Volume 2, Issue 3 of Tropical Plant Research

www.tropicalplantresearch.com 168 Received: 16 June 2015 Published online: 31 October 2015

ISSN (E): 2349 – 1183

ISSN (P): 2349 – 9265

2(3): 168–171, 2015

Research article

Morpho-taxonomy of Hydrodictyon reticulatum (L.) Lagerheim and

Pediastrum tetras var. tetraodon (Corda) Hansgirg,

Hooghly, West Bengal, India

Nilu Halder*

Department of Botany, Raja Peary Mohan College, Uttarpara-712258, Hooghly, West Bengal, India *Corresponding Author: [email protected] [Accepted: 25 September 2015]

Abstract: The present paper was communicated with the morpho-taxonomic descriptions of two

freshwater members viz. Hydrodictyon reticulatum (L.) Lagerheim and Pediastrum tetras var.

tetraodon (Corda) Hansgirg belonging to the family Hydrodictyaceae of the class Chlorophyceae.

The two taxa were collected from aquatic ecosystems in Hooghly district, West Bengal, India. The

limnological characteristics of the water bodies where they occurred were recorded. The above

stated taxa were new taxonomic reports from this district and Pediastrum tetras var. tetraodon

(Corda) Hansgirg was new report from West Bengal, India.

Keywords: Morpho-taxonomy - New report - Hydrodictyaceae - Limnology - West Bengal

[Cite as: Halder N (2015) Morpho-taxonomy of Hydrodictyon reticulatum (L.) Lagerheim and Pediastrum

tetras var. tetraodon (Corda) Hansgirg, Hooghly, West Bengal, India. Tropical Plant Research 2(3): 168–171]

INTRODUCTION

Algae are photoautotroph, replenish oxygen content in water, dominant primary producer and contribute

much to the productivity of freshwater ecosystems. On the other hand, physico-chemical parameters affect the

composition and diversity of algal flora in aquatic bodies. Therefore, morpho-taxonomic study for

documentation of algae and assessment of water quality are of utmost importance in taxonomic and ecological

investigation.

Hydrodictyon reticulatum known as "water net" is widely distributed in Asia, North Africa, Europe, America

and New Zealand. This alga is characteristic of the northern warm-temperate zone (Pocock 1960). It is a

macroscopic, free-floating and filamentous green alga that forms a closed, pentagonal or hexagonal net of

coenocytic cells in water bodies. At maturity, the alga turns into slightly yellowish colour and the coenocytic

cells are detached from each other or the coenobium is disintegrated and can form vegetative daughter nets.

Whereas, the genus Pediastrum was established by Meyen in 1829 and it is an interesting coenobial

chlorococcalean algal genus which grows commonly as planktonic form or in periphytic association (grows on

submerged plants or other water logged objects) of freshwater habitats. This is a microscopic green alga and

occurs commonly in natural freshwater bodies like ponds, lakes, moats, rivers and other aquatic reservoirs. This

alga was found generally in post monsoon season in the district Hooghly, West Bengal. It should be

mentionable that after the publication of the monograph "Chlorococcales" by Philipose (1967), several authors

added many algal taxa in the order Chlorococcales from the Indian sub-continent. Singh (1973), Patel & George

(1982), Pal & Santra (1984), Sharma et al. (1985), Pal et al. (1986), Banerjee & Santra (2001), Jena & Adhikary

(2007), Mallick & Keshri (2008, 2009) and Sau & Gupta (2008), Kumar et al. (2012), Rai & Misra (2012) and

Keshri & Mallick (2013) were some contributors who had worked earlier on the taxonomy of these algae.

MATERIALS AND METHODS

The algae had been collected in glass containers from different places viz. Tribeni (N 22°99' E 88°40'),

Chinsurah (N 22°90' E 88°39'), Khamargachi (N 23°05' E 88°43'), Dumurdaha (N 23°03' E 88°43'), Magra (N

23°12' E 88°28'), Kamarkundu (N 23°83' E 88°20') and Hooghly river at Kalichar Ghat (N 23°03' E 88°26') of

Hooghly district, West Bengal. Detailed study was made by examining specimens under Olympus microscope

(Model-CH20i) for identification of species. Samples were preserved in 4% formalin. Identifications of these

Halder (2015) 2(3): 168–171

.

www.tropicalplantresearch.com 169

taxa were accomplished with the help of authentic literatures viz. Phlipose (1967), Singh (1973), Coffey& Miller

(1988), Sharma et al. (1985), Kumar et al. (2012) and Rai & Misra (2012).The pH and temperature of the water

bodies were determined at the site immediately after collection with the help of portable pH meter (Model No.

PP9046 Philips, India) and Zeal’s mercury thermometers (UK).The other limnological parameters such as

nitrate-nitrogen (NO3-N), phosphate (PO43-), dissolved oxygen (DO), biochemical oxygen demand (BOD),

chemical oxygen demand (COD), total suspended solids (TDS)and sulphate (SO42-) of waters were estimated by

UV-VIS Spectrophotometry (CECIL CE- 7200) following the standard method (APHA 2005). All the physico-

chemical parameters in ecological notes are expressed in mg/l except pH and temperature.

RESULTS AND DISCUSSION

A total number of two algal species viz. Hydrodictyon reticulatum (L.) Lagerheim & Pediastrum tetras var.

tetraodon (Corda) Hansgirg under the genera Hydrodictyon Roth and Pediastrum Meyen of the family

Hydrodictyaceae belonging to the order Chlorococcales of the class Chlorophyceae were recorded for the first

time from different aquatic ecosystems in Hooghly district of West Bengal, India. Each currently accepted name

has been provided with its author (s) name. They were described below:

Morpho-taxonomic description

1. Hydrodictyon reticulatum (L.) Lagerheim in K. Svenska.Vetenskakad. Förhandl. 40: 71, 1883; Biswas,

Rec. Bot. Surv. India 15 (1): 68. Pl. 3. fig. 29, 1949; Philipose, Chlorococcales 134. fig.48, 1967; Coffey &

Miller, New Zealand Journal of Botany, 26: 319.figs. 2-5, 1988; Anand, Indian freshwater microalgae 32.fig.

90, 1998; Kant & Gupta, Algal Flora of Ladakh 81.pl.19. fig. 6, 1998. (Figs. 1 A-B)

Conferva reticulate L. 1753; Hydrodictyon utriculatum Roth 1800.

Plant macroscopic, grass green; free floating, saccate reticulum, colonial; colonies reticulate; 6 cells adjoined

together end to end walls repeatedly forming hexagonal mesh and whole structure of the alga appears as

cylindrical net; net may vary in size; cells coenocytic, elongate, cylindrical, 51.2–54.8 µm long, 9.1–10.8µm

broad; cell wall smooth, double layered; chloroplast reticulate; pyrenoids many; asexual reproduction by auto

colony formation.

Habitat: Ponds water at Tribeni and Chinsurah; canal water at Khamargachi, moat water at Dumurdaha and

rice fields in Magra.

Collection No: 138, 808; Dated: 20.03.06, 03.01.11

Ecological Notes: Grows as weed & forming net in pond at Tribeni; water temperature: 20ºC; pH: 7.6; NO3-

N: 0.17; PO43-: 0.20; DO: 8.0; BOD: 7.2; COD: 68.0; TDS: 124.0; SO4

2-: 7.0

Significance: Good source of nutrients in rice fields after decomposition; provides shelter to aquatic

zooplanktons and used as food by grass carp fishes when grows in ponds.

Figure 1. A-B, Hydrodictyon reticulatum (L.) Lagerheim; C, Pediastrum tetras var. tetraodon (Corda) Hansgirg

2. Pediastrum tetras var. tetraodon (Corda) Hansgirg in Prodr. Alg. Böhmen 1: 112, 1888; Phlipose,

Chlorococcales pl. 129.figs. 45 d, e, g, 1967; Kumar, Seth and Suseela, Phykos 42 (2): 38, fig. 18, 2012; Rai and

Misra, Our Nature 10: 172-173, fig. 4, 2012. (Fig. 1C)

Euastrum tetraodon Corda 1839.

Planktonic, colonial; colony entire, discoid to slightly rectangular of 8 cells; cells more or less straight;

peripheral cells crenate or angular, outer margins of peripheral cells with deep incision and pronounced

Halder (2015) 2(3): 168–171

.


projections; cell wall smooth; diameter of 8 celled colony 32.0–34.0 µm; inner cell also with straight sides but

one margin deeply incised; lateral margins of peripheral cells adjoined along their length; vegetative cells 7.2–

14.0 µm long, 2.4–13.0 µm broad.

Habitat: Pond water at Kamarkundu, canal water at Khamargachi and Hooghly River water.

Collection No: 349, 1194; Dated: 15.07.06, 25.11.11

Ecological Notes: Planktonic in a canal at Khamargachi; water temperature: 23ºC; pH: 7.5; NO3-N: 0.10;

PO43-: 0.18; DO: 6.6; BOD: 3.8; COD: 90.0; TDS: 72.0; SO4

2-: 6.0

Significance: Primary producer & a component of aquatic food chain in freshwater ecosystems.

Documentation of species and varieties as new records or their recollection from a particular habitat have a

significant importance from the taxonomical point of view in the floristic study of algal flora (Bajpai et al. 2013;

Singh et al. 2014; Srivastava et al. 2014; Halder 2015). In the present study, two freshwater green algal

members of the family Hydrodictyaceae under the class Chlorophyceae had been morpho-taxonomically

described first time from Hooghly district, West Bengal, India. Among them, Pediastrum tetras var. tetraodon

(Corda) Hansgirg was the new report from this state. The results of the analyses of physico-chemical

characteristics of studied water bodies were indicated that water was alkaline (pH: 7.5–7.6), although the range

of pH of different water bodies was reported from 5.5–7.0 in Bankura district by Mallick & Keshri (2009) which

was slightly lower than the present investigation. The values of the different physico-chemical parameters like

NO3-N, PO43-, DO, BOD, COD, TDS and SO4

2- were found within the permissible limit prescribed by WHO

(2011) and favoured algal growth in these studied water bodies. The morpho-taxonomic study and

documentation of algal species or new records will explore algal biodiversity of an area and physico-chemical

characterization of water will reveal water quality status in respect of pollution and species evenness (E) of a

particular aquatic body. Moreover, such kind of work will provide baseline information and sufficient

knowledge for the future studies on algal taxonomy and freshwater ecology.

ACKNOWLEDGEMENTS

The author is grateful to Dr. S. N. Sinha, Dept. of Botany, University of Kalyani, Nadia, West Bengal for

providing opportunity to work under his guidance. The author is also thankful to Dr. R.K. Gupta, BSI, Howrah

for his kind co-operation.

REFERENCES

Anand N (1998) Indian freshwater microalgae. Bishen Singh Mahendra Pal Singh Publishers, Dehra Dun. pp. 1–

200.

APHA (2005) Standard methods for the examination of water and waste water (21sted.). American Public Health

Association, Washington, DC. New York.

Bajpai O, Mishra S, Mohan N, Mohan J & Gupta RK (2013) Physico chemical charecteristics of Lakhna Devi

temple water tank, Lakhna, Bakewar, Etawah, U.P. with reference to Cyanobacterial diversity. International

Journal of Environment 1(1): 20–28.

Banerjee A & Santra SC (2001) Phytoplankton on the rivers of Indian Sunderban Mangrove estuary. Indian

Biologists 33(1): 67–71.

Coffey BT & Miller ST (1988) Hydrodictyon reticulatum L. Lagerheim (Chlorophyta) a new genus record from

New Zealand. New Zealand Journal of Botany 26: 317–320.

Halder N (2015) Two species of Zygnemopsis (Skuja) Transeau from West Bengal, India. Tropical Plant

Research 2(2): 82–84.

Jena M & Adhikary SP (2007) Chlorococcales (Chlorophyceae) of Eastern and North-eastern states of India.

Algae 22(3): 167–183.

Kant S & Gupta P (1998) Algal flora of Ladakh. Scientic publishers, Jodhpur, India. pp.1–341.

Keshri JP and Mallick P (2013) On the occurrence of the genera Pediastrum Meyen & Stauridium (Ehrenberg)

E. Hegewald (Sphaeropleales, Chlorophyta) in West Bengal, India with the description of four new taxa.

Phycological Society of India 43 (2): 9–17.

Kumar R, Seth MK & Suseela MR (2012) Chlorophyceae of district Kangra of Himachal Pradesh. Phycological

Society of India 42 (2): 35–38.

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Mallick P & Keshri JP (2008) New record of Pediastrum Meyen from West Bengal, India. Journal of Applied

BioSciences 34(1): 83–86.

Mallick P & Keshri JP (2009) A study on Chlorococcalean algae and their associate plants in Bankura district,

West Bengal. International Journal of Plant Sciences 4(1): 285–288.

Pal TK&Santra SC (1984) New additions to algal flora of Murshidabad, West Bengal. Phycological Society of

India 23(1&2): 139–141.

Pal TK, Adhya TK & Santra SC (1986) Algal flora of Murshidabad district, West Bengal I. A survey from

Berhampore and adjoining areas. Bulletin of Botanical Society of Bengal 40: 33–43.

Patel RJ & George I (1982) A new variety of Pediastrun- P. integrum Näg.var. undulatum var. nov.

Phycological Society of India 21: 129–130.

Philipose MT (1967) Chlorococcales. Indian Council of Agricultural Research, New Delhi. pp. 1–365.

Pocock MA (1960) Hydrodictyon: a comparative biological study. Journal of South African botany 26: 167–

319.

Rai SK & Misra PK (2012) Taxonomy and diversity of genus Pediastrum Meyen (Chlorophyceae, Algae) in

East Nepal. Our Nature 10: 167–175.

Sau A & Gupta RK (2008) A contribution to some fresh water Chlorococcales of Howrah district, West Bengal,

India. Journal of Economic & Taxonomic Botany 32(1): 186–191.

Sharma SP, Saxsena DN & Agarkar MS (1985) A note on two new species of Pediastrum from Gwalior India.

Phycological Society of India 24: 1–3.

Singh PK (1973) Occurrence of green algae Pithophora sp. and Hydrodyctyon reticulatum as weed in Rice

fields of Cuttack. Phycological Society of India 12 (1-2): 82–85.

Singh A, Tiwari V & Mohan J (2014) Chroococcales in River Ganga at JajmauGhat, Kanpur. Tropical Plant

Research 1(1): 28–30.

Srivastava N, Suseela MR &Toppo K (2014) Fresh water cyanobacteria of Sai River near Lucknow, Uttar

Pradesh. Tropical Plant Research 1(2): 11–16.

WHO (2011) Guidelines for drinking water quality. 4th edition. World Health Organization, Geneva,

Switzerland.

www.tropicalplantresearch.com 172 Received: 25 July 2015 Published online: 31 October 2015

ISSN (E): 2349 – 1183

ISSN (P): 2349 – 9265

2(3): 172–174, 2015

Research article

Effect of red laterite soil and vermicompost on growth and

development of chilli and brinjal grown under polypot conditions

Hruda Ranjan Sahoo, Madhuchhanda Sahoo, Mayeetreyee Baboo and Nibha Gupta*

Division of Plant Pathology and Microbiology, Regional Plant Resource Centre,

Bhubaneswar-751015, Odisha, India

*Corresponding Author: [email protected] [Accepted: 29 September 2015]

Abstract: The effect of vermicompost on growth enhancement and productivity of vegetable

crops like chilli and brinjal grown in polypots was observed under greenhouse conditions and

compared with red laterite soil treated as control. Periodical observations noted at 30 days interval

up to 120 days exhibited increasing order of growth enhancement in both the crop plants with

vermicompost mixed soil. Although the effect of vermicompost on growth of chilli could not be

significant; its impact on plant productivity is quite evident. However, significant difference could

be observed in Brinjal plants grown in both the normal soil and vermicompost treated soil.

Enhancement in growth parameters such as leaf area, fresh weight and shoot fresh weight was

noticed but no direct effect of vermicompost in fruit productivity and total weight was observed.

Keywords: Vegetable crops - Growth parameters - Productivity - Vermicompost

[Cite as: Sahoo HR, Sahoo M, Baboo M & Gupta N (2015) Effect of red laterite soil and vermicompost on

growth and development of chilli and brinjal grown under polypot conditions. Tropical Plant Research 2(3):

172–174]

INTRODUCTION

Vegetables are important food components of dietary systems as they provide essential nutrients for human

health. However, they require high amounts of nutrients for its luxuriant growth and development. Brinjal and

chilli are widely cultivated vegetable crops. Brinjal (Solanum melongena L.) is most popular vegetable crop

grown in the world and Chilli (Capsicum annuum L.) is also an important vegetable crop with high consumption

rate (Ahmed et al. 2000, Datta et al. 2011). The economic, nutritious and pharmacological significance is

responsible for its high demand. Although India is the largest producer of chilli in the world but lower yield in

terms of area used for cultivation (Bharathi et al. 2004, Khan & Raj 2006). As a result of which large amount of

chemical fertilizer is applied to enhance productivity of vegetable crops. There is enormous use of fertilizers that

has led to major environmental and health concerns due to its deleterious effect on aquatic ecosystem.

Mesophilic processes such as vermicomposting better known as vermiculture biotechnology refers to the

breeding and propagation of earthworms for cost-effective and eco-friendly organic manure (Beffa et al. 1998,

Masciandaro et al. 2000, Aalok et al. 2008, Perera & Nanthakumaran 2015).Vermicompost can be used to

improve soil health and enhance plant growth without causing damage to the environment. Vermicompost plays

a major role in improving growth and yield of different field crops, vegetables, flower and fruit crops such as

sorghum (Patil & Sheelavantar 2000), sunflower (Devi & Agarwal 1998), coriander (Vadiraj et al. 1998), brinjal

(Babu et al. 2010) etc. The present investigation was carried out to observe the effect of vermicompost on

vegetative growth and fruiting of chilli and brinjal under the pot culture conditions.


In order to study the impact of vermicompost on growth of vegetable crops, a pot culture experiment was set

up at the green house in Polybags of size 10×10" capacity containing 5kg of red laterite soil. Only soil was

treated as control whereas test experimental sets were supplemented with vermicompost at the rate of 250g/pot.

Seeds of chilli and Brinjal were sown into polypots containing control soil and soil supplemented with

vermicompost. The pots were maintained in the green house at an adequate temperature and water was supplied

Sahoo et al. (2015) 2(3): 172–174

.


daily to maintain the moisture level of the soil. Data on growth parameters of both the vegetable crops was

recorded for Plant height, fruiting pattern and biomass periodically. The fruit development and harvest was

recorded after 120 days.


Data recorded on leaf number and plant height of chilli is presented in figure 1. Periodical observations

noted at 30 days interval up to 120 days exhibited the increasing order of growth enhancement. Vermicompost

treated plants showed better growth than simple soil. 38.9% enhancement in shoot height was observed at 90

days of growth in vermicompost added plants of chilli. Data recorded on fruit harvest of chilli at 120 days has

been depicted in figure 2. Effect of vermicompost on plant growth could not be significant; its impact on plant

Figure 1. Effect of Vermicompost on Chilli: A, Leaf number; B, Shoot height.

Figure 2. Effect of vermicompost on Chilli fruit at 120 days: A, Number, weight & length; B, Total weight.

productivity is quite evident. Four times more number of fruits and their weight was observed in vermicompost

treated plants as compared to the untreated plants in first harvest. Fruit length and weight per fruit was also

observed in the chilli plants grown in vermicompost added soil. Second harvest of the vegetable product showed

similar pattern of growth enhancement in plants under treated condition. Data recorded on growth of Brinjal has

been presented in figure 3. Very significant difference could be observed in plants of both the normal soil and

Figure 3. Effect of Vermicompost on Brinjal: A, Leaf number; B, Shoot height.

Sahoo et al. (2015) 2(3): 172–174

.


vermicompost treated soil. The growth performance of brinjal under polypot conditions was influenced by the

vermicompost treatment. Enhancement in leaf area, fresh weight and shoot fresh weight as presented in figure 4

is clearly evident from the present study. No direct effect of vermicompost in fruit productivity and total weight

Figure 4. Effect of Vermicompost on Brinjal: A, Leaf area; B, Leaf fresh weight; C, Shoot fresh weight.

was observed. However, (40.37±20.69) and (51.83±18.53) g/fruit was observed in normal soil and

vermicompost treated soil, respectively. The effect of vermicompost has been clearly demonstrated in the

present study. To formularize the general usage of vermicompost for these crops more in depth study is required

as crop productivity and improvement is also dependent upon agro climatic zones and its microhabitats.

ACKNOWLEDGEMENTS

The financial assistance obtained through Forest and Environment Department, Govt. of Odisha (State Plan

Project 2014-15) and INSPIRE programme (No. DST/INSPIRE Fellowship/2013/506) DST, Govt. of India is

gratefully acknowledged.

REFERENCES

Aalok A, Tripathi AK & Soni P (2008) Vermicomposting: A better option for organic solid waste management.

Journal of Human Ecology 24: 59–64.

Ahmed J, Shivhare US & Raghavan GSV (2000) Rheological characteristics and kinetics of colour degradations

of green chilli puree. Journal of Food Engineering 44: 239–244.

Babu NS, Panicker S & Clarson D (2010) The effect of Vermicompost with different microbial fertilizers on the

growth and yield of Brinjal. Baselius Researcher 11(2): 168–177.

Beffa T, Staib F, Fisher JL, Lyon PF, Gumowski P, Marfenina OE, Dunoyergeindre S, Georgen F, Roch-Susuki

R, Gallaz L & Latge P (1998) Mycological control and surveillance of biological waste and compost.

Medical Mycology 36:137–145.

Bharathi R, Vivekananthan R, Harish S, Ramanathan A & Samiyappan R (2004) Rhizobacteria-based

bioformulations for the management of fruit rot infection in chillies. Crop protection 23: 835–843.

Datta M, Palit R, Sengupta C, Pandit MK & Banerjee S (2011) Plant growth promoting rhizobacteria enhance

growth and yield of chilli (Capsicum annuum L.) under field conditions. Australian Journal of Crop Science

5(5): 531–536.

Devi D & Agarwal SK (1998) Performance of sunflower hybrids as influenced by organic manure and fertilizer.

Journal of Oilseeds Research 15(2): 272–279.

Khan MS & Raj SK (2006) First report of molecular detection of an Aster yellows phytoplasma (‘Candidatus

Phytoplasma asteris’) isolate infecting chilli (Capsicum annum) in India. Plant Pathology 5: 822.

Masciandaro G, Ceccanti B & Garcia C (2000) ‘‘In situ’’ vermicomposting of biological sludges and impacts on

soil quality. Soil Biology and Biochemistry 32: 1015–1024.

Patil SL & Sheelavantar MN (2000) Effect of moisture conservation practices, organic sources and nitrogen

levels on yield, water use and root development of rabi sorghum [Sorghum bicolor (L.)] in the vertisols of

semiarid tropics. Annals of Agricultural Research 21(21): 32–36.

Perera KIM & Nanthakumaran A (2015) Technical feasibility and effectiveness of vermicomposting at

Household level. Tropical Plant Research 2(1): 51–57.

Vadiraj BA, Siddagangaiah D & Potty SN (1998) Response of coriander (Coriandrum sativum L.) cultivars to

graded levels of vermicompost. Journal of Spices and Aromatic Crops 7(2): 141–143.


ISSN (E): 2349 – 1183

ISSN (P): 2349 – 9265

2(3): 175–179, 2015

Research article

Ethnopharmacological investigation and antibacterial evaluation of

the methanolic extract of Asparagus racemosus (Shatavari)

T. Kumaran1, 2

* and T. Citarasu1

1Centre for Marine Science and Technology, Manonmaniam sundaranar University, Rajakkamangalam,

Kanyakumari, Tamilnadu 2PG and Research Department of Zoology, Muslim Arts College, Thiruvithancode, Kanyakumari, Tamilnadu

*Corresponding Author: [email protected] [Accepted: 02 October 2015]

Abstract: Plants are the base of sophisticated traditional medicine systems including Ayurvedic,

Unani and Siddha. Phytochemical analysis of Asparagus racemosus revealed that numerous

compounds of plants traditionally used for medicinal purposes have several therapeutical

properties. The result of the phytochemical studies revealed the presence of saponins, tannins,

alkaloids, steroids and other biochemicals. Saponins, tannins and alkaloids are well known for

their antibacterial properties. Experiments demonstrating the pharmacological properties of

saponins have aroused considerable clinical interest in these substances. The concentrations of the

plant used were 25 mg ml-1, 50 mg ml-1 and 100 mg ml-1 respectively. At these concentrations, the

extract inhibited the growth of Escherichia coli, Pseudomonas aeruginosa and Vibrio

parahaemolyticus and produced percentage inhibition ranging from 72.4–86.5 %. The anti-

bacterial activity demonstrated by the plant extract may due to the presence of the phytochemicals

present in the plant.

Keywords: Asparagus racemosus - Phytochemical - Saponins - Antibacterial - Pharmacology

[Cite as: Kumaran T & Citarasu T (2015) Ethnopharmacological investigation and antibacterial evaluation of

the methanolic extract of Asparagus racemosus (Shatavari). Tropical Plant Research 2(3): 175–179]

INTRODUCTION

Herbal medicine is a powerful method of disease treatment. Western drugs are usually used to control

symptoms, but do not alter the disease process Citarasu (2010). Many infectious diseases have been known to

be treated with herbal remedies throughout the history of mankind. Natural products, either as pure compounds

or as standardized plant extracts, provide unlimited opportunities for new drug leads because of the unmatched

availability of chemical diversity Harbone (1973). There is a continuous and urgent need to discover new

antimicrobial compounds with diverse chemical structures and novel mechanisms of action for new and re-

emerging infectious disease.

Plants have formed the base of sophisticated traditional medicine systems that have been in existence for

thousands of years and continue to provide mankind with new remedies. Green plants are the indispensable

storehouse of many chemical metabolites which are grouped into two categories namely: primary and secondary

metabolites. Secondary metabolites are the substances produced by plants as defence chemicals. They include

alkaloids, flavonoids, essential oils, phenols, saponins etc. In India, different regions have specific features

according to the climatic conditions (Kumaran & Citarasu 2015). These plants including medicinal plants are

also used as a feeding for animals. They are indirectly shown by their effects by which animals do not suffer by

any types of diseases. Growing plants are one of the cheapest sources of feeding for animals having crude

proteins of 14–25% (Babu et al. 2011).

Saponins are secondary metabolites and play a role in the protection of plants against microorganism. Many

saponins show strong antibacterial activities. As saponins are probably a part of plants’ defence systems, they

have been included in a group of protective molecules in plants called phyto-protectant (Francis et al. 2002).

Saponins are used antioxidant, antimicrobial, and anti-inflammatory etc. according to medical field. It is a

Kumaran & Citarasu (2015) 2(3): 175–179

.


bioactive antibacterial agent of plants (Yoshiki 1998). The present study was designed to evaluate the

fundamental phytochemical constituents and antimicrobial activities of the Asparagus racemosus (Shatavari).


Collection and Extraction

Asparagus racemosus Willd. (Shatavari) roots were purchased from the commercial market at Nagercoil,

kanayakumari district, Tamilnadu, India. Dried powder plant materials were boiled at above 100°C with two

hour. After filtered the extracts, the supernatant was collected and the residue were discarded. The supernatant

was condensed in the water bath and the condensate was extracted again by methanol. The methanolic extract

was concentrated in rotatory evaporator under reduced pressure at the room temperature of 45°C to 50°C in

order to avoid the evaporation of plant materials. Aqueous extract was concentrated using Lyophilizer and

stored at 4°C as described by Andrea et al. (2012).

Phytochemical screening

The Phytochemical screening was determined by the method (Sofowora 1993; Trease 1989). This screening

was carried out with the methanolic extracts using chemical methods and thin-layer chromatography (TLC) as

per standard protocol (Wagner & Bladt 1996).

Saponin Estimation Procedure

Weigh accurately 1.5 to 2 gm of the material in a beaker add 50 ml of petroleum ether and gently heat to

40°C on a water bath for 5 minutes with regular shaking. Filter the petroleum ether repeat the operation with

further 2×50 ml of petroleum ether. Discard petroleum ether and preserve the marc. Extract the marc obtained in

the previous test with 4×60 ml of methanol with mild heating. Filter the methanol layer to another beaker.

Concentrate the combined methanol layer to about 25 ml. Add 150 ml of dry acetone to precipitate the saponins.

Filter the saponins through a filter paper and dry at 100°C for constant weight.

Calculation

Bioautography

A TLC Bioautographic method was used to detect active components. After application of the extract on a

silica gel plate, thin layer chromatography (TLC) was developed using ethylacetate : methanol (9:1) as the

eluent system for Asparagus racemosus. Observe the bands- the TLC plates were dried for complete removal of

solvents. Then the fractions of TLC were spotted on already swabbed agar plates by bioautography method to

evaluate the activity of the different essential compounds, and the plates were incubated at 35ºC for 24 hours.

The activity of compound can detect by its zone formation.

Antibacterial Screening

Test Organisms

The test organisms were standard laboratory strains of Escherichia coli, Pseudomonas aeruginosa and

Vibrio parahaemolyticus. The organisms were obtained from the Department of marine Science (CMST),

Manonmaniam Sundaranar University, Rajakkamangalam, Kanyakumari district, Tamilnadu, India.

Antibacterial activity

Muller-Hinton agar was poured on to sterile Petri plates. When the media solidified, 0.1ml of inoculums

with 0.5 OD was poured over feeder layer and spread evenly with a sterile spreader. A well of 6mm diameter

was made by using a sterile cark borer. Each well-received the extract was tested in a different concentration (25

mg ml-1, 50 mg ml-1 and 100 mg ml-1. Distilled water was used as negative control while ampicillin was used as

positive control. And the commercial antibiotics like as Ampicillin and Tetracycline tested against pathogens.

They were incubated at 37ºC for 24 hours. After incubation, the diameter of the inhibition zone was measured.

RESULTS

Phytochemical Screening

The phytochemical screening of methanolic extracts showed the presence of different types of active

constituents, namely alkaloids, anthraquinones, cardiac glycosides, flavonoids, terpenoids, tannins, saponins,

sterols and triterpenes. These compounds were present in almost all the plants extracts. The details were given in


.


the (Table 1). The total percentage of saponin was estimated from the Asparagus racemosus, and it was found

that 2 g of Asparagus racemosus contains 40% of saponin molecule.

Table 1. Summary of The Results Phytochemical Analysis of Asparagus racemosus.

S.No. Phytochemical group Result

1. Alkaloids +

2. Saponins +

3. Flavonoids +

4. Tannins +

5. Steroids +

6. Terpenoides +

7. Titerpenoides +

8. Anthraquinones +

9. Cardiac glycosides +

Note: +, Present.

TLC Studies on Asparagus racemosus

On TLC analysis for the hot water extract Asparagus racemosus was revealed that, the single spot were

obtained, and it observed under UV-illuminator. The fraction obtained having the Rf values of 0.86 and it shows

on figure 1.

Figure 1. Thin layer Chromatography (TLC) for the steroidal saponin from Asparagus racemosus.

Bioautography

Bioautography method was used to detect active components by its zone formation. The maximum zone of

inhibition is measured in 6.1 mm in dm. The minimum zone of inhibition for the fraction of Asparagus

racemosus is 1.8 mm against Vibrio parahaemolyticus (Table 2).

Table 2. Bioautography of the saponin activities of Asparagus racemosus against some pathogenic bacteria.

S. No. Concentration

(g ml-1

)

Zone of Inhibition in pathogenic bacteria (mm)

Escherichia coli Pseudomonas aeruginosa Vibrio parahaemolyticus

1 1.0 4.2 3.8 4.5

2 2.0 5.9 5.4 6.1

3 3.0 2.7 1.4 1.8

Antimicrobial activity

The antimicrobial activities of the plant extracts against the three bacteria strains examined were assessed by

the presence or absence of inhibition zones. The aqueous extract of Asparagus racemosus exhibited moderate

level antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa and Vibrio parahaemolyticus the

test organisms. Methanol extract of Asparagus racemosus was active against all the test organisms except

Pseudomonas aeruginosa. On the other hand, it was found that the methanol extract of Asparagus racemosus

exhibited high activity against Escherichia coli and Vibrio parahaemolyticus (Fig. 2)

0.86


.


Figure 2. Antibacterial activity Asparagus racemosus against pathogenic microorganism (E. coli

= Escherichia coli; P. aerug = Pseudomonas aeruginosa; V. para = Vibrio parahaemolyticus).

To screen the antibacterial activity against tested organisms, ampicillin and tetracycline were used as a

standard. It was found that tetracycline (5 µg ml-1) standard showed higher activity than ampicillin (30 µg ml-1)

standard against tested microorganisms (Fig. 3).

Figure 3. Antibacterial activity Ampicillin and Tetracycline against pathogenic microorganism.

DISCUSSION

Saponins may beconsidered a part of plants’ defence systems, and as such have been included in a large

group of protective molecules found in plants (Morrissey & Osbourn 1999). The present study focuses on both the

phytochemical analysis and antimicrobial potential of Asparagus racemosus. In the present investigation, different

extracts of Asparagus racemosus was evaluated for exploration of their antimicrobial activity against certain

bacteria, which was regarded a pathogenic microorganism. Susceptibility of plant extract was tested by agar

well diffusion method was determined.

The results of our studies have shown that Asparagus racemosus contains saponins, tannins, flavonoids,

steroids, alkaloids and cardiac glycosides. The plant extract also showed antibacterial activity at concentrations

of 25 mg ml-1, 50 mg ml-1 and 100 mg ml-1 respectively. At these concentrations, the extract inhibited the

growth of Escherichia coli, Pseudomonas aeruginosa and Vibrio parahaemolyticus and produced percentage

inhibition ranging from 72.4% to 86.5%. Therefore, the ethnomedical application of the plant in the treatment of

bacterial infections is justified.

The antibacterial activity of aqueous extract (25 mg ml-1, 50 mg ml-1 and 100 mg ml-1) showed that the

extract has activity against Escherichia coli, Pseudomonas aeruginosa and Vibrio parahaemolyticus. At 100 mg

ml-1, it produced the highest percentage inhibition of 80.8% on Vibrio parahaemolyticus; at 25 mg ml-1 and least

percentage inhibition of 72.4% on Vibrio parahaemolyticus. The percentage inhibition of growth produced by

the extract on the other bacterial strains also tested ranges from 65% to 75% (Table 3). It may be deduced that

Asparagus racemosus showed a remarkable antibacterial activity (Table 2) when compared with Ampicillin

0

1

2

3

4

5

6

7

8

E.coli P.aerug V.para

Zon

e o

f in

hib

itio

n (

mm

)

Methanol Aqueous

0

1

2

3

4

5

6

7

E.coli P.aerug V.para

Zon

e o

f in

hib

itio

n (

mm

)

Ampicillin Tetracycline


.


(positive control) which produced a percentage inhibition of 98.8% (Table 3). Therefore, the antibacterial

activity exhibited by this plant extract may be due to the presence tannins, saponins and flavonoids in plant

which have been reported to have antibacterial properties. In conclusion, of the present investigation Asparagus

racemosus contain potential antimicrobial components that may be of great use for the development of

pharmaceutical industries as a therapy against various diseases.

Table 3. Summary of results of antibacterial activities of the aqueous extract of Asparagus racemosus.

Test organisms

Percentage inhibition of growth (%)

Asparagus racemosus Ampicillin

(100 mg ml-1

)

Distilled

water (25 mg ml-1

) (50 mg ml-1

) (100 mg ml-1

)

Escherichia coli 65.0 70.6 73.0 98.8 0.0

Pseudomonas aeruginosa 65.0 65.2 75.0 98.7 0.0

Vibrio parahaemolyticus 72.4 68.0 86.5 98.8 0.0

Note: Results are means of triplicate values.

ACKNOWLEDGEMENTS

The authors are thankful to Centre for Marine Science and Technology, Manonmaniam Sundaranar

University, Tamilnadu, India for providing necessary facilities.

REFERENCES

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Morrissey JP & Osbourn AE (1999) Fungal resistance to plant antibiotics as a mechanism of pathogenesis.

Microbiological and Molecular Biological Reviews 63: 708–724.

Sofowora A (1993) Medicinal plants and Traditional Medicine in Africa. Spectrum Books, Ibadan, pp. 150.

Trease GE & Evans WC (1989) Pharmacognosy, 13th edition. Bailliere Tindall, London, pp. 176–180.

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www.tropicalplantresearch.com 180 Received: 02 August 2015 Published online: 31 October 2015

ISSN (E): 2349 – 1183

ISSN (P): 2349 – 9265

2(3): 180–191, 2015

Research article

Bryophyte diversity in Terai regions of Uttar Pradesh, India with

some new additions to the state

Vinay Sahu and A. K. Asthana*

Bryology Laboratory, CSIR-National Botanical Research Institute Lucknow- 226 001, India


Abstract: An investigation of the Bryophytes from Terai region of Uttar Pradesh has revealed the

occurrence of 29 species of bryophytes: 21 species belonging to 16 genera of 11 Families of

Mosses; 6 species belonging to 3 genera of 3 families of Liverworts and 2 species belonging to 1

genus of 1 family of Hornworts. This includes several new reports viz., Notothylas kashyapii D.K.

Singh from Gangetic Plains, Dicranella macrospora Gangulee, Entodontopsis tavoyense (Hook. ex

Harv.) W.R. Buck & R.R. Ireland, Trachyphyllum inflexum (Harv.) Gepp., Weissia controversa

Hedw., Fissidens crenulatus Mitt., Fissidens flaccidus Mitt. and Fissidens zollingeri Mont. from

Uttar Pradesh.

Keywords: Uttar Pradesh - Terai region - Liverworts - Hornworts - New record

[Cite as: Sahu V & Asthana AK (2015) Bryophyte diversity in Terai regions of Uttar Pradesh, India with some

new additions to the state. Tropical Plant Research 2(3): 180–191]

INTRODUCTION

Uttar Pradesh (U.P.) can be divided into three topographical zones (i) The sub Himalayan Terai region in the

North (ii) The Gangetic Plain in the centre (iii) The Vindhya Hills and plateau in the South. In the state average

temperature ranges from 0o to 46 °C and average annual rainfall is around 65 to 70 cm

(https://en.wikipedia.org/wiki/Climate_of_Uttar_Pradesh). A total of 9.26% of the state's geographic area is

under forest/tree cover in U.P. Thus area is not much favourable for the growth of bryophytes. Various workers

from time to time made an attempt to provide consolidated account of bryophytes of Uttar Pradesh (Ahmad

1942, Pande & Ahmad 1944, Pande et al. 1954, Sahai 1962, Sahai & Sinha 1972, Srivastava 1964, Sinha et al.

1990, Singh & Kumar 2003, Singh et al. 2005, Lal 2007, Nath et al. 2010, Kumar & Kazmi 2004, 2006). Singh

(2013) has listed 27 species of liverworts, 3 species of hornworts and 24 species of mosses from Uttar Pradesh.

During the course of bryological exploration in that region, the authors come across some interesting taxa of

bryophytes. In the present study, an attempt has been made to provide an enumeration of bryophytes of this area

with ecology and distribution, range in India and abroad along with details of specimens examined.


Plant exploration trip has been undertaken to Lakhimpur, Pilibhit and their neighbouring areas in Uttar

Pradesh. Bryophyte plant samples has been collected from Gola forest, Salempur Forest Division, Malani

Range, Lalapur, near forest nursery, Lakhimpur-Kheri and Pilibhit Tiger Reserve, Barahi range. Collected

specimens have been deposited in Herbarium, CSIR-National Botanical Research Institute, Lucknow (LWG).


During the present investigation on the bryophytes of Pilibhit Tiger Reserve and its neighbouring areas

(Lakhimpur-Kheri, Gola, Shahjahanpur) about 30 taxa has been identified and enumerated. It includes an

account of 22 species belonging to 17 genera of 11 Families of Mosses, 6 species belonging to 3 genera of 3

families of Liverworts and 2 species belonging to 1 genus of 1 family of Hornworts. During the study one

Hornwort, Notothylas kashyapii D.K. Singh from Gangetic plains; and 8 mosses, Dicranella macrospora

Gangulee, Entodontopsis tavoyense (Hook. ex Harv.) W.R. Buck & R.R. Ireland, Trachyphyllum inflexum

(Harv.) Gepp., Weissia controversa Hedw., Fissidens crenulatus Mitt., Fissidens flaccidus Mitt. and Fissidens

zollingeri Mont. are reported from Uttar Pradesh for the first time.

mailto:[email protected]

Sahu & Asthana (2015) 2(3): 180–191

.


It has been observed that terricolous forms are dominant than epiphytic ones and out of 30 taxa investigated

from the area, mosses viz., Barbula indica, Philonotis mollis, Fissidens zollingeri and Hyophila nymaniana

were more frequent in occurrence. Among liverworts Riccia billardieri and Cyathodium cavernarum have been

frequently found in the area. Among the mosses, family Pottiaceae seems to be more dominant in the region

with 5 taxa and Genus Fissidens has maximum number (5) of species. As far as the liverworts are concerned,

family Ricciaceae exhibits maximum number of 4 species.

Enumeration

Liverworts

Family - Aytoniaceae

1. Plagiochasma appendiculatum Lehm. et Lindenb; Pag. IV. 14 (1832). (Fig. 1A)

Habitat: On stony wall, soil; Altitude- 452–536 ft.

Distribution: Central India (Amarkantak, Pachmarhi), Eastern Himalaya (Assam, Darjeeling, Sikkim), Gangetic

Plains (Uttar Pradesh), Punjab and West Rajasthan plain, South India (Maharashtra, Munnar, Tamil Nadu),

Western Himalaya (Corbett National Park, Dhanolti, Himachal Pradesh- GHNP, Kangra, Kullu, Simla, Nainital,

Mussoorie, Ranikhet); Afghanistan, Africa, China, Myanmar, Nepal, Pakistan, Philippines, Vietnam.

