities
Xanthomonas campestris [MTCC-2286], Pseudomonas syringae [MTCC-1604], Agrobacterium tumefaciens
[MTCC-431], Klebsiella pneumonia [MTCC-7028], Escherihia coli [MTCC-1559], Salmonella typhi [MTCC-
734], Pseudomonas aeruginosa [MTCC-1934], Staphylococcus aureus [MTCC-4734] and Streptomyces
pneumoneae [MTCC-4734].
Fungi used to check antifungal activities
Candida albicans [MTCC-1637], Chrysosporium merdarium [MTCC-4608], Trichophyton rubrum [MTCC-
3272], Chrysosporium keratinophilum [MTCC-1367], Fusarium solani [MTCC- 1040], Penicillium chrysoge-
num [MTCC-947], Aspergillus flavus [MTCC-1783] and Aspergillus niger [MTCC-514].
Preparation of extract
100% = 400 mg in 4 ml of DMSO, 50% = 200 mg in 4 ml of DMSO, 25% = 100 mg in 4 ml of DMSO.
Agar Well diffusion method
The antibacterial and antifungal activity of the mushroom extracts were tested using Agar well diffusion
method (Sridhar et al. 2011). The culture plates were prepared by inoculating with different bacteria and fungi.
Wells were made with 6 mm cork borer. The wells were loaded with extracts which were dissolved in dimethyl
sulfoxide (DMSO) of different concentration (100%, 50% and 25%) using micro pipette. Ciprofloxacin for
bacteria, Terbinafine for fungi were used as standard and DMSO was used as control for test microorganisms.
The plates were incubated at 272 C for 24 hrs for bacterial activity and 48 hrs for fungal activity (Das et al.
2010). The zone formation was observed in plates around the wells and calculated by measuring the diameter of
the inhibition zone around. The readings were taken in 4 replicates and the average values were tabulated.
Physicochemical analysis
a. Determination of Foreign Matter: One gram of sample was weighed and foreign matter was carefully
separated. The matter differing in colour and texture were considered as foreign. The separated matter was
weighed and subtracted from one gram and percentage was calculated.
b. Determination of Moisture Content: One gram of powder was weighed and dried at 80C for 24 hrs in hot air
oven. After 24 hrs, the powder was weighed again and the difference in the weight was determined. The
percentage of moisture was calculated.
c. Determination of pH: The 5% (w/v) (5 g in 100 ml of water) powder was kept on shaker for 5 h with 140
rpm and filtered. The filtrate was analysed for the pH using pH meter (Elico, India) (Iqbal et al. 2010).
d. Determination of Water Soluble Extractive: Five Grams of powder was weighed and added into a 100 ml
conical flask, 25 ml of distilled water is added into to it and kept on a rotator shaker (140 rpm) for 24 hrs. After
24 hrs it was filtered and dried in hot air oven at 80C for 24 hrs and weighed again. The difference in weight
was determined and percentage of water soluble extractive were calculated (Gupta 2003).
e. Determination of Alcohol Soluble Extractive: Five grams of powdered material is taken in a 100 ml conical
flask, 25 ml of absolute alcohol is added to it and kept on rotator shaker at 140 rpm for 24 hrs. After 24 hrs it
was filtered and dried in hot air oven set at 80C for 24 hrs and weighed again. The difference in weight was
determined and percentage of Alcohol soluble extractive were calculated (Gupta 2003).

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Kodiyalmath & Krishnappa (2017) 4(2): 351357

f. Determination of Total Ash Content: The clean and dried silica crucible was weighed, 10 g of powder was
taken and kept in muffle furnace and heated up to 300C for 34 hrs until the whole powder turns into ash. The
crucible was cooled and weighed again. The difference in the weight was calculated. (Gupta 2003, Indrayan et
al. 2005).
g. Determination of Water Soluble Ash: 1 g of powder was weighed and 10 ml of distilled water is added into
it. The mixture was kept on a shaker with 140 rpm for 8h and filtered through ash less filter paper. The ash
remained in the paper was kept in a crucible (silica) and burnt ash again in a muffle furnace for 34 hrs. The
weight of ash obtained was noted and percent of water soluble ash was determined (Ahmad & Sharma 2001).
h. Determination of Acid Insoluble Ash: 1gm of ash was weighed and 10 ml of distilled water is added into it.
The mixture was kept on a shaker with 140rpm for 8 h and faltered through ash less filter paper. The ash
remained in the paper was kept in a crucible (Silica) and burnt to ash again in a muffle furnace for 34 hrs. The
weight of ash obtained was noted and percentage of acid insoluble ash was determined. (Ahmad & Sharma
2001).

