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ISSN (E): 2349 1183

ISSN (P): 2349 9265


3(2): 249252, 2016

Research article

Persicaria perfoliata (L.) H. Gross (Polygonaceae):


A species new to Eastern Ghats of India
J. Prakasa Rao1* and K.V. Satish2
1
Department of Botany, Andhra University, Visakhapatnam - 530 003, Andhra Pradesh, India
2
Forestry and Ecology Group, National Remote Sensing Centre, Balanagar, Hyderabad, Telangana, India
*Corresponding Author: jprakasarao@gmail.com [Accepted: 17 June 2016]

Abstract: Persicaria perfoliata [Polygonaceae] is popularly known as Mile-a-minute weed. It was


recorded for the first time from forests of Paderu hills Eastern Ghats. As of now, no reports were
observed from Eastern Ghats of India. It forms new angiospermic addition for biodiversity of
Eastern Ghats. It is being described along with field photographs to make an easy identification.
Adjoining floral association and importance value of the present species were discussed.
Keywords: Biodiversity - Flora - Mile-a-minute - Paderu hills - Andhra Pradesh.

[Cite as: Prakasa Rao J & Satish KV (2016) Persicaria perfoliata (L.) H. Gross (Polygonaceae): A species new
to Eastern Ghats of India. Tropical Plant Research 3(2): 249252]

INTRODUCTION
Mile-a-minute weed [Persicaria perfoliata (L.) H. Gross], is a member of the family Polygonaceae. It is a
native plant of Asian countries, distributed including India, Bangladesh, Nepal, China, Japan, Korea, Siberia,
Turkey, Philippines, Malay Peninsula, Indochina Peninsula and Indonesia (Wu et al. 2002, Kantachot et al.
2010, Robbins et al. 2014). India, it was reported only from Darjiling and Sikkim parts of the Eastern Himalaya
(Das et al. 2010), Koch Bihar district of West Bengal (Bandyopadhyay & Mukherjee 2010) and Gori Valley of
Meghalaya (https://sites.google.com/site/efloraofindia/species) nevertheless, this taxon not been reported from
Eastern Ghats (Gamble & Fischer 19151936, Pullaiah & Moulali 1997, SubbaRao & Kumari 2002, Reddy et
al. 2008) (Fig. 1). However, this species is a noxious weed in USA, Mexico, England and other adjacent
countries (http://www.cabi.org/, Driesche et al. 2002). It spreads very rapidly, grows up to 15 cm per day
(Kumar & DiTommaso 2005) and reach up to forest sub canopies. It has the capability to overlap on other
native vegetation (Poindexter 2010) and curbs forest regeneration (Wu et al. 2002). In China, very few records
are showing Persicaria perfoliata as a weed in the agricultural environment (Wang et al. 1990). Such kind of
severe threat has not been recorded in India.

MATERIALS AND METHODS


Botanical explorations were conducted in forests of Eastern Ghats of Andhra Pradesh in July 2015. In this
processes, we have collected Persicaria sp. in degraded forests of Kantavaram village, Paderu hills. Few
individuals were collected for taxonomic study and to make herbarium. While making herbarium specimens, we
followed standard herbarium methods (Jain & Rao 1977). Field and voucher numbers were allocated. Habitat
condition, adjoining plant communities and other taxonomic notes were recorded for specimens and deposited in
Andhra University Herbarium (AUH).

RESULTS AND DISCUSSION


Enumeration of species:
Persicaria perfoliata (L.) H. Gross, Beih. Bot. Centralbl. 37(2): 113. 1919. (Fig. 2)
Amplelygonum perfoliatum (L.) Roberty and Vautier, Chylocalyx perfoliatus (L.) Hassk., Echinocaulon
perfoliatum (L.) Hassk., Echinocaulos perfoliatus (L.) Meisn. Fagoparum perfoliatum (L.) Rafine., Fagoparum
perfoliatum (L.) Rafine., Polygonum perfoliatum (L.) L. Tracaulon perfoliatum (L.) Greene, Truellum
perfoliatum (L.) Sojak (http://www.cabi.org)

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Received: 10 May 2016 Published online: 30 June 2016
Prakasa Rao & Satish (2016) 3(2): 249252
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Climbing annual vine, usually grows up to 615 m; Stems scandent, slightly ribbed, glabrous, and glaucous;
prickles covers both stem and petioles. Prickles 0.412 mm long, slightly recurved. Ocrea, round glaucous,
perfoliate, present at both base of the each petiole and inflorescence. Leaves alternate, petiole 3.57 cm; leaves
glaucous, ocrea green, foliaceous, peltate, not prickled, margins entire, eciliate, surface glabrous; leaf shape
triangular, base truncate, apex acute. Inflorescences spike, 512 510 mm; peduncle 1050 mm, retrorsely
prickly; ocrea overlapping, margins eciliate. Pedicels ascending, 13 mm. Flowers 14 per ocreate fascicle end
of the spike has 4 to 11 flowers; perianth creamy greenish white, glabrous, accrescent, becoming fleshy and pale
blue in fruit; tepals 5, broadly elliptic, 23.5 mm long, apex acute to obtuse; stamens (6)8, filaments distinct,
free; anthers ovate; styles 3, connate proximally. Seeds shiny black spherical, peanut sized.
Flowering: Mid-April to late July.

Figure 1. Location map of reporting site of Persicaria perfoliata (L.) H. Gross.


Common names: Devil's-tail or giant climbing Asiatic tearthumb, mile-a-minute weed.
Local name: Chepamullu kura (Telugu)
Uses: Fruits are edible (He et al. 1984) also used as herbal medicine (He et al. 1984, Yang & Kim 1993). In the
present study investigations showing that local tribes are being used as a leafy vegetable.
Identification of taxon: After our critical examination and perusal of literature and web sources these
specimens were identified as Persicaria perfoliata (L.) H. Gross owing to the following distinct features: the
presence of short prickles on stem and petioles, leaf surface glaucous, faintly veined, leaves are peltate,
triangular, leaf bracts round, perfoliate. Stem very thin, scandent, green in color later turn into dark pink.
Inflorescence capitates, flowers creamy greenish white in color. Perianth is accrescent and becomes fleshy while
fruiting. Fruits are blue, seeds are round and black.
Specimen examined: INDIA, Andhra Pradesh, District Visakhapatnam, Kantavaram village, Paderu hills.
25.07.2014, J. Prakasa Rao 20490 (AUH).
Site description: The Eastern Ghats are discontinuous hill ranges in peninsular India and geographically older
than the Himalayas and the Western Ghats (Abe et al. 2013, Reddy et al. 2014) extending over 1750 km with
average width of about 30N latitude05 to 22 100 km and extends from 10 50E longitude23 to 86 and 76.
Altitude ranges between 1001572 (Reddy et al. 2014) Due to high elevation and rainfall, the valleys have
luxuriant habitats consisting of evergreen, semi-evergreen, moist deciduous, dry deciduous, dry evergreen and
thorn forests. (Reddy et al. 2014). However, the Eastern Ghats region is relatively under-explored. Among the
Eastern Ghats, Visakhapatnam hills are one of the ecologically sensitive and biologically rich regions (Rao et al.
2015) with strict global endemics like Argyreia arakuensis Balakr, Phyllanthus narayanaswamii Gamble,
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Argyreia srinivasanii Subba Rao & Kumari, Bupleurum andhricum Nayer & Banerji, Kalanchoe
cherukondensis Subba Rao & Kumari, Leucas mukherjiana Rao & Kumari and Ophiorrhiza chandrasekharanii
Rao & Kumari (SubbaRao & Kumari 2002). Several plant species are being reported from the Eastern Ghats.
Rao et al. (2015) have reported Balanophora fungosa as new distributional record for the flora of Andhra
Pradesh and Achyranthes longifolia as a new record for Indian flora (Rao et al. 2016). The study is proven that
there is a need to conduct extensive botanical explorations in the Eastern Ghats.

Figure 2. Persicaria perfoliata (L.) H. Gross: A, Habit; B, Young branch; C, Node is showing perfoliate ocrea and sprickles
on both stem and petiole; D, Leaf; E, Fruiting.
Habitat and plant communities: Persicaria perfoliata is growing along the road sides where moist soils are
available with less tree cover. However, we recorded scattered trees, understory and herbaceous diversity of
present habitat and details are presented below.
Ageratina adenophora, Bidens pilosa, Chromolaena odorata, Lantana camara, Chrysopogon aciculatus,
Clematis gouriana, Cissampelos pareira, Rubia cordifolia, Stemona tuberose, Smilax zeylanica and Zingier
roseum are the major herbaceous associates of the species.
Indigofera cassioides, Rubus ellipticus, Zanthoxylum armatum, Solanum torvum, Cipadessa baccifera,
Colebrookea oppositifolia, Pogostemon benghalensis and Barleria strigosa are main shrubs while, Diospyros
melanoxylon, Phyllanthus emblica, Alstonia venenata, Terminalia chebula, Syzygium sp., Ttrichilia
connaroides, Gardenia latifolia, Pittosporum nepalense, Callicarpa tomentosa and Mallotus philippensis are the
tree associates of the species in the Eastern Ghats

CONCLUSION
Persicaria perfoliata is a new addition to the flora of the Eastern Ghats. This species is growing very well at
the higher altitudinal ranges of Paderu hills. P. perfoliata is reported as a weed in USA, Mexico and other
adjoining countries. Although we observed very few individuals growing normally in degraded forest areas of
present habitat. However, this species has the potential to grow even in higher altitudes and may suppress native
tree regeneration.

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Prakasa Rao & Satish (2016) 3(2): 249252
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ACKNOWLEDGEMENT
Authors are thankful to Dr. C. Sudhakar Reddy, Forestry & Ecology Group, NRSC, Hyderabad, Dr. S.B.
Padal, Andhra University, Dr. P. Hari Krishna and Dr. S.L. Meena Arid Zone Regional Centre, Botanical
Survey of India, Jodhpur, for their support and encouragement. We are also grateful to local people for their
help during the field study.

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Pullaiah T & Moulali DA (1997) Flora of Andhra Pradesh, Vol. II. Scientific Publishers, Jodhpur, pp. 813818.
Rao JP, Satish KV, Sankar BS, Reddy CS & Kumar OA (2015) On the occurrence of parasitic plant
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Threatened Taxa 7(2): 69436946.
Rao JP, Satish KV, Reddy CS & Kumar OA (2016) Achyranthes longifolia (Makino) Makino (Amaranthaceae)
- An angiosperm new to India. National Academy Science Letters-Springer (In press).
Reddy CS, Jha CS & Dadhwal VK (2014) Spatial dynamics of deforestation and forest fragmentation (1930-
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Robbins WHG, Lovero CDA, Mayer M & Beetle J (2014) Rhinoncomimus latipes (Coleoptera: Curculionindae)
As a Biological Control Agent For Mile-a-minute, Persicaria perfoliata in New Jersey. Annual Report.
SubbaRao GV & Kumari GR (2008) Flora of Visakhapatnam district, Andhra Pradesh, Vol-II. Botanical
Survey of India, Kolkata, India, pp. 106113.
Wang Z, Xin M, Ma D, Song S, Wang X, Yan C, Zhang D, Feng W, Ma E & Chen J (1990) Farmland weeds in
China. A collection of colored illustrative plates. Agricultural Publishing House, Beijing, China.
Wu Y, Reardon R C & Ding J (2002). Mile-a-minute weed. Biological Control of Invasive Plants in the Eastern
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
3(2): 253262, 2016

Research article

Natural regeneration dynamics of dominant tree species along an


altitudinal gradient in three different forest covers of Darhal
watershed in north western Himalaya (Kashmir), India
M. Hanief*, A. Bidalia, A. Meena and K.S. Rao
Department of Botany, University of Delhi, Delhi-100007, India
*Corresponding Author: haniefdu@gmail.com [Accepted: 17 June 2016]

Abstract: Regeneration is a cost effective natural process by which plants re-establish themselves
and this strategy help the plants to maintain their diversity and genetic identity. This study was
undertaken in three different forest cover types which were dominated by Quercus incana, Persea
duthiei and Abies pindrow, respectively. The aim of the present study was to investigate the
regeneration pattern of dominant tree species along the altitudinal gradient in Darhal watershed,
situated in the Pir Panjal mountain region of Jammu and Kashmir State. A total of 14 tree species
were recorded. The tree density varied between 492 to 1325 individuals/ ha, whereas the total
basal area ranged between 25.88 to 188.90 m2 ha-1. In general the density of seedlings, saplings
and trees increased with increase in elevation. All the three forest types have poor regeneration.
The recruitment of Q. incana seedlings and the rate of conversion of A. pindrow seedlings to
saplings and then saplings to young trees were very low. Proper care needed to prevent excessive
exploitation of these forests and livestock grazing should be controlled in all these forest types to
conserve these forests. Our study results could help for better management plans for sustainable
management and conservation of Himalayan mountain forests, especially in Pir Panjal mountain
region.
Keywords: Darhal watershed - Density - Elevation gradient - Regeneration status.

[Cite as: Hanief M, Bidalia A, Meena A & Rao KS (2016) Natural regeneration dynamics of dominant tree
species along an altitudinal gradient in three different forest covers of Darhal watershed in north western
Himalaya (Kashmir), India. Tropical Plant Research 3(2): 253262]

INTRODUCTION
The Himalayas are one of the richest and vast mountain ecosystems on earth covering India, Pakistan,
Afghanistan, Nepal, Bhutan, Bangladesh, China and Myanmar (Semwal et al. 2004). Himalayas are diverse with
a variety of forest covers due to elevation and climatic gradients as the vegetation community had a direct
relationship with altitude (Mani 1978). The Himalayan forest cover plays a significant role in providing many
ecological services to the human population and their livestock; however management of these forest types have
not been addressed properly due to inadequate reliable information on watershed and forest distinctiveness. A
majority of watersheds in the Himalayan region are experiencing a decline in different forest covers and
agricultural land use type has happened to be the main constituent of the current landscape (Sundriyal et al.
1994). Studies of demographic structure and population dynamics are important for understanding coexistence
mechanism of the tree species and long-term ecological status of forests (Miura et al. 2001). Population
structure determines the dominant status of tree species and development within the forest community (Gairola
et al. 2014). The species existence and recruitment process in a forest mostly depends on its regeneration
potential under varied climatic factors, competition between species, predation and anthropogenic disturbances.
Based on the demographic structure, the regeneration processes of tree species may reveal the seral stage of the
community and understand the prospective climax vegetation of a particular region (Mishra et al. 2013, Gariola
et al. 2014). The presence of the sufficient number of young trees, saplings and seedlings in a specific forest
population indicates that the tree species are able to regenerate successfully (Khan et al. 1987). Jammu &
Kashmir (J&K) State is a part of the north western Himalaya and has rich phytodiversity like rest of western

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Received: 15 May 2016 Published online: 30 June 2016
Hanief et al. (2016) 3(2): 253262
.
Himalaya. These forest types are susceptible to variations in climatic conditions. Moreover, the impacts of
different types of anthropogenic disturbances such as over grazing, lopping, tree extraction, fodder and fuel
wood extraction, road building, defense and other development activities are severe and damaged the tree
diversity in the region (Barik et al. 1996, Gupta 2004, Kumar & Sharma 2014). These forest covers are
considered valuable as they are rich in biodiversity and a sensitive indicator towards climate alterations and
anthropogenic influences (Rawal & Dhar 1997). Inadequate regeneration is the main problem of forests in
mountain regions (Krauchii et al. 2000). Sustainable conservation of forests involves proper planning and
management of seedlings, saplings and young trees that ensure maintenance of forest community structure and
ecological stability (Moravie et al. 1997). Successful management of forest covers needed reliable research data
on aspects such as structural attributes and demographic profile of tree species. Therefore, it is important to
study the population structure and regeneration potentials of different natural forest trees in Kashmir Himalaya
to determine the probable trends of vegetation in the future. Several studies were conducted in western
Himalaya on phytodiversity and phytosociology aspects by various workers (Saxena & Singh 1982, Bankoti
1990, Rawal & Pangtey 1994, Singh et al. 1994, Baduni 1996, Dhar et al. 1997, Baduni & Sharma 1999,
Ghildiyal et al. 1998, Kumar & Ram 2005, Gairola et al. 2008, Gairola et al. 2012; Pala et al. 2012, Gairola et
al. 2014, Kumar & Sharma 2014, Dar & Sundarapandian 2015). However, information related to the
regeneration dynamics of different forest covers in this area is still lacking. Taking into consideration this gap,
we explored the population structure of tree species along the elevation gradients in subtropical, temperate and
subalpine forests of Pir Panjal mountain region of western (Kashmir) Himalaya and to suggest some strategies
for successful regeneration of dominant tree species in this sensitive ecological mountain region.

MATERIALS AND METHODS


Study area
The present study was carried out in Darhal watershed which is situated (latitude 33o233233o2418 N and
longitude 74o183874o3020 E) in Rajouri district of the Jammu and Kashmir state, in the north western
Himalayan biogeographic zone. The watershed lies entirely in the Pir Panjal mountain range (lesser Himalaya)
and is characterized by undulating topography with moderate to very steep slopes towards south and southwest
directions. The area of watershed is about 116.27 km2 and with an elevation ranging from 9003700 m above
mean sea level (m asl). The climate of watershed varies from subtropical to alpine agro climatic zones. The
main drainage in the area flows from the north east to south west discharging into Munawwar Tawi River and is
a part of the Chenab sub basin. The watershed area is inhabited by 11 revenue villages with a population of
about 36227 people.
The age of rocks in the Pir Panjal Mountain region ranges from precambrian to the quaternary period. The
Rajouri district is a part of Autochthonous Folded Belt, one of the three geological structural units in poonch
area of Jammu and Kashmir Himalaya (Wadia 1931). On the northern side, it is bounded by the Panjal thrust
and on the southern side by Murree Thrust (Wadia 1928). Approximately 80% of the area is comprised of
Murree rocks of late Eocene to early Miocene, and are unconformably underlined by Subathu rock Formation.
Murree rock group mainly consists of pink sandstone and clay. Northern part of the Rajouri district consists of
metamorphic and older crystalline rocks, and southern side is composed of a medium to coarse sandstone, light
grey and clay stones. The soils in the study area are generally classified into three types such as Red yellow and
Brown Soils (Utisols), Bhabar Soil (Entisols) and Sub-Mountainous Soil (Alfisols) (Anonymous 2013).
Vegetation survey and data analysis
After the reconnaissance survey, the study area was found to be dominated by three dominant forest types
viz. oak forest (OF), mixed broadleaf forest (MF) and fir forest (FF). Three sites of about 1 ha size were selected
along the altitudinal gradient for each forest type (Table 1). Levels of disturbance in selected sites was assessed
by the degree of anthropogenic pressure in terms of ground grazing, extraction of timber, fuel and fodder,
lopping of trees, frequency of fire incidence and ease of accessibility. Based on a normal scale, the level of
disturbance were grouped into three classes, i.e., very high, moderate and low. For assessing the population
structure of dominant tree vegetation at each site, ten 10 10 m quadrats were randomly laid down, thus a total
of 90 plots were established. In each quadrat, trees with >30 cm CBH (circumference at breast height i.e., 1.37
m above the ground) were enumerated and their circumference was measured individually. Each 10 10 m
Quadrat was divided into four 5 5 m sub quadrats for enumeration of the saplings and seedlings. Individuals

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having circumference between 1030 cm were considered as saplings and those having circumference <10 cm
were considered as seedlings (Knight 1963). The rate of progression of density of seedlings, saplings and adult
trees is considered as the indicator of regeneration potential. The regeneration status of dominant trees in a given
forest type was categorized as:
(i) Good regeneration i.e. if number of seedlings > saplings >adults.
(ii) Fair regeneration i.e. if number of seedlings > or < saplings < adults.
(iii) Poor regeneration i.e. if the species was found as sapling, but no seedlings (Number of saplings maybe
more, less or equal that of adults).
(iv) No regeneration i.e. if individuals of species are present only as adults.
(v) New regeneration i.e. if individuals of species have no adults but present as seedlings or saplings.
Table 1. General details of selected sites of three forests cover types in Darhal watershed.
Forest sites Forest cover type (Dominant) Altitude (m a.s.l.) Disturbance level
OF1 Mainly Quercus incana 1650 High
OF2 Mainly Quercus incana 1800 Moderate
OF3 Mainly Quercus incana 2100 Low
MF1 Mixed broad leaved 1900 High
MF2 Mixed broad leaved 2000 Moderate
MF3 Mixed broad leaved 2150 Low
FF1 Mainly A. pindrow 2250 High
FF2 Mainly A. pindrow 2470 Moderate
FF3 Mainly A. pindrow 2630 Low
Note: OF oak forest, MF mixed broad leaf forest, FF fir forest.
The dominance of a plant species was determined using the Importance Value Index (IVI) of these species.
Vegetation composition was evaluated by analysing the frequency, density, abundance and IVI, using the
standard methods suggested (Mishra 1968, Curtis & McIntosh 1950). The basal area of each tree was calculated
by dividing the square of CBH (circumference at breast height) with 4. The mean basal area was multiplied
with the density of each species to obtain total basal area (TBA) (m2 ha1). The ratio of abundance to frequency
(A/F) for different species was derived to elicit the distribution pattern. The distribution pattern is considered
regular if the A/F ratio is (< 0.025), random (0.0250.050) and contiguous (> 0.050) (Whitford 1949).The
importance value index (IVI) was calculated with the following formulae: IVI = RF + RD + RDo, where RF is the
relative frequency, RD relative density and RDo relative dominance (Phillips 1959).

RESULTS
Phytosociological analysis of tree species
A total of 14 tree species were recorded in tree stratum in three different forest types in the study area. Q.
incana, P. duthiei and A. pindrow were the dominant tree species at all the sites of oak forest (OF), mixed
broadleaf forest (MF) and fir forest (FF) in terms of their Importance Value Index (IVI), respectively. In OF1,
the highest importance and tree density was recorded for Q. incana (IVI = 147.06, D = 529 individuals ha-1,
TBA = 30.60 m2 ha-1) followed by Pinus roxburghii, Pyrus pashia, Ficus palmata and Acacia nilotica (Table 2).
Likewise Q. incana also showed the highest importance and density in OF2 (IVI = 191.56, D = 608 individuals
ha-1, TBA = 24.40 m2 ha-1) and OF3 (IVI = 234.58, D = 780 individuals ha-1, TBA = 28.23 m2 ha-1) followed by
Olea cuspidata and other tree species (Table 2). Maximum value of frequency was observed for Q. incana (100
%) at all the three oak forest stands while the lowest Frequency was observed for F. palmata (40%), Punica
granatum (40 %) and O. cuspidate (30 %) in OF1, OF2 and OF3, respectively. The abundance to frequency
(A/F) ratio of different tree species ranged from 0.02 to 0.05, 0.01 to 0.06 and 0.03 to 0.11 for OF1, OF2 and
OF3, respectively. Among three sites of oak forest, the maximum total tree density and total basal area was
observed for OF1 (988 individuals ha-1 and 49.14 m2 ha-1) followed by OF3 (934 individuals ha-1 and 30.61 m2
ha-1) and OF2 (817 individuals ha-1 and 28.78 m2 ha-1) (Table 2).
In mixed broadleaf forest type stands studied, a total of 6 tree species were encountered during study period.
In MF1, the highest importance and tree density was observed for P. duthiei (IVI = 182.50, D = 784 individuals
ha-1, TBA = 20.40 m2 ha-1) followed by Q. incana, P. pashia and E. umbellate (Table 3). In MF2 and MF3, the
highest values of importance and tree density were also recorded for P. duthieii (IVI = 224.57, D = 739
individuals ha-1, TBA = 23.00 m2 ha-1 and IVI = 169.42, D = 910 individuals ha-1, TBA = 30.50 m2 ha-1)
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followed by Buxus wallichiana (IVI = 44.35, D = 70 individuals ha-1, TBA = 0.69 m2 ha-1 and IVI = 84.8, D =
319 individuals ha-1, TBA = 14.67 m2 ha-1), respectively. The least values of importance and density were
recorded for Persea odoratissima both in MF2 and MF3. P. duthiei showed the maximum frequency of 90% in
MF1 and 100% each in MF2 and MF3, respectively. In MF1, the A/F ratios for different tree species ranged
from 0.02 to 0.10 whereas in MF2 and MF3 it ranged from 0.02 to 0.08 and 0.02 to 0.09 respectively (Table 3).
The highest tree density and total basal area among three stands of mixed broadleaf forest were observed for
MF3 (1325 individuals ha-1 and 48.97 m2 ha-1) followed by MF1 (1129 individuals ha-1 and 27.00 m2 ha-1) and
MF2 (839 individuals ha-1 and 25.88 m2 ha-1), respectively.
Table 2. Phytosociological analysis of trees at three different sites of Oak forest.
Density
Forest sites Tree species Frequency % Ab A/F TBA (m2 ha-1) IVI
(individuals ha-1)
OF1 Q. incana 100 529 5.29 0.05 30.6 147.06
P. roxburgii 70 250 3.57 0.05 11.9 71.39
A. nilotica 50 60 1.20 0.02 1.54 24.83
P. pashia 60 78 1.30 0.02 1.70 30.10
F. palmata 40 71 1.78 0.04 3.40 26.61
Total 988 49.14
OF2 Q. incana 100 608 6.08 0.06 24.4 191.56
O. cuspidata 50 71 1.40 0.03 1.91 31.34
P. granatum 40 38 0.95 0.02 0.50 19.30
P. pashia 60 51 0.85 0.01 1.12 29.50
M. philiphinensis 60 49 0.82 0.01 0.85 28.31
Total 817 28.78
OF3 Q. incana 100 780 7.8 0.08 28.23 234.58
O. cuspidata 30 99 3.3 0.11 1.66 33.66
P. pashia 40 55 1.38 0.03 0.72 31.76
Total 934 30.61
Note: Ab: Abundance, A/F: Abundance/Frequency ratio, IVI: Importance Value Index, TBA: Total Basal Area.

