Inventory Message about Bat Species of Kalakad Mundanthurai Tiger Reserve (Tamil Nadu, India)...

ISSN: 0972-9720

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Theoretical and Experimental Biology (An International Journal of Basic and Applied Biology)

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Editor-in-Chief:

Dr. E. JOHN JOTHI PRAKASH, Elias Academic Publishers, ELMA-ZION,

214-B3/1A-Punnai Nagar, Nagercoil-629004, INDIA ([email protected]).

Executive Editor: Dr.M.JAYAKUMAR, Department of Botany, VHNSN College, Virudunagar-626001, INDIA ([email protected]).

Editors: Dr.M.VIVEKANANDAN, Department of Biotechnology, Bharathidasan University, Tirchirappalli-620024, INDIA ([email protected]).

Dr. V. B. HOSAGOUDAR, Tropical Botanic Garden and Research Institute, Palode-695562, Thiruvananthapuram, Kerala, INDIA ([email protected]).

Dr. JOSEPH A. J. RAJA, Department of Plant Pathology (Unit of Molecular Virology), College of Agriculture and Natural Resources, National Chung Hsing University, Taichung, TAIWAN (R.O.C). ([email protected]).

Dr. M. JAYASHANKARA, Department of Microbiology, Mangalore University PG Center, Cauvery Campus, Madikeri-571201, Karnataka, INDIA. ([email protected])

Dr.H.C.LAKSHMAN, P.G.Department of Botany, Karnatak University, Dharwad- 580003, Karnataka, INDIA ([email protected]).

Dr.C.VIJAYALAKSHMI, Department of Crop Physiology, Tamil Nadu Agricultural University, Coimbatore-641003, INDIA. ([email protected]).

Dr. APN LIPTON, Central Marine Fisheries Research Institute, Vizhingam, Trivandrum-695521, INDIA ([email protected]).

Dr.M.EYINI, Department of Botany, Thiyagarajar College, Madurai-625009, INDIA. ([email protected]).

Dr.P.K.JHA, Department of Botany, Tribuvan University, Kirtipur, Kathmandu, NEPAL. ([email protected]).

Dr.RUP KUMAR KAR, Department of Botany, Visva-Bharati, Santiniketan-731235, INDIA ([email protected]).

Dr. B. REDDYA NAIK, Department of Zoology, Osmania University, Hyderabad-500007, Andhra Pradesh, INDIA ([email protected]).

Dr.G.ANNIE JULIET, Department of Molecular Genetics and Microbiology, University of Texas at Austin, Texas 78712, USA ([email protected]).

Dr. A. THANGA RAJ, Global engineering Systems, FZC, P6-073, SAIF Zone, P.O. Box No. 7913, Sharjah, UNITED ARAB EMIRATES ([email protected]).

Dr. JULIET VANITHARANI, Department of Animal Sciences, Sarah Tucker College, Palayamkottai-627007, INDIA ([email protected]).

Dr. NIKKY THOMAS, Harrison Institute, Bowerwood House, 15-St.Botoophs Road, Sevenoaks, Kent TN12 3AQ, UNITED KINGDOM ([email protected])

Dr. K.S.JAGADEESH, Department of Agricultural Microbiology, College of Agriculture, University of Agricultural Sciences, Dharwad-580005, Karnataka, INDIA ([email protected]).

Dr. SM. SUNDARAPANDIAN, Department of Ecology and Environmental Sciences, School of Life Sciences,

Pondicherry University, Puducherry -605014, INDIA ([email protected]).

Dr. VATSAVAYA S RAJU, Department of Botany, Kakatiya University, Warangal-506 009, Andhra Pradesh INDIA. ([email protected]).

Dr. Md. GOLAM MORTUZA, Department of Biochemistry and Molecular Biology, Bangladesh Agricultural University, Mymensingh 2202, BANGLADESH. ([email protected]).

Dr.S.NATARAJAN, Department of Plant Biology and Plant Biotechnology, Guru Nanak College, Chennai-600042, Tamil Nadu, INDIA. ([email protected]).

Dr. V.A.J. HUXLEY, Biotechnology Research Laboratory, Department of Zoology, Thiru. Vi. Ka. Government Arts College, Tiruvarur-610003, Tamil Nadu, India. ([email protected]

Journal of Theoretical and Experimental Biology is an international journal for current research in

Basic and Applied Biology and is issued quarterly. It is published by Elias Academic Publishers, ELMA-ZION,

214-B3/1A-Punnai Nagar, Nagercoil-629004. INDIA. Email address: [email protected].

Journal of

Theoretical and

Experimental Biology (An International Journal of Basic and Applied Biology)

ISSN: 0972-9720 www. jteb.webs.com

Volume 10 No. 1 and 2 August and November 2013

Dr. E. John Jothi Prakash Editor-in-Chief

Dr. M. Jayakumar Executive Editor

Elias Academic Publishers

India

*Corresponding author; Email address: [email protected]

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Journal of Theoretical and Experimental Biology (ISSN: 0972-9720), 10 (1 and 2): 01-20, 2013 © 2013 Elias Academic Publishers

www.jteb.webs.com

Inventory Message about Bat Species of Kalakad

Mundanthurai Tiger Reserve (Tamil Nadu, India) through Global Positioning System (GPS)

Juliet Vanitharani

1*, Nikky Thomas

2, L. Jeyapraba

1 , C. Mercy

1, P. Selva Ponmalar

1 and

Gladrene Sheena Basil1

1Bat Research Laboratory, Department of Zoology and Research Centre, Sarah Tucker College (Autonomous),

Tirunelveli-627007, Tamil Nadu, India. 2Harrison Institute, Bowerwood House, 15-T. Bottphs Road, Kent TN12 3AQ, United Kingdom.

Received: 24 June, 2013; revised received: 14 July, 2013

Abstract

The Global Positioning System (GPS) is a space-based satellite navigation system that provides location and time information in all weather conditions, anywhere on or near the Earth where there is an unobstructed line of sight to four or more GPS satellites. The application of GPS brings an inventory message about the distribution, roosting pattern and other behavioral activities of bat species present in Kalakad Mundanthurai Tiger Reserve. GPS distribution data help to manage and give species specific protection. GPS location details of feeding roost from forest interiors help forest managers, in recovery of seeds and seedlings for afforestation and habitat improvement programmes. Keywords: Kalakad Mundanthurai Tiger Reserve, GPS, GIS.

Introduction

The only flying mammalian species, bats are one among the diverse species existing in the forests of Kalakad Mundanthurai Tiger Reserve [KMTR], Southern Western Ghats, South India. KMTR lies in one of the hotspots for biodiversity and declared world heritage centre by UNESCO. The present paper brings an inventory message about the distribution, roosting pattern and other behavioural activities of bat species present in Kalakad Mundanthurai Tiger Reserve through Global Positioning system (GPS).

The Global Positioning System (GPS) is a space-based satellite navigation system that provides location and time information in all weather conditions, anywhere on or near the Earth where there is an unobstructed line of sight to four or more GPS satellites. The system provides critical capabilities to military, civil, commercial users and decimation of field research activities around the world (Rodgers 2008, insidegnss.com, blurtit.com/ 2012, wikipedia.org/GPSystem, 2012). It is maintained by the United States government and is freely accessible to anyone with a GPS receiver. NAVSTAR is the official U.S. Department of Defence name for GPS.

The operations of GPS satellite system is done by the 24 satellites that make up the GPS space segment are orbiting the earth about 12,000 miles above us. They are constantly moving, making two complete orbits in less than 24 hours. These satellites are travelling at speeds of roughly 7,000 miles an hour. Each satellite is built to last about 10 years. Replacements are constantly being built and launched into orbit. A GPS satellite weighs approximately 2,000

Vanitharani and Basil / Inventory Message about Bat Species of KMTR, India through (GPS)

Journal of Theoretical and Experimental Biology (ISSN: 0972-9720), 10 (1 and 2): 01-20, 2013

2

pounds and is about 17 feet across with the solar panels extended. Transmitter power is only 50 watts or less.GPS satellites are powered by solar energy. They have backup batteries onboard to keep them running in the event of a solar eclipse, when there's no solar power. Small rocket boosters on each satellite keep them flying in the correct path. GPS has wide application in various day-to-day activities of human beings like cell phone with GPS is a tracking device (Maass, 2012), helps in navigation to take lanes during peak hours (en.wikipedia.org/wiki/San_Mateo%Hayward_Bridge), air navigation and ground navigation (Maddison, 2009; Jwo et.al., 2012), useful in finding missing persons, (askville.amazon.com/find-missing-person-phone-gps) to solve kidnapping cases (en.wikipedia.org/wiki/Kidnapping and Mitasova 2003) etc. Similar application is possible to study the various behavioural activities of wild animals (en.wikipedia.org/GPS _ wildlife_tracking).



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Map 1: Distribution of the fruit bats under family Pteropodidae Megachiroptera) in Kalakad Mundanthurai Tiger Reserve.

In the present paper, GPS is used as an excellent tool for plotting the distribution of bat diversity, occurrence of endemic endangered bat species in Kalakad Mundanthurai Tiger Reserve. The researcher has used GPS to map bat-interacting trees in the study area and to locate the diurnal roosts and feeding roost of fruit bats.



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Map 2: Distribution of microbats under families Rhinopomatidae, Emballonuridae, Megadermatidae, Rhinolophidae, Hipposideridae and Molossidae (Microchiroptera) ) in Kalakad Mundanthurai Tiger Reserve.



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Map 3: Distribution of microbats under family Vespertilionidae (Microchiroptera) in Kalakad Mundanthurai Tiger Reserve.

Methodology GPS satellites circle the earth twice a day in a very precise orbit and transmit signal information to earth. GPS receivers take this information and use triangulation to calculate the user's exact location. Essentially, the GPS receiver compares the time a signal was transmitted by a satellite with the time it was received. The time difference tells the GPS receiver how far away the satellite is. Now, with distance measurements from a few more satellites, the receiver can determine the user's position and display it on the unit's electronic map. GPS satellites transmit two low power radio signals, designated L1 and L2. Civilian GPS uses the L1 frequency of 1575.42 MHz in the UHF band. The signals travel by line of sight, meaning they will pass through clouds, glass and plastic but not through most solid objects such as buildings and mountains. A GPS signal contains three different bits of information - a pseudorandom code, ephemeris data and almanac data. The pseudorandom code is simply an I.D. code that identifies which satellite is transmitting information.

A GPS receiver can lock on to the signal of at least three satellites to calculate a 2D position (latitude and longitude) and track movement. If it is with four or more satellites in view, the receiver can determine the user's 3D position (latitude, longitude and altitude). Once the user's position has been determined, the GPS unit can calculate other information, such as speed, bearing, track, trip distance, distance to destination, sunrise and sunset time and more. Garmin 12 GPS unit is used for positioning various data of the present study.

Pseudorandom code I.D from the locked satellites by the Garmin12 GPS is viewed on the unit's satellite page, as it identifies which satellites it's receiving. Garmin 12 with parallel channel receivers capable of quickly locking six satellites even in dense foliage of the forest habitats as well as urban settings with tall buildings has been used to locate various data under the present study. Garmin 12's receivers are extremely accurate, because of their parallel multi-channel design.

Map 4: Distribution and roost location of Latidens salimalii, endemic, endangered fruit bat in Kalakad Mundanthurai Tiger Reserve.

Results



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The Global Positioning System data about the different bat species occurrence, foraging, and

diurnal roost location helps to map the distribution of bat species in KMTR [Map1-4]. The fruit

bats interaction and propagating biodivesity sustaining various fruiting tree location detail is

presented in Map 5. The bat species diurnal roosts available in the KMTR forests starting from

the foothill to the highest mountain peaks are given in Table1a,b. Six fruit species belonging to

the family Pteropodidae namely Rousettus leschenaulti, Pteropus giganteus, Cynopterus

brachyotis, Cynopterus sphinx, Latidens salimalii and Eonycteris spelaea, distributed in KMTR

(Table1a). The bat diversity studies made with the bat detectors and mist nets collection from

the foraging area and hand net collections from the available bat roost has revealed the

presence of 30 insect eating bat species belonging to the family Rhinopomatidae

(1),Emballonuridae (3) Megadermatidae (2) , Rhinolophidae (4) , Hipposideridae (4) ,

Molossidae (1) and Vespertilionidae (13) (Table1b), in KMTR. The location of feeding roosts

of the fruit bats with bat-treated seeds and the germinated seedlings of the rare endemic and bat-

propagated trees are illustrated in Table 2.



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Map 5: Fruit bats interaction and propagating biodivesity sustaining various fruiting tree locations in Kalakad Mundanthurai Tiger Reserve.

Table 1a: Roost location and characters of Fruit eating bat species of Kalakad Mundanthurai Tiger Reserve

Family - Pteropodidae

Rousettus lechenaulti (Cave and Temple Abandoned Dark Rooms -Corridors)

Roost Description GPS location Details

Arulmigu Ariyanatha Swamy Thirukoil Arikesavanallur (Foot hills) Ele : 229 ft, N: 8°42.776', E: 77°30.959'

Arultharum Gomathy Ambal Sametha Arulmigu Narum Poonathar Swamy Thirukovil

Thirupudai maruthur (Foot hills) Ele : 246 ft, N: 8°43.662', E: 77°29.892'

Sri Vanamamalai Perumal Thirukovil

Nanguneri (Foot hills) Ele : 335 ft, N: 8°29.523', E: 77°39.489'

Arulmigu Shenbagavallithayar Sametha Sri Jeganatha Perumal Thirukovil (Madapalli).

Shenbagaramanallur (Foot hills) Ele : 256 ft, N: 8°29.617', E: 77°43.279'

Arulmigu Aramvalartha Nayaki Udannurai Arulmigu Kulasekaramudaiyar Thirukovil

Kallidaikurichi (Foot hills) Ele:213ft, N: : 8°41.332', E: 77°28.187'

Arulmigu Kottiappar Thirukovil

Oorkadu (Foot hills) Ele: 177ft, N: 8°42.388', E: 77°28.167'

Sri Kanthimathiammal Nellaiyappar Thirukovil Tirunelveli (Foot hills) Ele : 193 ft, N: 8°44.012', E: 77°42.208’

Sri Alagiya mannar Raja Gopalasamy Thirukovil Sivankovil

Palayamkottai (Foot hills) Ele : 145 ft, N: 8°43.372', E: 77°44.238’

Pandurangan vittileshwarar

Vittilapuram (Foot hills) Ele: 146 ft N: : 8°41.083', E: 77°49.783'

Kowthalaiyar pudavu-cave

Karaiyar (Mundanthurai hills) Ele : 750 ft, N: 8°39.662', E: 77°20.181'

Vannathi parai

Chengammal estate (Mahendragiri hills) Ele : 1927 ft, N: 8°21.549', E: 77°30.789'

Ayiraperi cave

Kadayam hills Ele : 1110 ft, N: 8°54.205', E: 77°15.380

Thalaiyanai Muthaliruppan cave Sengaltheri (Kalakad hills) Ele : 2814 ft, N: 8°31.589' E: 77°26.759'

Pteropus giganteus (Open foliage-Tree branches) Ficus benghalensis (3 trees) near Sudalaimada swamy Thirukovil on road side

Padmaneri (Foot hills) Ele : 366 ft, N: 8°32.416', E: 77°34.053'

Terminalia arjuna on road side Nanguneri (Foot hills) Ele : 335 ft, N: 8°29.523', E: 77°39.489'

Terminalia arjuna near Police Station Panakudi (Foot hills) Ele : 316 ft, N: 8°19.485', E: 77°34.759'

Aegle marmelos – Sacred groove (Arultharum Gomathy Ambal Sametha Arulmigu Narumpoo Nathar Swamy Thirukovil)

Thirupudai maruthur (Foot hills) Ele : 246 ft, N: 8°43.662', E: 77°29.892'

Mangifera indica, Tarmarindus indicus, Madhuca indica (Thiruvaduthurai Atheenam madam- Sacred groove)

Kallidai kurichi (Foot hills) Ele : 222 ft, N: 8°41.332', E: 77°28.187'

Terminalia arjuna (4 trees- Sacred groove) on river edge Sivasilam (Foot hills) Ele : 220 ft, N: 8°47.077', E: 77°20.456'

Terminalia arjuna (Arulmigu Poorani Ambal Sametha Sri Poosan Perumal Sastha Thirukovil - Sacred groove)

Pattamudukku (Foot hills) Ele : 218 ft, N: 8°46.622', E: 77°25.241'

Terminalia arjuna in agricultural field Murappanadu (Foot hills) Ele : 208 ft, N: 8°42.170', E: 77°49.081'



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Table 1a: (Contd.) Roost location and characters of Fruit eating bat species of Kalakad Mundanthurai Tiger Reserve


Terminalia arjuna (3 trees) on railway lines

Melapalayam (Foot hills) Ele : 208ft, N: 8°42.456', E: 77°40.092'

Thambirabarani river side Athalanallur (Foot hills) Ele : 193 ft, N: 8°43.371', E: 77°29.270'

Cynopterus brachyotis (Tent-palm tree foliage and wild creeper bushes) Palmyra tree foliage Mundanthurai hills

Ele : 678 ft, N: 8°40.818', E: 77°20.768' Palmyra tree foliage Karaiyar (Mundanthurai hills)

Ele : 750 ft, N: 8°39.662', E: 77°20.181 Palmyra tree foliage Servalar hills

Ele : 857 ft, N: 8°41.008', E: 77°18.750' Kitul palm tree foliage & fruit strings Ambalam pudavu (Pothigai hills)

Ele : 1460 ft, N: 8°36.42', E: 77°18.38' Kitul palm tree foliage & fruit strings Therkumalai (Kadayam hills)

Ele : 2211 ft, N: 8°53.899', E: 77°16.289' Thick wild creeper bush Therkumalai (Kadayam hills)

Ele : 1872 ft, N: 8°54.674', E: 77°15.631' Kitul palm tree foliage & fruit strings Chenkammal estate (Mahendreagiri hills)

Ele : 1909 ft, N: 8°21.566', E: 77°30.789' Thick wild creeper bush Chenkammal estate (Mahendreagiri hills)

Ele : 1918 ft, N: 8°21.595', E: 77°30.733' Thick wild creeper bush Chenkammal estate (Mahendreagiri hills)

Ele : 1927 ft, N: 8°21.549', E: 77°30.789' Kitul palm tree foliage & fruit strings Thaipatham (Thirukurankudi hills)

Ele : 2139 ft, N: 8°41.891', E: 77°44.532' Kitul palm tree foliage & fruit strings Narakadu (Mahendreagiri hills)

Ele : 2633 ft, N: 8°27.75', E: 77°23.701' Thick wild creeper bush Kannikatti (Pothigai hills)

Ele : 2658 ft, N: 8°37.918', E: 77°16.195 Thick wild creeper bush Kuthiraivetti (Pothigai hills)

Ele : 3609 ft, N: 8°41.567' E: 77°44.208' Thick wild creeper bush Sengaltheri (Kalakad hills)

Ele : 2814 ft, N: 8°31.589' E: 77°26.759'

Cynopterus sphinx (Tent-palm tree foliage and wild creeper bushes) Palm tree foliage Thalavai puram (Foot hills)

Ele : 527 ft, N: 8°22.503', E: 77°33.507'

Palm tree foliage Rajapudhur (Foot hills) Ele : 526 ft, N: 8°25.179', E: 77°33.766'

Polyalthia longifolia branched foliage Mavadi (Foot hills) Ele : 456 ft, N: 8°27.998', E: 33.498'

Palm tree foliage Idaiyankulam (Foot hills) Ele : 450 ft, N: 8°34.917', E: 77°33.616'

Polyalthia longifolia branched foliage Kadayam (Foot hills Ele : 415 ft, N: 8°49.934', E: 77°22.406'

Palm tree foliage Pottal pudhur (Foot hills) Ele : 411 ft, N: 8°47.932', E: 77°23.594'



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Table 1a: (Contd.) Roost location and characters of Fruit eating bat species of Kalakad Mundanthurai Tiger Reserve


Palm tree foliage Poothathan kudiyiruppu (Foot hills) Ele : 367 ft, N: 8°36.029', E: 77°33.342'

Palm tree foliage Padmaneri (Foot hills) Ele : 366 ft, N: 8°32.416', E: 77°34.053'

Palm tree foliage Vallioor (Foot hills) Ele : 363 ft, N: 8°22.963', E: 77°34.750'

Palm tree foliage Panakudi (Foot hills) Ele : 316 ft, N: 8°19.483', E: 77°34.150'

Polyalthia longifolia branched foliage Manimuthar hills (Dam) Ele : 308 ft, N: 8°39.664', E: 77°26.106'

Palm tree foliage Shenbagaramanallur (Foot hills) Ele : 268 ft, N: 8°30.172', E: 77°42.559'

Polyalthia longifolia branched foliage Pappakudi (Foot hills) Ele : 241 ft, N: 8°45.098', E: 77°30.033'

Polyalthia longifolia branched foliage Ambasamuthram (Foot hills) Ele : 260 ft, N: 8°41.774', E: 77°27.268'

Polyalthia longifolia branched foliage Pathamadai (Foot hills) Ele : 252 ft, N: 8°40.058', E: 77°35.081'

Palm tree foliage Chingikulam (Foot hills) Ele : 220 ft, N: 8°42.779', E: 77°30.959'

Polyalthia longifolia branched foliage Alwarkurichi (Foot hills) Ele : 220 ft, N: 8°46.253', E: 77°24.6'

Palm tree foliage Pattamudukku (Foot hills) Ele : 218 ft, N: 8°46.622', E: 77°25.241'

Palm tree foliage Kallidaikurichi (Foot hills) Ele : 222 ft, N: 8°41.332', E: 77°28.187'

Palm tree foliage Cheranmahadevi (Foot hills) Ele : 212 ft, N: 8°40.964', E: 77°33.907'

Palm tree foliage Mukkudal (Foot hills) Ele : 201 ft, N: 8°44.669', E: 77°31.418'

Palm tree foliage Polyalthia longifolia branched foliage

Palayamkottai (Foot hills) Ele : 195 ft, N: 8°42.052', E: 77°44.274'

Latidens salimalii (Cave) Eluthukal pudavu Kodamadi (Pothigai hills)

Ele : 1080 ft, N: 8°43.083', E: 77°41.354' Ambalam pudavu Mundanthurai hills

Ele : 1460 ft, N: 8°36.42', E: 77°18.38' Udumbukal Servalar hills

Ele : 1804 ft, N: 8°43.936', E: 77°16.556' Ainthalai pudavu Inchiikuli (Pothigai hills)

Ele : 1960 ft, N: 8°37.242', E: 77°16.782' Deserted British building feeding roost Therkumalai (Kadayam hills)

Ele : 2412 ft, N: 8°53.838', E: 77°15.248' View point pudavu Sengaltheri (Kalakad hills)

Ele : 2814ft, N: 8°32.030', E: 77°26.877' Arangadu cave Kuthiraivetti (Kothaiyar hills)

Ele : 3343ft, N: 8°41.283', E: 77°31.098' Naga pudavu Nagapothigai (Pothigai hills)

Ele : 3476ft, N: 8°35.878', E: 77°16.556' Vellachi pudavu Poongulum (Pothigai hills)

Ele : 3712ft, N: 8°36.46', E: 77°15.243'



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Table 1b: Roost location and characters of insect eating bat species of Kalakad Mundanthurai Tiger Reserve.

Family - Rhinopomatidae

Rhinopoma hardwickii ( Cave)


Sivanthipatti Cave

Sivanthipatti (Palayamkottai) (Foot hills) Ele: 648 ft N: 8°40.421', E: 77°45.986'

Mullampudavu Cave Servalar hills Ele : 1157 ft, N: 8°41.891', E: 77°18.750'

Family - Emballonuridae

Taphozous melanopogon (Cave, Man Made Structure)

Ayiraperi Cave


Kilavi odai pudavu Cave Kunnathur (Tirunelveli town) (Foot hills) Ele : 145 ft, N: 8°43.372', E: 77°44.238’

Sri Alagiyamannar Raja Gopalaswamy Thirukoil

Palayamkottai (Foot hills) Ele : 145 ft, N: 8°43.372', E: 77°44.238’

Pandurangan vittileshwarar Thirukoil Vittilapuram (Foot hills) Ele: 146 ft N: 8°41.083', E: 77°49.783'

Venkatachalapathi Thirukovil Krishnapuram (Foot hills) Ele: 199 ft N: 8°41.329', E: 77°48.268'

Taphozous longimanus (Man Made Structure and Tree Bark) Arulmigu Kailasanathar Alayam Murappanadu (Vallanadu hills)

Ele: 298 ft N: 8°42.896', E: 77°49.948' Trunk of Palmyra tree near the crown Karaiyar (Mundanthurai hills)

Ele : 750 ft, N: 8°39.662', E: 77°20.181' Taphozous kachhensis (Cave)

Paraseri pothai Cave Keela Devanallur (Foot hills of Kalakad) Ele : 263 ft, N: 8°35.229', E: 77°37.968'

Family - Megadermatidae Megaderma lyra ( Man Made Structure)

Abandoned stone building Pathamadai (Foot hills) Ele : 252 ft, N: 8°40.058', E: 77°35.081'

Water Well in Arulmigu Shenbagaballi Thayar Sametha Sri Jeganatha Perumal Thirukoil, Sivan Koil

Sri Shenbagaramanallur (Foot hills) Ele : 256 ft, N: 8°29.617', E: 77°43.279'

Abandoned Stone building Arultharum Gomathy Ambalsametha Arulmigu Narum Poonathar Swamy Thirukoil

Thirupudaimaruthur(Foot hills) Ele : 246 ft, N: 8°43.662', E: 77°29.892'

Hindu temple: Arulmigu Ariyanatha Swamy Thirukoil. Arikesavanallur (Foot hills) Ele : 229 ft, N: 8°42.776', E: 77°30.959'

Gandhi Kathar Production unused buildng Idaikal (Foot hills) Ele : 214 ft, N: 8°45.242', E: 77°27.920'

Appan Koil – Feeding roost Cheranmahadevi (Foot hills) Ele : 212 ft, N: 8°40.964', E: 77°33.907'

Stone building - Mandabam on road side Kalloor (Foot hills) Ele : 210 ft, N: 8°41.900', E: 77°33.940'

Abandoned building Mukkudal (Foot hills) Ele : 201 ft, N: 8°44.669', E: 77°31.418'

ArulmiguKailasanathar Alayam Abandoned building

Murappanadu (Foot hills) Ele : 208 ft, N: 8°42.170', E: 77°49.081'



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Table 1b (Contd.): Roost location and characters of insect eating bat species of Kalakad Mundanthurai Tiger Reserve.