Specimens examined: INDIA, Uttar Pradesh, Lakhimpur-Kheri Dist., Near Radha Swami Inter College,

Kevalpurva, N 27º53.194' E 80º56.046', 08.09.2014, Vinay Sahu 257465A (LWG); Pilibhit Dist., Chimtiya

Puranpur, Barahi range, N 28º37.006' E 080º11.5', 11.09.2014, Vinay Sahu 258207A (LWG).

Family - Cyathodiaceae

2. Cyathodium cavernarum Kunze in Lehm., Pugillus 6: 17 (1834) Mont. in Ramon de la Sagra, Hist. de Cuba;

Crypyt. 491, tab. 19, Syn. Hepat. 577 (1846). (Fig. 1B)


Distribution: Central India (Gujarat, Madhya Pradesh), Eastern Himalaya (Darjeeling, Khasi Jaintia Hills,

Shillong), Gangetic Plains (Uttar Pradesh – Bareilly, Lucknow), South India (Mumbai, Elephanta caves,

Khandala, Mahabaleshwar, Malabar Hills, Panchagani, Pratabgarh), Western Himalaya (Dehradun, Gumkhal,

Karn Prayag, Mussoorie, Salkuli); Africa, America, Myanmar, Java.

Specimens examined: INDIA, Uttar Pradesh, Lakhimpur-Kheri Dist., Near Radha Swami Inter College,

Kevalpurva, N 27º53.194' E 80º56.046', 08.09.2014, Vinay Sahu 257464A, 257465D (LWG); Gola Forest, N

28º03.533' E 80º29.538', 09.09.2014, Vinay Sahu 257470A (LWG); Salempur Forest Division, Malani Range,

N 28º27.337' E 80º29.556', 09.09.2014, Vinay Sahu 257481A (LWG); Pilibhit Dist., Near Forest Office, Mala

range, Rechaula, N 28º37.070' E 79º54.894', 11.09.2014, Vinay Sahu 257495A (LWG); Barahi Range 72, N

28º37.006' E 080º11.5', 11.09.2014, Vinay Sahu 258204 A (LWG); Chimtiya Puranpur, Barahi range, N

28º37.006' E 080º11.5', 11.09.2014, Vinay Sahu 258213A (LWG).

Family - Ricciaceae

3. Riccia billardieri Mont. et Nees in Gottsche, Lindenberg & Nees, Syn. Hepat. 4: 602. (Fig. 1C)

Habitat: On soil; Altitude- 485–547ft.

Distribution: Central India (Gujarat, Jharkhand, Madhya Pradesh, Rajasthan), Eastern Himalaya (Assam,

Manipur, Sikkim), Gangetic Plains (Uttar Pradesh, West Bengal), South India (Andaman, Karnataka, Kerala,

Maharashtra, Tamil Nadu), Western Himalaya (Himachal Pradesh, Uttarakhand); Australia, Bangladesh,

Indonesia, Nepal, Philippines, Sri Lanka, Thailand.

Specimens examined: INDIA, Uttar Pradesh, Lakhimpur-Kheri Dist., Gola Forest, N 28º03.533' E 80º29.538',

09.09.2014, Vinay Sahu 257467B, 257468B, 257471A (LWG); Salempur Forest Division, Malani Range, N

28º27.337' E 80º29.556', 09.09.2014, Vinay Sahu 257476A, 257477E (LWG); Pilibhit Dist., Barahi Range 72, N

28º37.006' E 080º11.5', 11.09.2014, Vinay Sahu 258203A, 258210A (LWG).

4. Riccia gangetica Ahmad Ahmad ex L.Söderstr., A. Hagborg et von Konrat, Phytotaxa 65: 57 (2012). (Fig.

1D)

Habitat: On soil; Altitude- 530–600 ft.

Distribution: Central India (Gujarat, Madhya Pradesh Rajasthan), Eastern Himalaya (Meghalaya), Gangetic

Plains (Uttar Pradesh, West Bengal), Western Himalaya (Himachal Pradesh, Uttarakhand), South India (Kerala,

Maharashtra, Tamil Nadu); Australia, Bangladesh, Indonesia.

Sahu & Asthana (2015) 2(3): 180–191

.


Specimens examined: INDIA, Uttar Pradesh, Pilibhit Dist., Pipariya, N 28º32.471' E 79º54.905', 10.09.2014,

Vinay Sahu 257493A (LWG); Barahi Range 69, N 28º38.207' E 80º10.465', 11.09.2014, Vinay Sahu 258201A

(LWG).

Figure 1. A, Plagiochasma appendiculatum; B, Cyathodium cavernarum; C, Riccia billardieri; D, Riccia gangetica; E,

Riccia discolor; F, Riccia stricta; G, Notothylas indica; H–I, Archidium birmannicum; J–K, Philonotis mollis.

Sahu & Asthana (2015) 2(3): 180–191

.


5. Riccia discolor Lehm. et Lindenberg. in Lehmann, Nov. Stirp. Pug. 4: 1. (1832). (Fig. 1E)

R. himalayensis Kashyap, J. Bombay Nat. Hist. Soc. 24: 349. (1916).

Habitat: On soil; Altitude- 600 ft.

Distribution: Central India (Chattishgarh, Gujarat, Madhya Pradesh, Punjab, Rajasthan) Eastern Himalaya

(Assam, Sikkim, Meghalaya), Gangetic Plains (Uttarakhand, Uttar Pradesh, West Bengal), Western Himalaya

(Himachal Pradesh, Jammu & Kashmir), South India (Karnataka, Kerala, Maharashtra, Tamil Nadu); Africa,

Australia, Bangladesh, Myanmar, Nepal, Pakistan, Sri Lanka.

Specimens examined: INDIA, Uttar Pradesh, Lakhimpur-Kheri Dist., Barahai range 69, N 28º38.207' E

80º10.465', 11.09.2014, Vinay Sahu 257497A, 257500A (LWG).

6. Riccia stricta (Gottsche, Lindenb. & Nees) Perold, Bothalia 20: 197 (1990). (Fig. 1F)


Distribution: Central India (Madhya Pradesh), Eastern Himalaya (Sikkim), South India (Kerala, Tamil Nadu),

Gangetic Plains (West Bengal); Africa.

Specimens examined: INDIA, Uttar Pradesh, Shahjahanpur Dist., Near Khutar Forest, N 28º18.768' E

80º13.827', 12.09.2014, Vinay Sahu 258220E (LWG).

Family - Archidiaceae

7. Archidium birmannicum Mitt. ex Dix. in J. Ind. Bot., 2: 175 (1921). (Fig. 1H, I)


Distribution: Eastern Himalaya (Assam), Gangetic Plains (West Bengal Plains -Ramnagar, Hooghly), South

India (Nilgiri and Palni Hills); Myanmar, Nepal.

Specimens examined: INDIA , Uttar Pradesh, Lakhimpur-Kheri Dist., Gola Forest, N 28º03.533' E 80º29.538',

09.09.2014, Vinay Sahu 257467A, 257469A (LWG); Pilibhit Dist., Barahi Range 69, N 28º38.207' E

80º10.465', 11.09.2014, Vinay Sahu 257498A, 257500B, 258201B (LWG); Shahjahanpur Dist, Near Khutar

Forest, N 28º18.768' E 80º13.827', 12.09.2014, Vinay Sahu 258220C (LWG).

Family - Bartramiaceae

8. Philonotis mollis (Dozy & Molk.) Mitt. in Musci Ind. Or.: 60 (1859). (Fig. 1J, K)


Distribution: Eastern Himalaya (Sikkim), South India (Coorg, Kanara, Andaman Is.), Bhutan, Borneo, Celebes,

Japan, Java, Madagascar, Philippines, Sri Lanka, Sumatra, Taiwan, Tonkin.

Specimens examined: INDIA, Uttar Pradesh, Lakhimpur-Kheri Dist., Salempur Forest Division, Malani Range,

N 28 27.337' E 80º29.556', 09.09.2014, Vinay Sahu 257480A (LWG); Lalapur, near forest nursery, N 28º10.254'

E 80º19.395', 10.09.2014, Vinay Sahu 257485A, 257491A (LWG); Pilibhit Dist., Barahi Range 69, N

28º38.207' E 80º10.465', 11.09.2014, Vinay Sahu 257499A (LWG); Chimtiya, Puranpur, Barahi range, N

28º37.006' E 080º11.5', 11.09.2014, Vinay Sahu 258208A, 258210B, 258211B, 258212A, 258214B (LWG);

Shahjahanpur Dist., Near Khutar Forest, N 28º18.768' E 80º13.827', 12.09.2014, Vinay Sahu 258219A (LWG).

Family - Dicranaceae

9. Dicranella macrospora Gangulee in Nov. Hedwigia, 8: 145 (1964). (Fig. 2A)


Distribution: Assam, Uttar Pradesh (Lakhimpur Kheri).


N 28º27.337' E 80º29.556', 09.09.2014, Vinay Sahu 257484A (LWG); Lalapur, near forest nursery, N

28º10.254' E 80º19.395', 10.09.2014, Vinay Sahu 257491B (LWG). New record to Uttar Pradesh.

Family - Ditricaceae

10. Ceratodon purpureus (Hedw.) Brid. in Bryol. Univ., 1: 480 (1826). (Fig. 2D)


Distribution: Eastern Himalaya (Assam, Darjeeling), Gangetic plains (Raebareli), South India, Western

Himalaya (Kashmir); Antarctica, Australia, Brazil, China, Chili, East Nepal, Europe, Japan, Java, Madagascar,

New Zealand, North America (including Green Land, Alaska) North, Central and South Africa, Oceania,

Siberia, Sri Lanka, Tajikistan, Thailand.

Sahu & Asthana (2015) 2(3): 180–191

.


Specimens examined: INDIA, Uttar Pradesh, Pilibhit Dist., Barahi Range 72, N 28º37.006' E 080º11.5',

11.09.2014, Vinay Sahu 258202A (LWG).

Figure 2. A, Dicranella macrospora; B–C, Erpodium mangiferae; D, Ceratodon purpureus; E, Fissidens bryoides; F,

Fissidens crenulatus; G, Fissidens flaccidus; H–I, Fissidens involutus; J–K, Fissidens zollingeri; L, Physcomitrium eurystomum.

Sahu & Asthana (2015) 2(3): 180–191

.


Family - Erpodiaceae

11. Erpodium mangiferae C. Muell in Linnaea 37: 178. (1873). (Fig. 2B, C)

Habitat: Epiphytic; Altitude- 452 ft.

Distribution: Central India (Gujarat), Eastern Himalaya (Assam), Gangetic plains (U.P.-Allahabad, Saharanpur;

W. Bengal-Kolkata, Hooghly), Western Himalaya (Uttarakhand), South India (W. Ghats of Karnataka,

Maharashtra and Tamil Nadu); Bangladesh, Nepal.

Specimens examined: INDIA, Uttar Pradesh, Lakhimpur-Kheri Dist., Sankarpur police station, N 27º54.352' E

80º54.259', 08.09.2014, Vinay Sahu 257466A (LWG); Bira Salempur Forest Division, N 28º27.337' E

80º29.556', 09.09.2014, Vinay Sahu 257473A (LWG).

Family - Fissidentaceae

12. Fissidens bryoides Hedw. in Sp. Musc.: 153 (1801). (Fig. 2E)


Distribution: Eastern Himalaya (Khasi Hills), Gangetic Plains (Lower Bengal); South India (Coonoor, Nilgiri

Hills, Western Ghats), Western Himalaya (Nainital, Ranikhet, Simla), Africa, Caucasus, China, East Nepal,

Europe, Japan, Java and Philippines, Malay, N. & S. America, Sri Lanka, Siberia, Taiwan.

Specimens examined: INDIA, Uttar Pradesh, Pilibhit Dist., Chimtiya Puranpur, Barahi range, N 28º37.006' E

080º11.5', 11.09.2014, Vinay Sahu 258214A (LWG).

13. Fissidens crenulatus Mitt. in Musc. Ind. Or.: 140 (1859). (Fig. 2F)


Distribution: Gangetic Plains (Orissa), South India; East Nepal, Upper Burma.

Specimens examined: INDIA, Uttar Pradesh, Pilibhit Dist., Salempur Forest Division, Malani Range, N

28º27.337' E 80º29.556', 09.09.2014, Vinay Sahu 257474B, 257482A, 257483A, 257484B (LWG); Lalapur,

near forest nursery, N 28º10.254' E 80º19.395', 10.09.2014, Vinay Sahu 257490A, 257491C (LWG); Pilibhit

Dist., Near Gajraula, N 28º31.307' E 79º56.937', 12.09.2014, Vinay Sahu 258215A (LWG); Shahjahanpur Dist.,

Near Khutar Forest, N 28º18.768' E 80º13.827', 12.09.2014, Vinay Sahu 258219B (LWG). New record to Uttar

Pradesh.

14. Fissidens flaccidus Mitt. in Transactions of the Linnean Society of London 23: 56. 6 f. 18 (1860). (Fig. 2G)

Fissidens splachnobryoides Broth. in Schum. et Lauterb in Fl. Deutsch Schutzgeb. Südsee 81 (1900).


Distribution: Central India (Pachmarhi), Gangetic Plains (Lower Bengal), South India (Bombay, Khandala),

Western Himalaya (Kalka); Africa, Brazil, Borneo, Burma, China, Japan, Java, Nepal, New Guinea, Philippines,

Sri Lanka, Vietnam.

Specimens examined: INDIA, Uttar Pradesh, Lakhimpur Kheri Dist., Salempur Forest Division, Malani Range,

N 28º27.337' E 80º29.556', 09.09.2014, Vinay Sahu 257478A, 257480B (LWG); Lalapur, near forest nursery, N

28º10.254' E 80º19.395', 10.09.2014, Vinay Sahu 257488B (LWG); Pilibhit Dist., Barahi Range 72, N

28º37.006' E 080º11.5', 11.09.2014, Vinay Sahu 258204C (LWG); Chimtiya Puranpur, Barahi range, N

28º37.006' E 080º11.5', 11.09.2014, Vinay Sahu 258213C LWG). New record to Uttar Pradesh.

15. Fissidens involutus Wilson ex Mitt. in J. Proc. Linn. Soc. Botany, Supplement 2: 138. 1859. (Fig. 2H, I)


Distribution: Central India (Bastar, Chhotanagpur), Eastern Himalaya (Darjeeling, Sikkim), Gangetic Plains

(Saharanpur), South India (Bombay, Khandala), Western Himalaya; Borneo, Burma, China, Nepal, Thailand,

Vietnam.

Specimens examined: INDIA, Uttar Pradesh, Pilibhit Dist., Chimtiya Puranpur, Barahi range, N 28º37.006' E

080º11.5', 11.09.2014, Vinay Sahu 258209A (LWG).

16. Fissidens zollingeri Mont. in Ann. Sci. Nat. ser.3, 4: 114 (1845). (Fig. 2J, K)

Fissidens xiphioides Fleisch. in Hedwigia 38: 125 (1899).


Distribution: Central India (Pachmarhi), Gangetic Plains (Lower Bengal plains), South India (Bombay,

Khandala, Western Ghats, Kanara, Andamani Is.), Western Himalaya (Nainital, Simla); widely distributed in

Southwest Asia and Oceania, Borneo, Burma, China, Fiji, Indonesia, Nepal, Thailand, New Guinea, New

Sahu & Asthana (2015) 2(3): 180–191

.


Zealand, North and Central Vietnam, Philippines, Ryuku Is., Sri Lanka, Samoa, South and North America,

Tahiti.


N 28º27.337' E 80º29.556', 09.09.2014, Vinay Sahu 257474D, 257475A, 257477B, 257478B, 257483B,

257485B, 257486B (LWG); Lalapur, near forest nursery, N 28º10.254' E 80º19.395', 10.09.2014, Vinay Sahu

257490B, 257491D (LWG); Pilibhit Dist., Chimtiya Puranpur, Barahi range, N 28º37.006' E 080º11.5',

11.09.2014, Vinay Sahu 258208C (LWG); Shahjahanpur Dist., Near Khutar Forest, N 28º18.768' E 80º13.827',

12.09.2014, Vinay Sahu 258217B, 258218A, 258220B (LWG). New record to Uttar Pradesh.

Family - Funariaceae

17. Physcomitrium eurystomum Sendtn. in Denkschr. Bayer Bot. Ges. Regensb., 3: 142 (1841). (Fig. 2L)


Distribution: Gangetic plains (Lower Bengal, Hooghly, Allahabad), Western Himalaya (Kumaon); Britain,

Central and South Africa, China, France, North Vietnam, Taiwan.


N 28º27.337' E 80º29.556', 09.09.2014, Vinay Sahu 257474C (LWG); Pilibhit Dist., Pipariya, N 28º32.471' E

79º54.905', 10.09.2014, Vinay Sahu 257493B (LWG).

18. Entosthodon wichurae M. Fleisch. in Musci Fl. Buitenz., 2: 481 (1904). (Fig. 3C)


Distribution: Eastern Himalaya (Meghalaya- Khasi and Jaintia Hills), Western Himalaya (Ranikhet); Burma,

Java, Sri Lanka.


09.09.2014, Vinay Sahu 257496B (LWG); Chimtiya Puranpur, Barahi range, N 28º37.006' E 080º11.5',

11.09.2014, Vinay Sahu 258212B (LWG). New record to Uttar Pradesh.

Family - Pottiaceae

19. Barbula indica (Hook.) Spreng. Steud. Nomencl., 2: 72 (1824). Saito J. Hattori Bot. Lab., 39: 488 (1975).

(Fig. 3 A, B)

Semibarbula orientalis (Web.) Wijk et Marg., Taxon, 8: 75 (1959); Gangulee, Moss. E. Ind., 3: 717 (1972).

Habitat: On soil, stony wall; Altitude- 353–547 ft.

Distribution: Widely distributed plain area in the country and up to 1000 m in the Himalayas; Nepal, Southeast

Asia, Japan, South Africa, New Guinea.

Specimens examined: INDIA, Uttar Pradesh, Lakhimpur-Kheri Dist., Sankarpur police station, Near Radha

Swami Inter College, Kevalpurva, N 27º54.352' E 80º54.259', 09.09.2014, Vinay Sahu 257460A, 257462A,

257463A, 257465B (LWG); Gola Forest, N 28º03.533' E 80º29.538', 09.09.2014, Vinay Sahu 257470B,

257472B (LWG); Khalsa Public School, N28º24.987' E 80º11.207', 10.09.2014, Vinay Sahu 257492A (LWG);

Salempur Forest Division, Malani Range, N 28º27.337' E 80º29.556', 09.09.2014, Vinay Sahu 257477C,

257481B, 257486A, (LWG); Near Forest Office, Mala range, Rechaula, N 28º37.070' E 79º54.894', 11.09.2014,

Vinay Sahu 257494B, 257495C (LWG); Pilibhit Dist., Barahi Range 72, N 28º37.006' E 080º11.5', 11.09.2014,

Vinay Sahu 258204B (LWG); Chimtiya Puranpur, Barahi range, N 28º37.006' E 080º11.5', 11.09.2014, Vinay

Sahu 258211A (LWG); Near Amritsar dhabha, Ashram road, 12.09.2014, Vinay Sahu 258216A (LWG).

20. Gymnostomum calcareum Nees et Hornsch., Bryol. Germ., 1: 153 10f.15 (1823); Gangulee, Moss. E. Ind.,

3: 641 (1972). (Fig. 3D)

Habitat: On soil, Altitude- 547 ft.

Distribution: Eastern Himalaya, Western Himalaya (Uttarakhand-Mussoorie, Corbett National Park); Africa,

Australia, China, Europe, Japan, New Zealand, North Central and South America.


11.09.2014, Vinay Sahu 258202A (LWG); Near Gajraula, N 28º31.307' E 79º56.937' 12.09.2014, Vinay Sahu

258215B (LWG). New record to Uttar Pradesh.

21. Hydrogonium arcuatum (Griff.) Wijk et Marg., Taxon, 7: 289 (1958); Gangulee, Moss. E. Ind., 3: 725

(1972).

Habitat: On soil, Altitude- 353 ft.

Sahu & Asthana (2015) 2(3): 180–191

.


Figure 3. A–B, Barbula indica; C, Entosthodon wichurae; D, Gymnostomum calcareum; E, Hyophila spathulata; F–G,

Hyophila nymaniana; H, Weissia controversa; I, Splachnobryum obtusum; J, Trachyphyllum inflexum; K–L, Entodontopsis

tavoyense.

Sahu & Asthana (2015) 2(3): 180–191

.


Distribution: Central India (Madhya Pradesh, Jharkhand, Orissa), Eastern Himalaya (Assam, Arunachal

Pradesh, Meghalaya West Bengal), South India (Tamil Nadu), Western Himalaya (Himachal Pradesh, Kashmir,

Uttarakhand); Bhutan, China, East Nepal, Japan, Java, Malaya, Moluca, Myanmar, New Guinea, Philippines,

Oceania.

Specimens examined: INDIA, Uttar Pradesh, Lakhimpur-Kheri Dist., Sankarpur police station, N 27º54.352' E

80º54.259', 08.09.2014, Vinay Sahu 257461A (LWG).

22. Hyophila nymaniana (Fleisch.) Menzel 22: 198 (1992); Nair M. C. et al., Moss Waynad in W. Ghats 117

(2005). (Fig. 3F, G)

Hyophila rosea Williams, Bull. N.Y Bot. Gard., 8: 341 (1914); H. comosa Dix. et P. Verd., Arch. Bot., 1:

166 (1927).

Habitat: On soil, stony wall; Altitude- 452–600 ft.

Distribution: Central India (Gujarat), Eastern Himalaya (Meghalaya), Gangetic Plain (Allahabad), Western

Himalaya (Uttarakhand, Corbett National Park), South India (Karnataka, Kerala, Tamil Nadu); Nepal,

Philippines.

Specimens examined: INDIA, Uttar Pradesh, Lakhimpur Kheri Dist., Near Radha Swami Inter College,

Kevalpurva, N 27º53.194' E 80º56.046', 08.09.2014, Vinay Sahu 257463B, 257465C (LWG); Salempur Forest

Division, Malani Range, N 28º27.337' E 80º29.556', 09.09.2014, Vinay Sahu 257479A (LWG); Lalapur, near

forest nursery, N 28º10.254' E 80º19.395', 10.09.2014, Vinay Sahu 257487A, 257489A (LWG); Pilibhit Dist.,

Khalsa Public School, Ruria Puranpur, N 28º24.987' E 80º11.207', 10.09.2014, Vinay Sahu 257492B (LWG);

Near Forest Office, Mala range, Rechaula, N 28º37.070' E 79º54.894', 11.09.2014, Vinay Sahu 257494A

(LWG); Barahi Range 69, N 28º38.207' E 80º10.465', 11.09.2014, Vinay Sahu 257498B (LWG).

23. Hyophila spathulata (Harv.) A. Jaeger Ber. S. Gall. Naturw. Ges., 1871-72: 353 (1873). Gangulee, Moss. E.

Ind., 3: 687 (1972). (Fig. 3E)


Distribution: Gangetic Plain (Allahabad, Delhi), Western Himalaya (Uttarakhand) South India (Tamil Nadu);

China, Japan, East and West Nepal, Myanmar, Sri Lanka.


N 28º27.337' E 80º29.556', 09.09.2014, Vinay Sahu 227477D (LWG); Shahjahanpur Dist., Near Khutar Forest,

N 28º18.768' E 80º13.827', 12.09.2014, Vinay Sahu 258217A, 258219C, 258220A (LWG).

24. Weissia controversa Hedw., Spec. Musc., 67 (1801). Saito J. Hattori Bot. Lab., 39: 426 (1975). (Fig. 3H)


Distribution: Western Himalaya (Kashmir, Uttarakhand), South India (Tamil Nadu); Australia, China, Europe,

Japan, New Zealand, North Central and South America, Pakistan, Sri Lanka, West Indies.

Specimens examined: INDIA, Uttar Pradesh, Shahjahanpur Dist., Near Khutar Forest, N 28º18.768' E

80º13.827', 12.09.2014, Vinay Sahu 258220D (LWG). New record to Uttar Pradesh.

Family - Splachnaceae

25. Splachnobryum obtusum (Brid.) Mull. Hal., Verh. K. K. Zool. Bot. Ges. Wien 19: 503 (1869). (Fig. 3I)

S. indicum Hamp. et C. Muell. In Linnaea 37: 174 (1872).

Habitat: On rock, stony wall; Altitude- 485 ft.

Distribution: Gangetic plains (Gangetic Southern Bengal, Howrah, Hooghly, Kolkata, Delhi, Allahabad,

Orissa), Western Himalayas (Tehri), South India (Western Ghats); Java, Africa, America, Australia, Burma,

Europe, Indonesia, Macaronesia, Philippines, Papua New Guinea. Thailand.


09.09.2014, Vinay Sahu 257467C (LWG); Pilibhit Dist., Near Gajraula, N 28º31.307' E 79º56.937', 11.09.2014,

Vinay Sahu 258216B (LWG).

Family - Thuidiaceae

26. Trachyphyllum inflexum (Harvey) Gepp. in Hiern: Cat. Weln. Afr. Pl. 2, 21: 299 (1901). (Fig. 3J)


Distribution: Central India (Madhya Pradesh-Amakantak), Eastern Himalaya (Darjeeling, Khasi Hills, Sikkim),

Gangetic plains (Orissa), South India (Palni Hills, Kanara), Western Himalaya (Valley of Flowers, Corbett

Sahu & Asthana (2015) 2(3): 180–191

.


Figure 4. Notothylas kashyapii: A–C, Thalli; D, Sporophytes in the apical region; E, Lamellae present on involucres; F,

Capsule wall; G, chloroplast; H–I, Spores.

Sahu & Asthana (2015) 2(3): 180–191

.


National Park); Australia, Bangladesh, Burma, Central Vietnam, China, Combodia, Java, Madagascar,

Moluccas, Nepal, New Caledonia, Philippines, Thailand.

Specimens examined: INDIA, Uttar Pradesh, Pilibhit Dist., Barahi range 72, N 28º37.006' E 080º11.5',

11.09.2014, Vinay Sahu 258206B (LWG). New record to Uttar Pradesh.

Family - Stereophyllaceae

27. Entodontopsis tavoyense (Hook. ex Harv.) W.R. Buck. & R.R. Ireland, Nova Hedwigia 41: 105 (1985).

(Fig. 3K, L)

Sematophyllum tavoyense (Hook.) Jaeg.


Distribution: Gangetic plain (Bihar), Western Himalaya (Dehradun, Corbett National Park); South India

(Kerala); Bangladesh, East Nepal, Moulmein, Penang, Tavoy.

Specimens Examined: INDIA, Uttar Pradesh, Pilibhit Dist., Barahi range 72, N 28º37.006' E 080º11.5',

11.09.2014, Vinay Sahu 258205A (LWG). New record to Uttar Pradesh.

Hornworts

Family - Notothylaceae

28. Notothylas indica Kash. in Kashyap and Dutt in Proc. Lahore Phill. Soc. 4: 49-56 (1925); Asthana &

Srivastava in Bryophyt. Biblioth. 42:94 (1991). (Fig. 1G)


Distribution: Gangetic plain (Lucknow, Allahabad), Central India (Pachmarhi, Tikamgarh), South India

(Mumbai, Nagpur), Western Himalaya (Dehradun, Mussoorie); Pakistan (Parachhinar), Myanamar (Yangong).


09.09.2014, Vinay Sahu 257468A, 257471B, 257472A (LWG).

29. Notothylas kashyapii D.K. Singh, in Singh and Semwal in India J. For. 23(4): 386 (2000).

Habitat: On soil; Altitude- 418–600 ft. (Fig. 4A-I)

Distribution: Western Himalaya (Uttarakhand-Dehradun).


N 28º27.337' E 80º29.556', 09.09.2014, Vinay Sahu 257474A, 257477A, 257478C (LWG); Lalapur, near forest

nursery, N 28º10.254' E 80º19.395', 10.09.2014, Vinay Sahu 257488A (LWG), Pilibhit Dist., Barahi Range 69,

N 28º38.207' E 80º10.465', 11.09.2014, Vinay Sahu 257496A, 257497B (LWG); Barahi Range 72, N 28º37.006'

E 080º11.5', 11.09.2014, Vinay Sahu 258203B (LWG); Chimtiya Puranpur, Barahi range, N 28º37.006' E

080º11.5', 11.09.2014, Vinay Sahu 258213B (LWG); Shahjahanpur Dist., Near Khutar Forest, N 28º18.768' E

80º13.827', 12.09.2014, Vinay Sahu 258218B (LWG). New record to Uttar Pradesh.

ACKNOWLEDGEMENTS

Authors are grateful to the Director, CSIR-National Botanical Research Institute, Lucknow for

encouragement and providing the facilities and work has been carried out under in house project OLP-0083.

REFERENCES

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Kumar A & Kazmi S (2006) Leaf area indices of mosses from Unchahar, Raebareli, U. P. Geophytology 36: 23–

26.

Lal J (2007) Mosses of Gangetic plains – a neglected Biogeographic Zone of India. In: Nath V & Asthana AK

(eds.) Current trends in Bryology. Bishen Singh Mahendra Pal Singh, Dehradun, India, pp.131–147.

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mosses of Lucknow (U.P.). Indian Journal of Applied & Pure Biology 25(1): 25–29.

Pande SK & Ahmad S (1944) Liverworts of Lucknow and its neighbourhood. Proceedings of Indian Science

Congress 31(3): 80.

Pande SK, Misra KC & Srivastava KP (1954) A species of Riella Mont., R. vishwanathii Pande, Misra et

Srivastav, sp. nov., from India. Revue Bryologique et Lichenologique 23:165–172.

Sahai R (1962) Occurrence of Anthoceros crispulus (Mont.) Douin in Gorakhpur. Current Science 31: 519–520.

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Sahai R & Sinha AB (1972) Riccia perssonii Khan - a new record from India. Current Science 41: 226–227.

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river Sai (Raebareli), India. Proceedings of National Academy of Science India 75(B): 41–50.

Singh SK (2013) A Checklist of liverworts, hornworts and mosses of Uttar Pradesh, India. Geophytology 42(2):

163–167.

Singh SK & Kumar S (2003) A note on bryophytes of Ram Nagari (Ayodhya, Faizabad, Uttar Pradesh, India.

Phytotaxonomy 3: 108–111.

Sinha AB, Singh US & Shukla MS (1990) Genus Riccia (Mich.) L. of district Gorakhpur. Journal of Economic

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ISSN (E): 2349 – 1183

ISSN (P): 2349 – 9265

2(3): 192–203, 2015

Research article

Contribution of environmental factors

on in vitro culture of an endangered and endemic mangroves

Heritiera fomes Buch.-Ham. and Bruguiera gymnorhiza (L.) Lam.

Abdul Kader1, Sankar Narayan Sinha2, Parthadeb Ghosh1*

1Cytogenetics & Plant Biotechnology Research Unit, Department of Botany, University of Kalyani, West

Bengal, India 2Environmental Microbiology Research Laboratory, Department of Botany, University of Kalyani, West

Bengal, India *Corresponding Author: [email protected] [Accepted: 19 October 2015]

Abstract: Importance and destruction of mangroves have appeared in some recent surveys. So

their restoration through tissue culture study is urgently required because in vivo propagation is

plagued with unforeseen obstacles. This study describes for the first time in vitro approach for

threatened species Heritiera fomes and Bruguiera gymnorhiza through callus. For initiation of

callus modified MS medium was formulated for each species which correlated with soil conditions

of Sundarban mangrove forest. For both species the auxin NAA, nodal or shoot tip explants and

rainy season were found to be most suitable for callusing. NaCl at the concentration of 20 mM and

60 mM promoted growth for H. fomes and B. gymnorhiza callus respectively which was found to

be comparative for their growth in vivo as in Sundarban. Histological study indicated

morphogenicity of callus. Previous in vitro studies on mangroves were mostly based on the effect

of variety of hormones and different sea salts. However this present study clearly indicates that the

in vitro studies of mangroves not only depend on these factors but g reatly influence by soil

condition of their habitual environment, seasonal condition etc. From this study it seems that more

and more in vitro studies of mangroves are possible if researchers focus on their habitual

environmental conditions as many mangrove species remains recalcitrant for in vitro study. The

present research clearly indicated that the species may be restored in low saline or non -saline land

as land destruction is another vital reason for mangrove extinction.

Keywords: Bruguiera gymnorhiza - Heritiera fomes - Mangrove - Callus culture - Seasonal

influence

[Cite as: Kader A, Sinha SN & Ghosh P (2015) Contribution of environmental factors on in vitro culture of an

endangered and endemic mangroves Heritiera fomes Buch.-Ham. and Bruguiera gymnorhiza (L.) Lam. Tropical

Plant Research 2(3): 192–203]

INTRODUCTION

Mangrove ecosystems are found in tropical and subtropical muddy beaches worldwide. The importance and

threats to mangrove ecosystem have been discussed by various authors (Al-Bahrany & Al-Khayri 2003, Ren et

al. 2009). Because of their importance and destruction, mangroves have attracted attention for their conservation

and preservation (Al-Bahrany & Al-Khayri 2003). Problems for restoration of mangroves arise mostly in the

form of shortage of seeds or viviparous seedlings and the disturbed soil conditions (Ohnishi & Komiyama 1998,

Feller et al. 2003). Mangrove species are physiologically unique in their adaptations to such water logged and

saline condition. Crop scientists, studying the unique adaptation pattern of mangrov es, are keen to impart these

unique characters in food crops by breeding or biotechnological means (Fukomoto et al. 2004) as salinity and

water logging are among the major environmental threats with serious implication on food, fuel and fibre

production, especially in arid and semiarid regions (Dagar 2005). Besides, about one-third of all agricultural

lands are becoming saline (Dagar 2005). To understand the salt and water logging tolerance theoretically or

biochemically, callus or cell culture of mangroves may provide promising result. However, detailed knowledge

of the plant material and its requirements for callus initiation is necessary before mass in vitro propagation can

Kader et al. (2015) 2(3): 192–203

.


become a reality. However scanty literature is available for few mangrove species because it is recalcitrant to

tissue culture study (Al-Bahrany & Al-Khayri 2003, Fukomoto et al. 2004, Kawana & Sasamoto 2008). During

in vitro culture of mangroves explants frequently turn brown or black and eventually die shortly after

inoculation (Kawana & Sasamoto 2008, Arumugam & Panneerselvam 2012) as it excretes high tannin and

phenol or phenolic compound. Besides, it is very difficult to maintain their habitual environment. Moreover,

success of in vitro response does depend not only on the plant genotype but also is strongly affected by

environmental conditions.

Preventing or avoiding microbial contamination is the basis of successful plan t tissue cultures. Endogenous

microbial contamination is known to be one of the most serious threats in plant tissue culture, especially in

tropical species (Kneifel & Leonhardt 1992). Various literatures are available indicating the association of

microbes and mangroves in root, bark, leaves etc (Gupta et al. 2009). Among them Uchino et al. (1984)

identified some entophytic microbes in Bruguiera gymnorhiza species from its aerial parts.

Bruguiera gymnorhiza (L.) Lam. (Rhizophoraceae) is a multipurpose true mangrove species found in all

over the world. The fruits and bark of the whole plant have been used for treating diarrhoea, fever, malaria,

shingles and eye diseases (Naskar & Bakshi 1987, Bandaranayake 1998). The durable wood is used for making

boat, house, poles, beams etc (Naskar & Bakshi 1987). Natural products of this plant have anti-tumor activity

and antibacterial activity (Naskar & Bakshi 1987, Bandaranayake 2002).

Heritiera fomes Buch.-Ham. is a true mangrove tree from family Sterculiaceae, known as Sundari in

Bengali, found mainly in Southeast Asia (Naskar & Bakshi 1987, Ali et al. 2011). The largest deltaic mangrove

forest, Sundarban is derived from its Bengali name (Naskar & Bakshi 1987, Gopal & Chauhan 2006). The wood

of this species is used for making boat, raft, house and charcoal (Naskar & Bakshi 1987, Ali et al. 2011).

Besides, various parts of the tree are used as folk medicine for heart diseases, diabetes, pain, diarrhoea, skin

disorders, hepatic disorders, and goiter (Ali et al. 2011). Ethanolic extract of stem bark showed antioxidant,

lipoxygenase inhibitory, antihyperglycemic, antinociceptive effects and antibacterial activities (Wangensteen et

al. 2009, Ali et al. 2011). Due to its medicinal and economical values and increasing environmental stress

(Various salt concentrations, global warming etc), this species is being exploited indiscriminately since a very

long time and it is considered as a threatened plant according to IUCN red list 2013 (Naskar & Bakshi 1987,

Gopal & Chauhan 2006).

Keeping the deforestation, tissue culture problem and multiple utility of these two species in mind, we

describe here a preliminary study of micropropagation through callus culture for preservation and production of

micropropagated plant for future restoration of degraded mangrove forest areas. To our knowledge, this is the

first report of callus initiation as well as in vitro investigation of Heritiera fomes and Bruguiera gymnorhiza.


Plant materials

Different explants were collected from an respective 8–10 years old tree at various seasons all over the year

from Gosaba region (88° 39΄46" East and 22° 15΄45" North) of Indian Sundarban Mangrove forest.

Preparation of explants

Firstly explants were washed with running tap water, then dipped in 2% teepol solution for 8 min and

washed two to four times with sterile distilled water. The explants were then surface sterilized with 0.1–0.2%

HgCl2 (w/v) solution for different time duration to standardize the surface sterilization protocol. Th ereafter they

were dipped in 70% ethanol for 1–2 minute and finally they washed three times with sterile distilled water to

remove any traces of the HgCl2 and ethanol.

Culture Media and Conditions

For Heritiera fomes surface sterilized segments (1.0–1.5 cm long) were cultured on modified MS medium

(Murashige & Skoog 1962) having complete omission of ammonium nitrate and half concentration of potassium

nitrate with 3% (w/v) sucrose for callus initiation and further experiments. On the other hand for Bruguiera

gymnorhiza surface sterilized segments (1.0–1.5 cm long) were cultured on another type of modified MS

medium having slight modifications including thrice addition of micro salts and addition of beef extracts, yeast

extracts and casein hydrolysates into medium at the concentration of 50 mg l-1

each with 3% (w/v) sucrose for

callus initiation. For further experiments the sucrose concentration were altered to design the experiment. The

Kader et al. (2015) 2(3): 192–203

.


pH of the medium was adjusted to 5.6–5.8 before autoclaving. To eliminate browning problem polyvinyl

pyrrolidone (PVP) was used to treat explants at the concentrations of 1gm l-1

. The explants initially were

implanted vertically on the culture medium and plugged tightly with non -absorbent cotton. All the cultures were

kept under cool fluorescent light (16 h photo period 40 μmol m–2

s–1

at 25±2 °C) and 60–70 % relative humidity.

For this study 2, 4-D and NAA in combination with BAP were used. For induction of callus and determining the

degree of salt tolerant, NaCl was added in the medium at various concentration of this experime nt. The callus

initiation rate (the ratio of the number of explants pieces having calli to total number of explants pieces planted

in the same culture) was scored about one month after planting. Callus growth was measured by increment in

fresh weight. In the present study the callus growth was measured after 2 months of inoculation. Culture tube

containing medium was weighed just before and after inoculation. The difference in weight gave the fresh

weight of the inoculated tissue. In successive cultures the total amount of tissues were transferred to pre-

weighed fresh medium. 50% excess was taken for sacrificing due to contamination though all the experiments

were done under aseptic condition in laminar air flow chamber.

Figure 1. Callus initiation, in vitro sprouting of different explants, histological section and media discoloration of Heritiera

fomes: A, Callus initiation site after 10 days of explants inoculation using NAA and BAP combination of H. fomes; B,

Yellow and light brown callus formation after 2 weeks of inoculation of explants of H. fomes; C, General view of 3 week old

protuberance produced at the proximal part of the explant (Vetical section), VC- Large vaculated cells, Black arrow indicates

calli at inner region containing both small meristematic cells with highly-stained nucleus in mitotic cells zone (MCZ), Green arrow indicates embyogenic cells where red arrow indicates non embryogenic cells; D, Close view of embryogenic cells on

callus where black arrow shows the embryogenic cells; E, Media discoloration caused by secretion of tannin or phenolic

compounds after five days of inoculation; F, In vitro sprouting of leaf using NAA and BAP in combination; G, Deep brown

small callus formation in combination of 2, 4-D and BAP.

Kader et al. (2015) 2(3): 192–203

.


Histological preparation

For histological studies, the explants were fixed in FAA (formaldehyde: acetic acid: ethanol; 100:50:50)

solution for 10 days. The fixed samples were washed for 40 min, twice with double distilled water. After

washing, the fixed samples were dehydrated through the ethanol series (30%, 50%, 70%, 80% and 90%) for 30

min at each stage. The samples were embedded in paraffin wax (melting point 58°C) and section vertically at

10μM thickness on a rotary microtome. The sections were mounted onto slides and allowed to dry for at least 10

min before staining. The specimens were stained with hematoxylin-eosin and counter stained with safranin

solutions. The sections were then examined under phase contrast microscope.

Statistical Analysis

Experiments were set up in completely randomized design. Each experiment was repeated three times with

10 - 13 replicates. Data were analyzed by one way analysis of variance (ANOVA) and the difference between

means were scored using Duncan’s Multiple Range Test P ≤ 0.05 (Duncan 1955) on the statistical package of

SPSS (Version 10).

RESULTS

Selection of explants for callus initiation

Among the different explants used, leaves were not found to be suitable for callusing as it showed only in

vitro sprouting for both the species . For Heritiera fomes callus was obtained from nodal and internodal segments

only (Fig. 1) whereas for Bruguiera gymnorhiza, the callus formation was best observed with shoot tip (Fig. 2).

Figure 2. Callus initiation, histological section and media discoloration of Bruguiera gymnorhiza: A, Media browning by

secretion of phenolic compounds of explants of B. gymnorhiza; B, Showing fungal contamination by explants; C, Showing

bacterial contamination; D, White callus formation after 2 weeks of inoculation of explants; E, General view of 4-week-old

protuberance produced at the proximal part of the explants (Vertical section) with wound callus formation; F, Showing extensive enlargement of scutellar parenchyma cells and the formation of peripheral pockets of dividing scutellar cells. Calli

inner region containing both small meristematic cells with highly -stained nucleus in mitotic cells zone (MCZ) and

vacuolated large cells; G, Higher magnification of the MCZ: Blue arrow indicates accumulation of soluble carbohydrate

(sucrose and/or glucose) or tannin and Black arrow indicates 4 thick-walled proembryonic cells.