Table 3. Phytosociological analysis of trees at three different sites of Mixed broadleaf forest.
Density
Forest sites Tree species Frequency % Ab A/F TBA (m2 ha-1) IVI
(individuals ha-1)
MF1 Q. incana 50 223 4.46 0.10 4.50 57.25
P. duthiei 90 784 8.71 0.09 20.40 182.50
E. umbellata 50 42 0.84 0.02 0.90 27.89
P. pashia 40 80 2.00 0.05 1.20 28.20
Total 1129 27
MF2 P. duthiei 100 739 7.39 0.07 23.00 224.57
P. odoratissima 40 30 0.75 0.02 2.19 31.09
B. wallichiana 30 70 2.33 0.08 0.69 44.35
Total 839 25.88
MF3 P. odoratissima 80 96 1.20 0.02 3.80 45.77
B. wallichiana 80 319 3.99 0.05 14.67 84.80
P. duthiei 100 910 9.10 0.09 30.50 169.42
Total 1325 48.97
Note: Ab: Abundance, A/F: Abundance/Frequency ratio, IVI: Importance Value Index, TBA: Total Basal Area
In fir forest, only 2 tree species were encountered and A. pindrow has highest values of Importance and
density at all the three sites studied i.e., FF1 (IVI = 231.62, D = 392 individuals ha-1, TBA = 156.30 m2 ha-1),
FF2 (IVI = 240.47, D = 463 individuals ha-1, TBA = 159.60 m2 ha-1) and FF3 (IVI = 238.89, D = 619
individuals ha-1, TBA = 163.50 m2 ha-1) (Table 4). The least values of frequency, density and TBA were
observed for A. smithiana at the all three sites studied (Table 4). Maximum total tree density was recorded for
FF3 (690 individuals ha-1) followed by FF2 (526 individuals ha-1) and FF1 (492 individuals ha-1), whereas the
highest value for total basal area was observed for FF3 (188.90 m2 ha-1) followed by FF1 (183.20 m2 ha-1) and
FF2 (178.30m2 ha-1).

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Table 4. Phytosociological analysis of trees at three different sites of Fir forest.
Forest Density
Tree species Frequency % Ab A/F TBA (m2 ha-1) IVI
sites (individuals ha-1)
FF1 A. pindrow 100 392 3.91 0.04 156.3 231.62
A. smithiana 50 100 2.00 0.04 26.9 68.38
Total 492 183.2
FF2 A. pindrow 100 463 4.60 0.05 159.6 240.47
A. smithiana 60 63 1.00 0.02 18.7 59.53
Total 526 178.3
FF3 A. pindrow 100 619 6.19 0.06 163.5 238.89
A. smithiana 50 71 1.40 0.03 25.4 61.11
Total 690 188.9
Note: Ab: Abundance, A/F: Abundance/Frequency ratio, IVI: Importance Value Index, TBA: Total Basal Area
Regeneration status of dominant tree species
All the 14 tree species present in the study area were also recorded as juveniles in the studied plots (Table 5).
The density of adult trees, saplings and seedlings varied greatly among dominant tree species along the elevation
gradient. In OF1, the highest density for saplings and seedlings were recorded for P. roxburghii (143 and 190
individuals ha-1) followed by Q. incana (91 and 188 individuals ha-1), whereas the lowest density of saplings and
seedlings were observed for F. palmate (16 and 33 individuals ha-1) and A. nilotica (18 and 32 individuals ha-1).
In OF2, the maximum density for saplings and seedlings were recorded for Q. incana (287 and 210 individuals/
ha) whereas the lowest density for saplings and seedlings were obtained for P. granatum (20 and 9 individuals
ha-1) and P. pashia (11 and 36 individuals ha-1) and O. cuspidata (11 and 33 individuals ha-1). Likewise
maximum density for saplings and seedlings in OF3 were observed for Q. incana (371 and 312 individuals ha-1).
However, the minimum density for saplings and seedlings were recorded for P. pashia (30 and 19 individuals
ha-1).
In MF1, the maximum and minimum density for saplings and seedlings were recorded for P. duthiei (503
and 375 individuals ha-1) and Elaeagnus umbellata (33 and 40 individuals ha-1). Similar to MF1, in MF2 the
maximum density for saplings and seedlings were observed for P. duthiei (863 and 453 individuals ha-1) and
MF3 (1230 and 534 individuals ha-1). The lowest density for saplings and seedlings were obtained for P.
odoratissima both in MF2 (23 and 13individuals/ haand MF3 (33 and 20 individuals ha-1).In case of subalpine
forest cover, the maximum density of saplings and seedlings was recorded for A. pindrow followed by A.
smithiana at all the three sites of fir forest cover i.e., FF1, FF2 and FF3 (Table 5). The maximum and minimum
density of saplings and seedlings for A. pindrow was recorded in FF3 (64 and 231 individuals ha-1) and FF1 (20
and 98 individuals ha-1).
Table 5: Regeneration status of tree species in selected sites of three forest cover types.
Tree species OF1 OF2 OF3 MF1 MF2 MF3 FF1 FF2 FF3
Q. incana Sed. 188 210 312 121 - - - - -
Sap. 91 287 371 160 - - - - -
Trees 529 608 780 223 - - - - -
Status F P P P
P. roxburgii Sed. 190 - - - - - - - -
Sap. 143 - - - - - - - -
Trees 250 - - - - - - - -
Status F
A. nilotica Sed. 32 - - - - - - - -
Sap. 18 - - - - - - - -
Trees 60 - - - - - - - -
Status F
P. pashia Sed. 41 36 19 49 - - - - -
Sap. 22 11 30 40 - - - - -
Trees 78 51 55 80 - - - - -
Status F F P P
F. palmata Sed. 33 - - - - - - - -
Sap. 16 - - - - - - - -
Trees 71 - - - - - - - -
Status F
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O. cuspidata Sed. - 33 14 - - - - - -
Sap. - 11 42 - - - - - -
Trees - 71 99 - - - - - -
Status F P
P. granatum Sed. - 9 - - - - - - -
Sap. - 20 - - - - - - -
Trees - 38 - - - - - - -
Status P
M. philipinensis Sed. - 15 - - - - - - -
Sap. - 34 - - - - - - -
Trees - 49 - - - - - - -
Status P
E. umbellata Sed. - - - 40 - - - - -
Sap. - - - 33 - - - - -
Trees - - - 42 - - - - -
Status P
P. duthiei Sed. - - - 375 453 534 - - -
Sap. - - - 503 863 1230 - - -
Trees - - - 784 739 910 - - -
Status P P P
P. odoratissima Sed. - - - - 13 20 - - -
Sap. - - - - 23 33 - - -
Trees - - - - 30 96 - - -
Status P P
B. wallichiana Sed. - - - - 0 11 - - -
Sap. - - - - 7 95 - - -
Trees - - - - 70 319 - - -
Status P P
A. pindrow Sed. 98 154 231
Sap. - - - - - - 20 32 64
Trees - - - - - - 391 460 619
Status P P P
A. smithiana Sed. - - - - - - 28 29 37
Sap. - - - - - - 9 7 11
Trees - - - - - - 100 60 70
Status P P P
Note: P= poor regeneration and F= fair regeneration.

DISCUSSION
Phytosociological analysis of tree species
Forest structure and species composition are determined and regulated by a variety of ecological and
anthropogenic factors (Dolezol & Srutek 2002). Regular human interventions like overgrazing, lumbering and
encroachments of forest areas are among the key regulatory factor controlling the distribution of species (Muller
& Ellenberg 1974). Furthermore altitude is also one of the most important factors which determine the
distribution of tree species due to its direct effect on the microclimatic conditions of the habitat (Rawal &
Pangtey 1994, Singh et al. 2009). In the present study the tree density of the dominant species increased with
increase in altitude. Such trends were also reported by Rawat & Singh (2006) in various forest communities in
Great Himalayan National Park (GHNP) in north western Himalaya. However, opposite trends were also
reported from other parts of north western Himalaya where the density of trees decreased with increase in
altitude (Gairola et al. 2014, Kumar & Sharma 2014). In the present investigation the total density of dominant
tree species in OF, MF and FF types ranged from 529 (OF1) to 780 individuals per ha (OF3), 784 (MF1) to 910
individuals per ha (MF3) and 392 (FF1) to 619 individuals per ha (FF3) in oak, mixed and fir forests,
respectively. These values are similar to the tree density values reported by other studies in the north western
Himalaya such as for Quercus species (Singh et al. 1994, Ghildiyal et al. 1998, Kumar & Ram 2005, Sharma et
al. 2001), A. pindrow (Baduni et al. 1996, Dhar et al. 1997, Sharma et al. 2010, Gairola et al. 2010, Gairola et
al. 2014) and P. duthiei (Pant & Samant 2012). Total basal area of trees did not showed any trend with change
in altitude. In the present study total basal area of OF ranged from 28.78 to 49.14 m2 ha-1 which was similar to

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the values recorded in other oak forests of western Himalaya (Saxena & Singh 1982, Ghildiyal et al. 1998,
Kumar & Ram 2005). Total basal area of MF ranged from 25.88 to 48.97m2 ha-1 which was lower than the
values reported from other temperate broadleaved Himalayan forests (Pant & Samant 2012). We assume that,
this may be due to greater number of trees in smaller CBH classes in the study sites. Total basal area of FF
ranged from 156.30to 178.3 m2 ha-1 which was comparatively higher than the values reported for similar forest
types in western Himalaya (Baduni et al. 1996, Dhar et al. 1997, Gairola et al. 2010, Dar & Sundarapandian
2015). This may be due to the fact that the A. pindrow trees were more in larger CBH classes in our study area.
Regeneration status of dominant tree species
The occurrence of a sufficient number of young trees, saplings and seedlings in a given forest population
indicates successful regeneration (Saxena & Singh 1984).The above three life stages for various tree species
signify the probable forest structure in future. In general all the 14 tree species analysed in this study were found
to be more or less regenerating. The overall regeneration status was poor in these forests as survival from
saplings to poles was found to be almost non-existing in all the studied sites. Poor regeneration of Quercus
species in Himalayan mountain forests was also reported by other workers from time to time (Saxena & Singh
1982, Thadani & Ashton 1995) and anthropogenic disturbance was attributed for this. However, good
regeneration of Quercus species was also observed in similar forests of north western Himalaya (Gairola et al.
2012, Pala et al. 2012) when the extent of such forests was large or stricter management regimes were in place.
In the present study P. duthiei in all the studied sites (MF1, MF2 and MF3) shows poor regeneration with low
rate of conversion to trees from sapling stage in MF2 and MF3. Inadequate regeneration pattern of P. duthiei
was also observed by Pant and Samant (2012) in Khokhan Wildlife Sanctuary (KhWLS) in north western
Himalaya. B. wallichiana, a multipurpose high value tree species was present in MF2 and MF3, but shows very
poor to no regeneration. Uses of B. wallichiana in wood craft industry, extraction for fodder and fuel are the
possible reasons for its very poor conversion from lower CBH to higher CBH classes. Similar results were also
reported by Pant (2011) and Ahmed et al. (2015) in Rajouri and Poonch districts of J&K State. A. pindrow, an
important timber yielding species in the study area also showed inadequate representation in young stages. In
addition to livestock grazing, there are many other factors which hamper the regeneration of A. pindrow such as
undecomposed layer of litter (due to low temperature) (Troup 1921), growth of noxious weeds and release of
hydrophobic substances from litter decomposition (Jha et al. 1984). Moreover, drainage conditions in the A.
pindrow dominated forests also limit the wetting of soil which hampered its natural regeneration (Troup 1921).
Therefore, A. pindrow forests in the study area along with those in the region needed proper care from the forest
department officials and local people so that the seedlings and saplings could survive and replace adult trees in
future. We believe there are possibilities of destruction of this important forest tree species if present state of
affairs continues for longer period of time.
The anthropogenic disturbance in the study area is high and hence inadequate regeneration is expected for
Quercus species. OF1 and OF2 were situated in the vicinity of villages (Dodaj & Chowkian) and thus
anthropogenic disturbances such as ground grazing by cattle, tree lopping, and extraction of fuel wood, fodder
and timber were also observed during the present study. According to Singh (1992), the grazing by cattle is the
basic cause of poor regeneration of Quercus species in the Central Himalayan forest. However, OF3 located at
higher elevation (2100 m asl.) and comparatively away from the village settlements and was subjected to lesser
human disturbances and shows fair regeneration. Sharma et al. (2008) also observed the impacts of human
interferences on forest degradation in Birhun watershed, which is situated in district Udhampur of J&K State.
Kumar & Sharma (2014) also reported that the impacts like tree felling, looping, grazing by domestic cattle, fire
incidents, extraction of fodder and fuel wood are responsible for inadequate regeneration of forest trees in
Padder valley area of northwest Himalaya in J&K State. Similar impacts of human interference on forest plant
species composition was also reported by Kiran et al. (1999) and Kumar & Hamal (2009) from Poonch and
Kishtwar district of J&K State. In the present study the effect of fire was found to be low to moderate. Forest
litter was used as bedding material in the animal sheds and soil erosion was observed in the forests due to
human disturbances and steep slopes in the area. Overall the livestock grazing pressure in the study area was
found to be high and transhumant communities such as Gujjar, Bakerwal and some Paharis in the area migrate
to alpine pastures during summer for grazing their cattles, goats and sheeps. The grazing, browsing and
trampling by livestock cause a considerable mortality of saplings and seedlings and hence hamper the natural

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regeneration of forest trees in the study area. Krzic et al. (2006) who studied the effects of cattle grazing on
composition of plant species in central interior British Columbia also reports similar results.

CONCLUSIONS
The present study revealed that the tree density of dominant tree species showed marginal increase with
increase in altitude. Most of the dominant tree species showed poor regeneration which warrants the immediate
attention of forest managers and local forest dependent communities. In case of oak forest if current state of
recruitment of seedlings and saplings stage does not improve, its tree population composition might change in
the long run. In mixed broad leaved forest (MF2 and MF3) comparatively greater number of individuals in
saplings stage indicates that the rate of conversion of saplings to tree CBH size classes was not proportional.
Moreover, it also suggests that if present state of seedling recruitment continues the tree population may decline
in future. The results of this study also revealed that A. pindrow, an important timber yielding tree species of the
region showed poor regeneration. Therefore, if the current pattern of conversion of saplings to trees in this
subalpine forest continues, the fir forest might be replaced in future. Present study also revealed that the studied
forest types are under different anthropogenic pressures from growing population of neighboring human
settlements for higher demands of timber, fuel, fodder and ground grazing and therefore needs some
management and silvicultural intervention. Some of the prescription may include implementation of controlled
extraction and grazing, enrichment planting of tree species in protected forest compartments, proper lopping,
less use of wood for house construction and fuel by providing alternative options. In addition the formulation
and implementation of effective conservation strategies are required for proper regeneration of some of the
ecologically and economically important tree species such as A. Pindrow, B. wallichiana, P. duthiei and Q.
incana.

ACKNOWLEDGEMENTS
Authors would like to thank university grant commission (UGC) for providing financial support. We are
grateful to the forest department of district Rajouri, J&K for their support during study. We also thank Dr. J.
Deka, post-doctoral fellow, University of Guwahati, Assam and Dr. Nirmala Pandey and Krati Vikram,
University of Delhi, Delhi for fruitful discussions.

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ISSN (E): 2349 1183
ISSN (P): 2349 9265
3(2): 263271, 2016

Research article

Phytosociological assessment & distribution patterns of tree species


in the forests of Doon Valley, Shivalik hills of lower Himalaya
Mohommad Shahid* and Shambhu Prasad Joshi
Ecology Research Laboratory, Department of Botany, D.A-V. (P.G.) College, Dehra Dun, Uttarakhand, India
*Corresponding Author: mdshahid07@yahoo.com [Accepted: 18 June 2016]

Abstract: Study was conducted in the three forest ranges (Barkot, Lachchiwala and Thano) of
Dehra Dun forest division, Doon Valley, Uttarakhand, India. Phytosociological studies of the sites
were conducted for tree species. Fifty quadrats of 10 10 m2 size were laid on each site for
studying the trees composition and structure. Diversity Indices were calculated for each site.
Species richness ranged between six species in Thano to 15 species in Barkot. Two Way Indicator
Species Analysis was performed and classified tree layer into eight groups depending on the eigen
value.
Keywords: Dominance - Diversity - TWINSPAN - Classification.

[Cite as: Shahid M & Joshi SP (2016) Phytosociological assessment and distribution patterns of tree species in
the forests of Doon Valley, Shivalik hills of lower Himalaya. Tropical Plant Research 3(2): 263271]

INTRODUCTION
Vegetation is an essential part of the ecosystem that interprets the effects of the total environment (Billings
1952). Vegetation complex fluctuates from one season to another season in a cyclic manner over the years in a
successional way and the fluctuations suggest a response by each species population to prevailing heat, moisture
and light as modified by the vegetation itself (Heady 1958). The development and deterioration of plant species
alters the pattern of the species distribution in community (Watt1964). Vegetation emphasis on study of
composition, development, geographic distribution and environmental relationships of plant communities
(Legendre & Fortin 1989, Kolasa & Rollo 1991).
Plants growing together have mutual relationship among themselves and with the environment (Mishra et al.
1997). These interactions among different plants and between plants and their environment result in the outcome
of different vegetation types in different areas. The quantitative relationship between rare and profusely growing
species is a significant structural property of a community. Phytosociological analysis of a plant community is
the first and foremost basis of the ecological study of any piece of vegetation and this study is important to
understand the functioning of any community. Oosting (1956) suggested the importance of phytosociological
parameters for spatial problems in sociological behaviour of plants.
The Sal (Shorea robusta) forests of Doon Valley are one of the most frequently studied vegetation tracts.
The wooded communities have been described in detail, and studies on Sal regeneration are the standards in the
ecological and forestry literature of India (Champion 1923, Bhatnagar 1960, Champion & Seth 1968). The
ground layer has also received attention (Rajvanshi et al. 1983). The Doon Valley forests are however
increasingly noted for their wildlife values and ecological and forestry studies.
Urbanization, a new sign of prosperity, and decrease in forest cover are altering the temperature and
reducing the rainfall of Doon Valley (Negi & Chauhan 2002). The anthropogenic disturbances have created
wide openings reducing the soil moisture of the forest floor and the Sal regeneration. Doon Valley is
experiencing the wide range of anthropogenic disturbances like cattle grazing, fuel and fodder collection,
increase in the population near the villages of the study sites. In recent years, after the inception of Uttarakhand
in year 2000, Dehra Dun city (Doon Valley) is expanding exponentially. Other sources of disturbances in this
region are the National Highway connecting Dehra Dun and the holy cities of Haridwar and Rishikesh, forest
fires, collection of fodder and fuelwood, grazing of cattle, etc. The climate changes in the valley have forced the
locals to go for cooler places like Lachchiwala, putting extra pressures on the nearby forest. All these
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.
perturbations have put impact on the dominance and diversity of the forests of Doon Valley. Therefore, the
study is an attempt to address the following issues: (1) the current status of different plant species in the Doon
Valley; (2) the frequency, distribution patterns, and abundance of tree species in the valley.

MATERIALS AND METHODS


Study Site
The Doon valley is situated in the Siwalik Himalayas, between latitudes 2955 to 3030 N and 7735 to
7824 E. It is saucer-shaped and ca. 20 km wide & 80 km long valley with 2100 km2 geographical area (Fig.
1). In east the area is bounded by river Ganga and in West by river Yamuna. Mussoorie hills formed its northern
boundary whereas the Shiwalik Mountains formed the southern boundary of the valley. The climate of the area
is sub-tropical; the average maximum and minimum temperatures are 27.65C and 13.8C, respectively, and the
average rainfall is 202.54 cm. The soils are developed on the deep alluvial deposits with the parent material
derived from the Doon Alluvium. In these studied subtropical deciduous forests, dominant species is Shorea
robusta (Sal), with associates such as Anogeissus latifolia, Terminalia tomentosa, Adina cordifolia,Terminalia
bellirica etc.

Figure 1. Location of Study Sites.


Methodology
The study was conducted in Barkot forest range, Lachchiwala forest range and Thano forest range of Dehra
Dun forest division in Doon Valley. Forests of Doon Valley were thoroughly surveyed for its topography,
microclimate and biotic stress conditions. Phytosociological studies of the selected sites were conducted once in
the year 201011. The vegetation was analysed by means of random sampling to give most representative
composition of vegetation. Fifty quadrats of 10 10 m2 size were laid on each site for studying the trees
composition. In each tree quadrat cbh (circumference at breast height i.e., at 1.37 m above ground level) of each
tree (>10 cm cbh) was measured. Vegetation composition was evaluated by analyzing the frequency, density,
abundance and importance value index (IVI) according to Curtis & McIntosh (1951) and Mishra (1968) and as
given below:
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( )

( )

( )

Importance Value Index (IVI) = Relative Frequency + Relative Density + Relative Dominance

Diversity Indices and Evenness


The Shannon and Wiener diversity index (H) was calculated using the formula (Shannon &Wiener 1963)
s
H = pi ln pi
i 1
where, s = the number of species, pi = the proportion of individuals or abundance of the ith
species expressed as a proportion of total cover, ln = log base n
Alpha diversity () is within area diversity, measured as the number of species occurring within an area of
given size (Huston 1994). It is therefore a measure of richness of a potentially interactive assemblage of species.
Beta diversity () was introduced by Whittaker (1960) to designate the degree of species change along a
given habitat as such it is a measure of between area diversity. It indicates rate of proportion and is normally
represented in terms of similarity index or of a species turnover rate.
()
where, Sc is the total number of species encountered in all communities and S is the average
number of species per community.
Concentration of dominance (Cd) was measured by Simpsons index (Simpson 1949)on the basis of their
density.
s
Cd = (p )
i 1
i
2

where, pi is the proportion of ith species and s is the number of individuals of all the species.
Equitability or Evenness was calculated by as given by Pielou (1969).
s

H' p ln pi i
Equitability (J) = i 1
H'max ln s
where, s = the number of species, pi = the proportion of individuals or abundance of the ith
species expressed as a proportion of total cover, ln = log base n.
Dominance-Diversity Curves (D D curves)
Dominance-Diversity curves exhibit the community organization in terms of resource share (Saxena &
Singh 1982, Pande et al. 2001). The logarithm values of IVI of the species have been ordinated against the
species sequence to draw D-D curves (Dominance-Diversity curves) for different studied sites to interpret the
community organization in terms of resource share and niche-space.
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TWINSPAN
In the present study, TWINSPAN ordination, an indirect ordination was done. The classification of species
was done according to their ecological preferences. The classifications were used together to obtain an ordered
two-way table that expresses the synecological relations of species as succinctly as possible. TWISNPAN for
Windows Version 2.3 (Hill & Smilauer 2005) was used for the analysis.