Megaderma spasma ( Man Made Structure, Cave and Tree Hole)

Abandoned quarters – Forest buildings Mundanthurai hills Ele : 678 ft, N: 8°40.818', E: 77°20.768'

Thottapirai - Abandoned Godown Servalar hills Ele : 857 ft, N: 8°41.008', E: 77°18.750'

Forest Guest house Kodamadi (Pothigai hills) Ele : 1080 ft, N: 8°43.083', E: 77°41.354'

Mangifera indica tree hole Sengeltheri Ele : 2814 ft, N: 8°31.589' E: 77°26.759'

Vairakkal pudavu Kadayam hills Ele : 744 ft, N: 8°53.838', E: 77°15.248'

Pallivasal pudavu

Sivasilam Ele : 972 ft, N: 8°47.077', E: 77°20.456'

Family - Rhinolophidae Rhinolophus rouxii (Cave)

Ambalam cave Mundanthurai hills Ele : 1460 ft, N: 8°36.42', E: 77°18.38'

View point cave Sengaltheri(Kalakad hills) Ele : 2814 ft, N: 8°31.589' E: 77°26.759'

Kothaiyar Damsite tunnel Kothaiyar Ele : 3879ft, N: 8°41.283' E: 77°41.098'

Kuravankuli cave Kannikatti (Pothigai hills) Ele : 2634 ft, N: 8°37.922', E: 77°16.411'

Vannathi parai cave Chengammal estate (Mahendragiri hills) Ele : 1918 ft, N: 8°21.595', E: 77°30.733

Karumandi amman cave Sengaltheri (Kalakad hills) Ele : 3414 ft, N: 8°31.772', E: 77°26.762'

Rhinolophus beddomei (Cave and Man Made Structure)

Unused building

Sengaltheri (Kalakad hills) Ele : 3103 ft, N: 8°31.932' E: 77°26.932' Ele : 4050 ft, N: 8°31.127' E: 77°26.886'

Kilamalai Athupudavu

Kilamalai (Kothiyar hills) Ele : 3547 ft, N: 8°32.144' E: 77°20.302'

Kuthiraivetti Cave

Kuthiraivetti (Kothiyar hills) Ele : 3609 ft, N: 8°41.567' E: 77°44.208'

Narakadu Donavur fellowship building Narakadu Donavur (Mahendragiri) Ele : 1224 ft, N: 8°27.162' E: 77°30.544'

Kuravankuli cave Kannikatti (Pothigai hills) Ele : 2634 ft, N: 8°37.922', E: 77°16.411'

Rhinolophus lepidus (Cave)

Karadipudavu Milaru (Mundanthurai) Ele : 1800 ft, N: 8°54.205', E: 77°15.38'

Ambalam cave Mundanthurai (Pothigai hills) Ele : 1460 ft, N: 8°36.42', E: 77°18.38'

Kuravankuli cave Inchikuli Ele : 1645 ft, N: 8°37.438', E: 77°17.636'

Ayiraperi cave


Narakad Donavur cave

Narakadu Ele : 3282 ft, N: 8°27.162' E: 77°30.544'



12

Table 1b (Contd): Roost location and characters of insect eating bat species of Kalakad Mundanthurai Tiger Reserve.


View point cave

Sengaltheri (Kallakad hills) Ele : 3282 ft, N: 8°31.932' E: 77°26.886'


Rhinolophus pusillus (cave)

Kuravan kuli Pudavu Mundanthurai (Pothigai hills) Ele : 1648 ft, N: 8°37.546' E: 77°17.532'

Family - Hipposideridae Hipposideros pomona (Cave and Man Made Structure)

Narakad Donovur-Wood house in forest interior Narakad (Mahendragiri hills) Ele : 2767 ft, N: 8°27.162' E: 77°30.544'

Kowdalair Cave Kowdalair hills Ele : 1200 ft, N: 8°42.603' E: 77°35.394'


Hipposideros speoris (Cave and Man Made Structure)

Sri Arulmigu Nithya Kalyani Ambal Udanurai Thirukoil

Kadayam (Foot hills) Ele : 250 ft, N: 8°49.934', E: 77°21.406'

Arulmigu Sathyavageeswarar Thriukoil - Temple Tower

Kalakad (Foot hills) Ele : 220 ft, N: 8°43.083', E: 77°41.084'

Sri Vanumamalai Perumal Thirukoil – Temple Tower Nanguneri (Foot hills) Ele : 256 ft, N: 8°29.523', E: 77°39.489'

Arulmigu Somanatha Swamy Gomathy Ambal Sivankoil and Paraseri hillock

Keela Devanallur (Foot hills) Ele : 263 ft, N: 8°35.229', E: 77°37.968'

Arulmigu Udhravagini Alayam Kailasanathar Thirukoil

Chingikulam (Foot hills) Ele : 220 ft, N: 8°42.779', E: 77°30.959'

Hindu temple: Sivan Koil Sri Nataraja Sannathi Thirukoil

Shenbagaramanallur (Foot hills) Ele : 256 ft, N: 8°29.617', E: 77°43.279'

Annai Maragathambigai Sametha Arulmigu Kasinathaswamy Thirukoil

Ambasamuthram (Foot hills) Ele : 256 ft, N: 8°29.617', E: 77°43.279'

Arulmigu Narampoo Nathar Swamy Thirukoil Thirupudaimaruthur (Foot hills) Ele : 246 ft, N: 8°41.775', E: 77°27.468'

Arulmigu Vilvanathar Swamy Thirukoil Pathamadai (Foot hills) Ele : 252 ft, N: 8°40.058', E: 77°35.081'

Arulmigu Manthiappar Thirukoil Kallidaikurichi (Foot hills) Ele : 222 ft, N: 8°41.332', E: 77°28.187'

Unused well motor room N.G.O. Colony (Palayamkottai) Ele : 195 ft, N: 8°42.052', E: 77°44.274'

Arulmigu Avudaippan Thirukoil Alwarkurichi (Foot hills) Ele : 220 ft, N: 8°46.253', E: 77°24.6'

Sri Ramasamy Thirukoil Chariot Pappankulam (Foot hills) Ele : 210 ft, N: 8°46.611', E: 77°24.233'

Appan Koil Cheranmahadevi (Foot hills) Ele : 212 ft, N: 8°40.964', E: 77°33.907'

Arulmigu Karutheeswarar Alayam Madavarvilagam (Foot hills) Ele : 205 ft, N: 8°47.933', E: 77°23.593'

Arulmigu Kailasanathar Alayam Murappanadu (Foot hills) Ele : 208 ft, N: 8°42.170', E: 77°49.081'

Unused building Mukkudal (Foot hills) Ele : 201 ft, N: 8°44.669', E: 77°31.418'

Arulmigu Pusphavaneswarar Swamy Thirukoil Thenthirupuvanam (Foot hills) Ele : 202 ft, N: 8°43.493', E: 77°31.088’



13

Table 1b (Contd.): Roost location and characters of insect eating bat species of Kalakad Mundanthurai Tiger Reserve


Arulmigu Kailasanathar Swamy Thirukoil Ariyanayagipuram (Foot hills) Ele : 198 ft, N: 8°43.234', E: 77°32.707’

Arulmigu Agneeswarar Alayam Valudhur (Foot hills) Ele : 193 ft, N: 8°44.545', E: 77°28.779’

Jothimaan Godown Tirunelveli town (Foot hills) Ele : 193 ft, N: 8°43.372', E: 77°44.238’

Abandondoned quarters (Secondary roost) Mundanthurai hills Ele 863ft, N: 8°40.390', E: 77°20.116'

Sanamparai cave Karaiyar hills Ele : 1221 ft, N: 8°39.611', E: 77°20.081'

Servalar Dam site under ground tunnel Servalar hills Ele : 1074 ft, N: 8°41.427', E: 77°18.394'

Hipposideros ater (Man Made structure)

Old building

Kadayam (Foot hills) Ele : 376 ft, N: 8°49.901', E: 77°21.596'

Abandoned building

Jameen Singampatti (Foot hills) Ele : 291 ft, N: 8°39.671', E: 77°26.060'

Old house

Pappankulam (Foot hills) Ele : 220 ft, N: 8°39.666', E: 77°27.559'

Arulmigu Udhravagini Alayam Kailasanathar Thirukoil


Arulmigu Sivasilanathar Paramakalyani Ambal Thirukoil

Sivasilam (Foot hills) Ele : 220 ft, N: 8°47.077', E: 77°20.456'

Stone building on road side

Kalloor (Foot hills) Ele : 210 ft, N: 8°41.900', E: 77°33.940'

Old house Murappanadu (Vallanadu hillock) Ele : 208 ft, N: 8°42.170', E: 77°49.081'

Community hall – Small room Kurukkuthurai (Foot hills) Ele : 208ft, N: 8°42.406', E: 77°40.192'

Lakshmi Trinkering workshop

Tirunelveli (Foot hills) Ele : 200 ft, N: 8°44.152', E: 77°42.254'

Abandoned house Palayamkottai (Foot hills) Ele : 195 ft, N: 8°42.052', E: 77°44.274'

Old building, Sarah Tucker College Tirunelveli-7

Palayamkottai (Foot hills) Ele : 264 ft, N: 8°41.892', E: 77°44.533'

Arulmigu Kottaippar Thirukoil

Oorkadu (Foot hills) Ele: 177ft, N: 8°42.388', E: 77°28.167'

Hipposideros fulvus (cave)

Sivanthipatti Cave

Sivanthipatti (Palayamkottai) hillock Ele: 648 ft N: 8°40.421', E: 77°45.986'

Near Servalar Dam Servalar Hills Ele: 789 ft N: 8°41.442', E: 77°18.451'

Family - Molossidae Tadarida aegyptiaca (Man Made structure)

Crevices - Arulmigu Udhravagini Alayam Kailasanathar Thirukoil


Crevices - Venkatachalapathy Thirukoil Krishnapuram (Foot hills) Ele: 199 ft N: 8°41.329', E: 77°48.268'



14



Family - Vespertilionidae

Motifs montivagus (Cave)

View point cave ;Karumandi amman Cave Sengaltheri (Kalakad hills) Ele : 3332ft, N: 8°31.932', E: 77°26.877' Ele : 34204 ft, N: 8°31.387', E: 77°21.430'

Myotis horsfieldii (Cave and Dam site Tunnels)

Mahendragiri arukadu Cave Chenkammal estate (Mahendragiri hills) Ele : 1909 ft, N: 8°21.566', E: 77°30.789'

View point Cave Sengaltheri (Kalakad hills) Ele : 2814ft, N: 8°32.030', E: 77°26.877'

Rocky tunnel of Dam site Kothaiyar hills Ele : 3879ft, N: 8°41.283' E: 77°41.098'

Scotophilus heathii (Ttree trunk - Crown foliage, Temple unused Building) Tree Temple unused building

Murappanadu (Vallanadu hillocks) Ele : 208 ft, N: 8°42.170', E: 77°49.081'

Scotophilus kuhlii (Palm tree trunk near crown foliage, Temple unused Building) Palm tree Temple unused building

Murappanadu (Vallanadu hillocks) Ele : 208 ft, N: 8°42.170', E: 77°49.081'

Palm tree near the crown Karaiyar hills Ele : 750 ft, N: 8°39.662', E: 77°20.181'

Pipistrellus ceylonicus (Crevices and Cavities in Abandoned House Buildings)

Crevices in old building and Kailasanathar Thirukoil Murappanadu (Foot hills) Ele : 208 ft, N: 8°42.170', E: 77°49.081'

Crevice in Udravagini Alayam Kailasanathar Thirukoil Chingikulam (Foot hills) Ele : 376 ft, N: 8°35.229', E: 77°37.967'

Cavity in abandoned house buildings Pottal puthur (Foot hills) Ele : 400 ft, N: 8°47.932', E: 77°23.594'

Cavity in old house in abandoned house buildings

Mukkudal (Foot hills) Ele : 201 ft, N: 8°44.669', E: 77°31.418'

Pipistrellus tenuis (Crevices and Cavities in Abandoned House Buildings) Switch board crack in guest quarters Mundanthurai hills

Ele : 678 ft, N: 8°40.818', E: 77°20.768' Roof of the old house-- Cavity and Crevices Pottal pudhur (Foot hills)

Ele : 400 ft, N: 8°47.932', E: 77°23.594' Roof of the old building-- Cavity and Crevices Servalar hills

Ele : 857 ft, N: 8°41.008', E: 77°18.750' Roof of the old building- Cavity and Crevices Pappan kulam (Foot hills)

Ele : 350 ft, N: 8°39.666', E: 77°27.559' Crevice Arulmigu Udravagini Alayam Kailasanathar Thirukoil

Chingi kulam (Foot hills) Ele : 376 ft, N: 8°35.229', E: 77°37.967'

Hindu temple: Crevice Krishna puram (Foot hills) Ele: 199 ft N: : 8°41.329', E: 77°48.268'

Crevice – Old building Palayamkottai (Foot hills) Ele : 195 ft, N: 8°42.052', E: 77°44.274'

Roof of the old building-- Cavity and Crevices Mukkudal (Foot hills) Ele : 201 ft, N: 8°44.669', E: 77°31.418'

Roof of the house-- Cavity and Crevices Karaiyar (Servalar hills) Ele : 750 ft, N: 8°39.662', E: 77°20.181'



15



Roof of the house- Cavity and Crevices Therkumalai (Kadayam hills) Ele : 1872 ft, N: 8°54.674', E: 77°15.631'

Crevices in old building and Kailasanathar Thirukoil Murappanadu (Foot hills) Ele : 208 ft, N: 8°42.170', E: 77°49.081'

Switch board crack of krishnapuram temple Krishnapuram (Foot hills) Ele: 199 ft N: 8°41.329', E: 77°48.268'

Pipistrellus dormeri (Man Made Structures- Cavity and Crevices) Man made structure- Cavity and Crevices Mukkudal (Foot hills)

Ele : 201 ft, N: 8°44.669', E: 77°31.418' Man made structure- Cavity and Crevices Palayamkottai (Foot hills)

Ele : 195 ft, N: 8°42.052', E: 77°44.274' Man made structure- Cavity and Crevices Karaiyar hills

Ele : 750 ft, N: 8°39.662', E: 77°20.181' Pipistrellus pipistrellus( Man Made Structures- Cavity and Crevices)

Man made structures- Cavity and Crevices Karaiyar (Servalar hills) Ele : 750 ft, N: 8°39.662', E: 77°20.181'

Man made structures- Cavity and Crevices Chingikulam (Foot hills) Ele : 376 ft, N: 8°35.229', E: 77°37.967'

Pipistrellus coromandra( Man Made Structures- Cavity and Crevices) Crevices- Old building Murappanadu (Vallanadu hillock)

Ele : 208 ft, N: 8°42.170', E: 77°49.081' Switch board crack in abandoned building Mundanthurai

Mundanthurai hills Ele : 678 ft, N: 8°40.818', E: 77°20.768'

Miniopterus schreibersii ( Cave – crevice)

Kuravan kuli cave – crevice Inchikuli (Pothigai hills) Ele : 1646 ft, N: 8°37.471', E: 77°17.572'

Miniopterus pusillus ( Cave – Crevice)

Riverside rock crevices Kannikatti (Pothigai hills) Ele : 2634 ft, N: 8°37.922', E: 77°16.411'

Vellachi pudavu Poongulam (Pothigai hills) Ele : 3712ft, N: 8°36.46', E: 77°15.243'


Murina cyclotis (Cavity – Crevice-Tree foliage ) Riverside palm tree bark crevices and dry foliage Narakadu (Mahendragiri hills)

Ele : 2522 ft, N: 8°27.780' E: 77°30.068' Riverside palm tree bark crevices and dry foliage Poongulam (Pothigai hills)

Ele : 1646 ft, N: 8°37.471', E: 77°17.572' Kerivoula lenis ( Cavity – Crevice-Tree) Deserted British building - palm tree bark crevices and dry foliage

Therkumalai (Pothigai hills) Ele : 1872 ft, N: 8°54.674', E: 77°15.631'

The bat diversity studies made with the bat detectors and mist nets collection from the

foraging area and hand net collections from the available bat roost has revealed the presence of 30 insect eating bat species belonging to the family Rhinopomatidae (1) (Table1b), Emballonuridae (3) (Table1c), Megadermatidae (2) (Table1d), Rhinolophidae (5) (Table1e), Hipposideridae (4) (Table1 f), Molossidae (1) (Table1g). and Vespertilionidae (13) (Table1 h) in KMTR. These fruit bat roost location are given in Table1 b .The location of feeding roosts of the fruit bats with bat-treated seeds and the germinated seedlings of the rare endemic and bat-propagated trees are illustrated in Table 2.



16

Table 2: Sites for the recovery of seeds and seedlings of key plant species- Bat feeding roost.

Seeds and Seedlings of Plant

Species Recovery Possibility

Bat Feeding Roost Location

Details

Area of

Collection

Ele:1044ft N: 8°48.099' E: 77°17.481' Ele:1194ft N: 8˚31.944' E: 77˚26.899'

Ele:1890ft N: 8˚43.083' E: 77˚41.854'

Ele:1920ft N: 8˚54.559' E: 77˚15.616'

Ele:2334ft N: 9˚17.206' E:77˚18.722'

Ele:2040ft N: 8˚43.083' E: 77˚41.854'

Ele:2412ft N: 8°53.838' E: 77°15.248' Ele:2442ft N: 8°53.083' E: 77°41.854' Ele:3024ft N: 8˚53.920' E: 77˚15.190'

Ele:3174ft N: 8˚53.909' E: 77˚15.351'

Ele:3107ft N: 9˚20.002' E: 77˚18.876'

Ele:3281ft N: 8˚41.567' E: 77˚44.208'

Kadayam

range

Ele: 757ft N: 8°41.171' E: 77°18.757' Servalaru

Ele:1947ft N: 8°37.318' E: 77°16.778' Ingikuli

Ele:1185ft N:8˚27.161' E:77˚30.546' Naraikadu

Ele:2876ft N: 8˚41.567' E: 77˚44.208'

Ele:3343ft N: 8˚41.283' E: 77˚31.098'

Ele:4063 ftN: 8˚41.829' E: 77˚26.150'

Kudhiraivetti

Ele:3810ft N: 8˚36.458' E: 77˚15.215' Poongulam

Ele:2947ft N: 8˚21.849' E: 77˚30.660' Valaiyar

Ele:2657ft N: 8˚26.206'E: 77˚24.546' Mahenragiri

Ele:2184ftN:0 8°32.514'E: 77°27.986' Ele:2814ft N: 8°32.030' E: 77°26.877' Ele:2852ftN: 8°31.773' E: 77°26.802' Ele:2911ft N: 8°32.138' E:77°27.447' Ele:1918ftN: 8°32.514' E:77°27.986' Ele:3021ft N: 8°31.974' E 77°27.256' Ele:3030ft N: 8˚31.892' E 77˚27.389'

Ele:3112ft N: 8°32.163' E 77°27.488' Ele:3118ft N: 8˚31.960' E:77˚27.205'

Ele:3146ft N: 8˚32.061 'E:77˚26.859'

Ele:3147ft N: 8°32.061' E:77°26.859' Ele:3165ft N: 8˚31.589' E: 77˚26.757'

Ele:3118ft N: 8˚31.960' E:77˚27.205'

Ele:3266ftN: 8°31.787'E: 77°26.727' Ele:3392ftN: 8°31.699'E: 77°26.729' Ele:3351ftN: 8°31.658'E: 77°26.690' Ele:3429ft N: 8°43.083' E:77°41.854' Ele:3541ft N: 8°31.596' E: 77°26.748' Ele:3595ft N: 8°31.421' E: 77°26.362' Ele:4063 ftN: 8˚31.829' E: 77˚26.150'

Ele:4111ft N: 8˚31.240' E: 77˚26.860'

Shengaltheri

Ficus asperima, F. beddomei,

F.callosa, F.racemosa,

F.retusa, F.guttata,

Elaeocarpus tuberculatus

E.serratus, E.munroii, Ensete

superbum, Syzygium cumini,

S.mundagam, S. jambos,

Pallaquium ellipticum, Musua

ferrea, Nephelium longana,

Dichapetalum jelonioides,

Mangifera indica, Strychnos

cinnnamomifolia or Strychnos

minor, Cullenia exallirata,

Erythroxylum monogynum,

Atalantia monophylla,

Diospyros foliolosa,

Eriodendron penandrum,

Diospyros sylvatica,

Achronychia pedunculata,

Careya arborea, Acronychia

pedunculata or laurifolia,

Erythrina indica, Madhuca

longifolia,Nephelium longana,

Myristica dactyloides,

Psychotria sp, Tarena

asiatica,Dimocarpus longan,

Polyalthia longifolia,

Tamarindus indica, Carica

papaya, Drypetes rozburghii,

Croton klotzschianus, Aglaia

elaeagnoidea, Azardiracta

indica, Psidium guajua,

Morinda tinctoria, Chassalia

curviflora, Sapindus

emarginatus.



17

Discussion Knowing the 3D positions (latitude, longitude and altitude) of the field data is very supportive in the monitoring as well as conservation works of wild animals in the forest. Estimating the home range behaviour of deer (Vercauteren, et al., 1993 ; Pellerin et al., 2008), Bornean elephant, Elephas maximus borneensis (Alfred et al., 2012), African buffalo (Naidoo et al.,

2012) in a tropical environment helps a lot to do the conservation management activities. The GPS location data about the bat roosts really helps to view the bat shows variation in the distribution based on elevation.

GPS technology, satellite telemetry and modern 3D acceleration measurements are very helpful to scientists in mapping the migration routes. The only endemic, endangered fruit bat L.salimalii since the year 2000 for a very long period was known from only one location in the high wavey mountains of Theni division of southern Western Ghats (Vanitharani et al., 2013). The first author located their roost distribution for the first time in KMTR and made the record of the home range extension of L.salimalii down south (Vanitharani 2005, 2007a; Vanitharani et al., 2003, 2004, 2005). The GPS distribution data manifested in the mapping aids species specific conservation of the Shedule I endemic species (narrow distribution range within Tamil Nadu part of southern Western Ghats, India) L.salimalii in Tamil Nadu. Similar usage of GPS technology and modern 3D acceleration measurements are pertained by Åkesson et al .(2012) to study the wintering migration in swifts of tropical Africa; Krementz et al.(2011) to study spring migration phenology, routes, stopover regions, and nesting sites of mallards (wild ducks); the annual migration of the Galapagos giant tortoise and the Bar-headed Geese crossing the Himalayas (redorbit.com) .

Most of the fruit bat interacting as well as propagating trees are endemic and endangered to southern Western Ghats. These tree distribution not only supports the fruit bat community including the endangered Latidens salimalii but also support the endangered diversity of birds (Horn bills) and mammals ( Lion tailed macaque). The GPS location details of these fruiting trees helps the forest managers to navigate in the forest interiors to locate the trees and to recover the seeds for propagation and afforestation programmes in the affected areas (Vanitharani and Pandian, 2012; Paulina, 2013). Similar GPS mapping technology helps the in-situ conservation of biodiversity in the protected area (Malleshappa, 2011; Vanithrani et al. 2011; Bharathi, 2012; Margret, 2012). According to Roberts (2002) and Garrison (2013), similar GPS mapping technology is used by fishermen to locate the fish shoals, to keep up with key spots, to avoid known hazards and to share information

The literature review on GPS telemetry in bat roosting behavior are highly useful to study the habitat requirements (Vanitharani 2006, 2007b, Daniel et al.2007 Knight and Jones 2009 , and Ruczynski et al.2010), roosting ecology of reproductive (pregnant or lactating) adult female Veilleux et al. 2003), roost intra-annual and interannual fidelity to summer roost areas (Britzke et al. 2003, Veilleux et al. 2004), communal roosts (Watts et al. 2012), the characteristics of tree roosts between forest structure (Perry and Thill 2008, Trousdale et al. 2008, Nixon et al. 2009, Johnson et al. 2011) and the sex-specific roost selection (Borkin and Parsons 2011). The GPS roost location details of KMTR is helpful to study the behavioral details like acoustics studies, home range radio telemetry studies, reproductive behavior, chemical signaling behavior, roost preference, spatial orientation and communal interaction in both individual species as well as colony. Such data is more applicable in the case of endemic, endangered bat species which are restricted in the distribution to KMTR. The location data are more helpful for the forest managers to legislate the management action plan to protect the species specific roost conservation.



18

Conclusion Geographic Information System (GIS) system which is designed to capture, store, manipulate, analyze, manage, and present all types of geographical data. GIS as a tool helped to deliver inventory message about bat species of Kalakad Mundanthurai Tiger Reserve. There is very little information about the impact assessment of bat diversity in forest conservation. To address this deficiency, these monitoring programmes were carried out. The data through GIS expressions helps monitoring habitat assessment, floral resource availability for bat diversity maintenance. Global Positioning System (GPS) being a tool closely associated with GIS, functions to map data with the aid of space-based satellite navigation system. The analysis of bat distribution and impact assessment helps in forest management especially natural regeneration and pest management. GPS location message brings hand proof data for bat conservation in the country like India where they are considered as ill omen.

Acknowledgements

The authors specially thank the Whitley Foundation, UK for the award of the Rufford Small Grants and Ministry of Environment and Forest (Government of India) for the bat survey project in Agasthiyamalai Biosphere Reserve. Special acknowledgements are due to University Grants Commission, New Delhi for the endorse of the Major Research Project to study the distribution and ecology of Latidens salimalii the Endemic, Endangered bat species, which serves to support continuing bat studies and conservation initiatives in the southern Western Ghats. The authors are grateful to The Geomatics division of Tamil Nadu Forest Department, Chennai for helping in drawing the distribution maps. We submit our indebted gratitude to the Harrison Institute, UK for their help in the identification of bat species. The study has been continuously encouraged by Tamil Nadu Forest Department, State Government of India. We thank the Principal Chief Conservator of Forests and Chief Wildlife Warden for the special permission and support to work in the forest interiors.

References

Åkesson, S., Klaassen, R., Holmgren, J., Fox, J. W., and Hedenström, A. 2012. Migration Routes and

Strategies in a Highly Aerial Migrant, the Common Swift Apusapus, Revealed by Light-Level Geolocators. PLoS ONE 7(7): e41195. doi:10.1371/ journal.pone.0041195.

Alfred, R., Ahmad, A. H., Payne, J., Williams, C., Ambu, L.N. 2012. Home Range and Ranging Behaviour of Bornean Elephant (Elephasmaximus borneensis) Females. PLoS ONE 7(2): e31400. doi:10.1371/journal.pone.0031400.

Bharathi, B. K. 2012. Enhancement of biodiversity amplification through the ecosystem services of pollinators and seed dispersers in Srivilliputhur Grizzled Squirrel Wildlife Sanctuary - an evaluation. Ph.D. Thesis. Manonmanium Sundaranar University Tirunelveli, India.

Borkin Kerry, M., Parsons and Stuart. 2011.Sex-specific roost selection by bats in clear fell harvested plantation forest: improved knowledge advises.

Britzke, E. R., Harvey, M. J., and Loeb, S. C. 2003. Indiana Bat, Myotis sodalis, Maternity Roosts in the Southern United States. South eastern Naturalist, 2(2):235-242.