RESULTS AND DISCUSSION


Table 1. Result of physicochemical analysis of extract.
Physicochemical Parameters %
Foreign Matters 0.80
Moisture Content 7.30
Water Soluble Extractive 8.88
Alcohol Soluble Extractive 3.20
PH 6.64
Determination Of Ash 21.60
Water Soluble Ash 85.00
Acid Insoluble Ash 21.00
Physicochemical analysis showed 0.8% of foreign matter, 7.3% of moisture content 8.88% of water soluble
extractive, 3.2% of alcohol soluble extractive; pH is 6.4, 21.6% of determination of ash, 85% of water soluble
ash and 21% of acid insoluble ash (Table 1). The extracts were tested for presence of secondary metabolite.
Petroleum ether showed the presence of alkaloids and tannins. Chloroform extracts showed positive to alkaloids,
tannins, steroids, glycosides and phenols. Methanol showed the presence of flavonoids, glycosides triterpenoids
and phenols (Table 2).
Table 2. Phytochemicals test of Phellinus linteus (Berk. & M.A Curtis.).
Tests Pet, ether Chloroform Methanol
Alkoloids + + +
Saponins _ _ _
Tannins + + +
Flavonoids _ _ +
Steroids _ + +
Glycosides _ + +
Triterpenoids _ _ +
Phenols _ + +
The extracts showed varied antimicrobial result when tested against pathogens tables 2. The petroleum ether
extract inhibited Salmonella typhi and Staphylococcus aureus (12 mm) at maximum. Agrobacterium
tumefaciens, Klebsiella pneumonia, Pseudomonas aeruginosa were (10 mm) moderately inhibited.
Xanthomonas campestris, Pseudomonas syringae, Escherichia coli, Streptomyces pneumoneae were (80) least
inhibited by petroleum ether extracts. The fungal pathogens like Trichophyton rubrum, Fusarium solani,
Aspergillus flavus and Aspergillus niger were completely resistant showing no inhibition whereas
Chrysosporium keratinophilum was inhibited maximum that of other test organism. Candida albicans and
Chrysosporium merdarium were inhibited moderately.
Chloroform extracts inhibited test organisms better than petroleum ether. K. pneumonia and S. aureus were
inhibited maximum by showing 16 and 17 mm zone. The chloroform extract inhibited P. syringae, E. coli, P.
aeruginosa, S. pneumoneae, A. tumefaciens and S. typhi moderately by showing 1314 mm inhibition zone.
Whereas X. campestris were least inhibited with 8 mm (Table 3, Fig. 1). F. solani (22 mm) had the maximum

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effect of chloroform extract followed by A. niger and C. keratinophilum (20 mm). C. merdarium, T. rubrum and
P. chrysogenum were moderately affected with 1819 mm inhibition zone. A. flavus and C. albicans were least
inhibited by chloroform extract (Table 4, Fig. 2).
Table 3. Antibacterial activity of Phellinus linteus (Berk. & M.A Curtis.) at different concentration and different solvent.
Zone of inhibition in mm
Name of Standard
S.N. Petroleum ether Chloroform Methanol
pathogen
100 % 50% 25% 100 % 50% 25% 100 % 50% 25%
1 Xc 080 0 0 080.5 061.0 0 201.0 152.0 091.5 191.0
2 Ps 080 0 0 140.6 102.0 71.0 241.5 161.0 101.5 251.0
3 At 100 0 0 130.5 101.6 81.0 300.5 241.0 151.0 361.0
4 Kp 100 0 0 161.0 100.7 60.8 252.0 201.5 151.0 282.0
5 Ec 090 0 0 151.0 082.0 0 241.5 201.5 181.0 242.0
6 St 120 81 0 130.5 101.4 61.0 242.3 201.3 182.0 261.5
7 Pa 100 0 0 141.0 101.0 81.0 281.2 201.5 102.0 281.0
8 Sa 120 81 0 170.8 130.7 91.5 282.0 191.5 102.0 301.2
9 Sp 080 0 0 140.8 100.5 61.0 221.2 151.3 91.4 251.0
Note: Mean of 3 replicates for each concentration; XC= Xanthomonas campestris, Ps= Pseudomonas syringae, At=
Agrobacterium tumefaciens, Kp= Klebsiella pneumonia, Ec= Escherihia coli, St= Salmonella typhi, Pa= Pseudomonas
aeruginosa, Sa= Staphylococcus aureus, Sp= Streptomyces pneumoneae.
35
Petroleum Ether Chloroform Methanol Standard
Zone of inhibition in mm