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Standardisation of surface sterilization protocol for explants

Approximately 70% of successful sterilization was achieved from 0.1% HgCl2 (w/v) solution treatment for

10- 15 minute incubation period for H. fomes. However for surface sterilization of B. gymnorhiza 0.2% HgCl2

(w/v) solution treatment for 15-20 minute incubation period were required (Table 1) for efficient sterilization.

Although initially some other treatments showed promising result however after 2 to 3 weeks of incubation,

some cultures that had showed no contamination was found to be contaminated (Fig. 2).

Table 1. The effect of different concentration of HgCl2 solution for different periods of time for removal of microbial

contamination for Bruguiera gymnorhiza.

Percentage of HgCl2

Solution (w/v)

Incubation Period (minutes) Aseptic inoculation (% )

0.10 10 5.58±0.00d

0.10 15 7.68±4.43cd

0.10 20 30.25±5.12cd

0.15 10 25.38±4.43cd

0.15 15 30.50±2.56c

0.15 20 51.02±2.56b

0.20 10 46.15±7.69b

0.20 15 76.66±6.78a

0.20 20 76.66±6.78a

Note: Values in the last column are Mean ± SE of Mean followed by the letters within the column indicating the

level of significance at P≤0.05 by Duncan’s Multiple Range Test (same letter within the column of the

treatments indicates the absence of difference; different letters indicate the significant difference; and

combination of letters indicate no significant difference).

Elimination of browning problem

Generally media browning is caused by the secretion of phenolic compounds and its callus inhibition activity

was discussed by various authors (Gill et al. 2004). Before inoculation and after sterilization the explants were

treated with PVP solution at the concentration of 1gm/L (W/V) for 45 min and kept the culture after inoculation

was kept in darkness for seven days which was found to remove media discoloration sufficiently (Figs. 1 & 2).

Effect of sucrose on callus initiation

To check the effect of sucrose concentration on callus initiation, we incubated the cultures on different

sucrose concentration containing medium like 1%, 2%, and 3%. Among them, 3% (w/v) sucrose containing

medium gave best results for H. fomes while for B. gymnorhiza gave best result on 2% (w/v) sucrose containing

(Fig. 3).

Figure 3. Effect of sucrose concentration on callus response of Bruguiera gymnorhiza.

Callus initiation rate between different hormone concentrations

For both the species, callus initiation was observed within 2 to 3 weeks after inoculation. Higher rates of

callus initiation were obtained at combinations of 2 mg l-1

and 0.5 mg l-1

BAP and NAA respectively for H.

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fomes (Table 2) and for B. gymnorhiza it was 1.5 mg l-1

and 0.5 mg l-1

NAA and BAP respectively (Table 3).

Before fixing in FAA for histological preparation, the B. gymnorhiza callus showed white, compact and almost

smooth nature while the H. fomes callus was yellow or light brown in colour and compact and nodular in nature

in respective hormone combination (Figs. 1 & 2). It was observed that 2, 4-D failed to initiate callus on B.

gymnorhiza. The action of 2, 4-D on callus initiation showed slow growth rate for H. fomes (Table 3), the callus

being deep brown in colour (Fig. 1) and dormant in nature. From these result it seems that 2, 4-D was not

suitable for callus culture for these species. During this study it was observed that the rate of callusing response

of mangroves were very low and generally initiated callus showed slow growth than other territorial plants and

it may be because of their fluctuating and extreme environment of their habitat. Similar results were obtained by

Mimura et al. (1997).

Table 2. Rate of callus initiation of Heritiera fomes in modified MS medium at different concentrations of p lant

hormones (mg l-1).

Hormone Concentrations (mg l-1) Callus response (% ) Nature of Callus

NAA 2, 4-D BAP

0 0 0 0.00±0.00h -

0.5 - 0.5 17.94±5.12defg

Soft, Granular, Yellow

1.0 23.07±6.28def


2.0 69.25±4.44a Soft, Granular, Yellow

1.0 - 0.5 23.22±4.30def


1.0 25.63±2.56cde


2.0 15.38±4.43efgh


1.5 - 0.5 28.19±2.56cde


1.0 41.02±6.78bc


2.0 20.50±5.12def


2.0 - 0.5 33.32±6.78cd


1.0 17.94±2.56defg


2.0 28.20±6.78cde


2.5 - 0.5 28.20±5.13cde


1.0 51.27±2.56b Soft, Granular, Yellow

2.0 23.07±4.43def


- 0.5 0.5 0.00±0.00h -

1.0 7.69±4.43fgh

Hard, Deep brown

2.0 2.56±2.56gh

Hard, Deep brown

- 1.0 0.5 17.94±6.78defg

Hard, Deep brown

1.0 23.07±4.43def

Hard, Deep brown

2.0 15.38±4.43efgh

Hard, Deep brown

- 1.5 0.5 28.20±5.13cde

Hard, Deep brown

1.0 41.02±6.78bc

Hard, Deep brown

2.0 23.07±4.43def

Hard, Deep brown

- 2.0 0.5 20.51±9.24def

Hard, Deep brown

1.0 28.20±5.13cde

Hard, Deep brown

2.0 20.50±6.78def

Hard, Deep brown

- 2.5 0.5 19.22±3.84defg

Hard, Deep brown

1.0 28.20±5.13cde

Hard, Deep brown

2.0 23.22±4.30def

Hard, Deep brown

Note: Values in the second last column are Mean ± SE of Mean followed by the letters within the

column indicating the level of significance at P≤0.05 by Duncan’s Multiple Range Test (same letter

within the column of the respective hormone indicates the absence of difference; different letters

indicate the significant difference; and combination of letters indicate no significant difference).

We also tried to initiate callus on different media like MS, Woody Plant medium (WPM, Lloyd & McCown

1981), Linsmaier and Skoog (LS) medium (Linsmaier & Skoog 1965), X medium (Rao et al. 1998) and Amino

Acid (AA) medium (Thompson et al. 1986) but there were no response found for these species.

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Histological study of initiated callus

The histological study showed the production of protuberance at the proximal part of the explant (horizontal

section) of H. fomes. This study of both species showed the formation of mitotic cells zone (MCZ) i.e.,

parenchymatous cells with nucleus and dense cytoplasm divid ing actively (Figs. 1& 2). It also noted that in the

intercellular spaces of callus tissue there was a more or less dense fibrillar or reticular network which is

complexes of polysaccharide polymers having a microfibrillar network texture (Fig. 2) or accumulation of

tannin. B. gymnorhiza callus also indicated extensive enlargement of scutellar parenchyma cells and the

formation of peripheral pockets of dividing scutellar cells. Two types of cells present in callus might be

distinguished: embryogenic and nonembryogenic (Figs. 1 & 2).

Effect of salt concentration on callus initiation and growth

Bruguiera gymnorhiza gave maximum callus response and growth at the concentrations of 60 mM NaCl

(Fig. 4) concentrations whereas endangered and endemic H. fomes gave best callus response and growth at the

concentration of 20 mM NaCl (Fig. 5).

Figure 4. Effect of salt concentration on callus initiation and growth of Bruguiera gymnorhiza.

Table 3. Rate of callus initiation of Bruguiera gymnorhiza in modified MS medium at different concentrations of plant

hormones (mg l-1).

NAA

(mg l-1)

BAP

(mg l-1)

Callus Response (%) Nature of Callus

0 0 0.00±0.00f -

0.5 0.5 12.81±5.12e Hard, Compact, White

1.0 5.12±2.56bcef

Hard, Compact, White

1.0 0.5 53.84±7.69b Hard, Compact, White

1.0 28.20±5.13d Hard, Compact, White

1.5 0.5 74.35±2.56a Hard, Compact, White

1.0 56.40±5.12b Hard, Compact, White

2.0 0.5 56.40±2.56b Hard, Compact, White

1.0 38.88±2.77cd


2.5 0.5 51.27±2.56bc


1.0 43.58±2.56bc


Note: Values in the second last column are Mean ± SE of Mean followed by the letters within the column

indicating the level of significance at P≤0.05 by Duncan’s Multiple Range Test (same letter within the column

of the respective hormone indicates the absence of difference; different letters indicate the significant

difference; and combination of letters indicate no significant difference).

Kader et al. (2015) 2(3): 192–203

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Seasonal effect on callus formation

This investigation was carried out in different seasons viz., rainy season, winter season and summer season

to check the seasonal effect for callus formation. From this experiment it was found that for callus culture of this

two species rainy season was best time (Fig. 6) as compared to other seasons which generally showed explants

dormancy and excretion of phenolic compound vigorously.

Figure 5. Effect of salt concentration on callus initiation and growth of Heritiera fomes.

Figure 6. Effect of various seasons on callus initiation in modified MS medium: A, Bruguiera gymnorhiza; B, Heritiera

fomes.

DISCUSSIONS

Literature studies indicated that the leaves of mangrove species are excellent source for callusing or cell

culture studies (Mimura et al. 1997, Hayashi et al. 2009). However in this study both the species failed to

produce callus from leaf. They gave callusing response either on shoot tip or nodal and intermodal segments

which was not previously reported for any mangrove species. This may be due to the selection of explants, their

size, age and ecological factors, which greatly influence the success of the in vitro culture which varies widely

from plant to plant (George 1993).

In this study mercuric chloride was used as surface sterilant as it is the most ly used surface sterilant for

tissue culture. Here for surface sterilization of Bruguiera gymnorhiza 0.2% HgCl2 (w/v) solution treatment for

Kader et al. (2015) 2(3): 192–203

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15- 20 minute incubation periods was required.

In vitro response of most of the mangrove species till now reported maximum were done with MS medium

viz Yasumoto et al. (1999) reported for Sonneratia alba; Al-Bahrany & Al-Khayri (2003) reported for Avicennia

marina; Ogita et al. (2004) reported for Kandelia candel; Kawana & Sasamoto (2008) reported for Sonneratia

alba and Bruguiera sexangula; Hayashi et al. (2009) reported for Avicennia alba, Avicennia marina, Sonneratia

alba and Bruguiera sexangula. In this present work for callus culture of Bruguiera gymnorhiza and Heritiera

fomes, the MS medium was modified. Similar findings of use of modified MS medium for cultivation of

mangroves were also reported by Rao et al. (1998), formulated a new medium named ‘X’ for Excoecaria

agallocha; Vartak & Shindikar (2008) for Bruguiera cylindrica. Besides for optimum growth of B. gymnorhiza

yeast extract and casein hydrolysates were added. It may happen because mangroves are unique in their

physiological adaptations. They can tolerate various nutrient statuses, water logging and various levels of sea

salts (Feller et al. 2002). Similarly plant tissue culture depends on media composition with special emphasis to

growth regulators, carbon source and organic additives, the genotype and the source of explants. In this study

we found that B. gymnorhiza gave no callus response in MS media but when the media was formulated with

thrice micro nutrients and addition of organic substances i.e., yeast extracts and casein hydrolysate the callus

initiation occurred. It may have happened because they are naturally adapted to higher micronutrients and

organic matter, particularly for Sundarban forest (Naskar & GuhaBakshi 1987, Ramanathan et al. 2008). In

Sundarban mangrove region sulphates are higher (Naskar & GuhaBakshi 1987, Ramanathan et al. 2008). In our

study, addition of thrice concentration of micro nutrient was actually the addition of higher sulphates as

micronutrient to MS. It is well known that in plant tissue culture media, casein hydrolysate is a rich source of

different amino acids (Shahsavari 2010, Talapatra & Raychaudhuri 2012). Addition of casein hydrolysate in the

medium is required because Mimura et al. (1997) showed that the Amino Acid medium (Thompson et al. 1986)

enhanced the rate of callus initiation of Bruguiera sexangula which is another species of the Bruguiera genus.

From this study and from the study of Mimura et al. (1997) it seems that somehow amino acids play a crucial

role for in vitro response particularly for this genus and casein hydrolysate was found to be fit for this type of

study.

The physiological state of the donor plants on growth depends on environment, which affects the response of

explants under in vitro conditions (Jahne & Lorz 1995). The MS medium presented here for H. fomes had low

nitrogen level as compared to other plant medium which supports the low nitrogen level in mangrove region

(Feller et al. 2002, Feller et al. 2003) as well as Sundarban mangrove forest as there is no humus deposition in

the soil (Naskar & Bakshi 1987, Ramanathan et al. 2008). So it seems that mangrove species can grow in vivo in

low level of nitrogen, which was also retained in tissue culture conditions. Similarly in vitro response to low

level of nitrogen was also reported by Mimura et al. (1997), Rao et al. (1998) and Arumugam & Panneerselvam

(2012). The complete omission of ammonium nitrate may be essential as it has toxic effect in many higher plant

species which inhibit plant growth (Britto & Kronzucker 2002, Shanjani 2003).

In this study B. gymnorhiza respond and grew better in 2% sucrose concentration in respective medium. In

mangrove ecosystem carbon source are varied and in Sundarban the parcentage of organic carbon source was

too low (Naskar & GuhaBakshi 1987). In the present study with B. gymnorhiza we found that callus initiation

rate was highest in 2% (w/v) sucrose containing medium which correlate with nutrient status of Sundarban

mangrove ecosystem.

From this experiment it was found that the NAA could alone or in combination with BAP initiate callus

from mangrove species. However in case of 2,4-D it failed to initiate callus alone or in combination with with

BAP from Bruguiera gymnorhiza. It may be because recent study showed that 2,4-D has toxic effect on plants

i.e., it alters the chlorophyll, protein and phenol content (Peixoto et al. 2007). Generally high concentrations of

cytokinin and low concentrations of auxins favour shoot response. Here in case of Heritiera fomes this type of

ratio too favoured callus initiation from this species.

In this experiment two different mangroves showed different pattern of NaCl tolerance. Based on this study

it was found that the Bruguiera gymnorhiza gave best callus initiation and growth at 60 mM NaCl

concentration. Mimura et al. (1997) found that seedling callus of Bruguiera sexangula gave highest growth at

100 mM NaCl. Sundarban this species is very common on the side of creeks and river beds and plays a

dominant role for its better adaptation to the higher degree of salinity and tidal influences (Naskar &

GuhaBakshi 1987). Heritiera fomes calli showed better response and growth at 20 mM NaCl concentration.

Kader et al. (2015) 2(3): 192–203

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Hossain et al. (2014) showed that Heritiera fomes prefers extremely low saline condition for their survival and

growth. Recent studies on palynological evidence and salinity influences have clearly showed that Heritiera

fomes has declined relatively recently as the salinity has increased in Indian Sundarban regions which are also

retained in this experiment (Naskar & GuhaBakshi 1987, Gopal & Chauhan 2006, Mitra & Banerjee 2010).

Under natural conditions, mangroves exhibit clear tolerance of salts differences among species. Findings of the

present study are comparable with naturally relative tolerance levels of two different mangrove species.

From this experiment it was found that, for tissue culture of these species rainy season was best time as

compared to other seasons which generally showed explants dormancy and excretion of phenolic compound

vigorously. Many tree species which are collected during rainy season (active growth time) shows tremendous

growth in in vitro conditions because physiological state of tissue of tree species varies due to variation of

season (El-Morsey & Millet 1996). During winter season the explants showed low viability i.e., dormant in

nature and exuded maximum phenolic compounds. This may be because the cytosolic ribosome contents are

altered in winter metabolism at cellular level in tree species (Haggman 1986).

Land destruction in coastal region is another important factor for mangrove extinction (Ohnishi &

Komiyama 1998). In Indian Sundarban region approximately 50% of land has been destroyed for human

habitation and settlement, agricultural development and brackish water fisheries (Ramanathan et al. 2008). The

present study clearly indicates that these species may be restored in low saline or none of saline land as callus as

it is being extensively used for afforestation programmes (Ahuja 1991) and tapping useful compounds from

plants. The present investigation is a primilary study for micropropagation of mangrove species as most

mangrove species remain very hard to be established through cell culture systems or callus culture (Kawana &

Sasamoto 2008). The totipotent character of plant cells and tissues can be expressed by their ability to

regenerate into plants via embryogenesis or organogenesis and for this histological study of callus is very much

needed. Both processes lead to in vitro regeneration and are a major prerequisite for genetic transformation

study. However, the widespread application of gene transfer techniques for crop improvement cannot be

successfully achieved if the processes leading to morphogenesis are not well understood. Callus culture give

tools for genetic cell transformation by somaclonal variation, induced mutagenesis and genetic engineering

which are not only much more rapid than conventional breeding but can also give rise to novel genes and

genotypes rather than other traditional methods like mass selection, inbreeding and hybridization which is

laborious and time consuming depending on environmental conditions and existing g ene pool(s) for plant

development (Ahmad et al. 2010). This study can thus provide opportunities of micropropagation of these

multipurpose mangrove plants.

According to UNESCO there are approximately 50 different types of true mangrove species are found

worldwide. Among them less than 10 true mangrove species were reported for in vitro study. It seems from this

study that different environmental condition or stress which promotes the growth of mangroves in vivo greatly

influences the in vitro culture of mangroves. Previous in vitro studies on mangroves were mostly based on the

effect of variety of hormones with their different concentration taken and different effect of sea salts taken with

their different concentration. However this present study clearly indicates that the in vitro studies of mangroves

not only depend on variety hormones or different sea salts but greatly influence by soil condition of their

habitual environment, seasonal condition etc. From this study it also seems that more and more in vitro studies

of mangroves are possible if researchers focus on their habitual environmental conditions. The results presented

here give an insight into the in vitro studies suitable for mangrove species which is correlated with various

environmental factors of mangrove ecosystems. Potentially, higher callus efficiency may be achieved through

investigating medium components other than this study, hormones other than used in this study and use of

various sea salts other than used in this study.

ACKNOWLEDGEMENTS

The authors are grateful to University Grant Commission (UGC), New Delhi, for providing financial

assistance to the first author. The authors extended their thanks to the head of the department of botany,

University of Kalyani for providing DST-FIST central equipment facility. The authors are thankful to Dr.

Soumen Saha for help in statistical analysis. They are also thankful to Subhas Bhaumik for help in histological

analysis.

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ISSN (E): 2349 – 1183

ISSN (P): 2349 – 9265

2(3): 204–214, 2015

Research article

Plant community structure and composition in secondary

succession following wildfire from Nuèes Ardentes of

mount Merapi, Indonesia

Sutomo1, 2

*, Richard J. Hobbs1 and Viki A. Cramer

1

1School of Plant Biology the University of Western Australia, 35 Stirling Hwy, Crawley, Perth, Western Australia 2Bali Botanic Garden, Indonesian Institute of Sciences (LIPI), Candikuning, Baturiti, Tabanan, Bali, Indonesia


Abstract: Patterns of plant community structure and composition during secondary succession

following volcanic-fire induced disturbance of nuées ardentes was examined in Mount Merapi

National Park, Indonesia. Five sites with different age (time since fire) and one undisturbed site

were sampled. Species richness, diversity, turnover and importance value index (IVI) were

calculated. Sixty one species belonging to 29 families were recorded in the study sites. The highest

number of species belonged to the Poaceae (10), followed by Fabaceae (9) and then Asteraceae

(6). The number of species present varied as time progressed with a rising trend of species richness

and diversity over time and significant differences in species richness and diversity among sites

(ANOVA, p = 0.05). Species turnover was highest between the 2006 and 1998 sites, and then

between the 1997 and 1994 sites. Species turnover between the 1998-1997 sites was similar to the

turnover between the 1994 site and the reference site. In terms of vertical structure, four strata

were identified in the fire sites whereas in the reference site, all five stratums (A, B, C, D, and E)

were present. In terms of quantitative structure based on IVI, each site had different dominating

species for tree, groundcover and seedling layers. Non metric multidimensional scaling (NMDS)

ordination of plots and analysis of similarity (ANOSIM) test results showed that there were

significant differences in species composition between sites (Global RANOSIM = 0.93, P < 0.001). In

the Mount Merapi succession, the changes in abundance of some invasive species such as I.

cylindrica, Brachiaria spp., and Eupatorium spp. are important to note. These invasive species

have different timing in entering the system, but Imperata cylindrica was noted almost constantly

in every stage of succession except in the undisturbed site.

Keywords: Plant community - Volcanic fire - Disturbance - Secondary succession

[Cite as: Sutomo, Hobbs RJ & Cramer VA (2015) Plant Community Structure and Composition in Secondary

Succession Following Wildfire from Nuèes Ardentes of mount Merapi, Indonesia. Tropical Plant Research 2(3):

204–214]

INTRODUCTION

In active volcanoes, volcanic activity remains the most significant threat to forest vegetation (Lavigne &

Gunnell 2006, Whitten et al. 1996). Fire is an integral part of volcanic disturbance and has shaped community

composition in montane forests of Java (van Steenis 1972, Whitten et al. 1996). On Mt. Merapi, the intense heat

(often more than 700° C) released from nuées ardentes ignites wildfires (Bardintzeff 1984). A nuèe ardente

(French for “glowing cloud”) is the “rapid movement of extremely hot (often more than 700° C) turbulent gases

and fragmental material across a land surface from a volcanic vent. The denser part of a pyroclastic flow, hugs

the ground and follows topography and moving with great force and speed (up to 200 km h-1)” (Dale et al.

2005a).

The montane forests of Java and Bali are not resistant to fire (Marrinan et al. 2005). The forests are easily

ignited under conditions of prolonged drought, and when lightning strikes oil-rich species such as Vaccinium

spp. On Mt. Merapi, nuées ardentes are the primary cause of forest fire (Simon 1998, Whitten et al. 1996).

Recovery of the montane forest following fire is usually slow (Horn et al. 2001). Fire destroys the aboveground

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part of shrubs and some surviving species may be covered with ash, which could slow the rate of the secondary

succession (Antos & Zobel 2005, Whitten et al. 1996). Severely burned areas on mountains in Java and Bali are

usually characterized by the increase in abundance of invasive species, such as alang-alang grass (Imperata

cylindrica), and also white-leaved „edelweis‟ (Anaphalis longifolia) and bracken fern (Pteridium aquilinum)

(Whitten et al. 1996). Homalanthus giganteus is also a common pioneer tree species that occurs during

secondary succession in these areas (van Steenis 1972).

Li et al. (1999) stated that many succession theories were based on intensive work in temperate forests.

Gomez-Pompa and Vasquez-Yanes (1981) and Chazdon et al. (2007) studied secondary succession that occurs

in the tropics; however their findings were based on work on old fields or in lowland tropical forests. Other

forest types such as volcanic tropical montane forest have received little attention (Tsuyuzaki & Hase 2005,

Whittaker et al. 1999). Furthermore, it is increasingly acknowledged that „one model fits all‟ is not appropriate

for all communities and ecosystems due to the complexity of each system (Hobbs et al. 2007).

The objective of this study was to describe plant community structure and composition in secondary

succession using a chronosequence of sites that had been burnt by fires caused by nuées ardentes in the tropical

montane forest of the Merapi Volcano National Park. We were particularly interested in whether there are any

differences in species diversity, turnover, and community structure and composition across sites of different

ages, and which species contributed the most to these differences.


Site description

Mt. Merapi (7º 35' S and 110º 24' E) is administratively located in two provinces, Central Java (Magelang,

Boyolali and Klaten Districts) and Yogyakarta (Sleman District). In Yogyakarta Province, Mt. Merapi (± 2,900

m asl) is located approximately 30 kilometres north of Yogyakarta. Mt. Merapi is representative of the

landforms, soils and vegetation on a volcanic mountain that typify a large portion of montane ecosystems in

Java (Whitten et al. 1996). Based on Schmidt and Fergusson‟s climate classification (1951), the Merapi area is

classified as type B - tropical monsoon area, which is characterized by a high intensity of rainfall in the wet

season (November–March) with a dry season that can often be very dry without any rainfall (April–October).

Annual precipitation varies from 2,500–3,500 millimetres (Anonym 2004). The variation of rainfall on Mt.

Merapi‟s slope is influenced by orographic precipitation. As in many other tropical monsoon areas, there are

minor temperature and humidity variations during the year. Relative humidity on Mt. Merapi varies from 70%–

90%, with daily average temperatures from 19° to 30° C (Dinas Kehutanan DIY 1999). Soils of the study area

are mainly of young volcanic-ash origin (regosol) with shallow and/or deep, low to medium fertility solums with

a profile not yet developed (Anonym 2004, Darmawijaya 1990). The soil textures are granulated, whereas the

structures are crumbly (Anonym 2004, Dinas Kehutanan DIY 1999).

On Mt. Merapi, areas which have been completely buried by the nuées ardentes deposits undergo primary

succession. These areas usually occur along the streams, channels or valleys created by the solid material flow

paths of nuées ardentes. The secondary succession areas were located adjacent to the primary succession areas.

These areas are the adjacent forest on either side of the valley or deposit channel which escapes burial and is

mainly scorched by the extreme temperature of the nuées ardentes. Sites or areas of different ages (years since

last nuée ardente) were selected to obtain a chronosequence. Identification of site age was conducted by

studying aerial photographs, topography maps, and nuée ardente history maps (obtained from the Merapi

Volcanology Observatory) to date sites affected by recent nuées ardentes. Identification was also conducted by

reconnaissance study, interviewing long-term residents of the surrounding villages, personal communication

with the national park‟s ranger and managers, and also field site visits. Sites also had to show no obvious signs

of human disturbance and be at least 50 metres from any human activities or structures. Based on these, we

chose four sites that were affected by nuées ardentes at different times (2006, 1998, 1997 and 1994) and one

forest area that was mostly undisturbed and had not been affected by nuées ardentes for at least 50 yr as a

reference site (Fig. 1). The five sites were located in a lower montane zone and were located at a range of

altitudes from 1,000 to 1,600 metres. Chronosequence assumptions were met within these sites as they had

similar environmental conditions such as climate, substrate, topography and geomorphology, although we

acknowledge the limitations of the chronosequence approach and the potential for site-specific factors to be

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important. The fieldwork was conducted from March to August 2008. Average environmental conditions in

each site are summarized in table 1.

Figure 1. Map of mount Merapi National Park‟s eruption deposit sites (Circular symbols refer to the position of sampling

sites in each deposit. The rectangle refers to the site position of an undisturbed forest in Kaliurang).

Table 1. Site location, nuées ardentes history and environmental information in each study sites at Mt. Merapi National Park.

Location Year of nuée ardente Site age (years) Soil type Elevation (m)

Kaliadem 2006 2 Regosol 1,220

Kalilamat 1998 10 Regosol 1,579

Kalibedog 1997 11 Regosol 1,207

Kalikuning 1994 14 Regosol 1,180

Kaliurang - - Regosol 1,000

Vegetation sampling

In April 2008, vegetation was sampled in each of the four sites burnt by fire from nuées ardentes in 1994,

1997, 1998 and 2006. One area of unburnt forest (the reference site) was also sampled. The position and altitude

of each site were recorded using a GPS, and slope was measured using a clinometer. At each site, an area of

approximately 2.5 hectares was chosen and five circular plots (diameter range approximately 60 metres) were

randomly placed in the chosen area. In each of these larger plots, three sets of circular plots of 10, 5 and 2

metres diameter were nested within each other to measure trees (10 metre plots), groundcover (5 meter plots)

and seedlings (2 metre plots) (Isango 2007, Supriyadi & Marsono 2001). The species name, height and

diameter of trees (dbh ≥ 10 cm) and young trees (dbh 2.0–9.9 cm, height ≥ 1.3 m) were recorded. Understorey

plants and seedlings were counted (Kent & Coker 1992). All plants were identified to species level when

possible. Identification was conducted at the dendrology laboratory, Faculty of Forestry, Gadjah Mada

University Yogyakarta, Indonesia. Identification was done using flora books such as “The Flora of Java”

(Backer & van den Brink 1963) and “Mountain Flora of Java” and the results were confirmed by a botanist in

the Faculty.

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Data analysis

Species diversity at each site was calculated using the Shannon-Wiener diversity index. Differences in

diversity between sites were tested for significance using a one-way ANOVA in SPSS package V.11.5. To

examine short term species turnover (beta diversity), a modified Sorensen‟s community correspondence index

or CCI was used (Barbour et al. 1980, Cook et al. 2005) with the formula as follows:

ba

2c CCI

Where, a = the number of species present in the first community, b = the number of species present in the

second community, and c = the total number of species found in both communities.

I then calculated D, which is an index of how much a species list changes across sites with the formula as

follows (Cook et al. 2005):

CCI 1D

This index ranges from 0 to 1, and a low value indicates little change in the species composition between

sites whereas a high value indicates the opposite.

In order to examine the vertical structure, forest vegetation was divided into five strata (A, B, C, D and E), as

recognized for humid tropical forests (Simon 1996). Stratum A consisted of emergent trees more than 35 metres

tall. Stratum B was the main canopy layer with trees 18–35 metres in height. Stratum C consisted of young trees

8–18 metres tall. Stratum D consisted of shrubs and sapling (of trees) with height ranges from 1.5–5.0 metres.

Stratum E was the groundcover layer, including grasses, herbs, tree seedlings and fern allies (Simon 1996). The

number of trees, young trees (poles), sapling and shrubs that have the characteristics of stratum A, B, C and D

were noted, while the number of groundcover species was noted for the E stratum in each of the study sites.

Importance Value Index (IVI) (Curtis & McIntosh 1950, Kent & Coker 1992) was used to describe the

quantitative structure of the community. This statistic represents the contribution that a species makes to the

community in terms of the number of plants within the plots (density), its contribution to the community

through its distribution (frequency), and its influence on the other species through its dominance. Importance

Value Index was calculated for each species of tree and groundcover in each of the study sites. The formula for

tree IVI is as follow:

IVI = RD + RF + RDm

Where, RD = relative density of a species, RF = relative frequency of a species and RDom = relative

dominance of a species.

Relative Density of species A = 100% species all of individualnumber Total

speciesA of individual ofNumber

Relative Frequency of species A = 100% species all of valuefrequency Total

speciesA of valueFrequency

Relative Dominance of species A = 100% species all of valuedominance Total

speciesA of valueDominance

Dominance values for a tree species were obtained by dividing the basal area of the tree with the size of the

plot (Simon 1996, Supriyadi & Marsono 2001). The IVI formula for groundcover species (including seedlings)

was similar to the tree layer but without the calculation of relative dominance (Kusmana 1995), and so the

formula is as follow:

IVI = RD + RF

Where, RD = relative density of a species, and RF = is relative frequency of a species.

Species abundance data were square root transformed prior to all multivariate analyses. A resemblance

matrix based on a Bray-Curtis similarity index was generated as a basis for the subsequent ordination and cluster

analyses. Plant species composition and abundance at each site were compared using non-metric

multidimensional scaling ordination (NMDS) (Clarke 1993). Statistically significant differences in species

composition and abundance between the sites were determined by analysis of similarity (ANOSIM), which tests

the null hypothesis that there is no difference in species composition and abundance among groups (Clarke

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1993). SIMPER, an analysis that calculates the average Bray-Curtis dissimilarity between all samples, was used

to identify the species that differentiate sites (Clarke 1993). These analyses were done using PRIMER V.6

(Clarke & Gorley 2005).

RESULTS

Sixty one species belonging to 29 families were recorded in the study sites. The highest number of species

belonged to the Poaceae (10), followed by Fabaceae (9) and then Asteraceae (6). There were significant

differences in species richness between sites (ANOVA P = 0.05, table 2). Species richness was lowest at the

2006 site and highest at the 1994 site. Species richness in the reference site (undisturbed site) was much lower

when compared to the 1994 site. Species richness in the reference site was significantly lower than in all but the

2006 site.

Table 2. Species richness and diversity in the burnt sites and reference site in mount Merapi National Park. Superscript

letters (a-c) after mean values (±SD) indicates significant difference between sites as assessed with Tukey‟s HSD test. Dates

are those in which the site was burnt by fire generated by nuées ardentes.

ANOVA group/years since fire Species richness Species diversity

2006 site 9.20 (±1.48)a 1.91 (± 0.19)a

1998 site 14.0 (±3.39)b 2.13 (± 0.27)ab

1997 site 15.4 (±1.51)b 2.41 (± 0.19)bc

1994 site 19.4 (±2.96)c 2.7 (± 0.21)c

Reference site 10.6 (±1.67)a 2.21 (± 0.27)ab

The changes in species diversity are not as distinct as the changes in species richness over time (ANOVA P

= 0.05). The reference site is significantly different to the 1994 site, but not significantly different from 2006,

1998, and 1997. The 1998 site is not significantly different from 2006 and 1997 sites and also the 1997 site is

not significantly different from the 1994 site.

Species turnover was highest (lowest species similarities) between the 2006 and the 1998 sites, and then

between the 1997 and 1994 sites (Table 3). Species turnover between the 1998–1997 sites was similar to the

turnover between the 1994 site and the reference site.

Table 3. Species turnover rates (D) between pairs of sites in the chronosequence on mount Merapi.

2006-1998 1998-1997 1997-1994 1994-Ref site

D 0.89 0.63 0.83 0.66

Sorenson Index 0.11 0.37 0.17 0.34

In terms of vertical structure, the number of individuals found in each stratum indicates the presence of the

particular stratum in each site (Table 4). In the 2006 site, stratums B, C, D, and E were recorded. The 1998,

1997 and 1994 sites also had four stratums (B, C, D, and E) whereas in the reference site, all five stratums (A,

B, C, D, and E) were present.

Table 4. Number of individuals in each stratum for each site of secondary succession at mount Merapi. Stratum A refers to

the number of trees that are more than 35 m in height. Stratum B is number of trees that are 18 to 35 m in height. Stratum C

comprises of trees that are 8 to 18 m tall. Stratum D is the total number of saplings and Stratum E is the total number of

groundcover species.

Stratum 2006 site 1998 site 1997 site 1994 site Ref site

A - - - - 41

B 3 5 16 25 28

C 5 45 18 17 1

D 4 10 15 6 11

E 12 20 23 25 16

In terms of quantitative structure, tree and groundcover species in the sites were compared on the basis of the

Importance Value Index, (IVI) (Table 5). In the 2006 site, the tree layer was dominated by Pinus merkusii,

whereas in the 1998 and 1997 site, Homalanthus giganteus and Paraserianthes falcataria were the most

important tree species. In the oldest site (1994), Schima wallichii and P. merkusii were the most important tree

species whereas in the reference site, Altingia excelsa was the most important tree species. In the groundcover

layer, the 2006 site was dominated by Imperata cylindrica, whereas Eupatorium riparium was the most

important species in the 1998 and 1997 sites. In the oldest site (1994) Brachiaria reptans was the most

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important species, while in the reference site, Selaginella doederleinii was the most important species in the

groundcover layer. In the tree seedling layer, Acacia decurrens was the most dominant tree seedlings species in

the 2006 site, followed by P. merkusii. Albizia lopantha dominated the seedling layer in the 1998 site, while in

the 1997 and 1994 sites Calliandra callothyrsus was the most important seedling. In the reference site, A.

excelsa was the dominant seedling.

Table 5. Importance Value Index (IVI), and shade tolerance for the most important species in each stratum at each of the

study sites. Asterisks indicate exotic species.

Species Shading tolerance 2006 site 1998 site 1997 site 1994 site Reference site

Trees

Acacia decurrens* Intolerant 17.27 6.17 - 58.88 -

Albizia lopantha Intolerant - 33.68 - - -

Altingia excelsa Tolerant 60.63 - - 8.44 217.51

Erythrina sp Intolerant - 12.51 - - -

Homalanthus giganteus Intolerant - 148.65 - - -

Macaranga javanica Intolerant - 31.55 - - -

Paraserianthes falcataria Intermediate - - 116.20 46.83 -

Parkia sp Intolerant - 6.53 29.32 - -

Pinus merkusii Intermediate 222.09 - - 87.85 54.05

Schima wallichii Intermediate - - 104.92 89.0 21.80

Groundcover

Brachiaria reptans* Intermediate - 2.19 19.90 16.54 2.23

Eleusine indica Intermediate - - 8.82 14.49 3.11

Eupatorium riparium* Intermediate - 55.35 57.03 - 13.43

Eupatorium odoratum* Intermediate - 8.69 4.70 1.95 -

Imperata cylindrica Intolerant 73.77 14.18 23.51 9.55 -

Polygala paniculata* Intermediate 26.83 - 2.35 7.60 -

Selaginella doederleinii Tolerant - - 0.87 0.56 61.80

Seedling

Acacia decurrens* Intolerant 99.04 13.88 - 13.57 -

Albizia lopantha Intolerant - 77.77 - - -

Altingia excelsa Tolerant 9.52 - 11.05 7.46 33.35

Calliandra callothyrsus Intermediate - - 146.56 84.04 -

Pinus merkusii Intermediate 62.85 - - 41.31 -

Schima wallichii Intermediate - - 17.10 19.69 22.40

In addition to the IVI, table 5 also shows the presence and absence of the most important (dominant) species

in each layer throughout the succession. In the tree layer, A. excelsa and P. merkusii were present at the

youngest site (2006) and then absent in the next two older sites (1998 and 1997), and then reappeared in the

oldest (1994) and the reference site. Erythrina sp., H. giganteus, Albizia lopantha and Macaranga javanica were

only present at the 1998 site. In the groundcover layer, I. cylindrica was recorded in all four of the burnt sites,

but was more dominant in 2006, 1998 and 1997 sites than in the 1994 site, and was absent in the reference site.

In contrast, Brachiaria reptans was absent in the 2006 site and then present throughout the rest of the

chronosequence and was at a very low abundance in the reference site. Selaginella doederleinii started to appear

in the 1997 and, 1994 sites and became dominant in the reference site.

There was clear separation between the sites as shown by the NMDS ordination result (Fig. 2). Plots from

the youngest site (2006) were separated from plots from the older sites (1998, 1997 and 1994), and from the

undisturbed site. An analysis of similarity (ANOSIM) test confirmed that there were significant differences in

Bray-Curtis species similarities between sites (Global RANOSIM = 0.93, P < 0.001).

Six pairwise comparison tests between sites (2006 and 1998, 2006 and 1997, 2006 and 1994, 2006 and

reference site, 1998 and 1994, and 1994 and reference site) had an R value of 1.0 (Table 6). The comparison

between the 1997-1998 sites and 1997-1994 sites had R-values of 0.72 and 0.86 and also, the comparison

between 1997-undisturbed and 1998 undisturbed had R-value of 0.98 (Table 6).

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Figure 2. NMDS of sites based on vegetation composition and abundance: 2006 site (triangles), 1998 site

(inverse triangles), 1997 site (squares), 1994 site (diamonds) and reference site (circles).

Table 6. ANOSIM pairwise test of NMDS vegetation plots ordination. Sample statistic

(Global R): 0.93, significance level of sample statistic P < 0.001, number of permutation: 999.

Groups R Statistic

2006, 1998 1

2006, 1997 1

2006, 1994 1

2006, Reference site 1

1998, 1997 0.72

1998, 1994 1

1998, Reference site 0.98

1997, 1994 0.86

1997, Reference site 0.98

1994, Reference site 1

In table 7, Eupatorium riparium contributed most to the dissimilarity between the 2006 and 1998 sites

(21.27%), 2006 and 1997 sites (20.96%), 1998 and 1994 sites (16.31%), and 1997 and 1994 sites (21.39%).

Brachiaria reptans contributed most to the dissimilarity between the 2006 and 1998 sites (9.96%) and 1998 and

1997 sites (9.89%). Dichantium caricosum contributed most to the dissimilarity between the 2006 and 1994

sites (10.60%). Selaginella doederleinii was the most important species contributing to dissimilarities between

the reference site and the burnt sites. Imperata cylindrica was the second most important species in the

comparison between 2006 and 1998 sites and 2006 and the reference sites.

DISCUSSION

In the first decade after disturbance by fire there was a rapid recovery at the sites, with 54 species belonging

to 23 families recorded in the secondary forest at the study sites. The highest number of species belonged to the

Poaceae (10), followed by Fabaceae (9) and then Asteraceae (6). Species richness and diversity increased with

time since the fire, however species richness and diversity in the reference site was not significantly different

from the youngest (2006) site. This pattern was similar to that reported in other studies where species diversity

reached its peak in older succession sites after most of the climax species had entered the system, and then

decreased with the loss of the species present in early successional stages (Magurran 1988, Peet 1992, Zhu et al.

2009). The results support the hypothesis of Aubert et al. (2003) that species diversity will increase during the

early succession stage, reach a maximum in the mid-succession stage and decrease in the late succession stage.

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Table 7. SIMPER result. Percentage contribution of species to average Bray–Curtis dissimilarities in all pairs of sites. Only

those species with a contribution to average dissimilarity of >5% are included. The average dissimilarity value (%) is also

shown for each pair of the sites. Asterixis indicates exotic species.

Site comparison

Species

20

06 a

nd

199

8

20

06 a

nd

199

7

19

98

an

d 1

99

7

20

06

an

d 1

99

4

19

98

a

nd

19

94

19

97

an

d 1

99

4

20

06

an

d R

ef s

ite

19

98

an

d R

ef s

ite

19

97

an

d R

ef s

ite

19

94

an

d R

ef s

ite

Brachiaria paspaloides* 6.21 - 6.51 - 5.07 - - 5.98 - -

Brachiaria reptans* - 9.96 9.89 8.01 5.42 8.45 - - 8.30 5.89

Calliandra callothyrsus - 8.01 8.68 - - 6.31 - - 6.80 -

Dichantium caricosum* - - - 10.60 7.81 7.22 - - - 8.08

Eleusine indica - - - 8.96 6.53 - - - - 5.97

Eupatorium odoratum* - - - 9.84 5.29 6.42 - - - 7.42

Eupatorium riparium* 21.27 20.96 5.76 - 16.31 21.39 6.15 13.94 13.60 -

Imperata cylindrica 9.89 - 7.41 9.02 - 5.63 15.14 5.83 9.89 5.13

Polygala paniculata* 6.82 - - - - - 6.81 - - -

Selaginella doederleinii - - - - - - 18.45 16.94 13.77 14.33

Average dissimilarity (%) 88.98 79.38 61.51 75.50 85.38 60.75 95.56 83.35 80.67 87.82

A decrease in the light availability at the forest floor as the succession proceeds might be the cause of the

decline of species diversity in the reference site (Gomez-Pompa & Vazquez-Yanes 1981). Direct shading of

overstorey species inhibits the existence and regeneration or growth of less tolerant and intolerant understorey

species in the reference site (Lepš 1990).