Dah-Jing Jwo, Chi-Fan Yang, Chih-Hsun Chuang and Kun-Chieh Lin. 2012. A Novel Design for the Ultra-Tightly Coupled GPS/INS Navigation System. Journal of Navigation, 65: 717-747. doi:10.1017/S0373463312000161.

Johnson, J. S., Kiser, J. D., Watrous, K. S., and Peterson, T. S. 2011. Day-Roosts of Myotis leibii in the Appalachian Ridge and Valley of West Virginia. Naturalist, 18(1):95-106.

Krementz, D. G., Asante, K., and Naylor, L. W. 2011. Spring Migration of Mallards from Arkansas as Determined by Satellite Telemetry. Fish and Wildlife Management, 2(2):156–168.

Maddison, R., and Ni Mhurchu, C. 2009. Global positioning system: a new opportunity in physical activity measurement. International Journal of Behavioral Nutrition and Physical Activity, 6:73.



19

Maass P., and Rajagopalan, M. 2012. That’s No Phone. That’s My Tracker. The New York Times Sunday

Review, News Analysis. Malleshappa, H. 2012. Investigation on the interaction of pollinators and seed dispersers in forest

restoration and conservation of biodiversity in Kalakad Mundanthurai Tiger Reserve, southern Western Ghats. PhD. Thesis. Manonmanium Sundaranar University Tirunelveli.

Margret, V. I. 2012. Investigation on biodiversity augmentation through the interaction of bat, bird and insect species in the propagation of wild trees in Megamalai Wildlife Sanctuary. Ph.D. Thesis Manonmanium Sundaranar University, Tirunelveli, India.

Mitasova, H., Bernstein, D., Drake, T.G., Harmon R., Miller, C., and McNinch , J. 2003. Spatio−Temporal Analysis of Beach Morphology using LIDAR RTK−GPS And Open Source

Grass GIS. Naidoo, R ., Du Preez, P., Stuart-Hill, G., Jago, M., and Wegmann, M. 2012. Home on the Range:

Factors Explaining Partial Migration of African Buffalo in a Tropical Environment. PLoS ONE 7(5): e36527.

Nixon, A. E., Gruver, J. C., and Barclay, R. M. R. 2009.Spatial and temporal patterns of roost use by western long-eared bats (Myotis evotis). Naturalist, 162(1):139-147.

Paulina, A. 2013. Investigation on chemical signaling in Bat Propagated Plants and its impact on forest restoration. Ph.D. Thesis. Manonmanium Sundaranar University, Tirunelveli, India.

Pellerin, M., Saïd, S., and Gaillard, J.M. 2008: Roe deer Capreolus capreolus home-range sizes estimated from VHF and GPS data. Wildl. Biol., 14: 101-110.

Perry, R. W., and Thill, R. E. 2008. Roost selection by Big Brown Bats in Forests of Arkansas: Importance of Pine Snags and Open Forest Habitats to Males. Naturalist, 374-385.

Rodgers, A. R. 2001. Tracking Animals with GPS: The First 10 Years. An International Conference Held At The Macaulay Land Use Research Institute, Aberdeen, Washington 12-13, March 2001.

Ruczynski, I ., Nicholls, B., MacLeod, C. D., and Racey, P. A. 2010. Selection of roosting habitats by Nyctalus noctula and Nyctalus leisleri in Białowiez. Forest Ecol. and Manag., 259 :1633–1641.

Trousdale, A. W., Beckett, D. C., and Hammond. S. L. 2008. Short-term Roost Fidelity of Rafinesque's Big-eared Bat (Corynorhinus rafinesquii) Varies with Habitat. J. of Mamm., 89 (2): 477-484.

Vanitharani, J., Jeyapraba, L., and Annamalai, R. 2003. New record of distribution and roosting in Salim Ali’s fruit bat Latidens salimalii Thonglongya 1972. In : Proc. 28th Conf. Ethol. Soc. of India.

Edited by: R. Annamalai. M. Narayanan and J. Vanitharani. pp. 60-62. Vanitharani, J., Pearch, M. J., Jeya Praba, L., and Annamalai R. 2004. A review of the distribution and

status of Latidens salimalii (Chiroptera : Pteropodidae) with new records from the Western Ghats, India. Lutra, 47 (1):21-32.

Vanitharani, J. 2005. Special Report about Latidens salimalii of Kalakad Mundanthurai Tiger Reserve. BAT NET Newsletter-CCINSA., 6 (1). www.zooreach.org/ ZoosPrintNewsLetter/Bat.

Vanitharani, J., Vijaya, M., and Arul Sundari, A. 2005. Role of Fruit Bats in Forest Management of Agasthiyamalai Biosphere Reserve. In: Proc. of State Level Conf. on The Changing Environment. Edited by: G. Kulandaivelu, A. Thangaraj, E. John Jothi Prakash, M. Jeyakumar and Juliet Vanitharani. Department of Plant Biology and Plant Biotechnology, Tirunelveli Dakshina Mara Nadar Sangam College,T.Kallikulam. pp. 43-47.

Vanitharani, J. 2006. Noteworthy representatives of bat species in Agasthiyamalai biosphere reserve, Tamil Nadu. J. Theo. and Expt. Biol., 2(2): 47-59.

Vanitharani, J. 2007a. Conservation of Latidens salimalii and other red listed bats in Tamilnadu BAT NET Newsletter- CCINSA., 8 (1-2).

Vanitharani, J. 2007b. Status of Bats in Tamil Nadu and Integration of Bat Conservation in Management Work Plans. J. Theo. and Expt. Biol ., 3 (4): 175-188 .

Vanitharani, J., Viji Margaret, I., and Kavitha Bharathi, B. 2011. Interaction of Big Five ‘Bs’ (Bat, Bird, Butterflies, Beetles, Bees) a boon for Biodiversity Conservation in Megamalai Wildlife Sanctuary. J. Adav. Biotech., 10(10): 90-95.

Vanitharani, J., and Pandian. 2012. A Probe into Chemical Signaling in Fruit Bats for Quick Forest Restoration in Foot Hill Reserves of Southern Western Ghats. , In : Recent Advances in Biodiversity of India. Zoological survey of India, Kolkata. pp. 529.

Vanitharani, J., Singaravelan, N., and Marimuthu, G. 2013. Population, Ecology and Conservation of Salim Ali’s Fruit Bat (Latidens Salimalii) . IN E- BOOK Rare Animals of India, Bentham Science Publishers, Sharjah, UAE. pp.156-174

Vercauteren, K.C., and Hygnstrom, S.E. 1993 White-tailed Deer Home Range Characteristics and Impacts Relative to Field Corn Damage. Great Plains Wildlife Damage Control Workshop Proceedings. Paper No. 354.



20

Veilleux, J. P., Whitaker Jr, J. O., and Veilleux, S. L. 2003. Tree-Roosting Ecology of Reproductive Female Eastern Pipistrelles, Pipistrellus subflavus, in Indiana. Mammalogy, 84(3): 1068-1075.

Veilleux, J. P., and Veilleux, S. L. 2004. Intra-annual and Interannual Fidelity to Summer Roost Areas by Female Eastern Pipistrellus, Pipistrellus subflavus . Naturalist, 152:196-200.

Watts, B. D., and Mojica, E. K. 2012. Use of Satellite Transmitters to Delineate Bald Eagle Communal Roosts within the Upper Chesapeake Bay. Raptor Research, 46(1):121-128.

Sitography blurtit.com/ 2012 http://en.wikipedia.org/wiki/Kidnapping_of_Jaycee_Lee_Dugard http://askville.amazon.com/find-missing-person-phone-gps/AnswerViewer.do?requestId=88234015 wikipedia.org/GPSystem 2012 (en.wikipedia.org/GPS_wildlife_tracking redorbit.com/ migration of theGalapagosgiant tortoise and the Bar-headed Geese. http://en.wikipedia.org/wiki/San_Mateo%E2%80%93Hayward_Bridge http://www.insidegnss.com/auto/0706%20Benefits.pdf

*Corresponding Author, Email: [email protected] 21


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Sodium Azide: a Chemical Mutagen for Enhancement of

Agronomic Traits in Phaseolus vulgaris (Linn)

Girma Mosisa, Manikandan Muthuswamy * and Yohannes Petros

Department of Biology, Faculty of Natural and Computational Sciences, Haramaya University,

Haramaya , Dire Dawa, Ethiopia.

Received: 2 July, 2013; revised received: 7 September, 2013

Abstract

In bioassay studies the analysis of variance showed highly significant difference in seed germination of Haricot bean (Haramaya variety) treated with chemical mutagen (Sodium azide) when compared with the control plants. The agronomic traits variations based on green house experiment showed that the plant height, number of branch and internodes length were significantly increased in 0.01% concentration of sodium azide in Haramaya variety of haricot bean. Days required for flowering and days required for maturity were found to be significantly decreased in the lower doses of chemical mutagen.

Key Words: Agronomic Traits, Chemical Mutagen, Haricot Bean, Phaseolus vulgaris, Sodium Azide.

Introduction

Haricot bean (Phaseolus vulgaris L.) (2n = 22) belongs to the order Rosales, family Leguminosae, subfamily Papilionoideae, tribe Phaseoleae. The growth habit is classified as determinate or indeterminate based on whether a terminal reproductive or vegetative stem is formed though growth habit is strongly influenced by the environment (Graham and Ranalli, 1997).

Haricot bean is a well-established component of Ethiopian agriculture system and it is among the five most important food legumes (Abebe and Kefene, 1989). It has been an export crop for more than 40 years, and grown as a food crop for a much longer period. In many low lands of Ethiopia, haricot bean is considered as the main cash crop and protein source for the farmers (Abebe, 1987). At present, different verities of haricot beans are grown in the country. The major haricot bean production zone is central Rift Valley, southern, eastern, and western parts of Ethiopia (Habtu Aseffa, 1994) where beans are grown mainly for export purpose.

Despite its high economic importance in Ethiopia, the national average yield of dry bean is 861kg/ha. This yield is far less than the attainable yield (2500-3000kg/ha) under good management condition in Ethiopia (Abebe A and Kefene H, 1989). The average yield of haricot bean in Ethiopia is low, when compared to other African countries like Egypt where average yield is about 2,500kg/ha. This may be due to the lack of improved varieties for different agro-ecological zones (Abebe and Kefene , 1989).

The chemical mutagenic agents can have positive or negative effects on living organisms especially in quantitative characters. Many of these chemicals have clastogenic (chromosome damaging) effects on plants via reactive oxygen-derived radicals (Yuan and Zhang, 1994). These effects can occur both spontaneously and artificially following induction by mutagens. Chemical mutagens generally produce induced mutations which lead to base pair

Girma et al / Sodium Azide, a Mutagen, Enhances Agronomic Traits in Phaseolus vulgaris


22

substitutions, especially GC→AT resulting in amino acid changes, which change the function of

proteins but do not abolish their functions as like deletions or frame shift mutations. These chemo mutagens induce wide variations in morphological characters when compared to normal plants.

The chemical mutagen, sodium azide, is one of the strongest mutagenic agents inducing variations in quantitative characters. The mutagenic effects of this mutagen have been observed on tomato and it was very effective in inducing mutations with respect to germination percentage, root length, seedling height, seedling survival, number of branches per plant, and yield per plant (Adamu and Aliyu, 2007). Mutation techniques have been used almost exclusively for plant breeding from the 1960’s to 1990’s. The outcome was indeed remarkable:

about 2000 new varieties were developed in almost all countries except Africa, where progress was limited (Ahloowalia et al., 2004).

Therefore, the present work envisages the possibility of increasing the productivity of haricot bean by chemical mutation and selection for desirable yield traits.

Materials and Methods The selection of the crop plant was based on economic importance as well as availability in Haramaya University Ethiopia. Accordingly, the Haramaya variety (G-843) of haricot bean was selected.

Preparation of Chemical Mutagen

The induction of chemical mutation was achieved by using sodium azide. The different concentrations of sodium azide (0.01, 0.02, 0.03, 0.04 and 0.05 % V/V) were prepared in plastic beakers with distilled water.

Method of Mutagenesis

Seeds of haricot bean variety of Haramaya (G-843) were used for inducing mutation by sodium azide. The seeds of the selected varieties were surface sterilized with 0.1% mercuric chloride for 1 minute to remove the fungal spores on the surface of the seeds. Then the seeds were washed with distilled water several times to remove the mercuric chloride. After this the seeds were pre soaked in plastic beaker which contains distilled water for six hours and then treated with sodium azide at different concentrations (0.01, 0.02, 0.03, 0.04 and 0.05%) for 6 hrs (Dhanavel et al., 2008). Bioassay Studies

The bioassay studies were carried out following the method of Heisey (1990) in seed laboratory of Plant Science department Haramaya University, Ethiopia. Five seeds from each of the treatments were placed on whatman filter paper in petriplates (9cm X 2cm). Each petriplate was moistened with 2ml/ plate of distilled water and the control were normal seeds (untreated) and incubated at room temperature. The germination percentage, root and shoot length were measured after 8 days. The germination percentage was calculated by using the following formula:

Germination (%) = 100min

minx

ationgerforplacedseedsofNumber

atedgerseedsofNumber

Green House Experiments

The seeds that were treated with chemical mutagenic agents in the laboratory were sown in earthen pots (24 cm x 24 cm) in green house by CRD factorial design with three replications along with their respective control. Then the experimental plants were grown under the green house condition in Haramaya University, Ethiopia. The control plants were grown from the untreated seeds. The agronomic traits such as plant height, number of branches on the main axis, seed length, internode length, days to 50% flowering, days to 90% maturity, pods per plants and above ground biomass were measured and tabulated.



23

Results and Discussion

Mutagenic Effects of Sodium azide on Seed Germination and

Seedling Growth of Haricot Bean (Haramaya variety)

The effect of sodium azide on seed germination was observed to be concentration dependent. Seed germination percentages were decreased as concentration increased and significance difference was observed in all concentration when compared with control. But a maximum of 60% reduction in seed germination was observed in 0.05%. The result indicated highly significance difference was observed at (P<0.0001) level when compared with the control (Table 1). Similar results were also cited by (Siddiqui et al., 2007). Accordingly, the mutagenic chemical such as sodium azide decreases the seed germination percentage and increases the chromosomal aberrations in root tip mitotic cells of plant which was a dose-dependent manner. Reduction in seed germination in mutagenic treatments has been explained due to delay or inhibition of physiological and biological processes necessary for seed germination which include enzyme activity (Kurobane et al., 1979). Maximum root growth was observed in 0.01% concentration and the minimum the root growth in 0.05%. There was observed a significant variation between the treatments with the least concentration of sodium azide and one with the highest concentration. Mahalik and Routray. (2009) reported that maximum root length was recorded on mutant lines developed through ethyl methane sulfonate in blackgram. Highly significant difference was observed in shoot length between the treatments with 0.01%, 0.02% sodium azide and the control (Table 1). Table 1: Bioassay study of sodium azide on seed germination and seedling growth of the Haramaya variety haricot bean.

Concentration Germination (%) Root Length(cm) Shoot Length(cm)

Control 93.33a 7.59defgh 6.31fg 0.01% 80.00ab 13.79a 10.28abc 0.02% 66.67bcd 9.81dc 9.40abcde 0.03% 66.67bcd 8.15def 8.87abcdef 0.04% 66.67bcd 7.99defg 7.20defg 0.05% 60.00cd 4.84ij 5.89fg

LSD 14.03 2.33 2.99

Means in the same column followed by the same letters are not significantly different at 5% p level Effects of Sodium azide on Agronomic Traits of Haramaya Variety Haricot Bean

The results indicated that plant height and internodes lengths were significantly increased in 0.01% concentration of sodium azide treated plants. The effect was significant at (P<0.05) level, when compared with the control plants (Table 2). Similar result was seen on plant height at lower doses in Capsicum annuum L. (Kumar and Tripathi , 2004). Numbers of branches were highly stimulated at 0.01% concentration when compared to control. In the mean time differences in root nodules number was observed in all the treatments. Root nodule number was significantly increased in 0.01%, 0.02% and 0.03% concentration when compared with the control. Accordingly, the effect was highly significant at (p< 0.0001) level (Table 2).

In the lower doses of mutagenic treatments, days required for flowering and days required for maturity were found to be significantly decreased. Significant decrease in days to flowering and days to maturity were observed in 0.01%, 0.02% , 0.03% and 0.04% concentration of sodium azide when compared with the control plants and highly significance difference was observed at (p< 0.0001) level (Table 3). Considerable increase in pod number was observed at 0.01%, 0.02% and 0.03% concentrations of sodium azide when compared with control plants and highly significance difference was observed at (p< 0.001) level (Table 3).



24

Similar result was cited by Sureja and Sharma (2000) in lentil. No significant variations were observed in seed length. The aerial plant biomass weight was increased significantly in 0.01% concentration of sodium azide. Table 2 : Effects of various concentrations of sodium azide on plant height, number of branches on main axis, internodes length, and root nodules of the haramaya variety haricot bean

Concentration PH(cm) NBMA IL(cm) RN

Control 29.77de 3.20bcde 2.73def 130.10cd 0.01% 36.03ab 4.07a 4.16a 160.50b 0.02% 30.63cd 3.40abcd 3.06cde 181.00a 0.03% 31.80cd 3.40abcd 3.10bcde 155.53b 0.04% 31.63cd 3.13bcdef 3.12bcde 138.00c 0.05% 31.03cd 3.13bcdef 2.55defg 122.00de LSD 3.62 0.74 0.95 15.89

Means in the same column followed by the same letters are not significantly different at 5% p level PH= Plant height, NBMA=Number of branches on main axis, IL= Internode length, RN = root nodules. Table 3: Effects of various concentrations of sodium azide on days to flowering, pods per plant, days to maturity, seed length and above ground biomass of the haramaya variety haricot bean.

Concentration DF PPP DM SL(mm) AGB (g)

Control 54.33bc 2.47ghi 107.33a 10.23 26.39def 0.01% 48.33fg 3.67cdef 88.33j 10.17 34.28a 0.02% 48.67fg 3.60cdef 90.67ij 10.27 28.82cd 0.03% 50.67ef 3.73cde 93.33hi 10.73 27.99cde 0.04% 49.67f 2.80efgh 94.33gh 10.63 27.55cdef 0.05% 52.67cde 2.50ghi 98.00cdef 10.33 26.83defg

LSD 2.82 0.98 3.38 0.87 3.36

Means in the same column followed by the same letters are not significantly different at 5% p level. DF=Days of flowering, PPP= Pods per plant, DM=Days of maturity SL=Seed length, AGB= above ground biomass

Conclusions The effectiveness of chemical mutagen, Sodium azide, in haricot bean is associated with the chemical concentration. The results showed that, chemical mutagens showed positive effects on haricot bean (Haramaya variety) on some agronomic traits and this shows that chemical mutagens are essential in the improvements of crop plants.

References

Abebe, A. 1987. Haricot Bean varieties performance and recommended methods of production.

In:Institute of Agricultural Research Proceedings of the 19th National Crop Improvement Conference. Addis Ababa, Ethiopia. pp. 229-251.

Abebe, A., and Kefene, H.1989. Country reports of Eastern Africa, Ethiopia. In: The proceeding of a workshop on Bean varietal Improvement in Africa. CIAT African Works shop, 30th Jan.-2nd Feb., 1989. Maseru, Lesotho. pp. 110-121.

Adamu, A.K., and Aliyu, H. 2007. Morphological effects of sodium azide on tomato (Lycopersicon esculentum Mill). Science World Journal, 2(4): 9-12.



25

Ahloowalia, B.S., Malunszynski, M. and Nichterlein K., 2004. Global impact of mutation- derived varieties. Euphytica, 135: 187-204.

Dhanavel, D., Pavadi, P., Mullainathan,L., Mohana, D., Raju, G., Giaija, M., and Thilagavathi C., 2008. Effectiveness and efficiency of chemical Mutagenes in cowpea (Vagina uguiculata L.). African Journal of Biotechnology, 5 (22): 4116-4117.

Graham, P., and Ranalli, O.1997. Common bean (Phaseolus vulgaris L.). Field Crops Res., 53: 131-146. Habtu Aseffa, 1994. Epidemiology of bean rust in Ethiopia. Ph.D. Thesis. Wageningen Agricultural

University, Department of Phytopathology, The Netherlands. Heisey, R.M. 1990. Allelophatic and herbicidal effects of extracts from tree of heaven (Ahanthus

attissima). Amer.J.Bot., 77: 662-670. Kumar, J.S., and Selvaraj, R., 2003. Mutagenic effectiveness and efficiency of gamma rays and ethyl

methane sulphonate in sunflower (Helianthus annus L.). Madras Agric. J., 90(7-9): 574-576. Kurobane, I., Yamaguchi, H., Sander, C., and Nilan, R. 1979. The effects of gamma irradiation on the

production and secretion of enzymes and enzymatic activities in barley. Env. Exp. Bot., 19: 75-84. Mahalik J. K., and Routray, B. N. 2009. Changes in growth traits in Blackgram mutant lines induced by

different mutagenic treatments towards root Kknot Nematode infection. Assam University Journal of Science and Technology: Biological Sciences, 56-60.

Siddiqui, S., Meghvansi, M.K., and Hasan, Z. 2007. Cytogenetic changes induced by sodium azide (NaN3) on Trigonella foenum graecum L. seeds. South African Journal of Botany, 73: 632–635.

Sureja, A.K., and Sharma, R.R. 2000. Genetic variability and heritability studies in garden pea. Indian J. Hort., 57: 243-47.

Yuan, H.Y and Zhang, Z., 1993. Effect of free radicals and temperature on sister chromatid exchanges in Hordeum vulgare L. Acta Botanica Sinica, 35: 20-26.



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In vitro Antibacterial Activity and Phytochemical Analysis of

Three Aquatic Plants

Mofanato S.K. Kala Kumari

1* and B. Vasantha Kumari

2

1Department of Plant Biology and Plant Biotechnology, Women’s Christian College,

Nagercoil - 629 001, Tamil Nadu, India. 2Department of Plant Biology and Plant Biotechnology, Sree Ayyappa College for Women,

Chunkankadai - 629 807, Tamil Nadu, India.

Received: 24 June, 2013; revised received: 30 August, 2013.

Abstract

The antibacterial activity of three selected aquatic plants was evaluated on bacterial strains like Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes,

Klebsiella pneumoniae and Escherichia coli. The solvents used for extraction of plants were petroleum ether, chloroform and methanol. The presence of chemical constituents in the extracts was studied by preliminary phytochemical analysis. The antibacterial activity was evaluated by agar well diffusion method. The most susceptible Gram-positive bacterium was Staphylococcus aureus while the most susceptible Gram - negative bacterium was Klebsiella pneumoniae. Significant antibacterial activity of active extracts was compared with the standard drug chloramphenicol. Out of the three plant extracts, methanolic extract of Salvinia natans showed the best antibacterial activity. This study is unique, as it is the first of its kind in which antibacterial activity and phytochemical constituents of few aquatic plants were studied in detail. Keywords: Antibacterial activity, methanolic extracts, petroleum ether, chloroform, phytochemical.

Introduction

Medicinal plants are a source of great economic value in the Indian subcontinent. Nature has been bestowed on us a very rich botanical wealth and a large number of diverse types of plants grow in different parts of the country (Rana and Jain, 2011). In India, thousands of species are known to have medicinal value and the use of different parts of several medicinal plants to cure specific ailments has been in vogue since ancient times. The World Health Organization (WHO) has estimated that 80% of the world population relies on herbs for its primary health care needs. It has also observed that more than 35,000 plant species are being used around the world for medicinal purposes in traditional and ethnomedicinal practices (Kaushal and Singh, 2001). Now a days multiple drug resistance to antibiotics has developed due to indiscriminate use of commercial antimicrobial drugs commonly used to treat infectious diseases (Davis, 1994 and Service, 1995). In addition to this problem, antibiotics are sometimes associated with adverse effects on the host including hypersensitivity, immune suppression and allergic reactions (Ahamad et al., 1998). Therefore, there is a need to develop alternative antimicrobial drugs for the treatment of infectious diseases derived from medicinal plants (Clark, 1996;

27

Mofanato and Vasantha / In vitro Antibacterial Activity and Phytochemistry of Aquatic Plants


Cordell, 2000). Several reports on the antimicrobial activity of different herbal extracts have been carried out in different parts of the world (Nair and Chanda, 2004; Nair et al., 2005). All plants containing active compounds are important. The beneficial medicinal effects of plant materials typically result from the combinations of secondary products present in the plants, these compounds are mostly secondary metabolites such as alkaloids, steroids, tannins and phenolic compounds, which are synthesized and deposited in specific parts or in all parts of the plant (Balandrin et al., 1985). Consideration to all the mentioned facts, the present study was carried out to evaluate the phytochemicals and the bioactivity of extracts from three aquatic plant species against three Gram-positive and two Gram- negative bacterial strains in vitro.

Materials and Methods

Collection and Identification of Samples Fresh leaves of Jussiaea repens, Hydrilla verticillata and Salvinia natans were collected from five ponds at Aloor village in Kanyakumari District, Tamil Nadu, India and were identified with the help of Standard Flora and available literature. Extraction of Plant Material

Fresh plant materials were washed under running tap water, shade dried and powdered. 50gm of the powdered sample from each plant was successively extracted with petroleum ether, chloroform and methanol (40°C - 60°C) till the extracts became colourless using soxhlet apparatus. Each extract was concentrated by distilling off the solvent and then evaporating to dryness on a water bath, finally the trace solvents were removed in a vacuum desiccator. The extracts obtained were stored at 4°C until further use. Preliminary Phytochemical Analysis

Chemical tests were carried out on the petroleum ether, chloroform and methanol extracts to identify the constituents utilizing standard methods of analysis (Brinda et al., 1981). Bacterial strains

In vitro antibacterial activity was examined for petroleum ether, chloroform and methanol extracts from three water plants. Microorganisms were obtained from the Vivek Scientific Laboratory, Nagercoil, Kanyakumari district, Tamil Nadu, India. Among five microorganisms investigated, three Gram - positive bacteria were Staphylococcus aureus, Staphylococcus

epidermidis and Streptococcus pyogenes, while two Gram-negative bacteria were Klebsiella

pneumoniae and Escherichia coli.

Media Preparation and Antibacterial Activity

The antibacterial assay was performed by agar well diffusion method for solvent extract. The bacterial isolates were first grown in nutrient broth and inoculum was prepared. The Muller Hinton Agar medium was prepared, sterilized and the molten medium at 50°C was poured into sterile petriplates and the medium was allowed to solidify. The organisms were uniformly swabbed on the plates and wells were made in the agar medium using a sterile 6 mm cork borer. The wells were then filled with the extract at a concentration of 50ml. The plates were allowed to stand on for one hour to allow proper diffusion of the extract and to prevent spillage on the surface of the agar medium and then incubated overnight at 37°C for 24 hours. Microbial growth was determined by measuring the diameter of zone of inhibition. Chloramphenicol was used as positive control. For each bacterial strain, negative controls were maintained where pure solvents were used instead of the extract.