30
25
20
15
10
5
0
Ca Cm Tr Ck Fs Pc Af An
Test organisms
Figure 1. Antifungal activity of Phellinus linteus (Berk. & M.A Curtis.) at 100% concentration of different solvent extract.
[Ca= Candida albicans, Cm= Chrysosporium merdarium, Tr= Trichophyton rubrum, Ck= Chrysosporium
keratinophilum, Fs= Fusarium solani, Pc= Penicillium chrysogenum, Af= Aspergillus flavus, An=Aspergillus
niger]
Table 4. Antifungal activity of Phellinus linteus (Berk. & M.A Curtis.) at different concentration and different solvent.
Zone of inhibition in mm
Name of Standard
S.N. Petroleum ether Chloroform Methanol
pathogens
100% 50% 25% 100% 50% 25% 100% 50% 25%
1 Ca 111.6 102.0 081.0 160.0 120.5 100.6 291.5 222.0 191.5 302.3
2 Cm 100.0 081.0 0 181.0 100.5 080.5 251.5 191.5 151.0 301.0
3 Tr 0 0 0 191.2 161.0 140.5 211.0 191.2 140.4 231.2
4 Ck 141.0 080.5 0 201.3 130.5 060.0 282.0 252.2 201.7 280.5
5 Fs 0 0 0 221.8 191.8 151.0 231.2 201.7 151.0 251.4
6 Pc 082.0 060.5 0 190.4 140.5 090.7 201.0 151.0 110.2 291.2
7 Af 0 0 0 161.0 141.5 120.6 250.5 202.0 151.0 251.0
8 An 0 0 0 202.2 140.5 080.5 200.5 150.8 100.4 221.1
Note: Mean of 3 replicates for each concentration; Ca= Candida albicans, Cm= Chrysosporium merdarium, Tr= Trichophyton rubrum,
Ck= Chrysosporium keratinophilum, Fs= Fusarium solani, Pc= Penicillium chrysogenum, Af= Aspergillus flavus, An= Aspergillus niger.
The GCMS analysis of methanol extract showed the presence of ergosterol. This agrees with the result of
(Reis et al. 2014) which is anti-tumor component (Chen et al. 2016). A. tumefaciens S. aureus and P.
aeruginosa showed more susceptibility to methanol extract followed by K. pneumonia, S. typhi, P. syringae,
whereas X. campestris was resistant to methanol extract. C. keratinophilum and C. albicans were maximum
inhibited by methanol extracts, almost equal to inhibition zone of standard drug. C. merdarium, F. solani, A.
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flavus showed less susceptibility than the C. keratinophilum and C. albicans but T. rubrum, P. chrysogenum and
A. niger were least inhibited by methanol extract compared to another test organism (Table 3 and 4). The
relative decrease in the inhibition with the concentration of the extract shows that amount of bioactive
components in extracts play an important role in inhibition of pathogens. The number of organisms inhibited
decrease with the decrease in concentration of extracts. The observed result of the effectiveness of extracts,
methanol proved best and petroleum ether showed less inhibition effect which is favourable with the findings of
(Ehssan & Saadabi 2012). S. aureus and E. coli are inhibited maximum by methanol extract this agrees with
the result of (Bala et al. 2011). Sonawane et al. (2012) reported methanol showed better activity than ethyl
acetate, in present work methanol showed good result than chloroform. Antifungal activity of extracts was lower
compared to bacteria this agrees with the result of Jonathan & Fasidi (2003, 2005). Oyetayo (2009) studied the
effect of ethanol extracts of Termitomyces sp. and Lentinus sp. against C. albicans and S. aureus, ethanol extract
showed good inhibition against C. albicans but S. aureus was inhibited hardly by 8 mm inhibition zone.
Methanol extract showed good inhibition zone for E. coli, S. typhi, P. aeruginosa and S. aureus, this agrees with
the result of (Balakumar 2010). When (Iftekhar et al. 2011) worked on the antibacterial activity of Ganoderma
lucidum, Auricularia auricula and Pleurotus florida against S. aureus and E. coli none of the three mushroom
inhibited E. coli but in present study E. coli is inhibited in all the concentration by all solvent extracts.
40
Petroleum ether Chloroform Methanol Standard
35
Zone of ihibition in mm