RESULTS
Dominance (Importance Value Index) of trees at Barkot range
Shorea robusta was the dominant species in the Barkot range, with the IVI value 141.32. The Co-dominant
species are Mallotus philippensis and Ehretia laevis with IVI value 33.53 and 31.68 respectively. A total of 15
species are recorded from the Barkot range. 20% frequency was reported by the three species viz. Bauhinia
variegata, Syzygium cumini and Cassia fistula. 30% frequency was recorded for the only one species Adina
cordifolia. The minimum IVI value (3.60) was reported for the Casearia tomentosa. 10% frequency was
observed by the eight species. Shorea robusta has the 47% share in the forest of the Barkot range. Density of 15
trees 100 m-2 was recorded in the Barkot range (Table 1).
Table 1. Distribution analysis of tree species in Barkot range.
Species F D A A/F IVI
Shorea robusta Roxb. ex Gaertner f. 100 9.0 9.00 0.09 141.32
Mallotus philippensis (Lam.) Muell.-Arg 60 2.1 3.50 0.06 33.53
Ehretia laevis Roxb 70 1.8 2.57 0.04 31.68
Terminalia bellirica (Gaertner) Roxb. 10 0.2 2.00 0.20 14.40
Adina cordifolia (Roxb.) Hook.f. ex Brandis 30 0.4 1.33 0.04 11.56
Syzygium cumini (L.) Skeels 20 0.4 2.00 0.10 10.21
Cassia fistula L. 20 0.2 1.00 0.05 9.48
Ougeinia oojeinensis (Roxb.) Hochre. 10 0.1 1.00 0.10 9.04
Bauhinia variegate L. 20 0.2 1.00 0.05 8.34
Miliusa velutina (Dunal) Hook.f. & Thom 10 0.1 1.00 0.10 6.52
Terminalia alata Heyne 10 0.1 1.00 0.10 6.10
Anogeissus latifolia Wall. 10 0.1 1.00 0.10 5.35
Ficus benghalensis L. 10 0.1 1.00 0.10 4.56
Litsea glutinosa (Lour.) Robinson 10 0.1 1.00 0.10 4.30
Casearia tomentosa Roxb. 10 0.1 1.00 0.10 3.60
Note: F= Frequency (%), D=Density (tree 100m-2), A=Abundance, IVI= Importance Value Index.
Dominance (Importance Value Index) of trees at Lachchiwala range
The maximum IVI (126.36) was for the Shorea robusta. Mallotus philippensis and Syzygium cumini has the
IVI value of 33.28 and 29.84 respectively. 100% Frequency was recorded for the Shorea robusta. 10 %
Frequency was observed for the seven species. Terminalia alata, Adina cordifolia and Anogeissus latifolia had
the frequency of 20% in the Lachchiwala range. A Density of 11.70 tree 100m-2 was recorded in the
Lachchiwala range. The A/F ratio was in the range from 0.04 for the Mallotus philippensis to 0.20 for Miliusa
velutina. The minimum IVI Value (4.02) was recorded for the Caseaseria tomentosa (Table 2).
Table 2. Distribution analysis of tree species in Lachchiwala range.
Species F D A A/F IVI
Shorea robusta Roxb. ex Gaertner f. 100 6.5 6.50 0.07 126.36
Mallotus philippensis (Lam.) Muell.-Arg 60 1.6 2.67 0.04 33.28
Syzygium cumini (L.) Skeels 40 1.4 3.50 0.09 29.84
Terminalia bellirica (Gaertner) Roxb. 20 0.2 1.00 0.05 28.51
Ehretia laevis Roxb 30 0.8 2.67 0.09 17.18
Adina cordifolia (Roxb.) Hook.f. ex Brandis 20 0.2 1.00 0.05 9.63
Anogeissus latifolia Wall. 20 0.2 1.00 0.05 9.28
Miliusa velutina (Dunal) Hook.f. & Thomson 10 0.2 2.00 0.20 9.01
Cordia dichotoma G.Forst 10 0.1 1.00 0.10 7.67
Terminalia alata Heyne 10 0.1 1.00 0.10 7.67
Bauhinia variegate L. 10 0.1 1.00 0.10 6.57
Cassia fistula L. 10 0.1 1.00 0.10 6.57
Flacourtia indica (Burm.f.) Merrill 10 0.1 1.00 0.10 4.42
Caseaseria tomentosa Roxb. 10 0.1 1.00 0.10 4.02
Note: F= Frequency (%), D=Density (tree 100m-2), A=Abundance, IVI= Importance Value Index
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Dominance (Importance Value Index) of trees at Thano range
The Shorea robusta has the maximum IVI (187) in the Thano range. The Mallotus philippensis and
Syzygium cumini had the IVI value 38.97 and 25.36 respectively. A total of six species are recorded in the
Thano range. The maximum share was by the Shorea robusta. 20 % Frequency was recorded for the three
species viz. Syzygium cumini, Tectona grandis and Terminalia alata. The A/F ratio was in the range of 0.04 to
0.36. The minimum IVI Value (15.69) was of the Terminalia alata (Table 3).
Table 3. Distribution Analysis of Tree species in Thano range.
Species F D A A/F IVI
Shorea robusta Roxb. ex Gaertner f. 100 10.7 10.70 0.11 187.00
Mallotus philippensis (Lam.) Muell.- Arg. 50 1.4 2.80 0.06 38.97
Syzygium cumini (L.) Skeels 20 0.2 1.00 0.05 25.36
Ehretia laevis Roxb 30 0.4 1.33 0.04 17.28
Tectona grandis L. f. 20 0.2 1.00 0.05 15.71
Terminalia alata (Gaertner) Roxb. 20 0.2 1.00 0.05 15.69
Note: F= Frequency (%), D=Density (tree 100m-2), A=Abundance, IVI= Importance Value Index.
Diversity Measurements
-diversity (Species Richness) was the maximum 15 from the Barkot range. Lachchiwala and Thano ranges
have reported the -diversity (Species Richness) 14 and 06 respectively. () Beta Diversity 5.83 was recorded
from Thano range. The highest Concentration of Dominance (Cd) (0.4213) was recorded from the Thano range
while the lowest Concentration of Dominance (Cd) (0.2175) was recorded from the Lachchiwala range. The
Barkot range has 0.255 Concentration of Dominance (Cd). The highest H (2.023) was recorded in the
Lachchiwala range while the lowest H (1.242) was recorded in Thano range. 2.023 H was recorded from
Lachchiwala range. The maximum value of Evenness (J) (0.577) was recorded in the Thano range while the
minimum (0.4683) was recorded in the Barkot range (Table 4).
Table 4. Diversity index of trees at various study sites.
Indices Barkot range Lachchiwala range Thano range
Richness () 15 14 06
Diversity () 2.33 2.50 5.83
Dominance (Cd) 0.255 0.2175 0.4213
Shannon(H) 1.949 2.023 1.242
Evenness (J) 0.4683 0.5401 0.577
Dominance-Diversity (D-D) Curve

Figure 2. Dominance-Diversity curve of the tree species at various study sites. (BR- Barkot range, LR-
Lachchiwala range, TR- Thano range)
Importance values of plants were plotted against the species sequence to draw the Dominance-Diversity
curves (D-D curves) and interpret the community organization in terms of resource share and niche-space. A D-
D curve for the tree layer is shown in figure 2. In the present study, all the sites represented either geometric or
log normal distributions. Log normal series indicative of the highly mixed nature of vegetation. The geometric
form is often shown by vascular plants having lower density (Whittaker 1975).
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TWINSPAN- (Two-way Indicator Species Analysis)
Species Classification: A total of 18 tree species were analysed with TWINSPAN. Division number 1 classified
the 18 species with an Eigen Value 0.364 into 12 species in the Left Hand side and 06 species in the Right Hand
Side (RHS). A total of eight groups are formed after the classification with the TWINSPAN (Fig. 3).

Figure 3. Classification of Tree Species on the basis of TWISNPAN.


Group III is the largest groups with five tree species Anogeissus latifolia, Bauhinia variegata, Caseaseria
tomentosa, Cassia fistula, Miliusa velutina. One species each is present in the four groups (Group II, V, VII and
VIII). Group I is having the three species namely Ficus benghalensis, Litsea glutinosa, Ougeinia oojeinensis.
Shorea robusta along with Ehretia laevis, Mallotus philippensis and Syzygium cumini forms the group VI. Two
species Cordia dichotoma, Flacourtia indica are present in the Group IV (Table 5).
Table 5. Clustering of Tree species at various study sites.
Group No. of Species Name of Species
I 03 Ficus benghalensis, Litsea glutinosa, Ougeinia oojeinensis
II 01 Adina cordifolia
III 05 Anogeissus latifolia, Bauhinia variegata, Caseaseria tomentosa, Cassia
fistula, Miliusa velutina
IV 02 Cordia dichotoma, Flacourtia indica
V 01 Terminalia alata
VI 04 Ehretia laevis, Mallotus philippensis, Shorea robusta, Syzygium cumini
VII 01 Terminalia bellirica
VIII 01 Tectona grandis

DISCUSSION
The mosaics of species distribution in any forest are governed by various environmental factors (Bajpai et al.
2012). Bliss (1963), Douglas & Bliss (1977) and Billings (1973) have reported that vegetation of any place is
the outcome interaction of many factors like meso-topographic gradients, the elevation, soil, species
composition and biotic interferences. It is also reported that the regional patterns of species richness are
consequences of many interacting factors, such as plant productivity, competition, geographical area, historical
or evolutionary development, regional species dynamics, regional species pool, environmental variables and
human activity (Woodward 1988, Palmer 1991, Eriksson 1996, Zobel 1997, Criddle et al. 2003). The values of
vegetation parameters obtained for most of the sites in the present study fall within a comparable range of values
reported for Western Himalaya (Kala & Uniyal 1999).
The site is a natural Sal forest and lopping of these trees for fuel and fodder, along with extraction of
medicinal (Zingiber roseum) and ethanobotanical (Pterospermum acerifolium, Calamus tenuis etc.) plants are
the major disturbances prevailing in the region. In addition to this, in the recent years, over mature Sal, and
those infested by Hoplocerambyx spinicornis (Sal borers) were also removed by the forest department. All these
disturbances have resulted in large canopy gaps and tampering of forest soils, which has made the forest floor
vulnerable to runoff during rains. With the runoff of the top soil all the nutrients are washed away and the soil
becomes oligotrophic. Evergreen species are better adapted to oligotrophic soils as they can amortilize the
nutrients over a longer period of time (Grime 1973) with their greater tissue longevity (Hireman et al. 2002) and
greater nutrient storage and recycling. Dominance of perennial grasses shows their competitive success under

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stress conditions (Pandey &Singh 1987) as they have extremely large and complex root systems, which enables
them to collect water and essential nutrients over a wide area.
The presence of some opportunistic species such as Eupatorium adenophorum in forest are taking advantage
of canopy opening on the one hand and changing environmental conditions from mesic to xeric on the other
hand. Distribution pattern analysis shows species dispersion across a span of time at any given site. The pattern
of distribution depend both on physico-chemical natures of the environment as well as on the biological
peculiarities of the organisms themselves. Among the sites, under present study, an overview of distribution
patterns (A/F ratio) for tree layer shows that contiguous pattern was the most common followed by random
pattern. Odum (1971) contiguous distribution is common in nature and formed due to small but significant
variation in the ambient environmental conditions. Contiguous distribution of varying degree has been observed
in tropical forests of India (Shanmughavel 1994). Similar findings have also been reported by Kumar et al.
(2004) for tropical forest of Garhwal Himalaya. Variation in distribution pattern among sites and vegetation
composition are associated with micro-environmental and biotic factors (Singhal & Soni 1989).
Importance value of Sal in tree layer ranged between 123.36 and 187.00, which is well within the limits of
earlier studies (Rawat & Bhainsora 1999). Dominance of Sal depends upon the age, available resources,
associate species, disturbance regime and successional changes. The Site (Thano range) in which the IVI
exceeds more than 150.00 can be predicted that the Sal forest is progressing towards the climax stage, where as
in other two sites (Barkot and Lachchiwala ranges), having IVI values are ranging between 100.00 and 150.00
denotes that these sites were under heavy disturbances.
The value of Concentration (Cd) for Tree species ranges between 0.2175 and 0.4213. The values of species
diversity (H') reported in the present study are towards higher side, this may be due to the anthropogenic
disturbances prevailing in these sites, which can increase species diversity by lowering the dominance of a few
species, which make resources available to early successional species and by increasing environmental
heterogeneity provides a basis for specialization and resource partitioning (Grubb 1977, Denslow 1980).
In tropical forests, values of species diversity (H) are generally high, between 5.06 and 5.40 (Knight
1975)as compared to Indian forests, between 0.00 and 4.21 (Agni et al. 2000). The value of H reported in the
present study are well within these limits. Species richness of species in Sal forests of Doon valley is generally
low. This may be ascribed to false management practices adopted during the British rule to get the monoculture
of Sal trees by the removal of Kokats. Huge demand for wood during the two World Wars was catered to from
Sal forests of the valley. In the recent years, wide canopy openings have resulted due to various anthropogenic
reasons and infestation of Hoplocerambyx spinicornis. These canopy gaps have proven to be fruitful for the
invasion of large number of opportunistic plants.
Sal forests of Doon Valley are characteristically homogenous in distribution due to various silvicultural
operations carried out in the past, but the values of Cd in the present study are much less than the previous study
(Chauhan et al. 2001). This concludes the moving of Sal forests of the valley towards heterogeneity and may be
because of various disturbances prevailing in these forests viz. collection of fodder and fuel wood, grazing of
cattle, tremendous increase in the population of the Doon Valley after becoming the Capital of the State
Uttarakhand. The urbanization along the periphery of the forest has the great impacts on the forest structure. -
diversity for Trees varies from 2.33 to 5.83 in the Barkot and Thano respectively. Importance values of trees
were plotted against the species sequence to draw the Dominance-Diversity curves (D-D) curves and interpret
the community organization in terms of resource share and niche-space. D-D curves for tree species showed the
log series and log normal distributions for Barkot, Thano and Lachchiwala ranges respectively. The log series
distribution indicates that moderately common species reflect most closely the nature of environment and
fluctuate violently from time to time than the most abundant species. On the other hand, log normal distribution
gives the best distribution of species abundant pattern (Preston 1948). The assumption being that individuals
were distributed between species in accordance with Normal or Gaussian distribution and population growth is
Geometric (William 1964).
The 18 Trees species are classified into VIII groups by the TWINSPAN. Shorea robusta, the main species of
the Doon Valley is associated with the Ehretia laevis, Mallotus philippensis, and Syzygium cumini in the Group
VI. The dichotomies are primarily based on the eigen values depicting overlapping of species in different sites.
These results indicate the relationship among sites and among species on the basis of TWINSPAN and these
relationships ranked the sites and species in the vegetation accordingly.

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CONCLUSIONS
It can be concluded that species diversity is being regulated by factors like community stability and
evolutionary time as heterogeneity of both micro and macro environment affects the diversification among
different communities. Amongst major factors that influenced vegetation structure are human disturbance,
extensive grazing, trampling, invasion of opportunistic species and soil erosion. Human disturbance, extensive
grazing has resulted in the formation of highly fragmented vegetation type, which in turn has critical impact on
community structure.

ACKNOWLEDGEMENTS
We are thankful to Principal and Head, Botany Department, DAV (PG) College for providing the necessary
facilities for giving permission to do research.

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Whittaker RH (1975) Communities and Ecosystems (2nd ed). Macmillan Publishing Co., New York, 385 p.
William CB (1964) Patterns in the Balance of Nature and Related Problems in Quantitative Ecology.Academic
Press, London and New York, 324 p.
Woodward FL (1988) Temperature and the distribution of plant species and vegetation. In: Long SP &
Woodward FI (eds) Plants and Temperature. Vol 42. Society of Experimental Biology by the Company of
Biologists Limited, Cambridge, pp. 5975.
Zobel M (1997) The relative role of species pools in determining plants species richness: an alternative
explanation of species coexistence. Trends in Ecology and Evolution 12(7): 266269.

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Research article

Status of the Genus Pisonia L. (Nyctaginaceae) in Andaman &


Nicobar Islands, India
Debasmita Dutta Pramanick1*, G. G. Maiti2 and M. S. Mondal3
1
Botanical Survey of India, Publication Section, CGO Complex, 3rd MSO Building, DF Block, Sector-1, Salt
Lake City, Kolkata-700064, West Bengal, India
2
Department of Botany, Kalyani University, Kalyani-741235, West Bengal, India
3
Ex-Addl. Director, Botanical Survey of India, Central National Herbarium, Botanical Survey of India, Howrah-
711103, West Bengal, India
*Corresponding Author: debasmita.bot@gmail.com [Accepted: 20 June 2016]

Abstract: The family Nyctaginaceae is represented by 6 genera and 14 species in India of which
the genus Pisonia L. is the most significant genus due to its woody, arborescent habit, differential
leaf arrangement pattern, presence of unisexual, hermaphrodite and polymorphic flowers in same
and or different plants, unique characters of anthocarps and distinct types of pollen grains. During
carrying out the taxonomic revision of the family Nyctaginaceae in India, the first author has
emphasized the genus Pisonia with detailed account of infra-generic taxa. The present paper deals
with current status of the species of Pisonia in Andaman & Nicobar Islands, along with their
correct nomenclature, diagnostic characters, phenology, ecology, distribution and uses. A key has
been provided to help in easy identification of the species.
Keywords: Pisonia L. - Status - Andaman & Nicobar Islands.

[Cite as: Pramanick DD, Maiti GG & Mondal MS (2016) Status of the Genus Pisonia L. (Nyctaginaceae) in
Andaman & Nicobar Islands, India. Tropical Plant Research 3(2): 272282]

INTRODUCTION
The Andaman and Nicobar Islands, situated in the Bay of Bengal within 9294 E and 614 N is
characterized by tropical rain forests enriched with mangrove vegetation. The genus Pisonia L. comprises ca. 40
species in world (Mabberley 2008), mostly distributed in tropical America, few in continental South-east Asia;
only one species has reported in east Africa and two others in Madagaskar. In India, the genus is represented by
only 3 species, viz. P. aculeata L., P. grandis R.Br. and P. umellifera (Forst.) Seem. All the species are reported
from Andaman and Nicobar Islands (Fig. 1) of which P. umbellifera is endemic to this region.

MATERIALS AND METHODS


The preset study is primarily based on thorough scrutiny of herbarium specimens of 3 species of the genus
Pisonia L. deposited at the herbarium of Botanical Survey of India, Andaman and Nicobar Circle, Port Blair
(PBL), Southern Circle (MH) and Central National Herbarium (CNH), Howrah, W.B. The cibachrome
photographs of types provided from Royal Botanic Garden Herbarium (K) were consulted. Identification of taxa
was done with the help of local and regional literatures (Hook 1885, Parkinson 1923). For studied species, a key
to the species along with correct nomenclature, diagnostic features, phenology, ecology, distribution and uses
are provided.

RESULTS
Observation
The genus Pisonia L. is characterized by dioeceous, or monoecious shrubs, small trees or vines, sometimes
overhanging climbers, unarmed or with axillary recurved thorns, up to 30 m high. Bark soft, brittle, pale cream
in colour. Leaves opposite or alternate, or ternate or conferted to the end of the twigs, chartaceous or leathery or
papery or membraneous. Inflorescence many-flowered in umbelliform or corymbosely thyrsiform, pedunculate
cymes. Flowers unisexual or bisexual or polymorphic, bracteates; bracts caducous. Male and female flowers of

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.
different shapes, Stamens 610 in male flowers. Carpel rudimentary. Staminode as long as ovary, with
rudimentary anthers in female flowers. Anthocarp (fruit) dry, indehiscent, utricle with coriaceous perianth base,
obscurely or distinctly 5-angled, with or without monoserial to biserial prickles.

Figure 1. Distribution of the genus Pisonia L. in Andaman & Nicobar Island.


Key to the studied species
1a. Overlapping woody climber, mostly with recurved axillary thorns; male and female flowers of different
shape; anthocarps with 5 biserial rows of viscid prickles... 1. P. aculeata
1b. Unarmed shrubs or trees; dioecious plants, male and female flowers of similar shape; anthocarp
otherwise..... 2
2a. Leaves with distinct dark veins contrasting with lighter coloured intercostal veins; lateral and intercostal
veins hairy beneath; anthocarps with monoserial prickles... 2. P. grandis
2b. Leaves without distinctly contrasting dark veins; veins glabrous beneath; anthocarps glabrous, neither
prickled or muricate... 3. P. umbellifera
Enumeration of the studied species
1. Pisonia aculeata L., Sp. Pl. ed. 1: 1026. 1753; Parkinson, Forest Fl. Andaman Is. 222. 1923; Banerjee et al.,
Diversity Coastal Plant Comm. India 326. 2002. (Fig. 2, 3, 4)
Tall woody overhanging thorny climber, or shrub, to 6 m tall with smooth, olive green bark. Branches
pubescent, with numerous thorns as abortive shoots, 0.51.0 cm long, axillary, recurved, sharp, glabrous or rusty
pubescent. Inflorescences dense axillary and terminal corymbose cymes; male flowers in compact corymbose
cymes; female flowers in lax divaricate cymes, brown, short hairy. Flowers unisexual. Fruit (anthocarp)
narrowly oblong or clavate-oblong or clavate, 130.40.6 cm, with 5-biserial rows of viscid prickles,
tomentose between the ribs, rounded at apex, narrowed at base, thinly coriaceous.
Phenology: Fl.- DecemberJanuary; Frt.- FebruaryMarch.
Ecology: The plant is found to grow along coasts, hedges, rain forests and semi dry places forming impenetrable
messes on forest edges; from low land up to 500 m.
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Figure 2. Pisonia aculeata L. (Male plant): A, A portion of flowering twig; B, Floral bract; C, Flower; D, Stamens
surrounding rudimentary carpel; E, Stamen; F, Rudimentary carpel; [AF: R.K. Premnath 8451(CAL)].
Distribution: India (Andaman Islands, Andhra Pradesh, Bihar, Jharkhand, Karnataka, Kerala, Maharashtra,
Orissa, Tamil Nadu, West Bengal), Africa, America, Australia, Malagasy, Mauritius, Myanmar, Seychelles, Sri
Lanka, Vietnam.
Uses: The bark and leaves of the plant are used as a counter-irritant for swellings and rheumatic pains. The juice
of the leaves, mixed with pepper and other ingredients is given to children, suffering from pulmonary
complaints. Decoction of the fresh leaves is used to wash scabies. The plant makes an excellent hedges (Kirtikar
& Basu 1918, Anonymous 1962).
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Figure 3. Pisonia aculeata L. (Female plant): A, A portion of fruiting twig; B, Floral bract; C, Flower; D, Carpel; E,
Infructescence [AE: D. Hooper & M.S. Ramaswami 39273(CAL)].
Specimens examined: INDIA, Nicobars, Battimalo, March 1891, Prain s.n. (CAL); Andaman Is., Long Island,
sea level, 5.12.1915, Parkinson 760 (CAL, DD); Andaman, Little Andaman beach, 2 m, 10.03.1959, Thothathri
9266 (CAL, MH); South Andamans, Havelock Island, Camp no. 4, 24.03.1980, Rao, Chakraborty & Premnath
7664 (CAL); South Andamans, Havelock Island, Camp no. 3, Sea level, 27.03.1980, Rao & Premnath 7939
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(CAL); Little Andaman, Hut Bay, 11.02.1981, Premnath 8451 (CAL); Little Andaman, Hut Bay, 16.02.1981,
Premnath 8479 (CAL).

Figure 4. Pisonia aculeata L.: Photograph of the portion of fruiting twig.


2. Pisonia grandis R. Br., Prodr. Fl. Nov. Holl. 1: 422. 1810. (Fig. 5, 6)
P. alba Span. in Linnaea 15: 342. 1841; Hook. f. in Hook. f., Fl. Brit. India 4: 711. 1885. P. morindifolia R. Br.
ex Wight, Ic. t. 1765.1852.
Terrestrial, evergreen, arboreous, unarmed, branched shrub, or a small tree, 812 m tall with exposed roots.
Bark white-grey with conspicuous furrows, large leaf-scars and conspicuous lenticels. Leaves lettuce green at
maturity, yellowish-white at younger stage. Inflorescences terminal, dense corymbose cymes with polygamous
flowers. Fruits (anthocarp) narrow, elongated to club-shaped, or clavate, 5-ribbed or angled; each angle with
monoserial row of prickles; hairy between ribs.
Phenology: Fl. & Frt.- JanuaryMarch.
Ecology: The plant grows on dry to semi-dry places, along coasts, sandy or rocky situation, up to 50 m, on
oceanic islets and often dominant. This Species is also cultivated as an ornamental plant in gardens.
Distribution: India (Andaman Islands, Dadra, Daman, Diu, Goa, Gujarat, Kerala, Maharashtra, Nagarhaveli,
Nicobar Islands, Tamil Nadu), Australia, China, Laccadive, Malagasy, Maldives Island, Malesia, New
Caledonia, Polynesia, Pakistan, Sri Lanka.
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Figure 5. Pisonia grandis R.Br.: A, A portion of flowering twig; B, Flower; C, Flower splitted open; D, Stamen; E, Carpel;
F, Fruit [AF: V. Balasubramanium 1359 (CAL, MH)].
Uses: The fresh leaves, moistened with Eau-de-colognue, are used to subdue inflammation of a filarioid nature
in the legs and other parts. Plant is used as diuretic (Anonymous 1969). The root is considered as purgative.
Leaves are taken as green vegetable. The plant also serves as a good hedge. Native people sometimes use the
sticky fruits to catch birds. In several islands the leaves are used as vegetables specially the cultivated land race
with creamy or yellowish chlorotic leaves described as so-called Moluccan Cabbage or Lettuce tree.
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Specimens examined: INDIA, South Andaman, Narcondam, 1891, Prain s.n. (CAL); Little Andaman, Way to
Harmander Bay, sea level, 07.01.1976, Bhargava 3284 (CAL, PBL); Andaman Islands, 10.02.1979, Krishanlal,
Bramoh 33 (DD).

Figure 6. Pisonia grandis R.Br.: Photograph of the plant [Inset Portion of flowering twig (right below); portion of
fruiting twig (left top)].

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3. Pisonia umbellifera (Forst.) Seem. in Bonplandia 10: 154. 1862. (Fig. 7, 8)
Ceodes umbellifera Forst., Char. Gen. 71, t. 71. 1776. Pisonia excelsa Bl., Bijdr. 735. 1825; Choisy in DC.,
Prod. 13(2): 441. 1849; Parkinson, Forest Fl. Andaman Is. 222. 1923; P. brunoniana Endl., Prodr. Fl. Norfolk
43. 1833. Heimerliodendron brunonianum (Endl.) Skottsb. in Svensk. Bot. Tidskr. 35: 364. 1941. Bugainvillia
racemosa Blanco, Fl. Filip. 307. 1837; Merr., Sp. Heimerl Oest. Bot. Z. 63: 284. 1913.