28

Ta

ble

1:

Res

ults

of

prel

imin

ary

phyt

oche

mic

al a

naly

sis

of t

hree

aqu

atic

pla

nts.

Co

nst

itu

ents

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ed

Ju

ssia

ea r

epen

s H

ydri

lla

ver

tici

lla

ta

Sa

lvin

ia n

ata

ns

Pet

role

um

eth

er

Ch

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form

M

eth

an

ol

Pet

role

um

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er

Ch

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form

M

eth

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ol

Pet

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um

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er

Ch

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M

eth

an

ol

Ste

roid

s +

+

-

+

+

+

+

+

-

Tri

terp

enoi

ds

+

- -

+

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- +

-

Sug

ars

+

+

+

+

+

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+

+

+

Alk

aloi

ds

+

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+

- -

- -

-

Phe

noli

c C

ompo

unds

+

-

+

+

- +

+

+

+

Fla

vono

ids

- -

+

- -

- -

- -

Cat

echi

ns

+

+

+

+

+

+

+

+

+

Sap

onin

s +

+

-

+

+

- +

+

-

Tan

nins

-

+

+

- +

+

+

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+

Ant

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nes

- +

+

-

+

- -

+

-

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Abs

ent

+ P

rese

nt



29




The antibacterial activity of three aquatic plant species extract was assayed in vitro by agar well diffusion method against 5 bacterial species. Table 2 shows the microbial growth inhibition of plant extracts in petroleum ether, chloroform and methanol. The maximum antibacterial activity was shown by Salvinia natans followed by Hydrilla verticillata and Jussiaea repens

respectively. The methanol extracts of the investigated plants showed maximum antibacterial activity against Gram-negative bacterium Klebsiella pneumoniae. Similar results were also reported by Stainer et al. (1986); Prescott et al. (1999); Venkatesan et al. (2005); and Jigna (2007), who reported diseases such as pneumonia, urinary and respiratory tract infections caused by Klebsiella species. The petroleum ether extracts of Hydrilla verticillata and chloroform extracts of Jussiaea repens could not inhibit any of the bacterial strains studied. The significant antibacterial activity of the three plant extracts were compared with the standard antibiotic, chloramphenicol. Preliminary phytochemical analysis revealed the presence of steroids, sugars, phenolic compounds, catechins, saponins and tannins. The other secondary metabolites like triterpenoids, alkaloids, flavonoids and anthroquinones were also present in trace amounts in all the three plants (Table 1). Table 2: Antibacterial Activity (Zone of inhibition in mm)

Plant Extracts Staphylococcus

aureus

Staphylococcus

epidermidis

Streptococcus

pyogenes

Klebsiella

pneumoniae

Escherichia

coli

Jussiaea repens

Pet. ether Ch. Me.

- -

12

- - -

12 - -

- -

13

- - -

Hydrilla

verticillata

Pet. ether Ch. Me.

- -

12

- -

11

- - -

- -

12

-

11 -

Salvinia natans

Pet. ether Ch. Me.

11 -

13

- -

11

- -

15

11 13 16

-

12 11

Chloramphenicol 28 25 23 22 21

Pet.ether - Petroleum ether; Ch - Chloroform; Me – Methanol

Conclusion

The present study was conducted to evaluate phytochemical and antibacterial activities of Jussiaea repens, Hydrilla verticillata and Salvinia natans. From this study, it is concluded that the investigated water plants contain potent phytochemicals like steroids, triterpenoids, sugars, alkaloids, phenolic compounds, catechins, saponins, tannins and anthroquinones. Amongst the plant species investigated, methanolic extracts of Salvinia natans showed the maximum antibacterial activity against all the microorganisms, probably due to the presence of the phyochemical constituents. This study is unique, as it is the first of its kind in which

30



antibacterial activity and phytochemical constituents of three aquatic plants (Jussiaea repens,

Hydrilla verticillata and Salvinia natans) were studied in detail.

References

Ahamad, I., Mehmood, Z. and Mohammad, F. 1998. Screening of some Indian medicinal plants for their antimicrobial properties. J. Ethnopharmacol, 62: 183-193.

Balandrin, M.F.J., Kjocke A. and Wurtele, E. 1985. Natural plant chemicals: sources of industrial and medicinal materials. Science, 228: 1154-1160.

Brinda, P., Sasikala B., and Purushothaman, K.K. 1981. Pharmacognostic studies on Merugan Kilzhangu, BMEBR, 3(1):84-96.

Clark A.M. 1996. Natural products as resource for new drugs Phar. Res., 13: 1133-1141. Cordell, G.A. 2000. Biodiversity and drug discovery a symbiotic relationship. Phytochemistry, 55: 463-

480. Davis, J. 1994. Inactivation of the antibiotics and the dissemination of resistance genes. Science, 264:

375-382. Jigna, P. 2007. In vitro Antimicrobial Activity and Phytochemical Analysis of some Indian medicinal

plants. Turk J.Biol., 31:53-58. Kaushal Kumar and Singh, 2001. Urgent need for preservation of the cultural heritage of Ethnoherbology.

Current Science, 81(3):791-794. Nair, R. and Chanda, S.V. 2004. Antibacterial activity of some medicinal plants of Saurashtra region.

J.Tissue Res., 4:117-120. Nair, R., Kalariya, T. and Chanda, S. 2005. Antibacterial activity of some selected Indian medicinal flora.

Turk. J.Bio., 29: 41-47. Prescott, L.M., Harley, J.P. and Klein D.A. 1999. Microbiology, 4th ed. Boston: The Mc Graw Hill

Companies Inc. pp. 685. Rana, P.S. and Jain, D.A. 2011. Evaluation of Antimicrobial Activity of Alcoholic and Aqueous Extracts

of Five plants used in Traditional medicine in North India. International Journal of Pharm. Tech. Research, 3(1):376-380.

Service, R.F. 1995. Antibiotics that resist resistance. Science, 270: 724-727. Stainer, R.Y., Ingraham, J.L. and Wheelis, M.L. 1986. General Microbiology. 5th ed. London: The Mac

Millan Press Ltd. Venkatesan, M., Vishwanathan, M.B. and Ramesh, N. 2005. Antibacterial potential from Indian Suregada

angustifolia. J.Ethnopharmacol, 99: 349-352.

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*Corresponding author; Email address: [email protected] 33


www.jteb.webs.com

Phytochemical Analysis and in vitroAntimicrobial Activity of

Various Extracts of Euphorbia nivulea Ham

S. Selvadhas and S. Natarajan*

Department of Plant Biology and Plant Biotechnology, Guru Nanak College,

Chennai – 600 042, Tamil Nadu, India.

Received: 24 July, 2013; revised received: 20 August, 2013.

Abstract

Anti-microbial potency in different solvents and pharmacognostic study of the medicinal plant which has the tamil name Akujemudu,paalaich, chetthai, milakaychchakkalattiin Sanskrit aspatrasnuhi,patta-karie in Hindi, and in Bengali as Sij.Traditional information possess that the juice of the leaf is used as a purgative, diuretic etc. The paste of the leaf made with neem oil is applied externally in rheumatism. Plant latex is used for treating jaundice, dropsy, enlargement of liver and spleen, and applied to hemorrhoids. Coagulated latex is used for bronchitis. Phytochemical studies indicated that the methanol and ethyl acetate extracts contain a broad spectrum of secondary metabolites like terpenes, flavonoids phenolic compounds, alkaloids, and tannins.Phenol, tannins and flavonoids were predominantly found in ethyl acetate and methanol solvent extracts and petroleum ether and chloroform exhibits phytosterols, fixed oils and terpenoids. In the present study the in-

vitro anti-bacterial and anti-fungal activities of petroleum ether, chloroform, ethyl acetate and methanol extracts of Euphorbia nivulea Ham (Euphorbiaceae) was evaluated against various strains of bacteria and fungi. The aerial parts of the plant extracts were tested for the anti-bacterial activity against gram positive (Staphylococcus aureus, Micrococcus

luteus, Bacillus subtilis) and Gram negative (Escherichia coli, Salmonella paratyphi, Pseudomonas aeroginosa, Klebsiella pneumoniae, Vibrio cholera) bacteria. The anti-fungal potency was tested against Aspergillus fumigatus, Aspergillus niger, Monococcus

purpura and Candida albicans. The preliminary anti-microbial activities were done by agar well diffusion method. Petroleum ether and chloroform extracts displayed very less anti-microbial activity; whereas ethyl acetate and methanol extracts showed very good anti-microbial activity with widest zone of inhibition which was comparable to standard drug. Hence these two extracts were further tested for their MIC by micro broth dilution method. From the study it was found that ethyl acetate and methanol extracts exhibited remarkable anti-microbial activity against the tested micro-organism. Key words: Euphorbia nivulea, Fluorescence characteristics, Pharmacognostic studies, Phytochemical screening, Antibacterial activity.

Introduction

People of inaccessible villages and tribal areas are reliant upon the practice of folk medicines (Nadkarni,1982). Ethnobotanically plant latex has a great potential with respect to its medicinal value. Latex has been reported to occur in 12000 plant species belonging to 900 genera. A common feature that can be found in the latex of Euphorbia ceaeis the presence of obvious digestive enzyme activity. Euphorbia is a large genus consisting of about over 2000 species distributed all over the world. Approximately 195 species of Euphorbia have been

Selvadhas andNatarajan / Antimicrobial Activity of Various Extracts of Euphorbia nivulea

Journal of Theoretical and Experimental Biology (ISSN: 0972-9720),10 (1 and 2): 37-44, 2013

34

recorded from India (Basak et al., 2009). This genus includes herbs, shrubs and trees in widely diverse habitats. Euphorbia nivulea Buch– Ham, a member of this family Euphorbiaceae is a wild, thorny, xerophytic, succulent plant, commonly used in fencing of the agricultural field and also in dry barren areas. It has different biological activities for the treatment of several ailments of man. One such plant, Euphorbia nivulea Buch.-Ham. invites attention of the researchers worldwide for its biological activities.There is not much literature available on the biological activities of Euphorbia nivulea. Also, the earlier reviews on Euphorbia plants lack satisfactory information regarding its biological activities. The aim of the present review is to provide the updated information on the biological uses of Euphorbia nivulea. Emphasis is being laid on the areas of the most recent interest and those which have not been presented in earlier reports.

In the present study the in-vitro anti-bacterial and anti-fungal activities of petroleum ether, chloroform, ethyl acetate and methanol extracts of Euphorbia nivulea Buch.-Hamwas evaluated against various strains of bacteria and fungi. The aerial part of the plant extracts were tested for the anti-bacterial activity against gram positive (Staphylococcus aureus, Micrococcus

luteus, Bacillus subtilis) and Gram negative (Escherichia coli, Salmonella paratyphi, Pseudomonas aeroginosa, Klebsiella pneumoniae, Vibrio cholera) bacteria. The anti-fungal potency was tested against Aspergillus fumigatus, Aspergillus niger, Monococcus purpura

Candida albicans and Tinea rubrum.The preliminary anti-microbial activities were done by agar well diffusion method. Petroleum ether and chloroform extracts displayed very less anti-microbial activity; whereas ethyl acetate and methanol extracts showed very good anti-microbial activity with widest zone of inhibition which was comparable to standard drug. Hence these two extracts were further tested for their MIC by micro broth dilution method. From the study it was found that ethyl acetate and methanol extracts exhibited very good anti-microbial activity against the tested micro-organism.


Plant collection, Drying, Pulverizing and Preparation of Extract

The Euphorbia nivulea Ham shrub was collected from the moist places near Thirunelveli District, Tamil Nadu and identified and authenticated by Mr.V.Chelladurai Research Officer of Botany, Central Council for Ayurveda and Siddha, Government of India. The voucher specimen was preserved in our laboratory for future reference. Euphorbia nivuleais is a tall shrub with cylindrical stem and branches. Stipular spines glabrous, straight, paired, often blackish. Leaves appear only during rainy season, 8.5 – 20 ×3.5 – 6.5 cm, crowded at the end of branches, obovate oblong or spathulate, glabrous. Cymes – 3- flowered, born from above the leaf scars on the tubercles. Capsules are glabrous, trigonous, seeds globose, dorsally lined and smooth.Flowering and fruiting period is March to July (Khare, 2007; Patil, 2003). After the collection of the plant, the root was removed; the aerial part was washed thoroughly in tap water and dried in shade for about 10 days under controlled temperature (25± 2 ºC), powdered and passed through a 40 mesh sieve and stored in a well closed container for further use. Foreign matter, loss on drying, ash value and extractive values were determined as per the standard procedures (Anonymous, 1996; Anonymous, 1998). Coarsely powdered dried aerial plant (1.1 kg) was successively soxhlated using petroleum ether, chloroform, ethyl acetate and methanol for 72 h at room temperature respectively. The extracts were filtered and the solvents evaporated to dryness under reduced pressure in an Eyela rotary evaporator at 40 to 45 ºC. The percentage yield was noted as 1.28 for petroleum ether, 2.68 for chloroform, 5.91 for ethyl acetate and 7.83 for methanol (Table 3). Analysis of Fluorescence PharmacognosticCharacters

Fluorescence analysis was carried out with powders prepared from shade dried plants as well as in petroleum ether, chloroform, ethyl acetate and methanol extracts as described by Thomas et al. (2008). The powders were treated separately with 1N aqueous NaOH, 1N ethonolicNaOH, 1 N H2SO4 and 1N HNO3. The supernatants were examined under ultraviolet light and ordinary



35

day light. Pharmacognostic characters of Euphorbia nivulea were analyzed by employing standard method as described in Pharmacopeia of India(Anonymous, 1996). Phytochemical Screening

Phytochemical screening was carried out to assess the qualitative chemical composition of crude extracts using commonly employed precipitation and coloration to identify the major natural chemical groups such as steroids, reducing sugars, alkaloids, phenolic compounds,saponins, tannins, flavonoids, amino acids, anthrquinone ,cardiac and iridoide glycosides (Harborne 1998; Kokate 2001). General reactions in these analysis revealed the presence of these compounds in the crude extracts tested byBrindhaet al.(1981). Crude extracts in various solvents were prepared and stored in a refrigerator were used for the phytochemical tests. Test Organisms

Gram positive bacteria, Micrococcus luteus NCIM 2169, Staphylococcus aureus NCIM 2079,Bacillus subtilis NCIM 2063 and Gram negative bacteria; Escherichia coli NCIM 2065, Salmonella paratyphi NCIM 2501, Pseudomonas aeroginosa NCIM 2200, Klebsiella

pneumonia NCIM 2707, were used as test organisms. The strains were obtained from Nation Collection of Industrial Microorganism, Pune. The fungal strains Aspergillus fumigates MTCC1811, Aspergillusniger NCIM 1207 Monococcuspurpura MTCC 1090 and the yeast Candida albicans MTCC 3100, were collected from Microbial type culture collection, Chandigarh. Preparation of Culture Media

Dehydrated media were purchased from Hi-Media Laboratories Ltd, India. All the media were prepared in sterile glass petri plates (4 mm thickness) according to the manufacturer’s

instructions. Chloramphenicol (10 μg/ml) and Griseofulvin (25 μg/ml) was used as standard drug for comparison of anti-bacterial and anti-fungal activity respectively. DMSO was used as a solvent. Determination of Antimicrobial Activities of Extract

The anti-microbial activities of petroleum ether, chloroform, ethyl acetate and methanol extracts were determined by agar well diffusion method (Huffordet al., 1975). All bacterial and fungal strains were grown in nutrient broth (NB) and Sabouraud dextrose broth (SDB) for 4-6 hours at specified temperatures. The turbidity of the broth culture was adjusted to 0.5 McFarland units. This gives a suspension containing approximately 1-2 x 106cfu/ml (Mackie and Mac Cartney, 1996). An aliquot of microbial culture was added to agar medium at 45°C and poured into the petri plate. After solidification of the agar, medium was punched with a sterile corkborer (5.0 mm diameter) to cut uniform wells. Different concentrations of the extracts (125, 250 and 500 μg/ml) were prepared using DMSO as solvent and added to the wells. Bacterial cultures were incubated at 37°C for 24 hours and fungal cultures at 25°C for 48 hours. Anti-microbial activity was determined by measuring the zone of inhibition surrounding the well. The zones of inhibition were then measured, recorded and compared with positive standard controls, Chloramphenicol (10 μg/ml) and Griseofulvin (25 μg/ml) for anti-bacterial and anti-fungal activity respectively. The assays were carried out under aseptic conditions. DMSO was used as a negative control.

Minimum inhibitory concentration (MIC) The minimum inhibitory concentration (MIC) of the extracts was determined by micro broth dilution method (Eloff, 1999; McGawet al., 2002; Rabe et al., 2002; Eloff et al., 2005). For MIC, four fold serial dilutions of the extracts were prepared (15.625, 31.25, 62.5 and 125 μg/ml) in microtire wells. Incubation of the microtire plates was carried out at 37

oC for 24 hours for bacteria and at 25°C for 48 hours for fungi. After incubation, microtire wells were observed for any visible growth. The bacterial suspensions were used as positive control and extracts in



36

broth were used as negative control. The MIC was interpreted as the lowest concentration of the extract that did not show any visible growth when compared to control tubes.

Results

The results of quality control evaluation of aerial parts of the plant of Euphorbia nivuleawere presented in Table 1 and were helpful in evaluating the pharmacognostic value of the medicinal plant. The result of fluorescence analysis of the powder and extracts in visible and UV range has been shown in Table 2. The Loss on drying, total ash, acid insoluble ash, water soluble ash contents were found to be 78.4 % ,66.3 %; 5.6 % and 19.3 % for crude plant materials respectively. The percentage yield was noted as 1.22 for petroleum ether, 4.66 for chloroform, 5.77 for ethyl acetate and 7.46 for methanol (Table 3). Higher extractive value was found in methanol extract when compared to other solvents. Table 1: Quality control evaluation of aerial parts of the plant of Euphorbia nivulea Ham.

Quality control Parameters Amount in

percentage

Foreign matter 3.4% Loss on drying 5.8% Total ash 31.8% Acid insoluble ash 3.82% Water soluble ash 19.3% Ethanol soluble extractive value 5.28% Water soluble extractive value 22.62%

Phytochemical Screening

The phyto chemical screening of aerial part of the plant different extracts revealed the presence of phytosterols and fats and fixed oils in petroleum ether extract, slight reaction for alkaloids in chloroform extract, gave positive results for flavonoids, phenolic acids and tannins in ethyl acetate and methanol extracts. Table 2: Analysis of fluorescence characters of powder and extracts of Euphorbia nivuleaHam.

Sl.

No. Treatment category Under Ordinary Day Light Under UV Light

1 Powder as such No colour change No colour change 2 Powder + 1N NaOH

(aqueous) No colour change No colour change

3 Powder + 1N NaOH (alcoholic )

Light yellow colour Yellowish green fluorescence

4 Powder + 1N Hcl Dark brown colour Green fluorescence 5 Powder + H2SO4(1:1) Black colour Green fluorescence 6 Powder + HNO3(1:1) Yellowish brown yellowish fluorescence 7 Powder + Ammonia(1:1) No colour change Green fluorescence 8 Powder + Iodine(1:1) No colour change No fluorescence 9 Powder + 5% FeCl2(1:1) Dark brown colour Brown fluorescence 10 Powder + Acetic acid Brown colour No fluorescence 11 Petroleum ether extract 60º-80

ºC Green colour Green fluorescence

12 Chloroform extract Dark greencolour Green fluorescence 13

Ethyl acetate extract Greenish colour Light yellowish fluorescence

14 Methanol extract Greenish brown Brown fluorescence



37

Table 3: Extract value of LochnerapusillaK.Schum

Name of the extract Percentage of extractive

value

Petroleum ether 60°-80°C 1.28 Chloroform 2.68 Ethyl acetate 5.91 Methanol 7.83

Antimicrobial Activities The results of the preliminary anti-microbial activities (zone of inhibition) are presented in Table 5. All test strains of bacteria were found to be sensitive to Chloramphenicol and fungal strains were sensitive to Griseofulvin. DMSO was used as the negative control which not showed any zone of inhibition against tested bacteria and fungi.

Table 4:Phyto chemical screening variousextracts of Euphorbia nivuleaHam.

Constituent name Petroleum ether

(60-80˚C) extract

Chloroform

extract

Ethyl acetate

extract Methanol extract

Alkaloid --- --- --- --- Phytosterol ++ + --- --- Fixed oil and fats ++ -- --- --- Proteins and free amino acids

--- --- --- ++

Flavonoids --- --- +++ +++ Phenolic acids and tannins

--- --- ++ +++

Note : + Traces,++ Positive ,+++ Strongly positive,--- Absent.

Petroleum ether extract does not displayed any anti-microbial activity against tested micro-organism at all three tested concentration; whereas chloroform extract displayed mild activity against Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumonia, Aspergillus

fumigatus, Monococcus purpura, Candida albicans and Trychophyton rubrumat all three tested concentration. More over chloroform extract showed activity at 250 and 500 μg/ml

concentration against Pseudomonas aeruginosa and Aspergillus niger. In addition, the chloroform extract did not exhibit any activity against Micrococcus luteus, Escherichia coli, Salmonella paratyphi and Vibrio cholera even at 500 μg/ml concentration.

Ethyl acetate and methanol extracts displayed anti-bacterial and anti-fungal activity against all the tested bacteria and fungi at all three tested concentrations (125, 250 and 500 μg/ml). Against all the tested micro-organism ethyl acetate extracts displayed better activity (highest zone of inhibition) than methanol extracts. Highest activity was observed at 500 μg/ml

concentration for ethyl acetate and methanol extracts. Both the extracts displayed more anti-fungal activity than anti-bacterial activity.

Minimum inhibitory concentration (MIC) was tested for the ethyl acetate and methanol extracts of Euphorbia nivulea and the results are presented in table 6. The MIC was considered as the lowest concentration of the extract that did not show any visible growth when compared to control tubes.

MIC of ethyl acetate extract was found to be 15.625 μg/ml against Bacillus subtilis,

Salmonella paratyphi, Aspergillus niger, Candida albicans and Tinea capitis. The MIC for the same extract against Staphylococcus aureus, Klebsiella pneumonia, Aspergillus fumigatus, and Monococcu spurpura was found to be 31.25 μg/ml. The MIC was found to be 62.5 μg/ml for

ethyl acetate extract against Micrococcus luteus and Escherichia coli. 125 μg/ml was found as

MIC fore ethylacetate extract against Pseudomonas aeruginosa and Vibrio cholerae .MIC of



38

methanol extract was found to be 15.625 μg/ml against Salmonella paratyphi and Candida

albicans. The MIC for the same extract against Bacillus subtilis, Aspergillus fumigatus, Aspergillus niger and Tinea capitis was found to be 31.25 μg/ml. The MIC was found to be 62.5

μg/ml for ethyl acetate extract against Staphylococcus aureus, Escherichia coli, Klebsiella

pneumonia, and Monococcus purpura. 125 μg/ml was found as MIC for ethyl acetate extract

against Micrococcus luteus, Pseudomonas aeruginosa and Vibrio cholerae.

Table 5: Antimicrobial activity of four extracts of aerial part extracts of Euphorbia nivulea Ham.

Bacteria

gram

(-)/(+)

Extract

concentration

(µg/ml)

Growth inhibition zone diameter(mm)

Peroleum

ether

Chloro-

form

Ethyl

acetate Methanol

Antibiotic

control

(Chloram-

phenicol

10 µg/ml)

Fungal

control25

mcg/ml

s. aureus 125 -- -- 16 19 22 -- 250 -- 7 15 20 20 -- 500 -- 9 17 20 21 --

M.luteus 125 -- 6 11 12 20 250 -- 6 10 11 -- -- 500 -- 7 12 15 -- --

B.subtilis 125 -- -- 16 17 19 -- 250 -- -- 14 17 -- -- 500 -- 9 15 18 -- --

E.coli (-) 125 -- -- 16 18 23 -- 250 -- -- 17 19 -- -- 500 -- 10 16 22 -- --

S.paratyphi (-)

125 -- -- 15 17 20 -- 250 -- -- 16 18 -- -- 500 -- 9 16 19 -- --

P. aeroginosa 125 -- -- 15 17 23 -- 250 -- -- 16 18 20 -- 500 -- 8 17 19 21 --

K.pneumonia

125 -- -- 15 18 24 --

250 -- -- 17 18 -- -- 500 -- 10 18 19 -- --

A. fumigates

125 -- -- 18 17 -- 21

250 -- 16 14 -- 23 500 9 14 12 --

A.niger

125 -- -- 16 15 -- 22 250 -- -- 17 13 -- 500 -- 8 16 9 --

M. purpura

125 -- -- 18 18 -- 23 250 -- -- 15 18 -- 500 -- 8 10 17 --

C. albicans

125 -- -- 11 12 -- 18 250 -- -- 13 12 -- -- 500 -- 6 11 10 -- --

T.rubrum

125 6 -- 11 15 -- 18 250 7 -- 12 16 -- -- 500 9 -- 14 17 -- ---

Discussion

The anti-microbial potentials of substances are useful tools in the control of various infections caused by micro-organisms especially fungal infections. Antibiotic resistance is a major concern and development of new agents from plants could be useful in meeting the demand for new



39

anti-microbial agents with improved safety and efficacy (Srivastava et al., 2000). Newer anti-microbials from plant extracts may be useful in many (food, dairy and pharmaceutical) industries to prevent contamination by limiting the microbial growth. In this study, it was found that the ethyl acetate and methanol extracts of aerial parts of Euphorbia nivulea exhibited highest anti-microbial activity than the petroleum ether and chloroform extracts. The difference in the anti-microbial efficacy could be due to the presence of variable phytochemical compounds in different extracts. Table 6: Minimum inhibitory concentration (in μg/ml) of ethyl acetate and methanol extracts of aerial part extractsof Euphorbia nivulea Ham.

Microorganism

Ethyl

acetate

extract

Methanol

extract

S. aureus62.5 62.5 125 125

M. luteus 125 125

B. subtilis 125 125

E.coli 31.25 62.5

S. paratyphi 31.25 31.25

P. aeruginosa 62.5 62.5

K. pneumonia 15.625 15.625

A. fumigates 62.5 62.5

A. niger 62.5 62.5

M. purpura 31.25 31.25

C. albicans 31.25 31.25

T.rubrum 62.5 62.5

The bacterial organisms found in this study to be susceptible include E. coli, S.

Aureusand B. subtilis which have been implicated in many systemic infections such as respiratory and genitourinary tract infections. The phytochemical screening of aerial part of the plant different extracts revealed the presence of phytosterols and fats and fixed oils in petroleum ether extract, slight reaction for alkaloids in chloroform extract, gave positive results for flavonoids, phenolic acids and tannins in ethyl acetate and methanol extracts. The phytochemical screening of this plant showed the presence of flavonoids in both of the ethyl acetate and methanol extracts which have been shown to possess anti-microbial properties (Hostettmanet al., 1995; Obohet al., 1998). Flavonoids are known for their anti-inflammatory, anti-arthritic and anti-microbial proper-ties (Trease and Evans, 1989). Therefore, the anti-microbial activities of this plant may be ascribed to the presence of flavonoids. These results support the popular ethno pharmacological use of this plant for the treatment of a scaly fungi infection. Bioassay directed fractionation of the most active extract is in progress to isolate and identify the compounds responsible for the anti-microbial activity.