There was progressive development of forest structure over time. Although all of the burnt sites had four

strata (B, C, D, E), the number of individuals in each stratum differed. The number of individuals of stratum B

(tall trees 18–35 m) was lowest in the 2006 site, greater in the older sites, but was the greatest in the reference

site. The reference site had five strata (A, B, C, D, and E) with the lowest number of individuals of stratum E

compared with the proportion of stratum E in the burnt sites. There were also differences in the patterns of

abundance of the most important species with different light requirement characteristics (shade

tolerant/intolerant) in the groundcover layer. The gradual decrease in Imperata cylindrica (shade-intolerant

species) abundance over time contrasted with the gradual increase in the abundance of Selaginella doederleinii

(shade-tolerant species), suggesting that there was a decrease in the light availability at the forest floor as the

canopy developed and the succession proceeded.

Over the course of succession, the characteristics of species found at a site will change (Wills 2002). In the

Mt. Merapi sites, the younger sites were characterized by shade-intolerant colonizer species with good dispersal

capability. I. cylindrica is a widely distributed invader grass that has a long record of colonising cleared lands in

Indonesia (Eussen & Soerjani 1975, Soerjani et al. 1983). I. cylindrica has wide-spread rhizomes and its seeds

are wind-dispersed (Jonathan & Hariadi 1999), making it an effective colonizer following fire (Murniati 2002).

Acacia decurrens, however, is a nitrogen-fixing shrub that is usually recruited after fire. At Mt. Merapi, it may

have regenerated following the nuées ardentes fire from a soil seed bank (Hardiwinoto et al. 1998, Spurr &

Barnes 1980). I. cylindrica and A. decurrens can also be found in other degraded areas on volcanoes in Java,

such as in Mt. Bromo-Tengger and Mt. Semeru (Anonym 2009, Whitten et al. 1996). The species that occurred

in the older sites and reference site on Mt. Merapi were characterized as intermediate to shade-tolerant species

with greater longevity. In the older sites, A. decurrens was replaced by the leguminous tree, Calliandra

callothyrsus, which occurred with other tree species such as Altingia excelsa. A. excelsa is a native emergent

tree species and its seedlings are shade tolerant. Older sites were also characterized by the presence of the fern

Selaginella doederleinii and the exotic invasive Eupatorium spp. in the groundcover layer. Eupatorium spp. is a

fast growing species, usually found on steep slopes in a wide range of soil conditions and light availability

(Heyne 1987).

Many studies have shown that generally species composition changes with time after a fire (Clearly et al.

2006, Reilly et al. 2006, Ross et al. 2002, Spencer & Gregory 2006). The result of NMDS ordination was

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notable in that species composition differed among all sites, suggesting that the species composition changes

with time after a fire. The 2006 and 1998 sites were different in terms of floristic composition and abundance

with highest species replacement rate when compared with the replacement rate in the other sites (D = 0.89).

Altingia excelsa, Pinus merkusii, and Polygala paniculata which were present in the 2006 site, dropped out in

the 1998 site whereas there was an increase in the number of species from the Fabaceae family in the 1998 site

with the colonization of Albizia lopantha, Erythrina sp and Parkia sp. The differences in species composition

between the 1998 and 1997 sites (short interval) was the lowest in all of the site pair-wise comparisons, but they

were still significantly different from each other. Consistent with this, the species replacement rate was also

lower (D = 0.63) when compared with the replacement rate in the other site comparisons. Although ANOSIM

showed that the reference site and the 1994 site were significantly different, the turnover rate between these sites

was more or less the same as the rate in the 1998-1997 sites (D = 0.66). This result indicates that some of the

species that characterized the reference site, such as Altingia excelsa, Schima wallichii and Selaginella

doederleinii, had appeared earlier in the 1994 site and thus suggested convergence of floristic composition in

these sites.

In the Mt. Merapi succession, the changes in abundance of some invasive species such as I. cylindrica,

Brachiaria spp., and Eupatorium spp. are important to note. I. cylindrica is an invasive native of south-east

Asia. I. cylindrica dominated the early succession sites, but was not recorded in older sites as it was most likely

shaded out by increasing canopy cover. In contrast, the invasive exotic species Eupatorium spp. remained in the

system long after the fire had occurred and forest structures had developed. Eupatorium is native to South

America, and this noxious and highly competitive species has become a problem elsewhere in Asia, such as in

Nepal (Kunwar 2003). In the longer term, domination of invasive exotic species may limit the chance of

recruitment of other native species including seedlings of woody species, thereby reducing diversity and even

changing the successional trajectory and ecosystem functioning (Dale et al. 2005b, Hobbs & Huenneke 1992,

Raghubanshi & Tripathi 2009, Standish et al. 2009).

This study suggested that the Merapi forest exhibited a high resilience for site recovery following nuées

ardentes-induced wildfire with the rapid re-colonisation of plant species. It is also important to consider the

potential problems of invasive species Eupatorium spp. as weeds, as these species remain abundant even in the

much more developed sites. These findings may have important consequences for forest management as there is

still much to learn about the capability of alien invasive species to change soil chemical properties, which can be

crucial factors in driving the successional trajectory.

ACKNOWLEDGEMENTS

We would like to thank AusAid for funding this research. We are very grateful to Kuspriyadi Sulistyo from

the Merapi National Park for granting the research permit to our project. Gratitude also goes to Mbah Maridjan

the gatekeeper and caretaker of Mt. Merapi and also for kind helps of the fieldwork team, Gunawan, Ali, Indri

and Iqbal.

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ISSN (E): 2349 – 1183

ISSN (P): 2349 – 9265

2(3): 215–223, 2015

Review article

Application of information technology and GIS in agroforestry

Rajesh Kumar Mishra1* and Rekha Agarwal

2

1Tropical Forest Research Institute, P.O. RFRC, Mandla Road, Jabalpur - 482021, Madhya Pradesh, India 2Department of Physics, Government Model Science College, Jabalpur - 482021, Madhya Pradesh, India


Abstract: Computer-based Decision Support Tools (DST) helps to integrate information to

facilitate the decision-making process that directs development, acceptance, adoption, and

management aspects in agroforestry. Computer-based DSTs include databases, geographical

information systems, models, knowledge-base or expert systems, and „hybrid‟ decision support

systems. Although agroforestry lacks the large research foundation of its agriculture and forestry

counterparts, the development and use of computer-based tools in agroforestry have been

substantial and are projected to increase as the recognition of the productive and protective

(service) roles of these tree-based practices expands. The utility of these and future tools for

decision-support in agroforestry must take into account the limits of our current scientific

information, the diversity of aspects (i.e. economic, social, and biophysical) that must be

incorporated into the planning and design process, and, most importantly, who the end-user of the

tools will be. Incorporating these tools into the design and planning process will enhance the

capability of agroforestry to simultaneously achieve environmental protection and agricultural

production goals. This paper highlights the relevance of information technology (IT) in

Agroforestry. Existing areas of applications such as forest and environmental management, specie

identification and research publications are identified. The paper also looked into future possible

usage of information technology and concludes that while the application of information

technology to Agroforestry practices nowadays is of tremendous importance it is important to

know that there are still more areas where information technology would be applicable in

Agroforestry which are yet to be discovered.

Keywords: Forest management - Information technology - Agroforestry education

[Cite as: Mishra RK & Agarwal R (2015) Application of information technology and GIS in agroforestry.

Tropical Plant Research 2(3): 215–223]

INTRODUCTION

Within half a century, computers and information technology have changed the world and affected millions

of lives in ways that no one could have foreseen (Heathcote 2000). The great impacts, contributions to

knowledge, importance and economic achievements that have emerged from the fields of computer science

(information science) and electronic engineering, in the 21st century, are revolutionary and mind boggling

(Bamgbade 2011). The extent to which IT applications have improved agro-forestry practices in recent times

cannot be over emphasized. Area of application includes:

Forestry and environmental management, species identification, research publication, Information

Communication Technology ICT in agro forestry education, plant pathology studies, wood anatomy, biometrics,

Data management, modelling, analysis and mining. The list is infinite; however some of these applications

would be discussed in the present paper.

Advances in information and communications technology (ICT) and knowledge management (KM) have

changed the way people learn and e-learning is increasingly recognized as a viable and learner-friendly

approach that can complement, or even replace, more traditional training and education approaches. Agriculture

however is a very practical subject and not all of it can be generalized at a global level since local context will

largely determine success or failure of agricultural and natural resources management innovation. The

management of plant genetic resources for example involves practices that are almost impossible to teach in an

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online environment and transmission of knowledge is often better achieved through peer-to-peer learning

(Baena et al. 2007). Likewise, many agricultural practices need to be adapted to local biophysical and

socioeconomic conditions if they are to be successfully adopted by those they intend to serve. Blended learning,

combining an online, more general learning experience with more practical face-to-face problem solving

activities, has the potential to include more learners while dealing with the issues of practicality and

contextualization.

Agroforestry, the deliberate integration of trees into crop and livestock operations, has the potential to

achieve many of the environmental, economic, and social objectives being demanded from working landscapes

by landowners and society (Fig. 1). By adding structural and functional diversity to the landscape, these tree-

based plantings can perform ecological functions that have significance far greater than the relatively small

amount of land they occupy (Guo 2000, Nair 2001, Ruark et al. 2003). Realizing this potential is, however, a

complex task of determining what opportunities, limitations, and trade-offs exist in each situation, and of

designing an agroforestry practice that achieves the best balance among them. There are numerous impacts

created by agroforestry plantings, ranging from intended to non-intended and, therefore, ranging from

detrimental to advantageous, occurring both on- and off-site, and varying over time. Consequently, if

agroforestry is to be a viable strategy in promoting agro-ecosystem sustainability, the decision making process

must incorporate many considerations, not only at the practice scale but also at the larger scales of farm,

landscape, and watershed (Schoeneberger et al. 1994). Simply putting, agroforestry creates a complex system of

interactions that must be managed for multiple objectives, multiple alternatives and multiple social interests and

preferences, while being applied over a wide range of landscapes and landscape features.

Figure 1. Wheat based agro-forestry system with: A, Walnut; B, Salix.

The decision-making process involved in agroforestry research, development and application is composed of

several components: the person or group making the decision, the problem, the approach or method to solve the

problem, and the decision. Decision support tools (DST) are a wide variety of technologies that can be used to

help integrate diverse and large sets of information. DSTs do not replace the decision-making by the landowner

or natural resource manager, but they do facilitate the decision-making process by making the planning process

more informed and more objective (Grabaum & Meyer 1998). Although agroforestry, like most natural-resource

management sciences, is characterized by high complexity of which we have limited understanding and data

(Sanchez 1995, Nair 1998), the science and application of agroforestry can be greatly enhanced through the use

of these tools.

FOREST AND ENVIRONMENTAL MANAGEMENT

Mathematical and computational programming in Forest Management

Mathematical and computational programming remains a viable approach for strategic planning in forestry

and agriculture worldwide. One of such computational based system is the SPECTRUM used by the United

State government to carry out strategic planning in agroforestry. The system which evolved from an earlier

system (FORPLAN) has the following key attributes:

1. Multi-resource modeling

The system provides a generic framework for modelling any resource. A basic configuration depends on

user-defined analysis units, management actions, activities and outputs, resource coefficients, and economic

information.

A B

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2. Spatial and temporal scales

Spectrum applications are not scale-specific. Up to 90 time periods of any length may be used to support

analysis at relevant spatial and temporal scales.

3. Multiple options for mathematical programming

Spectrum supports numerous combinations of optimization techniques and objective functions. Optimization

techniques includes,

• Linear programming (optimization of a single criterion).

• Mixed-integer programming (optimization with categorical outcomes).

• Multi-objective goal programming (simultaneous optimization of multiple goals).

• Stochastic programming to account for random events such as fires, pest epidemics, and uncertainty about

data.

Specifications for objective functions

Options for objective functions in traditional linear programming include maximizing or minimizing a single

outcome or measure of performance. Objective Functions for goal programming include minimizing under-

achievement of goals, minimizing over- achievement of goals beyond thresholds, or minimizing both.

Two additional options for objective functions are MAXIMIN (maximizing the minimum level of

occurrence for a critical resource) and MIN/MAX (minimizing the highest level of occurrence of an undesirable

outcome).

Simulation of ecological processes and modelling natural disturbance

Spectrum allows embedding simulation of ecological processes and modelling of natural disturbances by

means of state, flow, and accessory variables in dynamic equations.

The Regional Ecosystems and Land Management (RELM) system extends the utility of Spectrum solutions

by apportioning forest-wide, strategic planning solutions to tactical sub-units of the forest such as watersheds.

Cumulative effects and connected actions can be analysed both within and between sub-units, allowing

planners to evaluate how alternative management scenarios affect neighbouring units.

Research publication

Information dissemination is a prominent activity in any research institute as it is the means through which it

could be adjudged whether it is living up to the mandate and purpose for which, it was established. Also,

research publications play a pivotal role in any academics environment. Paper publication is a useful instrument

through which research discoveries and breakthroughs are disseminated to the stakeholders. However,

publishing research papers in a manual format is attached with great difficulties and problems, which includes

ineffective and inefficient delivery system of the journal as at when due, prone to natural disasters, lost, theft,

mutilation. Sequel to the aforementioned problems, a software (FRIN –eJOURNAL: An Electronic Submission

Platform for Research papers) has been developed in FRIN which would allow for easy electronic retrieval,

storage and efficient research information delivery system. It will go a long way to automate the existing manual

journal with easy search tool and navigation properties, providing researchers, administrator and FRIN editors

with separate interface with hands on functionality and notification capability also creating a proper record of

subscribers and records of subscription. It is cost effective and is not regional bound.

Species identification

Although automated species identification for many reasons is not yet widely employed, efforts towards the

development of automated species identification systems within the last decade is extremely encouraging; that

such an approach has the potential to make valuable contribution towards reducing the burden of routine

identification.

There are many factors influencing the taxonomic impediment to the study of biodiversity. A major one

being that the demand for routine identification in biodiversity studies extends beyond the available resources.

In many spheres the volumes of plant or animal specimens that can usefully be obtained, particularly using

modern sampling methods, vastly outstrip any capacity to identify this material. This has limited the progress in

some aspect of biodiversity research. These demands are likely to steadily increase as the proportion of

previously un-described species in local, national or regional floras and fauna declines and as requirement or

desirability of biodiversity inventories and other such survey grows.

Mishra & Agarwal (2015) 2(3): 215–223

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This has led to several solutions being preffered to reduce the burden of routine identification. One of the

preffered solutions is automating the identification process in some way. This is generally referred to as

Computer Assisted Taxonomy (CAT). However, the development and application of an automated approach to

taxonomic identification has remained a minority interest till date.

Among reasons for this are the notions that it is too difficult, too threatening, too different or too costly. It is

most encouraging to know that despite these limitations, efforts towards the development of automated species

identification systems have been progressive.

From the evidences witnessed in this area, it buttresses the present minority notion that the automation of

species identification process is possible and achievable. A system that uses binary codes generated based on the

morphological characters of trees to uniquely identify tree species has been developed. Though this is not the

first time an attempt is made to automate species identification using their morphological characters, our

approach is far simpler and less expensive to implement. For instance while previous approaches are centred

round the need for a computerized pattern recognition system, ours does not require such. We were able to

easily prove the effectiveness of the system by restricting our study to the over one thousand Nigerian Trees

species. All the user need is a functional computer system, a ruler and personal ability to supply answers to the

questions asked by the system and the tree identification process is complete.

INFORMATION TECHNOLOGY IN AGRO FORESTRY EDUCATION

Information technologies (ITs) have the potential to enhance access, quality, and effectiveness in education

in general and to enable the development of more and better teachers (Fig. 2). As computer hardware becomes

available to an increasing number of schools, more attention needs to be given to the capacity building of the

key transformers in this process, namely, teachers.

While societies undergo rapid changes as a result of increased access to information, the majority of the

school going youth continues to undergo traditional rote learning. Very little is done to take advantage of the

wealth of information available on the Internet. Whereas the processing of information to build knowledge is

one of the essential literacy skills vital for the workforce in the 21st century, it is often overlooked in current

educational practices. The Computers for Schools Program appears to be doing valuable work and in the process

has become an unwitting champion of ITs in education. Its experiences are real, its challenges huge, and the

lessons valuable for the future resource for poor countries. In order to function in the new world economy,

students and their teachers have to learn to navigate large amounts of information, to analyse and make

decisions, and to master new knowledge and to accomplish complex tasks collaboratively. Overloaded with

information, one key outcome of any learning experience should be for learners to critically challenge the

material collected in order to decide whether it can be considered useful input in any educational activity.

This is the basis for the construction of knowledge. The use of ITs as part of the learning process can be

subdivided into three different forms: as object, aspect or medium.

As object, one refers to learning about ICTs as specific courses such as 'computer education.' Learners

familiarize themselves with hardware and software including packages such as Microsoft Word, Microsoft

Excel, and others. The aim is computer literacy.

As aspect, one refers to applications of ICTs in education similar to what obtained in industry. The use of

ITs in education, such as in computer-aided design and computer aided agroforestry technology, are examples.

ITs are considered as a medium whenever they are used to support teaching and learning.

The use of IT as a medium is rare where the availability of resources is a major obstacle to the widespread

integration of ITs in education. In order to sustain what has already been done and expand into areas still

unreached. Sequel to this is the need to explore the use of ITs in education, such as in computer-aided design

and computer-aided agroforestry technology, are examples.

Need of applications of ITs in Agroforestry Education

With the advent of IT, it is found that IT forms the "backbone" of several industries and is today a dominant

force in enabling companies to exploit new distribution channels, create new products, and deliver differentiated

value added services to customers. IT is also an important catalyst for social transformation and national

progress. Disparities in levels of IT readiness and usage could translate into disparities in levels of productivity

and, hence, different rates of economic growth. It is also important to observe that ICTs can lead to economic

growth but not development. The social exclusion of large groups of persons, especially women, children, and

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people living in rural areas, is a common phenomenon when adequate planning has not accompanied IT

exploitation. Education faces a number of problems. These problems include the shortage of qualified teachers,

very large student populations, high drop-out rates of students and teachers, and weak curricula.

All of these negative aspects result in poor delivery of education. The education crisis is worsened by the

devastating effects of increasing poverty, a brain drain in the teaching community, budgetary constraints, poor

communication, and inadequate infrastructure. Technology is not new to education. However, contemporary

computer technologies, such as the Internet, allow new types of teaching and learning experiences to flourish.

Many new technologies are interactive, making it easier to create environments in which students can learn by

doing, receive feedback, and continually refine their understanding and build new knowledge.

Access to the Internet gives unprecedented opportunities in terms of the availability of research material and

information in general. This availability of research material and information happens to both inspire and

threaten teachers. ITs are one of the major contemporary factors shaping the global economy and producing

rapid changes in society. They have fundamentally changed the way people learn, communicate, and do

business. They can transform the nature of education – where and how learning takes place and the roles of

students and teachers in the learning process.

Figure 2. Application of information technology in agro forestry.

IT application in agroforestry

Diagnosis and Design methodology is a methodology for the diagnosis of land-management problems and

the design of agro forestry solutions. There is a need to develop programmes to assist agroforestry researchers

and fieldworkers to plan and implement effective research and development projects. From on-farm research

trials, more rigidly controlled on-station investigations, and eventual extension trials in an expanded range of

sites. It provides a basis for prompt feedback and complementarities between different project components. In

an integrated agroforestry research and extension program, pivotal decisions can be made in periodic meetings

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of the various project personnel who evaluate new results and revise the action plan accordingly. The process

should be continue until the design is optimal and further refinement is deemed unnecessary.

Research and development

To advance agro forestry, research is needed both on basic, process-level questions and on applied

management techniques that are appropriate for commercial farm or forest operations. While basic research

may, for example, investigate the long-term biological interactions between the components of an agro forestry

practice, applied research should seek to maximize the tangible short and intermediate term benefits. Agro

forestry practices should be tailored to readily integrate into existing farming or forestry enterprises, minimize

the displacement of existing crops, use equipment and technical skills that are readily available, and allow some

harvesting of products within conservation agro forestry practices (e.g. hardwood timber from riparian buffer

strips). There is the potential to expand the participation of state, community and institutions, through their

agriculture and forestry programs, in agro forestry research.

The greatest research need is to develop farm-level analyses of the potential economic costs, benefits, and

risks associated with agro forestry practices. This information is a vital prerequisite to the objective comparison

of both production-and conservation-driven agro forestry practices with alternative land use options.

Furthermore, attention should be given to evaluations of future price trends in regional, national and

international markets for commodities that can be produced using agro forestry (e.g. hardwood lumber or high-

value, wind-sensitive crops). Research on tree-crop-animal-environment interactions should be pursued to

provide a scientific basis for optimizing agro forestry designs.

GIS and Remote Sensing in Environmental Management

Use of geographic information systems (GIS), a collection of computer hardware and software used to

analyse and display geographically referenced information, can facilitate planning process. A GIS can be

defined as a data management system designed to input, store, retrieve, manipulate, analyse, and display spatial

data for the purposes of research and decision-making (DeMers 1997). In a GIS, a database is associated with

map features, and data values are geographically referenced, so resource managers can spatially represent

information such as soil types or plant communities. Since land use and a diversity of related disciplines (i.e.

agriculture, forestry, rural planning, and conservation) all deal with spatial characteristics of landscapes (Lacher

1998), GIS has gained considerable use in land use planning and natural-resource management, providing a

spatial framework to aid in the decision-making process (Zeiler 1999).

Additional technologies are often associated with GIS, such as Global Positioning Systems (GPS) and

remote sensing. GPS is a means for inputting spatial data with real world coordinates into a GIS and has become

an important tool for researchers locating and recording information in the field. Remote sensing involves using

spatial data from photographic and satellite images, and software tools to analyse and interpret these data. Rhind

(1988) defined GIS as “a computer system for collecting, checking, integrating and analysing information

related to the surface of the earth”. As it were, there is an ever increasing recognition of the need to perform

large scale mapping and map analysis operations for a wide variety of traditionally manual tasks. Furthermore,

forests see GIS (a computer based application) as an efficient management tool for their day –to-day operations.

A wide variety of software applications are available to support decision making in forest management,

including databases, growth and yield models, wildlife models, silviculture expert systems, financial models,

geographical information systems (GIS), and visualization tools (Schuster et al. 1993). Typically, each

application has its own interface and data format, so managers must learn each interface and manually convert

data from one format to another to use combinations of tools. Considering the scope of topics that may need to

be addressed in a typical ecosystem management problem, and consequently the need to run several to many

applications, manual orchestration of the entire analysis process can quickly become a significant impediment.

Learning Management System (LMS) relieves this problem by managing the flow of information through

predefined pathways that are programmed into its core component. LMS integrates landscape-level spatial

information, stand-level inventory data, and distance independent individual tree-growth models to project

changes on forested landscapes over time.

Spatial data layers like soil type, slope, and land cover can be used to develop suitability assessments that

can identify optimal locations for agroforestry practices to solve landowner and community concerns. By

selecting data with the appropriate spatial resolution, this assessment process can be used at any scale for

Mishra & Agarwal (2015) 2(3): 215–223

.


planning agroforestry practices. The most significant benefit of using GIS-guided suitability assessments is the

ability to combine different assessments to determine locations where multiple objectives can be achieved.

Suitability assessments have been used for several decades to identify locations for different land uses such

as landfills, wildlife reserves, and residential development (McHarg 1995). Some of the first examples of

suitability assessments in the United States were prepared by the Natural Resources Conservation Service

(previously the Soil Conservation Service), which ranked soil types based on suitability for different engineering

and agricultural functions (Soil Survey 1993). Although GIS and the suitability process have been used for

many environmental protection applications, this technology has yet to be used extensively in agroforestry (Ellis

et al. 2000, Bentrup & Leininger 2002).

Considering that GIS technology is widely available and affordable today and the fact that agroforestry is

directly dependent upon spatial characteristics, it is logical to expect to have several agroforestry-specific GIS

DSTs; but the reality is that only a few are available. An early GIS application compiled information on 173

species including their descriptions, soil and climate preferences, and management characteristics for Africa

(Booth et al. 1989). This application allowed users to query the database and generate maps showing the

climatic suitability for different species. At a regional scale, Booth et al. (1990) created a similar application for

Zimbabwe, demonstrating how GIS applications can be done at many scales. Unruh & Lefebvre (1995)

performed a similar GIS application for sub-Saharan Africa to determine areas suitable for different agroforestry

systems. Integrating ICRAF‟s agroforestry database with spatial data on geographic regions, climate and land

uses in the region, their application was able to map out potential regions for 21 specific types of agroforestry

systems.

Most of the past agroforestry GIS applications mentioned above have been research-oriented. The

Southeastern Agroforestry Decision Support System (SEADSS), developed recently by the Center for

Subtropical Agroforestry (CSTAF) at the University of Florida brings on-line GIS capabilities directly to

extension agents and landowners; it offers county soils, land use and other spatial data for selecting suitable tree

and shrub species in a specified location (Ellis et al. 2003). The USDA National Agroforestry Center (NAC) is

currently using GIS to facilitate conservation buffer planning in the Western Corn Belt eco-region in the central

United States (Bentrup et al. 2000). GIS guided assessments, derived from publicly available datasets, are being

used to evaluate four key issues of the Western Cornbelt: biodiversity, soil protection, water quality, and

agroforestry products. By combining these assessments, information is generated for use in identifying

opportunities and constraints on the landscape where multiple benefits from conservation buffers, especially

agroforestry plantings, can be achieved (Bentrup et al. 2000). Utilizing the agroforestry product assessments

(Bentrup & Leininger 2002) in conjunction with the riparian buffer connectivity assessments, areas were

identified where riparian forest buffers could be located to improve habitat connectivity while offering

landowners the option to grow woody floral for profit (Bentrup & Kellerman 2003). GIS-guided agroforestry

suitability analysis will only improve as spatial data and computer resources become more accessible. Many

states and countries already are assembling internet-accessible GIS data clearinghouses to facilitate the use of

spatial information.

Information and technology transfer

Technical information must be developed locally or regionally for application within that region. Information

which is too general or which is based on studies conducted in dissimilar regions or climate zones is not likely

to convince landowners to adopt agro forestry practices, or provide relevant skills and knowledge to ensure their

success. On-farm demonstrations and field days are keys to the understanding and appreciation of agro forestry

practices by landowners. Education and training in agro forestry are needed both for natural resource

professionals and college students.

In addition to the traditional model for the transfer of technology from researcher to extension agent to

practitioner, landowners should have greater involvement in all phases of this process. With the assistance of

research and extension personnel, local groups of landowners may analyse their own needs for agro forestry

development, conduct on-farm experiments under real-life conditions, and then choose the practices most

appropriate for their individual properties. Rather than accusing landowners of causing environmental

degradation, they should be approached from a "win-win" perspective. Emphasis should be placed on

participatory decision-making including landowner advisory groups. Research and information development

Mishra & Agarwal (2015) 2(3): 215–223

.


should focus on agro forestry practices that afford economic opportunities, increase production efficiency, and

provide cost-effective and pro-active solutions to conservation problems.

CONCLUSION AND FUTURE PROSPECTIVE

The relevance and application of information technology to Agro forestry practices in these days is of

tremendous importance. There are still more areas where IT would be applicable in agroforestry which are yet to

be discovered, but in the immediate future. Virtually, all other human endeavours have come to know that the

benefits of IT is far outstripped its disadvantages. It is therefore suggested that IT should be a tool that all

professions should embraced. Successful application of agroforestry systems depends upon pulling together

diverse sources of information, in a manner that responds to users‟ needs and resources. Computer-based DSTs

that accommodate these tasks can greatly facilitate the decision-making process that seeks to simultaneously

balance environmental and production goals that meet landowner and societal needs. We must go beyond

providing tools that only address the ecological and economic aspects of sustainability and provide those that

also enhance the cultural sustainability of agroforestry systems; that is, it must elicit sustained human attention

over time or else the benefits may be compromised as land ownership changes, as development pressure

increases

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www.tropicalplantresearch.com 224 Received: 05 August 2015 Published online: 31 October 2015

ISSN (E): 2349 – 1183

ISSN (P): 2349 – 9265

2(3): 224–229, 2015

Research article

Variability and germination divergence in seed traits of

Stereospermum chelonoides DC.

Anita Tomar

Centre for Social Forestry and Eco-rehabilitation, 3/1, Lajpat Rai Road, New Katra, Allahabad, U.P, India


Abstract: The investigation was carried out in two different seed sources viz. Uttarakhand and

Uttar Pradesh of Stereospermum chelonoides. The aim of the study was to determine variability

and germination divergence in seed traits of Stereospermum chelonoides collected from two states.

A variation was observed in germination percent, mean daily germination, peak value, germination

energy and germination value and seed growth parameters (capsule/seed length, capsule/seed

width and seed weight) of two states. The seeds from Uttarakhand found better as per selected

parameters in comparison to the seeds from Uttar Pradesh.

Keywords: Seed sources - Peak value - Mean daily germination - Germination value

[Cite as: Tomar A (2015) Variability and germination divergence in seed traits of Stereospermum chelonoides

DC. Tropical Plant Research 2(3): 224–229]

INTRODUCTION

Stereospermum chelonoides, DC. is a large sized tree, deciduous, branches and usually 9 to 10 m tall and

distributed in sub Himalayan tract, central parts of India. It is commonly called as "Patla and "Padri" and

belongs to the "Bignoniacea" family (Troup 1986, Masoumeh & Deokule 2013).The decoction of the root is

antipyretic and it is useful in asthma, cough and excessive thirst. The bark and all parts contain a napthaquinone

and lepachol (Sandermann & Dietrichs 1957, Joshi et al. 1977). Flowers are used in bleeding disease, sore throat

and diarrhoea; fruits are useful in blood diseases. The root-bark is an ingredient of Dashmoola (Tomar et al.

2013) and Chywanprash (Yashoda et al. 2004). It is regarded as cooling, astringent cardio tonic, bitter, diuretic

and generally used in combination with other medicine; the ashes of this plant are used in the preparation of

alkaline water and caustic pastes. Fruits are useful in hic cough and blood diseases (Negi 2000).

Seeds of different species and of the same species from different provenances behave differently in their

germination response. Similarly a species may be found in a wide variety of climatic regions, but the

germination behaviour may differ according to provenance. Germinability is a measure of the ability of

population of seeds to germinate or the maximum percentage of seeds that will germinate under favourable

conditions. (Bewley & Black 1978). Variation in seed germination is due to a complex of environmental and

genetic factors during seed formation and subsequent handling of treatments (Wang et al. 1982).

Destructive harvesting practices have seriously reduced seed production and caused gradual erosion of its

natural populations. The species is mainly propagated through seeds and collecting them becomes a laborious

process as their pericarps are winged. Another difficulty it faces is poor germination rate and thus propagation

through seeds in the wild is limited (Baul 2006). Hence, steps have to be taken to conserve this tree of great

economic value therefore, its planting and conservation is recommended for future conservation. Keeping this in

view the present study was conducted to study the variability and germination divergence in seed traits of

Stereospermum chelonoides.


A reconnaissance field survey was carried in five sites (Lakhimpur Kheri, Faizabad, Chitrakoot, Allahabad

and Mirzapur) bearing Stereospermum chelonoides trees in the state of Uttar Pradesh for undertaking the present

study (Fig. 1). The Uttarakhand collection was done from only one site which falls in Dehradun. The latitudinal

and longitudinal ranges of all the six sites have been given in table 1.

Tomar (2015) 2(3): 224–229

.


Fig. 1: studied sites of Stereospermum chelonoides.

Mature capsules were collected during 2012–2013 from all the six sites from minimum eight to ten selected

plants of each seed sources and packed in marked polythene bags. For Uttar Pradesh, a composite sample of

seed was drawn by mixing the seed collected from different sites for seed studies. Capsule and seeds were

randomly drawn from the pool in order to determine their size and shape. For each individual seed, three

principal dimensions: length, width, and weight were measured.

Table 1. Geographic information of the studied sites of Stereospermum chelonoides forests. State Forest Divisions Altitude (m) Latitude Longitude

Uttar Pradesh Lakhimpur Kheri 174.0 28° 28’ 29.94″ N 80° 41’ 56.32″ E

Faizabad 113.0 26° 47’ 00.00″ N 82° 12’ 00.00″ E

Chitrakoot 92.1 25° 14’ 00.00″ N 81° 28’ 00.00″ E

Allahabad 102.6 25° 15’ 53.90″ N 81° 37’ 18.20″ E

Mirzapur 167.0 24° 49’ 16.88″ N 82° 18’ 57.71″ E

Uttarakhand Dehradun 640.0 30° 19’ 48.00″ N 78° 03′ 36.00″ E

Germination test were conducted in 10 cm diameter petri dishes lined with Whatman filter papers. Distilled

water was added whenever moisture loss was detected. There were 4 treatments in this experiment including the

control. The experiment was undertaken in completely randomized block design with four replication in each

treatment and twenty five seeds per replication. Results were expressed as germination percentage which was

the percentage of live seeds that had germinated at the end of test. The seeds were inspected every day and were

considered to be germinated when the radicle penetrated the seed coat and reached about 1mm in length

(Teketay 1996). The data of seed germination was recorded and quantified as per ISTA (1976). The parameters

studied were germination percent (%), germination value (GV) calculated as per Czabator (1962) procedure,

mean daily germination (MDG) according to Bonner (1983), germination energy and germination value (Grouse

& Zimmer 1958).

RESULT AND DISCUSSION

Seed traits, namely seed length, width, weight, and germination parameters vary significantly among both

the state seed sources. The capsule and seed characteristics of Stereospermum chelonoides from Uttarakhand

state have been described in table 2. The highest coefficient of variation (CV) of 38.19% was observed in the

capsule length as the capsule length varies from 14.40 to 49.20 cm with mean value 33.91 cm. The number of

seeds per kg varied from 25,641–40,000 as this depends on size of the capsules. Lowest coefficient of variation

Tomar (2015) 2(3): 224–229

.


was observed in seed length with and without wings (6.60–6.09%). However seed width shared a variation

10.12%.

The characteristics of Stereospermum chelonoides capsule and seed from Uttar Pradesh have been provided

in table 3. The highest coefficient of variation (CV) of 31.9 % was observed in no. of seeds /capsule. This is due

to the fact that the actual values varied from a minimum of 21 seed in one capsule to a maximum of 56 seeds per

capsules.

Capsule length varies from 34.3–50.0 cm with mean value 43.64 cm. The lowest co-efficient of variation

observed in number of seeds per kg (0.2) as it varied from 25870–26000. The variation in seed size may be due

to both internal (maternal, hereditary) and external (environmental) conditions operating at the time of seed

development (Harper et al. 1970) and advantageous for wide range of adaptability. Seed size has been found to

regulate germination and subsequent seedling growth in many species (Baldmin 1942, Langdon 1958, Williams

1967, Kandya 1978, Devagiri 1997, Singh 1998). Both seed sources of S. suaveolens varied significantly in

respect of capsules and seed traits.

Comparatively wider variations were observed in case of capsule characters, number of seeds per capsule

and seed weight. Such genetic variations have been reported in Acacia catechu (Ramachandra, 1996), Acacia

nilotica (Bagchi & Dobriyal, 1990), Dalbergia sissoo (Gera et al. 2000).

The commencement of germination in Uttarakhand started eight day onwards after sowing and continued up

to 15 days. The seed germination varied significantly (ANOVA; p < 0.01) during the study period. The peak

germination (11.0%) was observed on 10th day and the total germination under laboratory conditions recorded

was 90.0%. (Table 4). Seeds sown achieved 2.43 peak value and 2.33 as mean daily germination, 45.0

germination energy and 5.66 as germination value (Fig. 2).

In Uttar Pradesh the peak germination was observed on 12th day and total germination under laboratory

conditions recorded was 65% (Table 4). Seeds sown achieved 1.73 as mean daily germination, 1.75 peak value,

42.5 germination energy and 3.02 as germination value (Fig. 2).

Table 4. Variation in Uttarakhand and Uttar Pradesh Germination.

States

Seed germination % Germination

8th

day

9th

day

10th

day

11th

day

12th

day

13th

day

14th

day

15th

day Total %

Period

(days) Energy Value

Uttarakhand 3 3 11 7 5 3 3 1 90 ±1.41 8–15 45.0 5.66

Uttar Pradesh 4 4 4 4 5 1 0 4 65 ±0.58 8–15 42.5 3.02

Note: The values refer to mean Standard deviation, (n = 25 x 4).

Germination energy is a measure of speed of germination and is assumed to given an idea of the vigour of

seed and seedlings which it produces (Willan 1985). Germination value, an index combining speed and

completeness of germination was influenced by seed size and weight (Baldwin 1942, Czabator 1962, Dunlop &

Barnett 1984). Differences observed for germination percent, germination value and germination energy could

Table 2. Variation in Capsules, seed traits of Stereospermum chelonoides of Uttarakhand state.

No. of seeds

/capsule

No. of seeds

/kilogram

Capsule Character Seed Character

Length (cm) Width

(mm)

Weight (gm) Length with

wings (cm)

Length without

wings (cm)

Width

(mm)

Mean ±SD 49.71 ±16.60 33080±5320.06 33.91 ±12.95 12.21±3.42 32.43±7.09 3.09±0.20 1.83±0.11 4.42±0.45

Range 22–65 25,641–40,000 14.40–49.20 7.32–16.24 24–42 2.9–3.5 1.7–2.0 3.88–5.03

C.V. 33.39 16.08 38.19 28.00 21.9 6.60 6.09 10.12

Note: S.D. = Standard deviation; C.V. = Coefficient of variation. (n = 25 x 4)

Table 3. Variation in Capsules, seed traits of Stereospermum chelonoides of Uttar Pradesh state.

No. of seeds

/capsule

No. of seeds

/kilogram

Capsule Character Seed Character

Length (cm) Width

(mm)

Weight (gm) Length with

wings (cm)

Length without

wings (cm)

Width

(mm)

Mean ±SD 42.4313.55 25906 43.51 43.645.50 18.60 0.93 55.5713.21 3.34 0.60 1.1 10.12 10.070.85

Range 21–56 25870–26000 34.3–50.0 17.11–19.75 41.6–83.1 2.1–3.9 0.98–1.30 8.20–10.75

C.V. 31.9 0.2 12.6 5.00 23.8 17.9 11.0 8.4

Note: S.D. = Standard deviation; C.V. = Coefficient of variation. (n = 25 x 4)

Tomar (2015) 2(3): 224–229

.


be genetic in nature because environmental deviations are negligible for experimental conditions and seeds of

both states were stored in similar conditions. This is supported by the reports of Gera et al. (2000) and

Vakshasya et al. (1992) for Dalbergia sissoo and Arya et al. (1995) for Prosopis cineraria. Since the seeds were

germinated under similar condition, variations among the seed sources may be attributed to genetic differences.

Such variations in nursery performances have reported in Acacia albida (Sniezko & Stewart 1989), Acer rubrum

(Townsend 1977) and Prosopis cineraria (Hooda & Bahadur 1996).

Figure 2. Germination values of Stereospermum chelonoides under laboratory conditions.

Variation in germination of seed sources has been reported in Acacia mangium (Salazar 1989), Pinus brutia

(Isik 1986), Betula ermanii (Shembreg & Protemkin 1987), Pinus greggi (Dvorak et al. 1996) Acacia catechu

(Ramachandra 1996) and Pinus roxburghii (Roy et al. 2004). In general pod, seed and germination traits are

supposed to be inherited characters influenced by age, growth, micro and macro habitats of the parent tree (Isik

1986). Larger seed germinate faster and more completed than smaller one probably due to more endosperm

nutrient pool (Kandya 1978). Aldhous (1972) opined that only those seeds which germinate rapidly and

vigorously under favourable conditions, are likely to be capable of producing vigorous seedlings in field

conditions which is of immediate interest, whereas, week or delayed germination is often fatal. Isik (1986)

stated that populations with high germination rate are more vigorous in terminal and root growth. Khalil (1986)

also recommended the detection of fast growing provenances based on germination traits.

CONCLUSION

It emerged from the present study that a large variability exists in the Stereospermum chelonoides growing

naturally in Uttar Pradesh and Uttarakhand particularly for number of seeds/capsule, capsule character and seed

character. The variability of different characters could be utilized for selection of genotypes suitable for the

plantation and utilization. In this study, Uttarakhand seed source had shown better germination as compared to

Uttar Pradesh. However, more comprehensive survey of Stereospermum chelonoides habitat areas of

Uttarakhand is required to select some promising forms of Stereospermum chelonoides.

This study helps to identify the better seed source of S. chelonoides having better yield therefore, the best

seed source selected may improve the poor sites for agroforestry systems and energy plantations in the

wastelands.

ACKNOWLEDGEMENTS

This work was financially supported by Indian Council of Forestry Research and Education (ICFRE),

Dehradun, India.

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ISSN (E): 2349 – 1183

ISSN (P): 2349 – 9265

2(3): 230–239, 2015

Research article

Diversity and carbon stock assessment of trees and lianas in

tropical dry evergreen forest on the Coromandel Coast of India

P. Vivek1 and N. Parthasarathy

1*

1Department of Ecology and Environmental Sciences, Pondicherry University, Puducherry - 605014, India


Abstract: The diversity and carbon stock of all woody plants were investigated in ten tropical dry

evergreen forest (TDEF) sites on the Coromandel Coast of India. All trees ≥ 10 cm girth at breast

height and all lianas ≥ 1 cm diameter, measured at 1.3 m from the rooting point were enumerated.