Figure 7. Pisonia umbellifera (Forst.) Seem.: A, A portion of flowering twig; B, Floral bract; C, Flower; D, Flower splitted
open; E, Stamen; F, Carpel; G, Infructescence (In portion) [A-F: Dr. Prains collector 47(CAL); G: N.P. Balakrishnan and
N.C. Nair 3591(CAL)].

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Evergreen, perennial shrub, or a small-sized tree, up to 30 m high with spreading, unarmed branches. Bark
smooth, grayish with soft, cream-coloured sap wood. Inflorescences terminal, compact, multi-branched, many-
flowered, compound umbel, 39 cm across, sericeous or glabrous. Flowers polygamous. Fruits (athocarps)
cylindrical or clavate, subterete, 240.30.5 cm, slightly curved, indistinctly 5-ribbed, coriaceous, enclosed
within persistent calyx, black-brown; ribs very viscid, without prickle-like glandular structures.
Phenology: Fl.- JanuaryFebruary; Frt.- MarchMay

Figure 8. Pisonia umbelliera (Forst.) Seem.: Photograph of the plant growing in the garden, Hadda, Port Blair, BSI
[Inset- Portion of the flowering twig with buds (left below) and portion of flowering twig (right below)].

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Ecology: This plant is found to grow often in coastal areas, from low to medium elevations, exposed to winds,
both in ever-wet and monsoon forests; also grows along river banks, creeks, on sandy clay, sand and rocks under
xeric habitat. It is known to grow up to the altitude of 243 m.
Anthocarps of this species being too much viscid and becomes adhered with feathers of birds and thus they
are known to have fallen victim. This species has been commonly named as Bird Lime tree.
Distribution: India (Andaman Islands and Great Nicobar Islands), South Africa (Cape of Good Hope);
Christmas Island (S. of Java), Formosa, Hainan, Malagasy, Malesia, Mascarenes, Mauritius, New South Wales,
North Australia, Pacific (Bonin Island), Micronesia (Palau, Yap, Truk), Melanesia (Bismarck Arch., Fiji, Lord
Howe Island, Mangareva, Marquesas, North Island Of New Zealand, Norfolk Island, Pitcairn, Samoa, Solomon
Island, Tanna, Tubuai Island), Queensland.
Uses: In Pacific region the sticky-viscid anthocarps of Pisonia umbellifera (Forst.) Seem. have been noted to
use as bird catcher. The fruits or infructescences hang as fly or bird catcher. The birds disseminate the sticky
fruits but due to excessive accumulation of fruits on feathers of small birds render them incapable of further
flight and cause their eventual death (Govett 1884, Kirk 1884, John 1951, Stemm. 1964, White 1924). The wood
of this plant is soft and full of sap, eaten with relish by elephants (Parkinson 1923) and is said that the sheep,
which eat it get over their teeth a golden colour and appeared just like gold.
Specimens examined: INDIA, Andaman Islands, 1884, Kings Collector Y (DD); S. Andamans, Namuna ghar-
hilly jungle, 13.12.1890, King s.n. (CAL); S. Andaman, Part monat jungle hill, 28.02.1891, King s.n. (CAL); S.
Andaman, Tea garden, January 1893, Kings collector s.n. (CAL); S. Andaman, Hikk jungle at Hobdaypur,
04.03.1893, Kings collector s.n. (CAL); S. Andaman, Perseverance Point-Hill jungle, 19.12.1894, Kings
collector s.n. (CAL); S. Andaman, Peseverance by, Kurz, s.n. (CAL); Andamans, 23.01.1901, Prains collector
42 (CAL); Andaman Islands, Mount Harett, 800 ft., 02.01.1916, Parkinson 836 (CAL); Andamans, Betapur
valley, sea level, 25.03.1916, Parkinson 1141 (CAL, DD); Andaman Islands, Havelock Islands, 2 m,
20.01.1959, Thothathri 9097 (MH); Andaman Islands, Colinpur, Mount Harriet, 50 m, 16.01.1974, Nair 780
(CAL); S. Andamans, Kalapathar, Havelock Islands, 1 m, 02.09.1977, Premnath 6128 (CAL); S. Andamans,
inside the nalli on the way to Bishnunali, Baratang, 20 m, 28.01.1978, Basu 6850 (PBL); Little Andaman, 22
km from Hut Bay, 28.01.1981, Premnath 8322 (CAL, PBL).

DISCUSSION AND CONCLUSION


The present study indicates that the genus Pisonia L., represented by three species in India, viz. P. aculeata
L., P. grandis R.Br. and P. umbellifera (Forst.) Seem., is of woody habit and have very restricted distribution.
Among the three species, P. umbellifera is found to grow only in Andaman & Nicobar Islands, not yet reported
from mainland of India.
In regards to growing region, P. aculeata attains an extended range of distribution from sea level to 500 m
followed by P. umbellifera and P. grandis growing from low to 250 m and upto 50 m respectively, can be
correlated with their differential phenological characters. Regarding analysis of phenological characters in
respect to growing region and altitudinal range, some distinctive features have been noticed. The maximum
range of both flowering and fruiting period of P. aculeata has been correlated to the extended altitudinal range
of distribution while short flowering and fruiting period of P. grandis and P. umbellifera can be correlated to the
low altitudinal range of distribution. The distinct woody habit in combination with unisexual flower and unique
nature of anthocarp made the genus unique representative of the family Nyctaginaceae.

ACKNOWLEDGEMENTS
First author is thankful to the former Director of BSI, Dr. M. Sanjappa for the award of fellowship under
Flora of India Project. Authors are grateful to the former Indian Liaison Officer, Royal Botanic Garden, Kew
Herbarium, U.K., Dr. S.K. Srivastava for providing cibachrome photographs and relevant literatures on the
genus Pisonia L.

REFERENCES
Anonymous (1962) The Wealth of India-Raw Materials VI: 392393. Council of Scientific and Industrial
Research, New Delhi.
Govett RH (1884) A bird-killing tree. Transactions & Proceedings of the New Zealand Institute 16: 364366.

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Hooker JD (1885) Nyctaginaceae. In: Hooker JD (ed) The Flora of British India (Vol. 4). L. Reeve & Co.,
London, pp. 708711.
John St H (1951) The distribution of Pisonia grandis (Nyctaginaceae): Pacific Plant Studies No. 10. Webbia
8(1): 225228.
Kirk T (1884) Botanical Notes. Transactions & Proceedings of the New Zealand Institute 16: 367368.
Kirtikar KR & Basu BD (1918) Indian Medicinal Plants (Vol. 3., Reprint ed.). Allahabad, India, pp. 2049.
Mabberley DJ (2008) The Plant-Book. A Portable Dictionary of the Vascular Plants (3rd edition). Cambridge
University Press, pp. 590591.
Parkinson CE (1923) A Forest Flora of the Andaman Islands. Superintendent, Government Central Press,
Shimla, pp. 221223.
Stemmarik JF (1964) Flora Malesianae Precursores. 38. Notes on Pisonia in the old world (Nyctaginaceae).
Blumea 12: 275284.
White CT (1924) An insect-catching tree. Australian Forestry Journal 7: 36.

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Research article

Genus Brachymenium Schwgr. (Bryophyta) at Pachmarhi


Wildlife Sanctuary (Madhya Pradesh), India
Reesa Gupta, Virendra Nath and A. K. Asthana*
Bryology Laboratory, CSIR-National Botanical Research Institute Lucknow- 226 001, India
*Corresponding Author: drakasthana@rediffmail.com [Accepted: 22 June 2016]

Abstract: The present work describes the genus Brachymenium Schwgr. at Pachmarhi Sanctuary
that is a protected unit of Pachmarhi Biosphere Reserve in central India. Brachymenium belongs to
family Bryaceae which is a dominant family of mosses in India in terms of species diversity as
well as their occurrence. Four species of the genus were encountered at the Sanctuary which have
been collected from terrestrial habitats. Three species provided in the present study are new
additions to the moss flora of central India viz. B. bryoides Hooker ex. Schwgr., B. acuminatum
Harv. and B. ptychothecium (Besch.) Ochi. The account provides the current status of the genus at
the Pachmarhi Sanctuary.
Keywords: Moss - Brachymenium - Central India - New additions.

[Cite as: Gupta R, Nath V & Asthana AK (2016) Genus Brachymenium Schwgr. (Bryophyta) at Pachmarhi
Wildlife Sanctuary (Madhya Pradesh), India. Tropical Plant Research 3(2): 283288]

INTRODUCTION
Pachmarhi Biosphere Reserve (PBR) situated at Madhya Pradesh (India) is eminent for its bryophyte
vegetation with commendable bryophyte species being scattered throughout the region mostly in dense, damp
and shady pockets. PBR acquires an expanse of 4987.38 km2 and offers a perfect niche to cryptogams of central
India. The geological significance of being a Gondwanaland region and connecting link between Himalayan and
Western Ghats biodiversity belts further adds to the importance of this conservation unit. The reserve area has
three conservation units viz. the Pachmarhi Sanctuary, Bori Sanctuary and Satpura National Park. Presently, the
current status of Brachymenium Schwgr. at Pachmarhi Sanctuary is being explicated. The Sanctuary occupies
an area of approx. 417.78 km2 and lies between 22o 11 to 22o 56 N latitudes and 77o 47 to 78o 52 E longitudes.
Among the bryophyte flora of Pachmarhi Sanctuary, mosses form a major and dominant part.
Previous workers have listed bryophyte flora of Pachmarhi and neighbouring areas (Kaul 2001, Singh &
Kaul 2002, Handoo et al. 2009, Sharma & Alam 2011) but only few detailed accounts of genera and family
have been provided (Nath & Gupta 2009, Nath et al. 2011, 2011a). The urgent need to provide comprehensive
illustrated account of the important families and genera at Pachmarhi region is apparent. The dominant families
of the reserve area include Bryaceae, Pottiaceae, Dicranaceae and Hypnaceae.
Bryaceae is a prominent family of acrocarpous mosses that is well distributed throughout the world. The
family is recognized among bryologists for its substantially divergent morphological characters. Genus
Brachymenium Schwgr. belongs to family Bryaceae and is represented by nearly 70 species worldwide (Ochi
1992, 1994), while in India 14 species were reported by Gangulee (197477) and Lal (2005) and most recently
Dandotiya et al. (2011) have reported 26 species. The genus is well adapted to hot and humid climate and
species are abundantly found in the tropical belt (Gangulee 197477). The plants are characterized by erect,
green habit forming dense mats, branched by 24 subfloral innovations, matted together at base. Leaves are
erectopatent, ovate lanceolate to oblong lanceolate densely arranged on stem, acuminate, with flat to recurved
margin that may be dentate at apex, may be bordered or non-bordered with shortly excurrent or long costa.
Brachymenium is a difficult genus in terms of taxonomy and identification. It was initially divided into four
sections (Brotherus 1924) and later workers have sporadically revised the status of this genus (Ochi 1972, 1980,
1992, 1994, Spence 1987). Some recent revisions based on molecular approach have also been provided by
Spence (2005). The present work is an attempt to describe the current status of the genus at Pachmarhi
Sanctuary. During the present investigation, four species of the genus namely Brachymenium bryoides Hooker
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Gupta et al. (2016) 3(2): 283288
.
ex. Schwgr, B. sikkimense Renauld & Cardot, B. acuminatum Harv. and B. ptychothecium (Besch.) Ochi have
been unveiled from the study area. Among these, three taxa viz. B. bryoides, B. acuminatum and B.
ptychothecium are new additions to central India. Previously Singh & Kaul (2002) have reported two other
species viz. B. exile (Dozy & Molk.) Bosch & Sande Lac. and B. nepalense Hook Schwgr. from Pachmarhi
region. Thus total six species of Brachymenium are now known from the Wildlife Sanctuary. Four are being
described in the present contribution.

MATERIALS AND METHODS


The specimens were collected during field excursions in different years from localities of Pachmarhi
Sanctuary in an altitudinal range of approximately 880 to 1050 meters a.s.l. The plants were collected from
terrestrial habitats such as soil covered rocks and wet rocks, and have been deposited in the Bryophyte
herbarium, CSIR-National Botanical Research Institute, Lucknow (LWG), India. The material was studied in
detail and the taxonomic observations were recorded to perform the identification and proper illustrations were
made.
TAXONOMIC OBSERVATIONS
Key to the species of genus Brachymenium at Pachmarhi Sanctuary:
1. Plants smaller (up to 4mm), not much changed when dry, leaves bordered ................................ 2
Plants larger (up to 10 mm), contorted when dry, leaves not distinctly bordered .... 3
2. Leaves apressed to stem, leaf margin flat .... B. bryoides
Leaves erectopatent, leaf margin irregularly recurved ........ B. sikkimense
3. Plants smaller (up to 6 mm), pale yellowish, leaves small, margin entire .... B. acuminatum
Plants larger (up to 10 mm), glossy green, branched, leaves larger, margin dentate .... B. ptychothecium
Brachymenium bryoides Hooker ex. Schwgr., Sp. Musc. Suppl. 2(1): 134. 1824. (Fig. 1AL)
Plants green, forming dense mats, stem short, +3 mm, erect, branched by 23 subfloral innovations, matted
together at base. Stem ovate-circular in cross section, irregularly sized cells without any distinction of cortical
or medullary region. Leaves erectopatent, ovate lanceolate, dense, +0.85 0.185 mm in size, acuminate, margin
flat, entire; costa excurrent in a short arista, +1.7 mm long. Leaf cells elongated, rhomboidal +35 6.4 m at
apex, 37 6.4 m at middle sub quadrate to rectangular at base, +32 7 m, marginal row of cells narrower,
sporophyte not seen.
Ecology & Distribution: Plants growing on dry rocks at Pandav Caves and Rajat Prapat, 9881004 m.
Range of Distribution: Antarctica, Brazil, India: Central India (Pachmarhi), Eastern Himalaya (Darjeeling,
Khasia hills), South India (Nilgiri, Sherveroy hills), Western Himalaya (Almora, Simla), Papua New Guinea,
Phillipines, Sri Lanka, Nepal.
Specimens examined: India, Madhya Pradesh, PBR: Pandav Caves, elv. 1004 m, on dry rocks, 07.11.2011,
A.K. Asthana & R. Gupta 263114 (LWG); near Rajat Prapat, elv. 988 m, on rocks, 07.11.2011, A.K. Asthana &
R. Gupta 263133A (LWG).
Brachymenium sikkimense Renauld & Cardot, Bull. Soc. R. Bot. Belg., 38(1): 12. 1900. (Fig. 1MW)
Plants small, glossy green, densely tufted, stem erect, up to 4 mm long, branched by several (2-4) sub floral
innovations, matted together by rhizoids at base. Stem broadly circular in cross section, uniformly medium
sized cells without any demarcation of cortical and medullary regions. Leaves erectopatent, ovate-lanceolate,
0.82 0.38 mm in size, broad at base, acuminate, margin flat to recurved, entire; costa brown, strong, excurrent
in an arista 0.18 mm in size. Leaf cell rhomboidal +28 6 m at apex, +28.5 6 m at middle, sub-rectangular
to sub rectangular at base, +36 9.25 m; marginal row of cells narrower, forming indistinct border.
Sporophyte not seen.
Ecology & Distribution: Plants growing at Pandav Caves, near Rajat Prapat and Pachmarhi Lake, on dry rocks
from 9881050 m.
Range of Distribution: India: Central India (Gujarat, Pachmarhi), Eastern Himalaya (Darjeeling).
Specimens examined: India, Madhya Pradesh, PBR: Pandav Caves, elv. 1004 m, on dry rocks, 07.11.2011,
A.K. Asthana & R. Gupta 263119 (LWG); near Rajat Prapat, elv. 988 m, on rocks, 07.11.2011, A.K. Asthana &
R. Gupta 263133B (LWG); Pachmarhi Lake, elv. 1050 m, on dry rocks, 09.11.2011, A.K. Asthana & R. Gupta
264827A (LWG).

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Figure 1. AL, Brachymenium bryoides Hooker ex Schwgr. (A,B, vegetative plants; C, cross section of axis; DI, leaves;
J, apical leaf cells; K, median leaf cells; L, basal leaf cells); MW, Brachymenium sikkimense Renauld & Cardot. (M,N,
vegetative plants; O, cross section of axis; PT, leaves; U, apical leaf cells; V, median leaf cells; W, basal leaf cells).

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Figure 2. AI, Brachymenium acuminatum Harv. (A,B, vegetative plants; C, cross section of axis; DF, leaves; G, apical
leaf cells; H, middle leaf cells; I, basal leaf cells); JS, Brachymenium ptychothecium (Besch.) Ochi. (J,K, vegetative plants;
L, cross section of axis; MP, leaves; Q, apical leaf cells; R, middle leaf cells; S, basal leaf cells).

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Brachymenium acuminatum Harv., Icon. Pl. Rar, 1:19. 1836. (Fig. 2AI)
Plants erect, terrestrial, pale to yellowish green plants in dense mats, up to 6 mm high, branched by several
subfloral innovations, matted by tomenta. Stem circular in cross section, outer cells smaller, getting slightly
larger towards centre, innermost cells again smaller. Leaves densely and uniformly arranged on stem, imbricate,
erect, oblong-lanceolate up to 1.2 mm long and 0.42 mm wide, acuminate, margin entire; costa strong, excurrent
in an arista +0.11 mm long. Leaf cells rhomboid, up to 75 12 m at apex, gradually getting rectangular at
base, 50 16 m in size; marginal cells narrower but no distinct margin seen. Sporophyte not seen.
Ecology & Distribution: Plants growing on soil and soil over rocks, at Jambu Dweep at 900m.
Range of Distribution: Argentina, Australia, Bolivia, Botswana, Chile, China, Cong, India: Central India
(Pachmarhi), Eastern Himalaya (Darjeeling, Sikkim), South India, Lesotho, Madagascar, Malawi, Mauritius,
Mexico, Myanmar, Namibia, Nepal, Nicaragua, Peru, Philippines, Reunion, South Africa, Swaziland,
Zimbabwe.
Specimens examined: India, Madhya Pradesh, PBR: Jambu Dweep, elv. 900 m, growing on soil, 17.12.1993,
V. Nath & A.K. Asthana 205598 (LWG); 17.12.1993, Jambu Dweep, elv. 900 m, growing on soil over rock, V.
Nath & A.K. Asthana 205599 (LWG).
Brachymenium ptychothecium (Besch.) Ochi, Adv. Front. Pl. Sc., 4: 108. 1963. (Fig. 2JS)
Plants, erect, terrestrial, tufted, glossy, green, reddish at base, up to 10 mm high, with subfloral innovations.
Stem oval in cross section, outer cortical cells smaller, inner cells larger. Leaves distantly arranged on the stem,
erectopatent, curled when dry, oblong-spathulate, bordered and apiculate, +1.93 mm long and +65 mm broad at
middle, margin entire below, slightly dentate at apex, usually revolute from base to mid leaf; costa strong, deep
brownish, excurrent in a slightly denticulate arista, +0.27 mm in size. Leaf cells rhomboid to hexagonal at apex,
+37.5 m long and +17.7 m wide; basal cells sub rectangular, +41.7 19.8 m; marginal cells elongated,
narrow, forming an indistinct border of 23 rows. Sporophyte not seen.
Ecology & Distribution: Plants growing on moist rocks, at Down Fall at 900 m.
Range of Distribution: China, India: Central India (Pachmarhi), Eastern Himalaya (Darjeeling, Sikkim).
Specimens examined: India, Madhya Pradesh, PBR: Down Fall, elv. 884m, growing on moist rock,
28.11.2006, V. Sahu & V. Awasthi 227601 (LWG).

DISCUSSION
Among the four species of Brachymenium, B. bryoides and B. sikkimense have distinctly bordered leaves
whereas in case of B. acuminatum and B. ptychothecium leaves are less clearly bordered. B. bryoides and B.
sikkimense both have very short stems (up to 4 mm). The primary distinguishing point between these two
species is that the leaves are closely appressed to the stem and margin is flat in former while in the latter, leaves
are erctopatent and leaf margin is irregularly recurved. In B. acuminatum and B. ptychothecium the stem is
larger, ranging from 6 to 10 mm or more. In case of B. acuminatum, the leaves are densely and uniformly
arranged on stem, larger in size and the leaf margin is revolute towards the apex which can be discriminated
from B. ptychothecium as its leaves are distantly arranged on stem, smaller in size and have entire margin. In the
former species the marginal cells do not form a distinct border while in the latter, indistinct border cells can be
identified.
Table 1. Habitat and geographical distribution of the species of Brachymenium at Pachmarhi Sanctuary.
Habitats Distribution in India
S.No. Plant Name
S DR WR SCR E WH EH PR GP CI SI
1. Brachymenium bryoides Hooker ex. Schwgr. - + - - - + + - + - +
2. Brachymenium sikkimense Renauld & Cardot - + - - - - + - -- +
3. Brachymenium acuminatum Harv + - - + - - + - + - +
4. Brachymenium ptychothecium (Besch.) Ochi - - + - - - + - -- +
5. Brachymenium exile (Dozy & Molk.) Bosch Not available + + + + - +
& Sande Lac.*
6. Brachymenium nepalense Hook Schwgr.* Not available - - - - + -
Note: * Species reported previously from Pachmarhi region, but not found during present study; S= soil; DR= dry rocks;
WR= wet rocks; soil covered rocks; E= epiphytic; WH= western Himalaya; EH= eastern Himalaya; PR= Punjab &
Rajasthan Plains; GP= Gangetic Plains; CI= central India; SI= South India.

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Among the species studied, all are distributed in eastern Himalaya where as two taxa viz. B. bryoides and B.
acuminatum are found in South India also (Table 1). Brachymenium bryoides is most widely distributed in
India. The central Indian bryogeographical zone has been under explored for mosses till recent years and this
account adds to the knowledge of the mosses of this region.

ACKNOWLEDGEMENTS
The authors are grateful to the Director, National Botanical Research Institute, Lucknow, India for
encouragement and providing the laboratory facilities, the Ministry of Environment, Forests and Climate
Change and CSIR, New Delhi, for providing financial assistance and the Chief Conservator of Forests, Madhya
Pradesh, for extending the necessary help during the course of field excursions.

REFERENCES
Brotherus VF (1924) Musci In: Engler A & Prantl K (eds.) Die Natrlichen Pflanzenfamilien, Leipzig. Vol. 10
(ed. 2) pp. 243302.
Dandotiya D, Govindapyari H, Suman S and Uniyal PL (2011) Checklist of the bryophytes of India. Archive for
Bryology 88: 1126.
Gangulee HC (19741977) Mosses of Eastern India and Adjacent Regions Vol-II Books and Allied (P) Ltd,
Calcutta, India.
Handoo ON, Dhabade GT, Rai S & Rai PK (2009) Mosses of Pachmarhi. Indian Journal of Applied & Pure
Biology 24(2): 505520.
Kaul A (2001) An assessment of the present pesition of Hepatic flora of Pachmarhi. In: Nath V & Asthana AK
(eds.) Prespectives in Indian Bryology. Bishen Singh Mahendra Pal Singh, Dehra Dun, India. pp. 7585.
Lal J (2005) A Checklist of Indian Mosses. Bishen Singh Manhendra Pal Singh, Dehra Dun, India.
Nath V & Gupta R (2009) A Detailed Taxonomic Assessment of Family Bryaceae (Musci) of Pachmarhi
Biosphere Reserve (M.P.) India. Nelumbo 51: 161174.
Nath V, Asthana AK & Gupta R (2011) An overview of family Pottiaceae (Bryopsida) in central India with
special reference to Pachmarhi Biosphere Reserve (PBR). Lindbergia 34: 3039.
Nath V, Asthana AK & Gupta R (2011a) Genus Fissidens Hedw. (Fissidentaceae, Bryopsida) at Pachmarhi
Biosphere Reserve (Madhya Pradesh), India. Taiwania 56 (1): 7180.
Ochi H (1972) A revision of African Bryoideae, Musci (First Part). Journal of Faculty Education Tottori
Uniersity of Natural Sciences 23: 126.
Ochi H (1980) A revision of the Neotropical Bryoideae, Musci (First Part). Journal of Faculty Education Tottori
Uniersity of Natural Sciences 29: 49154.
Ochi H (1992) A revised infrageneric classification of the genus Bryum and related genera (Bryaceae, Musci).
Bryobrothera 1: 231244.
Ochi H (1994) Bryum and Brachymenium. In: Sharp AJ, Crum H & Eckel PM (eds), The Moss Flora of Mexico.
Memoirs New York Botanic Garden 69: 454501.
Sharma D & Alam A (2011) Present status of liverwort diversity at Pachmarhi (Madhya Pradesh). Journal of
Indian Botanical Society 90(3&4): 332338.
Singh VP & Kaul A (2002) Biodiversity and Vegetation of Pachmarhi Hills. Scientific Publishers, Jodhpur,
India.
Spence JR (1987) A proposed reclassification of Bryum, Anomobryum and Brachymenium (Musci, Bryaceae).
Journal of Bryology 14(4): 659676.
Spence JR (2005) New genera and combinations in Bryaceae (Bryales, Musci) for North America, Phytologia
87: 1528.