Conclusion

Crude extracts from Euphorbia nivuleahave medicinal applications from ancient days and very little work has been done on the biological activity and credible medicinal applications of isolated compounds of Euphorbia nivulea. Hence a drug development program undertaken to investigate the anti-microbial potency Euphorbia nivulea. From the study it was found that ethyl acetate and methanol extracts exhibited remarkable anti-microbial activity against the tested micro-organism. The good quality activity may be attributed to the presence of flavonoids and phenolic compounds in these extracts.



40

References

Anonymous. 1996. Indian Pharmacopoeia. In: Appendix Physical test and determinations. Ministry of Health and Family welfare, Government of India, New Delhi.Vol II : A-89.

Anonymous. 1998. WHO Quality control methods for medicinal plant materials. AITBS Publishers and Distributors, New Delhi. pp.08-73.

Basak, S.K., Bakshi, P.K., Basu, S., Basak, S. 2009. Keratouveitis caused by Euphorbia plant sap, Indian journal of Ophthalmology, 57(4): 311-313.

Brindha, P., Sasikala, B., and Purushothman, K.K. 1981. Pharmacognostic studies on Murugan kizhangu. Bull. Medic.Ethen.Botanic.,3 (1): 84 -96.

Eloff, J.N. 1999. A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Medica, 64:711–713.

Eloff, J.N., Famakin, J.O., and Katerere, D.R.P. 2005.Combretum woodii (Combretaceae) leaf extracts have high activity against Gram-positive and Gram-negative bacteria. African Journal of Biotechnology, 4(10):1161-1166.

Harborne, J.B. 1973. Phytochemical Methods. Chapman and Hall, London. pp. 52-59. Hostettman, K, Martson, A.J., Wolfender, I., and Maillard, M. 1968. Screening for flavonoids and related

compounds in medicinal plants by LC-UV-MS and subsequent isolation of bioactive compounds. Akademiai, Kiaho, Budapest. pp. 35-52.

Hufford, C.D., Funderburk, J.M., Morgan, J.M., and Roberts, L.W. 1975. Two antimicrobial alkaloids from heartwood of Liriodendron tulipifera. Journal of Pharmaceuticals Sciences, 64: 789-792.

Khare, C.P. 2007. Indian medicinal plants (An illustrated dictionary), Springer Science, New York, USA. Kokate, C.K. 2001.Pharmacognosy.16th Edn., NiraliPrakashan, Mumbai, India. Mackie and Mac Cartney. 1996. Practical Medical Microbiology. 14th Edn., Churchill Livingstone, New

York. McGaw, L.J., Jäger, A.K., and Van Staden, J. 2002. Variation in antibacterial activity of Schotiaspecies.

South African Journal of Botany, 68:41-46. Nadkarni, A.K. 1982. K.M. Nadkarni’s Indian Materia Medica, Bombay Popular Prakashan Pvt. Ltd,,

Bombay, India. Oboh, G., Akindahunsi, A.A., Famutimi, R., and Adetuyi, F.C. 1998. Anti-microbial activity of saponin

extracts from two wild yams (Dioscorea spp.). Nig J BiochemMol Biol., 13: 1998, 47-50. Patil, D.A. 2003. Flora of Dhule and NandurbarDistricts.Bishen Singh Mahendra Pal Singh, Dehradun. Rabe, T., Mullholland, D., and Van Staden, J. 2002.Isolation and identification of antibacterial

compounds from Vernonia colorata leaves.Journal of Ethnopharmacology, 80:91-94. Srivastava, A., Shukla Kumar, Y.N. 2000. Recent development in plant derived antimicrobial constituents

A review. J Med Arom Pl Sci., 20: 717-772. Thomas, S., Patil, A.G., and Naresh, C. 2008.Pharmacognostic Evaluation and physiochemical analysis

of Averrhoa carambola L. fruit. Journal of Herbal Medicine and Toxicology, 2 (2): 51-54. Trease GE, Evans WC. 1989. Pharmacognosy English Language Book Society. 13th Edn., Bailliere

Tindall, London. pp. 683-684.


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Enhancement of Tolerance of Faba Bean-nodulating

Rhizobial Isolates to High Temperature

Andarge Zelalem , Ameha Kebede and Manikandan Muthuswamy*

Department of Biology, Faculty of Natural and Computational Sciences, Haramaya University,

Haramaya , Dire Dawa, Ethiopia.

Received: 24 June, 2013; revised received: 20 August, 2013.

Abstract

A total of 50 isolates were obtained from nodules of Faba bean planted on soils collected from eastern Hararghe high lands (Gorogutu, Deder and Meta) and western Hararghe high lands (Chiro and Tulu). After isolation, the isolates were subjected to different presumptive tests to get pure culture. The result indicated that all isolates were gram negative and did not absorb Congo red from YEMA-CR media. All isolates were able to grow well within the temperature range of 20-35°C. 45 (90%) of the isolates grew at the temperature of 15°C, whereas 12 (24%) of the isolates grew at the temperature of 40°C. None of the isolates grew at the temperature of 4°C. 11 (22%), were able to grew at the temperature of 10°C. The ten selected isolates were subjected to chemical mutagenesis with sodium azide and hydroxyl amine hydrochloride, after the mutagenesis only eight isolates were survived. The survived mutant isolates were exposed to high temperature tolerance test. The mutant isolates such as HUFBR50M1 and HUFBR 23 M5 treated with sodium azide and hydroxyl amine hydrochloride respectively showed highest temperature tolerance that is 50°C. In addition to this mutant isolates were performed very well in symbiotic effectiveness then compared with respective parent’s isolates or wild types isolates. Therefore, it can be concluded that the mutagenic agent such as sodium azide and hydroxyl amine hydrochloride is a tool to enhance the Faba bean - nodulating rhizobial isolates to higher temperature tolerance. Key Words: Chemical mutagen, Rhizobial isolates, Temperature tolerance, Sodium azide, Hydroxyl Amine Hydrochloride.

Introduction In agriculture, leguminous biological nitrogen fixation is used to improve infertile soils, especially those affected by salinity (Brockwell et al., 1995). Rhizobial strains are very sensitive to soil environmental factors like high salt, water potential, pH, and temperature stresses, which affect their dinitrogen fixation capacity and hence the productivity of legumes (Abdelmoumen et al., 1999).

Leguminous plants can obtain most of nitrogen they need from the vast supply of gaseous nitrogen in the air by working symbiotically with special bacteria (rhizobia) in nodules on their roots. The behavior of some nitrogen-fixing systems under severe environmental conditions such as salt stress, drought stress, acidity, alkalinity, nutrient deficiency, fertilizers, heavy metals, and pesticides was reviewed (Zahran, 1999). These major stress factors suppress the growth and symbiotic characteristics of most rhizobia (Gálvez, 2005). Therefore, isolation of rhizobia strains capable to tolerate these stresses is essential for efficient nitrogen fixation (Woldeyohannes et al., 2007).

Zelalem / Tolerance of Faba Bean-nodulating Rhizobial Isolates to High Temperature


42

Rhizobia are mesophiles and most have a poor growth at temperature below 10OC or above 37°C (Graham, 1992). Although response to temperature is strains dependent, rhizobia are found to tolerate between 4 to 42. 4°C. However, growth at 4OC is rare, and only R. meliloti

can grow at 42.5°C. R.leguminosarum isolates from lentil plants in Southern Nile Valley of Egypt were tolerant to 35°C to 40°C inducing less effective symbiosis with their legume host (Moawad and Beck., 1991). For most rhizobia, the optimum temperature range for growth in culture is 28°C to 31°C. (Bordeleau and Prevost., 1994). Changes in temperature strongly affect bacterial infection and N2 fixation in several legume species (Kishinevsky et al., 1992). Nodulation and symbiotic nitrogen fixation depend on the nodulating strain in addition to plant cultivars. Elevated temperature may delay nodule initiation and development, and interfere with nodule structure and functioning in temperate legumes, whereas nitrogen fixation efficiency is mainly affected in tropical legumes. Furthermore, temperature changes affect the competitive ability of Rhizobium strains (Bordeleau and Prevost, 1994). High soil temperature in tropical and subtropical areas is a major problem for biological nitrogen fixation of legume crops. Maximum soil temperatures in the tropics regularly exceed 40°C at 5cm and 50°C at 1cm depth (Eaglesham and Ayanaba, 1984). Because high temperatures decrease rhizobial survival and establishment in tropical soils, repeated inoculation of grain legumes and higher rate of inoculation may frequently be needed (Thies et al., 1991). Therefore, this research was initiated to enhance the tolerance to high temperature of Faba bean- nodulating rhizobial isolates through chemical mutagenesis.

Materials and Methods Sample Collection

A total of 50 soil samples were collected from five major Faba bean growing districts of west and east Hararghe highlands, Ethiopia. From each district 10 farm lands were selected, and from each farm land bulk samples from 10-15 cm depth were pooled, merged and collected in alcohol sterilized plastic bags. The collected samples were then transported to Haramaya University greenhouse for pot experiment. Upon arrival the soil samples were used for isolation of indigenous rhizobia by the host trap method (Somasegaran and Hoben, 1994).

Collection of Root Nodules

Five seeds of Vicia faba (Faba bean), Gachena variety, which were obtained from Haramaya University research centre, were sown for each bulk sample in a sterile plastic pot at Haramaya University greenhouse. The pots were watered with distilled water every three days for 45 days. Then after 45 days the plants were uprooted from the pots and intact, pink, multi-lobed, and large nodules of plant were separated from the taproot with a portion of the root attached to the nodule and transported to the lab using a vial containing silica gel. The nodules were crushed with sterile glass rods in a large drop of water. The crushed nodule suspension was streaked on YEMA plates that contained Congo red. Then the plates were inverted and incubated at 28OC for 3- 5 days (Lupwayi and Haque, 1994).

Presumptive tests were done by re-culturing the primary isolates into YEMA containing CR, peptone glucose agar (PGA), and acid alkaline production on BTB. Plates were examined for growth and single colonies were picked up and periodically purified by re-streaking on new YEMA plates. Pure isolates were then preserved on YEMA slants containing 0.3% (w/v) CaCO3 and stored at 4OC (Vincent, 1970). Finally ten highly performed isolates were subjected to chemical mutagenesis.

Chemical Mutagenesis

Hydroxylamine hydrochloride and sodium azide were used as chemical mutagenic agents to induce mutation in ten selected Rhizobial isolates as described by O’Connel et al. (1990). Late- exponential phase cultures (2x108CFUml-1) were used in all mutagenesis experiments. The cells of rhizobial isolates were first pelleted in an eppendorf microcentrifuge, washed once with phosphate buffered saline (0.876gm NaCl, 0.522gm K2HPO4, 0.136gm KH2PO4 per 100ml), and re-suspended to the original volume in phosphate- buffered saline solution. From the stock



43

solution of each mutagenic substance (1.17g/ml), 0.0, 100, 200 and 300µl was added into each ml of rhizobial suspension. After mixing with a vortex, the cells were incubated at room temperature for 60 minutes. The mixture was then diluted and spread on TYEA medium without salt. The surviving colonies were transferred to other fresh TY slant media for preservation and further subjected to temperature tolerance test. The mutants were selected based on their survival conditions. Symbiotic Effectiveness of Mutants on Sterilized Sand

In the determination of symbiotic effectiveness of mutants, the parameters such as number of nodules, nodule color, nodule dry weight and shoot dry weight were determined. A well washed, sulfuric acid immersed and autoclaved sand soil was filled into surface sterilized plastic pots (Subba Rao., 1988). Surface sterilized (dipped into 95% ethanol and 35% H2O2 for 3 minutes) seeds of faba bean were planted (5 per pot). After a week, they were thinned to three and inoculated with 3 day’s old 1ml of YEM broth culture (about 10

8 to 109 cells). The experiment was carried out in Haramaya Univeristy Horticulture laboratory using

growth pouch and was laid out in complete randomized design (CRD) and replicated three times with two controls; the negative control (without N and Rhizobium) and the positive control (with 70 mg N liter-1 of distilled water). N was given as 0.05% KNO3 (w/v) by applying 120 ml during inoculation and 21 days later (Singleton and Tavares, 1986). All the pots were irrigated every day with distilled water and fertilized with quarter strength of Broughton and Dilworth N-free nutrient solution week-1 as described by Somasegaran and Hoben (1994). Forty five days after planting, screening was made using the parameters such as nodule number plant-1, nodule color, nodule dry weight (mg plant-1), shoots dry weight (g plant-1) and symbiotic effectiveness (%) base. Nodule dry weight and shoot dry weights were determined by drying nodules and shoots in air and oven at 70OC to constant weight and reported as mg and g plant-1, respectively. The percent symbiotic effectiveness (SE) of the isolates was computed using the formula (Beck et al., 1993):

SE (%) = X 100

Finally, the symbiotic effectiveness (SE) values of the isolates was rated as highly effective (>80%), effective (50-80%), less effective (35-50%), and ineffective for SE < 35% (Beck et al., 1993). Temperature Tolerance

The ability of bacterial strains to proliferate at varying temperatures was assessed on YEMA plates incubated at the temperatures of 4, 10, 15, 20, 25, 30, 35 and 40°C (Lupiwayi and Haque., 1994). Growth qualitatively recorded as (+) for growth and (-) for no growth. The ability of mutants at extreme temperatures was tested on TY medium incubated at the high temperatures of 40°C to 60°C at 5°C interval.


Isolation and presumptive Identification of the Isolates A total of fifty isolates were obtained from the nodules of faba bean plants grown at Haramaya University greenhouse. All isolates were found to be gram negative and did not absorb Congo red from YEMA-CR media. None of the isolates in this study showed growth on peptone-glucose agar (PGA). Failure of these isolates to absorb Congo red from YEMA-CR media and to grow on peptone glucose agar media presumptively indicated that they were root nodule bacteria (Lupway and Haque., 1994). All isolates turned Yeast Extract Mannitol Agar containing bromothymol blue (YEMA-BTB) into deep and moderate yellow color after the incubation of 3-5 days at 28°C. This showed that all were fast growers and acid producers (Table 1). The fact that almost all isolates displayed acid production on YEMA – BTB medium



44

confirms the characteristics of fast growing rhizobia (Somasagaran and Hoben 1994). Similarly, a study made by Aynabeba Adamu et al. (2001) on faba bean rhizobia isolated from Northern Shoa confirmed that all faba bean nodulating rhizobia were acid producing.

Temperature Tolerance of Wild Isolates

The ability of the isolates to grow at different incubation temperature was tested, all isolates were able to grow well within the temperature range of 20-30OC. The result is within the range of T max: 32.5-34.5°C reported for R.leguminosarum var. viceae HAMBI499, HAMBI 1125, and MPI 6001 isolated from faba bean and field pea from USA, UK, and the Netherlands (Lindstrom and Lehtomaki., 1988). 45 (90%) of the isolates grew at the temperature of 15°C, whereas 12 (24%) of the isolates grew at the temperature of 40°C. Among the 50 isolates none of the isolates grew at the temperature of 4°C. 11 (22%) isolates such as HUFBR11 and HUBFR18 (Tullo district), HUFBR22, HUFBR24, HUFBR26, and HUFBR29 (Gorgotu District); HUFBR35 (Deder District) and HUFBR44, HUFBR46, HUFBR48 and HUFBR50 (Meta District) were able to grow at the temperature of 10°C (Table 1). These isolates can be used as bio inoculants in very low temperature areas. Increased temperature optima of some isolates may be beneficial for the application of temperature stress condition. Thus, some of the isolates can overcome high soil temperature, which is one of the major problems for biological nitrogen fixation in tropical and subtropical areas (Michiel et al., 1994). Finally 10 highly performed isolates were selected and subjected to mutagenesis and high temperature tolerance test. (The naming of isolates, HU= Haramaya University, FB= Faba bean, R= Rhizobium, Number denotes = Number of Isolates, M= Mutataion, M1, 2, 3, 4, 5,6,7,8 = Number of mutated isolates.) Temperature Tolerance of Mutant Isolates

As shown in the table 2, 2 (20%) of mutants HUFBR50M1 and HUFBR23M5 have shown growth at the temperature of 50°C. 8 (80%) of the mutants have shown temperature sensitivity after 40°C like that of the wild isolates. Most parental isolates of rhizobia showed growth within a temperature range of 25°C – 40°C, hence most of them were mesophiles as described by (Pelczar et al., 1983). According to Brock et al. (1984) thermophiles grow at above 45°C – 50°C, so the mutants which grew at the temperature of 45°C and 50°C were thermphiles which were genetically modified. In agreement with this result Bolatito (2011) found Rhizobium species CWP G34A mutant (MU70) that could grow at 60°C for 24hrs. With regard to this, the mutagen (EMS) changed the gene(s) in the variant MU70 to support its survival and growth at 60°C. The change appeared to have endowed the mutant with ability to synthesize into its membrane, certain substances, possibly lipids rich in saturated fatty acids that enable thermophiles to live at high temperatures (Prescott et al., 2005). Similarly, in the current study two chemical mutagens sodium azide and Hydroxyl amine hydrochloride changed the gene(s) of HUFBR50 and HUFBR23 isolates, respectively which enables their mutants to grow at the temperature of 50°C. Furthermore, Kulkarni and Nautiyal (1999) described that out of 2500 rhizobial strains 405 strains were selected that had similar growth patterns after 72hrs on YEM plates incubated at 30 and 45°C. The second screening resulted in 24 tolerant strains which were able to grow at 47. 5°C. In this study no mutants were able to grow at low temperature. Contrary to this study, Beringer et al. (1977) have found two mutants of Rhizobium leguminosarum which were temperature sensitive and they were ineffective at 26°C but were effective at 13°C. The mutant isolates such as HUFBR50M1 and HUFBR 23 M5 treated with sodium azide and hydroxyl amine hydrochloride respectively showed highest temperature tolerance that is 50 °C.

Table 1: Growth of the 50 selected wild rhizobial isolates at different temperature levels



45

Isolates Temperature (°C)

4 10 15 20 25 30 35 40 HUFBR1 - - + + + + + - HUFBR2 - - + + + + + - HUFBR3 - - + + + + + - HUFBR4 - - + + + + + - HUFBR5 - - + + + + + + HUFBR6 - - - + + + + + HUFBR7 - - + + + + + + HUFBR8 - - + + + + + - HUFBR9 - - + + + + + + HUFBR10 - - + + + + + - HUFBR11 - + + + + + + - HUFBR12 - - + + + + + - HUFBR13 - - - + + + + - HUFBR14 - - + + + + + - HUFBR15 - - + + + + + - HUFBR16 - - + + + + + - HUFBR17 - - - + + + + + HUFBR18 - + - + + + + - HUFBR19 - - + + + + + - HUFBR20 - - + + + + + - HUFBR21 - - + + + + + - HUFBR22 - + + + + + + - HUFBR23 - - + + + + + - HUFBR24 - + + + + + + - HUFBR25 - - + + + + + - HUFBR26 - + + + + + + + HUFBR27 - - + + + + + + HUFBR28 - - + + + + + + HUFBR29 - + + + + + + - HUFBR30 - - + + + + + - HUFBR31 - - + + + + + + HUFBR32 - - + + + + + + HUFBR33 - - + + + + + - HUFBR34 - - + + + + + + HUFBR35 - + + + + + + - HUFBR36 - - + + + + + - HUFBR37 - - - + + + + - HUFBR38 - - + + + + + - HUFBR39 - - + + + + + + HUFBR40 - - + + + + + - HUFBR41 - - + + + + + - HUFBR42 - - + + + + + - HUFBR43 - - + + + + + - HUFBR44 - + + + + + + - HUFBR45 - - + + + + + - HUFBR46 - + + + + + + - HUFBR47 - - + + + + + - HUFBR48 - + + + + + + - HUFBR49 - - + + + + + - HUFBR50 - + + + + + + -

(+) is for growth and (-) is for no growth.

Table 2: Effect of temperature on the growth of selected mutant isolates



46

Mutagen Mutant code Temperature (°C)

4 5 40 45 50 55 60 Sodium azide

HUFBR50M1 - - + + + - - HUFBR31M2 - - - - - - - HUFBR12M3 - - - - - - -

Hydroxylamine hydrochloride

HUFBR12M4 - - - - - - - HUFBR23M5 - - + + + - - HUFBR39M6 - - - - - - - HUFBR50M7 - - - - - - - HUFBR18M8 - - - - - - -

Symbiotic Effectiveness of Rhizobium Mutated with Sodium Azide

The data indicated (Table 3) that all mutants were able to form nodules upon inoculation. Faba bean plants inoculated with mutant HUFBR31M2 produced a maximum nodule number of 180 per plant. But the same isolate wild type produced only 113 root nodules. This isolate showed a statistically significant difference from other mutants at (P <0.05). The minimum nodule number (45/plant) was produced by plant inoculated with mutant HUFBR23M5. The mutagenic chemical sodium azide treated isolate showed minimum nodule number of 113%, even though this number was higher than the wild type isolates. Similar to this result William (1981) reported that some mutants of cow pea Rhizobium produced more nodule numbers than plants inoculated with the wild type isolates. Mutants showed variation in nodule dry weight. Plants inoculated with HUFBR12M3 showed significant difference than other isolates at (P< 0.05). The maximum nodule dry weight was produced by plants inoculated with mutant HUFBR12M3 (133mg/plant). The maximum nodule dry weight of the plants inoculated with mutant was more than the maximum nodule dry weight of plants inoculated with parental type Rhizobium isolate HUFBR12 (94.4 mg/plant). Therefore, sodium azide may be a potential mutagenic agent to induce the nodulation activity in Faba bean nodulating Rhizobium. Table 3: Effect of chemical mutagen sodium azide and hydroxyl amine hydrochloride on nodulation and symbiotic effectiveness of Faba bean rhizobium isolates.

Mutagen Mutant

Code

Nodule

number

mean ± Std

Dev

NDW

(mg/pl)

mean ± Std

Dev

SDW (g/pl)

mean ± Std

Dev

Nodule

Color

SE

(%)

Sodium Azide

HUFBR50M1 139±10b 85±7.9b 1.79±0.78b 2 79.5

HUFBR31M2 180±13a 63.3±1.5cd 1.61±0.1bcd 2 71.6

HUFBR12M3 113±11cd 133±2.6a 0.58±0.03g 1 25.8

Hydroxyl amine Hydro chloride

HUFBR12M4 102±36d 58.3±3.5cd 0.44±0.05g 1 19.6

H HUFBR23M5 45±22e 49.3±21.5ed 1.19±0.08def 2 2.9

HUFBR39M6 65±7e 48±6.6ed 1.1±0.2ef 1 48.9

HUFBR50M7 123±7bcd 72.3±4.9bc 2±0.15ab 4 88.9

HUFBR18M8 52±3e 55.7±24cd 1.2±0.2efd 2 53.3

In this study only one mutant treated with sodium azide (HUFBR50M7) showed higher shoot dry weight as compared to its parental type isolate of HUFBR50, but all other mutants recorded lower shoot dry weight than plants inoculated with its parental type. In this regard,



47

Sharma and Yadav (2012) reported that shoot dry weight of pigeon pea plants infected with PRODH- mutants of Rhizobium sp. (Cajanus) was significantly lower for all mutants than the plants inoculated with parental strain.

Symbiotic Effectiveness of Rhizobium Mutated with Hydroxyl Amine Hydrochloride

Only five isolates showed the nodulation among the selected isolates treated with hydroxyl amine hydrochloride. The mutant isolate (HUFBR50 M7) showed maximum nodule number, but the value was lesser than the mutant treated with sodium azide as well as the respective wild type (Table 3).The overall correlation in the sand experiment revealed that nodule number was associated positively and significantly (r = 0.63, P < 0.0001) with nodule dry weight. Shoot dry weight showed a positive correlation with nodule number at (r = 0.1, p > 0.05). Similar to this finding, Tejera et al. (2005) showed a positive and significant correlation of SDW with nodule numbers upon mutant inoculation on Phaseolus vulgaris.

The mutants were also evaluated by nodule color examination scaled visually from 1 to 4 (Table 2). According to the scale 2- 4, i.e., pink to dark red colored nodules of plants displayed positive symbiosis (N fixation) while white nodules showed that poor nitrogen fixation. Seven (70%) of mutants displayed pink to dark red colored nodules hence, these mutants showed a positive symbiosis. Consequently, shoot dry weight accumulation of the treated plants in reference to N supplied positive control is used to evaluate the symbiotic effectiveness (SE) of the mutants. Accordingly, 10% of mutant HUFBR50M7 was highly effective, 60% of mutants (HUFBR50M1, HUFBR31M2, HUFBR23M5 and HUFBR18M8) were effective.

References Abdel-Wahab, S.M., El-Mokadem, M.T., Helemish, F.A., and Abou El-Nour, M.M. 1991. The symbiotic

performance of Bradyrhizobium japonicum under stress of salinized irrigation water. Ain Shams Sci Bull., 28:469–488.

Beck, D.P., Materon, L.A., and Afandi, F. 1993. Practical Rhizobium-legume technology manual, Technical Manual No: 19. ICARDA, Aleppo, Syria.

Beringer, J.E., Johnston, A.W.B., and Wells, B. 1977. The isolation of conditional ineffective mutants of Rhizobium leguminosarum. Journal of General Microbiology, 339-343.

Bolatito, E.B., Babatunde, A.O., and Helen, E. 2011. Mutational search for high temperature (60°C) tolerant variant of Rhizobium species CWP G34A, Advances in Bioscience and Biotechnology, 255-262.

Bordeleau, L.M., and Prevost, D.1994. Nodulation and nitrogen fixation in extreme environments. Plant Soil, 161:115-125.

Brock, T.D., Smith, D.W., and Madigan, M.T. 1984. Biology of microorganisms. 4th Edition, Prentice-Hall International, Inc., London. pp. 244 , 310.

Brockwell, J., Bottomly, P.J., and Thies, J.A. 1995. Manipulation of rhizobia microflora for improving legume productivity and soil fertility. A critical assessment. Plant Soil, 174: 143-180.

Eaglesham, A. R. T., and Ayanaba, A. 1984. Tropical stress ecology of rizobia, root nodulation and legume nitrogen fixation. In: Root Nodulation and Legume Nitrogen Fixation. Edited by: N.S Subba Rao. Oxford IBH publishing, New Delhi. pp. 1-35.

Gálvez, M.D. 2005. Nodule metabolism in Pisum sativum L. in response to water stress: carbon/nitrogen interactions and the possible molecules involved in the modulation of the response, Ph.D. thesis, Public University of Navarre.

Graham, P. H. 1992. Stress tolerance in Rhizobium and Bradyrhizobium, and nodulation under an adverse soil conditions. Can. J. Microbiol. 38:475-484.