A total of 81 tree species (26.3±6.7 species ha-1) and 52 liana species (23.4±5.7 species ha-1) that

belonged respectively to 34 and 28 families were inventoried from the ten study sites. The

abundance of woody plants in the ten study sites totaled 18705 individuals (9466 trees and 9239

lianas) and the average tree density was 946.6±298.9 stems ha-1 and liana density was 923.9±403.3

stems ha-1. Trees contributed 61 % and 51 % respectively to the total woody species richness and

abundance. The basal area of trees in the ten study sites ranged from 8.23 m2 ha-1 to 29.48 m2 ha-1

and that of lianas ranged from 0.2 m2 ha-1 to 1.76 m2 ha-1. The aboveground biomass (AGB) of

trees totaled 3025.8 Mg and ranged from 96.9 Mg ha-1 to 576.4 Mg ha-1 across the ten sites. The

liana aboveground biomass ranged from 2.24 Mg ha-1 to 42.13 Mg ha-1 and totaled 153.76 Mg in

the ten sites. The woody plants in the present study sites stocked 1978.24 Mg Carbon and it ranged

from 62.2 Mg C ha-1 to 365.4 Mg C ha-1. Trees accounted for a maximum share of 95 % and lianas

contributed just 5 % to the total woody-plant carbon stock in the study sites. The extent of woody

species diversity and estimated carbon stock of the TDEF sites, underlines the need for biological

conservation of this unique forest type which are fast vanishing due to anthropogenic pressure.

Keywords: Biomass - Allometric equation - Anthropogenic pressure - Wood specific density -

Carbon flux

[Cite as: Vivek P & Parthasarathy N (2015) Diversity and carbon stock assessment of trees and lianas in

tropical dry evergreen forest on the Coromandel Coast of India. Tropical Plant Research 2(3): 230–239]

INTRODUCTION

Forests are one of the major pools of carbon, where plants fix atmospheric carbon into the biological system.

Indeed it is the tropical forest ecosystems that have the potential to hold and sequester large amounts of carbon

than the other forest biomes (Metz et al. 2001). Tropical forests comprise about 40 % of the total terrestrial

carbon stock (Dixon et al. 1994), but uncertainty prevails in their quantitative contribution to the global carbon

cycle (Chave et al. 2005). This uncertainty is largely due to the lack of standard methods for converting field

measurements into biomass estimates (Hall 2012, Liu et al. 2014). The woody biomass of trees and lianas, their

standing crop of litter including the soil organic matter together comprise the key carbon pools in tropical forest

ecosystems (Gibbs et al. 2007). The carbon stocked as aboveground and belowground biomass in woody plants

is impacted directly by human-mediated disturbances. Deforestation currently accounts for about 18 % of the

global carbon emissions (IPCC 2007). Several other factors including selective logging, forest fragmentation

and shifting cultivation are expected to play a major role in altering forest biomass (Houghton 2005). Under the

present scenario of global climate change and increasing deforestation rates, it has become crucial to quantify

the carbon stocks and fluxes particularly in the tropics. Currently, data presented on forest biomass is available

from wide array of sources (e.g. quantitative inventories, output of ecological models and through satellite

products). However, it is the direct assessment of biomass through destructive sampling is likely to give better

estimates on forest carbon stocks. It could also be approached by applying generalized allometric equations

(Brown 1997, Chave et al. 2005) using variables such as diameter, height and wood specific density. Allometric

Vivek & Parthasarathy (2015) 2(3): 230–239

.


equations applied for forest inventory data, relate these inventory data to measurements made from destructive

sampling by statistical means, and available for most forest types (Brown 1997, Chave et al. 2005).

Tropical dry evergreen forest (TDEF) is a unique and geographically restricted forest type distributed as

numerous patches along the Coromandel Coast of peninsular India. All the TDEF sites are protected as sacred

forests owing to the religious and traditional believes of the local people. However, the recent developments and

erosion in the belief system, questioned the concept of sacred forests and as a result, some TDEF sites are now

subjected to unprecedented levels of anthropogenic pressure. Meher-Homji (1974) estimated that just 4–5 % of

the original TDEFs remain. Hence, it is of prime importance to estimate the diversity and carbon stocking

potential of TDEF ecosystem for conservation with the aid of scientific data. Although, there have been many

studies that reported the estimation of carbon stocks of trees in tropical forests, only few studies have included

the liana life-form. Hence, the present study is aimed to investigate woody species (trees and lianas) diversity

and their carbon stocks in TDEF ecosystem.


Study area

Figure 1. Map showing ten tropical dry evergreen forest sites distributed in Cuddalore, Pudukottai and Nagapattinam

districts on the Coromandel Coast of India.

The present study was conducted in ten tropical dry evergreen forest (TDEF) sites located in Cuddalore (11°

44ʹ 57.88ʹʹ N latitude and 79° 44ʹ 50.99ʹʹ E longitude), Nagapattinam (10° 45ʹ 56.28ʹʹ N latitude and 79° 50ʹ

31.68ʹʹ E longitude) and Pudukottai (10° 22ʹ 46.63ʹʹ N latitude and 78° 49ʹ 18.35ʹʹ E longitude) districts of Tamil

Nadu, India (Fig. 1). The tropical dry evergreen forest that occurs as patches along the Coromandel Coast of

peninsular India is characterized by two to three-layered, tree-dominated forests with short stature and sparse


.


ground flora (Parthasarathy et al. 2008, Vivek & Parthasarathy 2015). The rainfall here is tropical dissymmetric

type with most rains received during the north-east monsoon and a little, inconsistent rainfall during the south-

west monsoon. The mean annual rainfall is 1184 mm, 1346 mm and 919 mm in Cuddalore, Nagapattinam and

Pudukottai respectively. The length of dry season is 6–8 months annually. The mean annual maximum and

minimum temperature are 36.9°C and 20.8°C in Cuddalore, 34.9°C and 22.3°C in Nagapattinam and 36.1°C and

21.6°C in Pudukottai. Soil type varies from hard lateritic, red ferralitic and alluvium to coastal sandy. All the

study sites are community-managed, except the sites Point Calimere 1 and 2, which are a part of Point Calimere

Wildlife Sanctuary (RAMSAR site) and is probably the largest existing TDEF site in India. The community-

managed sites are protected by the local people as sacred groves (sacred forests) dedicated to Gods, based on the

traditional belief system. However, the concentration of human settlements near these study sites makes them

more vulnerable to anthropogenic pressure.

Field inventory

Field work was carried out between April 2013–May 2014 in ten 1-ha study plots. Each one-hectare study

plot was further divided into one-hundred 10 × 10 m sub-grids to facilitate the inventory. During inventory, all

trees ≥ 10 cm girth at breast height (gbh) were measured at 1.3 m from the ground level and all lianas ≥ 1 cm

diameter were measured at 1.3 m from the rooting point. All the inventoried tree and liana species were

recognized to species-level using regional floras (Gamble & Fischer 1915–1935, Matthew 1991) and confirmed

with the specimens lodged in the herbarium of Department of Ecology and Environmental Sciences,

Pondicherry University. Diversity indices such as Shannon, Fisher’s alpha and Simpson index were computed

following Magurran (2004).

Allometric equation and biomass estimation

We used the forest inventory data (dbh values) to estimate the aboveground (AGB) and belowground biomass

(BGB). The aboveground biomass of trees was estimated following the allometric equation of Chave et al.

(2005) using two variables, the diameter and wood specific density (WSD):

AGB est = ρ × exp (-1.499 + 2.148 ln (D) + 0.207(ln (D))2 - 0.0281(ln (D))3)

where D is the diameter and ρ is the wood specific density of tree species.

The wood specific density of each tree species was taken from available literature (Mani & Parthasarathy

2007) and also from global wood density database. We used the generalized allometric equation (Pearson 2005)

for few species for which WSD value was not available, using diameter as the only variable.

For lianas, the allometric equation of Schnitzer et al. (2006) was used:

AGB = exp [– 1.484 + 2.657 ln (D)]

where D is the diameter.

The belowground biomass of trees and lianas was calculated by multiplying the aboveground biomass value

with 0.26 (Cairns et al. 1997, IPCC 2003). The carbon stock was estimated to be 50 % of the total biomass

(AGB + BGB) (IPCC 2005).


Woody species diversity

Woody species inventory yielded a total of 133 species including 81 tree species and 52 liana species from ten

study sites (Table 1) and it ranged from a minimum of 29 species ha-1 at site MK to the maximum of 64 species

ha-1 at site PC 1. Trees in the study sites comprised 60 % of the total woody species richness and lianas the rest.

A total of 18705 woody individuals were enumerated from the study sites, of which trees and lianas shared 51 %

and 49 % of the total abundance respectively. Site VV harbored maximum density of trees and lianas (3351

individuals ha-1) and it was minimum at site MK (1194 individuals ha-1). Site PC 1 with greater species richness

had the highest Fisher’s α value and site MK, the lowest (Table 1). Shannon and Simpson index values were

higher for sites PR and SN respectively. Memecylon umbellatum (2318 individuals), Glycosmis mauritiana (740

individuals) and Albizia amara (700 individuals) were the top three abundant tree species forming 42 % of the

total tree species abundance. Among lianas, Strychnos lenticellata (1920 individuals), Combretum albidum (987

individuals) and Reissantia indica (747 individuals) were the predominant species which together accounted for

40 % of the total liana abundance.


.


Forest biomass

The total woody species basal area in the ten study sites was 176.75 m2 and it varied from a maximum of 30.16

m2 ha-1 at site PC 1 to a minimum of 8.43 m2 ha-1 at site MK (Table 2). Trees in the study sites comprised 96 %

of the total woody species basal area. The total biomass of the woody species recorded in study sites was 3956.4

Mg with the maximum contribution from trees (95 %). The mean woody species carbon stock in the ten study

sites was 197.8±81.8. Whereas, Tiwari & Singh (1987) estimated 68.5–122.5 Mg ha-1 biomass carbon in

Table 2. Basal area (BA), aboveground biomass (AGB), belowground biomass (BGB), total biomass (TB) and carbon stock (CS) of

all woody plants followed by trees and lianas in ten TDEF sites on the Coromandel Coast of India: Karukkai (KA), Kothattai (KT),

Maanadikuppam (MK), Point Calimere 1(PC1), Point Calimere 2 (PC2), Purangani (PR), Silattur (SL), Sunayakkadu (SN), Suran

Viduthi (SV) and Vanniyan Viduthi (VV).

Variable Study site Total

for 10 ha KA KT MK PC1 PC2 PR SL SN SV VV .

BA (m2 ha-1) 13.99 16.15 8.43 30.16 20.97 11.29 15.13. 19.02. 20.25 21.35. 176.74

Trees 13.38 15.92 8.23 29.48 20.53 10.64 14.36. 18.07. 19.19 19.59. 169.39

Lianas 0.61 0.23 0.2 0.68 0.44 0.65 0.77. 0.95. 1.06 1.76. 7.35

AGB (Mg ha-1) 271.35 300.31 98.84 580.02 315.67 138.07 270.26 391.5. 390.26 383.83. 3140.11

Trees 262.2 296.8 96.6 569.6 307.4 128.1 256.9 353.2. 373.9 341.7. 2986.4

Lianas 9.15 3.51 2.24 10.42 8.27 9.97 13.36 38.30. 16.36 42.13. 153.71

BGB (Mg ha-1) 70.551 78.080 25.698 150.80 82.074 35.898 70.267 101.79 101.46 99.795. 816.41

Trees 68.172 77.168 25.116 148.09 79.924 33.306 66.794 91.832 97.214 88.84. 776.45

Lianas 2.379 0.9126 0.5824 2.7092 2.1502 2.5922 3.4736 9.958 4.2536 10.95. 39.96

TB (Mg ha-1) 341.90 378.390 124.53 730.82 397.74 173.96 340.52 493.29 491.72 483.62. 3956.49

Trees 330.37 373.96 121.71 717.69 387.32 161.40 323.69 445.03 471.11 430.54. 3762.82

Lianas 11.529 4.4226 2.8224 13.129 10.420 12.562 16.833 48.258 20.613 53.083. 193.67

CS (Mg ha-1) 170.95 189.19 62.2692 365.41 198.87 86.984 170.26 246.64 245.86 241.81. 1978.24

Trees 165.18 186.98 60.858 358.84 193.66 80.703 161.84 222.51 235.55 215.27. 1881.39

Lianas 5.7645 2.2113 1.4112 6.5646 5.2101 6.2811 8.4168 24.129 10.306 26.541. 96.8356

Himalayan region of Uttar Pradesh. Ravindranath et al. (1997) reported the average of 63 % Mg C ha-1 from the

values for few forest types studied using harvest method. Thus, the estimated carbon on per hectare basis in the

present study is much higher than the values reported in the previous studies in India. The increased interests in

estimating the biomass and carbon stocks resulted in the evolution of new methods that confounded

comparisons across the different studies. For example, Mani & Parthasarathy (2007) obtained two contradictory

results on aboveground biomass using two different allometric equations for the same dataset. It is of paramount

importance to obtain more accurate estimates on carbon stocks for tropical forests to understand the role of

tropical ecosystems in the global carbon cycle (Brown et al. 1989, Kale et al. 2004, Kuller et al. 2001). The

Table 1. Consolidated details of woody plant (trees and lianas) diversity in ten tropical dry evergreen forest (TDEF) sites

distributed 1-ha each at: Karukkai (KA), Kothattai (KT), Maanadikuppam (MK), Point Calimere 1(PC1), Point Calimere 2 (PC2),

Purangani (PR), Silattur (SL), Sunayakkadu (SN), Suran Viduthi (SV) and Vanniyan Viduthi (VV) on the Coromandel Coast of

India.

Variable Study site Total

for 10 ha KA KT MK PC1 PC2 PR SL SN SV VV

TWSR (number of spp.) 42 45 29 65 58 42 47 55 64 50 133

Trees 20 25 18 37 27 20 22 27 37 30 81

Lianas 22 20 11 28 31 22 25 28 27 20 52

TWSD (individuals ha-1) 1786 1213 1194 1462 1523 1867 2424 1542 2343 3351 18705

Trees 845 661 786 790 803 948 1211 841 888 1693 9466

Lianas 941 552 408 672 720 919 1213 701 1455 1658 9239

Note: TWSR- Total woody species richness; TWSD- Total woody species density.


.


choice of equation is therefore much important when comparing the biomass estimates in regional scale. Among

all the study sites, the relatively undisturbed site PC 1 stocked maximum carbon (365.41 Mg C ha-1). Tree

species such as Manilkara hexandra with 481 individuals stocked maximum carbon (399.4 Mg C), followed by

Drypetes sepiaria (192.20 Mg C) and Albizia amara (165.29 Mg C) (Table 3). The predominant tree species

Memecylon umbellatum and Glycosmis mauritiana contributed at least four-fold lower value (92.12 Mg C and

58.64 Mg C respectively) than that of Manilkara hexandra, possibly due to their major representation in smaller

girth classes. Ventilago madraspatana, an unarmed scrambler was the highest contributor of carbon stock

among the 52 liana species, followed by Acacia caesia (16.72 Mg C) and Derris scandens (11.95 Mg C) (Table

3). Although lianas continue to increase in biomass in tropical forests (Schnitzer & Bongers 2011), they have

not been figured in most forest biomass assessment studies. It is estimated that lianas can add up to 30% of the

total aboveground biomass in tropical forests with dense liana population (Schnitzer & Bongers 2011).

However, in the present study sites, lianas with almost equal abundance as that of the trees, comprised just 5 %

of the total forest biomass. Yet, they may play a major role in reducing the whole forest carbon stock and

sequestration potential by competing aggressively with trees for aboveground and belowground resource

(Schnitzer & Bongers 2011). Lianas usually capitalize and grow well on the disturbed environments and reduces

tree growth and increases the tree mortality rates. This may not be a good sign as lianas do not compensate for

the tree biomass that they displace (van der Heijden & Phillips 2009, Schnitzer & Bongers 2011).

Table 3. Species abundance (Ab), aboveground biomass (AGB), belowground biomass (BGB), total biomass (TB) and total

carbon stock (TCS) of all the 81 tree species and 52 liana species enumerated from 10-ha area distributed 1-ha in each of ten

tropical dry evergreen forest sites

S.No. Woody species Ab

(10-ha) )

AGB

(kg)

BGB

(kg)

TB

(kg)

TSC

(kg)

Tree species

1 Acacia leucophloea (Roxb.) Willd. 4 228.6 59.4 288.0 144.0

2 Alangium salvifolium (L.f.) Wangerin 6 1416.6 368.3 1784.9 892.5

3 Albizia amara (Roxb.) Boivin 700 262366.6 68215.3 330581.9 165290.9

4 Allophyllus serratus (Roxb.) Kurz 7 65.4 17.0 82.3 41.2

5 Anacardium occidantale L. 9 35973.4 9353.1 45326.4 22663.2

6 Atalantia monopylla (L.) Correa 363 44060.3 11455.7 55516.0 27758.0

7 Azadirachta indica A. Juss. 87 73078.3 19000.4 92078.6 46039.3

8 Bauhinia racemosa Lam. 1 213.5 55.5 269.0 134.5

9 Benkara malabarica (Lam.) Tirven. 14 339.6 88.3 427.9 213.9

10 Borassus flabellifer L. 51 64176.7 16685.9 80862.6 40431.3

11 Breynia vitis-idaea (Burm. f.) Fischer 3 7.9 2.1 9.9 5.0

12 Cadaba trifoliata (Roxb.) Wight & Arn. 143 2439.5 634.3 3073.8 1536.9

13 Canthium coromandelicum (Burm.f.) Alston 18 17.1 4.4 21.5 10.8

14 Canthium dicoccum (Gaertn.) Teijsm.& Binn 230 28752.3 7475.6 36227.9 18114.0

15 Carmona retusa (Vahl) Masm 6 22.8 5.9 28.7 14.3

16 Cassia auriculata L. 1 3.8 1.0 4.7 2.4

17 Cassia fistula L. 146 26408.0 6866.1 33274.1 16637.1

18 Cassia roxburghi DC. 8 2631.8 684.3 3316.1 1658.1

19 Cassine glauca (Rottb.) Kuntze 17 710.2 184.7 894.9 447.5

20 Catunaregam spinosa (Thunb.) Tirven 13 43.8 11.4 55.2 27.6

21 Chionanthus zeylanica L. 48 9929.4 2581.6 12511.0 6255.5

22 Chloroxylon sweitenia DC. 398 61783.0 16063.6 77846.6 38923.3

23 Clausena dendata (Willd.) Roemer 248 1627.7 423.2 2050.8 1025.4

24 Commiphora berryi (Arn) Engler 2 329.0 85.5 414.6 207.3

25 Commiphora caudata (Wight & Arn.) Engl. 26 7268.7 1889.9 9158.6 4579.3

26 Cordia obliqua Willd. 7 666.1 173.2 839.2 419.6

27 Dalbergia coromandeliana Prain 2 234.9 61.1 296.0 148.0


.


28 Dalbergia paniculata Roxb. 46 81653.5 21229.9 102883.5 51441.7

29 Dichrostachys cinerea (L.) Wight & Arn. 4 54.5 14.2 68.7 34.3

30 Diospyros ebenum J. Koenig ex Retz. 149 28684.8 7458.0 36142.8 18071.4

31 Diospyros ferrea (Willd.) Bakh. var. buxifolia

(Rottb.) Bakh.

103 22402.1 5824.6 28226.7 14113.4

32 Diospyros montana Roxb. 85 6368.3 1655.8 8024.1 4012.0

33 Dodonea angustifolia L. f. 47 1837.2 477.7 2314.8 1157.4

34 Drypetes sepiaria (Wight & Arn.) Pax & Hoffm. 390 305091.4 79323.8 384415.2 192207.6

35 Ehretia pubescens Benth. 2 5.0 1.3 6.3 3.1

36 Ehretia wightiana Wall. ex G.Don 2 973.4 253.1 1226.5 613.3

37 Eugenia bracteata (Willd.) Roxb. ex DC. 4 41.5 10.8 52.3 26.2

38 Euphorbia antiquorum L. 176 - - - -

39 Ficus amplissima Sm. 1 4238.4 1102.0 5340.4 2670.2

40 Ficus benghalensis L. 17 172077.9 44740.3 216818.1 108409.1

41 Ficus microcarpa L.f. 7 8950.6 2327.1 11277.7 5638.8

42 Ficus religiosa L. 1 84.4 21.9 106.3 53.1

43 Garcinia spicata (Wight & Arn.) J. D. Hook. 161 88635.4 23045.2 111680.5 55840.3

44 Gardenia resinifera Roth 17 3441.7 894.8 4336.5 2168.2

45 Glycosmis mauritiana (Lam.) Yuich. Tanaka 940 9308.1 2420.1 11728.2 5864.1

46 Gmelina asiatica L. 33 2951.0 767.3 3718.2 1859.1

47 Ixora pavetta T.Anderson 36 10299.4 2677.8 12977.2 6488.6

48 Jatropha gossipyfolia L. 1 1.8 0.5 2.3 1.2

49 Lannea coromandelica (Houtt.) Merr. 32 3094.8 804.6 3899.4 1949.7

50 Lepisanthes tetraphlla (Vahl.) Radlk. 313 153606.5 39937.7 193544.2 96772.1

51 Mallotus phillipensis (Lam.) Muell.-Arg. 20 2693.5 700.3 3393.8 1696.9

52 Manilkara hexandra (Roxb.) Dubard 481 634041.8 164850.9 798892.7 399446.3

53 Maytenus emarginata 43 599.5 155.9 755.3 377.7

54 Memecylon umbellatum Burm.f. 2318 146235.1 38021.1 184256.2 92128.1

55 Morinda coreia Buch.-Ham. 14 1162.7 302.3 1465.1 732.5

56 Muntingia calabura L. 4 417.0 108.4 525.4 262.7

57 Ochna obtusata DC. 33 4957.3 1288.9 6246.2 3123.1

58 Pamburus missionis (Wight) Swingle 4 13.8 3.6 17.4 8.7

59 Phyllanthus polyphyllus Willd. 2 87.9 22.9 110.8 55.4

60 Pleiospermium alatum (Wall. ex Wight. & Arn.)

Swingle

48 12944.2 3365.5 16309.7 8154.8

61 Polyalthia korintii (Dunal) Thw. 10 904.4 235.1 1139.6 569.8

62 Polyalthia longifolia (Sonn.) Thw. 4 137.8 35.8 173.7 86.8

63 Pongamia pinnata (L.) Pierre 65 86160.0 22401.6 108561.6 54280.8

64 Premna serratifolia L. 14 7548.2 1962.5 9510.7 4755.4

65 Prosopis juliflora (Sw.) DC. 131 119681.9 31117.3 150799.1 75399.6

66 Pterospermum canescens Roxb. 86 105674.5 27475.4 133149.9 66575.0

67 Pterospermum xylocarpum (Gaertn.) Sant. &

Wagh.

8 13920.2 3619.2 17539.4 8769.7

68 Salvadora persica L. var. wightiana

(Thwaites) Verdc

15 6820.1 1773.2 8593.4 4296.7

69 Sapindus emarginatus Vahl 3 21.0 5.5 26.5 13.2

70 Sapium insigne (Royle) Trimen 16 140.1 36.4 176.5 88.2

71 Securenega leucopyrus (Willd.) Muell.-Arg. 3 12.5 3.2 15.7 7.9

72 Strebulus asper Lour. 5 60.7 15.8 76.5 38.2


.


73 Strychnos nux-vomica L. 2 126.0 32.8 158.8 79.4

74 Syzygium cumini (L.) Skeels 51 211847.6 55080.4 266928.0 133464.0

75 Tamarindus indica L. 4 62402.1 16224.5 78626.6 39313.3

76 Tarenna asiatica (L) kuntz ex Schumann. 510 4990.5 1297.5 6288.1 3144.0

77 Tricalysia sphaerocarpa (Dalz.) Gamble 372 81809.3 21270.4 103079.7 51539.8

78 Vitex altíssima L.f. 10 7734.7 2011.0 9745.8 4872.9

79 Walsura trifolia (A. Juss.) Harms 6 683.1 177.6 860.7 430.4

80 Wrightia tinctoria (Roxb.) R. Br. 4 75.5 19.6 95.2 47.6

81 Zizyphus mauritiana Lam. 13 603.0 156.8 759.8 379.9

Liana species

82 Abrus precatorius L. 9 4.4 1.1 5.6 2.8

83 Acacia caesia (L.) Willd. 196 16722.9 4348.0 21070.9 10535.5

84 Adenia wightiana (Wall.exWight & Arn.) Eng. 1 0.3 0.1 0.4 0.2

85 Aristolochia indica L. 5 1.7 0.4 2.1 1.1

86 Asparagus racemosus Willd. 82 70.2 18.3 88.5 44.2

87 Azima tetracantha Lam. 25 357.6 93.0 450.6 225.3

88 Canavalia virosa (Roxb.) Wight & Arn. 7 4.7 1.2 6.0 3.0

89 Cansjera rheedii Gmel. 74 1150.1 299.0 1449.2 724.6

90 Capparis brevispina DC. 141 1971.9 512.7 2484.5 1242.3

91 Capparis divaricata Lam. 1 0.3 0.1 0.4 0.2

92 Capparis rotundifolia Rottl. 28 104.4 27.1 131.5 65.7

93 Capparis sepiaria L. 4 26.8 7.0 33.7 16.9

94 Capparis zeylanica L. 58 1463.9 380.6 1844.5 922.3

95 Carissa spinarum L. 575 2224.3 578.3 2802.6 1401.3

96 Cissus quadrangularis L. 254 409.3 106.4 515.7 257.8

97 Cissus vitiginea L. 293 4972.1 1292.7 6264.8 3132.4

98 Clerodendrum inerme (L.) Gaertn. 13 60.7 15.8 76.4 38.2

99 Coccinia grandis (L.) Voigt 182 960.0 249.6 1209.6 604.8

100 Combretum albidum G.Don 987 10042.2 2611.0 12653.1 6326.6

101 Derris ovalifolia (Wight & Arn.) Benth. 192 9014.1 2343.7 11357.8 5678.9

102 Derris scandens (Roxb.) Benth. 343 11951.2 3107.3 15058.5 7529.3

103 Dioscorea oppositifolia L. 1 0.2 0.1 0.3 0.1

104 Gloriosa superba L. 4 3.0 0.8 3.7 1.9

105 Grewia rhamnifolia Heyne ex Roth 445 11772.3 3060.8 14833.1 7416.6

106 Gymnema sylvestre (Retz.) R.Br. ex Schultes 180 4053.4 1053.9 5107.3 2553.7

107 Hugonia mystax L. 488 6329.6 1645.7 7975.3 3987.6

108 Ichnocarpus frutescens (L.) R.Br. 162 158.9 41.3 200.2 100.1

109 Ipomoea staphylina Roemer & Schultes 5 36.4 9.5 45.9 23.0

110 Jasminum angustifolium (L.) Willd. 309 1123.2 292.0 1415.2 707.6

111 Jasminum sessiliflorum Vahl 61 34.8 9.1 43.9 21.9

112 Olax scandens Roxb. 18 138.4 36.0 174.4 87.2

113 Pachygone ovata (Poir) Miers ex Hook. 56 391.1 101.7 492.7 246.4

114 Plecospermum spinosum Trecul. 30 1061.2 275.9 1337.1 668.6

115 Premna corymbosa (Burm.f.) Rottl. & Willd. 137 459.5 119.5 578.9 289.5

116 Pterolobium hexapetalum (Roth.) Sant.&Wag. 74 208.3 54.2 262.5 131.2

117 Pyrenacantha volubilis Wight 191 209.5 54.5 264.0 132.0

118 Reissantia indica (Willd.) Halle 747 10709.3 2784.4 13493.7 6746.8

119 Rivea hypocrateriformis (Desr.) Choisy. 39 922.1 239.7 1161.8 580.9


.


120 Salachia chinensis L. 8 66.0 17.1 83.1 41.6

121 Sarcostemma acidum (Roxb.) Voigt 57 103.0 26.8 129.8 64.9

122 Scutia myrtina (Burm. f.) Kurz 110 2892.3 752.0 3644.3 1822.1

123 Secamone emetica (Retz.) R. Br. 186 505.1 131.3 636.4 318.2

124 Strychnos lenticellata (Dennst.) Hill 1920 7554.2 1964.1 9518.2 4759.1

125 Symphorema involucratum Roxb. 62 6260.5 1627.7 7888.2 3944.1

126 Tiliacora acuminata (Lam.) Hook.f. & Thoms. 16 23.2 6.0 29.2 14.6

127 Tinospora cordifolia (Willd.) Hook.f.&Thoms. 87 87.0 22.6 109.6 54.8

128 Toddalia asiatica (L.) Lam. 40 301.9 78.5 380.4 190.2

129 Trichosanthes tricuspidata Lour. 2 5.5 1.4 6.9 3.5

130 Tylophora indica (Burm. f.) Merr. 7 4.4 1.1 5.5 2.8

131 Ventilago madraspatana Gaertn. 87 26031.1 6768.1 32799.2 16399.6

132 Wattakaka volubilis (L.f) T. Cooke 102 1048.4 272.6 1321.0 660.5

133 Zizyphus oenoplia (L.) Mill. 136 9759.3 2537.4 12296.7 6148.4

Size-class distribution and carbon stock

Overall, in the ten study sites, 62 % of trees and 70 % of lianas fell within the lowest dbh class (Fig. 2 & 3)

and this observed pattern could have resulted from the greater recruitment and mortality rates in the lowest dbh

class (Vivek & Parthasarathy 2015). The highest dbh class comprised just 7 % of the total tree abundance, yet,

managed to represent 75 % of the total tree carbon stock, suggesting the role of large trees in maintaining the

carbon pools in TDEF ecosystem. Similarly in lianas, the highest dbh class represented by 3 % of the total liana

abundance across the study sites, accounted for 67 % of the total carbon stocked by lianas in the study sites. In

general, the carbon stock of the trees and lianas increased with increasing size-class irrespective of the number

of individuals.

Figure 2. Girth-class wise distribution of tree abundance and their corresponding carbon stocks in tropical dry evergreen

forests on the Coromandel Coast of India.

CONCLUSION

This study provides valuable data on biomass carbon of woody plants, thereby emphasizes the role of TDEF

ecosystem in maintaining carbon pool of the local forest environment and will be helpful in framing

conservation strategies and action plans. The present study also indicates the role of trees, particularly the large

trees in maintaining the carbon stock, but in recent years, the TDEFs on the Coromandel Coast are experiencing

immense pressure that result in reduced tree counts (Baithalu et al. 2012), but the lianas on the other hand

increased drastically (Khadanga 2015). Therefore, we recommend long-term monitoring studies to estimate


.


carbon stocks and dynamics in TDEF ecosystem under the current scenario of climate change and anthropogenic

disturbance.

Figure 3. Diameter-class wise distribution of liana abundance and their corresponding carbon stocks in tropical dry

evergreen forests on the Coromandel Coast of India.

ACKNOWLEDGEMENTS

We thank the Ministry of Environment and Forests for funding this study through a project (No.22/16/2011-

(SG)-RE).

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ISSN (E): 2349 – 1183

ISSN (P): 2349 – 9265

2(3): 240–245, 2015

Research article

Vitamin C content of commonly eaten green leafy vegetables in

fresh and under different storage conditions

Umaramani Mathiventhan1* and Sivakanesan

Ramiah

2

1Senior Lecturer, Department of Botany, Faculty of Science, Eastern University, Sri Lanka 2Senior Professor, Department of Biochemistry, Faculty of Medicine, University of Peradeniya, Sri Lanka


Abstract: This study was an attempt to determine the consumption of popular green leafy

vegetables (GLVs) available in Batticaloa district, Sri Lanka and to determine vitamin C content of

fresh and stored (under room temperature 30±2ºC and at 4ºC for 4 days) GLVs. Vitamin C content

was estimated in aqueous extracts using dichloroindophenol titrimetric method. Thirty one species

of GLVs were consumed commonly by the subjects with an average consumption of 59%. Vitamin

C content of fresh GLVs ranged from 5.25 mg/100 g for Centella asiatica to 433.13 mg/100 g wet

weight for Drgea volubilis. Drgea volubilis, which had the highest amount of vitamin C, is

consumed by 80% of the consumers followed by Delonix elata, which is consumed by 52% of the

consumers interviewed. Murraya koenigii, which is used by almost all the consumers interviewed,

is a poor source of vitamin C. Similarly Centella asiatica, which was claimed to be consumed by

90% of the consumers, too was a poor source of vitamin C. The decline in vitamin C content of all

GLVs, ranging from 18% for Aerva lanata to 100% for Moringa oleifera, was higher and

significant (p = 0.000 at 95% confident interval) when stored at room temperature for 4 days than

stored at 4ºC (ranging from 2.5% for Sauropus androgynus to 70% for Alternanthera sessilis)

except Pisonia grandis. Both Drega volubilis and Delonix elata showed 22.3 and 6.1% increase

in vitamin C content respectively when stored at 4ºC.

Keywords: Vitamin C - Green leafy vegetables - Drgea volubilis

[Cite as: Umaramani M & Sivakanesan R (2015) Vitamin C content of commonly eaten green leafy vegetables

in fresh and under different storage conditions. Tropical Plant Research 2(3): 240–245]

INTRODUCTION

Green leafy vegetables (GLVs) are rich sources of many nutrients and form a major category of vegetable

group that are rich in health promoting phytochemicals. Their high antioxidant contents have attracted attention

of several investigators (Boxin et al. 2002, Ismail et al. 2004, Gupta et al. 2005).

More than 90% of vitamin C in human diet is supplied by fruits and vegetables (Latif & El-Aal 2007).

Vitamin C is required for the prevention of scurvy and has many biological activities in the human body

(Antonelli 2002). Unfortunately, this vitamin has low thermo stability and high water solubility (Anna 2007). In

view of this, refrigerated storage recommended for increased shelf life of fresh vegetables (Vina & Chaves

2006) would be helpful in retention of their vitamin C content. Latif & El-Aal (2007) reported that simple

packaging of fresh green leafy vegetables in polythene was very effective in reducing weight and moisture

losses during cold storage.

Inadequate number of studies, shortage of primary data and many information gaps exist regarding the

consumption pattern of GLVs as well as the vitamin C content of the GLVs under the existing practice of

storage. Therefore this study is an attempt to find the answers for the above concerns.


Determination of percentage of consumption

Ten popular market places in Batticaloa district namely Arayampathy, Batticaloa, Chenkalady, Eravur,

Kaluwanchikudy, Kallar, Kattankudy, Kiran, Oddamavadi and Valaichenai were selected for the study. During

Umaramani & Sivakanesan (2015) 2(3): 240–245

.


the preliminary study it was found that around 20,000 people regularly used these markets. Interviews were

conducted for a period of 10 months and 5 consumers (subjects) were interviewed, on a random basis, during

each visit to the selected market. A total of one thousand subjects were interviewed at the end of the survey (5

consumers × 2 times per month × 10 places × 10 months). Types of GLVs and percentage consumption of each

GLV based on the interview was calculated

Preparation of GLVs, storage and packing

The GLVs collected from field were cleaned and shoots separated. The shoots were washed thoroughly

under running tap-water for 5 minutes to remove soil particles and dirt. Then the washed shoots were placed in a

plastic container with paper towel and allowed to drain for 5 minutes. The cleaned, washed, GLVs were left at

room temperature to dry, and thereafter each GLV was wrapped with paper and then placed in polyethylene

bags. They were stored both at room temperature (30± 2ºC) and 4ºC (refrigerator) for 4 days.

Estimation of vitamin C (Ascorbic acid)

All chemicals used for the study were of analytical grade and distilled water was used for the preparation of

reagents. Samples of GLVs (10 g of each) were accurately weighed and ground using a mortar and pestle in 20

ml of metaphosphoric acid-acetic acid solution. The mixture was strained through a muslin cloth and the extract

was made up to 100 ml with the metaphosphoric-acetic acid solution. The extracts were prepared using 3

different samples of each GLV and all analyses were carried out in triplicates.

Ascorbic acid content was estimated by the 2, 6-dichloroindophenol (DCP) titrimetic method (Nielsen 2010).

Metaphosphoric acid-acetic acid solution (5 ml) was pipetted separately into three 50 ml Erlenmeyer flasks

followed by 2 ml of the sample extract. The samples were titrated separately with the indophenol dye solution

until a light rose pink colour persisted for 5 seconds. The 2, 6-dichlorophenolindophenol dye was standardized

against standard ascorbic acid. The results were expressed as mg ascorbic acid/100 g wet weight (WW).


Consumption of GLVs

Thirty one species of GLVs were consumed commonly as food by the subjects, which ranged from 15% for

Lactuca sativa to 98% for Murraya koenigii and the average consumption was 59.35%. Among the GLVs, 16

species were consumed for more than 50 % and 15 species were consumed 50% or less by the subjects

interviewed (Table 1). GLVs consumed more than 50% by the subjects were selected to determine the vitamin C

content. The consumption of 16 selected GLVs ranged from 52 to 98% with an average of 79.1% (Table 1).

Table 1. Overall consumption of commonly consumed leafy vegetables for food purposes.

No Name of GLVs Vernacular names

(T-Tamil, S- Sinhala, E-English)

Consumption

(%)

1 Aerva lanata* Polpala (s) /Thaenkaipukerai (T) /Hongone 81

2 Allmania nodiflora Kumatiya (S),Kumatti (T) 44

3 Alternanthera sessilis* Mukunuwenna/Ponnangani/Alligator weed 97

4 Amaranthus caudatus* Rana-Tampala/Kerai/Pendant amaranth 94

5 Amaranthus spinosus Mudkerai(T), Spiny amaranth (E) 42

6 Amaranthus viridis* Kura/Kuppaikerai/Green amaranth 76

7 Argyreia pomacea* Manpanchan (T) 60

8 Asteracantha longifolia Neer Mulli(T), Hydrophylla (E) 22

9 Basella alba Nivithi kola (S),Pasali (T), Indian spinach (E) 60

10 Borreria hispida Nathaisuri (T) 38

11 Canthium parviflorum Kara (S),Kaarai (T), Wild jasmine (E) 28

12 Cardiospermum halicacabum* Penela-wel (S)/Mudakottan (T)/ Wintetcherry 82

13 Centella asiatica* Gotukola (S)/Vallarai (T), Indian penny ward (E) 88

14 Cucurbita maxima Kumbala (S),Poosani (T), Pumpkin (E) 50

15 Delonix elata* Vatham-Nairaini (T),Yelow Gul-Mohur (E) 52

16 Drega volubilis* Anguna kola (S) Kurinja (T)/Sneez ward (E) 80


.


17 Gymnema sylvestre Bin –nuga (S),Sirukurinja (T), Gymnema (E) 37

18 Ipomoea aquatica Kangkong (S),Vallal (T), Water spinach (E) 49

19 Lactuca sativa Salada (S), Salathu (T), Lettuse (E) 15

20 Mollugo oppositifolia* Hinipala (S)/Thirai (T)/Itch flower (E) 84

21 Moringa oleifera* Murunga (S)/Murungai (T)/ Drum stick (E) 95

22 Murraya koenigii* Karapincha (S)/Karuveppilai (T)/Curry leaf (E) 98

23 Passiflora edulis Kodimathulai (T) 46

24 Pisonia grandis* Wathabanga (S) Ledchakaddai (T), Lettuse tree (E) 67

25 Premna obtusifolia Sihin-Midi (S), Kananthi (T) 39

26 Premna serratifolia Midi (S,) Earumai mullai (T) 24

27 Premna latifolia Maha midi (S), Pasumullai (T) 40

28 Rivex ornate Musutai (T), Midnapore creeper (E) 41

29 Sauropus androgynus* Japanbatukola (S)/ Thavasi murungai (T) 54

30 Sesbania grandiflora* Katuru murunga (S)/ Ahaththi (T) 76

31 Solanam trilobatum* Wel-tibbatu (S), Thuthuvilai(T), Heliotrope (E) 81

Average 59.35

Note: Selected plant species used for determining vitamin C content.

Vitamin C (Ascorbic acid) content

The sixteen GLVs showed varying ascorbic acid content in fresh state and under two temperature storage

conditions for 4 days (Table 2). The vitamin C content of fresh GLVs ranged from 5.25 mg/100 g for Centella

asiatica to 416.2 mg/100 g for Drega volubilis on wet weight basis (W/W). Gupta & Prakash (2009) reported

the ascorbic acid content of C. asiatica was 15.18 mg/100 g (W/W) using the same method as in the present

study. However, the vitamin C content of Centella asiatica in the present study is much lower than that reported

by Gupta & Prakash (2009).

The vitamin C content of 14 common vegetables in Nigeria estimated by the Nielsen (2010) procedure was

found to range from 400 to 692 mg/100 g dry weight and Amaranthus caudatus was reported to contain 400

mg/100 g on dry weight (Akindahunsi & Salawu 2005). Amaranthus caudatus was the only GLV investigated

by Akindahunsi & Salawu (2005) and in the present study and a comparison could not be made because of the

different procedures used. Oboh (2005) reported the vitamin C content of Amaranthus cruentus and Solanum

Table 2. Vitamin C contents of fresh and stored (for 4 days) GLVs.

No GLVs Vitamin C content (mg/100g)

Fresh Room temp. 30±2 ºC Refrigeration 4 ºC

1 Aerva lanata 53.67±5.10a 43.75±3.21c

49.29±4.50b

2 Alternanthera sessilis 36.17±3.42a 6.71±1.91c

10.79±1.58b

3 Amaranthus caudatus 51.63±4.15a 5.83±2.55c

28.88±5.41b

4 Amaranthus viridis 35.58±4.37a 6.42±2.32c

24.50±4.73b

5 Argyreia pomacea 7±1.86a 0.29±0.88c

3.21±1.16c

6 Cardiospermum helicacabum 85.17±11.07a 27.71±3.96c

44.63±5.08c

7 Centella asiatica 5.25±1.86a 2.63±1.31b

4.38±2.27a,b

8 Delonix elata 183.17±13.43a 77±13.32b

194.83±16.54a

9 Drega volubilis 416.2±38.6b 371±29.14c

505.46±7.08a

10 Mollugo oppositifolia 39.38±10.08a 7.29±2.19c

27.13±10.66b

11 Moringa oleifera 135.33±5.10a 0.00c

123.08±4.03b

12 Murraya koenigii 22.75±5.57a 16.04±5.16b

23.92±6.49a

13 Pisonia grandis 17.50±3.71a 11.08±2.55b

12.54±3.15b

14 Sauropus androgynus 66.79±6.58a 47.71±5.79b

65.04±5.53a

15 Sesbania grandiflora 134.75±14.07a 42.88±7.54c

83.13±9.64b

16 Solanam trilobatum 10.5±2.94a 3.5±2.27b

6.42±2.32b

Note: Values are means of 3 replicates ± Standard deviation; Statistically significance at 5% level

(Superscripts of letters a, b, c denotes statistically significant, superscripts of same letter denotes statistically

not significant).