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Short communication

Eleocharis spiralis (Rottb.) Roem. & Schult. (Cyperaceae): An


addition to the flora of Andaman and Nicobar Islands
Sanjay Mishra*, C.P. Vivek and Lal Ji Singh
Botanical Survey of India, Andaman and Nicobar Regional Centre, Port Blair, India
*Corresponding Author: sanjayalld74@gmail.com [Accepted: 22 June 2016]

[Cite as: Mishra S, Vivek CP & Singh LJ (2016) Eleocharis spiralis (Rottb.) Roem. & Schult. (Cyperaceae):
An Addition to the Flora of Andaman and Nicobar Islands. Tropical Plant Research 3(2): 289291]

Eleocharis R.Br. is a cosmopolitan genus of the family Cyperaceae which comprises about 200 species
distributed in tropical, subtropical, and temperate regions of the world (Gonzalez-Elizondo & Peterson 1997,
Mabberley 2009). Prasad & Singh (2002) mentioned about 21 species of this genus from India. During field
expedition in Andaman Islands, the authors came across an interesting species of Eleocharis from Mangroove
area behind the Corbyn's cove beach in Port Blair (South Andaman). On Critical study, perusal of literature and
consultation of herbarium at PBL, the specimens were identified as Eleocharis spiralis (Rottb.) Roem. &
Schult., a species widely distributed in the tropical regions of the world and in Indian states Karnataka, Kerala,
Maharashtra, Madhya Pradesh, Tamil Nadu and West Bengal (Cook 1996, Lakshminarasimhan 1996, Verma
2001, Prasad & Singh 2002). A thorough scrutiny of the literature (Rao 1986, Lakshminarasimhan & Rao 1996,
Mathew 1998, Pandey & Diwakar 2008, Singh & Murugan 2014, Rasingam 2015) revealed that this species has
so far not been reported from Andaman and Nicobar Islands. Hence, it is reported and described here as new
record for Andaman and Nicobar Islands.
Eleocharis spiralis (Rottb.) Roem. & Schult., Syst. Veg. 2: 155. 1817. (Fig. 1)
Scirpus spiralis Rottb., Descr. Icon. Rar. Pl. 45. 1773; Clarke in Hook. f., Fl. Brit. India 6: 627. 1893.
Perennial herbs with short rhizome. Stolons slender, with scales. Culms erect, pale green, densely tufted, 30
60 cm tall, 24 mm thick, firm, triquetrous, without transverse septa and nodes. Leaf sheaths membranous, 3 or
4, 316 cm long, smooth, slightly shiny; basalmost sheath purplish red to dark brown; cauline sheaths pale red,
elongate, mouth obliquely truncate and parted, apex shortly acuminate with a setaceous appendage. Spikelets
cylindrical, pale yellow, slightly broader than apex of the stem, 23.5 cm 35 mm, many flowered, apex acute
to obtuse. Glumes tightly and spirally arranged, basalmost glume empty, broadly triangular, amplexicaul for
whole spikelet base; fertile glumes densely imbricate, subquadrate, ca. 3 3 mm, pale and brownish red
puncticulate striate, medially leathery and with a midvein, margin hyaline and minutely brown puncticulate,
apex truncate to subtruncate. Perianth bristles 46, reddish purple, linear, unequal in length, as long as or shorter
than nutlet, laxly retrorsely spinulose. Stamens 3; anthers linear oblong, ca. 0.5 mm long. Stigmas 3. Nutlet
yellowish green, turning dark brown when mature, obovoid to broadly obovoid, 1.21.5 ~1 mm, compressed
biconvex, obscurely cancellate with 1720 rows of transversely oblong epidermal cells, margin narrow, apex not
constricted but with an obscure annular thickening; persistent style base conic, basally gradually narrowed.
Flowering & fruiting: AugustFebruary.
Habitat: It is a perennial herb, grows in shallow water, restricted to brackish and saline localities towards coastal
areas.
Distribution: India; Andaman and Nicobar Islands (Corbyn's cove beach, Port Blair, South Andaman),
Karnataka, Kerala, Maharashtra, Madhya Pradesh, Tamil Nadu and West Bengal. Worldwide distributed in
Bangladesh, Myanmar, Sri Lanka, Malesia, Thailand, South China, Australia, New Caledonia, Papua New
Guinea, tropical Africa and tropical America.
Specimens examined: INDIA, Andaman and Nicobar Islands, Port Blair, behind the Corbyn's cove beach, 8 m
asl, N 11 38'16.06" E 92 40'00.01", 01.11.2015, Sanjay Mishra 32424 (PBL).

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Received: 12 February 2016 Published online: 30 June 2016
Mishra et al. (2016) 3(2): 289291
.

Figure 1. Eleocharis spiralis (Rottb.) Roem. & Schult.: A, Habit; B, Cross section at distal end of culm below the spikelet;
C, Culms with spikelets; D, Glume, adaxial view; E, Glume, abaxial view; F, Flower; G, Nut.

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ACKNOWLEDGEMENTS
The authors are thankful to Dr. Paramjit Singh, Director, Botanical Survey of India, Kolkata, for providing
facilities and constant support. Authors are also thankful to the Ministry of Environment, Forests and Climate
Change for providing necessary facilties and support through the Director BSI, Kolkata.

REFERENCES
Cook CDK (1996) Aquatic and Wetland Plants of India. Oxford University Press, Oxford.
Gonzalez-Elizondo MS & Peterson PM (1997) A classification of and key to the supraspecific taxa in
Eleocharis (Cyperaceae). Taxon 46: 433449.
Lakshminarasimhan P & Rao PSN (1996) Supplementary list of angiosperms recorded (19831993) from
Andaman and Nicobar Islands. Journal of Economic and Taxonomic Botany 20: 175185.
Lakshminarasimhan P (1996) Flora of Maharashtra State Monocotyledons. In: Sharma BD, Karthikeyan S &
Singh NP (eds). Botanical Survey of India, Calcutta.
Mabberley DJ (2009) Mabberley's plant book, a portable dictionary of plants, their classification and uses, 3rd
ed. Cambridge University Press.
Mathew SP (1998) A supplementary report on the flora and vegetation of Bay Islands, India. Journal of
Economic and Taxonomic Botany 22: 249272.
Pandey RP & Diwakar PG (2008) An integrated checklist of plants in Andaman & Nicobar Islands, India.
Journal of Economic and Taxonomic Botany 32: 403500.
Prasad VP & Singh NP (2002) Sedges of Karnataka (India). Reprinted from Journal of Economic and
Taxonomic Botany, Additional Series No. 21. Sci. Publ., Jodhpur.
Rao MKV (1986) A preliminary report on the angiosperms of Andaman and Nicobar Islands. Journal of
Economic and Taxonomic Botany 8: 107184.
Rasingam L (2015) Five new additions to the flora of Andaman and Nicobar Islands, India. Journal of
Threatened Taxa 7(3): 70377041.
Singh L J & Murugan C (2014) Seed plant species diversity and conservation in dhanikhari Experimental
Garden cum Arboretum in Andaman and Nicobar Islands. In: Nehera S, Gothwal RK & Ghosh P (eds)
Biodiversity in india: Assesment, scope and conservation. Lambert Academic Publishing Heinrich-Booking-
str.Saarbruken, Germany, pp. 253280.
Verma DM (2001) Cyperaceae. In: Singh NP, Khanna KK, Mudgal V & Dixit RD (eds) Flora of Madhya
Pradesh. Botanical Survey of India, Calcutta.

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Research article

Plant diversity and community analysis of the vegetation around


Tulu Korma project centre, Ethiopia
Zewdie Kassa1,2*, Zemede Asfaw1 and Sebsebe Demissew1
1
Department of Plant Biology and Biodiversity Management, the National Herbarium, Addis Ababa University,
P.O. Box: 3434, Addis Ababa, Ethiopia
2
Department of Biology, Mizan-Tepi University, P.O. Box, 260, Tepi, Ethiopia
*Corresponding Author: zewdiekasa@gmail.com [Accepted: 23 June 2016]

Abstract: A study on plant diversity and community analysis was conducted between October
2014 to September 2015 with the objective of documenting plant species diversity together with
the status and trends of biodiversity change. Common ecological methodologies and techniques
were applied. Aerial photographs using Google earth and ground survey using GPS, compass and
clinometers supported by ArcGIS based mapping was done to identify the clear distinction
between vegetation and habitat heterogeneity before vegetation sampling. R statistical Software
was used to run cluster analysis and ordination. Four plant community types were identified in the
study. The Shannon-Weiner Diversity index was calculated to see the overall diversity of the study
area. The area is with relatively high plant species diverse at H= 3.37 with overall species
evenness, J= 0.61 confirming the fact that Shewa floristic region is among the most species rich
habitats. Ordination results showed that the major environmental variables structuring the floristic
composition and plant community types and altitude was found to be the most influential factor at
95% confidence interval (P=0.005) using Canonical Correspondence Analysis. Comparison of
historical records from literature and practical observation of the current status of the study area
confirmed that there is an increasing trend of biodiversity and vegetation cover. This is justified by
observing the positive impacts of the project centre for indigenous trees propagation and
biodiversity development for Ethiopia located at Tulu Korma. The study showed that the area is
rich in plant species and floristic composition, hence, experiences adopted by the Project Centre
should be the lessons learned for the restoration and rehabilitation of similar environments.
Keywords: Cluster analysis - Ordination - Multipurpose plants - Indigenous species - Restoration.

[Cite as: Kassa Z, Asfaw Z & Demissew S (2016) Plant diversity and community analysis of the vegetation
around Tulu Korma project centre, Ethiopia. Tropical Plant Research 3(2): 292319]

INTRODUCTION
Biodiversity is the variability among living organisms from all sources including inter alia, terrestrial,
marine and other aquatic ecosystems and the ecological complexes of which they are part; this includes diversity
within species, between species, and of ecosystems. In a broad sense it is to mean the abundance and
distributions of and interactions between genotypes, species, communities, ecosystems and biomes. The term
inter alia above is to mean among other things (Reynolds 2000, Gaston et al. 2004, Leadley et al. 2010, CBD
2011).
For countries like Ethiopia, it is important to learn the success histories from other countries as well as
certain ongoing project trials in some parts of the country to rehabilitate degraded habitats, ecosystems and
biodiversity loss. Ecological restoration is an attempt to return a system to some historical state, although the
difficulty or impossibility of achieving the aim is widely recognized (Guyton 2001, Palmer et al. 2006, Naeem
et al. 2009, Ferwerda 2012). A more realistic goal may be to move a damaged system to an ecological state that
is within some acceptable limits relative to a less disturbed system. Ethnoecological approaches are among the
key options in integrating academic disciplines and traditional ecological knowledge for sustainable
environment (Albuquerque et al. 2014, Berkes et al. 1995, Berkes et al. 2000, Gmez-Baggethun et al. 2013).

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Kassa et al. (2016) 3(2): 292319
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This research project focused on studying the status and trends of biodiversity change in Tulu Korma and its
surrounding areas of Ejere District. Tulu Korma and its surrounding where the study was carried out is the
Center for Indigenous Trees Propagation and Biodiversity Development in Ethiopia. The formerly known as
Center for Indigenous Trees Propagation and Biodiversity Development in Ethiopia and currently named as
Center for the Restoration of Ethiopias Biodiversity and Key Natural Resources was founded by Professor
Legesse Negash and established on 10 July 2004 with its primary objectives which envisages providing a
platform for research and development on indigenous trees, shrubs, biodiversity, watersheds, and key natural
resources including water and soils (Legesse Negash 2010).

MATERIALS AND METHODS


Location
Tulu Korma is located at 5055 km West of Addis Ababa on the high way running from Addis Ababa to
Ambo between 0901.188 N and 03821.570 E within altitude range of 2,1632,267m. (Legesse Negash,
2010). Four neighboring kebeles bordering Tulu Korma are Chiri to the north, Kimoye to the west, Hora to the
south and Endode to the east. Addis Alem is the nearest town about 3 kilometers from the center and it is with
weather station from where the Ethiopian National Meteorological Service Agency (ENMSA) record weather
data (Zewdie Kassa et al. 2016) (Fig. 1).

Figure 1. Location of the study area.

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Climate
According to Zewdie Kassa et al. (2016), the annual average minimum and average maximum temperature
for 16 years data is 7.4C and 26.2C respectively. The annual average temperature and average rainfall for the
same years data are 16.9C and 1099 mm respectively (Fig. 2).

Figure 2. Climate diagram of the study area (data from ENMSA).


Vegetation
Areas between altitudes of 1800 and 3000 meters have been marked as the Dry evergreen Afromontane
forest and grassland complex with the exception of high annual rainfall areas of 1700 millimeters and above
(Friis et al. 2011). The vegetation of Tulu-korma and its surrounding belongs to such vegetation type and
characterized by a canopy dominated by Juniperus procera (Cupressaceae), Podocarpus falcatus
(Podocarpaceae), Olea europaea subsp.Cuspidata (Oleaceae), Croton macrostachyus (Euphorbiacea) and Ficus
spp. (Moraceae). Shrubs and bush lands, woodlands and plantations are also available.
GIS data collection
It was noted that geographical information systems and models are among Computer-based Decision
Support Tools integrating information (Mishra & Agarwal 2015). Hence, both aerial photographs using Google
earth and ground survey using GPS, compass and clinometers supported by ArcGIS based mapping was done to
identify the clear distinction between vegetation and habitat heterogeneity before vegetation sampling. Due to
habitat heterogeneity, preferential sampling technique was used to collect vegetation and floristic data.
Vegetation and Ecological Data Collection
Vegetation data were collected using preferential sampling techniques and cover abundance values were
calculated using presence/absence method (Kent & Cocker 1992). Cover was calculated as the area of the
ground within a quadrate which is occupied by the above ground parts of each species when viewed from above
(canopy cover) visually estimated as percentage. A 30m 30m square quadrate was used to sample plant
species. Sub-quadrates of 2m 2m for grasses and sedges, 5m 5m for herbs, 10m x 10m for shrubs were used
to estimate the cover abundance values of different plant habits. The layouts of the quadrates were in such a way
that one from each corner of the large quadrate and one at the center for the respective life forms. Species
abundance values were converted following the Braun-Blanquet 19 scales (Van Der Maarel 2005) by
calculating the Ordinal Transformed Values (OTV) as,
OTV = 1.415
Where, ln is the natural logarithm and C is the cover abundance value in percentage. The vegetation data were
arranged in a three column format (sites, species and abundance) of creating ecological data tables for ease of
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analysis using R statistical software following (Zerihun Woldu 2012). The data were then carefully inspected in
the three column table to check for possible errors before analysis.
Ecological data such as altitude, latitude, longitude, slope and aspect were collected by setting the GPS on
the projected coordinate system format (UTM_Adindan_Zone 37). Aspect and slope were also carefully
recorded by using SILVA compass and SILVA Ranger Clinometers instruments respectively right during
specimen collection. Geophysical features, presence/absence and intensity of disturbance were recorded (Milgo
2011). Assessment of the type and levels of disturbance were determined by carefully observing the presence or
absence of types of disturbance such as charcoal burning, fire, pole cutting, grazing, firewood collection,
agriculture, construction, bee harvesting and any other. Then disturbance scales were set as: 0= no disturbance,
1= 020% of the quadrate disturbed, 2= 2140% of the quadrate disturbed, 3= 4160% of the quadrate
disturbed, 4= 6180% of the quadrate disturbed, 5= 81100% of the quadrate disturbed.
Specimen Collection and Identification
Voucher specimens were collected together with all the necessary information about the specimens carefully
recorded at the spot. Specimen identification and deposition was made at the National Herbarium, Addis Ababa
University using taxonomic keys, characters and published volumes of the flora of Ethiopia and Ertrea as well
as other reference sources (Fitchl & Admasu Adi 1994, Hedberg & Edwards 1989 1995, Edwards et al. 1995,
Edwards et al. 1997, Edwards et al. 2000, Judd et al. 2002, Hedberg et al. 2004, Hedberg et al. 2006, Azene
Bekele 2007, Friis 2009).
Data Analysis
Vegetation data was analyzed using R statistical software (R Core Development Team 2013). The Shanon-
Weiner diversity index was used to analyze vegetation data following Zerihun Woldu (2012). Hence the
Shannon Diversity Index (H) is the average uncertainty per species in an infinite community made of S species
with known proportional abundance and the Pi are the population parameters:

where ni = the number of individuals belonging to the ith of S species in the sample, n= the number of
individuals in the sample, Pi = , the probability of sampling species i, = the natural logarithm of the
probability of sampling species i. Shannons equitability (J) was calculated to test species evenness as:

where S is species richness, is the natural logarithm of the species richness and J assumes the values
between 0 and 1 with 1 being with complete evenness. Diversity, richness and evenness values were computed
by running a data frame in R statistical software for diversity analysis. Srensens coefficient of similarity index
was used to compare the general floras with respect to other flora areas in Ethiopia (Wildi 2010) and the output
of the analysis interpreted. This, Srensens Similarity Index (I) calculated as:

Where, Ss is Srensens coefficient of similarity, a is the number of species common to both samples, 1 and 2,
b is the number of species in sample 1 and c is the number of species in sample 2.

RESULTS
Plant Diversity and floristic composition
A total of 304 plant species belonging to 216 genera and 78 families were identified (Appendices 1, 2, 3).
About four major life forms were identified of which 165 (54.28%) of the species were herbs, 80 (26.32%) were
shrubs, 43 (14.14%) were trees and the remaining 16 (5.26%) were climbers. Top five plant families with the
highest percentages of the total recorded were Asteraceae 50 (64.10%), Fabaceae 27(34.62%), Lamiaceae
25(32.05%), Poaceae 18(23.04%) and Solanaceae 15 (19.23%). About 47.44% of the families were represented
by more than one species and 52.56% of the families were represented by single species each accounting 1.28%
of the total (Appendix 2).

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Visual classification of vegetation into sub sites was made to get homogenous representative groups for
sampling. Five sub sites namely BERGA (BG), GAWO (GW), SAFARA (SF), SOROR (SR) and TECHISA
(TC) localities were identified based on Google Earth images, their vegetation composition as well as visually
observed ecological factors such as altitude, aspect slope, intensity of grazing and disturbance. Plot distribution
among visually classified vegetation types and sub sites for sampling frame is indicated below (Table 1).
Table 1. Plot distribution among visually classified vegetation groups.
S.N. Locality Plots Size Percentage
1 BERGA 1,2,3,5,6,7,8,28,29,30,31,32 12 24
2 GAWO 9,10,11,22,38,39,40,41 8 16
3 SAFARA 34,35,36,37 4 8
4 SORORO 4,11,12,24,26,27, 6 12
5 TECHISA 13,14,15,16,17,18,19,20,21,23,33,42,43,44,45,46,47,48,49,50 20 40
Total 50 100
Plant Community
The result of visual vegetation classification indicated five visually classified vegetation. Vegetation
sampling and plots per the visual classification were taken proportionally (Fig. 3).

Figure 3. Frequencies of plots per visually classified vegetation.


Clustering along elevation range was also done and the result was compared with the dendrogrm output. The
dendrogram output merged the five visually identified vegetations into four major clusters after proper
inspection of the dendrogram structure and partitioning methods (Table 2).
Table 2. Cluster distribution along elevation range.
C Cluster Groups/Sub sites/Plots S % Ele. (m) Diff. (m)
1 BG,BG,TC,TC,GW,GW,GW,BG,SF,SF,SF,TC,SF 13 26 21422269 127
5,6,13,18,22,9,41,32,34,37,36,33,35
2 BG,BG,SR,BG,TC,TC,TC,TC,TC,TC,TC,TC 12 24 21212306 185
3,7,27,28,19,20,21,23,47,45,46,43,
3 BG,TC,TC,SR,TC,TC,TC,BG,TC 9 18 21112237 126
1,16,49,12,44,14,15,2,48
4 SR,SR,BG,TC,TC,BG,GW,GW,SR,TC,BG,GW,SR,BG,GW,GW 16 32 21222338 216
4,26,29,42,50,8,10,38,25,17,31,39,24,30,11,40
Note: BG = Berga, GW = Gawo, SF = Safara, SR = Sororo, TC = Techisa; C = Cluster, S = Cluster size,
% = Percentage, Ele. = Elevation, Diff. = Elevation difference.
Results of vegetation data analysis using R Statistical Software version 3.0.2 (R Core Development Team
2013) revealed four clusters that could be recognized as plant community types. Division of the dendrogram out
put into specific number of cluster was done after inspection. The result of the inspection indicated that there are
four numbers of possible clusters with K-value equal to 4 clusters. The K value was also confirmed for
consistency by partitioning method which is obtained by plotting within groups sum of squares versus number

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of clusters and observing where there is a sharp break in the graph. The value on the x-axis where there is sharp
break of the graph represents the optimal number of clusters in the dendrogram (Fig. 4).

Figure 4. Optimal number of clusters.


The result was plotetted in the dendrogram as indicated below. From the dendrogram, the following sites
belong to the respective cluster number and community type. The four community groups were indicated in the
dendrogram output of agglomerative hierarchical cluster analysis using similarity ratio (SR) in R version 3.0.2
(R Core Development Team 2013) (Fig. 5).
uster1(C1)= Community1: 1,16,49,12,44,14,15,2,48 with cluster size = 9
Cluster2(C2)= Community2: 3,7,27,28,19,20,21,23,47,45,46,43 with custer size = 12
Custer3(C3)= Community3: 4,26,29,42,50,8,10,38,25,17,31,39,24,30,11,40 with cluster size =16.
Cluster4(C4)= Community4: 5,6,13,18,22,9,41,32,34,37,36,33,35 with cluster size = 13
Community 3 comprises about 32% of the clusters followed by community 4 about 26%, followed by
community 2 about 24% followed by community 1 about 18% of the clusters.

Figure 5. Agglomerative Hierarchical Cluster Analysis using similarity ratio (SR).


A combination of dominant or characteristic species with high synoptic values in each community types
were used to name these plant community types (Table 3). A range of possible environmental variables such as

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altitude, aspect, slope, grazing and disturbance were analyzed and presumed to be structuring plant species
composition and community types were indicated by ordination graph (Fig.6).
Table 3. Synoptic table of species reaching value of >1 in at least one community type.
Cluster Number Cluster 1 Cluster 2 Cluster 3 Cluster 4
Cluster Size 13 12 9 16
Acacia abyssinica 4.56 0 0 0
Cordia africana 2.11 0 0 0
Sesbania sesban 1.56 0 0 0
Acacia negrii 1.44 0 0 0
Andropogon abyssinicus 1.33 0 0 0
Caesalpinia decapetala 1.22 0 0 0
Xanthium spinosum 1 0 0 0
Olea europaea 0 3 0 0
Rhus glutinosa 0 2.42 0 0
Croton macrostachyus 0 2.25 0 0
Capparis tomentosa 0 2 0 0
Rhus vulgaris 0 1.92 0 0
Pittosporum viridiflorum 0 1.33 0 0
Clematis simensis 0 1.08 0 0
Podocarpus falcatus 0 0 5.38 0
Ficus sur 0 0 3.31 0
Calpurnia aurea 0 0 2.06 0
Phoenix reclinata 0 0 1.75 0
Vernonia amygdalina 0 0 1.63 0
Juniperus procera 0 0 1.25 0
Bersama abyssinica 0 0 1.19 0
Ficus palmata 0 0 1.13 0
Salix subserrata 0 0 1.06 0
Albizia schimperiana 0 0 0 5.38
Carissa spinarum 0 0 0 1.62
Dovyalis abyssinica 0 0 0 1.54
Pterolobium stellatum 0 0 0 1.31
Euclea divinorum 0 0 0 1.31
Premna schimperi 0 0 0 1.15

Figure 6. CCA: Displaying sites constrained by environmental factors using clusters.


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However, analysis of soil as one of the possible environmental variables did not included in this study. The
description of these plant community types is shown as follows. Therefore, it is worth considering soil as one of
the variable structuring plant species composition thereby community type although the analysis is not included
here.
Cordia africana-Acacia abyssinica type
This community type is found between elevation ranges of 21422269 m and dominated by Acacia
abyssinica and Cordia africana plant species. Sesbania sesban, Acacia negrii, Andropogon abyssinicus,
Caesalpinia decapetala and Petrolobium stellatum are also common species on this community type. Similarly,
Carissa spinarum, Croton macrostachyus, Pittosporum virdiflorum, Capparis tomentosa, Olea europaea, Salix
subserrata, Juniperu procera, Podocarpus falcatus, Premna schimperi, and Pterolobium stellatum are relatively
rare species in this community type (Fig. 7).

Figure 7. AB, Cordia africana-Acacia abyssinica community type.


Olea europaea- Rhus glutinosa type
This community type is found between elevation ranges of 21212306 m and dominated by Olea europaea,
Rhus glutinosa, Croton macrostachyus, Albizia schimperiana and Capparis tomentosa plant species. Common

Figure 8. AD, Olea europaea-Rhus glutinosa community type.