Kishinevsky, B. D., Sen, D., and Weaver, R. W. 1992. Effect of high root temperatures on Bradyrhizobium peanut symbiosis. Plant soil, 143:275-282.

Kulkarni, S., and Nautiyal, C.S. 1999. Characterization of high temperature-tolerant rhizobia isolated from Prosopis juliflora grown in alkaline soil. J Gen Appl Microbiol., 45: 213–220.

Lupwayi, N. Z., and Haque., I.1994. Legume-Rhizobium Technology Manual. Environmental Science Division, International Livestock Center for Africa Addis Ababa, Ethiopia. pp. 97.

Michiels, J., Verreth, C., and Vanderleyden, J.1994. Effects of temperature stress on bean nodulating Rhizobium strains. Appl. Environ. Microbiol, 60: 1206-1212.



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Moawad, H., and Beck. 1991. Some characteristics of Rhizobium leguminosarum isolate from uninoculated field-grown lentil. Soil Biol. Biochem., 23:917-925.

O’Connell, K. P., Araujo, R.S., and Handelsman, J. 1990. Exopolysaccharide deficient mutants of Rhizobium sp. strain CIAT899 inducechlorosis in common bean (Phaseolus vulgaris). Mol. Plant-Microbe Interact, 3: 424-428.

Pelczar, M.J, Reid, R.D., and Chan, E.C.S. 1983. Microbiology. 4th Edition, Tata McGraw-Hill Publishing Company, Delhi. pp. 111.

Sharma, P., and Yadav, A.S. 2012. Symbiotic characterization of mutants defective in proline dehydrogenase in Rhizobium sp. Cajanus under drought stress condition. European Journal of Experimental Biology, 2 (1):206-216.

Somasegaren, P., and Hoben., H.J. 1994. Hand book for rhizobia. Methods in legume Rhizobium

Technology. Springer Verlag, New York. pp. 1- 441. Subba Rao, N.S. 1988. Bio-fertilizers in agriculture. Oxford and IBH Publishing Co. Pvt. Ltd, New Delhi. Tejera, N. A., Campos, R., Sanjuan, J. and Lluch, C. 2005.Effect of sodium chloride on growth, nutrient

accumulation and nitrogen fixation of common bean plants in symbiosis with isogenic strains. Journal of Plant Nutrition, 28: 1907-1921.

Thies, J. E., Singleton, P. W., and Bohlool, B. B. 1991. Influences of the size of indigenous rhizobial populations on establishment and symbiotic performance of introduced rhizobia on field grain legumes. Appl. Environ. Microbiol., 57:19-28.

Vincent, J.M.1970. A Manual for the Practical Study of Root-Nodule Bacteria. IBP Handbook15. Blackwell Scientific Publications, Oxford. pp.164-190.

Woldeyohannes, W.H., Dasilva, M.C., and Gueye, M. 2007. Nodulation and Nitrogen Fixation of Stylosanthes hamata in Response to Induced Drought Stress. Arid Land Research and Management., 21: 157-163.

Zahran, H.H. 1999. Rhizobium–legume symbiosis and nitrogen fixation under severe conditions and in an arid climate. Microbiol Molecul Biol Rev., 63: 968-989.


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Isolation and Characterization of Pathogenic Bacteria

Infecting Cultured Koi Carp (Ciprinus carpio)

D. S. Nasaran and V.A.J. Huxley*

Biotech Research Laboratory, Department of Zoology, Thiru. Vi. Ka. Government Arts College,

Tiruvarur – 610 003, Tamilnadu, India.

Received: 3 August, 2013; revised received 31 August, 2013.

Abstract

Present study determines the causative organism of ornamental koi carp Ciprinus carpio bacterial disease; the study also isolated the bacteria and their experimental transmission studies were carried out using standard techniques. The results of biochemical and 16S rRNA studies confirmed that the pathogen which is responsible for these infections are Aeromonas hydrophilla with 1038 nucleotides. The phylogenetic tree clearly displayed that this strain has some close relatives with the earlier strains of Aeromonas jandaei and Aeromonas hydrophila sub sp dhakensis strain. Key words: Koi carp, Ciprinus carpio, Aeromonas hydrophilla, pathogenesis.

Introduction Infectious diseases are the major problems in ornamental fish aquaculture, causing heavy loss to the fish farmers. The recent expansion of intensive aquaculture practices has led to high interest in understanding the various fish diseases. The range scale settings of aquatic animal husbandry have resulted in an increased antibiotic resistant bacterium potentially pathogenic to fish and related environment (Alcaide et al., 2005). So, the prevention, diagnosis and treatment may be given more priority than others. It is widely demonstrated that the occurrence of diseases in fish farm is due to several factors concerning with the rearing methods, environmental conditions and processes in intensive aquaculture field trials such as handling, crowding, transport, grading and poor water quality lead to more stress. Consequently, cultivated fish can become more susceptible not only to pathogenic but also to opportunistic bacteria. Diseases in intensive freshwater aquaculture have assumed greater importance in India due to economic loss observed in recent years. In this study, the bacterial strains were isolated from diseased fishes, the experimental transmission studies and median lethal dose studies were carried out in cultured Koi carp C. carpio, the real causative organism was characterized with various methods including biochemical/physiological tests and 16s rRNA markers.


Collection of Experimental Fish

A total number of 500 apparently healthy koi carps Ciprinus carpio were collected from a private fish farm (Vijai meen pannai) near Thiruvarur, Tamilnadu. The average body weight of the fish was 10 ± 2g. They were transported alive to the laboratory with air bags and stored in one ton Fiber Reimposed Plastic (FRP) tanks with adequate aeration.

Nasaran and Huxley / Pathogenic Bacteria Infecting Cultured Koi Carp Ciprinus carpio


50

Water Quality Analysis

Analysis of water quality for temperature, pH, dissolved oxygen, free carbon dioxide, total alkalinity and ammonia was done at 15 days intervals, collecting samples from the experimental tanks between 09.00 and 10.00 hr. Water temperature was recorded using a digital thermometer, while pH was measured with a digital pH meter (LI-120, ELICO, India). The rest of the parameters like dissolved oxygen, free carbon dioxide, total alkalinity (CaCO3) and ammonia were determined following standard procedures (APHA 1998).

Isolation of Pathogenic Bacteria from the Infected Fish

The moribund and infected fishes were collected from ARL Aquarium and Aqua world fish hatcheries in Tiruvarur. The samples were brought to the lab in live conditions in aerated bags. Initial bacterial isolations were made in nutrient broth (HI-MEDIA, Mumbai) from the parts such as tail, infected body tissue, heart, liver, spleen and intestine. Subsequent purifications were carried out by streak plate method. To determine the pathogenic Aeromonas, a swab were taken from each isolates and streaked onto Aeromonas isolation specific medium plates and incubated overnight at 37oC. Procedure for the preparation of specific medium was briefly given as follows, Sheep blood (2ml) was collected aseptically, using sterile vacutainers with anticoagulant. Sterilized nutrient agar medium was cooled to luke warm condition. Sheep blood was added to this medium, mixed by swirling, plated onto sterile petriplates. This blood agar media plates were used for demonstration of hemolytic activity, following the method described by Chandran et al., (2002). The colony which was grown in selective agar plate was then transferred to Aeromonas isolation specific medium and was used for further studies.

Experimental Transmission and LD50 Analysis

The healthy experimental fishes were segregated from the stock and maintained at the rate of 10 fishes per group in to glass aquaria. The length and weight of fish were measured before starting the experiment. Prior to the infection experiments, random sampling was made to detect any external lesions or other perceptible symptoms so as to ensure that the fishes were free from diseases/ infections. Initially the bacterial slant culture was activated in nutrient broth and kept overnight at 30 ± 20C in shake culture condition in an orbital shaker at 80 ± 5 rpm. The bacteria was centrifuged at 4000 rpm for 15 min and washed twice in normal saline (NS). The pellet was serially diluted on NS and enumerated using pore plate method. The concentration of 103 to 108 cfu / ml of the bacterium per fish were taken in 0.1 ml saline and inoculated intramuscularly using a 1 ml tuberculin syringe (25 gauge) at the peritoneal cavity of the fishes. Parallel control groups received 0.1 ml of normal saline. The mortalities were noted upto 7th day of post injection. Characterization of Pathogenic Bacteria

Clinical signs were recorded and the fish were anaesthetised with 100 mg/l of tricaine methane sulfonate (MS222). The fish were killed by transecting the spinal cord behind the skull for postmortem examination and the gross lesions were recorded. The Gram staining and biochemical tests were followed after Mac Faddin (1981). Classification followed as per Baumann and Schubert (1984) and Colwel and Grimes (1984).

16S rRNA Characterization

Genomic DNA was extracted by Genome DNA isolation Kit (Biogeno, India). The PCR amplification was carried out in a DNA thermo cycler (Vector, Japan) with universal primers: 16F27 (5’ to AGAGTTTGATCCTGGCTCAG to 3’) and 16R1522 (5’ to

AAGGAGGTGATCCAGCCGCA to 3’). The method was used according to the manufacturer’s

instructions. The PCR conditions consisted of 35 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 2 min. Nucleotide sequencing was commercially serviced by Bangalore Geni (Banalore, India). The obtained 16S rDNA sequence was compared with those in the database and the phylogenic tree was made using Neighbor Joining tree, PHYLIP Version 3.5 (http://evolution.genetics. washington.edu/phylip.html).



51

Results Water Quality

The ranges of water quality parameters monitored over the experimental period were given in the table 1. Based on the results the water temperature 26 - 27°C, pH 7.26 - 7.81, dissolved oxygen 3.97- 4. 82 ppm, free carbon dioxide 0- 9.4 ppm, total alkalinity (CaCO3) 50 - 80 ppm and ammonia below 0.5 mg/lit were noted. The microbial load varied from 2.8 x102 to 2.8 x 103 CFU/ ml, increasing with the progress of the experiment.

Table 1: Hydrological parameters of rearing system.

Parameters Range

Day 1 Day 15 Day 30

Temperature (o C) 26 27 26 pH 7.26 7.52 7.81 DO (ppm) 3.97 4.60 4.82 Free carbon dioxide (ppm) 0 6.8 9.4 Total alkalinity (ppm) 50 80 80 Nitrite (mg/l) 0.25 0.28 0.25 Ammonia (mg/l) Below 0.5 Below 0.5 Below 0.5 Microbial Load (CFU/ ml) 2.8x102 2.8x103 3.4x102

Isolation of Pathogenic Bacteria from the Infected Fish

The initial evaluation of the virulence and pathogenicity of the isolates in healthy fishes indicated the following distinct clinical sign: erosions on the tail (tail erosions), reddish patches near the tail and skin regions. Initially seven different bacterial isolates were isolated from nutrient agar streak plate method. Among that only one (KBI-03) was grown in Aeromonas

specific media.

Table 2: Observations of mortality (%) and LD50 of experimental fish injected with different doses of A.

hydrophila.

Sl. No Dose Mortality

(%)

1 3.2 x 10-3 10 2 5.2 x 10-4 30 3 6.0 x 10-5 40 4 7.6 x 10-6 50 5 6.4 x 10-7 60 6 3.6 x 10-8 80 7 4.2 x 10-9 100

Each value is mean of three data

Experimental Transmission and LD50 Analysis

The results indicated that the higher dose of 4.2 x 109 CFU / fish, all the fishes died within 24 h of injection. However, the mortality was varying in the lower dilutions such as 3.2 x 103, 5.2 x 104, 6.0 x 105, 7.6 x 106, 6.4 x 107 and 3.6 x 108 produced 10, 30, 40, 50, 60 and 80% respectively (Table 2). So the LD50 value was 7.6 x 106 CFU / fish. The results are diagrammatically depicted in Figure 1. Characterization of Pathogenic Bacteria

Biochemical Characterization

The biochemical characterization of isolated pathogen Aeromonas (KBI-03) is given in Table 3.



52

Table 3: The biochemical characterization of isolated fish pathogen.

Test/characteristic features KBI - 03

Gram staining negative

Shape rod

Motility +

Growth on TCBS agar -

Sensitivity to 0/129 phosphate -

Luminescence -

indole +

Citrate -

Oxidase production +

Catalse production +

Oxidative-fermentive test +

Acid/gas production:

Glucose +

Sucrose +

Mannitol +

Maltose -

Xylose -

Nitrate reduction

Methyl red Variable

Voges-Proskauer -

Indole production +

Hydrogen sulfide production -

ONPG hydrolysis -

Decarboxylase of:

Arginine -

Lysine -

Ornithine +

Growth in:

4ºC - 40ºC +

Growth in peptone with NaCl

0% -

0.5% -

1% +

3% +

6% -

8% -

10% -

Production of exo-cellular enzymes:

Amylase +

Caesinase +

Gelatinase +

Chitinase -

Urease -



53

16S rRNA characterization Isolated pathogen was confirmed with 16s rRNA sequencing. Based on the results, the amplicons were noted approximately 54.72 positions and also confirms the sequences with 1038 nucleotides. The phylogenetic tree clearly displayed that these strain has some close relatives with the earlier strains of Aeromonas jandaei and Aeromonas hydrophila sub sp dhakensis strain. The results are given in Figure 2.

TTTCAGCGAGGAGGGAAAGGTCAGTAAGCTATTTCTGCTGACTGTGACGTTACTCGCAGAAGAA

GCACCG

GCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGC

GTAAAG

CGCACGCAGGCGGTTGGATAAGTTAGATGTGAAAGCCCCGGGCTCAACCTGGGAATTGCATTTA

AAACTG

TCCAGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCT

GGAGGA

ATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGC

AAACAG

GATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGATTTGGAGGCTGTGTCCTTGAGACG

TGGCTT

CCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAA

TTGACG

GGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGC

CTTGAC

ATGTCTGGAATCCTGCAGAGATGCGGGAGTGCCTTCGGGAATCAGAACACAGGTGCTGCATGGC

TGTCGT

CAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTGTCCTTTGTTGCCA

GCACG

TAATGGTGGGAACTCAAGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACGTCAA

GTCATCA

TGGCCCTTACGGCCAGGGCTACACACGTGCTACAATGGCGCGTACAGAGGGCTGCAAGCTAGCG

ATAGTG

AGCGAATCCCAAAAAGCGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGG

AATCGC

TAGTAATCGCAAATCAGAATGTTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCA

CACCAT

GGGAGTGGGTTGCACCAGAAGTAGATAGCTTAACCTTCGGGAGGGCGTTACCACGGTA

Figure 1: The nucleotide sequence 16S rRNA of pathogenic bacteria.

Discussion

Aeromonas hydrophila is a Gram-negative aerobic and facultative anaerobic, oxidase-positive motile bacterium that inhabits aquatic environments and the gastrointestinal tract of healthy fish. It also commonly occurs in foods like fish, milk, red meats and poultry (Roberts, 1993). It causes disease and mortality mainly in freshwater fish but sometimes in marine fish (Aoki 1999). The bacteria also infect human beings and cause lesions ranging from gastroenteritis to septicaemia (Sha et al., 2002). A. hydrophila possesses many factors related to its virulence, such as extracellular products including aerolysins, α- and β-haemolysins (Hirono and Aoki 1991), enterotoxins, proteases, haemagglutinins and adhesins (Sha et al., 2002).

In the present study cleared that the causative organisms for such infections in Koi is Aeromonas hydrophilla. It is proved by 16S rRNA characterization and experimental transmission studies which produced same symptoms. The external pathology produced by



54

A.hydrophilla includes haemorrhages at the base of the fins, around the vent, gills and inside the mouth. Aeromonas is an important bacterial pathogen as it infects both fish and humans (Santos et al., 1988). A. hydrophila infection in fish culture results in decrease in production and economic losses. Uses of antibiotics or chemicals to control A. hydrophila infections have an adverse effect on the environment and also to the host by developing resistant strains of bacteria (Thayumanavan et al., 2003). A. hydrophila is an opportunistic as well as primary pathogen of variety of aquatic and terrestrial animals including man. Diseases caused by A. hydrophila

(hemorrhagic septicemia, fin-tail rot, and epizootic ulcerative syndrome) have a major impact in aquaculture (Koeypudsa and Jongjareanjai, 2010). They also reported the opportunistic A.

hydrophila infections in catfish after the temperature stress. Temperature is also known to have strong influences on enzyme reaction, growth efficiency, reproduction and immune response in fish (Suzuki et al., 2003). However, the pathogenesis of the disease has not been fully elucidated in fish. Several studies have assessed the virulence virulence (LD50) of different strains of A. hydrophila (Khalil and Mansour, 1997), but the environmental stressors involved in the onset of disease, the possible route of entry of the bacteria (Roberts 1993), their distribution within the fish and the evolution of the pathological process (Grizzle and Kiryu, 1993), have rarely been assessed. On the other hand, the lethal effects of the extracellular products of A.

hydrophila have been reported in several species of fish without the description of any histological lesions (Khalil and Mansour, 1997).

Figure 2: The phylogenitic tree of 16S rRNA of pathogenic bacteria.



55

Acknowledgements

The authors are thankful to the Principal, Thiru. Vi. Ka. Govt. Arts College, Tiruvarur for his support and encouragement. These data are the preliminary doctoral work of D. S. Nasaran.

References

Alcaide, E., Blasco, M. D., and Esteve, C. 2005. Occurrence of drug-resistant bacteria in two European

eel farms. Appl. Environ. Microbiol., 71: 3348–3350.

Aoki, T. 1999. Motile Aeromonads (Aeromonas hydrophila). In: Fish Diseases and Disorders. Vol 3:

Viral, Bacterial and Fungal Infections. Edited by: P. Woo and D. Bruno. Wallingford, CABI

Publishing. pp. 427-453.

APHA. 1998. Standard Methods for the Examination of Water and Wastewater, 20th Edn. Edited by: L.S.

Clesceri, A.E. Greenberg and A.D. Eaton. American Public Health Association/American Water

Works Association/Water Environment Federation, Washington, DC, USA.

Baumann, P.S., and Schubert, R.H.W. 1984. The family II. Vibrionaceae veron. In: Bergey’s Manual of

Systematic Bacteriology. Vol.1. Edited by: N.R.Krieg. Williams and Wilkins. pp.515-538.

Chandran, M.R., Aruna, B.V., Logambal, S.M., and Dinakaran M.R. 2002. Immunisation of Indian major

carps against Aeromonas hydrophila by intraperitoneal injection. Fish and Shellfish Immunology,

13: 1-9.

Colwell, R. R., and Grimes, D. J. 1984.Vibrios in the environment. John Wiley and Sons, New York.

Grizzle, J. M., and Kiryu, Y. 1993. Histopathology of gill, liver and pancreas, and serumenzyme levels of

Channel catfish infected with Aeromonas hydrophila complex. Journal of Aquatic Animal Health,

5: 36-50.

Hirono, I., and Aoki, T. 1991. Nucleotide sequence and expression of an extracellular hemolysin gene of

Aeromonas hydrophila. Microbial Pathogenesis, 11: 189-197.

Khalil, A. H., and Mansour, E. H. 1997. Toxicity of crude extracellular products of Aeromonas

hydrophila in tilapia, Tilapia nilótica. Letters in Applied Microbiology, 25: 269-272.

Koeypudsa, W., and Jongjareanjai, M. 2010. Effect of WaterTemperature on hematology and Virulence

of Aeromonas hydrophila in Hybrid Catfish (Clariasgariepinus Burchell x C. macrocephalus

Gunther) The Thai Journal of Veterinary Medicine, 40: 179-186.

Mac Faddin, J. F. 1981. Biochemical tests for identification of medical" bacteria, 2nd Edition, Williams

and Wilkins, Baltimore. pp. 527.

Roberts, R. J. 1993. Motile aeromonad septicaemia. In: Bacteriology of Fish Disease. Edited by: V.

Inglis, R. Roberts, and N. Bromage. Blackwell Scientific Publications. pp. 143-155.

Santos, Y., Toranzo, A. E., Barja, J. L., Nieto, T. P., and Villa, T. G. 1988. Virulence properties and

enterotoxin production of Aeromonas strains isolated from fish. Infection and Immunity, 56: 3285-

3293.

Sha, J., Koslova, E., and Chopra, A. 2002. Role of various enterotoxins in Aeromonashydrophila-induced

gastroenteritis: generation of enterotoxin gene-deficient mutants andevaluation of their enterotoxic

activity. Infection and Immunity, 70: 1924-1935.

Suzuki, Y., Tasumi, S., Tsutsui, S., Okamoto, M., and Suetake, H. 2003. Molecular diversity of skin

mucus lectins in fish . Comp Biochem Physiol Biochem Mol Biol., 136: 723–730.

Thayumanavan, T., Vivekanandhan, G., Savithamani, K., Subashkumar, R., and Lakshmanaperumalsamy

P. 2003. Incidence of haemolysin-positive and drug-resistant Aeromonas hydrophila in freshly

caught finfish and prawn collected from major commercial fishes of coastal South India. FEMS

Immunology and Medical Microbiology, 36: 41-45.

*Corresponding author; Email address: [email protected] 57


www.jteb.webs.com

Role of Hormones in Differential Growth Responses of

Mung Bean Vigna radiata L. Wilczek

Seedlings under Water Stress

Satyajit Das and Rup Kumar Kar*

Plant Physiology and Biochemistry Laboratory, Department of Botany, Visva-Bharati,

Santiniketan - 731235, West Bengal, India

Received: 24 March, 2013; revised received: 2 September, 2013

Abstract Plants are predisposed to water deficit under natural condition and shoot and root growth is differentially responsive towards water stress as an adaptive response. Present investigation has been aimed to elucidate the hormonal basis for such discrimination using early stage seedlings of Vigna radiata as model system. Water stress simulated by exposing seedlings to PEG 6000 solutions (-0.5, -1.0 and -1.5MPa) induced growth inhibition except low level of stress (-0.5MPa) that promoted only root growth. Abscisic acid (ABA) application caused growth inhibition except lower concentrations (below 100 µM) that promoted root growth both under stressed and non-stress condition. Reverse responses occurred with the treatment with fluridone, ABA biosynthesis inhibitor. Ethylene application inhibited both root and shoot growth and treatment with cobalt chloride (CoCl2), ethylene biosynthesis inhibitor, could marginally improved growth rate either under stress or non-stress condition. Gibberellic acid (GA) promoted shoot growth but inhibited root growth, although treatment with paclobutrazole, GA biosynthesis inhibitor, inhibited both root and shoot growth. Neither ethylene nor GA appears to be involved in stress-induced growth responses of mung bean seedlings. Key words: Abscisic acid, ethylene, gibberellins, growth, Vigna radiata, water stress.

Introduction

Water stress (drought) is the major abiotic stress that limits crop productivity worldwide (Shao et al., 2009). Drought affects growth, yield, membrane integrity, pigment content, osmotic adjustment, water relations and photosynthetic activity of plants (Benjamin and Nielsen, 2006; Praba et al., 2009). The susceptibility towards drought stress depends on the degree of stress, different accompanying stress factors, plant species, and their developmental stages (Demirevska et al., 2009). Water stress induces changes in plant growth and physio-biochemical processes, such as changes in plant structure, growth rate, tissue osmotic potential and antioxidant defenses (Duan et al., 2007). Abscisic acid (ABA) accumulates in high concentration of plants experiencing drought and has been proposed to be involved in the regulation of differential root and shoot growth responses (Creelman et al., 1990; Munns and Sharp, 1993; Bacon et al., 1998). Root elongation at low water potential is considered to be an adaptive feature that promotes survival of plants under water limited conditions (Sharp and

Das and Kar / Growth of Mung Bean Seedlings under Water Stress


58

Davies, 1989; Spollen et al., 1993). In such condition, ABA accumulation is required for maintenance of primary root elongation in Zea mays seedling (Sabb et al., 1990; Sharp et al., 1994).

The mechanisms that determine the differential sensitivity of root-shoot growth to water stress are not well understood till date. The exact role of ABA accumulation in the maintenance of root elongation at low water potential is not much clear from the available reports. It has been demonstrated by several workers that the application of exogenous ABA can inhibit ethylene production (Gertman and Fuchs, 1972; Write, 1980; Tan and Thimann, 1989). ABA deficient mutant exhibits increased ethylene production from the shoot of Tomato plant (Tal et al., 1979). Wright (1980) suggested that endogenous ABA accumulation may limit ethylene production during water stress. Spollen et al. (2000) revealed that the role of endogenous ABA is to limit ethylene production in Zea mays and this interaction is involved in the effect of ABA status on root and shoot growth.

In the present study, the involvement of different hormones like ABA, Ethylene and GA in seedling growth of mung bean (Vigna radiata) under water stress condition induced by PEG 6000 has been investigated. The results indicate that endogenous ABA accumulated under water stress acts differentially to maintain primary root growth and inhibit shoot growth. Moreover, it also indicates that ethylene does not play any role in case of water stress-induced seedling growth responses. In non-stress (normal) condition, exogenous application of GA induces the shoot growth; however, it does not show any effect on shoot growth under water stress in Vigna

radiata.

Materials and Methods Seeds of Mung bean Vigna radiata (L.) Wilczek var. B1 were used as the experimental material. Seeds were first surface sterilized by 1% sodium hypochlorite solution followed by the twice rinsing in distilled water and finally incubated on the moistened Whatman No. 1 filter paper placed in a 9 cm diameter Petri dish and kept in a germinator at 30oC (±2˚C) for 20 h. After 20

h, the seedlings were transferred into transparent (for light treatment) and black coated (for dark treatment) plastic boxes containing Whatman No. 1 filter paper which was soaked with either distilled water (control) or test solutions under stress or non stress condition. Boxes were then kept under the same conditions mentioned above. In case of stress conditions PEG solutions of water potential -0.5 MPa (mild stress), -1.0 MPa (moderate stress), -1.5 MPa (severe stress) were used to simulate water stress condition. Different water potentials were adjusted by using particular concentrations of polyethylene glycol (PEG 6000) solution (Michel and Kaufman, 1973). The test solutions consisted of either hormones or their inhibitors. Growth of seedlings was observed by measuring root (radicle) and shoot (hypocotyl) length at one day intervals. Root growth was always observed under continuous dark condition while shoot growth was observed under continuous light condition. To establish the role of phytohormones on growth under mild water stress (-0.5 MPa PEG) and non stress condition, germinated seeds of Vigna radiata were treated under non stress condition with various concentrations of abscisic Acid (ABA; 1, 10, 100 and 1000 µM), fluridone, an ABA biosynthesis inhibitor (Flu; 1, 10 and 50 µM), ethylene (C2H4; 1, 10, 50, 100 µM), cobalt chloride, an ethylene synthesis inhibitor (CoCl2; 1, 10, 100, 1000 µM), gibberellic Acid (GA; 1, 10, 100 and 500 µM), paclobutrazol, a GA biosynthesis inhibitor (Pac; 1, 10, 50 and 80 µM). When the treatments with hormones and their inhibitors were combined with mild stress, optimum concentrations of hormones and their inhibitors were selected from range of concentration used in non stress condition. Combinations with stress were performed in case of fluridone (10 µM), CoCl2 (10 µM) and GA (100 µM). In the case of each experiment, axis growth was analysed using at least ten replicates and the average data were expressed in the form of figures. Data were statistically analysed and standard errors (SE) of the mean values were presented as vertical bars in the figures.