.


macrocarpon estimated by the Nielsen (2010) method as 70.0±0.5 mg/100 g and 43.57±0.6 mg/100 g

respectively. The Amaranthus varieties used in the current study had lower vitamin C content than reported by

Oboh (2005) and the difference could be due to different varieties studied. Similarly the vitamin C content of

Solanam trilobatum used in the present study too was lower than the levels reported by Oboh (2005) for

Solanum macrocarpon.

In general, ascorbic acid content of GLVs declined during 4 days of storage and the differences observed in

vitamin C content among selected GLVs in fresh and under two storage conditions are significant (p < 0.05)

(Table 2). Drega volubilis showed the highest content of ascorbic acid (416.2±38.6 mg/100 g, when fresh) in all

three conditions followed by Delonix elata, Sesbania grandiflora, Moringa oleifera and Sauropus androgynus.

Three species showed lower ascorbic acid content in all three conditions, such as Argyreia pomacea, Centella

asiatica and Solanam trilobatum (Table 2). Vitamin C content of fresh GLVs such as Centella asiatica, Delonix

elata, Murraya koenigii and Sauropus androgynus showed no notable changes when stored in a refrigerator

(Table 2).

The decline in vitamin C content in all the GLVs, ranging from 18% for Aerva lanata to 100% for Moringa

oleifera, was higher and significant when stored at room temperature for 4 days than when stored at 4ºC

(ranging from 2.5% for Sauropus androgynus to 70% for Alternanthera sessilis) except for Pisonia grandis

(Table 3). Both Drega volubilis and Delonix elata showed 22.3 and 6.1% increase in vitamin C content

respectively when stored at 4ºC (Table 3).

Table 3: Loss of vitamin C of GLVs stored for 4 days at different temperatures.

No GLVs

Loss during storage (%)

Room temp.

30±2 0C

Refrigeration

4 0C

1 Aerva lanata 18a 8.04b

2 Alternanthera sessilis 81.6a 70.06b

3 Amaranthus caudatus 88.93a 44.4b

4 Amaranthus viridis 82.3a 31.51b

5 Argyreia pomacea 96.30a 53.70b

6 Cardiospermum helicacabum 67.41a 47.43b

7 Centella asiatica 50.00a 14.81b

8 Delonix elata 58.23a -6.31b

9 Drega volubilis 10.75a -22.29b

10 Mollugo oppositifolia 81.48a 32.93b

11 Moringa oleifera 100.00a 9.03b

12 Murraya koenigii 30.13a -4.88b

13 Pisonia grandis 36.27a 28.27a

14 Sauropus androgynus 37.69a 2.47b

15 Sesbania grandiflora 68.37a 38.31b

16 Solanam trilobatum 67.96a 38.52b

Note: Values are means of 3 replicates ± Standard deviation; Statistically significance

at 5% level (Superscripts of letters a, b, c denotes statistically significant, superscripts

of same letter denotes statistically not significant).

The vitamin C content of Drega volubilis increased significantly by 22.3% when stored in a refrigerator for 4

days compared to the fresh state (Tables 2 & 3). This should be further investigate any biochemical support to

this increasing. Interestingly, Latif & El-Aal (2007) also found that the vitamin C content of fresh GLVs

Raphanus sativus and Anthum graveolans increased during first 4 days of storage at 4ºC. The same observation

was made by Latif & El-Aal (2007) for Allium kurrat stored at 4ºC for 4 days. M. oleifera showed complete loss

of vitamin C at room temperature when stored for 4 days (Table 2 and 3). Moringa oleifera turned yellow and

some decayed when stored under room temperature for 4 days. Pisonia grandis showed no notable changes in

vitamin C when stored under room temperature and in a refrigerator.

More than 50% of vitamin C was lost in 10 species when stored at room temperature (30±2ºC) such as

Alternanthera sessilis, Amaranthus caudatus, Amarantus viridis, Argyeria pomacea, Cardiospermum

halicacabum, Delonix elata, Mollugo oppositifolia, Moringa oleifera, Sesbania grandiflora and Solanam

trilobatum. Green leafy vegetables such as Alternanthera sessilis, Cardiospermum halicacabum, Delonix elata,


.


Moringa oleifera and Sesbania grandiflora turned yellow when stored at room temperature. Alternanthera

sessilis and Argyeria pomacea lost more than 50% of vitamin C when stored at 4ºC (Table 3).

The highest percentage vitamin C loss when stored at room temperature was observed for Moringa oleifera

(100%) whereas highest percentage of vitamin C retained when stored at 4ºC was 98% for Sauropus

androgynus. Seven GLVs namely Aerva lanata, Centella asiatica, Delonix elata, Drega volubilis, Moringa

oleifera, Murraya koenigii and Sauropus androgynus showed more than 80% of vitamin C retention (Table 3).

Latif & El-Aal (2007) also reported that 80% of total vitamin C is retained when stored for 8 days, at 4ºC±1ºC

packed in polyethylene bags. The rate of loss of vitamin C is low at cold temperature in the current study which

is in agreement with the observation of Latif & El-Aal (2007) and Favell (1998) under similar packing

conditions using polyethylene bags. The variation in decline in vitamin C content in GLVs appears to be due to

species difference, pre and post-harvest conditions, initial vitamin C concentration, auto-oxidation, storage and

enzymatic degradation (Latif & El-Aal 2007, Rivera et al. 2006, Howard 1999). Vitamin C loss continues

during post-harvest handling, processing, cooking and storage (Moreira et al. 2006).

Drega volubilis, which had the highest amount of vitamin C is consumed by 80% of the subjects interviewed

(Table 1). Murraya Koenigii, which is used by almost all the subjects interviewed, is a poor source of vitamin C.

Similarly Centella asiatica, which was claimed to be consumed by 90% of the subjects, too was a poor source of

vitamin C. Furthermore, packaging and storing the vegetables at 4ºC, which prevents wilting and retain the

freshness of GLVs, preserves the vitamin C content compared to packaging and storing at room temperature.

These observations are valuable in carrying out nutrition awareness among the public. Delonix elata, which was

next to Drega volubilis in ascorbic acid content, was stated to be consumed by 52% of the subjects.

CONCLUSION

Thirty one species of GLVs were commonly consumed by the subjects. The average consumption was about

59% and ranged from 28 to 98%. GLVs consumed for medicinal purpose, ranged from 1.7% for Amaranthus

caudatus to 48.9% for Cardiospermum halicacabum and the average consumption was 20.11%. GLVs

consumed more than 50% by the subjects interviewed, were selected to determine the vitamin C content and

such GLVs also showed a high consumption for their medicinal properties.

Vitamin C content of fresh GLVs ranged from 5.25 mg/100 g wet weight for Centella asiatica to 416.2

mg/100 g wet weight for Drega volubilis. Decline in vitamin C content was observed in GLVs stored at room

temperature. GLVs wrapped with paper and then in polyethylene bags and stored at cool temperature retained

more than 80% of vitamin C. Vitamin C content of Drega volubilis increased by 22% when stored in a

refrigerator for 4 days. This needs further investigation.

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www.tropicalplantresearch.com 246 Received: 17 August 2015 Published online: 31 December 2015

ISSN (E): 2349 – 1183

ISSN (P): 2349 – 9265

2(3): 246–252, 2015

Research article

Physiological response of broccoli exposed to RuO2 nanoparticle

Imtiyaz Hussain1, Ajey Singh

1, Himani Singh

1, S. C. Singh

2 and N. B. Singh

1*

1Plant Physiology Laboratory, Department of Botany, University of Allahabad, Uttar Pradesh, India

2Department of Physics, University of Allahabad, Uttar Pradesh, India

*Corresponding Author: [email protected] [Accepted: 28 November 2015]

Abstract: Nanotechnology no doubt is a boom for science but exposure of nanoparticles (NPs) in

the environment is a new concern. Application of NPs in agriculture to increase crop yield is still

debated. In the present study 10–40 µgml-1

concentrations of ruthenium oxide (RuO2) NPs were

exposed to Broccoli (Brassica oleracea var. italica) seedlings in hydroponic culture. RuO2 NPs are

synthesized via Laser ablation method and hence are contamination free. UV-visible spectroscopy

and field emission scanning electron microscope(FE-SEM) is used for the characterization of NPs.

Carotenoids, protein and sugar content decreased with increase in concentrations of RuO2 NPs.

Total chlorophyll content increased to maximum with highest content of Chlorophyll a & b at 10

µgml-1

of RuO2 NPs, but no stimulatory effect was recorded at highest doses. Lipid peroxidation,

was unaffected by exposure to 0–20 µgml-1

NPs, but at 40 µgml-1

malondialdehyde (MDA)

formation and SOD activity increased by two fold. It is concluded that RuO2 NPs significantly

inhibited the seedlings growth of broccoli by impairing the metabolism of the test plants.

Keywords: RuO2 NPs - Brassica oleracea var. italica - FESEM - Hydroponic culture -

Malondialdehyde

[Cite as: Hussain I, Singh A, Singh H, Singh SC & Singh NB (2015) Physiological response of broccoli

exposed to RuO2 nanoparticle. Tropical Plant Research 2(3): 246–252]

INTRODUCTION

Nanoparticles (NPs) in between 1–100 nm size act as bridge between bulk material and atomic or molecular

structure (Kaushik et al. 2010). They possess remarkable and interesting properties due to small size, large

surface area, free dangling bonds, high reactivity other than bulk material of same composition (Daniel &

Astruc 2004). The use of nanomaterials in industries such as medicine (Panatarotto et al. 2003) and agriculture

(Singh et al. 2014, Shekhawat et al. 2014) is increasing rapidly which leads to its exposure in the environment.

Application of NPs in agriculture to increase crop yield is still debated. It is now widely recognized that

sufficient amount of NPs exists in the soil which affects living systems. A broad and mechanistic understanding

of the risks is posed by NPs in the environment, including bioaccumulation through the food chain, thus it is

necessary to adequately protect human and environment.

Ruthenium (Ru) a rare earth element is known to possess useful catalytic properties and have imperative role

in nuclear reactors which become source of Ru in the environment. Cowser & Parker (1958) reported Ru as

radioactive waste in soil. Incorporation of Ru in by plants is already verified (Selders 1950, Klechkwosky 1956,

Goss & Romey1959). Menzel & Brown (1959) found that ruthenium at 0.0018 and 0.18 mgml-1

in hydroponic

solution had no effect on the metabolism of Trifolium pratense. Very limited studies have been done to explore

the response of RuO2 NPs on plants and the toxicity data regarding Ru and its oxides are also scarce. Ru NPs

synthesized using Gloriosa superb L. leaf extract shows antibacterial activity against gram positive & negative

bacterial strains (Kasi et al. 2014).

The essential processes leading to plant adaptation to any stress or toxicity include regulation of water loss

through stomata, metabolic adjustment, toxic ion homeostasis, and osmotic adjustment has been studied

(Hasegawa et al. 2000, Munns & Tester 2008). Imlay & Linn (1998) reported that the reactive oxygen species

(ROS) are responsible for various stress-induced damages to tissues of an organism. Consequently the role of

antioxidant enzymes viz. superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) are

responsible for the quenching of ROS becomes very important (Bowler et al.1992). Thus the present study

Hussain et al. (2015) 2(3): 246–252

.


aimed to evaluate the potential effects of RuO2 NPs on living systems especially on physiological metabolism of

broccoli plant.


Synthesis of RuO2 NPs

Procedure for the synthesis of NPs was adopted from Singh et al. (2010). In the experiment, 2g of ruthenium

oxide bulk powder was dispersed in 20 ml of double distilled water (DDW). Fundamental (1064 nm) output

from pulsed Nd:YAG laser operating at 40 mJ pulse-1

energy, 10 ns pulse width and 10 Hz rep rate was

bombarded at the centre of the solution column using 25 cm focal length quartz lens for half an hour with

continuous magnetic stirring . In order to avoid laser induced sedimentation or aggregation, ablation process was

carried out for 30 minutes. The weight of the source was measured before and after the ablation process to

estimate concentration of RuO2 NPs. This process was repeated several times to get colloidal solution of RuO2

NPs as stock solution.

Characterization of RuO2 NPs

Characterization of synthesized RuO2 NPs was carried out by UV-visible spectroscopy and field emission

scanning electron microscope (FE-SEM). Lambda 35 Perkin Elmer spectrophotometer was used to record of

UV-visible absorption spectra of colloidal solution of RuO2 NPs. Scanning electron microscopic analysis was

done by JEOL JXA-8230. Very few amount of precipitated powder of NPs was coated on the copper grid and

allowed to magnify the grid to record morphological characters and size of NPs.

Hydroponic culture

The experiment was conducted in the month of February, 2015 in the glass house in the Department of

Botany, University of Allahabad, Allahabad (24o47’ and 50

o 47’N latitude; 81

o 91’ and 82

o 21’E longitude; 78

m above sea level). The different concentrations viz. 0, 10, 20, 30 and 40 µgml-1

of RuO2 NPs considered for C,

R1, R2, R3, R4 treatments respectively were obtained by dilution of stock solution with appropriate amount of

double distilled water (DDW). The nursery of the test plants was raised in nursery beds (1×1m). Twenty one

days old seedlings were uprooted and washed with tap water followed by DDW until soil was totally removed

from the roots. The properly washed 10 seedlings were transferred in transparent plastic boxes (23×17×9 cm)

filled with 2 litre of half strength Hoagland solution (Hoagland & Arnon 1950). Aeration of medium was done

with the help of bubblers for 12 h daily. The plants were allowed to establish for a week. After a week the

Hoagland solution was replaced with fresh Hoagland solution and the respective concentration of NPs twice at

the interval of seven days were also added. Box containing only Hoagland solution was taken as control. The

boxes were covered with black paper to avoid the algal growth in the nutrient medium. The biochemical

parameters were recorded after 14 days of treatment.

Pigment, Protein and Sugar content

The photosynthetic pigments viz. chlorophyll a, chlorophyll b and carotenoids from the first fully expanded

leaves (10 mg) were extracted with 80% acetone and quantified following Lichtenthaler (1987). Optical density

of supernatant was measured with UV-visible spectrophotometer Lambda 35 PerkinElmer at 663, 646 and 470

nm. The amount of pigments was calculated as:

Chlorophyll a (µgml-1

) = 12.21 x A663 - 2.81 x A645

Chlorophyll b (µgml-1

) = 20.13 x A645 - 5.03 x A663

Carotenoid (µgml-1

) = [1000 x A470 - 2.27(Chl. a) – 81.4(Chl. b)] / 227

Where, A is the observed OD

Quantitative analysis of protein was done following Lowry et al. (1951). The absorbance was measured at

650 nm. The amount of protein was calculated with reference to standard curve of bovine serum albumin.

The quantification of total soluble sugars was done following Hedge & Hofreiter (1962). Fresh leaf tissue

(0.05 mg) was homogenized in 5 mL of 95% ethanol. After centrifugation, 1 mL of supernatant was mixed with

4 ml anthrone reagent and heated in boiling water bath for 10 min. After cooling, the absorbance was recorded

at 620 nm. The amount of sugar was determined using the standard curve prepared from glucose.

Lipid peroxidation and SOD activity

The lipid peroxidation (LP) in leaves was measured by determining the malondialdehyde (MDA) content

according to Heath & Packer (1968). The plant material (200mg) was homogenized in 5 ml of 0.1% w/v

Hussain et al. (2015) 2(3): 246–252

.


trichloroacetic acid and centrifuged at 10,000g for 10 min. One mL of supernatant was mixed with 4 mlof 0.5%

thiobarbituric acid made in 20% trichloroacetic acid. The mixture was then heated at 95 ºC for 30 min followed

by cooling and centrifugation. The absorbance of supernatant was measured at 532 nm and corrected by

subtracting the non-specific absorbance at 600 nm. The MDA concentration was calculated using the extinction

coefficient of 155 mM-1

and expressed as n mol g-1

FW.

The activity of SOD (EC 1.15.1.1) was estimated by the nitrobluetetrazolium (NBT) photochemical assay

following Beyer & Fridovich (1987). The reaction mixture (4 ml) consisted of 20 mM methionine, 1.3M

riboflavin, 0.15 mM ethylene diamine-tetra acetic acid (EDTA), 0.12 mM NBT, and 0.5ml supernatant. The test

tubes were exposed to fluorescent lamp for 30 min and identical unilluminated assay mixture served as blank.

One unit of enzyme was measured as the amount of enzyme which caused 50% inhibition of NBT reduction.

Statistical Analysis

Treatments were arranged in a randomized block design with three replications. Data were statistically

analyzed using analysis of variance (ANOVA) by using SPSS (Ver.10; SPSS Inc., Chicago, IL, USA. The

treatment means were analyzed by Duncan’s multiple range test (DMRT) at p < 0.05.


UV-visible absorption spectra of bulk as well as NPs produced after 5, 10 and 30 minutes of laser

irradiations are shown in figure 1. It is evident that with the increase of time of laser irradiation, band edge

absorption shifts towards the shorter wavelength side, which indicates laser induced reduction in the size of

particles. Colloidal solution of NPs produced after 30 minutes of laser irradiation is highly constant in colloidal

form for several months and is used for the experiment. The morphological studies of synthesized NPs have

been carried out by Field Emission Scanning Electron Microscopy magnifications (FE-SEM). It is observed,

NPs constructing nanostructures were in the range of 30–113 nm with spherical shape in the under 50,000 X

(Fig. 2). The FE-SEM images of NPs were assembled on to the surface due to the interactions such as hydrogen

bond and electrostatic interactions which is supported by the SEM images of Kathiravan et al. (2015).

Figure 1. UV-Visible spectra of ruthenium oxide NPs at time in terval of 0, 5, 10 and 30 minutes.

Ru exhibited adverse effect on both physiological and biochemical parameters of crop plants. Amounts of

chlorophyll a, chlorophyll b, carotenoids, protein and MDA content and SOD activity were considered as

indicators for the estimation of effects of different concentrations of ruthenium oxide NPs on the health of the

plant.

Hussain et al. (2015) 2(3): 246–252

.


Figure 2. FE-SEM image of ruthenium oxide NPs taken at 50,000 X, showing NPs in the range of 30-113 nm.

The present study confirms that the increase in RuO2 NPs concentrations on broccoli seedlings appeared to

have negative impacts, primarily which is evident from a reduced sugar and protein content and the increase in

MDA formation and SOD activity by 2 fold at highest concentration. A decrease in sugar and protein under

unfavourable conditions allows the conservation of energy, thereby launching the appropriate defence response

and also reducing the risk of damage. RuO2NP did not significantly influence the chlorophyll content of plant

seedlings but in all treatments carotenoid content decreased (Table 1). The total chlorophyll content improved

under lower concentrations of RuO2 NPs. The seedlings treated with 10µgml-1

of RuO2 NPs exhibited maximum

content of chlorophyll a and b, while no effect was recorded at the highest dose of NPs. This might be due to

increase in efficiency of photosystems by Ru NPs and the role of Ru in photosystem is in agreement with the

work of Xiaojun et a.l (2004). They found that Ruthenium tris-bipyridine complex act as photosensitizer, that

plays the role of the p680 chlorophyll in psII. The protein content of leaves decreased significantly, reaching the

minimum value in the plant treated with 30µgml-1

of RuO2 NPs (Table 2). The decrease of sugar was higher

under low treatments as compared to that of treatments with increased concentration. RuO2 at 20 µgml-1

concentration caused maximum decrease in sugar content over control (Table 2). Our results on biochemical

parameters are in agreement with the findings of Khuzihko et al. (2011). According to their report RuO2 in

Table 1. Effects of Ruthenium Oxide NPs on pigment content of broccoli seedlings.

Treatments Chlorophyll a

(mg g-1

FW)

Chlorophyll b

(mg g-1

FW)

Total Chlorophyll

(mg g-1

FW)

Carotenoids

(mg g-1

FW)

C 2.26±0.088b 0.56±0.032

c 2.83±0.120

c 0.52±0.017

a

R1 2.93±0.088a 0.85±0.034

a 3.78±0.070

a 0.44±0.008

b

R2 2.72±0.109a 0.68±0.037

b 3.41±0.140

b 0.33±0.017

c

R3 1.91±0.075c 0.45±0.033

d 2.36±0.041

c 0.25±0.014

d

R4 2.16±0.094bc

0.40±0.006d 2.56±0.100

c 0.20±0.005

e

Note: C= 0 µgml-1

; R1= 10 µgml-1

; R2= 20 µgml-1

; R3= 30 µgml-1

; R4= 40 µgml-1

.

Mean ± SE values followed by same letters within each column are not significantly different at

0.05 (ANOVA and Duncan’s multiple range test) n=3.

Hussain et al. (2015) 2(3): 246–252

.


lower concentration enhanced the photocatalytic activity by taking part in oxygen evolution reaction (OER)

while in excess amount exhibited adverse effect on photocatalytic activity. Moreover, RuO2 also acts as a water

oxidation catalyst (Trasatti & Buzzanca 1971).

In our study, H2O2 in connection with other signal molecules may contribute to the control of plant growth

and development at specific checkpoints of the cell cycle (Xiong et al. 2002). Malondialdehyde (MDA)

production, a measure of lipid peroxidation, was unaffected by exposure to 0–20 µgml-1

NPs, but at 40 µgml-1

MDA formation was increased by more than two fold (Table 2). Increase in H2O2 was observed only at higher

concentrations conditions which are in agreement with the significant increase in H2O2 observed in cultivated

tomato (Mittova et al. 2002) and pea plants (Hernandez et al. 2001) under stresses. The seedlings treated with

the lower concentrations R1 and R2treatment of RuO2 NPs exhibited minimum lipid peroxidation while the

highest dose of RuO2 NPs exhibited maximum 128% of lipid peroxidation.

SOD activity significantly enhanced under higher concentrations of RuO2 NPs (Table 2).Because of the

significant damage likely resulting from ROS production and associated toxicity of NPs exposure, the SOD

content of plant leaves was determined. Quantification of SOD activity confirms the results that at the10 µgml-

1exposure level had no impact but exposure at 40 µgml

-1 NPs resulted in significantly greater SOD activity.

SOD activity directly modulates the amount of ROS same from what was reported by Gómez et al. (2004)

which found an increase in all SOD isoenzymes of pea chloroplasts under stress. Deficiency of micronutrients

such as Mn and Zn also affects SOD activities in plants (Yu & Rengel 1999) but we provide appropriate

hogland solution as reported earlier (Singh et al. 2014). Thus in our results the activity of SOD under stress

depends on the toxicity level.

CONCLUSION

We have successfully demonstrated the toxic effect of RuO2NPs on broccoli plant. Purity of laser produced

RuO2 NPs with chemical contamination free surfaces helps in the real investigation of NPs effects on the

biological systems. The NPs at 40 µgml-1

resulted in drastic increase of antioxidant enzyme (SOD) and MDA

content and decrease in carotenoid, protein and sugar content in hydroponic culture. The decrease in sugar

content and protein content is due to diverting of maximum energy towards plants defence mechanism. Based

on findings we conclude that higher doses (>20 µgml-1

) of RuO2 NPs are toxic. Further study is required to

explore the minimum dose of RuO2 NPs which have least impact on biological system that ensures safe

environment release.

ACKNOWLEDGEMENTS

The authors are thankful to the CSIR and UGC, New Delhi, India for providing financial assistance to

Imtiyaz Hussain and IIT Kanpur for providing SEM facility.

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Table 2. Effects of Ruthenium Oxide NPs on protein and sugar content, lipid peroxidation and SOD

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lipid peroxidation

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Superoxide dismutase

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R1 24.39±0.265b 77.39±1.243b 12.98±0.193c 43.27±0.177e

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R4 20.14±0.098d 77.32±0.779b 27.62±0.285a 99.79±1.015a

Note: C= 0 µgml-1

; R1= 10 µgml-1

; R2= 20 µgml-1

; R3= 30µgml-1

; R4= 40 µgml-1

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ISSN (E): 2349 – 1183

ISSN (P): 2349 – 9265

2(3): 253–256, 2015

Research article

Three new records of dicotyledonous plants from Bangladesh

Md. Salah Uddin¹*, Mohammad Sajid Ali Howlader² and Shaikh Bokhtear Uddin³

¹Initiative for Biodiversity Research and Development (IBIRD), Dhaka, Bangladesh

²Ecological Genetics Research Unit, Department of Biosciences, University of Helsinki, Helsinki, Finland

³Department of Botany, University of Chittagong, Chittagong, Bangladesh

*Corresponding Author: [email protected] [Accepted: 02 December 2015]

Abstract: Three dicotyledon plant species belong to three different families of angiosperm include

Symphorema involucratum from Verbenaceae, Miliusa balansae from Annonaceae and

Spermacoce ocymoides from Rubiaceae have been recorded for the first time from Bangladesh.

They are collected from Chittagong and Chittagong hill tracts. Each species is presented with

updated nomenclature, taxonomic description, ecology, geographical distribution, places of

occurrence in Bangladesh and field photographs of these three new records are also provided.

Keywords: Bangladesh - Dicotyledon - Angiosperm - New records - Plants

[Cite as: Uddin MS, Howlader MSA & Uddin SB (2015) Three new records of dicotyledonous plants from

Bangladesh. Tropical Plant Research 2(3): 253–256]

INTRODUCTION

Dicotyledons plants are the most successful and dominant plant group (Heywood 1993) having seeds with

two cotyledons and an exogenous manner of growth (Cronquist 1981, Bewley & Black 1994). It is one of the

classes of the great subdivision of flowering plants, the angiosperms (Wagner et al. 1999, McKenna et al. 2009).

About 4,00,000 species of angiospermic plants have so far been recorded of which more than 2,50,000 are

dicotyledons and the remaining are monocotyledons (Leitch & Leitch 2008). Bangladesh is endowed with about

5,000 species of flowering plants (Pasha & Uddin 2013, Rashid et al. 2014) of which more than two third are

dicotyledonous (Khan 1972-1987). Dicot plants dominate the forests, village groves and woodlands of

Bangladesh (Khan & Banu 1969, Khan & Afza 1968, Khan 1972-1987).

Here, in this paper, three dicot plant species from three different families includes Verbenaceae, Annonaceae

and Rubiaceae are reported for the first time from Chittagong and Chittagong Hill Tracts for Bangladesh, placed

in Indo-Burma biodiversity hotspot.


The plant specimens of three families include Verbenaceae, Annonaceae and Rubiaceae have been collected

from different areas of Bangladesh, mainly the forest of Chittagong and Chittagong Hill Tracts (Fig. 1) through

repeated field trips.

The collected specimens were preserved at Chittagong University Herbarium (CUH) and examined by using

Microscope. Unnamed specimens were identified and described by consulting relevant floristic literature of

Roxburgh (1805), Gamble (1921-1935), Matthew (1991), Nair et al. (1983), Henry et al. (1987, 1989), Pasha &

Uddin (2013).

The photographs of fertile specimens in natural habitat were taken during field trips. A taxonomic

enumeration with these three newly recorded from three families are prepared. In the ennumeration, each

species is cited with current nomenclature, taxonomic description, ecology, geographical distribution,

occurrence in Bangladesh.

RESULTS

Taxonomic enumeration

Family: Verbenaceae J. St.-Hil. 1805

Uddin et al. (2015) 2(3): 253–256

.


Figure 1. Distribution of three species in Bangladesh.

Genus: Symphorema Roxburgh, 1805

Symphorema involucratum Roxburgh, 1805 (Fig. 2A)

A climbing shrub, with cylindrical branchlets which are hairy when young. Leaf blade is nearly elliptic to

ovate, densely velvety on the underside, and somewhat smooth above. Leaf base is rounded to slightly heart-

shaped, margin nearly entire to toothed. Flowers are borne in beautiful clusters carried on long, velvety stalks.

The bracts just below the flowers are oblong, enlarged in fruit. Sepal cup is tube-like, velvety outside. Flowers

are white, with 6-8 narrowly oblong petals. Fruit is nearly round.

Flowering: March–April.

Ecology: Forests or scrub in valleys.

Geographical distribution: India, Myanmar, Sri Lanka and Thailand.

Occurrence in Bangladesh: It was collected from Recha jhiri (22°10'56.4"N 92°11'18.0"E), Meghla, Sadar

upazila, Bandarban district, Bangladesh.

Family: Annonaceae Juss., 1789

Genus: Miliusa Leschenault ex Alph. de Candolle, 1832

Miliusa balansae Finet & Gagnepain, 1906 (Fig. 2B)

A shrub about 2–5 m tall, branchlets slightly pubescent. Leaves petiolate, leaf blade elliptic, elliptic-oblong,

or oblong, membranous, glabrous or sparsely puberulent on midvein and secondary veins but glabrescent,

secondary veins 10–12 on each side of midvein, base cuneate to rounded and oblique, apex acuminate to shortly

acuminate. Inflorescences axillary, flowers solitary. Pedicel filiform, pendulous, glabrous.Sepals ovate, slightly

pubescent. Petals red; outer petals slightly longer than sepals; inner petals ovate, apex reflexed. Anthers ovoid to

obovoid.Carpels oblong to lens-shaped, slightly pubescent; ovules 2 or 3 per carpel; stigmas terete,

puberulent.Fruiting peduncle slender; monocarps globose.Seeds 1–3 per monocarp.

Flowering: April–July; fruiting: July–December.

Ecology: Forests or scrub in vallyes.

Geographical distribution: China and Vietnam.

Occurrence in Bangladesh: The plant species was collected from Dulu jhiri (22°11'01.7"N 92°11'24.0"E),

Meghla, Sadar upazila, Bandarban district, Bangladesh

http://www.tropicos.org/LocationDetails.aspx?locationid=625

Uddin et al. (2015) 2(3): 253–256

.


Family: Rubiaceae Juss., 1789

Genus: Spermacoce L., 1753

Spermacoce ocymoides Burm. f., 1768 (Fig. 2. C & D)

An annual herb, stem erect rarely prostrate, hairy. Leaves are oppositely arranged. It should be relatively

easy to differentiate from other species with its broadly elliptic (sometimes lanceolate) leaves which have

depressed venation, either hairless or with scattered hairs on veins below and margin. Flower clusters appear at

the end of branches or in leaf axils. Sepals are narrowly triangular, length variable on individual flowers.

Flowers are white, petals much longer than the tube. Stamens protrude out, but are much shorter than the petals.

Style does not exceed the stamens.

Flowering: April–August.

Ecology: Plain land along roads, hill slopes and forest periphery.

Geographical distribution: Tropical Africa, Mauritius, India, Myanmar, Java, Peninsular Malaysia and

Philippines.

Occurrence in Bangladesh: This species was collected from two regions; one is Chittagong University Kata

Pahar road side (22°28'15.4"N 91°47'23.5"E), Hathazari, Chittagong, Bangladesh and another collection place is

Toitong High School road side (21°53'00.2"N 91°58'22.5"E), Pekua, Cox’s Bazar, Bangladesh.

Figure 2. A, Symphorema involucratum; B, Miliusa balansae; C & D, Spermacoce ocymoides.

DISCUSSION AND CONCLUSION

We have reported three species from three highly diverged families comprising several numbers of species.

The family Verbenaceae consists of about 100 genera and 2600 species mostly pantropical, a few are limited to

temperate regions. In Bangladesh, this family is represented by 19 genera and 68 species (Ahmed et al. 2009).

Uddin et al. (2015) 2(3): 253–256

.


Annonaceae family consists of about 130 genera and 2300 species are very largely confined to tropical regions,

15 genera and 42 species in Bangladesh (Ahmed et al. 2008). Rubiaceae includes about 450 genera and 6500

species occur in tropical and subtropical regions, in Bangladesh it has 56 genera and 170 species (Ahmed et al.

2009).

Herein we report three species which were available in other neighboring countries of Bangladesh. Our

findings may increase the possibility of finding more species which were reported from neighboring countries.

ACKNOWLEDGEMENTS

The authors expressed their deepest sense of gratitude and sincere thanks to Dr. Mostafa Kamal Pasha,

Department of Botany, University of Chittagong for his kind co-operation for article preparation.

REFERENCES

Ahmed ZU, Begum ZNT, Hassan MA, Khondoker M, Kabir SMH, Ahmad M, Ahmed ATA, Rahman AKA &

Haque EU (2008) Encyclopedia of Flora and Fauna of Bangladesh. Vol. 6. Angiosperms: Dicotyledons

(Acanthaceae-Asteraceae). Asiatic Society of Bangladesh, Dhaka, Bangladesh, pp. 122–142.

Ahmed ZU, Hassan MA, Begum ZNT, Khondoker M, Kabir SMH, Ahmad M & Ahmed ATA (2009)

Encyclopedia of Flora and Fauna of Bangladesh. Vol. 10. Angiosperms: Dicotyledons (Ranunculaceae-

Zygophyllaceae). Asiatic Society of Bangladesh, Dhaka, Bangladesh, 580 p.

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Cronquist A (1981) An integrated system of classification of flowering plants. Columbia University Press, New

York, United States.

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alkaloids from Garcinia kola, Borreria ocymoides, Kola nitida and Citrus aurantifolia. The Journal of

Applied Bacteriology 71: 398–401.

Gamble JS & Fischer CEC (1921-35) Flora of the Presidency of Madras. 3 Vols. Adlard and Son Ltd. London,

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Botanical Survey of India, Coimbatore, India, 171 p.

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Survey of India, Coimbatore, India, 258 p.

Heywood VH (1993) Flowering plants of the world (No. Ed. 2). BT Batsford Ltd.

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(Monocotyledons). Journal of Asiatic Society, Pakistan 14(2): 217–224.

Khan MS (1972-1987) Flora of Bangladesh. Fasc. 1–39. Bangladesh National Herbarium, Dhaka, Bangladesh.

Leitch A R & Leitch IJ (2008) Genomic plasticity and the diversity of polyploid plants. Science 320(5875):

481–483.

Matthew KM (1991) An Excursion Flora of Central Tamil Nadu, India. Oxford and IBH Publishing co., New

Delhi, India, 647 p.

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of weevils and flowering plants. Proceedings of the National Academy of Sciences 106(17): 7083–7088.

Nair NC & Henry AN (1983) Flora of Tamil Nadu, India. Series I: Analysis. Vol. 1. Botanical Survey of India,

Coimbatore, India, 184 p.

Pasha MK & Uddin SB (2013) Dictionary of Plant Names of Bangladesh. Janokalyan Prokashani, Chittagong,

Bangladesh, 434 p.

Rashid, MHU, Rashid M E & Rahman MA (2014) Inventory of threatened plants of Bangladesh and their

conservation management. International Journal of Environment 3(1): 141–167.

Roxburgh W (1805) Plants of the Coast of Coromandel. Vol. 2. W. Bulmer and Co., London, pp. 46.

Wagner WL, Herbst DR & Sohmer SH (1999) Manual of the Flowering Plants of Hawai'i, Vols. 1 and 2 (No.

Edn 2). University of Hawai'i and Bishop Museum Press, Honolulu Hawaii, USA.


ISSN (E): 2349 – 1183

ISSN (P): 2349 – 9265

2(3): 257–263, 2015

Research article

Genotypic variations in the inhibitory potentials of four combined

botanicals on mycelia growth of Macrophomina phaseolina of

cowpea [Vigna unguiculata (L) Walp.]

A. O. Akanmu1, O. J.

Olawuyi

1, O. B.

Bello

2*, O. A.

Akinbode

3,

T. Aroge

1, B.

Oyewole

1 and A. C.

Odebode

1

1Department of Botany, P.O. Box 128, University of Ibadan, Ibadan, Nigeria 2Department of Biological Sciences, Fountain University, Osogbo, Nigeria

3Plant Pathology Unit, Institute of Agriculture Research and Training, Apata, Ibadan, Nigeria


Abstract: The ethanol extracts of Ficus asperifolia, Mormordica charantia, Anacardium

occidentals and Psidium guajava were evaluated sole and in treatment combinations at 25, 50 and

75mg ml-1 concentration levels against the mycelial growth of Macrophomina phaseolina of

Cowpea. The pathogen was cultured on plates containing botanicals amended Potato Dextrose

Agar (PDA) in three replicates while only ethanol treated PDA tested plates served the control

experiment. The radial growths were recorded at 4th, 6th and 8th day after inoculation. Data

obtained were analysed using the SAS software program version 9.2. The extract of Mormordica

charantia was the most effective in the botanical treatments alone. The most significant inhibition

of Macrophomina phaseolina were observed from the combined treatments of Ficus asperifolia,

Mormordica charantia and Anacardium occidentals (3.11 cm), followed by Mormordica

charantia and Psidium guajava (3.29 cm), then combination of four extracts; Ficus asperifolia,

Mormordica charantia, Anacardium occidentals and Psidium guajava (3.53 cm), then

Mormordica charantia and Anacardium occidentals (3.84 cm). Other treatments, either alone or in

combination produced significant result compared to the control experiment (6.94 cm). However,

the efficacy of botanicals increased with concentration and also significantly correlated with time

and reduction in mycelia extension of the pathogen. More so, variability in the antifungicidal

potentials of the botanicals on Macrophomina phaseolina ranges from 15.93% to 34.06%

according to Eigen proportions. The treatment combinations of; Ficus asperifolia, Mormordica

charantia and Anacardium occidentals at 75mg ml-1 concentration level produced the most

inhibitory effect against Macrophomina phaseolina in vitro. However, the untreated plates did not

show inhibitory effect on the mycelial growth of the pathogen. Therefore, combined treatments of

botanicals could be a potential source in the practise of plant disease control.

Keywords: Macrophomina phaseolina - Mycelial growth - Correlation - Eigen proportion

[Cite as: Akanmu AO, Olawuyi OJ, Bello OB, Akinbode OA, Aroge T, Oyewole B & Odebode AC (2015)

Genotypic variations in the inhibitory potentials of four combined botanicals on mycelia growth of

Macrophomina phaseolina of cowpea [Vigna unguiculata (L) Walp.]. Tropical Plant Research 2(3): 257–263]

INTRODUCTION

Macrophomina phaseolina (Tassi) Goid. belongs to the family Botryosphaeriaceae. It is a highly polyphagus

necrotrophic fungal pathogen that infects more than 500 plant hosts (Wyllie 1988). The plants invaded by this

pathogen are; major food crops, pulse, fiber and oil crops (Su et al. 2001, Mayek-Pérez et al. 2001, Dinakaran &

Mohammed 2001, Aly et al. 2007, Khan 2007). Macrophomina phaseolina have a wide geographical

distribution, and is especially found in tropical and subtropical countries with arid to semi-arid climates in;

Africa, Asia, Europe, and North and South America (Wrather et al. 2001). Diseases caused by Macrophomina

phaseolina include ashy seedling blight, seedling damping-off, charcoal rot, stem rot, and root rot leads to

significant yield loss (Emechebe & Lagoke 2002, Wellington et al. 2011). Incidences of these diseases are

Akanmu et al. (2015) 2(3): 257–263

.


favoured at low soil moisture (Sandhu et al. 1999, Islam et al. 2012) and high temperatures (30–35°C). The

comparatively high capacity of Cowpea to withstand drought stress, poor soil conditions and high temperatures

made the crop very valuable especially in arid and Sub-Saharan regions of West Africa where cowpea is a crop

of major economic importance for resource poor farmers (Sanginga et al. 2003).

The persistence of sclerotia of Macrophomina phaseolina in the soil and plant debris (Short et al. 1980)

coupled with its wide host range has resulted in difficulty of the disease control especially on Cowpea where the

pathogen constitutes a major yield-suppressing factor even when under drought stress (Wyllie 1993). The

ineffective chemical control approach against Mormordica phaseolina necessitates a biological control measure

(Mark & Norman 2007). However, disease caused by fungi pathogens and their control with botanicals has been

found to be environmental friendly and effective against the targeted pathogen (Odebode 2006, Akanmu et al.

2013a, Abiala et al. 2013). This study investigates the efficacy of the interactive treatments of the extracts of

Ficus asperifolia, Mormordica charantia, Anacardium occidentals and Psidium guajava alone and in treatment

combinations against the pathogenic Macrophomina phaseolina of Cowpea.


Source of pathogen

The pathogenic strain of Macrophomina phaseolina isolated from Cowpea was obtained from the plant

pathology laboratory, International Institute of Tropical Agriculture (IITA) Ibadan, Oyo state, Nigeria.

Source of plant extracts

Fresh leaves of Ficus asperifolia, Mormordica charantia, Anacardium occidentales and Psidium guajava

were obtained from Botanical garden and authenticated at the herbarium laboratory, both of the Department of

Botany, University of Ibadan, Ibadan, Nigeria.

Preparation of plant extracts

The fresh leaves were washed in clean water to be freed from possible dirt and debris. The cleaned leaves

were soaked in 5% Sodium hypochlorite solution for 10 minutes and rinsed in three exchanges of distilled water

to neutralize the effect of Sodium hypochlorite. The leaves were then air dried for two week weeks after which

they were blended separately into powdery form. The ethanolic extraction was carried out by weighing 2.5 g,

5.0 g and 7.5 g of each powdered extracts separately into 100ml of 75% ethanol, to achieve 25 mg ml-1, 50 mg

ml-1 and 75 mg ml-1 concentration levels respectively.