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characteristic plant species belonging to this community include Rhus vulgaris, Carissa spinarum, Pittosporum
virdiflorum, Calpurnia aurea, Pterolobium stellatum, Caesalpinia decapetala and Clematis simensis. Other
relatively rare species belonging to this community include Podocarpus falcatus, Bersama abyssinica, Euclea
divinorum, Juniperus procera, Premna schimperi, Andropogon abyssinicus and Salix subserrata (Fig. 8).
Podocarpus falcatus-Ficus sur type
The dominant species in this community type are Acacia abyssinica, Podocarpus falcatus, Ficus sur and
Croton macrostachyus and it is found in the altitude ranges of 21112237 m. Other common characteristic
species belonging to this community include Calpurnia aurea, Phoenix reclinata, Olea europaea, Vernonia
amygdalina, Carissa spinarum, Rhus vulgaris, Bersama abyssinica, Juniperus procera, Ficus palmate and Salix
subserrata. Species such as Petrolobium stellatum, Premna schimperi, Dovyalis abyssinica, Pittosporum
viridiflorum, Euclea divinorum, Acacia negrii, Andropogon abyssinicus, Caesalpinia decapetala, Capparis
tomentosa, Clematis simensis and Rhus glutinosa are relatively rare species of this community type (Fig. 9).

Figure 9. AD, Podocarpus falcatus-Ficus sur community type.


Albizia schimperiana- Carissa spinarum
The dominant plant species of this community type are Acacia abyssinica, Albizia schimperiana, Croton
macrostachyus and Olea europaea. The community is found in the altitude range of 2122__2338m. Relatively
common species of this community type are Rhus glutinosa, Calpurnia aurea, Capparis tomentosa, Carissa
spinarum, Rhus vulgaris, Dovyalis abyssinica, Euclea divinorum, Premna schimperi and Pterolobium stellatum.
Species such as Juniperus procera, Podocarpus falcatus, Vernonia amygdalina, Acacia negrii, Andropogon
abyssinicus, Phoenix reclinata, Pittosporum viridiflorum, Bersama abyssinica and Clematis simensis are
relatively rare species of this community (Fig. 10).
Ordination
To see which environmental variables that were responsible for structuring the plant species composition and
community types, the results/values of test for significance of environmental variables were obtained for
Canonical Correspondence Analysis (CCA) by running the environmental data for the sites in R Statistical
Software (Table 4).
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Figure 10. Albizia schimperiana-Carissa spinarum community type.


Environmental factors for constraint analysis: CCA and ANOVA
Results of backward and forward selection of environmental variables for constrained analysis based on their
P-value and Akaike Information Criterion (AIC) where the variable with the lowest AIC value is the most
influential (Table 4).
Table 4. Results of test for significance of environmental variables, CCA
Variables Df AIC F_val. N.per Pr(>F) Significance Codes Significance
Altitude 1 307.10 1.6538 199 0.005 ** Yes
Disturbance 1 307.20 1.5529 199 0.005 ** Yes
Aspect 1 307.25 1.5055 199 0.010 ** Yes
Grazing 1 307.37 1.3910 199 0.010 ** Yes
Slope 1 307.51 1.2475 199 0.045 * Yes
Results for ANOVA & CCA test for the significance of each environmental variable prior to analysis,
sequential test for terms, test for axis and permutation test for CCA under reduced model marginal effects of
terms are given below (Table 5).
Table 5. Results for ANOVA in conjunction with CCA to filter out environmental variables.
Variables Df Sum of Sqs Mean Sqs F. model R2 Pr (>F) Signi. codes Significance
Slope 1 0.3941 0.39407 1.3522 0.02605 0.12 No
Aspect 1 0.4560 0.45598 1.5646 0.03107 0.03 * Yes
Grazing 1 0.4700 0.4700 1.6127 0,04471 0.02 * Yes
Altitude 1 0.6763 0.67628 1.3205 0.02028 0.01 ** Yes
Disturbance 1 0.3067 0.30671 1.0524 0.84775 0.32 No
Residuals 44 12.8282 0.29244 1.00000 No
Total 49 15.1262
Results for permutation test sequentially for CCA under reduced model (Table 6); results of ANOVA.CCA
test for availability of axes (Table 7); results for permutation test for cca under reduced model marginal effects
of terms (Table 8) and the values of CCA (Table 9) to display sites constrained by some environmental factors
using cluster groups have also been provided (Fig. 6).

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Table 6. Results of sequential test for terms for significance environmental variables.
Variables Df Chisq F N.perm Pr (>F) Signi.codes Significance
Slope 1 0.1738 1.2893 99 0.05 * Yes
Aspect 1 0.1922 1.4262 99 0.02 * Yes
Grazing 1 0.1807 1.3405 99 0.01 ** Yes
Altitude 1 0.2223 1.6489 99 0.01 ** Yes
Disturbance 1 0.1608 1.1929 99 0.09 Low
Residual 44 5.9309

Table 7. Results of test for availability of axis CCA.


Axis Df Chisq F N.perm Pr (>F) Signi.codes Significance
CCA1 1 0.2654 1.9688 99 0.01 ** Yes
CCA2 1 0.2221 1.6478 99 0.02 * Yes
CCA3 1 0.1736 1.2878 99 0.04 * Yes
CCA4 1 0.1507 1.1171 99 0.24 No
CCA5 1 0.1180 0.8757 99 0.85 No
Residuals 44 5.9309

Table 8. Results of anova.cca values for marginal effects of environmental variables.


Variables Df Chisq F N.perm Pr (>F) Signi.codes Significance
Slope 1 0.1581 1.1726 99 0.15000 No
Aspect 1 0.1671 1.2395 2699 0.06222 Low
Grazing 1 0.1361 1.0098 99 0.47000 No
Altitude 1 0.2221 1.6474 199 0.00500 ** Yes
Disturbance 1 0.1608 1.1929 99 0.13000 No
Residuals 44 5.9309

Table 9. Biplot scores for constraining variables to show colored cluster groups.
Available axis
Variables
CCA1 CCA2 CCA3 CCA4 CA1 CA2
Altitude 0.807 -0.19 -0.516 -0.22 0 0
Grazing -0.271 -0.80 -0.024 -0.54 0 0
Aspect -0.607 -0.37 -0.667 0.22 0 0
Disturbance -0.065 -0.97 0.188 -0.16 0 0
The Beta diversity of paired comparison of sites (Srrensen index of dissimilarity) was found to be 0.79
indicating beta diversity indexes for the sample plots. The Shannon-Wiener diversity and the Simpson diversity
indices were used to calculate alpha diversity. The result of the measures of combined information in multiple
taxa of plant species and their respective abundance values per plot in the whole data frame was represented in
(Appendix 4) and that of clusters is also indicated (Table 10).
Table 10. Shannon-Wiener and Simpson diversity indices for clusters. (H=Diversity)
Clusters Richness H (Shannon-Wiener) H (Simpson) ShannonEvenness Simpson Evenness
1 194 4.74333312 55.39280314 0.900429164 0.285529913
2 161 4.520059437 58.50900307 0.889529569 0.363409957
3 183 4.456467679 42.39296476 0.855452447 0.231655545
4 87 3.727884826 25.0698853 0.83474284 0.288159601

DISCUSSION
Plant Diversity and floristic composition
The result of vegetation data analysis revealed that Tulu Korma and its surrounding area is very rich in plant
species diversity and floristic composition. About 37 (47.44%) of the families recorded from the area were
represented by two and more species while about 41 (52.56%) of the families were represented by only a single
species (1.28%) each.
Top ten species rich families were Asteraceae (50, 64.10%), Fabaceae (27, 34.62%), Lamiaceae (25,
32.05%), Poaceae (18, 23.08%), Solanaceae (15, 19.23%), Acanthaceae (9, 11.34%), Amaranthaceae (8,
10.26%), Cucurbitaceae and Rubiaceae (7, 8.97%) each and Brassicaceae (6, 7.69%). The remaining families;
Apiaceae, Euphorbiaceae, Myrsinaceae, Polygonaceae, Ranunculaceae and Rosaceae were (5, 6.41%) each;
Boraginaceae, Flacourtiaceae; Myrsinaceae and Oleaceae (4, 5.13%) each; Anacardiaceae, Asparagaceae,
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Buddlejaceae, Celastraceae, Cucurbitaceae, Malvaceae, Moraceae, Sapindaceae and Urticaceae (3, 3.85%)
each; Asclepiadaceae, Balsaminaceae, Cactaceae, Cupressaceae, Rhmnaceae, Rutaceae, Tiliaceae and
Verbenaceae (2, 2.56%) each (Appendix 2).
The results also indicated that family Asteraceae (50, 64.10%) was represented by the largest number of
species in the area confirming the fact that family Asteraceae is among the species rich families in the flora
areas of the Flora of Ethiopia and Eritrea (Mesfin Tadesse 2004).
Environmental variables structuring the vegetation composition
Factors responsible for structuring the community types observed in this study will be best explained by the
outputs of ordination analysis pining to five main environmental variables namely altitude, aspect, disturbance,
grazing and slope of which only four of them were found to be significant variables. Moreover, stepwise
selection of environmental factors for constrained analysis using Analysis of variance (ANOVA) in conjunction
with Canonical Correspondence Analysis (CCA) based on their P-value and Akaike Information Criterion (AIC)
where the variable with the lowest AIC value (Table 4) is the most influential indicated that altitude was found
to be the most influential environmental variable at 95% confidence interval (P =0.005) using CCA ordination
(Table 4).
Analysis of diversity
Oksanen (2015) noted that species accumulation models and species pool models study collection of sites
and their species richness. It was further noted that the exact method of calculating site based species
accumulation curve yielded results that are very close to classical algorisms based on a random approach (Kindt
& Coe 2005, Kindt et al 2006, Oksanen 2015). The Beta diversity of paired comparison of sites (Srrensen
index of dissimilarity) for the vegetation of Tulu Korma and its surrounding areas was found to be 0.79
indicating beta diversity indexes for the sample plots. It was stated that beta diversity measures the change in
diversity of species among sets of habitats (Zerihun Woldu 2012). According to (Whittaker 1960 1972), high
beta diversity (w > 5) implies low level of similarity while low beta diversity (w < 1) implies high level of
similarity among sets of habitats. Moreover, it was said that since species turn over between cluster groups is
greater than species turnover between sample plots of the original data high species turnover will indicate high
beta diversity (Whittaker 1972). Hence, for this particular study, since the value of beta diversity was found to
be w = 0.79 and this value is less than one (w < 1), it indicates that there is low beta diversity with no species
turnover and hence high level of similarity among the five sets of vegetation groups that were visually classified
as discussed before.
Similarly, the overall Shannon-Wiener diversity and Simpson diversity indices were also computed to
measure the average degree of uncertainty in predicting to what species an individual chosen from a random
collection of S species and N individuals will belong. Hence, Shannon diversity (H) was found to be 3.37 with P
value = 0.04, then Simpson = 1-P which is about 0.96 on average. In other words, this value is the overall
diversity index of the study area which is relatively higher although it is lower than the diversity indices
reported by other studies like Ermias Lulekal (2014) which was H equal to 4.07 for Dense forest of Ankober
District in North Shewa Zone of Amhara Regional State, Ethiopia. The Shannon-Wiener equitability (J) =
H/Hmax=H/log(S). The overall equitability for S=251 species found to be, equitability (J) =0.61 which is the
average degree of equitability. It was emphasized that a larger Simpson Index will indicate lower diversity;
hence it is better to analyze the reciprocal value of the Simpson Index. An alternative approach is to report 1-
Simpson index (Kindt & Coe 2005). The overall Shannon-Wiener index (H= 3.37) and over all species
evenness (J = 0.61) indicate high species diversity. This high species diversity is in line with Friis (2009) that
the Shewa floristic region is one of the most species rich areas of the flora regions. The values of the Shannon-
Wiener diversity indices for the four clusters were also analyzed to show the diversity among cluster groups
(Appendix 4; Table 10).
Major threats to vegetation
The major threats identified during the course of the study were agricultural intensification, high population
pressure, charcoal making, firewood collection, grazing and demand of plant products for construction and
tools. There are also warning signs of invasive alien species such as Parthinium hystophorus, Argemone
mexicana and Lantana trifolia encroachment observed in the area although their distribution is very limited.

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Eucalyptus plantations are also almost replacing the indigenous trees and other natural vegetation around the
town margins of Addisalem Town.
Impact of centre for indigenous trees propagation project on the area
The Tulu Korma Centre for Indigenous Trees Propagation and Biodiversity Development project is an
interesting trial from where lessons has to be learnt in restoration and rehabilitation activities of similar
environments of other places. There is dramatic change in vegetation cover of the project area during the past
decade. A good justification is that practical observation of the current status of the area and historical records
that maintained by the founder of the centre before the project has centre has established. The centres impact is
not only restoring and rehabilitating the already dead habitat to its current living one but also has changed the
minds and levels of understating of the local community of the surrounding areas towards vegetation
conservation. Such positive impact was confirmed during individual as well as group discussion with local
informants. Farmers, for instance, maintained remnant trees standing in their farmlands and tree cutting is not
common practice in the area.
Moreover, in areas few kilometers away from the centre along the Berga River there are patches of
vegetation in some pocket places with better conservation status. Information from the local informants showed
that it is the farmers of the surrounding areas who kept the vegetation with the lowest possible disturbances.
Therefore, it is good implication that the surrounding communities have learnt a lot from the project activities
about vegetation conservation. The scenario also justifies the hypothesis that the Tulu Korma project centre for
indigenous trees propagation and biodiversity developments has positive impact on local communities
understating of vegetation conservation to come to be true resulting in increasing trend of biodiversity of the
area. That takes us to the conclusion that there is a need to knowledge integration from farmers traditional
ecological knowledge (FTEK) and scientific practices engaged in ecosystem conservation.
Floristic similarity
The Srensen index of similarity with respect to nine floral areas; Masha= 20% (Abreham Assefa et al.
2013), Adisalem-Ginchi= 35% (Abdi Shentema 2012), Ginchi-Ambo= 38% (Belete Kebede 2012), Jeldu= 60%
(Zewdie Kassa 2009), Bale Mountain= 15% (Haile Yinger et al. 2008), Cheliya= 33% (Endale Amenu 2007),
Gimbi = 16% (Etana Tolasa 2007), Wonago = 45% (Fiseha Mesfin 2007), Borena = 4% (Gemedo Dalle et al.
2005) revealed that the vegetation of Tulu Korma and its surrounding areas most similar to Jeldu at 60%
(Zewdie Kassa 2009) similarity and least similar to Borena at 4% (Gemedo Dalle et al. 2005) similarity. The
highest similarity index with respect to Jeldu is pertaining to the fact that these two places are located in the
same vegetation type, the Dry Evergreen Montane Forest and Grassland complex, (Friis et al. 2011) as well as
the same sampling method followed during the study. Similarly, the least similarity with respect to Borena is
pertaining to the fact that Borena lowland belongs to Combretum-Terminalia and Acacia-Commiphora
woodlands which is different vegetation when compared to that of Tulu Korma and its surrounding (Appendix
5).

CONCLUSIONS
The current study revealed that Tulu Korma and its surrounding environs are very rich in plant species and
floristic composition as well as indigenous traditional ethnobotanical knowledge systems. Comparison of
historical records from literature and practical observation of the current status of the study area with regard to
the trends of biodiversity changes in the area confirmed that there is an increasing trend of biodiversity and
vegetation cover. The increasing trend is justified by observing the positive impacts of the project centre for
indigenous trees propagation and biodiversity development located at Tulu Korma.
Although altitude, aspect, disturbance and grazing were found to be among the main environmental variables
structuring the plant species composition as well as plant community types of the study area, altitude is the most
influential environmental variable. This was justified by the ordination out puts of stepwise test for the
significance levels of all the environmental variables presumed to be significant effects.
In Tulu Korma and its surrounding areas, despite the fact that there is better understanding of the local
communities about vegetation conservation, there are also potential threats that need top priority for long-lasting
and sustainable vegetation conservation of the area. This will come to be true by integrating and implementing
effective natural resource management policies in connection with community based conservation projects at its
grass root level. This can be come to truth through understanding the need to build on the existing resources of
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indigenous plant communities by improving on their management sustaining the resources as well as further
domestication of highly valuable taxa.

ACKNOWLEDGMENTS
The authors gratefully acknowledge the local peoples of Tulu Korma and surrounding areas for their
unreserved collaboration during data collection, Addis Ababa University and Mizan-Tepi University for
financial and material support and IDEAWILD for materials and field tools assistance during the study. The
first author would like acknowledge the United Nations Economic Commission for Africa Library (UNECA-
Library) for freely availing digital resources and library facilities during the write up of the article.

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Supporting information
Appendix 1: List of species.
Appendix 2: List of Families.
Appendix 3: List of Genera.
Appendix 4: Values of the Shannon-Wiener diversity indices for each plot.
Appendix 5: Floristic similarity.

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Appendix 1: List of plant species collected from Tulu Korma and its surrounding areas of Ejere District. (Key: T- Tree, Sh-Shrub, H- Herb, Li- Liana, Cl- Climber, W- Willd, HG-
Homegarden, SW- Semi wild, Coll No.(ZKX)- Collection number, DSC_Code- Digital Scan_Code of fresh specimen during data collection)

Scan code/

Distribution
Vernacular

Range of
Coll. No.