59

Results

Differential Growth Responses of Root (Radicle) and Shoot (Hypocotyl) under Stress

Germinated seeds of Vigna radiata were exposed to three levels of water stresses (-0.5, -1.0 and -1.5MPa) induced by PEG-6000 and the length of root (radicle) and shoot (hypocotyl) was measured separately at 1 day intervals. Results (Fig. 1) showed that more or less similar rate of inhibition of shoot growth occurred at all levels of stress and inhibition was almost total (Fig. 1B). However, root growth slightly increased than the control set only in case of mild water stress (-0.5 MPa) while other levels (moderate and severe) of water stress (-1.0 and 1.5 MPa) showed proportionate inhibition of root growth (Fig. 1A).

0

20

40

60

80

100

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ControlPEG -0.5MPaPEG -1MPaPEG-1.5MPa

Days of incubation

2 3

0

20

40

60

80

100

1 2 3

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Control

PEG -0.5MPa

PEG -1MPa

PEG-1.5MPa

B

Figure 1: Effect of three different levels of water stress on seedling growth of Vigna radiata. Germinated seeds were incubated in mild (ψ = -0.5 MPa), moderate (ψ = -1.0 MPa), and severe (ψ = -1.5 MPa) water stress conditions induced by PEG 6000 and root (A) and hypocotyls (B) length (mm) was measured at 1 day intervals. SE shown as vertical bars. Role of ABA on Root (Radicle) and Shoot (Hypocotyl) Growth of Non-stress and

Stress Grown Seedlings Germinated seeds of Vigna radiata were treated with ABA (1, 10, 100 and 1000 µM) under non-stress condition showed proportionate inhibition of shoot growth at all the concentrations used (Fig. 2B), but in case of root growth, lower concentrations (1 and 10 µM) promoted growth significantly; however, higher concentrations (beyond 100 µM) inhibited root growth (Fig. 2A).

0

20

40

60

80

100

1 2 3

Days of incubation

Le

ng

th (

mm

)

Control1µM10µM100µM1000µM

1 2 3

Days of incubation

control1µM10µM100µM1000µM

Figure 2: Role of ABA in seedling growth of Vigna radiata under stress and non-stress condition.

Germinated seeds were incubated in presence of different concentrations of ABA under non-stress

condition and root (A) and hypocotyl (B) length was measured at 1 day intervals.

A B

A

1



60

0

20

40

60

80

100

1 2 3

Days of incubation

Len

gth

(m

m)

Control PEG ABA Flu PEG+FluPEG+Flu+ABA

1 2 3

Days of incubation

Control PEG FluridonPEG + Fluridone

Figure 2 (Contd): Root (C) and hypocotyl (D) length was also measured under PEG-induced stress (-

0.5MPa) in presence of fluridone (10 µM) and also exogenous ABA (10 µM) added with Flu + PEG

along with ABA, fluridone and PEG independently. SE shown as vertical bars.

Germinated seeds incubated with fluridone (10 µM) showed severe inhibition of root growth compared to untreated seedlings in stress condition as well as non-stress condition, while independent treatment with mild water stress (-0.5 MPa) and ABA (10 µM) rather accelerated such growth (Fig. 2C). Application of exogenous ABA (10 µM) in the fluridone treated seedling under stress slightly increased the root growth (Fig. 2C). In case of shoot growth, however, fluridone (10 µM) treatment caused a promotion of shoot growth over control under non-stress condition (Fig. 2D). PEG-induced stress (-0.5 MPa) severely inhibited shoot growth and addition of fluridone somewhat improved growth under stress (Fig. 2D).

A

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Control1 µM 10 µM 50 µM 100 µM 1000 µM

B

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100

1 2 3

Days of incubation

Control1 µM 10 µM 50 µM 100 µM 1000 µM

Figure 3: Role of Ethylene in seedling growth of Vigna radiata under stress and non stress conditions. Germinated seeds were incubated in presence of different concentrations of ethylene (ethrel) under non-stress condition and root (A) and hypocotyl (B) length was measured at 1 day intervals.

C D



61

C

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1 2 3

Days of incubation

Len

gth

(m

m)

Control

CoCl2

PEG

CoCl2+PEG

1 2 3

Days of incubation

ControlCoCl2 PEGCoCl2 +PEG

Figure 3 (Contd): Root (C) and hypocotyl (D) length was also measured under PEG-induced stress (-0.5MPa) in presence of cobalt chloride (10 µM) along with cobalt chloride and PEG independently. SE shown as vertical bars.

A

0

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Le

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th (

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)

Control 1µM 10µM100µM500µM

B

1 2 3

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Control 1µM 10µM100µM500µM

Figure 4: Role of GA in seedling growth of Vigna radiata under stress and non-stress condition.

Germinated seeds were incubated in presence of different concentrations of GA under non-stress

condition and root (A) and hypocotyl (B) length was measured at 1 day intervals.

D



62

C

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D

1 2 3

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Figure 4 (Contd): Length of root (C) and hypocotyl was measured keeping the seedlings in different

concentration of paclobutrazol (GA biosynthesis inhibitor).

0

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1 2 3

Days of incubation

Le

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th (

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)

ControlGA PEGGA +PEG

1 2 3

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F

Figure 4 (Contd): Root (E) and hypocotyl (F) length was also measured under PEG-induced stress (-0.5MPa) in presence of GA (100 µM) along with GA and PEG independently. SE shown as vertical bars.

Role of Ethylene in Normal and Stress-grown Seedlings Seedling treated with ethrel (ethylene) (1, 10, 50 and 100 µM) in non-stress condition inhibited both root and shoot growth (Fig. 3A and B) and such inhibition was proportionate with the concentration reaching almost a complete inhibition of growth at 100 µM. Cobalt chloride (10 µM CoCl2), an ethylene biosynthesis inhibitor, neither exhibited any root growth promotion nor could overcome hypocotyl growth inhibition under water stress (Fig. 3C and D). However, it could slightly promote hypocotyl growth in non-stress condition. Role of Gibberellic Acid (GA) on the Growth of Seedlings under Non-stress and

Stress Conditions

GA treatment on the germinated seeds clearly promoted shoot growth (Fig. 4B), while inhibited root growth (Fig. 4A) under non-stress condition and such effects were concentration dependent. All concentrations of paclobutrazol (GA biosynthesis inhibitor) severely inhibited shoot growth (Fig. 4D), but in case of root growth the inhibitory effect was concentration dependent (Fig. 4C). When GA treatment was given in stress condition, some increase in root growth occurred over GA treatment alone (Fig. 4E); however, GA treatment did not show any improvement over stress-induced shoot growth inhibition (Fig. 4F).

E



63

Discussion

Water stress limits plant production and the performance of crop plants, more than any other environmental stresses (Shao et al., 2009). Growth is the most vulnerable process first affected by water stress, since the cell elongation or expansion is dependent on turgor which in turn depends on the water status. Tolerant plants can maintain their water potential either by lowering water loss via stomatal regulation or by increasing water uptake. The latter is possible by extensive root growth. Thus root growth often has been found to be maintained or enhanced by mild water stress as an adaptive response along with a suppression of shoot growth. In the present investigation with Vigna radiata seedlings, mild water stress condition (-0.5MPa) induced by PEG-6000 rather promoted root growth compared to the control set (Fig. 1A), whereas more lower water potentials (- 1.0 and -1.5MPa) caused the growth inhibition as usual in case of both roots and shoots, being more effective in shoots (Fig 1. A and B). ABA, a plant hormone, is well known for growth inhibition and mostly ascribed for stress-induced growth inhibition. As a positive response to stress, plant tissues are commonly found to accumulate ABA, which ultimately brings about stress responses including inhibition of growth. However, endogenous ABA was reported to act differentially to maintain primary root growth and to inhibit shoot growth of maize seedlings at low water potential (Saab et al., 1990). Sharp et al. (1994) confirmed that ABA accumulation is required for maize primary root elongation at low water potential. Our result with Vigna radiata is also in agreement with such observation suggesting a dual role of endogenous ABA in the growth responses of seedling to low water potential, i.e. promotion of root growth and inhibition of shoot growth. Such role of ABA was further supported by the observation that fluridone (ABA biosynthesis inhibitor) treatment resulted in severe inhibition of root growth and slight promotion in shoot growth under water stress condition (Fig 2. C and D). Sharp et al. (1994) demonstrated that application of exogenous ABA restored the internal ABA level in the apical 10 mm of fluridone treated maize roots and this was associated with a gradual increase in the rate of root elongation under stress grown seedling. In case of Vigna radiata also exogenous application of ABA (10 µM) somewhat overcame root growth inhibition caused by fluridone in stress grown seedlings (Fig. 2C). In contrast to the stressed plants, it is reported that exogenous ABA application inhibits both root and shoot growth in well watered plants (Sharp and LeNoble, 2002). However, in the present study with Vigna radiata seedlings, although higher concentration of exogenous ABA (100 µM) inhibited both root and shoot growth under normal condition (well watered), but lower concentration (10 µM) promoted root growth (Fig 2. A and B) under the same condition. Moreover, inhibition of root growth by fluridone further supports the role of ABA in root elongation under normal condition.

It was proposed that the role of endogenous ABA accumulation in the maintenance of maize primary root elongation at low water potential is to prevent the excess ethylene production that may otherwise inhibits root growth (Spollen et al., 2000). But the same may not be true for shoots where ABA itself inhibits growth. We have tested the involvement of ethylene in ABA induced differential growth response under water stress. Ethylene was found to be inhibitory for both root and shoot under normal condition (Fig. 3A and B). On the other hand, treatment with ethylene synthesis inhibitor (cobalt chloride, CoCl2) did not affect much either root or shoot growth (Fig. 3C and D) indicating minimum involvement of ethylene during normal seedling growth. Under PEG-induced stress also CoCl2 had no effect on root and shoot growth suggesting that at least ethylene is not involved in water stress-induced shoot growth inhibition in Vigna radiata seedlings.

Gibberellins (GA) are well known for their role in shoot elongation. Thus, GA has been reported to regulate hypocotyl elongation, both in light and in the dark, via the effects on cellular elongation (Cowling and Harberd, 1999). In mung bean seedlings also hypocotyl was sensitive to exogenous GA (Fig. 4B) and treatment with paclobutrazole, GA biosynthesis



64

inhibitor, totally inhibited hypocotyl growth under non-stress condition (Fig. 4D) supporting the role of GA in shoot growth. However, strong inhibition of hypocotyl growth by PEG-induced water stress is not due to suppression of GA synthesis as simultaneous GA application could not overcome such inhibition (Fig. 4F). Unlike shoot growth, involvement of GA in root growth of Vigna radiata seedlings is very complicated, since both GA and paclobutrazole treatment caused growth inhibition in roots (Fig. 4A and C). Under water stress when root growth was enhanced possibly because of accumulation of ABA, GA treatment prevented such growth enhancement. This may be due to well known antagonistic effect between GA and ABA.

From the above results, it can be concluded that endogenous ABA is primarily responsible for the differential growth responses of Vigna radiata seedlings under water stress. ABA is also playing a natural role in root elongation under normal condition. Ethylene appears to be not involved in stress-induced growth responses. GA, although plays definitive role in hypocotyl elongation under normal condition, is not involved in stress-induced growth suppression. Interestingly, GA has been found to retard the enhancement of root growth under water stress.

References

Bacon, M.A., Wilkinson, S., and Davies, W.J. 1998. pH - regulated leaf cell expansion in droughted plants is abscisic acid dependent. Plant Physiol., 118: 1507–151.

Benjamin, J.G., and Nielsen, D.C. 2006. Water deficit effects on root distribution of soybean, field pea and chickpea. Field Crops Res., 97: 248-253.

Cowling, J.R., and Harberd, P.N. 1999. Gibberellins control Arabidopsis hypocotyl growth via regulation of cellular elongation. J. Exp. Bot., 50: 1351-1357.

Creelman, R.A., Mason, H.S., Bensen, R.J., Boyer, J.S., and Mullet, J.E. 1990. Water deficit and abscisic acid cause differential inhibition of shoot versus root growth in soybean seedlings: Analysis of growth, sugar accumulation, and gene expression. Plant Physiol., 92: 205–214.

Demirevska, K., Zasheva, D., Dimitrov, R., Simova-Stoilova, L., Stamenova, M., and Feller, U. 2009. Drought stress effects on Rubisco in wheat: changes in the Rubisco large subunit. Acta Physiol. Plant., 31: 1129-1138.

Duan, B., Yang, Y., Lu, Y., Korpelainen, H., Berninger, F., and Li, C. 2007. Interactions between water deficit, ABA and provenances in Picea asperata. J. Exp. Bot., 58: 3025-3036.

Gertman, E., and Fuchs, Y. 1972. Effect of abscisic acid and its interaction with other plant hormones on ethylene production in two plant systems. Plant Physiol., 50: 194–195.

Michel, B.E., and Kaufmann, M.R. 1973. The osmotic potential of Polyethylene Glycol 6000. Plant Physiol, 51: 914-916.

Munns, R., and Sharp, R.E. 1993. Involvement of abscisic acid in controlling plant growth in soils of low water potential. Aust. J. Plant Physiol., 20: 425–437.

Praba, M.L., Cairns, J.E., Babu, R.C., and Lafitte, H.R. 2009. Identification of physiological traits underlying cultivar differences in drought tolerance in rice and wheat. J. Agron. Crop Sci., 195: 30-46.

Saab, I.N., Sharp, R.E., Pritchard, J., and Voetberg, G.S. 1990. Increased endogenous abscisic acid maintains primary root growth and inhibits shoot growth of Maize seedling at low water potentials. Plant Physiol., 93:1329-1336.

Sharp, R.E., and Davies, W.J. 1989. Regulation of growth and development of plants growing with a restricted supply of water. In: Plants under Stress. Edited by: H. G. Jones, T. L. Flowers and M. B. Jones. Cambridge University Press, Cambridge, UK. pp. 71–93.

Sharp, R.E., and LeNoble, M.E. 2002. ABA, ethylene and the control of shoot and root growth under water stress. J. Exp. Bot., 53: 33-37.

Sharp, R.E., Wu, Y., Voetberg, G.S., Saab, I.N., and LeNoble, M.E. 1994. Confirmation that abscisic acid accumulation is required for maize primary root elongation at low water potentials. J. Exp. Bot., 45:

1743–1751. Shao, H.B., Chu, L.Y., Jaleel, C.A., Manivannan, P., Panneerselvam, R., and Shao, M.A. 2009.

Understanding water deficit stress-induced changes in the basic metabolism of higher plants - biotechnologically and sustainably improving agriculture and the ecoenvironment in arid regions of the globe. Crit. Rev. Biotechnol., 29: 131-151.



65

Spollen, W.G., Sharp, R.E., Saab, I.N. and Wu, Y. 1993. Regulation of cell expansion in roots and shoots at low water potentials. In: Water Deficits: Plant Responses from Cell to Community. Edited by: J. A. C. Smith and H. Griffiths. BIOS Scientific Publishers, Oxford, pp. 37–52.

Spollen, W.G., LeNoble, M.E., Samuels, T.D., Bernstein, N., and Sharp, R.E. 2000. Abscisic acid accumulation maintains maize primary root elongation at low water potentials by restricting ethylene production. Plant Physiol., 122: 967–976.

Tal, M., Imber, D., Erez, A., and Epstein, E. 1979. Abnormal stomatal behavior and hormonal imbalance in flacca, a wilty mutant of tomato. V. Effect of abscisic acid on indoleacetic acid metabolism and ethylene evolution. Plant Physiol., 63: 1044–1048.

Tan, Z.Y., and Thimann, K.V. 1989. The roles of carbon dioxide and abscisic acid in the production of ethylene. Physiol. Plant., 75: 13–19.

Wright, S.T.C. 1980. The effect of plant growth regulator treatments on the levels of ethylene emanating from excised turgid and wilted wheat leaves. Planta, 148: 381–388.


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Effect of Chosen Immunostimulant Induced Immunological

Changes in Common Carp (Cyprinus carpio)

D. S. Nasaran and V.A.J. Huxley*

Biotech Research Laboratory, Department of Zoology, Thiru. Vi. Ka. Government Arts College,

Tiruvarur –610 003, TamilNadu, India

Received: 10 June, 2013; revised received: 15 September, 2013

Abstract In the present study, the effects of chosen immunostimulants such as levamisole, vitamin C, and potent seaweed extracts on immunological parameters of common carp, Cyprinus

carpio were studied. Blood samples were collected from each group after feeding. The leukocyte (WBC) (x103/mm3) amounts and differential blood counts (%) were determined. In comparison with the control group, WBC amounts were increased in the fish fed with levamisole (500mg/kg). Lymphocytes were decreased, while monocytes and neutrophils were increasing among the leukocyte cell types. The immunological parameters like phagocytosis (PI), nitro blue tetrosolium assay (NBT), lysosymal assay and agglutination assay were performed in both control and experimental groups using standard protocols. The results clearly indicated that the extract of Gracilaria corticata (1000mg/kg) and levamisole (500mg/kg) possess significant immune enhancing property when compared with the other extracts Keywords: Common carp (Cyprinus carpio), Immunostimulant, levamisole, Vitamin C, Gracilaria

Introduction

Immunostimulants are substances that aid in bolstering the immune system through the activation of non specific immunity, and thereby gets more resistant to infections by a variety of biological agents (Raa, 2000). The compounds used as immunostimulants are vitamins trace elements, fatty acids, glucans, yeasts, nucleotides and others such as lactoferin, chitin, Levamisole, etc (Lall, 2003). Apart from that, the natural immunostimulants have been used as dietary supplements in aquaculture to reduce disease occurrence via activation of organism’s

innate immune responses as well as improving digestibility of various dietary substances. Ancient studies reveals that seaweeds used as food, fodder, fertilizer and as source of medicine today are the raw materials for many industrial productions like agar, algin and carageenan,but they are continued to be widely consumed as food in Asian countries .

There are numerous reports of compounds derived from macroalgae with a broad range of biological activities, such as anti bacterial, antifungal, antiviral, antitumoral, anticoagulant and antifouling (Athukorala et al., 2006), Such activities have been detected in green brown and red algae. The coast of TamilNadu bears luxuriant growth of sea weeds. More than two hundred species of sea weeds have been found in this area. Among them Gracilaria corticata, Ulva

fasciata and Sargassum weighti are the prominent sea weeds/macro alga with bio medical and pharmacological activities. Extracts of Ulva fasciata, a green sea weed, have been extensively used in shrimps as an antibacterial and immunostimulating agent (Huxley, 2002; Selvin et al.,

Nasaran and Huxley / Immunostimulant Induced Immunological Changes in Cyprinus carpio


68

2004). Among the non- specific defense mechanisms important in fish are the “barriers in

place” such as the skin and scales and lytic enzymes of the mucus and sera; cellular aspects include monocytes, macrophages, neutrophils and cytotoxic cells (Secombes, 1990). Thus, the present study aimed to investigate application of dietary vitamin C (ascorbic acid), Levamisole and chosen seaweeds such as Gracilaria corticata, Ulva fasciata and Sargassum weighti as an Immunostimulants on cultured koi carp Ciprinus carpio.


Dosing Regimens of Immune Evaluation

The efficacy of immunostimulants thorough different immune parameters evaluation were studied. The effective doses were selected (as per growth and challenge studies) and were subjected to immune index analysis. Six different groups with 100 fishes were separated and were fed with appropriate doses of immunostimulants for 30 days. The selected dosing regimens are given in Table 1.

Table1: Different doses immunostimulants used in this study.

Code Dose of Immunostimulants

Group A Control Group B 500mg/Kg fish body wt levamisole/day

Group C 1000mg/Kg fish body wt Gracilaria extract/day

Group D 500mg/Kg fish body wt Vitamin C/ day Group E 1000mg/Kg fish body wt Ulva/ day Group F 2000mg/Kg fish body wt Sargasam /day

Sampling and Blood Collection

Blood samples were collected on day 1, 7, 14, 21 and 28. Ten fishes were randomly collected from each treatment groups and control group, the blood samples were collected by heart puncture and caudal peduncle cut. Among that pooled sample, parts of the blood were mixed with anticoagulant (EDTA 10%) and the rest were used for serum collection without adding anticoagulant. Whole Blood Collection Whole blood was collected from the caudal vessels according to Rowley (1990) which used for Assessment of cellular immune response like Total Count, Differential count and NBT assay.

a. Total WBC count

Total WBC count was determined to find the effect of the vaccine on the immune system. The blood was diluted with the WBC diluting fluid. A well-mixed blood sample in the diluting fluid was charged into the counting well of the haemocytometer and the count was taken under 40X objective in the microscope.

b.Differential Blood Count

The differential blood count of the fish was made by using the method of Huxley et al., (2011). Blood was collected into heparinised vial and a small drop of the blood was placed on a clean slide. Using a spreader slide, the blood was made into a thin smear. The blood smear was air dried and fixed in methanol for three minutes. Later the smear was stained using Field's stain and observed under the microscope.

c. NBT Assay

Respiratory burst activity of neutrophils was assessed by using nitroblue tetrazolium test (Sigma Chemical Company). The blood is treated with NBT dye it combines to form NBT-heparin or



69

NBT-fibrinogen complex which is taken up by stimulated phagocytes. They incorporate the dye complex into phagosomes and after lysosomal fusion, intracellular reduction results in the formation of blue formazan granules. The blood from the fish was collected into a heparinised siliconized vial (Sigma, USA). 0.1ml of freshly prepared NBT solution was mixed with 0.1 ml of the blood and incubated at 37oC for 10 min and at 30oC for another 10 min. 50-70 l of this blood was transferred to a clean microscopic slide and a thick smear was made. The dried slide was then stained by Wright's stain and observed under 100X. The neutrophils with formazan granules in the cytoplasm were counted as positive ones. Percentage of positive cells was determined.

d. Lysozyme Assay

Lysozyme activity was measured by turbidimetric assay described by Parry et al., (1965). Lyophilized Micrococcus lysodekticus (0.2mg ml –1) in 0.04 M sodium phosphate buffer at pH 5.75 was used as a substrate for the serum lysozyme. Test serum (40 g) was added to 3 ml of the bacterial suspension and the reduction in absorbance at 540 nm was measured after 0.5 and 4.5 min at 22oC. One unit of lysozyme activity was defined as a reduction in absorbance of 0.001 min-1

e. Agglutination Assay

Agglutination assays were performed using blood plasma against bacterial cells. For agglutination titre the syringe bleeding was not effective for agglutination. So the blood was collected without anti coagulants (AC) in the clean eppendorf tubes. The eppendorf cup was incubated in the sliding position at 20oC for 1 h. After incubation, using a blunt end glass rod the clots was broken. The plasma was separated as earlier maintained procedure. The resultant plasma was diluted with PBS in a 74 well plate. A drop of different dilutions were mixed with a drop of heat or formalin killed pathogens (bacteria) and observed agglutination under 100X magnification. The index was estimated by the dilution factor.

Results Total WBC Count

The mean total WBC counts of the experimental groups (A - F) along with control groups are given in the Table. 2. The leucocyte population of all immunostimulated groups shows the enhanced counts; among these groups B (500 mg/Kg fish body wt levamisole exhibited maximum increment at 4.25 x 105, 4.20 x 105, 4.15 x 105, 4.25 x 105 and 4.35 x 105 in Day 1, Day 7, Day 14, Day 21 and Day 28 respectively. The other groups were also produced meager increment than the control. Table 2: WBC count (cells/mm3) in blood samples of control and experimental fishes.

Code Cells/ mm3

Day 1 Day 7 Day 14 Day 21 Day 28

Group A 2.50 x 105 2.35 x 105 2.55 x 105 2.85 x 105 2.50 x 105

Group B 4.25 x 105 4.20 x 105 4.15 x 105 4.25 x 105 4.35 x 105

Group C 3.35 x 105 3.55 x 105 3.65 x 105 3.30 x 105 3.25 x 105

Group D 3.15 x 105 3.10 x 105 3.00 x 105 3.05 x 105 3.15 x 105

Group E 2.85 x 105 2.75 x 105 2.80 x 105 2.95 x 105 3.05 x 105

Group F 2.95 x 105 2.45 x 105 2.65 x 105 2.90 x 105 2.85 x 105

Differential Count

The mean percentage values of the differential counts are given in Table-3. Based on the results there was an increase in the lymphocyte and neutrophil count in all test groups, In the case of group B the increment was very high than other groups. The monocyte counts remained almost the same in all groups.



70

Agglutination Assay

The agglutination titres of immunostimulated group of C. carpio are shown in the Table- 6. Based on the results of agglutination titre, Group B was considered as effective dose and it gave the maximum increment of 200 % over control was noted on 28 days of immunostimulation Table 3: Differential Blood cell count of control and immunostimulated Cyprinus carpio

Experiments Cells (%)

Types Day 1 Day 28

Group A

Monocytes Neutrophil Eosinophil Lymphocyte Thrambocytes

07.00 25.00 12.00 28.00 28.00

07.33 23.66 14.00 26.66 28.33

Group B


09.33 29.66 08.00 30.00 22.00

08.33 30.00 09.33 29.00 21.33

Group C


08.00 29.33 10.33 29.33 23.00

07.00 24.33 14.33 25.00 26.33

Group D


07.33 31.66 06.33 32.33 20.33

07.33 31.33 11.33 30.66 19.33

Group E


07.33 25.66 11.66 29.33 26.00

08.66 25.66 12.33 29.33 24.00

Group F


05.00 33.66 05.33 36.00 18.00

06.00 32.00 09.00 35.66 17.33

Nitro Blue Tetrazolium (NBT) Assay

The positive reaction shows the formazan granules inside the cytoplasm while the negative does not produce any granules. Based on the results of NBT assay of immunostimulated group of C. carpio were enhanced formazan granules inside the cytoplasm. The mean NBT assay values are given in the Table 4. Table 4: NBT levels of control and immunostimulated fishes.

Groups NBT levels (%)


Group A 18.0 20.0 18.6 21.0 21.0 Group B 27.0 28.0 39.3 48.0 50.0 Group C 22.0 28.3 33.0 43.3 45.3 Group D 24.0 23.0 23.3 25.0 23.3 Group E 21.0 21.0 22.0 22.0 24.0 Group F 20.0 22.6 21.6 21.6 24.0



71

Lysozyme Assay

Based on the results of Lysozyme level of all immunostimulated supplemented groups of C.

carpio were enhanced. Among that the Group B produced higher level of increment at a rate of 320, 340, 350, 360 and 375 units/ml in day 1, 7, 14, 21 and 28 respectively. The mean Lysozyme values are given in the Table 5. Table 5: Lysozyme level (units/ml) of different immunostimulated fishes.