In vitro control of Macrophomina phaseolina with plant extracts

The Macrophomina phaseolina culture obtained was asceptically subcultured on Potato Dextrose Agar

(PDA) and incubated for 7 days. The biocontrol experiment using plant extracts was carried out by adding 1ml

of the extracts to 9 ml of PDA poured on each plate. This was observed on each concentration level of the four

plant extracts used in this study. For the interactive treatment which involves the combination of two extracts,

0.5 ml of each extracts were added to 9ml of PDA poured per plate. The interaction of three extracts were

prepared by adding 0.33 ml of each extract to 9 ml of PDA while for the different four extracts used, 0.25 ml of

each were added to 9 ml of PDA. The extracts amended molten PDA was initially swirled gently for

homogenization before the plates were poured and allowed to solidify. Using a 5 mm cork borer, the mycelia

growth from the advancing edge of the 7 day old pure culture of Macrophomina phaseolina were picked and

inoculated at the centre of extracts treated PDA plates. The control experiment consisted of the treatments of 1

ml 75% ethanol with 9 ml of PDA. The plates were then incubated for 8 days, during which data on the radial

growth of the pathogen in each plate were taken at the 4th day, 6th day and 8th day of the experiment.

Percentage inhibition of mycelial growth by the leaf extracts was calculated using the formula:

Where: %MGI = % Inhibition of mycelial growth

DC = diameter of control

DT = diameter of test

Data analysis

Data obtained from the radial growths were subjected to the Analysis of Variance (ANOVA) using System

Akanmu et al. (2015) 2(3): 257–263

.


Analysis Software package (SAS) 9.1 2009 version, while means were separated by Duncan Multiple Range

Test (DMRT).

RESULTS

Highly significant (p<0.0) result was obtained on the radial growths of Macrophomina phaseolina by

activities of the plant extracts, with respect to period (day) of the study, also, in the interactions involving;

concentration x day, extracts x concentration, extracts x day, extracts x replicates, extracts x concentration x

day, also, extracts x replicate x concentration. While significant (p<0.05) effects were produced in the

interactions of concentration x day and extract x replicates x day (Table 1).

Table 1. Interactive effect of extract, replicate, concentration and day of data collection on the

mycelia growth of Macrophomina phaseolina.

Source df Radial Growth

Concentration 2 1.46ns

Day 2 1624.83**

Extracts 15 33.01**

Replicates 3 0.13ns

Concentration x Day 4 1.32*

Extracts x Concentration 30 8.10**

Replicates x Concentration 6 0.19ns

Extracts x Day 30 3.61**

Replicates x Day 6 0.03ns

Extracts x Replicates 45 1.33**

Extracts x Concentration x Day 60 1.23**

Replicate x Concentration x Day 12 0.51ns

Extract x Replicate x Concentration 90 1.18**

Extract x Replicate x Day 90 0.82*

Error 180 101.21

Corrected total 575 4527.88

Note: ** = Highly significant (p<0.01), * = Significant (p<0.05), ns = not significant.

Considering the sole treatment of the four botanicals tested, Mormordica charantia (4.34 cm) had the

highest mean inhibitory effect of 4.34 cm on the mycelial growths of Macrophomina phasolina, while F.

asperifolia (5.69 cm), Anacardium occidentals (5.59 cm) and Psidium guajava (5.55 cm) which showed slight

inhibitory effect on the pathogen, were not significantly (p>0.05) different from the control (5.69 cm). A more

significant (p<0.05) control of the pathogen was achieved by the effects of the combined treatments of the four

extracts, with the extracts of Mormordica charantia and Psidium guajava (3.29 cm) as well as Ficus asperifolia,

Mormordica charantia and Anacardium occidentales (3.11 cm) on Macrophomina phaseolina (Table 2).

Table 2. Inhibitory effect of interaction of extracts on the mycelia growth of Macrophomina phaseolina.

Extracts Radial growth (cm)

Anacardium occidentals 5.59b

Psidium guajava 5.55b

Ficus asperifolia and Mormordica charantia 4.79c

Ficus asperifolia and Anacardium occidentals 4.61dc

Ficus asperifolia and Psidium guajava 4.63dc

Mormordica charantia and Anacardium occidentals 3.84gh

Mormordica charantia and Psidium guajava 3.29ij

Anacardium occidentals and Psidium guajava 4.76c

Ficus asperifolia, Mormordica charantia and Anacardium occidentals 3.11j

Ficus asperifolia, Mormordica charantia and Psidium guajava 4.36de

Mormordica charantia, Anacardium occidentals and Psidium guajava 4.16efg

Ficus asperifolia, Anacardium occidentals and Psidium guajava 3.95fg

Ficus asperifolia, Mormordica charantia, Anacardium occidentals and Psidium guajava 3.53hi

Control 6.94a

Note: The significant difference (p<0.05) is indicated by different letters along each column.

The growth inhibition at 75 mg ml-1 concentration produced significant (p<0.05) effect, followed by 25 mg

ml-1, while the concentration of 50 mg ml-1 had the least mycelia inhibition. The radial growth of the pathogen

Akanmu et al. (2015) 2(3): 257–263

.


increases with time in which the most significant growth was recorded on the 8th day of data collection (7.32

cm), followed by the 6th day (4.63 cm) while the least was recorded on the fourth day (Table 3).

Table 3. Mean performance of botanical concentration, days of observation and replicated treatments

on the mycelia growth of Macrophomina phaseolina.

Observed characters Variables Radial growth (cm)

Extract concentration (mg ml-1

) 25 4.57a

50 4.49ab

75 4.40b

Days 4 1.51c

6 4.63b

8 7.32a

Replicates 1 4.50a

2 4.51a

3 4.48a

4 4.45a

Note: The significant difference (p<0.05) is indicated by different letters along each column.

The day of observation is positive and significantly (p<0.05) correlated with concentration of the extract and

mycelia growth with r=0.15 and 0.85. There was negative association between the extracts and the mycelia

growth with r=0.22 while non-significant and negative association occurs between the radial growth and

replicate as well as concentration, no association exists between extracts and days of observation, concentration

and the replicates (Table 4).

Table 4. Effect of Correlation of the extracts; extract concentration, days of observation and

replicates on the radial mycelia growth of Macrophomina phaseolina.

Correlation Extracts Replicates Concentration Day

Replicates 0.00ns

Concentration 0.00ns 0.00ns

Day 0.00ns 0.00ns 0.15*

Radial growth -0.22* -0.01ns -0.01ns 0.85**

Note: ** = Highly significant (p<0.01), * = Significant (p<0.05), ns = not significant.

The contribution of principal component analysis (PCA) in tables 5 showed variations in the Eigen values

and proportion of the mycelial growth. Prin 1 accounted for the highest variation with the highest Eigen vector

for extract, concentration and day of experimental observation at proportion of 34.06%. The first and fourth

PCA showed positive and more relatedness for the extracts treatments. The extract concentration of the second

PCA had the highest Eigen value which accounted for 25.01% of total variation while the third PCA had the

highest vector for the replicates (Table 5).

Table 5. Contribution of principal component analysis (PCA) to the variation in the radial growth

of Macrophomina phaseolina.

Radial growth Prin1 Prin2 Prin3 Prin4

Extracts 0.706 -0.067 -0.011 0.705

Replicates 0.022 0.389 0.920 0.003

Concentration 0.035 0.919 -0.390 0.047

Day 0.707 0.010 0.002 -0.707

Eigen values 1.360 1.000 1.000 0.637

Proportion (%) 34.06 25.01 25.00 15.930


Research has demonstrated that biological control is a potentially feasible alternative to the use of pesticides

(Jacobsen et al. 2004, Adandonon et al. 2006, Sobowale et al. 2008, Olawuyi et al. 2011, Akanmu et al. 2012,

2013a, Olawuyi et al. 2013). The combined use of biocontrol agents in the control of some important pathogens

affecting crops of economic importance is a new trend in plant disease control experiments. The integrated

approach of biocontrol research adopted in this research using botanicals of; Ficus asperifolia, Mormordica

charantia, Anacardium occidentals and Psidium guajava at different concentrations levels in sole and in

combinations towards the control of the mycelial growth of Macrophomina phaseolina of cowpea is in line with

the observations earlier made (Odebode 2006, Adandonon et al. 2006).

Akanmu et al. (2015) 2(3): 257–263

.


Macrophomina phaseolina had been reported as the cause of charcoal rot disease which is a major biotic

factor that limits cowpea productivity worldwide (Ma et al. 2010, Zveibil et al. 2012, Arshad et al. 2012). This

was demonstrated in the reaction of cowpea varieties to infection of Macrophomina phaseolina obtained from

six different leguminous plants in Nigeria, as was earlier investigated by Amusa et al. (2007). The possible

control of this pathogen using botanicals as investigated in this research showed the efficacy of all the extracts

tested when compared to the results obtained in the control experiment. The effectiveness of the botanicals

against Macrophomina phaseolina could be attributed to their antifungal properties as was also discovered by

Arshad et al. (2012) who reported the efficacy of the antifungal properties of different parts of Sorghum

halepense Pers. in the control of Macrophomina phaseolina of cowpea.

The efficacy of the extract of Mormordica charantia which showed the most inhibitoriest potential on the

growth of Macrophomina phaseolina had earlier been reported in the treatment of human and plant diseases

(Virdi et al. 2003, Burger et al. 2010, Jonathan et al. 2012). However, the most significant mycelial inhibition of

Macrophomina phaseolina which was recorded in the combination of the extracts of; Ficus asperifolia,

Mormordica charantia and Anacardium occidentals, compared to the control experiment conformed to the

findings of Akhter et al. (2006) who observed 91% to 100% inhibition of conidial germination of Bipolaris

sorokiniana by plant extracts amended with cow dung and cow urine. The efficacy of combined treatment of

plant extracts was also affirmed in the combined treatments of botanicals and antibiotics against some emerging

drug-resistance microorganisms investigated by Rakholiva & Chanda (2012).

The significant result in interactions of the biological agents with the pathogen is in accordance with

findings of Sobowale et al. (2009), Akanmu et al. (2013b), Olawuyi et al. (2011, 2013). More so, the efficacy of

botanicals increases with concentration while it was also significantly correlated with time and the reduction in

the mycelia extension of the pathogen as similar reported by Akinbode (2010).

The significant correlation of the days of observation with extracts concentration and mycelia growth is an

indication of the positive contribution of the extract and their concentration in inhibition of Macrophomina

phaseolina as similarly observed by Akanmu et al. (2013a) and Olawuyi et al. (2014). The contribution of the

principal component analysis in this study is an indication of variability in the inhibitory potential of the

botanical extracts on the mycelia growth of the pathogen in accordance with the findings of Olowe et al. (2013).

The mycelial growths of Macrophomina phaseolina showed variability which ranges from 15.93% to 34.06%

due to the interactive treatments of botanicals at varying concentration over a period of time according to Eigen

proportion. The variations in the extract performances could be attributed to differences in their anti-fungicidal

properties, as similarly confirmed by Burger et al. (2010), Olawuyi et al. (2010) and Fapounda et al. (2011).

This suggests that there are variations in bioactive of antifungal compounds with varying characteristics and

potentials in their modes of action. However, the effectiveness of botanicals in field condition could

subsequently affirm the findings of this study to prove further their potentials in plant disease control.

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www.tropicalplantresearch.com 264 Received: 03 September 2015 Published online: 31 December 2015

ISSN (E): 2349 – 1183

ISSN (P): 2349 – 9265

2(3): 264–270, 2015

Research article

Comparative evaluation of salicylic acid and EDTA chelant induced

phytoremediation of lead and nickel using Lemna minor L.

Leela Kaur1*, Kasturi Gadgil

2 and Satyawati Sharma

3

1Department of Environmental Science, Maharaja Ganga Singh University, Bikaner, Rajasthan, India 2ABES Engineering College, Ghaziabad, Uttar Pradesh, India

3Centre for Rural Development & Technology, Indian Institute of Technology (IIT) Delhi, New Delhi, India


Abstract: The objective of the present study was to study the influence of natural organic agent

SA (salicylic acid) and synthetic organic agent EDTA (ethylenediaminetetraacetic acid) on metal

uptake by Lemna minor L. in Pb and Ni contaminated water. L. minor was treated with Pb and Ni,

each at concentration of 10 mg l-1. EDTA and SA were added at 2.4 mM concentration. Samples

were collected at an interval of 7 days for four weeks i.e. 7, 14, 21 and 28 day. The nickel

accumulation capacity of L. minor for combined Pb+Ni treatment was lower than individual Ni

treatment accumulation capacity. Salicylic acid significantly enhanced the uptake of Pb and Ni in

single Pb and Ni treatments. However, addition of EDTA could not induce Pb and Ni

accumulation. Based on results it could be said that removal of metals depends on the type and

concentration of chelants and concentration of pollutants in water.

Keywords: Chelant - Duckweed - Heavy metals - Phytoremediation

[Cite as: Kaur L, Gadgil K & Sharma S (2015) Comparative evaluation of salicylic acid and EDTA chelant

induced phytoremediation of lead and nickel using Lemna minor L. Tropical Plant Research 2(3): 264–270]

INTRODUCTION

Heavy metals are persistent pollutants and they are of major concern in the environment due to their toxicity

and harm to plant and animal life. They are released either by natural or anthropogenic sources. Lead and nickel

heavy metals are detected in the waste streams emerging from mining operations, tanneries, electronic

industries, electroplating, batteries and petrochemical industries as well as textile mill products (Johnson 1998).

Lead is an extremely toxic metal and is having many important industrial applications. The permissible limit

of Pb in drinking water is 0.005 mg l-1 (Patterson et al. 1998). Pb effects kidney, nervous disorder and mental

deficiency (Panchandaikar & Das 1994, Axtell et al. 2003). Pb is the most abundant, globally distributed, best-

recognised dangerous among heavy metals that has been longest use since ancient times (Volesky & Holan

1995, Salt et al. 1998). Pb has been used as an anti-knocking gasoline additive since 1920 and remains an

important component in the manufacturing of the several commercial items such as storage batteries, cable

coverings, casting, sheet lead, pipes and ammunition. Pb has been tested and found to be carcinogenic,

mutagenic, and teratogenic. It affects the reproductive, nervous, muscular and haemopoeitic systems (Volesky &

Holan 1995). Ni causes dermatitis and bronchial problems (Gardea-Torresday et al. 1996).

Metal contamination in water can be treated using a variety of technologies that incorporate chemical,

physical and biological processes. Conventional remediation technologies such as chemical precipitation, ionic

exchange, filtration, electrochemical treatment, reverse osmosis, evaporative recovery and solvent extraction

(Rahmani & Sternberg 1999) are generally too expensive and are not able to remove heavy metals completely

and its byproducts (toxic sludge generation) again need disposal. Phytoremediation has emerged as a cost-

effective and environmental friendly sustainable technique in the last decades (Debusk et al. 1996). It is the use

of plants for pollutant removal from contaminated water or soil (Vujevic et al. 2000). Several studies indicate

that aquatic plants have large potential for the removal of organic and inorganic pollutants from wastewater.

Aquatic plants such as Eichhornia crassipes, Elode Canadensis, Myriophyllum spicatum, Potamogeton

pectinatus, Wolfia globosa, Lemna, Vellisnaria, Hydrilla verticilleta and Typha latifolia have been extensively

Kaur et al. (2015) 2(3): 264–270

.


used in phytoremediation research. Floating macrophytes (macro algae, duckweed, and water hyacinth) provide

advantages over emergent aquatic plants as they are much easier to harvest (Begonia et al. 2002).

Lemnoideae are limnic vascular plants that belong to the Araceae family and comprise the Landoltia spp.,

Lemna spirodela, Wolfia spp. and Wolffiella spp. They are commonly found in fresh water and brackish

ecosystems in temperate climates and serve as an important food source for various water birds and fish.

Additionally they provide habitats for invertebrates. Lamnaceae plants are easy to culture and handle, have a

high growth rate and are highly sensitive to different pollutants. Lemna minor L. is frequently used in

ecotoxicological research as a representative of higher aquatic plants. It can serve as a hyperaccumulator,

keeping the metal from continuous reintroduction into the ecosystem (Turgut et al. 2004). Lemna minor can be

used in phytotoxicity tests of contaminants, including heavy metals, phenolics and herbicides (Wu et al. 2004).

Studies have revealed that EDTA forms a metal-complex that enhances the mobility of the metal through the

plant (Raskin 1992, Dat et al. 1998, Janda et al. 2000). Salicylic acid, which may act as a component of the

signal transduction system important in defense mechanisms against pathogen attack (Lu et al. 2004), may also

provide protection against certain abiotic stresses e.g. heat stress in mustard seedlings (Saygideger & Dogan

2004) or chilling damage in maize (Janda et al. 1999).

The objective of this research was to investigate the ability of chelants (EDTA and SA) to enhance the

phytoremediation of Pb and Ni in contaminated water.


Duckweed plants were picked up from a stream of water from the main Indian Institute of Technology Delhi

campus. They were identified as Lemna minor L. and were cultured in a water tank in micro model IIT Delhi

(the experimental site). Stock solutions were prepared by using lead nitrate and nickel nitrate salt. Plastic

containers of ten litre capacity were filled with water. 6.0 g initial fresh weight of L. minor was used and treated

with Pb and Ni, each at concentrations of 10 mg l-1. EDTA and SA were amended at 2.4 mM concentration as

C10H14K2N2O8.2H2O (MERCK) and C6H4OH-COOH (Qualigens). A black line was drawn on the containers so

a six litre water level could be kept constant. Every 1-2 days, the plants were checked and tap water was added

to each container so the six litre water level line remained constant. Samples were collected in an interval of 7,

14, 21 and 28 days.

The biomass weight was taken by drying duckweed plants on filter paper for 10 minutes (fresh weight).

Plants were dried and their dried weights were noted in the lab notebook. Relative growth of control and treated

plants were calculated as follows (Lu et al. 2004):

( )

( )

The dried plant samples were heated in a muffle furnace at 500°C for 6 hours. The ash of each sample was

dissolved in 5 ml of 20% HCl to dissolve the residue. Samples were heated on a hot plate to boiling. Required

amount of HCl (20%) was added to avoid sample drying. The resulting solutions were filtered and diluted to 50

ml with deionized water in volumetric flasks. The Pb and Ni content of these plant samples and water samples

were determined using flame atomic absorption spectrophotometer (Electronics Corporation of India Limited

AAS4129) with the following settings: for Pb - wavelength 217 nm, lamp current 5 mA, slit 1 nm, fuel –

acetylene and oxidant air and for Ni - wavelength 232 nm, lamp current 5 mA, slit 0.2 nm, fuel – acetylene and

oxidant air.


Relative growth

Effect of chelants (EDTA and SA) on relative growth of Lemna minor L. treated to Pb and Ni is shown in

figure 1. Relative growth of L. minor increased with duration of time. In Pb experiment sets, the highest relative

growth was 2.54±0.13 in Pb+SA combination after 28 days. Furthermore, the lowest relative growth was

1.13±0.05 in individual Pb treatment after 7 days. However, in Ni experiment sets, the maximum value of

relative growth 1.99±0.19 in Ni+SA combination after 28 days and the minimum relative growth was observed

in Ni+EDTA combination after 7 days (1.07±0.025). Nevertheless, the highest and the lowest relative growth in

Pb+Ni experiment sets were measured in Pb+Ni+SA combination after 28 days (2.48±0.18) and in Pb+Ni

combination after 7 days (1.17±0.08) respectively.

Kaur et al. (2015) 2(3): 264–270

.


Figure 1. Effect of chelants on relative growth of Lemna minor L. treated with Pb and Ni concentrations (10+10 mg l-1).

Saygideger & Dogan (2004) observed that single and combined Cd and Pb with and without EDTA

concentrations affected growth in L. minor and Ceratophyllum demersum L. after 7 days exposure. Both

macrophytes were adversely affected in 50 mg l-1 Pb plus 0.5 mg l-1 Cd combination more than other tested

concentrations. Liu et al. (2004) reported that when Sedum alfredii Hance plants were treated with 200 µM

Pb+200 µM EDTA+1-100 µM IAA (Indole-3-acetic acid) for 12 days, EDTA and IAA had no effects on shoot

biomass.

Metal accumulation

Effect of chelants on Pb and Ni accumulation by Lemna minor L. is shown in figure 2–3. Pb and Ni

accumulation increased with time in all sets. The highest and the lowest Pb accumulation in Pb experiment sets

were determined as 2.56±0.12 mg kg-1 for Pb+SA combination after 28 days and 1.10±0.10 mg kg-1 for

individual Pb for 7 days respectively. Similarly, in Ni experiment sets, the highest Ni accumulation (910±101

mg kg-1) was found in the same treatment and same time as in case of Pb. However, the lowest Ni accumulation

(205±36 mg kg-1) was obtained in Ni+EDTA after 7 days.

Figure 2. Effect of chelants on accumulation in Lemna minor L.: A, Pb; B, Ni.

Figure 3. Effect of chelants on accumulation in Lemna minor L. by Pb+Ni treatments: A, Pb; B, Ni.

Kaur et al. (2015) 2(3): 264–270

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Effect of EDTA and SA on Pb and Ni accumulation in combined Pb+Ni treatment by L. minor is shown in

figure 3 respectively. The highest and the lowest Pb accumulation were in Pb+Ni+EDTA treated plants and

Pb+Ni treated plants respectively. The highest and the lowest Ni accumulation were in Pb+Ni+SA treated plants

and Pb+Ni+EDTA treated plants respectively. Pb and Ni accumulation increased with time. It shows that in

combined Pb+Ni treatment, Pb and Ni accumulation in L. minor was found to be the utmost in Pb+Ni+SA

treatment. However, addition of EDTA could not induce Pb and Ni accumulation much. Salicylic acid enhanced

the uptake of Pb and Ni in single Pb and Ni treatments. Nickel accumulation capacity of L. minor for combined

Pb+Ni treatment was lower than individual Ni treatment accumulation capacity.

The Pb concentrations in the leaves and roots of Typha orientalis plant increased with increasing of Pb level

in the nutrient solution of 0–500 mg l-1 (Li et al. 2008). Results from this study demonstrated that the plants had

the highest accumulation and translocation under the condition of 500 mg l-1 Pb+0.5 m moll-1 EDTA. Rahman et

al. (2008) reported that the uptake of inorganic arsenic species into the aquatic plant Spirodela polyrhiza L.

increased by EDTA when plants were exposed to different arsenic species at 6 µM and 50 µM EDTA for two

weeks.

Metal remained in the residual solution

SA and EDTA absorbed/extracted greater Pb/Ni than control. The best results of Pb and Ni removal were

obtained in SA treatments which may be due to the greater solubility of SA (0.2 g/100 ml at 20ºC) in water than

EDTA (0.05 g/100 ml at 20ºC) and metal-SA complexes may have been more available to plants than metal-

EDTA complexes.

In combined Pb+Ni treatments, Pb removal by EDTA was more than SA and Ni removal was best in SA.

This may be due to the competition between Pb and Ni to bind with chelants EDTA and SA. EDTA has strong

complexing capacity with Pb but in Pb treatment, uptake got reduced may be due to reduction in ion activity in

water. SA acts as defender and the protective function of SA includes absorption and distribution of elements.

SA is implicated in the high degree of cellular tolerance towards Ni in the genus Thalspi (Freeman et al. 2005).

SA may help in metal uptake by chelating Pb/Ni in the solution and then release Pb/Ni metal in the plant

system. The differences in uptake between the essential and non-essential metals and the effects of chelants on

their uptake can be explained by two parallel pathways: a selective symplastic pathway and a nonselective

apoplastic pathway (Tandy et al. 2006). Selective uptake of Ni is very efficient and uptake of Pb is very low

under these conditions. Apoplastic (passive) uptake of metal complexes is a function of the complex

concentration in the surrounding solution. This also suggests that higher chelate concentrations may lead to

increased uptake of essential metals. There were no literature on EDTA and SA effect in removal of Pb and Ni

from water. Based on presented results it could be said that removal of metals depends on the type and

concentration of chelants in water.

Ni uptake was inhibited by EDTA. On the other hand, the addition of SA in the medium increased the uptake

substantially. Our results are corroborated with Shi & Zhu (2008). They reported that addition of SA increased

Mn concentration in cucumber plants under excess Mn condition. Similarly, content of Cd in barley treated with

SA was higher than Cd alone treatment (Metwally et al. 2003).

Figure 4. Concentration of remained in the residual solution after treatments: A, Pb; B, Ni.

Concentrations of Pb and Ni (mg l-1) remained in the water samples after treatments are represented in

figures 4–5. Pb and Ni content in water were decreased with the passage of time. The lowest value of Pb

Kaur et al. (2015) 2(3): 264–270

.


(1.12±0.12 mg l-1) remained in the solution after 28 days was in treatment Pb+Ni+EDTA (97% removal) while

the highest value (2.58±0.34) in the same treatment was after 7 days. Removal potential of Ni was highest in

Pb+Ni+SA treatment for 28 days (99.5%).

Figure 5. Concentration of remained in the residual solution in Pb+Ni treatments: A, Pb; B, Ni.

Removal potential of L. minor with chelant in combined Pb+Ni treatment was better as compared to

individual Pb and Ni treatments (Fig. 6–7). Akin et al. (1994) observed that removal of lead was decreased by

increasing the concentration of EDTA in Eichhornia crassipes (water hyacinth). Kruatrachue et al. (2002)

studied the combined effects of Pb and humic acid on Pb uptake by L. minor. Humic acid did not significantly

decrease Pb uptake at 50 and 100 mg l-1 Pb treatment. In the 200 mg l-1 treatment, the lowest Pb contents were

observed in the presence of 160 mg l-1 humic acid. Therefore, a high concentration of humic acid could

significantly decrease the Pb uptake. Miretzky et al. (2006) investigated the mechanism of simultaneous metal

removal of Cd, Ni, Cu, Zn and Pb by Spirodela intermedia, L. minor and Pistia stratiotes. L. minor biomass

presented the highest mean removal percentage and P. stratiotes the lowest for all metals tested. Pb and Cd were

more efficiently removed by the three of them. No significant differences were observed in the metal exchange

amounts while using multi-metal or individual metal solutions.

Figure 6. Removal potential of Lemna minor L. after different days: A, Pb; B, Ni.

Figure 7. Removal potential of Lemna minor L. in Pb+Ni treatments with or without chelants: A, Pb; B, Ni.

Kaur et al. (2015) 2(3): 264–270

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CONCLUSION

The results showed that growth of Lemna minor helped in the accumulation of Pb and Ni. SA significantly

increased more Pb and Ni accumulation in L. minor as compared to EDTA when added as chelants in water.

This suggests that L. minor can be used for cleaning water polluted by heavy metals with the help of chelants.

ACKNOWLEDGEMENTS

This research work was financially supported by the University Grants Commission (U.G.C.), New Delhi,

India.

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2(3): 271–275, 2015

Review article

A comprehensive review of effects of water stress and tolerance in

wheat (Triticuma estivum L.)

Muhammad Bilal1*, Irfan Iqbal

1, Rashid Mehmood Rana

1, Shoaib Ur Rehman

2, Qurat-

ul-ain Haidery3, Farah Ahmad

3, Ammara Ijaz

3 and Hafiz Muhammad Imran Umar

4

1Department of Plant Breeding and Genetics, PMAS Arid Agriculture University, Rawalpindi, Pakistan

2Chinese Academy of Agriculture Sciences, Beijing, China

3Department of Biochemistry, PMAS Arid Agriculture University, Rawalpindi, Pakistan

4Department of Plant Breeding and Genetics, Bahauddin Zakariya University, Multan, Pakistan


Abstract: Wheat is regarded as one of the most important worldwide cereal crop and utilized as

staple food commodity. Its productivity restricted by several biotic and abiotic stresses but among

all of these,drought is most devastating stress which immediately affects the morphological and

physiological attributes of wheat crop and lead to severe reduction in overall production.

Keywords: Wheat - Staple - Biotic - Abiotic - Drought

[Cite as: Bilal M, Iqbal I, Rana RM, Shoaib Ur Rehman, Haidery Q-ul-A, Ahmad F, Ijaz A & Umar HMI

(2015) A comprehensive review of effects of water stress and tolerance in wheat (Triticuma estivum L.).


INTRODUCTION

Wheat (Triticum aestivum L.) is regarded as one of most vital cereal crop of world and mainly grow in rain

fed regions in which drought occur which cause serious yield reduction (Rana et al. 2013). Drought stress is a

globally widespread and ever growing environmental phenomenon encountered by wheat crop and long duration

of water deficit lead to severe reduction in overall production (Nezhad ahmadi et al. 2013). Drought stress can

be determined by three factors viz., intensity, time of incidence and duration. Under drought stress conditions

changeable nature of these factors make it complicated for plant breeder to decide which plant trait should be

improved first to improve plant production (Mujtaba & Alam 2002).

Recent research advances associated with crop responses to numerous biotic and abiotic stresses especially

water deficit stress is gaining significant emphasis, as global environment fluctuations situation prognosticates

water deficit conditions (Ullah et al. 2010). Better critical knowledge about drought stress tolerance related to

physiological and morphological characters helps in the screening of germplasm to evaluate genotypes against

drought. One of the superior goals of plant breeders is to make wheat genotypes suitable to drought stress

condition which ensures higher grain yield. Several efforts have been address to enhance the per acre

productivity of wheat crop under water deficit situation by improving the attributes damaged by drought stress.

Numerous previous reports exposed many morphological and physiological attributes correlated with drought in

cultivated wheat varieties. Reviewed article on the drought subject has discussed separately the studies on

morphological basis and studies on physiological basis. The comprehensive overview has been explained below.

1. Morphological Attributes

Wheat yield under drought stress suffer serious moisture deficit throughout its growth period from seedling

to full maturity (Bilal et al. 2015). Under drought condition decreasing pattern was experienced in

morphologically yield contributing characters like plant height (PH), grains per spike, spikes per plant, 1000-

grain weight (TGW) in wheat (Kilic & Yağbasanlar 2010). Blum & Pnuel (1990) reported that yield and yield

contributing traits of wheat crop were drastically decreased under least annual precipitation. Drought stress lead

to reduction in number of fertile tillers per plant, grains per spike and 1000-grain weight (TGW) which

ultimately cause noticeably low grain productivity. Relationship between plant height (PH), leaf area and wheat

Bilal et al. (2015) 2(3): 271–275

.


grain yield has been noticed at booting and anthesis phase which cause improvement in grain yield under water

deficit condition (Gupta et al. 2001). The decreasing graph in grain number was linked with reduced leaf area

and lower photosynthesis as outcome of drought stress (Fischer et al. 1980). Under drought stress condition

screening of wheat genotypes to evaluate these yield contributing characters are suggested in hybridization

programs for water deficit tolerance. Various research scientists have reported considerably positive relationship

for effective tillers plant1 in wheat which depicted negative relationship with 1000-grains weight (Ali et al.

2008). Considerable positive and worth mentioning association has been observed for grains per spike with total

grain production in diverse bread and durum wheat genotypes, and grains per spike was observed 50–68 in

irrigated condition while under stress 32–50 grains per spike were found (Jatoi et al. 2011). Thousand grains

weight (TGW) is essential yield enhancingtrait and has been given due consideration during varietal evaluating

procedure. Kilic & Yagbasanlar (2010) investigation depicted that under drought conditions grain filling period

and spikelets per spike were associated with high grain production. Therefore it is suggested that these

morphological attributes should be selected for screening diverse wheat genotypes under drought in successful

breeding schemes. The adverse and unfavorable outcomes of water deficit stress on wheat sensitive stages of

crop growth such as reproductive, booting and grain filling stagecan be minimized by preventing stress at these

stages to develop genotypes showing drought tolerant (Saini & Westgate 1999).

2. Physiological Attributes

Different types of plant physiological responses have been reported by various Plant physiologists in their

findings under drought stress situation. Zaharieva et al. (2001) reported that in globally drought affected areas

physiological mechanism is very handy approach in evaluating and screening the extraordinary genotypes

having drought resistant mechanism. Comprehensive information of physiological mechanisms permits plant

researcher to develop promising genotypes that would be utilized efficiently, continue his growth and

production under water deficit stage (Ashraf & Khan 1993).

Cell-membrane stability (CMS) is of vital important selection criteria of drought tolerant genotypes

(Tripathy et al. 2000). It has been reported that under water stress cell membrane integrity and stability confers

drought resistance (Bewley 1979). The water stress activates the reactive oxygen species which ultimately

decreases membrane stability caused by lipid peroxidation (Menconi et al. 1995). Although many reports

depicted lower lipid peroxidation and higher cell membrane stability (CMS) in drought tolerant wheat and maize

genotypes (Pastori & Trippi 1992). It has been reported by Sairam & Saxena (2000) that higher level of

accumulation of H2O2 under water stress leads to production of hydroxyl radicals, which cause lipid

peroxidation and consequently cell membrane rupture. Damage caused by water deficit stress to cell membrane

is negatively associated with increased activities of superoxide dismutase (SOD) and catalase (CAT) in drought

susceptible and tolerant genotypes (Dhindsa & Matowe 1981). Under drought stress assembly of lower levels of

H2O2 lead to lower damage of cell membrane in wheat drought tolerant genotypes.

Cell membrane stability (CMS) under drought stress depicts the ability of plant tissues to prevent

electrolytes leakage by keeping the cell membrane in safe mood (Sullivan 1971). Estimation of Cell membrane

stability (CMS) via in vitro includes dehydration of leaf tissues by means of polyethylene glycol (PEG) and then

assessment of electrolyte leakage from leaves. Leakage of various solutes, such as organic acids, amino acids,

saccharides, phenolic compounds and hormones from revealed cell membrane stability (CMS) after subject to

dehydration through polyethylene glycol has been reported (Leopold et al. 1981). CMS Values have immense

significance in hybridization programs because these Values predict the drought tolerant varieties (Dhanda et al.

2004). Genotypes having lower CMS value are vulnerable to water deficit condition while the genotypes

showing higher CMS values depicts drought tolerant behaviour. The genotypes having less than 50% values are

tremendously susceptible to drought while genotypes with 71–80% values are considered to grow with full

potential under water deficit. Farshadfar et al. (2011) noticed in investigation that under water deficit conditions

cell membrane stability (CMS) depicted positively considerable relationship with tillers per plant, grain yield,

but negative association 100 kernel weights (TGW).

Higher leaves chlorophyll contents is significantly correlated with photosynthesis and regarded as

encouraging selection trait in crop productivity (Teng et al. 2004). It has been reported in many studies that

under drought stress Photosynthesis exhibit direct relationship with wheat grain production because less stomata

opening frequency and low amount of CO2 fixation lead to reduction in photosynthetic amount (Mafakheri et al.

Bilal et al. (2015) 2(3): 271–275

.


2010). Severe water deficit stress restricts the photosynthesis by damaging the chlorophyll components (CC)

and changing the photosynthetic machinery (Iturbe-Ormaetxe et al. 1998). Decreased photosynthetic amount

under water deficit condition is an outcome of Inhibition of RuBisCO (ribulose-1, 5-bisphosphate

carboxylase/oxygenase) enzyme activity and development of ATP (Dulai et al. 2006). Many researchers

revealed in their investigations that photosynthesis of higher plants is extremely susceptible to drought stress

and Lower amount of chlorophyll cause chlorosis and reduction in crop growth (Khosh & Ando1995). Higher

concentration of chlorophyll is essential for plants because it depicts the low quantity of photo-inhibition of the

photosynthetic which prevents the carbohydrates losses and eventually enhances growth (Farquhar et al. 1989).

Relative water content (RWC) of leaves has been reported as direct indicator of plant water contents under

water deficit conditions (Lugojan & Ciulca 2011). Drought stress lead to reduction of water status during crop

growth, soil water potential and plant osmotic potential for water and nutrient uptake which ultimately reduce

leaf turgor pressure which results in upset of plant metabolic activities. Momentous pattern of divergence can be

observed in Relative water content (RWC) among diverse genotypes during various plant growth stages. The

highest Relative water content (RWC) might be calculated at crop vegetative stage (Almeselmani et al. 2011).

Under water stress condition decrease in water status and osmotic potential in plants is the ultimate outcome of

lower relative water content. Osmoregulation mechanism plays a phenomenal role in preserving turgor pressure

which helps in soil water absorption and continue plant metabolic activities for its survival.

Proline is well known to occur extensively in higher crop plants and accumulates in higher concentration in

response to different abiotic environmental stresses specially drought stress (Kavi-Kishore et al. 2005).

Accumulation of higher proline concentration in crop plant under water deficit condition is highly associated

with and drought tolerance genotypes depicts its concentration is much higher than drought sensitive genotypes.

It has been found by many scientists that in saline stress soil proline are mainly accumulated in leaves of many

higher halophytic plant (Briens & Larher 1982) but plants grown under drought stress showed much higher

concentration of proline in leaves, shoots, in desiccating pollen and in root apical regions Lansac et al. 1996).

Accumulation of higher concentration of proline permits plants to keep less amount of water potential which

cause accumulation of osmolytes in osmoregulation process which enables the plant to take up water to perform

growth and metabolic activities (Kumar et al. 2003).

Under water deficit condition proline perform many functions like act as osmolyte contribute s in the

maintenance of membrane and protein, scavenging free radicals. Moreover after the severe damage of stresses

proline contents provide adequate reducing agents that assist in mitochondrial oxidative phosphorylation and

production of adenosine triphosphate (ATP) for revival from damages of various stresses (Hare et al. 1998).The

primary site of proline contents accumulation in response to drought stress in crop plant is cytosol (Ketchum et

al. 1991).

CONCLUSION

Wheat (Triticum aestivum L.) being a most vital cereal crop has always been of area of interest to plant

breeders. Since several years numerous efforts have been made to boost up its productivity under various

conditions especially under drought stress condition. This review depicted that drought stress caused extensive

decline in all the studied attributes performance. So there is need to explore several helpful attributes and to

minimize the harmful effect of water stress on wheat crop productivity through development of genotype having

drought tolerant and better performance.

ACKNOWLEDGEMENTS

The authors would like to express their special thanks and grateful to acknowledge the Rashid Mehmmod

Rana for their assistance, guidance, and all kind of support for the successful completion of this review article.

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2(3): 276–281, 2015

Research article

Priming of Abelmoschus esculentus (L.) Moench (okra) seeds with

liquid phosphobacterium: An approach to mitigate drought stress

P. Pravisya and K. M. Jayaram*

Division of Plant Physiology and Biochemistry, Department of Botany,

University of Calicut, Calicut - 673635, Kerala, India


Abstract: The present investigation aimed to evaluate the effect of priming of Abelmoschus

esculentus (okra) seeds with liquid phosphobaterium (LPB) on water stress. The

phosphobacterium is known for its use as inoculants to increase the uptake of soil phosphorus as

well as crop yield. The availability of rain fall is getting decreased every year causing severe

drought and that may adversely affect the agricultural crops in the state of Kerala. So the aim of

the study is to provide a helping hand to the farming community to fight against drought stress. In

the present study the seeds of okra cv. Arka anamika were subjected to priming treatment with 5%

and 10% liquid phosphobaterium, and the parameters like biomass, relative water content,

chlorophyll content, total protein and yield were studied. Priming with liquid phosphobaterium

showed considerable variation in both the physiological and biochemical parameters. Among the

concentrations of liquid phosphobaterium tested seeds primed with 10% liquid phosphobaterium

were found to effective in mitigating the effect of water stress, stimulating early flowering and also

increase in yield.

Keywords: Drought - Re-irrigation - Biomass - Chlorophyll - Protein

[Cite as: Pravisya P & Jayaram KM (2015) Priming of Abelmoschus esculentus (L.) Moench (okra) seeds with

liquid phosphobacterium-An approach to mitigate drought stress. Tropical Plant Research 2(3): 276–281]

INTRODUCTION

Agricultural crop productions have been determined by the availability of soil water and which in turn

related to global climate changes (Cias et al. 2005). Drought is one of the major causes in the field of agriculture

all over the world. Depending upon the period of exposure to drought (water stress) and growth stage of plants,

water scarcity experienced plants responded differently. Drought stress leads to the commendable variation in

the morphological, anatomical, physiological and biochemical parameters of plants which is finally reflected on

yield potential (Kramer 1969, Shintu & Jayaram 2015). Drought, irrespective of the length of exposure of the

plant and severity, adversely affected photosynthesis and other metabolic activities of plants and ultimately the

growth productivity of such plants.

Phosphobacteria (Phosphate solublizing bacteria- PSB) is one of the most useful plant soil microorganisms,

widely used as bio-fertilizer. PSB plays an important role in enhancement of plant growth by improving texture

of soil by adding organic matter to the soil, solublizing the insoluble phosphorous in soil (Bhattacharya & Jain

2000, Ravikumar et al. 2010). Inorder to compensate phosphorus deficiency in soil phosphate fertilizer are

being used widely. However the increased use of chemical fertilizers, cause soil contamination. In such

condition PSB efficiently take part in the utilization of unavailable native phosphates (Lagreid et al. 1999). The

studies conducted by various authors revealed that priming or pre-sowing treatment of seeds with various

chemicals or even with water can enable the plants to improve the health and such plant may become resist

water stress (Chivasa et al. 2000, Harris et al. 2004, Shintu & Jayaram 2015). During drought stress these

microorganisms help to accumulate large amount of compatible solutes and accelerate the production of

antioxidant enzymes in plants and reduce the adverse effect of drought (Mayak et al. 2004). Considering all

these facts the authors made an attempt to study the effect of priming of Abelmoschus esculentus (L.) Moench

Pravisya & Jayaram (2015) 2(3): 276–281

.


(okra) seeds with liquid phosphobacterium (LPB), a cheapest method that adapt to overcome the adverse effect

of water stress.


For the present study, seeds of Abelmoschus esculentus (L.) Moench cv. Arka anamika (okra) were procured

from the Regional Agricultural Research Station, Mele Pattambi, Palakkad District, Kerala State. Healthy seeds

were manually selected from the seed lot and were divided to 3 sets. First set was not inoculated with Liquid

phosphobacterium (LPB) and considered as control, the second and third sets were inoculated with different

concentrations of LPB such as 5% and 10% respectively. The pre weighed seeds were surface sterilized with

teepol and 0.1% mercuric chloride solutions and were kept in respective concentrations of LPB solutions for six

hours with continuous shaking. Thereafter the seeds were air dried until the weight became equal to the initial

weight. All the seeds were sown in garden pots filled with potting mixture. The experimental setup was

maintained in the open field of the Department of Botany, University of Calicut (35±5ºC temperature). After 17

days of vegetative growth the control and LPB treated plants were again divided in to two sets, one set kept as

irrigated regularly and the other set as non-irrigated for 3 days. After 3 days of water stress the plants were re-

irrigated as in the other case and was continued until taking yield.