30
25
20
15
10
5
0
Xc Ps At Kp Ec St Pa Sa Sp
Test organisms
Figure 1. Antibacterial activity of Phellinus linteus (Berk. & M.A Curtis.) at 100% concentration of different solvent
extract. [Xc=Xanthomonas campestris, Ps=Pseudomonas syringae, At=Agrobacterium tumefaciens, Kp= Klebsiella
pneumonia, Ec=Escherihia coli, St=Salmonella typhi, Pa=Pseudomonas aeruginosa, Sa= Staphylococcus aureus,
Sp=Streptomyces pneumoneae]

CONCLUSION
Resistance to a wide variety of antibiotics by pathogens and multiple drug resistant organisms has become a
serious threat to growing population, there is a need to find new source of antibiotics which are capable of
combating several disease causing pathogens. Mushrooms are such a group of organisms which are rich in
useful metabolites so further identification and isolation of different active compounds from varieties of
mushroom and commercializing their products is necessary. The finding of present work reveals that Phellinus
linteus (Berk. & M.A. Curtis) proves its powerful antimicrobial activity. Candida albicans and Staphylococcus
aureus are significantly inhibited by chloroform and methanol extracts. Further work is required to culture
mycelia and extract metabolites from it so that there is no need to wait for the appropriate season for harvesting
fruiting bodies, for the extraction of secondary metabolites and other bioprospecting activities.

ACKNOWLEDGEMENTS
The authors are grateful to chairman department of Applied Botany Shankaraghatta for providing lab
facilities. They are also indebted to Dr. Syed Abrar, Guest Lecturer Department of Applied Botany, Kuvempu
University for identification of the species. We are also thankful to research scholars, Nandan Patel K.J., Vinu
K. and Gourish K.C. from Department of Applied Botany, Kuvempu University notably for his help and
support.

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ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(2): 358362, 2017
DOI: 10.22271/tpr.2017.v4.i2.047
Research article

Morpho-anatomical description of Lygodium hazaricum Haq a


new contribution to the fern flora of Pakistan
Faizul Haq
Department of Botany, Government Degree College, Battagram, Khyber Pakhtunkhwa, Pakistan
*Corresponding Author: faizulhaq80@yahoo.com [Accepted: 29 August 2017]

Abstract: Lygodium hazaricum a climbing fern was collected from Nandiar valley western
Himalayas during 2016. The rhizome is widely creeping, dichotomously branched and densely
clothed with dark brown hairs. The epidermal layer of rhizome is covered with dark brown hairs,
which is sheeting at base and acute at tip. Roots arise opposite to fronds and root branches are
bulbous. Rachis is with solid oval protostele. The cells of phloem are smaller than xylem cells.
The sporangia are 600 m long and 500 m wide, globular in cross section. Sporangium is very
short stalked and placed horizontally. The mature sporangium has upto 256 spores. Spores are
subtriangular. Sporangium also contains few red spores. L. hazaricum is used for the treatment of
diarrhea, dysentery and hepatitis. It is a new contribution to the fern flora of Pakistan.
Keywords: L. hazaricum Haq - Morphology - Anatomy - Sporangia - Spore - Nandiar valley.