collected
Alt. (m)
Family

Digital
Name

Photo
Habit

Plant

from
SN Species Name

1 Acacia abyssinica Hocst. ex Benth T Laaftoo Fabaceae ZK038 DSC01539/540 1500-280 W


2 Acacia albida Del. T Garbii Fabaceae ZK275 ~2600 W
3 Acacia mearnsii Del. Wild. T Amoozaa Fabaceae ZK136 DSC01922 elsewhere HG
4 Acacia melanoxylon R.Br. T Omedla (Am) Fabaceae ZK276 HG
5 Acacia negrii Pic.Serm. T Dodota Fabaceae ZK277 DSC00384/385 2000-3100 W
6 Acacia polyacantha Willd. T Fabaceae ZK278 DSC00430/431 500-1600 W
7 Acacia seyal Del. T Laaftoo Fabaceae ZK279 W
8 Acanthes sennii Chiov. S kosorruu Acanthaceae ZK225 DSC02419 1700-3200 W
9 Achyranthes aspera L. H Darguu Amaranthaceae ZK039 DSC01561-563 750-3050 W/HG
10 Acmella caulirhiza Del. H Gutichaa Asteraceae ZK047 DSC01572/573 550-2600 W
11 Aeschynomene abyssinica (A.Rich.) Vatke S Fabaceae ZK280 1300-3300 W
12 Agave sisalana Perrine ex Engel H qaaca Agavaceae ZK281 DSC07178/179 HG
13 Ageratum conyzoides L. H Asteraceae ZK089 DSC01408 1900-2400 W
14 Ajuga integrifolia Buch.-Ham. ex D. Don H Armaguusaa Lamiaceae ZK282 DSC00418 1500-3400 W
15 Albizia schimperiana Oliv. T Ambaltaa Fabaceae ZK092 DSC01571 1600-2600 W
16 Alchemilla pedata A.Rich. H Gurra hantuutaa Rosaceae ZK081 DSC01456/57 1800-4000 W
17 Alisma plantago-aquatica L. H Alismataceae ZK283 1300-2500 W
18 Allium sativum L. H Qulubbii adii Alliaceae ZK284 HG
19 Allophylus abyssinicus (Hochst) Radlk T Sarara Sapindaceae ZK285 1450-3000 W
20 Amaranthus caudatus L. H Iyyaasuu Amaranthaceae ZK286 DSC00433 500-2500 HG
21 Amaranthus dubius Thell. H Laamoyii Amaranthaceae ZK073 150-2300 HG/SW
22 Amaranthus hybridus L. H Laamoyii Amaranthaceae ZK287 DSC01798 1500-2400 HG/SW
23 Amaranthus spinosus L. H Laamoyii Amaranthaceae ZK288 DSC01485 400-2400 HG/SW
24 Amaranthus viridis L. H Laamoyii Amaranthaceae ZK289 600-2300 W
25 Andropogon abyssinicus Fresen. H Baallamii Poaceae ZK029 DSC01719/720 1800-2400 W
26 Apodytes dimidiata E.Mey. ex Arn. T Calalaqaa Icacinaceae ZK0243 DSCO7749-757 1300-2600 W
27 Argemone mexicana L. H Paperaceae ZK290 DSC00402/403 ~2400 W
28 Argyrolobium fischeri Taub. H Atara hantuutaa Fabaceae ZK291 1600-2400 W
29 Artemisia abyssinica Sch. Bip. ex A. Rich H Arittaa jaldesaa Asteraceae ZK254 DSC07154 1800-3500 W/HG
30 Artemisia afra Jaq. H Arittaa Asteraceae ZK292 HG
31 Arundo donax L. H Shambaqoo Poaceae ZK293 HG
32 Asparagus africanus Lam. S Saritii Asparagaceae ZK294 DSC01869/870 700-3000 W
33 Asparagus racemosus Willd. S Saritii Asparagaceae ZK114 1350-3100 W
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34 Asparagus setaceus (Kunth) Jassop S Saritii Asparagaceae ZK135 DSC02005 550-2400 W
35 Astragalus astropilosulus (Hochst.) Bunge H Fabaceae ZK119 DSC02067-071 1500-2500 W
36 Barleria grandis Hochst. ex Nees H Acanthaceae ZK018 900-1800 W
37 Barleria parviflora R. Br.ex T.Andres. S Acanthaceae ZK169 DSC01440/441 700-2400 W
38 Barleria ventricosa Hochst. ex Nees H Acanthaceae ZK165 500-2600 W
39 Bersama abyssinica Fresen. S Lolchisa Melianthaceae ZK021 DSC01875-877 1700-2715 W
40 Bidens ghedoensis Mesfin H Kelloo Asteraceae ZK002 DSC01402 1380-2600 W
41 Bidens pilosa L. H marfee seexana Asteraceae ZK037 DSC01987/988 750-2400 W
42 Bothriocline schimperi Oliv. & Hiern ex Benth S Asteraceae ZK295 1300-2800 W
43 Bougainvillea spectabilis Willd S Nyctaginaceae ZK260 W
44 Brassica carinata A.Br. H Raafuu Brassicaceae ZK296 DSC01644 1350-3000 HG
45 Brassica nigra (L.) Koch H Raafuu Brassicaceae ZK297 1600-2450 HG
46 Brassica oleracea L. H Raafuu Brassicaceae ZK298 100-3000 HG
47 Brassica rapa L. H tikilgomen Brassicaceae ZK299 ~3000 HG
48 Brucea antidysenterica J.F. Mill. S Qabanyoo Simaroubaceae ZK198 DSC01950-952 1650-2800 W
49 Buddeleja davidii Franch. S Qawisa faranjii Budlejacea ZK193 DSC01775 HG
50 Buddeleja polystachya Fresen. T Qawwisa Budlejacea ZK137 DSC01892/893 200-3300 W/HG
51 Caesalpinia decapetala (Roth) Alston. S Arangamaa faranjii Fabaceae ZK214 DSC01533 W/HG
52 Callistemon citrinus (Curtis) Skeels T Myrtaceae ZK300 DSC00441/442 1200-2500 HG
53 Calpurnia aurea (Ait.) Benth. S Ceekaa Fabaceae ZK009 DSC00378 1650-3000 W
54 Capparis tomentosa Lam. S Arangama guracha Capparidaceae ZK133 DSC01931-934 500-2200 W
55 Cardiospermum halicacabum L. CL Sapindaceae ZK301 W
56 Carduus nyassanus (S. Moore) R. E. Fr. H Qoree harree Asteraceae ZK302 DSC01858/859 1200-3350 W
57 Carissa spinarum L. S Agamsa Apocyanaceae ZK040 DSC00361/364 550-2500 W
58 Cassipourea malosana (Baker) Alston T Warallo Rhizophoraceae ZK303 W
59 Caylusea abyssinica (Fresen) Fisch & Mey H Resedaceae ZK304 DSC01786 1700-2750 W/HG
60 Centella asiatiaca (L.) Urban H Apiaceae ZK245 1000-3200 W
61 Chlorophytum tuberosum (Roxb.) Baker H Anthericaceae ZK027 550-1600 W
62 Cicer arietinum L. H shunburaa Fabaceae ZK048 HG/SW
63 Cirsium englerianum O.Heffm H Asteraceae ZK054 W
64 Cirsium schimperi (atke) C.Jeffrey ex Cufod. H Qoree harree Asteraceae ZK057 1900-3000 W
65 Clausena anisata (Willd) Benth. S ulumaa Myrsinaceae ZK010 DSC01853/854 1500-2300 W
66 Clematis simensis Fresen. Cl Hidda fiitii Ranunculaceae ZK023 DSC01984-986 1500-3350 W
67 Clerodendrum myricoides (Hochst.) Vatke S maraasisaa Lamiaceae ZK094 DSC01618/619 700-2600 W
68 Coffea arabica L. S Buna Rubiacea ZK058 HG
69 Commelina benghalensis L. H Commelinaceae ZKK003 DSC01403 400-2500 W
70 Conyza steudelii Sch. Bip. ex A. Rich. H Asteraceae ZK061 DSC00365 2200-3000 W
71 Cordia africana Lam. T Wadeesa Boraginaceae ZK069 700-2550 W
72 Cotula abyssinica Sch. Bip. ex A. Rich. H Asteraceae ZK077 1600-4600 W
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73 Crassocephalum macropappum (Sch. Bip. ex A. Rich.) H Mandillo(Sh) Asteraceae ZK024 DCS01442 870-2700 W
74 Crassocephalum rubens (Juss. ex Jacq.) S.Moore H Asteraceae ZK070 870-2700 W
75 Crassocephalum viellinum aut non (Bent) S.moore H Mandillo(Sh) Asteraceae ZK099 DSC01549/540 870-2700 W
76 Crotalaria rosenii (Pax) Milne-Redh. ex Polhill S atara qamalee Fabaceae ZK074 1350-2800 W
77 Croton macrostachyus Del. T Bakkaniisa Euphorbiaceae ZK011 DSC00407-409 500-2350 W
78 Cucumis dipsaceus Ehrenb. ex Spach Cl Hidda buqe seexana Cucurbitaceae ZK075 670-2000 W
79 Cucumis ficifolius A. Rich. H Hidii hooloo Cucurbitaceae ZK031 DSC01499 1300-2400 W
80 Cucumis prophetarum L. H Cucurbitaceae ZK076 200-1950 W
81 Cucurbita pepo L. Cl dabaaqula Cucurbitaceae ZK079 HG
82 Cupressus lusitanica Mill. T gaatira faranjii Cupressaceae ZK083 HG
83 Cyathula cylinderica Moq. H Darguu/cogogiitii Amaranthaceae ZK086 900-3100 W
84 Cyathula uncinulata (Schrad.) Schinz H Darguu/cogogiitii Amaranthaceae ZK088 1300-2500 W
85 Cymbogon citratus (D.C.) Stapf. H Xajisaara Poaceae ZK090 HG
86 Cynodon dactylon (L.) Pers. H coqorsa Poaceae ZK091 1600-2700 W
87 Cynoglossum amplifolium Hochst. ex A.DC. H qoricha michii Boraginaceae ZK093 1500-3500 W
88 Cynoglossum coeruleum Hochst. ex A.DC. H qoricha michii Boraginaceae ZK195 DSC01473/75 1750-2250 W
89 Cyphostemma adenocaule Desc. ex Wild & R.B.Drumm. Cl hidda dololaa Vitaceae ZK063 DSC01629-631 600-2650 W
90 Datura stramonium L. H manjii Solanaceae ZK095 DSC00380/381 600-2800 W
91 Datura suaveolens Humb. & Bonpl. ex Willd. S Tsadaqii Solanaceae ZK105 DSC00434 W
92 Delphinium wellbyi Hemsl. H Ranunculaceae ZK109 DSC01815/816 1520-3800 W
93 Dicranopteris linearis (Burn. f.) Underw. H kaarolee Gleicheniaceae ZK113 W
94 Dicrocephalia integrifolia H Q/Marzii Asteraceae ZK082 1750-3500 W
95 Dicrostachys cinerea (L.) Wight & Am. S Fabaceae ZK118 450-2000 W
96 Digitaria abyssinica (Hochst. ex A. Rich) H Poaceae ZK072 1400-2700 W
97 Discopodium penninervium Hochst. T coongii Solanaceae ZK122 1500-3500 W/HG
98 Dodonea angustifolia L. f. S ittacha Sapindaceae ZK244 500-2900 W
99 Dovyalis abyssinica (A. Rich.) Warb. T koshomii Flacourtiaceae ZK132 DSC01946-949 1700-3000 W
100 Dovyalis caffra (Hook. f. & Harv.) Hook. f. S koshomii Flacourtiaceae ZK123 1000-2700 W
101 Dovyalis verucosa (Hochst.) Warb. S mixmixaa Flacourtiaceae ZK168 1700-2200 W
102 Dregea schimperi (Becne.) Bullock Cl hidda gor'isaa Asclepiadaceae ZK111 DSC0220-022 1600-2400 W
103 Echinops hispidus Fresen. H shokolee Asteraceae ZK080 1700-3100 W
104 Echinops kebericho Mesfin S qabarichoo Asteraceae ZK126 DSC01606 2300-2600 W
105 Echinops macrochaetus Fresen. H shokolee Asteraceae ZK128 DSC01994/995 1350-3600 W
106 Echinops spinosus L. S shokolee Asteraceae ZK036 DSC01504 1350-2200 W
107 Echium plantaginum L. H Boraginaceae ZK134 DSC01917-919 2200-2350 W
108 Ekebergia capensis Sparrm T somboo Meliaceae ZK112 DSC01822/823 1680-3000 W
109 Eleusine floccifolia (Forssk.) Spreng H Cangeedara Poaceae ZK026 DSC01491 1800-3200 W
110 Embelia schimperi Vatke S haanquu Myrsinaceae ZK129 1700-2800 W
111 Ensete ventricosum (Welw.) Cheesman H warqee Musaceae ZK130 HG
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112 Epilobium hirsutum L. H Ciraaroo/Ibsaa Onagraceae ZK120 DSC02125-129 1200-3000 W
113 Equisetum ramosissimom Derf. H Equisetaceae ZK022 DSC01458-461 W
114 Eragrostis spp. H muriyyii Poaceae ZK071 W
115 Eragrostis teff (Zucc.) Trotter H xaafii Poaceae ZK210 DSC01595 HG/SW
116 Erucastrum arabicum Fisch & Mey H Jare seexanaa Brassicaceae ZK131 1350-2350 W
117 Erythrina brucei Schweinf. T waleenssuu Fabaceae ZK139 DSC01737/738 1400-2600 W
118 Ethulia gracilis Del. S Asteraceae ZK143 300-2150 W
119 Eucalyptus camaldunesis Dehnh. T bargamoo Myrtaceae ZK060 1250-2800 HG
120 Eucalyptus globulus Labill T bargamoo Myrtaceae ZK052 DSC01557/558 1700-2800 HG/SW
121 Eucalyptus terticornis Smith. T bargamoo Myrtaceae ZK144 DSC01647-649 1450-2350 HG
122 Euclea divinorum Hiern. S mi'essaa Ebenaceae ZK045 DSC01992/993 1000-2400 W
123 Euphorbia abyssinica Gmel. T adaamii Euphorbiaceae ZK145 DSC00421 1900-2400 W/HG
124 Euphorbia pulcherrima Willd. ex Klotzsch S Euphorbiaceae ZK146 DSC01722 HG
125 Ficus Palmata Forssk. S Luugoo Moraceae ZK148 100-2400 W
126 Ficus sur Forssk. T harbuu Moraceae ZK104 DSC02083/084 1400-2500 W
127 Ficus vasta Forssk. T qilxuu Moraceae ZK149 1000-2400 W
128 Flacourtia indica (Burn. f) Merr. S Flacourtiaceae ZK241 DSC01873/874 1100-2350 W
129 Foeninculum vulgare Miller H insilaalee Apiaceae ZK142 DSC01978/979 1600-2300 HG/SW
130 Galiniera saxifraga (Hochst.) Bridson S buniitii Rubiacea ZK154 1350-3000 W
131 Galinsoga paviflora Cav. H Asteraceae ZK025 DSC00365 950-2800 W
132 Galium simense Fresen. H saam'ee Rubiacea ZK234 1700-4000 W
133 Girardinia bullosa (Steudel) wedd. H Boobbii Urticaceae ZK155 DSC01905 1700-3000 W/HG
134 Glycine wightii (Wigh & Arn.) Verde Cl hidda hantuuta Fabaceae ZK216 DSC01711 380-2600 W
135 Gnaphalium rubriflorum Hilliard H Asteraceae ZK078 DSC01464/65 2000-3600 W
136 Grewia ferruginea Hochst. ex A. Rich. S dhoqonuu Tiliacea ZK235 1350-2700 W
137 Guizotia abyssinica (L. f.) Cass. H nuugii Asteraceae ZK055 DSC00432 1500-2400 HG/SW
138 Guizotia scabra (Vis.) Chiov. H adaa Asteraceae ZK151 DSC01598 1300-3300 W
139 Guizotia schimperi Sch.Bip. ex Walp. H adaa Asteraceae ZK158 DSC01555 1300-3300 W
140 Harpachne schimperi (Hochst.ex Rich.) H Marga/Qunnii Poaceae ZK166 1000-2400 W
141 Helminthotheca echioides (L.) Holub H Asteraceae ZK167 DSC00365 2000-2450 W
142 Heracleum abyssinicum (Boiss.) Norman H Ulululee Apiaceae ZK117 1800-3750 W
143 Hibiscus ludwigii Eckl. & Zeyh. H Malvaceae ZK087 1400-2800 W
144 Hygrophila shulli (Hamilt.) M. R. & S. M. H kachoo Acanthaceae ZK046 DSC01554 550-2600 W
145 Hyparrhenia anthistirioides (Hochst. ex Arich.) Stapf H Poaceae ZK170 1200-2400 W
146 Hypericum quartinianum A. Rich. S ulee foonii Gutiferae ZK150 DSC01817-819 1500-3000 W
147 Hypoestes triflera (Forssk.) Roem & Schult. H Acanthaceae ZK013 DSC01431 1200-3200 W
148 Impatiens rothii Hook. f. H burii Balsaminaceae ZK173 DSC01930 1800-3500 W
149 Impatiens spp. H ansosila Balsaminaceae ZK172 W
150 Ipomoea indica (Burn.f.) Merrill Cl Convolvulaceae ZK174 W
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151 Jasminum abyssinicum Hochst. ex DC. Cl idda ichilbee Oleaceae ZK012 DCS01940/941 1700-2800 W
152 Jasminum fluminense Vell. Cl Oleaceae ZK209 1300-2000 W
153 Jasminum grandiflorum L. Cl qamaxee Oleaceae ZK051 DSC01610-613 1600-2800 W
154 Juniperus procera Hochst. ex Endle. T gaatira habashaa Cupressaceae ZK125 DSC01920/921 W/HG
155 Justicia ladanoides La. H Acanthaceae ZK014 DSC01434-436 500-2700 W
156 Justicia schimperiana (Hochst. ex Nees) T. Anders. S dhumugaa Acanthaceae ZK043 DSC01576/577 1300-2700 W/HG
159 Kalanchoe densiflora Rolfe H bosoqee Crassulaceae ZK177 DSC01507 W
157 Kalanchoe marmorata Bak. H bosoqee Crassulaceae ZK035 1340-2500 W
158 Kalanchoe petitiana A. Rich H bosoqee Crassulaceae ZK175 DSC01555/556 2000-3000 W
160 Lactuca inermis Forssk H Asteraceae ZK176 W
161 Laggera alata (D.Don) Olive. H Asteraceae ZK041 DSC01426 1400-2700 W
162 Laggera crispata (Vahl) Hepper & Wood H Asteraceae ZK068 DSC01448 600-2650 W
163 Laggera tomentosa (Sch.Bip.ex A.Rich.) Oliv.& Heirn S Kaskasee Asteraceae ZK179 W
164 Lantana trifolia L. S Aqanciraa Asteraceae ZK180 W
165 Leucas martinicensis (Jacq.) R. Br. H bokkoluu adii Lamiaceae ZK050 DSC01488 ~2500 W
166 Linum usitatissimum L. H talbaa Liniaceae ZK217 DSC01599/600 HG/SW
167 Lippia adoensis var. adoensis Hochst. ex Walp S kusaayee Lamiaceae ZK006 DSC01414 1600-3000 W/HG
168 Lippia adoensis var. koseret S shokonota Lamiaceae ZK147 DSC01546 1600-3000 W/HG
169 Lycopersicon esculentum Mill. H timatima Solanaceae ZK181 800-1800 HG
170 Maesa lanceolata Forssk. T abbayii Myrsinaceae ZK102 DSC01561-563 1350-3000 W
171 Malva verticillata L. H liitii Malvaceae ZK182 1600-4000 W/HG
172 Maytenus arbutifolia (A. Rich) Wiczek. S kombolcha Celasteraceae ZK067 DSC01557/558 1200-3000 W
173 Maytenus gracilipes (Welw. ex Olive.) Exell S kombolcha Celasteraceae ZK184 DSC01823-825 1250-2800 W
174 Maytenus obscura (A.Rich) Cuf. S kombolcha Celasteraceae ZK185 DSC01925 1700-3100 W
175 Medicago polymorpha L. H Fabaceae ZK020 DSC01455 1400-3000 W
176 Milletia ferruginea (Hochst.) Bak. T birbirraa Fabaceae ZK186 DSC01899-901 1600-2500 W
177 Momordica foetida Shumach Cl Cucurbitaceae ZK226 530-3450 W
178 Myrica salicifolia A. Rich. T baroodoo Myricaceae ZK255 DSC07156 1750-3300 W
179 Myrsine africana L. S qacama Myrsinaceae ZK098 1900-3800 W
180 Nicandra physaloides (L.) Gaertn H Solanaceae ZK064 DSC00405/406 600-2100 W
181 Nicotiana tabacum L. H tamboo Solanaceae ZK053 DSC01640 300-2400 HG/SW
182 Nuxia congesta R. BT. ex Fresen. T qawwisa Budlejacea ZK249 110-3800 W
183 Ocimum basilicum L. S Bosobila Lamiaceae ZK187 DSC01980/981 ~2400 HG
184 Ocimum lamiifolium Hochst. ex Benth. S Lamiaceae ZK115 DSC02059/060 1200-2900 W/HG
185 Ocimum uritcifolium Roth S Lamiaceae ZK033 DSC01878/879 600-2100 W
186 Oenanthe palustris (Chiov.) Norman H qoricha lagaa Apiaceae ZK124 DSC02110-012 1500-3000 W
187 Olea europaea subsp. Cusp (Wall. ex G. Don) Cif T ejersa Oleaceae ZK127 DSC01923/924 1250-3000 W/HG
188 Opuntia cylinderica (Lam.) D.C. T adaamii Cactaceae ZK188 2300-2450 W/HG
189 Opuntia ficus-indica (L.) Miller T adaamii Cactaceae ZK189 1400-2400 W/HG
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190 Oreoshimperella verrucosa (A. Rich.) Rausder H Gosa dinbilaalaa Apiaceae ZK190 1900-3800 W
191 Orobanche minor Smith H Orobanchaceae ZK056 DSC01620 1100-3000 W
192 Osyris quadripartita Decn. S waatoo Santalaceae ZK096 DSC01621 1600-2900 W
193 Oteostegia tomentosa subsp. ambigiensis S Lamiaceae ZK191 DSC01820-822 1200-3000 W
194 Parthenium hysterophorus L. H Asteraceae ZK246 DSC00404 900-1800 W
195 Pavetta abyssinica Fresen. S Rubiacea ZK066 DSC01871/872 1350-2850 W
196 Pavonia urens Cav. S Malvaceae ZK192 DSC01433 1500-3500 W
197 Pelargonium multibracteatum Hochst. ex A. Rich. H Geraniaceae ZK242 1000-2700 W
198 Pennisetum sphacelatum (Nees) Th. Dur. & Schinz H Poaceae ZK196 1900-2800 W
199 Pennisetum thunbergii Kunth. H Poaceae ZK197 1700-3000 W
200 Pennisetum vellosum Fresen. H Poaceae ZK199 1800-2800 W
201 Pentas schimperiana (A. Rich.) Vatke H Rubiacea ZK247 1800-3200 W
202 Persicaria setosula (A. Rich) K.L. Wilson H kicuu Polygonaceae ZK201 500-2900 W
203 Phaulosis imbricata (Forssk.) Sweet H Acanthaceae ZK202 DSC01601 600-2700 W
204 Phoenix reclinata Jacq. T meexii Arecaceae ZK097 DSC01557 500-2400 W
205 Physalis peruviana L. H Awuxii Solanaceae ZK203 1200-2600 W
206 Phytolacca dodecandra L. 'Hrit. S handoodee Phytolacacea ZK204 DSC01990/991 1500-3000 W
207 Pisum sativum L. H atara Fabaceae ZK206 HG/SW
208 Pittosporum viridiflorum Sims S soolee Pittosporacea ZK205 DSC01799-803 1400-3000 W
209 Plantago lanceolata L. H qorxobbii Plantaginaceae ZK015 DSC01432 1200-3200 W
210 Plectocephalus varians (A. Rich) C,Jeffrey ex Cufod H Asteraceae ZK108 DSC02034-036 1900-3600 W
211 Plectranthes alpinus (Baker) J.K. Morton H Lamiaceae ZK194 1800-2800 W
212 Plectranthes assurgens Baker H Lamiaceae ZK207 1800-2800 W
213 Plectranthes barbatus Andrews H Lamiaceae ZK138 950-2750 W
214 Plectranthes cylindraceus Hochst. ex Benth. H dabasee Lamiaceae ZK274 1000-2600 W
215 Plectranthes orantus Codd H Lamiaceae ZK222 1300-3500 W
216 Plectranthes punctatus (L.f.) L' Hr. H Lamiaceae ZK084 DSC01406/407 1350-3200 W
217 Plectranthes schimperi Chiov. H Lamiaceae ZK116 DCS01582 W
218 Plumbago zeylanica L. H Plumbaginaceae ZK208 700-2200 W/HG
219 Podocarpus falcatus (Thunb.) R. B. ex. Mirb T birbirsa Podocarpaceae ZK042 DSC00412-416 W
220 Polygonium plebeium R.Br. H Polygonaceae ZK140 DSC01965/966 1000-2400 W
221 Premna schimperi Engl. S urgeessaa Lamiaceae ZK017 DSC01451 1350-2400 W
222 Prunus africana (Hook. f.) Kalkm. T gura'ee Rosaceae ZK212 DSC01906-911 1700-2500 W/HG
223 Pterolobium stellatum (Forssk.) Brenan S arangama diimaa Fabaceae ZK153 DSC01793/794 W
224 Pycnostachys abyssinica Fresen. H Lamiaceae ZK213 DSC01847 1100-2700 W
225 Pycnostachys eminii Grke H Lamiaceae ZK215 DSC01847 1700-2200 W
226 Ranunculus multifidus Forssk. H Ranunculaceae ZK106 DSC01943 1200-3800 W
227 Raphanus raphanistrum L. H Solanaceae ZK062 1800-2400 W
228 Rhamnus prinoides Lyterit. S geeshoo Rhamnaceae ZK218 DSC00370 1175-3200 W/HG
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229 Rhamnus staddo A.Rich S qadiidaa Rhamnaceae ZK110 DSC01614 1400-2900 W
230 Rhus glotinosa A. Rich T daboobesa Anacardiacea ZK159 DSC01535/536 1450-2700 W
231 Rhus vulgaris Meikle T Xaaxessaa Anacardiacea ZK059 DSC01466 W
232 Ricinus communis L. S qobbo Euphorbiaceae ZK121 DSC02117 W
233 Rorippa nasturtium-aquaticum (L.) Hayek H Brassicaceae ZK219 2200-2600 W
234 Rosa abyssinica Lindley S hangooxoo Rosaceae ZK030 DSC01500/501 1900-3300 W
235 Rosa x richardii Rehd S Rosaceae ZK220 1000-2100 HG
236 Rosmerinus officinalis L. S Lamiaceae ZK221 HG
237 Rubia cordifolia L. H Rubiacea ZK223 W
238 Rubus steudneri Schweinf S goraa Rosaceae ZK211 2300-3000 W
239 Rumex abyssinicus Jacq. H maqmaqoo Polygonaceae ZK101 DSC01547 1200-3300 W
240 Rumex nepalensis Spreng H shuultii Polygonaceae ZK001 DSC00368 1200-3900 W
241 Rumex nervosus Vahl S dhangaggoo Polygonaceae ZK224 DSC00395 400-3300 W
242 Ruta chalepensis L. H Rutaceae ZK227 HG
243 Rytigna neglecta (Hiern) Robyns S mixoo Rubiacea ZK267 W
244 Saccharum officinarum L. H shankoora Poaceae ZK228 DSC02088/089 HG
245 Salix subserrata Willd. S alaltuu Salicaceae ZK005 DSC01411/412 1250-2850 W
246 Salvia nilotica Jacq. H qoricha michii Lamiaceae ZKO16 DSC01438/39 1300-3800 W
247 Sapium elipticum (Krauss) Pax S Euphorbiaceae ZK229 1050-2100 W
248 Satureja abyssinica (Benth.) Briq. H Lamiaceae ZK230 DSC01567/568 900-2700 W
249 Satureja paradoxa (Vatke) Engl. ex Seybold H duufaa loonii Lamiaceae ZK103 DSC01581 1350-3500 W
250 Satureja punctata (Benth.) Briq. H naddoo in Sheka Lamiaceae ZK231 600-3840 W
251 Scadoxus multiflorus (Martyn) Raf H kuukuu/arfaasee Amaryllidaceae ZK232 W
252 Schefflera abyssinica (Hochst. Ex A. Rich) Harmon T gatamaa Araliaceae ZK233 1450-2800 W
253 Schinus molle L. T Anacardiacea ZK237 HG
254 Senecio ochrocarpus Oliv. & Hiern H Asteraceae ZK238 DSC01564/566 2800-4300 W
255 Senecio spp. H Asteraceae ZK164 W
256 Senecio subsessilis H Asteraceae ZK239 DSC01505 2400-4310 W
257 Senna didymobotrya (Fresen.) Irwin & Bar S ceekaa guraacha Fabaceae ZK019 DSC00390/391 1450-2400 W
258 Sesbania sesban (L.) Merr. T Fabaceae ZK236 300-2000 HG
259 Setaria incrassata (Hochst.) Hack. H Poaceae ZK240 W
260 Setaria pumela (Poir.) Roem & Schult. H Poaceae ZK085 500-2400 W
261 Sida schimperiana Hochst. ex A. Rich S Fabaceae ZK028 DSC01495/496 1500-2600 W
262 Sida tenuicarpa L. S Fabaceae ZK248 1550-2300 W
263 Silybum marianum (L.) Gaertn. H Asteraceae ZK251 2200-2600 W
264 Snowdenia polystachya (Fresen.) Pilg. H mujjaa Poaceae ZK252 1500-2700 W/HG
266 Solanacio gigas (Vatke) C. Jeffrey S dilu arbaa Asteraceae ZK253 W
267 Solanum anguivi Lam. H Solanaceae ZK163 500-2800 W
268 Solanum incanum L. S Solanaceae ZK162 DSC00396 ~2100 W
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269 Solanum indicum L. S hidii waraabesa Solanaceae ZK256 DSC01583/584 W
270 Solanum marginatum L. f. S Solanaceae ZK100 DSC00429 2000-3000 W
271 Solanum nigrum L. H Solanaceae ZK032 DSC01479/80 700-2350 W
272 Solanum tuberosum L. H dinicha Solanaceae ZK257 1800-2600 HG
273 Solanum vilosum Mill. H Solanaceae ZK258 1200-2600 W
274 Sonchus asper (L.) Hill H Asteraceae ZK259 DSC00352 1050-2850 W
265 Sorghum bicolor (L.) Moench H misngaa Poaceae ZK261 DSC01597 HG/SW
275 Sphaeranthus suaveolens (Forssk.) DC. H Q/Marzii Asteraceae ZK107 DSC01593 1600-2100 W
276 Spilanthes uliginosa Jacq. H moxomoxaa Asteraceae ZK262 W
277 Stephania abyssinica (Dillon & A. Rich) Walp. Cl hidda kalaalaa Menispermaceae ZK044 DSC01579 1450-3400 W
278 Syzygium guineense (Wild.) DC. S badeessaa Myrtaceae ZK004 DSC01409/410 1200-2500 W
279 Tacazzea conferta N.E.Br. Cl hidda annannoo Asclepiadaceae ZK141 DSC01959-952 W
280 Tagetes minuta L. S bari fidee Asteraceae ZK049 DSC01645-646 W
281 Teclea nobilis Del. S hadheesa Rutaceae ZK250 W
282 Thalictrum rhyncocarpum Dill. A. Rich H siraabizuu Ranunculaceae ZK200 DSC01761-763 1600-3050 W
283 Thalictrum schimperianum Hochst. ex Schweinf. H Ranunculaceae ZK263 DSC01826/827 1900-3800 W
284 Thymus spp. H Lamiaceae ZK183 W
285 Trifolium polystachyum Fresen H Fabaceae ZK008 DSC01416 W
286 Triumfetta rhomboidea Jaq. S Tiliacea ZK264 400-2750 W
287 Typha domingensis Pers. H shanbaqoo jaldesa Thyphaceae ZK171 DSC02122-124 W
288 Urera hypselodendron (A. Rich) Wedd H laanqisaa Urticaceae ZK265 1700-2800 W
289 Urtica simensis Steudel H saamaa Urticaceae ZK266 1500-3400 W/HG
290 Verbascum siniaticum Benth. H guraa harree Verbenaceae ZK034 DSC00397 W
291 Verbena officinalis L. H q/albaatii Verbenaceae ZK268 W
292 Vernonia adoensis Sch. Bip.ex Wlp H Asteraceae ZK269 500-2000 W
293 Vernonia amygdalina Del. T ebicha Asteraceae ZK152 DSC01541 W/HG
294 Vernonia auriculifera Hiern S reejii Asteraceae ZK161 W
295 Vernonia myrantha Hook. f. S reejii Asteraceae ZK156 W
296 Vicia faba L. H baqelaa Fabaceae ZK270 HG/SW
297 Xanthium abyssinica Walroth H Asteraceae ZK271 W
298 Xanthium spinosum L. H Asteraceae ZK272 DSC00428 W
299 Xanthium strumarium L. H Asteraceae ZK65 W
300 Ximenia americana L. S Olacaceae ZK273 500-2450 W
301 Zanthodeschia aethiopica (L.) K.P.J. Sprengel H Araceae ZK305 DSC02041/042 W
302 Zea mays L. H boqoolloo Poaceae ZK306 DSC01549/540 HG/SW
303 Zehneria scabra (Linn. f.) Sond. Cl hidda adii Cucurbitaceae ZK160 DSC02023-027 W
304 Zehneria spp. Cl hida xixiixa Cucurbitaceae ZK307 W
Note: *Commercial in a sense is to mean that people depend on these species to make a living by selling them to generate income for their livelihood and not referring
to large scale production. It is simply to mean marketability or how the poor people in the area depend on the species mentioned for their daily life