The following parameters were studied by using standard procedures: fresh and dry weight of plants

(biomass), relative water content (RWC) (Bars & Weatherly1962), total chlorophyll (Arnon 1949),total protein

(Lowry et al.1951) and yield parameters like fruit length, fruit fresh and dry weight, number of fruits per plant

and seed per pod. All the data were collected as detailed below: on the previous day of commencement of

water stress treatment (0th

day), 1stday (24hrs after water stress), 2

ndday (48 hrs after water stress), 3

rdday (72hrs

after water stress), 24 and 48 hrs after re-irrigation (1stand 2

ndday of recovery respectively).

Analysis of variance was performed using SPSS software 16. Means were compared using the Duncan test

at 5% probability level. The data is an average of three independent experiments each with three replicates

(n=9). The data represent Mean±Standard Error (SE).

RESULTS

Biomass

The plants showed significant reduction in biomass under water stressed condition and which was prominent

in untreated (control) water stressed plants than LPB treated plants subjected to drought stress (Fig. 1). Plants

treated with 10% LPB exhibited lesser biomass reduction compared to other treatment and control. During re-

irrigation LPB treated plants showed fastest recovery, compared to untreated plants given drought.

Figure 1. Effect of liquid phosphobacterium on Abelmoschus esculentus (L.) Moench (Okra): A, Fresh weight; B, Dry

weight. (C- Control; Cs- Control with drought stress; 5C- 5% LPB treated plants; 5Cs- 5% LPB treated plants with

drought stress; 10C- 10% LPB treated plants; 10Cs- 10% LPB treated plants with drought stress)

Relative water content (RWC)

RWC was observed high in all the irrigated set of plants and among the plants subjected to water stress

treatment, LPB treated plants exhibited high rate of RWC of which highest rate was observed in 10% LPB

c e e e e e

c f f f f

f b

c c c

c c

b d d d

d d

a

a a a a

a

a

b b b b

b

0

5

10

15

20

25

0th day 1st daystress

2 nd daystress

3rd daystress

1strecovery

2ndrecovery

Fre

sh w

eig

ht

of

pla

nt

(g)

Days

C Cs 5C 5s 10C 10s A

c e e

e

e

e

c f f f

f

f

b c c

c

c

c

b d d d

d

d

a a

a a

a

a

a b

b b

b b

0

1

2

3

4

5

6

7


2 nd daystress

3rd daystress

1strecovery

2ndrecovery

Dry

we

igh

t o

f p

lan

t (g

)

Days

C Cs 5C 5s 10C 10s B


.


treated plants (Fig. 2). The same pattern of increase in RWC was noticed during re-irrigation also.

Figure 2. Effect of liquid phosphobacterium on relative water content of Abelmoschus esculentus (L.) Moench (Okra). (C-

Control; Cs- Control with drought stress; 5C- 5% LPB treated plants; 5Cs- 5% LPB treated plants with drought stress; 10C- 10% LPB treated plants; 10Cs- 10% LPB treated plants with drought stress)

Total Chlorophyll

The total chlorophyll content of untreated water stressed plants was found decreased more significantly

throughout the period of water stress treatment compared to LPB treated water stressed plants (Fig. 3A). Among

the LPB treated plants highest rate of chlorophyll content was observed in 10% LPB treated plants. During re-

irrigation LPB treated plants showed fastest recovery, compared to untreated plants.

Total protein

The okra plants treated with LPB (5% and 10%) exhibited higher rate of accumulation of protein content

compared to control plants (Fig. 3B). But the plants exposed to drought stress the decrease of protein content

was greater in untreated water stressed plants compared to LPB treated plants. The recovery of protein content

was found faster in LPB treated water stressed plants compared to untreated water stressed plants.

Figure 3. Effect of liquid phosphobacteriumon Abelmoschus esculentus (L.) Moench (Okra): A, Chlorophyll; B, Protein. (C-

Control; Cs- Control with drought stress; 5C- 5% LPB treated plants; 5Cs- 5% LPB treated plants with drought stress; 10C- 10% LPB treated plants; 10Cs- 10% LPB treated plants with drought stress)

Yield

Drought stress exhibited a reduction of yield in okra, which was measured by using parameters like length of

fruit, fresh weight of fruit, dry weight of fruit, number of fruit per plant and number of seed per fruit but the

reduction was more prominent in untreated plants than LPB treated plants (Fig. 4). The plants treated with 10%

LPB showed significant increase in yield parameters when compared to 5% LPB treated plants and control

plants.

c b c b b e c e f

f f

d b c b c c c b f e

e e

f a a a a a a a

d d d

d b

0

10

20

30

40

50

60

70

80

90

100

0th day 1st day stress 2 nd day stress 3rd day stress 1st recovery 2nd recovery

Re

lati

ve w

ate

r co

nte

nt

(%)

Days

C Cs 5C 5s 10C 10s

c e e e e e c f f f

f f

b c c c c c b d d d d

f

a a a a a a a b b b b b

0

200

400

600

800

1000

1200

1400

1600


2 nd daystress

3rd daystress

1strecovery

2ndrecovery

Tota

l ch

loro

ph

yll (

µg/

gfw

)

Days

C Cs 5C 5s 10C 10s A

c e e e e

e

c

f f

f

f

f

b b b

b b

b

b c

c c

c

c

a a

a

a a

a

a d

d d

d

d

0

10

20

30

40

50

60

70

80

90

100


2 nd daystress

3rd daystress

1strecovery

2ndrecovery

Pro

tein

co

nte

nt

(mg/

fw)

Days

C Cs 5C 5s 10C 10s B


.


Figure 4. Effect of liquid phosphobacterium on yield parameters of Abelmoschus esculentus (L.) Moench (Okra): A, Length

of fruit; B, Fresh and dry weight of fruit; C, Number of fruit per plant and seed per fruit. (C- Control; Cs- Control with

drought stress; 5C- 5% LPB treated plants; 5Cs- 5% LPB treated plants with drought stress; 10C- 10% LPB treated plants;

10Cs- 10% LPB treated plants with drought stress)


The present study showed that the LPB inoculated plants exhibited an increased rate of fresh and dry weight

of which 10% LPB inoculation exhibited higher rate of fresh and dry weight (Fig. 1 A–B). The studies

conducted by Singh & Singh (2010) also revealed higher dry matter content in PSB treated fenugreek plants,

which reflected the tolerance of the plants due to bacterial inoculums. Similar pattern of dry matter increase was

observed in finger millet treated with agrochemicals like CaCl2 (Maitra et al. 1998), rice plants treated with

Rhizobacterium (Raj et al. 2012) and black gram treated with bio-fertilizers (Selvakumar et al. 2012). All these

results are in tune with the results obtained in the present study, which may lead to the conclusion that priming

has an important role in nutrient uptake and growth, that may resulted in the high rate of biomass in plants

treated with LPB.

Priming of okra seeds with LPB showed an increase in RWC which was very prominent in 10% LPB treated

plants (Fig. 2). Studies conducted by Yordanov et al. (2003) observed that mild drought helped plants to

regulate water loss and uptake, allowing maintenance of their leaf water content within the limits. According to

Velentovic et al. (2006) RWC in the leaves of maize plants at low water potential decreased significantly

compared to control. Similarly the RWC in drought affected leaves of okra was significantly lower than the

continuously irrigated control plants. These are in confirmation with the results obtained in fenugreek (Singh &

Singh 2010) and tomato (Shintu & Jayaram 2015) and the authors found that the plants inoculated with PSB

exhibited highest level of RWC as compared to non-inoculated plants under controlled condition. All these

observations revealed that the RWC in leaves of drought affected plants were significantly reduced as a result of

relative water uptake and storage by the plants. So it can be presumed that comparatively high RWC in the

leaves of LPB treated plants may be due to the higher activity of water uptake and restoration by these plants.

Thus LPB can be considered as a bio-priming agent in order to tolerate drought stress.

Total chlorophyll content of LPB treated plants was found decreased but the decrease was not lesser than

untreated plants (Fig. 3A). Drought stress produced changes in the total chlorophyll content (Farooq et al.

2009). The results obtained in the present study showed a high rate of chlorophyll pigment in plants raised from

10% LPB treated seeds. Estill et al. (1991) and Ashraf et al. (1994) observed same pattern of changes in

chlorophyll content in stressed alfalfa and wheat respectively. Similarly fenugreek plants treated with PSB also

showed identical results (Singh & Singh 2010). Moreover the studies conducted by Rupa (2007) also revealed

that, under moisture stress conditions there will be degradation in pigment composition which ultimately

induced a decrease in the chlorophyll content. In the present study total chlorophyll content of okra leaves

decreased with increased moisture stress, but it was found increased during the recovery period. Higher

persistence of chlorophyll content in plants under stress due to LPB may be attributed to decreased chlorophyll

degradation and increased chlorophyll synthesis, as suggested by Jayakumar & Thangaraj (1998). So in the

present study also higher level of photosynthetic pigment was obtained in LPB treated drought affected plants

that may be due to non-degradation of chlorophyll. The LPB’s regulatory effect on phosphate solubilisation may

be beneficial to the non-degradation of chlorophyll pigments and that may be the reason for getting high

chlorophyll content in the LPB treated drought affected plants.

c e

b d

a c

0

5

10

15

20

25

30

Length of fruit (cm)

C Cs5C 5s10C 10s

A

c

c f

e

b

b e d

a

a d

d

0

5

10

15

20

Fresh weightoffruit (g)

Dry weightoffruit (g)

C Cs 5C

5s 10C 10sB

d

c

e

f

c

b

d

e a

a

b

d

0

20

40

60

80

100

No.of fruit perplant

No.of seed perfruit

C Cs 5C

5s 10C 10sC


.


The leaves of LPB treated okra plants exhibited an increase in total protein content which was more

prominent in 10% LPB treatment (Fig. 3B). Similar results were obtained in fenugreek plants treated with

phosphobacterium (Singh & Singh 2010). According to those authors the non-inoculated plants exhibited a

lesser amount of protein than inoculated plants. Selvakumar et al. (2012) opined that double inoculation of

Rhizobium with Phosphobacteria yielded more protein content than single and non-inoculated in black gram.

Rhizobium increased protein content in sunflower by increasing nitrogen uptake (Shehata & EL-Khawas 2003).

Radin (1984) suggested that high phosphorous caused stomatal opening, and facilitate plants to accumulate

more protein in inoculated plants compared to non-inoculated plants. Accumulation of ABA caused by

deficiency of phosphorus is directly proportional to the degree of water stress and thus resulted in stomatal

closure and low photosynthetic rate (Singh & Singh 2010). So in the present study it can be presumed that LPB

may cause to enhance the uptake of insoluble soil phosphorous and thus resulted in increased stomatal opening

and ultimately enhanced the accumulation of protein in okra plants.

In addition to the beneficial effect on growth of plants, bio-priming is also known to increase the yield by its

significant effect during drought on yield parameters of crop under study (Casanovas et al. 2003). In the present

study, maximum yield was obtained in 10% LPB treated and water stressed okra plants which were followed by

its control plants (Fig. 4). These results revealed that the LPB has an important role in increasing the yield and

also to reduce the adverse effect on drought stress. According to Prabhakar & Saraf (1991) the interaction effect

of limited irrigation regimes and phosphorous fertilizers was significant on sorghum grain yield. Chauhan et al.

(1995) observed that inoculation of Azospirillum as a bio-fertilizer markedly increased pod number and seed

yield of Brassica napus plants over the non-inoculated plants. Zodape (2001) suggested that, the increase in

yield with bio-fertilizer application was due to micro-element and plant growth regulator contained in the

fertilizer. So it can be presumed that the increase in yield of LPB treated plants exposed to water stress may be

due to the positive effect of priming of okra seeds with LPB. Priming with LPB also enhanced the potential of

the plant to drought stress by influencing on various physiological as well as biochemical parameters studied. So

it can be concluded that LPB may beneficially affected plants to improve water status of okra plants that may

help them to tolerate water deficit condition to a certain extend and to give high productivity. So, liquid

phosphobacterium can be recommended to the farming community as a priming agent to their vegetable crops,

in order to fight against mild drought stress.

ACKNOWLEDGEMENTS

The first author (PP) gratefully acknowledged to the Govt. of Kerala for providing the financial support

and both the authors are thankful to the Head, Department of Botany, University of Calicut, for providing

laboratory facilities in order to complete the work.

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ISSN (E): 2349 – 1183

ISSN (P): 2349 – 9265

2(3): 282–287, 2015

Research article

Diversity of invasive alien species in Pantnagar flora

Jyotsna Rastogi*, D. S. Rawat and Satish Chandra

Department of Biological Sciences, College of Basic Sciences and Humanities

G. B. Pant University of Agriculture and Technology, Pantnagar - 263145, Uttarakhand, India


Abstract: Biological diversity faces many threats throughout the world and one of the major

threats is caused by invasion of alien species. The present study proves presence of 94 invasive

alien species in flora of Pantnagar, Uttarakhand, India. These 94 invasive alien species (IAS)

belong to 72 genera, under 33 families 85 species are dicotyledons while 9 species are

monocotyledons. On the basis of their nativity maximum IAS have their sourced region as

American continents (74), followed by Africa (8), Europe (5), Mediterranean (3) and Asia & Australia

(2). The taxonomic analysis of IAS reveals dominance of Asteraceae with 18 spp. followed by

Fabaceae, Amaranthaceae, Convolvulaceae, Malvaceae, Solanaceae, Poaceae etc. Among these, 78 IAS

are herbs followed by shrubs (8), grasses (4), sedges (2), trees (1), and climber (1). Such a large number

of invasive alien species in small area of Pantnagar, indicate miserable condition of natural vegetation.

Keywords: Invasive alien species - Diversity - Biological invasion - Nativity - Pantnagar

[Cite as: Rastogi J, Rawat DS & Chandra S (2015) Diversity of invasive alien species in Pantnagar flora.


INTRODUCTION

The plants that has been introduced by humans intentionally or accidentally from one region to another are

referred as exotic, introduced, foreign, non-indigenous or non-native (Reddy et al. 2008). Invasive alien species

(IAS) are those that occur outside their natural range, spread rapidly and cause harm to other species,

communities and entire ecosystem. Invasive alien species are one of the major causes of biodiversity loss in the

world and impose high cost to agriculture, forestry, and aquatic ecosystem. The introduction of invasive alien

species in the new habitat outside of their natural home range carries significant risk. Plant invasion is defined as

the whole process from the arrival of a new species into a community, its establishment and maintenance in that

community, to its further spread into neighbouring communities (Prieur-Richard & Lavorel 2000). Biological

invasions are posing a great threat to biodiversity, which is already threatened by habitat destruction due to

human population growth. Biological invasions have been recognized as one of the most serious global process

impacting the structure, composition and function of natural and semi natural ecosystems (Mooney & Hobbs 2000,

Vitousek et al. 1997).

The common characteristics of invasive species include rapid reproduction and growth, high dispersal

ability, ability to adapt physiologically to new conditions, the ability to survive on various food types and in a

wide range of environmental conditions and ability of association with humans. Many invasive alien species grow

faster than native plants and reproduce quickly and thus replace indigenous plants and completely alter the

composition of flora of the area they have colonized. The presence of invasive species in an area changes the soil

structure, its profile, composition, nutrient content of soil, moisture availability etc. It has been reported that

agriculture and grazing land, as well as protected areas, are threatened by rapidly growing species of plants that were

introduced during colonialism as garden plants and wind breakers (Hall 2003).

The present study is conducted in the land area covered by G. B. Pant University of Agriculture and Technology

Pantnagar in Udham Singh Nagar, district of Uttarakhand, India. The University campus at Pantnagar is spread in an

area of 12,661acre (51.24 K m2) between the latitudes N 28º 59' 36" – 29º 02' 34" and longitude E 79º 28' 33" – 79º

31' 12" with an altitude range of 213 to 238 m above the sea level. Geographically Pantnagar is situated in the Terai

belt near the outer hills of Kumaon Himalaya. The soil is quite rich in nutrients and soil pH is around 6.85. In

Rastogi et al. (2015) 2(3): 282–287

.


Pantnagar area the landscape is completely devoid of natural vegetation and the land is mainly used for agricultural

activities. Such an environment is congenial for invasion by invasive alien species and their presence in the area is

sporadically reported earlier (Gaur & Rawat 2013, Joshi & Rawat 2011, Nisha et al. 2015). However, a complete

account of invasive alien species of angiosperms is not yet available and therefore, attempted in this work.


Invasive alien species (IAS) were searched and collected from the different localities of study area

(Pantnagar). Plant specimen with flower and fruits were collected from different gardens, parks, and research

centres of the university regularly for further study. These localities were visited in different seasons to find out

the exact flowering and fruiting time of IAS. Collected specimens contain information on locality, date, and

other important information as suggested by Jain & Rao (1976). Plant specimen were identified with the help of

different floristic work like Duthie (1903–29), Bailey (1949), Maheshwari (1963), Raizada (1976), Babu (1977),

Sharma & Pandey (1984), Graf (1992), Gaur (1999), Khuroo et al. (2006), Negi & Hajra (2007), Reddy (2008),

Chandra Sekar (2012), Mehra et al. (2014), Bajpai et al. (2015) and volumes of Flora of India by BSI (Sharma

et al.1993, Sharma & Balakrishnan 1993, Sharma & Sanjappa 1993, Hajra et al. 1995a,b, Hajra et al. 1997,

Singh et al. 2000, Balakrishnan et al. 2012). Collected specimens were pressed and dried according to the

standard method suggested by Jain & Rao (1976) and submitted in the herbarium of G. B. Pant University of

Agriculture and Technology in the department of Biological Sciences, CBSH.


All the invasive alien species collected from Pantnagar area are enumerated in table 1. Each botanical name is

followed by family name, source region (nativity), growth forms and wild/cultivated status.

Table 1. Invasive alien species of Pantnagar flora.

S. No. Name of Species Family Nativity Growth

form

Cultivated

or Wild

1. Acacia farnesiana (L.) Willd. Mimosaceae Australia Tree Wild

2. Acanthospermum hispidum DC. Asteraceae Brazil Herb Wild

3. Acmellaradicans (Jacq.) R.K. Jansen. Asteraceae Trop. America Herb Wild

4. Ageratum conyzoides L. Asteraceae Trop. America Herb Wild

5. Ageratum houstonianum Mill. Asteraceae Trop. America Herb Wild

6. Alternanthera paronychioides St. Hill. Amaranthaceae Trop. America Herb Wild

7. Alternanthera philoxeroides (Mart.) Griseb. Amaranthaceae Trop. America Herb Wild

8. Alternanthera pungens Kunth Amaranthaceae Trop. America Herb Wild

9. Alternathera sessilis (L.) DC. Amaranthaceae Trop. America Herb Wild

10. Amaranthus spinosus L. Amaranthaceae Trop. America Herb Wild

11. Anagallis arvensis L. Primulaceae Europe Herb Wild

12. Antigonon leptopus Hook. & Arn. Polygonaceae Trop. America Climber Cultivated

13. Argemone mexicana L. Papaveraceae South America Herb Wild

14. Argemone ochroleuca Sweet Papaveraceae South America Herb Wild

15. Asclepias curassavica L. Asclepiadaceae Trop. America Herb Cultivated

16. Bidens pilosa L. Asteraceae Trop. America Herb Wild

17. Blainvillea acmella (L. f) Philipson Asteraceae Trop. America Herb Wild

18. Blumea lacera (Burm. f.) DC. Asteraceae Trop. America Herb Wild

19. Calotropis gigantea (L.) R.Br. Asclepiadaceae Trop. Africa Shrub Wild

20. Calotropis procera (Ait.) R.Br. Asclepiadaceae Trop. Africa Shrub Wild

21. Cannabis sativa L. Cannabaceae Central Asia Herb Wild

22. Cassia alata L. Caesalpiniaceae South America Shrub Wild

23. Cassia occidentalis L. Caesalpiniaceae South America Herb Wild

24. Cassia tora L. Caesalpiniaceae South America Herb Wild

25. Celosia argentea L. Amaranthaceae Trop. Africa Herb Wild

26. Chenopodium album L. Chenopodiaceae Europe Herb Wild

27. Chenopodium ambrosioides L. Chenopodiaceae Trop. America Herb Wild

28. Cleome viscosa L. Capparaceae Trop. America Herb Wild

29. Convolvulus arvensis L. Convolvulaceae Europe Herb Wild

30. Conyza canadensis (L.) Cronquist Asteraceae South America Herb Wild

31. Corchorus aestuans L. Tiliaceae Trop. America Herb Wild

Rastogi et al. (2015) 2(3): 282–287

.


32. Corchorus olitorius L. Tiliaceae Trop. Africa Herb Wild

33. Croton bonplandianum Baill. Euphorbiaceae South America Herb Wild

34. Cuscuta reflexa Roxb. Cuscutaceae Mediterranean Herb Wild

35. Cyperus difformis L. Cyperaceae Trop. America Sedges Wild

36. Cyperus iria L. Cyperaceae Trop. America Sedges Wild

37. Datura metel L. Solanaceae Trop. America Shrub Wild

38. Datura stramonium L. Solanaceae Trop. America Shrub Wild

39. Echinochloa colona (L.) Link. Poaceaea South America Grass Wild

40. Echinochloa crusgalli (L.) P. Beauv. Poaceaea South America Grass Wild

41. Eclipta prostrata (L.) L. Asteraceae Trop. America Herb Wild

42. Eichhornia crassipes (C.Martius) Solms Pontederiaceae Trop. America Herb Wild

43. Emilia sonchifolia (L.) DC. Asteraceae Trop. America Herb Wild

44. Euphorbia cythophora Murray Euphorbiaceae Trop. America Herb Wild

45. Euphorbia heterophylla L. Euphorbiaceae Trop. America Herb Wild

46. Euphorbia hirta L. Euphorbiaceae Trop. America Herb Wild

47. Evolvulus nummularius (L.) L. Convolvulaceae Trop. America Herb Wild

48. Gnaphalium pensylvanicum Willd. Asteraceae Trop. America Herb Wild

49. Gomphrena celosioides Mart. Amaranthaceae South America Herb Wild

50. Grangea maderaspatana (L.) Poir. Asteraceae Trop. America Herb Wild

51. Hyptis suaveolens (L.) Poit. Lamiaceae Trop. America Herb Wild

52. Impatiens balsamina L. Balsaminaceae Trop. America Herb Cultivated

53. Imperata cylindrica (L.) Raeusch. Poaceae Trop. America Grass Wild

54. Ipomoea eriocarpa R.Br. Convolvulaceae Trop. Africa Herb Wild

55. Ipomoea fistulosa Mart. ex Choisy Convolvulaceae Trop. Africa Herb Wild

56. Ipomoea hederifolia L. Convolvulaceae Trop. America Herb Wild

57. Ipomoea pes-tigridis L. Convolvulaceae Trop. E. Africa Herb Wild

58. Ipomoea quamoclit L. Convolvulaceae Trop. America Herb Wild

59. Lantana camara L. Verbenaceae Trop. America Herb Wild

60. Leucaena latisiliqua (L.) Gilli. Mimosaceae Trop. America Herb Wild

61. Ludwigia perennis L. Onagraceae Trop. America Herb Wild

62. Malvastrum coromandelianum (L.) Garcke Malvaceae Trop. America Herb Wild

63. Mecardonia procumbens (Mill.) Small Plantaginaceae Trop. America Herb Wild

64. Melilotus albus Medik. ex Desr. Fabaceae Europe Herb Wild

65. Melochia corchorifolia L. Sterculiaceae Trop. America Herb Wild

66. Mimosa pudica L. Mimosaceae Brazil Herb Wild

67. Mirabilis jalapa L. Nyctaginaceae Peru Herb Wild

68. Nicotiana plumbaginifolia Viv. Solanaceae Trop. America Herb Wild

69. Ocimum americanum L. Lamiaceae Trop. America Herb Cultivated

70. Opuntia vulgaris Miller Cactaceae South America Shrub Wild

71. Oxalis corniculata L. Oxalidaceae Europe Herb Wild

72. Parthenium hysterophorus L. Asteraceae North America Herb Wild

73. Peperomia pellucida (L.) Kunth Piperaceae South America Herb Wild

74. Physalis angulata L. Solanaceae Trop. America Herb Wild

75. Physalis minima L. Solanaceae Trop. America Herb Wild

76. Pistia stratiotes L. Araceae Trop. America Herb Wild

77. Portulaca oleracea L. Portulaceae South America Herb Wild

78. Portulaca quadrifida L. Portulaceae Trop. America Herb Wild

79. Prosopis juliflora (Sw.) DC. Mimosaceae Mexico Shrub Wild

80. Rorippa dubia (Pers.) Hara Brassicaceae South America Herb Wild

81. Saccharum spontaneum L. Poaceae Trop. W. Asia Grass Wild

82. Scoparia dulcis L. Plantiganeace Trop. America Herb Wild

83. Sida acuta Burm. f. Malvaceae Trop. America Herb Wild

84. Solanum nigrum L. Solanaceae Trop. America Herb Wild

85. Sonchus asper (L.) Hill Asteraceae Mediterranean Herb Wild

86. Sonchus oleraceus L. Asteraceae Mediterranean Herb Wild

87. Torenia fournieri Linden ex Fourn. Linderniaceae Australia Herb Wild

88. Tribulus terrestris L. Zygophyllaceae Trop. America Herb Wild

89. Tridax procumbens L. Asteraceae C. America Herb Wild

90. Triumfetta rhomboidea Jacq. Tiliaceae Trop. America Herb Wild

Rastogi et al. (2015) 2(3): 282–287

.


91. Typha angustifolia L. Typhaceae Trop. America Herb Wild

92. Urena lobata L. Malvaceae Trop. Africa Shrub Wild

93. Xanthium indicum Koenig Asteraceae Trop. America Herb Wild

94. Youngia japonica (L.) DC. Asteraceae South America Herb Wild

In the present study 94 invasive alien species under 72 genera, belonging to 33 families have been recorded

in Pantnagar area. In IAS flora of Pantnagar dicotyledons are represented by 85 species under 65 genera, and 28

families, whereas monocoyledons are represented by 9 species under 7 genera and 5 families (Table 2). On the

basis of their source regions IAS can be broadly categorized into six major groups viz., American, African,

European, Asian, Mediterranean, Australian. Almost 78% (74 spp.) IAS were introduced from the American

continent followed by Afican continent 8.5% (8 spp.), Europe 5.3% (5 spp.), Mediterranean 3.1% (3 spp.), Asian

and Australian by 2.1% (2 spp). These results are in tune of Reddy (2008), Singh et al. (2010), Chandra Sekar et

al. (2012) where Tropical American elements are recorded as the dominant part of IAS flora. Pantnagar is a hot

and moist subtropical habitat where, tropical American plants have found climatic conditions similar to their

native habitats and thus flourish well (Fig. 1).

Table 2. Families, Genera and species of IAS diversity in Pantnagar area.

Families Genera Species

Number % Number % Number %

Dicots 28 84.84 65 90.28 85 90.42

Monocots 5 15.15 7 9.72 9 9.57

Total 33 100.00 72 100 94 100

Figure 1. Source regions of Invasive Alien flora of Pantnagar.

Family Asteraceae dominates the invasive alien flora with 18 species in 16 genera, followed by Fabaceae (8

spp., 6 genera), Amaranthaceae (7 spp., 4 genera), Convovlvulaceae (7 spp., 3 genera), Malvaceae (7 spp., 6 genera),

Solanaceae (6 spp., 4 genera), Poaceae (4 spp., 3 genera), Euphorbiaceae (4 spp., 2 genera) constituting the eight

dominant families of IAS (Fig. 2). In other regions IAS flora also have the dominance of family Asteraceae

(Singh et al. 2010, Wagh & Jain 2015). The IAS flora of Uttarakhand and India also recorded the highest

member of species from family Asteraceae (Reddy 2008). The dominance of family Asteraceae may be

attributed to its prolific seed production and efficient seed dispersal mechanism. Habit wise analysis showed that

the herbs (78 species) were dominant, followed by shrubs (8 species), grasses (4 species), Sedges (2 species)

trees (1 species), and climbers (1 species) (Fig. 3). Herbs, shrubs and trees may have equal chances of dispersal

to non-native lands of these species but since herbs have shortest life cycle their further proliferation in non-

native land is easier. This seems to be the reason behind large percentage of herbs in IAS flora in Pantnagar and

elsewhere. The genera with the highest number of invasive alien species in Pantnagar region are Ipomoea (5

species), Alternanthera (4 species) and Cassia (3 species). These genera are also recorded having many species

in Reddy (2008), Chandra Sekar et al. (2012). In the recent years IAS have gained considerable notoriety as

being major threats to native species and ecosystem. These IAS proliferates easily because they find no natural

enemies in their new habitat and produce large numbers of propagules. Presence of IAS in any area be it

0

10

20

30

40

50

60

70

80

American African European Mediterranean

Nu

mb

er o

f Sp

ecie

s

Source regions

Asian Australian

Rastogi et al. (2015) 2(3): 282–287

.


country, state, district or local, indicates the disturbance in the vegetation of that area. More disturbed is the

vegetation more are the chances of occurrence of invasive alien species in the area.

Figure 2. Eight dominant families of Invasive Alien flora in Pantnagar.

Figure 3. Life forms of IAS in Pantnagar region

Pantnagar is a small land area devoid of natural stands of vegetation and suffer from continuous agricultural

operations and human activities, which seems to be the reasons behind the presence of large number of IAS

flora here. The challenge now is to find ways to manage the invasive aliens that are firmly entrenched in the

area. Though prevention is suggested as the most effective and feasible method for controlling the Invasive

species it cannot be applied after invasion. Rather, now steps should be taken to ensure no further spread of

these weedy species through planting materials, seeds carried from the university to adjacent areas.

ACKNOWLEDGEMENTS

Authors’ are thankful to Head of Department Biological Sciences, CBSH and incharge of MRDC Pantnagar,

G. B. Pant University of Agriculture and Technology, Pantnagar for providing us constant support during this

entire research.

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0 5 10 15 20

Asteraceae

Fabaceae

Amaranthaceae

Convolvulaceae

Malvaceae

Solanaceae

Poaceae

Euphorbiaceae

Number of Species

Fam

ilie

s

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ISSN (E): 2349 – 1183

ISSN (P): 2349 – 9265

2(3): 288–291, 2015

Research article

Phytoconstituents composition and in vitro antibacterial activity

of a blue green alga Anabaena variabilis Kütz. ex Born. et Flah.

Nilu Halder*

Department of Botany, Raja Peary Mohan College, Uttarpara - 712258, W. Bengal, India


Abstract: Cyanobacterial species produce various types of secondary metabolites which are used

in drug development as medicinal importance, dye and pigmentation and, food additives.The

preliminary screening of phytoconstituents analyses in various solvent extracts (benzene,

chloroform, acetone and methanol) of a microscopic blue green alga, Anabaena variabilis Kütz. ex

Born. et Flah., collected from a pond at Diara in Hooghly district, West Bengal, India was done

following standard methods and the results exhibited the presence of alkaloid, terpenoid, phenol,

flavonoid, flavonol and phycocyanin phytoconstituents in those solvent extracts. The antibacterial

activity of the said alga was detected using eight pathogenic bacterial strains out of which three are

Gram positive (Bacillus subtilis, Micrococcus luteus and Staphylococcus aureus) and five are

Gram negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Shigella dysenteriae, Shigella

flexneri and Vibrio cholerae) by agar well diffusion method with minor modifications. Maximum

inhibition zones were observed in acetone and benzene extracts against the same Gram positive

bacteria Bacillus subtilis. This study also indicated that acetone and benzene crude extracts were

best active solvents against most of the studied bacterial strains followed by methanol and

chloroform crude extracts. So, the present work suggested that this alga possessed several

bioactive phytoconstituents which showed antibacterial potentiality on the tested pathogenic

bacteria.

Keywords: Anabaena variabilis - Phytoconstituents screening - Antibacterial activity

[Cite as: Halder N (2015) Phytoconstituents composition and in vitro antibacterial activity of a blue green alga

Anabaena variabilis Kütz. ex Born. et Flah. Tropical Plant Research 2(3): 288–291]

INTRODUCTION

Algae are one of the most important richest sources of novel bioactive compounds which may be used in the

pharmaceutical industries for the development of pharmaceuticals. Cyanobacteria are well known incredible,

primitive and prokaryotic algal group which produce various types of phytoconstituents with biological

activities.

The search of cyanobacterial species with antimicrobial activities has gained much momentum in the recent

times due to growing worldwide concerns about increases in the emergence of antibiotic resistance among the

pathogenic bacteria (Chauhan et al. 2010). Cyanobacteria have been recognized as a good source of antibiotic

with antimicrobial activities during the last two decades (Bhattacharyya et al. 2013).They have potentiality to

produce an elaborate array of secondary metabolites with unusual structures and potent bioactivities. They also

produce industrially important secondary compounds like antibiotic, algicide, cytotoxic, immune suppressive

and enzyme inhibiting agents (Shaieb et al. 2014). The cyanobacterial algal group produces different

biologically active compounds which may be used in drug development and some of the active components

have potentially to inhibit anticancer, antimicrobial, antiviral, anti-inflammatory effects (Seal et al. 2014,

2015).The aims of the present study were preliminary screening of phytoconstituents and to investigate

antibacterial potentiality of four organic solvent extracts of this alga against human pathogenic bacteria.


Selected species

Halder (2015) 2(3): 288–291

.


Anabaena variabilis Kütz. ex Born. et Flah. is a blue-green alga which belongs to the family Nostocaceae

under the order Nostocales of class Cyanophyceae and it is grown in different types of aquatic bodies. The

trichomes contain heterocystous stuctures and the alga acts a bio-fertilizer due to having the capability to fix

atmospheric nitrogen in the soil.

Collection of algal sample

Algal material had been collected in plastic packets and sterilized glass containers from a pond of Diara (N

22° 79' E 88° 28') of Hooghly district (N 20° 30'–23° 1' E 87° 30'–80° 30'), West Bengal. Detailed study was

made by examining the specimen under Olympus microscope (Model-CH20i). Identification of taxon was

accomplished with the help of authentic literatures (Geitler 1932, Desikachary 1959).

Preparation of algal extracts

For extraction, the selected algal material, collected from pond was washed under running tap water to

remove adhering soil particles, epiphytes and associated debris, if any, and dried up at room temperature.

Benzene, chloroform, acetone and methanol were used for preparing the different solvent extracts. Extracts were

prepared by grinding the algal material in a mortar and pestle at 20±1°C. In a soxhlet extractor at 50–55°C,

extracts were concentrated under reduced pressure in a rotary evaporator and kept deep frozen until tests. Each

time before extracting the powdered drug (marc) was dried up in a hot air oven. The concentrated extracts were

obtained with each solvent were weighed. However, when the pH was out of range it was adjusted to 7.0 before

assay of antibacterial activity.

Preliminary qualitative phytoconstituent tests

All the extracts were subjected to preliminary phytoconstituents screening as described by Trease & Evans

(1989), Harborne (1998) and Silveira et al. (2007).

1. Used Gram positive bacteria

The tested bacterial strains were Bacillus subtilis MTCC441, Micrococcus luteus MTCC1538,

Staphylococcus aureus MTCC3160. These strains were maintained on nutrient agar slant at 4°C and sub-

cultured for 24 h before use.

2. Gram negative bacteria

The tested bacterial strains were Escherichia coli MTCC 443, Pseudomonas aeruginosa MTCC2581,

Shigella dysenteriae (clinically isolated), Shigella flexneri MTCC1457, Vibrio cholera MTCC3904. The

maintenance procedure of these bacteria was same as Gram positive bacteria.

Antibacterial assay

The antibacterial activity test of the above said alga was done using agar well diffusion method of Perez et

al. (1990). 0.1 ml of diluted inoculum (105CFU ml-1) of the bacterial strain was swabbed on the nutrient agar

plates. Wells of 5.0 mm size diameter were made into the agar plates with the help of sterilized cork borer (5.0

mm). Using a micropipette, each of 100 µl of the algal extract was added to the wells made in plates. The plates

were inoculated aerobically in an upright position at 37±2°C for 24–48 h. Antibacterial activity was evaluated

by measuring the zone of inhibitions (mm) against the bacterial strains. The tests were performed in triplicates

with controls.

Statistical analysis

The results were presented as mean values ± standard errors (SE). The standard errors were calculated using

statistical software package (SPSS v. 13, Inc.USA).


Qualitative screening of phytoconstituents of organic solvent extracts (benzene, chloroform, acetone and

methanol) of Anabaena variabilis Kütz. ex Born. et Flah. showed the conformity of different types of bioactive

compounds (Table 1). The result of the phytoconstituents screening revealed that this alga contained alkaloid,

tannin, terpenoid, phenol, flavonoid, flavonol and phycocyanin compounds.

In table 2, the result of antibacterial activity of the said algal species against three Gram positive and five

Gram negative bacterial strains was shown. The extracts of this alga confirmed antibacterial activities against

only six tested pathogenic bacteria out of eight bacterial strains. Acetone and benzene extracts exhibited better

antibacterial activities than other two organic solvent extracts. Regarding antibacterial activities, Bacillus

Halder (2015) 2(3): 288–291

.


subtilis and Escherichia coli were more susceptible whereas, Staphylococcus aureus and Pseudomonas

aeruginosa showed intermediate results. Micrococcus luteus and Vibrio cholera were comparatively less

susceptible against all the four solvent extracts. Both acetone and benzene extracts executed the higher

inhibition zones of 18.1 mm and 16.5 mm against the same bacteria Bacillus subtilis. Comparatively, somewhat

lower antibacterial activities were recorded by methanol and chloroform extracts of Anabaena variabilis. The

aqueous extract showed poor activities against the tested pathogenic bacteria (Table 2).

Table 1. Presence of phytoconstituents of Anabaena variabilis Kütz. ex Born. et Flah. in four solvents.

Alk

alo

id

Ta

nn

in

Ste

roid

Sa

po

nin

Gly

cosi

de

Ter

pen

oid

Ph

eno

l

Fla

vo

no

id

Fla

vo

no

l

Ph

yco

cya

nin

n

Benzene extract + - - - - + + + + +

Chloroform extract + - - - - + + + + +

Acetone extract + - - - - + + + + +

Methanol extract + + + - - + + + + +

Note: + = Presence or positive reactions; – = Absence or negative reactions.

Table 2. Antibacterial activity of different solvent extracts of Anabaena variabilis Kütz. ex Born. et Flah.

Solvents Zone of inhibition (mm) as (Mean ± SE ) in different bacteria

Bs Ml Sa Ec Sd Pa Vc Sf

Benzene 16.5±0.07 10±0.03 15±0.05 16.1±0.06 - 14±0.05 11±0.03 -

Chloroform 14.4±0.06 8.6±0.02 12±0.04 14±0.05 - 12±0.04 10±0.03 -

Acetone 18.1±0.08 12±0.04 16.1±0.06 15.5±0.07 14.2±0.06 13.8±0.04

Methanol 14.6±0.05 9.0±0.02 13.2±0.04 14.8±0.04 - 13±0.04 8.0±0.03 -

Water 9.0±0.04 - 7.0±0.03 8.0±0.03 - 7.0±0.02 - -

Note: Ec= Escherichia coli, Sf= Shigellaflexneri, Pa= Pseudomonas aeruginosa, Sd= Shigelladysenteriae, Vc=

Vibrio cholerae, Bs= Bacillus subtilis, Ml= Micrococcus luteus, Sa= Staphylococcus aureus; “-”= Not detected.

Chauhan et al. (2011) carried out in vitro antibacterial evaluation of Anabaena sp. against several clinically

significant pathogenic bacteria and HPTLC analysis of its crude extracts indicated that different solvents

possessed significant antibacterial effects on both Gram positive and Gram negative bacteria. In the current

scenario, similar observation was noticed.

Ethyl acetate solvent among three solvents (chloroform, ethyl acetate and n-butanol) was proved as a most

effective organic solvent for the extraction of the antibacterial compounds in five species of Anabaena namely

Anabaena solitaria, Anabaena variabilis, Anabaena cylindrical, Anabaena spiroides and Anabaena circinalis

(Abdel-Raouf & Ibraheem 2008). Among various solvent extracts (acetone, methanol, ethanol. diethyl ether,

chloroform and hexane) methanol extract of Anabaena circinalis appeared to be the most effective solvent by

showing maximum antibacterial activity against the selected bacterial pathogens viz. Escherichia coli,

Salmonella typhi, Proteus vulgaris, Streptococcus pyogens, Pseudomonas solanocearum (Sivakami et al. 2013).

In this study, it was noticed that acetone and benzene extracts were more effective over the other two solvent

extracts for extraction of antibacterial compounds.

Malathi et al. (2014) while working on the screening of three cyanobacterial strains observed significant

antibacterial activities of Anabaena variabilis against the bacteria Bacillus subtilis (ATCC-11774) and

Pseudomonas aeruginosa (ATCC-15442) in chloroform and methanol crude extracts respectively. In the present

work, in vitro antibacterial activities of this alga showed the similar result. Maximum size of inhibition zone

(9.67±0.57) of Anabaena BT2 was observed in methanol extract against Pseudomonas sb1 (Yadav et al. 2012)

but in this study, higher inhibition zone (18.1 mm) was observed in acetone extract against a Gram positive

bacteria Bacillus subtilis.

CONCLUSION

It is quite evident from the discussion that the above said alga possessed several bioactive compounds of

pharmaceutical interests and the formation of inhibition zones (mm.) depended on various factors like types of

Halder (2015) 2(3): 288–291

.


algal species, solvents used and the kind of tested pathogenic bacterial stains. Therefore, this study could be

used for future research and to produce antibacterial drugs of Cyanobacterial origin.

ACKNOWLEDGEMENTS

The author is indebted to Dr. S. N. Sinha, University of Kalyani, Nadia, West Bengal for his guidance and

supports. The author is also grateful to Dr. T. Seal and K. Chaudhuri, Plant Chemistry Department, BSI,

Howrah for their kind co-operations and suggestions.

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Documents

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