[Cite as: Haq F (2017) Morpho-anatomical description of Lygodium hazaricum. Haq a new contribution to the
fern flora of Pakistan. Tropical Plant Research 4(2): 358362]
INTRODUCTION
The genus Lygodium Sw. is placed in family Lygodiaceae comprises 45 species throughout the world.
Previously it was placed in family Schizaeceae (Murtaza et al. 2004). The plants of Lygodiaceae family are
terrestrial (Lott et al. 2003). Stems slender; branched, fronds several meters, alternately pinnate, climbing by
means of a twining rachis; primary blade divisions (pinnae) pseudo dichotomously forking with a dormant
apical bud in axils; pinnules entire to palmately or 1- or 2-pinnate or more divided; fertile and sterile pinnae
similar or fertile pinnae greatly contracted; veins free or anastomosing; sori on lobes of ultimate segments;
sporangia abaxial, solitary, 1 per sorus, each sporangium covered by an antrorse indusium-like subtending
flange; spores 128256 per sporangium, tetrahedral and trilete; gametophytes green, cordate, terrestrial. x = 29,
30 (Zhang & Hanks 2013). The species of Lygodium occur from tropical to temperate region of the world
(Schmitz et al. 1997, Pemberton 1998, Pemberton & Ferriter 1998, Murtaza et al. 2004).
In Pakistan there is no detailed study of fern flora. Stewart (1957) and Sheikh (1962) described certain fern
species from Kaghan Valley. In Pakistan only Lygodium japonicum (Thunb.) Sw. has been reported from
Kashmir and Mansehra districts (Stewart 1957, Sheikh 1962, Murtaza et al. 2004).The morphology, anatomy
and palynology of few ferns have been investigated (Murtaza et al. 2004, 2008).The current study is an effort to
add some information about the Lygodium hazaricum Haq collected from Nandiar valley district Battagram.

MATERIALS AND METHODS


The specimens of Lygodium hazaricum Haq were collected from Nandiar valley district Battagram during
2016. The material was preserved in acetic alcohol (1:3) till further use. The plant morphology was studied with
naked eye and USB digital microscope. For anatomical studies, the transverse sections of the, root, rhizome and
rachis were prepared. The clearing of leaflets/fronds was made according to the techniques used by Bhutta &
Sadiq (1987). The fronds were washed with distilled water. The sporangia were detached from the fertile fronds
with the help of needle under binocular microscope. For spore investigations, the sori were crushed with glass
rod and passed through 4 cm diameter funnel placed in a centrifuge tube and plucked with loosely placed glass
wool. The sieved spores were treated with 5% KOH solution to remove the oils and humic acid. The spores
were chlorinated as suggested by Bhutta & Sadiq (1987) and mounted in 2% glycerin jelly already stained with

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Received: 20 May 2017 Published online: 31 August 2017
https://doi.org/10.22271/tpr.2017.v4.i2.047
Haq (2017) 4(2): 358362

2% safranin (Erdtman & Sorsa 1971). The specimen were compared with the available literature (Stewart 1957,
Sheikh 1962, Murtaza et al. 2004, 2008, Zhang & Hanks 2013). The specimens were deposited in the
department of Botany, government degree college Battagram for future reference.

RESULTS AND DISCUSSION

Figure 1. Morphology of Lygodium hazaricum Haq: A, Full plant; B, Rhizome and rhizoid; C, Sporangia on leaf margin.
Morphology
Dark brown rhizome, 36 mm in diameter, widely creeping, dichotomously branched, densely clothed with
dark brown hairs. Roots arises opposite to fronds 38 mm apart, root branches bulbous and flattened 25 mm
apart, 612 mm long. Juvenile fronds erect, 310 mm apart, first branching an unequal dichotomy, main
branches of large fronds bipinnate, deltoid in outline, with palmatisect pinnae, pinna margins doubly serrate.
Rachis of climbing fronds 12 mm in diameter, upto 60 feet, slightly oval in outline, glabrous, hairs present on
flattened adaxial surface between wings; primary rachis branches 311 mm, dormant apex covered with pale
hairs; secondary branches of fronds on young or stunted plants pinnate, on well-grown fronds bipinnate or
tripinnate, deltoid in outline, commonly ca. 14 cm long and wide, rachises densely shortly hairy on adaxial
surface and with fewer longer hairs elsewhere; sterile tertiary pinnae of lower rachis branches., palmate with 3
7 lobes, middle lobe much longer than lateral lobes, tertiary pinnae higher up frond 3-lobed with an elongate
middle lobe or pinnate with small oblique and often lobed quaternary pinnae and a usually deltoid-pinnatisect