Groups Lysozyme level (units/ml)


Group A 250 260 240 250 265 Group B 320 340 350 360 375 Group C 290 320 300 320 335 Group D 250 300 320 290 305 Group E 230 310 310 330 325 Group F 260 300 320 310 315

Table 6: Agglutination titre of immunostimulated Cyprinus carpio.

Experiments Agglutination titre


Group A 1:12 1:13 1:12 1:12 1:12 Group B 1:20 1:21 1:20 1:18 1:18 Group C 1:24 1:24 1:22 1:21 1:18 Group D 1:16 1:18 1:18 1:16 1:15 Group E 1:18 1:18 1:16 1:16 1:16 Group F 1:15 1:14 1:14 1:14 1:15

Discussion

The total WBC count was used as an indicator. In the present study, the results have shown that there was an increase in the leucocyte counts in all groups. The results were similar to those reported by Siwicki (1989). The differential count was used to find the effect of the chemical on each group of leucocytes. There has been an increase in the neutrophil count in group B while a mere increase was seen in all other groups. Not much difference in the monocyte counts was observed when compared to the control in both the test groups in the present study. This is in agreement with the findings of Siwicki (1987).

Previous studies have shown great variability in the effect of ß -glucans on total WBC count. Siwicki et al., (1994) reported contradictory effects of ß -glucans on rainbow trout leucocyte numbers after an oral exposure. In their experiment they obtained an elevated leucocyte count in immunostimulated fish. In contrast, Jeney and Anderson (1993) observed a rise in the WBC after injection or bath administration of a barley-extracted glucan in rainbow trout. The results nearly agreed with the result obtained by Siwicki et al (1994) who observed an increase of some cellular activities in rainbow trout after feeding with S. cerevisiae at a dose of 27 g/kg diet for one week. The enhanced cellular activity could be attributed to the presence of glucan receptors on the cell surface of blood monocytes, macrophages and neutrophils which was mainly responsible for phagocytic activity and phagocytic index. These findings coincided with the results obtained by Yoshida et al., (1995) in African catfish who reported an increase in phagocytic activity and index after feeding with ß- glucan supplemented diet at a dose of 1 g/kg diet for 45 days. There was a marked increase in the respiratory burst activity as found from the number of positive cells in group B while there was an overall increase in other groups which



72

was lesser than that of the group D. The test results are similar to those observed by Siwicki (1987) in Common carp and Siwicki et al., (1989) in Salmonids. Mulero et al., (1998) reported that a dosage of 250-mg/Kg diet was ideal for enhancing the neutrophil activity in Gilthead seabream. It must be pointed out that, in the present work, the mean lysozyme activity values obtained from control groups varied according to the different sampling days. This remains unexplained, as all the measurements were performed on the same date upon defrosted plasma samples and according to a classical method used to determine lysozyme activity in salmonids (Jorgensen et al., 1993).

Ellis (2003) observed an increased lysozyme activity from Day 7 to Day 28 after a single IP injection of Macrogard in salmonids and Jorgensen et al., (1993) obtained the same result in rainbow trout 7, 14 and 21 d after M-glucan injection. An elevation in lysozyme activity occurred in the Vitamin C group only on Day 14, but in the IS+VAC group the increase was significant on Days 14 and 21. Increased lysozyme levels may occur after an antigenic stimulation and the present results showed that the use of glucan could potentiate the effect of the vaccine. The results showed that immunizing Chanos chanos with ECP from Micobacterium

spp stimulated the release of lysozyme into the serum of the fishes, this elevation is not likely to have been caused by the resting levels of lysozymal enzymes, but is more likely to be due to, firstly, increased amounts of neutrophils or macrophages and secondly increased synthesis and release of lysozyme, as was absorbed in mice treated with glucans. Ellis (2003) suggested that observed increases in serum lysozyme levels in fish may be induced by glucans associated with the infecting bacteria.

Agglutinins, which cause aggregation or agglutination of foreign particles, have been reported from a number of fish species. However, the effect was small, short-lived and non - specific. Recent findings relating to the effects of β-glucans, administered orally, on the immune response of certain species of Mediterranean fish, have lead to the question of their effect on European sea bass. There are only two reports available in the literature relating to β-glucans fed to sea bass. In the first report Bagni et al., (2000) fed glucans together with increased long-term doses of vitamins. They were able to demonstrate increased activation of the alternative complement pathway and increased serum lysozyme activity in these fishes, although they did not see any difference in their serum protein content, including albumin and globulin. Unfortunately, it was not possible to establish the effect of the β-glucans on the immune response of the fish in their study since the two immunostimulants were fed simultaneously. Bagni et al., (2005) compared the effects of Macroguard with Ergosan, a commercial algal extract containing alginic acid. Long term feeding of these substances did not appear to affect the innate or adaptive immune responses measured.

Acknowledgements The authors are thankful to the Principal, Thiru. Vi. Ka. Goverenment Arts College, Tiruvarur for his support and encouragement. These data are the preliminary doctoral work of D. S. Nasaran

Reference

Athukorala,Y., Lee, K. W.,Kim, S. K. and Jeon,Y. J. 2006. Anticoagulant activity of marine green and

brown algae collected from Jeju Island in Korea. Bioresource Technol., 98: 1711-1716. Bagni, M., Archetti, L., Amadori, M., and Marino, G. 2000. Effect of long-term administration of an

immunostimulant diet on immunity in sea bass (Dicentrarchus labrax). J Vet Med B, Infect Dis Vet Public Health, 47: 745-751.

Bagni, M., Romano, N., Finola, M.G., Abelli, L., Scapigliati, G., and Tiscar, P. G. 2005. Short- and long-term effects of a dietary yeast betaglucan (Macrogard) and alginic acid (Ergosan) preparation on immune response in sea bass (Dicentrarchus labrax). Fish Shellfish Immunol ., 18: 311-336.

Ellis, A.E. 2001. Innate host defense mechanisms of fish against viruses and bacteria. Develop.Comp.

Immunol., 25:827-839.



73

Huxley, V.A.J. 2002. Studies on Non-Specific immunomodulation in Penaeus monodon with special reference to protection against common bacterialpathogens. PhD. Thesis submitted to Manonmaniam SundaranarUniversity, Tirunelveli, Tamil Nadu.

Huxley,V.A.J., Jerold Xavier, S., Bhamini and Xavier Innocent, B. 2011. Immunomodulatory potential of crude Ulva Extract on the proction of bacterial Diseases in Malabar Grouper (Epinnephelus

malabaricus). Journal of basic and applied biology, 5(1&2): 271-277. Jeney, G., and Anderson, D.P. 1993b. Glucan injection or bath exposure given alone or in combination

with a bacterin enhance the non-specific defense mechanisms in rainbow trout (Oncorhynchus

mykiss). Aquaculture, 116: 315-329. Jorgensen, J.B., Lunde, H., and Robertsen, B. 1993. Peritoneal and head kidney cell response to

intraperitoneally injected yeast glucan in Atlantic salmon, Salmo salar L. Journal of Fish Diseases, 16: 313–325.

Lall, S.P. 2003. Role of nutrients in immune response and disease resistance infish. IHNV Research Workshop, Campell River, BC, January 10-12 (Dr.Lall is from the Institute for Marine Biosciences, National Research Council,Halifax, NS).

Mulero, V., Estaban, M.A., and Meseguer, J. 1998. In vitro levamisole fails to increase seabream (Sparusaurata L.) phagocyte functions. Fish Shellfish Immunol., 8:315-318.

Parry, R.M., Chandan, R.C., and Shahani, R.M. 1965. A Rapid Sensitive Assay of Muramidase. Proc Soc. Exp. Biol. Med., 119: 384-386.

Raa, J. 2000. The use of immunostimulants in fish and shellfish feeds. In: Advance Nutrition Acuicola V. Memories del V Symposium International de Nutrition A cucola cruz- Sua’rez, LE.Recque- Mario.

Rowley, A.F. 1990. Collection , Separation and Identification of fish leucocytes. In: Techniques in fish Immunology. Edited by: J.S. Stolen, T.C. Fletcher, D.P. Anderson , B.S. Robertson , W.B. van Muiswinkel. SOS. Publications, Fair Haven , USA.

Secombes, C.J. 1990. The nonspecific immune system: cellular defense. In. The fish immune system, fish physiology series vol.15. Edited by: G. Iama, and T. Nakanishi. Academic Press, San Diego, CA. pp.63-103.

Selvin, J., Huxley, A. J., and Lipton, A.P. 2004. Immunomodulatory potential of marine secondary metabolites against bacterial diseases of shrimp. Aquaculture, 230 (1-4): 241-248.

Siwicki, A.K., Anderson, D.P., and Rumsey, G.L. 1994. Dietary intake of immunostimulants by rainbow trout affects non-specific immunity and protection against furunculosis. Vet jour Immunopathol., 41(1–2): 125-139.

Siwicki, A.K. 1987. Immunomodulating activity of levamisole in carp spawner. J Fish Biol., 31(Suppl.

A): 245-246. Siwicki, A.K. 1989. Immunomodulating influence of levamisole on non-specific immunity in carp

(Cyprinus carpio). Develop Comp Immunol., 13: 87-91. Yoshida, T., Kruger, R., and Inglis, V. 1995. Augmentation of n on –specific protection in African

catfish, Clarias gariepinus (Burchell), by the long term oral administration of fish: Applications to Aquaculture. Annual Review of Fish Diseases, 2: 281-307.

*Corresponding author: Email address: [email protected]


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Wound Healing Potential of

Tarenna asiatica (L.) Kuntze Ex K.Schum. Leaves

N. Anjanadevi* and S.Menaga

Department of Botany, Vellalar College for Women, Erode-638012, Tamil Nadu, India.

Received: 10 June, 2013; revised received: 15 October , 2013

Abstract The qualitative screening using ethanol and aqueous leaf extract of Tarenna asiatica showed the presence of primary and secondary metabolites. The ethanol extract was used to formulate 5% and 10% test ointment. The 5% and 10% test ointment healed the excision and incision wound in wistar rat. 10% test ointment was found to be superior as compared to 5% test ointment in contracting the wound and increasing the tensile strength of the skin. Key words: Excision, incision , wound, healing, Tarenna, leaves.

Introduction Plants exert multifarious influence on the mode of human life in a myriad ways. Among them their medicinal use fetches greater significance in the human life since they provide materials which help in curing diseases. Because of this fact, people have been using plant products as medicine for curing variety of diseases since the time immemorial especially skin wounds. Natural remedies from medicinal plants are considered to be effective and safe alternative treatment for wounds. Wound is a break in the epithelial integrity of the skin and may result from contusion, haematoma, laceration or an abrasion (Enoch and Leaper, 2005).

Wound healing involves different phases such as contraction, epithelialization, granulation and collagenation (Purna et al., 1995). Wound healing is an anabolic process that requires both energy and nutritive substances. Fibroblasts produce a variety of substances essential for wound repair including glycosaminoglycans and collagen (Stadelmann et al. 1998). Nayak (1999) reported that Ixora coccinea flowers healing the wounds in rats. Participation of various inflammatory cells such as macrophages and neutrophils is extremely crucial to the repair process (Rasik et al., 1999). It is found that the leaves of Tarenna asiatica belonging to Rubiaceae are used as folk medicine in the treatment of wounds (Chopda and Mahajan, 2009). There is no scientific evidence to this effect. Hence, in the present study, leaves of Tarenna

asiatica was evaluated by using excision and incision wound model in terms of wound contraction and tensile strength.

Materials and Methods The leaves of Tarenna asiatica were collected from Mookanur hill in Dharmapuri district, shade dried and used for various activities. Qualitative Phytochemical Screening

Phytochemical screening of ethanol and aqueous leaf extracts was carried out following the methods of Harborne (1984) and Kokate et al. (1995). Carbohydrates, proteins and aminoacids, alkaloids, anthraquinones, flavonoids, glycosides, phenols and tannins, saponins, steroids and sterols, triterpenoids and volatile oil were qualitatively analyzed.

75

Anjanadevi and Menaga / Wound Healing Potential of Tarenna asiatica Leaves


Formulation of Ointment

5% (w/w) and 10% (w/w) test ointments were prepared by incorporating 5g and 10g of Soxhlet ethanol extract of Tarenna asiatica leaf separately in 100g of simple ointment base. Experimental Animals and Raising Environment

After approval of the Institutional Ethics Committee, pathogen free mice and wistar rats ranging from 25- 200g of either sex were used for toxicological study and wound healing activity. In vivo Toxicity Studies The acute dermal toxicity was performed according to OECD guidelines. For this, a limit test of 5000 mg/ kg did not show any sign of lethality and the gross behaviour of the animal was normal and there was no apparent sign of dermal toxicity. Excision Wound Model-Wound contraction

Wistar rats of either sex weighing between 150g and 200g were inflicted with excision wounds as described by Morton and Malone (1972) under light ether anesthesia. A circular wound of about 500 sq.mm was made on depilated ethanol sterilized dorsal thoracic region of the rats. The animals were divided in to four groups of four each. The animals of group I was considered as the control. Animals of group II and III were treated with 5% w/w and 10% w/w test ointment prepared from ethanol leaf extract of Tarenna asiatica and group IV served as standard and treated with 2% w/w Nitrofurazone ointment (standard drug).

The ointment was topically applied once a day, starting from the day of the operation, till complete epithelialization. The progressive changes in wound area were monitored planimetrically by tracing the wound margin on graph paper on 0 day, 2nd day, 4th, 8th, 12th, 16th, 18th and 20th day. The day on which wound was made considered as “0”day. This model was

used to monitor wound contraction. Wound contraction was calculated as percent reduction in wound area. Statistically analysed data are expressed as Mean ± SEM and subjected to ANOVA and DMRT by comparing with the control. Incision Wound Model (Ehrlich and Hunt, 1968) The animals were divided into 4 groups of 4 each and for the Incision wound model the animals in each group were anaesthetized with ether and incision wounds of about 6cm length were made through the skin and cutaneous muscles at a distance of about 1.5 cm from the midline on each side of the depilated back of the rats. After the skin incision was made, the parted skin was kept together and stitched at 0.5cm intervals continuously and tightly using suture threads (No.ooo) and a curved needle (No.11). The wounds of animals in the different groups were treated with topical application of the ointment as described in excision for a period of 10 days. The wounding day was considered as day 0. When the wounds were cured thoroughly, the sutures were removed on the 9th post wounding day and the tensile strength of the skin, that is the weight in gram required to break open the wound/skin, was measured by tensiometer on the 10th day. Statistically analysed data are expressed as Mean ± SEM.

Results

The preliminary phytochemical screening revealed the presence of carbohydrates, proteins and aminoacids, alkaloids, flavonoids, glycosides, phenols and tannins, saponins, steroids and sterols, anthraquinones, triterpenoids and volatile oil in ethanol extract of the leaves of Tarenna

asiatica. The aqueous extract showed negative response to saponins, steroid and sterol, volatile oil and anthraquinones. The results showed that the ethanol extract was more efficient than the aqueous extract (Table 1).

76

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3.2

(62.

8)

2.

II –

Tes

t oi

ntm

ent

5%

W/W

51

6 ±

3.1

a (0

.01)

43

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3.8a

b (1

6.6)

35

6.6±

4.4a

b (3

2.05

) 18

8.3±

1.8a

b (6

3.51

) 86

±2.

3ab

(83.

3)

21.2

±3.

96a

(95.

86)

42±

0.8a

(9

9.18

)

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III-

Tes

t oi

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10

% W

/W

512

± 5

.2b

416±

1.8a

b (1

8.75

) 32

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7ab

(36.

7)

149±

1.9a

b (7

0.89

) 21

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7ab

(95.

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00

(100

) -

4.

IV -

Nit

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razo

ne

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(st

anda

rd)

510

± 3

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(2

1.17

) 30

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(40.

39)

116±

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(7

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77



Table 1: Qualitative Phytochemical screening of leaf sample of Tarenna asiatica

S.No Constituents Ethanol Water

1 Carbohydrates + +

2 Proteins and aminoacids + +

3 Alkaloids + +

4 Flavonoids + +

5 Glycosides + +

6 Phenols and Tannins + +

7 Saponins + -

8 Steroids and sterols + -

9 Anthraquinones + -

10 Triterpenoids + +

11 Volatile oil + -

Excision Wound -Wound Contraction

A better healing pattern with complete wound closure was observed in wistar rat treated with Tarenna asiatica ointment within 20 days, while it took about 25-30 days in control rats. There was a significant reduction in wound area from day 8 onwards in treated animals and also on later days, the contraction rate was much faster than the control animals.

The simple ointment (control) gradually contracted the wounds, caused in wistar rats by excision method from 0 day to 20th day. As compared to 0 day, 62.8% wound contraction was observed on 20th day in the control group. Progressive changes in wound contraction (mm2) from 4th, 8th day, 12th day, 16th day, 18th day and 20th day were 478.9±11.21, 406.6±2.1, 340±6.2, 278.1±2.6, 230±3.7 and 190±3.2 respectively in simple ointment (control). The results of 5% (w/w) herbal extract ointment were significantly superior to simple ointment (control) and on 20th day 99.18% wound contraction was observed. As compared to simple ointment, 5% test ointment was significantly effective in reducing the wound area from the 4th day onwards. The wound at 5% test ointment treatment completely healed on 20th day. The 10% (w/w) test ointment was excellent in contracting the wounded area. As compared to 5% test ointment, 10% test ointment showed, a significant wound contraction.

The per cent wound contraction at 10% test ointment was 18.75, 36.7, 70.89, 95.89 and 100 respectively on 4th day, 8th day, 12th day, 16th day and 18th day. On 12th day, 70.89% wound contraction was observed in 10% test ointment application. Almost 96% wound contraction was achieved on 16th day. Nitrofurazone contracted the wound 100% on 16th day. The difference in wound contraction between 10% test ointment and Nitrofurazone ointment was insignificant on 16th day (Table-2). The effect of 10% test ointment was better than that of 5% test ointment and control and occurred in the order of magnitude10%>5%>control.

The results of this study revealed that the leaf of Tarenna asiatica has potentials for use in wound care due to the ability of its chemical constituents and the extract accelerates the wound healing by enhancing epithelialization and stimulating tissue proliferation.

78



Incision wound -Tensile Strength

The effect of herbal extraction on the tensile strength is given in Table-3. The tensile strength of control animal was 228.36±3.8g and the test ointment treated animals showed increased tensile strength. 5% test ointment significantly increased the tensile strength in treated animals as compared to the control animals. 10% test ointment increased the tensile strength 19.3% more than the 5% test ointment. Nitrofurazone effect on tensile strength is slightly better than that of 10% test ointment applied tensile strength. Table 3: Effect of topical application of test ointment of Tarenna asiatica on Incision wound model (tensile strength)

Sl.

No. Group Tensile strength(g)

1 I Simple ointment(control) 228.36±3.8

2 II Test ointment 5% w/w 298.3±1.9

3 III Test ointment 10% w/w 370±6.8

4 IV Nitrofurazone 0.2% (standard)

412.1± 5.7

Values are Mean ± SE (n=4)

Discussion

Excision wound study on animal model showed enhanced rate of wound contraction and drastic reduction in healing time (16 day in 10%w/w test ointment) than simple ointment (control) which might be due to enhanced epithelialization. Wound contraction is the process of mobilizing healthy skin surrounding the wound to cover the denuded area. This centripetal movement of wound margin is believed to be due to the activity of myofibroblast. Since Tarenna asiatica test ointment enhanced the wound contraction, it would have either enhanced contractile property of myofibroblasts or increased the number of myofibroblasts recruited in to the wound area as reported by Sidhu et al. (1999).

Significant wound contraction on 16thday at 10% test ointment of Tarenna asiatica may also be due to the participation of various inflammatory cells such as macrophages and neutrophils in the repair process and may also promote the migration and proliferation of endothelial cells, leading to neovascularization of connective tissue cells, which synthesize the extracellular matrices including collagen and of keratinocytes leading to reepithelialization of the wounded tissue as reported by Rasik et al. (1999). It is well accepted that secondary metabolites play an important role in wound healing process (Sang et al., 2001; Kerr, 2002 and Ghorbani, 2005).

In the present study the qualitative screening of leaf extract revealed the presence of alkaloids, triterpenoids, phenols, flavonoids, glycosides, anthraquinones, saponins and tannins. Wound contraction in animal model may be due to the presence of the above mentioned compounds. Similar report was made by Joshi et al. (2003). Scortichini and Piarossi (1991) also reported that tannins, flavonoids, triterpenoids and sesquiterpenes are known to promote the wound healing process mainly due to their antioxidant, astringent and antimicrobial properties which seem to be responsible for wound contraction and increased rate of epithelialization. The sesquiterpene lactones are known to possess antioxidant property which may also contribute to the wound healing process (Manjunatha et al., 2005).

79



Thus the wound healing potency of Tarenna asiatica may be attributed to the phytoconstituents present in it, which may be either due to their individual or additive effect that fastens the process of wound healing.

Incision wound-Tensile strength

The tensile strength of a wound represents the degree of wound healing. Usually wound healing agents promote a gain in tensile strength. The results on tensile strength revealed that upon application of 5% and 10% w/w test ointment, there was an increase in the tensile strength or breaking strength of skin as compared to control (simple ointment). The findings of present study showed a significant increase in tensile strength of animals treated with 10% test ointment. This is more or less comparable with standard drug nitrofurazone. In the present study, the animals treated with 5% and 10% test ointment showed significant results when compared with simple ointment treatment (control). 10% test ointment treated animals showed faster epithelialization of wound than the 5% test ointment treated animals. From the above result of excision and incision wound model, it is evident that 10% test ointment was found to be superior to 5% test ointment in wound contraction and increasing the tensile strength. The observation of the current study confirms the traditional use of Tarenna asiatica leaves for wound healing properties. The formulation of ethanol extract applied augments the healing process by strengthening the tensile strength and promoting the wound contraction.

References

Chopda,M.Z., and Mahajan, R.T. 2009. Wound healing plants of Jalgaon District of Maharashtra state, India. Ethnobotanical leaflets, 13: 1-32.

Ehrlich,H.P., and Hunt,T.K. 1968. Effect of cortisone and vitamin A on wound healing. Ann.Surg., 167:

324-328. Enoch,S., and Leaper,J.D. 2005. Basic science of wound healing. Surgery, 23: 37-42. Ghorbani,A. 2005. Studies on pharmaceutical ethnobotany in the region of Turkmen sahra, north of Iran,

J. Ethnopharmacol., 102: 58-68. Harborne,J.B. 1984. Phytochemical methods, 2nd Edn., Chapman and Hall, London . pp. 44. Joshi,S.D., Aravind,M.B., Ashok,k., Veerapur,V.P., and Shastry,C.S. 2003. Wound healing activity of

Dodonaea viscosa leaves. Indian Drugs, 40(9): 549-552. Kerr, J. 2002. The use of essential oils in healing wounds. Int. J. Aromather., 12: 202-206. Kokate,C.K., Khandelwal,K.R., Pawar,A.P., and Gohalz,S.B. 1995. Practical Pharmacognosy, 4th Ed.

Vallabh Prakashan, New Delhi, pp. 107. Manjunatha,B.K., Vidya,S.M., Rashmi,K.V., Mankani,K.L., Shilpa,H.J. and Single,S.D.J. 2005.

Evaluation of wound healing potency of Vernonia arborea. Ind. J. Pharmacol., 37(4): 223-226. Morton,J.J.P., and Malone,M.H. 1972. Evaluation of vulnerary activity by an open procedure in rats.

Arch. Int. Pharmacodyn., 196: 117-126. Nayak,B.S. 1999. Effects of Ixora coccinea flowers on dead space wound healing in rats. Fitoterapia,

70(3): 233-236. Purna,K.S., Neelakanta,P.R., and Babu,M. 1995. Investigations on wound healing by using amphibian

skin. Ind. J. Exp. Biol., 33: 673-676. Rasik,A.M., Raghubir,R., Gupta,A., Shukla,A., Dubey,M.P., Srivastava,S., Jain,H.K. and

Kulshrestha,D.K. 1999. Healing potential of Calotropis procera on dermal wounds in Guinea pigs. J. Ethnopharmacol., 68: 261-266.

Sang,S., Cheng,X., Zhu,N., Wang,M., Jhoo,J.W., Stark,R.E., Badme,V. Ghai,G., Rosen,R.T., and Ho,C.T. 2001. Iridoid glycosides from the leaves of Morinda citrifolia. J. Nat. Prot., 64(6): 799-800.

Scortichini,M., and Piarossi,M. 1991. Preliminary in vitro evaluation of the antimicrobial activity of terpenes and terpenoids towards Erwinia amylovora. J. Appl. Bacter., 71: 109-112.

Sidhu,G.S., Mani,H., Gaddipatti,J.P., Singh,A.K., Seth,P., Banaudha,K.K., Patnaik,G.K., and Maheswari,R.K. 1999. Curcumin enhances wound healing in Streptozotocin induced diabetic rats and genetically diabetic mice. Wound repair Regeneration, 7: 362-374.

Stadelmann,W.K., Digenis,A.G., and Tobin,G.R. 1998. Physiology and healing dynamics of chronic cutaneous wounds. Am. J. Surgery, 176: 26-38.

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ISSN: 0972-9720

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Volume 10 Number 1 and 2 August and November 2013

Contents

Inventory Message about Bat Species of Kalakad Mundanthurai Tiger Reserve (Tamil Nadu, India) through Global Positioning System (GPS) Juliet Vanitharani, Nikky Thomas, L. Jeyapraba , C. Mercy, P. Selva Pobmalar and

Gladrene Sheena Basil

01-20

Sodium Azide: a Chemical Mutagen for Enhancement of Agronomic Traits in Phaseolus vulgaris (Linn) Girma Mosisa, Manikandan Muthuswamy and Yohannes Petros

21-25

In vitro Antibacterial Activity and Phytochemical Analysis of Three Aquatic Plants Mofanato S.K. Kala Kumari and B. Vasantha Kumari

27-31

Phytochemical Analysis and in vitroAntimicrobial Activity of Various Extracts of Euphorbia nivulea Ham S. Selvadhas and S. Natarajan

33-40

Enhancement of Tolerance of Faba Bean-nodulating Rhizobial Isolates to High Temperature Andarge Zelalem , Ameha Kebede and Manikandan Muthuswamy

41-48

Isolation and Characterization of Pathogenic Bacteria Infecting Cultured Koi Carp Ciprinus carpio D. S. Nasaran and V.A.J. Huxley

49-55

Role of Hormones in Differential Growth Responses of Mung Bean Vigna radiata L. Wilczek Seedlings under Water Stress Satyajit Das and Rup Kumar Kar

57-65

Effect of Chosen Immunostimulant Induced Immunological Changes in Common Carp (Cyprinus carpio) D. S. Nasaran and V.A.J. Huxley

67-73

Wound Healing Potential of Tarenna asiatica (L.) Kuntze Ex K.Schum. Leaves N. Anjanadevi and S.Menaga

75-80

Printed at NANGIL Offset Printers, 67/1-Court Road, Nagercoil-629001,

Tamil Nadu, INDIA. Phone: +91 4652 233853. E-Mail: [email protected]

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