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Haq (2017) 4(2): 358362

terminal pinna ca. 3 cm, margins acutely biserrate, apex obtuse or subacute; stalks of pinnae up to 3 mm, never
articulate or thickened at apex; costae with long scattered hairs, veins and surfaces usually glabrous but
sometimes shortly hairy; fertile secondary branches tripinnate, pinnae smaller than sterile pinnae, sorophores 2
12 mm; indusia glabrous or with few hairs if lamina hairy (Fig. 1). Similar morphological characters were also
presented by Haq (2015) from Battagram district.

Figure 2. Anatomy of Lygodium hazaricum Haq: A, Rachis; BC, Rhizome; D, Root; E, Sporangium; F, Spores.
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Haq (2017) 4(2): 358362

Anatomy of rachis
The rachis is slightly oval. The cutinized epidermis was single layered, slightly hairy in young fronds. The
cells of the epidermis were broader than longer. Hypodermis consisted of 36 layers. Protostele, solid, almost
oval, covered by endodermis and pericycle. The cells of the phloem were smaller than xylem cells. The phloem
is protruded into bays formed by three angled protoxylum and protostele. The cortical region is parenchymatous
(Fig. 2A). Murtaza et al. (2004) also reported the anatomical structure of L. japonicum.
Anatomy of rhizome
The rhizome was dark brown, almost circular in outline. The epidermal layer was covered with dark brown
hairs, hair base was sheeting and with acute tip. Below epidermis there was a cortical region. The cortical layers
were differentiated with outer whitish parenchymatous tissue and inner brown collenchymatous tissue. Inner to
the cortex was distinct endodermis followed by 13 pericycle layers. Protostele was solid, almost rounded. The
cells of the phloem were smaller than xylem cells. The traces of rachis and root were almost opposite (Fig.
2B&C).
Anatomy of root
The root was rounded and dark brown in color. The outer most layer was damaged epidermis. The cortex
was differentiated into outer larger cells of 23 layers and inner smaller cells of brown in color of 57 layers.
Inner to the cortex was endodermis of usually 6 cells with unequal size. It was followed by a single layer
pericycle. The xylem was surrounded by phloem. The xylem cells are larger than phloem cells (Fig. 2D).
Sporangium
The sporangia was 600 m long and 500 m wide, globular in cross section. Sporangia was seated near each
vein dichotomy and was protected by the curled margin (Fig. 2E). Sporangium was very short stalked and was
placed horizontally. The annulus was clearly shown and having 65um thickness in cross section (Fig. 2E). The
mature sporangium has upto 256 spores. The sporangium morphology of similar fern Schizaea dichotoma was
also presented by Murtaza et al. (2008).
Spores
Spores were subtriangular, 6090 m equatorial diameter, isosporous, coatour anisomorphic, trilete, laesurea
almost to the length of the spore radius, commissure open, extremities of arm slightly arculate, proximal surface
slightly reticulate, distal surface heavily warty at equatorial line and variable in dimensions. At certain points
they are oriented in reticulate pattern. Outer layer was thick exine and inner was thin intine. Few red spores were
also seen in the same sporangium (Fig. 2F). Lott et al. (2003) also reported the reproductive biology of
Lygodium microphyllum (Cav.) R. Br. and L. japonicum and reported similar result.
Uses of the plant
The powder of whole plant is used for the treatment of diarrhea, dysentery and hepatitis. Similar results were
also presented by Haq et al. (2011) from same area.

CONCLUSION
Lygodium hazaricum Haq is a premative fern having protostele stem, dichotomous brancheed venation of
open type as these character are quite primative. I concluded from morphology, anatomy of rachis, rhizome,
root, sporangia and spore are the character that it was differentiated as a new species to the fern flora of
Pakistan. It is recommended that a complete biochemical investigation and genotype should be studied for
clarifification of its position in classification.

ACKNOWLEDGEMENTS
The author is thankful to the Head of Department and all other staff of Department of Botany, Government
Degree College, Battagram, Khyber Pakhtunkhwa, Pakistan for their valuable support during the study.

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