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Appendix 2: List of Families.
S.N. Families Number Percentage 27 Cucurbitaceae 7 8.97 54 Paperaceae 1 1.28
1 Acanthaceae 9 11.34 28 Cupressaceae 2 2.56 55 Phytolacacea 1 1.28
2 Agavaceae 1 1.28 29 Ebenaceae 1 1.28 56 Pittosporacea 1 1.28
3 Alismataceae 1 1.28 30 Equisetaceae 1 1.28 57 Plantaginaceae 1 1.28
4 Alliaceae 1 1.28 31 Euphorbiaceae 5 6.41 58 Plumbaginaceae 1 1.28
5 Amaranthaceae 8 10.26 32 Fabaceae 27 34.62 59 Poaceae 18 23.08
6 Amaryllidaceae 1 1.28 33 Gleicheniaceae 1 1.28 60 Podocarpaceae 1 1.28
7 Anacardiacea 3 3.85 34 Flacourtiaceae 4 5.13 61 Polygonaceae 5 6.41
8 Anthericaceae 1 1.28 35 Geraniaceae 1 1.28 62 Ranunculaceae 5 6.41
9 Apiaceae 5 6.41 36 Gutiferae 1 1.28 63 Resedaceae 1 1.28
10 Apocyanaceae 1 1.28 37 Icacinaceae 1 1.28 64 Rhamnaceae 2 2.56
11 Araceae 1 1.28 38 Lamiaceae 25 32.05 65 Rhizophoraceae 1 1.28
12 Araliaceae 1 1.28 39 Liniaceae 1 1.28 66 Rosaceae 5 6.41
13 Arecaceae 1 1.28 40 Malvaceae 3 3.85 67 Rubiacea 7 8.97
14 Asclepiadaceae 2 2.56 41 Meliaceae 1 1.28 68 Rutaceae 2 2.56
15 Asparagaceae 3 3.85 42 Melianthaceae 1 1.28 69 Salicaceae 1 1.28
16 Asteraceae 50 64.1 43 Menispermaceae 1 1.28 70 Santalaceae 1 1.28
17 Balsaminaceae 2 2.56 44 Moraceae 3 3.85 71 Sapindaceae 3 3.85
18 Boraginaceae 4 5.13 45 Musaceae 1 1.28 72 Simaroubaceae 1 1.28
19 Brassicaceae 6 7.69 46 Myricaceae 1 1.28 73 Solanaceae 15 19.23
20 Budlejacea 3 3.85 47 Myrsinaceae 4 5.13 74 Thyphaceae 1 1.28
21 Cactaceae 2 2.56 48 Myrtaceae 5 6.41 75 Tiliacea 2 2.56
22 Capparidaceae 1 1.28 49 Nyctaginaceae 1 1.28 76 Urticaceae 3 3.85
23 Celasteraceae 3 3.85 50 Olacaceae 1 1.28 77 Verbenaceae 2 2.56
24 Commelinaceae 1 1.28 51 Oleaceae 4 5.13 78 Vitaceae 1 1.28
25 Convolvulaceae 1 1.28 52 Onagraceae 1 1.28
26 Crassulaceae 3 3.85 53 Orobanchaceae 1 1.28

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Appendix 3: List of Genera.
S.N. Genera Number Percentage 38 Cassipourea 1 0.46 76 Eleusine 1 0.46
1 Acacia 6 2.78 39 Caylusea 1 0.46 77 Embelia 1 0.46
2 Acanthes 1 0.46 40 Centella 1 0.46 78 Ensete 1 0.46
3 Achyranthes 1 0.46 41 Chlorophytum 1 0.46 79 Epilobium 1 0.46
4 Acmella 1 0.46 42 Cicer 1 0.46 80 Equisetum 1 0.46
5 Aeschynomene 1 0.46 43 Cirsium 3 1.39 81 Eragrostis 2 0.93
6 Agave 1 0.46 44 Clausena 1 0.46 82 Erucastrum 1 0.46
7 Ageratum 1 0.46 45 Clematis 1 0.46 83 Erythrina 1 0.46
8 Ajuga 1 0.46 46 Clerodendrum 1 0.46 84 Ethulia 1 0.46
9 Albizia 1 0.46 47 Coffea 1 0.46 85 Eucalyptus 3 1.39
10 Alchemilla 1 0.46 48 Commelina 1 0.46 86 Euclea 1 0.46
11 Alisma 1 0.46 49 Conyza 1 0.46 87 Euphorbia 2 0.93
12 Allium 1 0.46 50 Cordia 1 0.46 88 Ficus 3 1.39
13 Allophylus 1 0.46 51 Cotula 1 0.46 89 Flacourtia 1 0.46
14 Amaranthes 4 1.85 52 Crassocephalum 3 1.39 90 Foeninculum 1 0.46
15 Andropogon 1 0.46 53 Crotalaria 1 0.46 91 Galiniera 1 0.46
16 Apodytes 1 0.46 54 Croton 1 0.46 92 Galinsoga 1 0.46
17 Argemone 1 0.46 55 Cucumis 3 1.39 93 Galium 1 0.46
18 Argyrolobium 1 0.46 56 Cucurbita 1 0.46 94 Girardinia 1 0.46
19 Artimesia 2 0.93 57 Cupressus 1 0.46 95 Glycine 1 0.46
20 Arundo 1 0.46 58 Cyathula 2 0.93 96 Gnaphalium 1 0.46
21 Asparagus 3 1.39 59 Cymbogon 1 0.46 97 Grewia 1 0.46
22 Astralagus 1 0.46 60 Cynodon 1 0.46 98 Guizotia 3 1.39
23 Barlerial 3 1.39 61 Cynoglossum 2 0.93 99 Harpachne 1 0.46
24 Bersama 1 0.46 62 Cyphostemma 1 0.46 100 Helminthotheca 1 0.46
25 Bidens 2 0.93 63 Datura 2 0.93 101 Heracleum 1 0.46
26 Bothriocline 1 0.46 64 Delphinium 1 0.46 102 Hibiscus 1 0.46
27 Bougainvillia 1 0.46 65 Dicranopteris 1 0.46 103 Hygrophila 1 0.46
28 Brassica 4 1.85 66 Dicrocephalia 1 0.46 104 Hyparrhenia 1 0.46
29 Brucea 1 0.46 67 Dicrostachys 1 0.46 105 Hypericum 1 0.46
30 Budeleja 2 0.93 68 Digitaria 1 0.46 106 Hypoestes 1 0.46
31 Caesalpinia 1 0.46 69 Discopodium 1 0.46 107 Impatiens 2 0.93
32 Callistemon 1 0.46 70 Dodonea 1 0.46 108 Ipomoea 1 0.46
33 Calpurnia 1 0.46 71 Dovyalis 3 1.39 109 Jasminum 3 1.39
34 Capparis 1 0.46 72 Dregea 1 0.46 110 Juniperus 1 0.46
35 Cardiospermum 1 0.46 73 Echinops 4 1.85 111 Justicia 2 0.93
36 Carduus 1 0.46 74 Echium 1 0.46 112 Kalanchoe 3 1.39
37 Carissa 1 0.46 75 Ekebergia 1 0.46 113 Lactuca 1 0.46
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114 Laggera 3 1.39 149 Phytolacca 1 0.46 184 Senna 1 0.46
115 Lantana 1 0.46 150 Pisum 1 0.46 185 Sesbania 1 0.46
116 Leucas 1 0.46 151 Pittosporum 1 0.46 186 Setaria 1 0.46
117 Linum 1 0.46 152 Plantago 1 0.46 187 Sida 2 0.93
118 Lippia 2 0.93 153 Plectocephalus 1 0.46 188 Silybum 1 0.46
119 Lycopersicon 1 0.46 154 Plectranthes 7 3.24 189 Snowdenia 1 0.46
120 Maesa 1 0.46 155 Plumbago 1 0.46 190 Soghum 1 0.46
121 Malva 1 0.46 156 Podocarpus 1 0.46 191 Solanacio 1 0.46
122 Maytenus 3 1.39 157 Polygonium 1 0.46 192 Solanum 7 3.24
123 Medicago 1 0.46 158 Premna 1 0.46 193 Sonchus 1 0.46
124 Milletia 1 0.46 159 Prunus 1 0.46 194 Sphaeranthus 1 0.46
125 Momordica 1 0.46 160 Pterolobium 1 0.46 195 Spilanthes 1 0.46
126 Myrica 1 0.46 161 Pycnostachys 2 0.93 196 Stephania 1 0.46
127 Myrsine 1 0.46 162 Ranunculus 1 0.46 197 Syzygium 1 0.46
128 Nicandra 1 0.46 163 Raphanus 1 0.46 198 Tacazzea 1 0.46
129 Nicotiana 1 0.46 164 Rhamnus 2 0.93 199 Tagetes 1 0.46
130 Nuxia 1 0.46 165 Rhus 2 0.93 200 Teclea 1 0.46
131 Ocimum 3 1.39 166 Ricinus 1 0.46 201 Thalictrum 2 0.93
132 Oenanthe 1 0.46 167 Rorippa 1 0.46 202 Thymus 1 0.46
133 Olea 1 0.46 168 Rosa 2 0.93 203 Trifolium 1 0.46
134 Opuntia 2 0.93 169 Rosmerinus 1 0.46 204 Triumfetta 1 0.46
135 Oreoshimperella 1 0.46 170 Rubia 1 0.46 205 Typha 1 0.46
136 Orobanche 1 0.46 171 Rubus 1 0.46 206 Urera 1 0.46
137 Osyris 1 0.46 172 Rumex 3 1.39 207 Urtica 1 0.46
138 Oteostegia 1 0.46 173 Ruta 1 0.46 208 Verbascum 1 0.46
139 Parthenium 1 0.46 174 Rytigna 1 0.46 209 Verbena 1 0.46
140 Pavetta 1 0.46 175 Saccharum 1 0.46 210 Vernonia 4 1.85
141 Pavonia 1 0.46 176 Salix 1 0.46 211 Vicia 1 0.46
142 Pelargonium 1 0.46 177 Salvia 1 0.46 212 Xanthium 3 1.39
143 Pennisetum 3 1.39 178 Sapium 1 0.46 213 Ximenia 1 0.46
144 Pentas 1 0.46 179 Satureja 3 1.39 214 Zanthodeschia 1 0.46
145 Persicaria 1 0.46 180 Scadoxus 1 0.46 215 Zea 1 0.46
146 Phaulosis 1 0.46 181 Schefflera 1 0.46 216 Zehneria 2 0.93
147 Phoenix 1 0.46 182 Schinus 1 0.46
148 Physalis 1 0.46 183 Senecio 3 1.39

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Appendix 4: Values of the Shannon-Wiener diversity indices for each plot.
PID H'(Diversity) Richness Evenness P17 3.060292 27 0.928533 P34 2.899514 22 0.938038
P1 3.329631 32 0.960729 P18 3.142136 29 0.933133 P35 3.054263 25 0.94886
P2 3.253272 28 0.976312 P19 3.184894 29 0.945831 P36 3.054912 26 0.937637
P3 3.389128 32 0.977896 P20 2.999674 25 0.931901 P37 2.813805 21 0.924219
P4 3.581226 41 0.964361 P21 3.092242 26 0.949095 P38 3.251435 31 0.94684
P5 3.370436 36 0.940538 P22 3.107949 28 0.932701 P39 3.271425 32 0.943934
P6 3.533461 41 0.951499 P23 2.74415 19 0.931977 P40 3.218976 28 0.96602
P7 3.295591 31 0.959698 P24 3.09061 24 0.972485 P41 3.091012 25 0.960277
P8 3.321857 34 0.942007 P25 3.144701 27 0.954143 P42 3.238892 30 0.95228
P9 3.260078 32 0.94066 P26 3.088121 28 0.92675 P43 3.504557 37 0.970545
P10 3.362857 37 0.931302 P27 3.122136 28 0.936958 P44 3.20823 31 0.934258
P11 3.288576 33 0.940532 P28 3.017692 24 0.949541 P45 2.796782 18 0.96762
P12 3.347116 34 0.94917 P29 3.165204 30 0.930615 P46 2.902043 21 0.953201
P13 3.504579 40 0.950039 P30 3.113305 26 0.95556 P47 2.797547 19 0.950112
P14 3.539896 45 0.929921 P31 3.005245 27 0.911831 P48 2.614161 15 0.96533
P15 3.616323 46 0.944545 P32 3.059186 26 0.938949 P49 2.653067 18 0.917898
P16 3.143009 30 0.924089 P33 2.689051 17 0.949117 P50 3.209445 28 0.96315

Appendix 5: Floristic similarity.


Locality Elevation Author Year a b c Ss %
Tulu Korma 2111-2338 Zewdie Kassa 2015 - - - - -
Masha 1700-3000 Abreham Assefa 2013 43 261 87 0.198 7.424
Addisalem-Ginchi 2137-2289 Abdi Shentema 2012 71 233 30 0.351 13.161
Ginchi-Ambo 2101-2200 Belete Kebede 2012 78 226 32 0.377 14.136
Jeldu 1200-3100 Zewdie Kassa 2009 165 139 77 0.604 22.647
Bale mountain 3010-3410 Haile Yinger et al. 2008 41 263 189 0.154 5.774
Cheliya 1300-3060 Endale Amenu 2007 82 222 106 0.333 12.486
Gimbi 1310-2100 Etana Tolasa 2007 42 262 169 0.163 6.112
Wonago 1350-2875 Fiseha Mesfin 2007 112 192 86 0.446 16.723
Borena 1252-1777 Gemedo Dalle 2005 13 291 314 0.041 1.537

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ISSN (E): 2349 1183
ISSN (P): 2349 9265
3(2): 320324, 2016

Research article

Identification of heterotic crosses for sesame breeding using


diallel matting design
Swapan K. Tripathy*, D.R. Mishra, S. Panda, N. Senapati, Pramod K. Nayak,
G. B. Dash, Sasmita Dash, Kartik C. Pradhan, A.P. Dash, D. Mishra, S. K. Mohanty,
P. M. Mohapatra and D. Lenka
Department of Agricultural Biotechnology, College of Agriculture, Orissa University of Agriculture and
Technology, Bhubaneswar, India
*Corresponding Author:swapankumartripathy@gmail.com [Accepted:23June 2016]

Abstract:Heterosis over better parent (Hb) was estimated in a set of diallel crosses comprising 12
diverse sesame varieties, for fourteen morpho-economic traits viz. days to initial flowering, days to
cessation of flowering, duration of flowering, days to maturity, height to first capsule, plant height,
number of primary branches/plant, number of capsules/plant, capsule length, capsule breadth,
number of seeds/capsule, 500-seed weight, oil content and seed yield/plant. Sixty out of 66 hybrids
revealed significant positive heterosis for seed yield. T13 x E8 was considered as the best heterotic
cross combination which had yield advantage of 93% over the best high yielding parent CST 785.
Besides, Pratap x RT 103 exhibiting second highest Hb value for number of capsules/plant,
resulted significantly higher seed yield with second highest positive heterosis. Similarly, CST 785
x E8 with first highest and second highest significant positive Hb value for capsule number and
number of primary branches, had also shown to be equivalent to third highest position in terms of
significant heterobeltiosis for seed yield/plant. Pratap x T13 and Pratap x Madhabi harbour per se
oil content more than 58% which resulted maximum heterobeltiosis for the trait among the crosses.
Keywords:Mean performance - Morpho-economic traits - Sesamum indicum L.

[Cite as:Tripathy SK, Mishra DR, Panda S, Senapati N, Nayak PK, Dash GB, Dash S, Pradhan KC, Dash AP,
Mishra D, Mohanty SK, Mohapatra PM &Lenka D (2016) Identification of heterotic crosses for sesame
breeding using diallel matting design.Tropical Plant Research3(2): 320324]

INTRODUCTION
Sesame (Sesamum indium L, Family: Pedaliaceae) is the oldest oilseed crop. India is the second largest
producer of sesame in the world and the crop is considered as the queen of high quality vegetable oils owing to
high levels unsaturated fatty acids and antioxidants e.g.sesamol, sesamin, sesamolin and sesaminol (Nupuret al.
2010). Sesame harbour comparatively high oil content (55% of dry seed) among the oilseed crops, but, it suffers
a serious setback in terms of productivity (368 kg ha-1) as compared to world average (489 kg ha-1). This is due
to large scale cultivation of low yielding varieties in marginal lands. High levels of morphological genetic
diversity do exist in sesame (Arrielet al. 2007) but this has not been fully harnessed for genetic improvement of
the existing cultivars through heterosis breeding. Heterosis for seed yield is due to simultaneous manifestation
of allelic and inter-allelic interactions of innumerable number of genes controlling important morpho-economic
component traits under certain environmental conditions. Hybrid vigour of even a small magnitude for
individual components may have an additive or synergistic effect on the end product (Sasikumar&Sardana
1990). Thus, extent of heterotic response of F1 hybrids largely depends on the breeding value and genetic
diversity of the parents involved in the crosses (Young &Virmani 1990). Heterosis over better parent
(heterobeltiosis: Hb) is relatively more important than relative heterosis for commercial exploitation of hybrids.
Heterobeltiosis for seed yield and yield components in sesame has been reported by many workers
(Saravannan&Nadarajan 2002, Prajapatiet al. 2010, Padmasundari& Kamala 2012). Besides, heterotic crosses
may be aminable for selection of high yielding transgressivesegregants in F2 and follow up selfing generations.

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.
Therefore, in the present investigation, an attempt has been taken to identify most desirable heterotic
combination(s) in sesame following half diallel mating design.

MATERIALS AND METHODS


Twelve popular parental genotypes of sesame collected from different states of India were tested along with
all possible cross combinations in RBD with three replications to raise F1 generation. Emasculation and
pollination were carried out in late afternoon using Fevicol method (Das 1990) for all possible combinations of
parental genotypes in a diallel mating design. Each parental genotype and cross was grown in five rows of 3.5m
length with a spacing of 30 x 10 cm. Observations on days to initial flowering, days to cessation of flowering,
duration of flowering, days to maturity, height to first capsule (cm), plant height (cm), number of primary
branches/plant, number of capsule/plant, capsule length (cm), capsule breadth (cm), number of seeds/capsule,
500-seed weight (gm), oil content (%) and seed yield/plant (gm) were recorded. Mean values per replication for
all traits were subjected to analysis of variance as per Panse&Sukhatme (1985).
Heterosis of 66 cross combinations in sesame was estimated over better parent for 14 morpho-economic
traits including oil content and seed yield as per cent increase of the F1 from higher or lower parent means
(Tumer 1953). The statistical significance of heterosis estimate was tested by t test (Wynne et al. 1970). The
test was performed on the difference of the F1 mean and the higher or lower parent mean for heterobeltiosis (Hb)
before expressing them in percentage.

RESULTS AND DISCUSSION


Heterosis is a common feature in most of the crop plants. It represents superiority over better parent
when F1 exceeded the higher parent or over lower parent if F 1 fell below the lower parent. A positive
estimate of heterobeltiosis would truly reflect hybrid vigour for important favourable yield related traits
and seed yield per se which are meant for improvement in positive direction. However, some of the traits
related with flowering and maturity; and also plant height are favoured in negative direction where a
negative estimate that referred as negative heterobeltiosis would be appropriate.
In the present investigation, a high level of heterosis in the desired direction was observed in several hybrids
for seed yield and yield components including oil content (Table 1). Estimates of Hb for days to initial
flowering (DIF), days to cessation of flowering (DCF) and days to maturity (DM) ranged from -17.62 to
14.29%, -14.05 to 13.13% and -10.44 to 5.12% respectively. A few crosses e.g.12, 6 and 4 crosses had
shown significant positive Hb values while, a large number of crosses i.e. 39, 27 and 31 crosses exhibited
significant negative Hb estimates for the above flowering and maturity traits. This indicates that there is
ample scope for recovery of transgressivesegregants in F 2 and onwards towards negative direction for the
said traits.Pratap x BS 5-18-6 had shown very high significant negative heterobeltiosis(-12.70) for both
days to initial flowering and days to cessation of flowering.
Hb values for period of flowering ranged from -34.29 to 12.90% (Table 1). Seven crosses exhibited
significant positive Heterobeltiosis for the trait with maximum value being observed in case of CST 785 x
T 13(14.77%). These crosses may pave the way for wider scope for capsule formation and seed
development. In contrast, as high as 42 crosses revealed significant negative Hb indicating tendency of
large number of crosses for early and synchronous flowering.
In the present investigation, 14 crosses showed positive heterosis for plant height. Heterosis for plant
height has been also reported by Dixit (1976) and Mishra et al. (1994). Singh et al. (2007), Raghunaiahet
al. (2008) and Krishnaiahet al. (2003) observed maximum positive heterosis of 8.67% and 20.23% and
35.7% for plant height. But, hybrids with shorter plant type can withstand lodging. Two cross combinations
e.g. BS 5-18-6 x Phuletil 1(-10.10%) and BS 5-18-6 x TMV 5(-9.52%) have been identified to have
significant negative heterobeltiosis for plant height (Table 1). On the other hand, moderately tall plants that
bore capsules from lower height can bring about more number of capsules/plant. As many as 27 cross
combinations revealed significant negative heterobeltiosis for height to first capsule. Three elite cross
combinations that exhibited lower most Hb values for the trait are RT 103 x Phule Til 1(-35.7%), BS 5-18-
6 x Phule Til 1(-33.5%), Pratap x RT 103 (-32.6%) which would have better scope in sesame breeding.
Number of primary branches, number of capsules/plant, capsule length, number of seeds/capsule and
500-seed weight are the important determinants for seed yield in sesame. A tremendously higher number of
crosses (40, 50, 41, 23 and 35) revealed significant positive heterobeltiosisfor above characters which ranged
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Table 1. Heterobeltiosis (Hb%) of possible crosses involving twelve parents of sesame following half-diallel mating design.
Character Range of Hb values (and Freq. of Most desirable F1s with high heterobeltiosis
mean performance) elite crosses
Days to initial -17.62 to 14.29 12(39) TC 25x Madhabi(14.29), PratapxPhuletil
floweringb (33.5-46.0) 1(13.11), RT 103x Madhabi(11.22)a
TC 25 x Pratap(-17.62), Pratap x BS 5-18-6 (-
12.70), CST 785 x Pratap (-11.89)
Days to cessation -14.05 to 13.13 6(27) RT 103x Phuletil 1(13.13), TMV5x
of flowering b
(58.67-73.0) Madhabi(9.62), RT 103x T 13(8.94)
TC 25x E 8 (-14.05), Pratap x BS 5-18-6(-
13.02), BS 5-18-6x TMV 5 (-11.50)
Period of flowering -34.29 to 12.90 7(42) Pratap x TMV 5(12.90), CST 785x T
(23.0-35.0) 13(12.22), B67xVinayaka(8.33),
b
Days to maturity -10.44 to 5.14 4(31) TC 25x RT 103(5.14), BS 5-18-6xMadhabi
(71.0-85.0) (4.59), TC 25x Madhabi(3.21)
TC 25x TMV 5 (-10.44), TC 25 x E 8 (-9.22),
BS 5-18-6 x TMV 5 (-8.63)
Plant heightb -10.10 to 31.86 14(2) T 13x Madhabi(31.86), Pratap x TMV
(91.67-130.4cm) 5(19.50), Madhabix E 8(19.14)
BS 5-18-6 x Phuletil 1 (-10.10), BS 5-18-6 x
TMV 5 (-9.52)
Height to first -35.7 to 24.99 6(27) T 13x Madhabi (24.99),
capsule b
(44.18-74.6 cm) B67xVinayaka(24.02), T 13x Madhabi(18.90),
RT 103 x Phule Til 1 (-35.7), BS 5-18-6 x
Phule Til 1 (-33.5), Pratap x RT 103 (-32.6)
No of primary -38.68 to 116.67 40(0) Pratap x T13(116.67), CST 785 x E 8(108.51),
branches (1.08-3.27) BS 5-18-6 x Phuletil 1(101.39)

No of capsules -15.6 to 102.56 50(0) CST 785x E 8 (102.56), Pratap x RT103


per plant (15.57-39.57) (96.06), T 13 x E 8 (81.75)

Capsule length -13.69 to18.84 41(0) CST 785 x Phuletil 1 (18.84), RT 103 x
(2.34-3.01cm.) Phuletil 1 (15.35), RT 103x T 13 (13.84),

Capsule breadth -26.81 to 1.61 5(0) TC 25 x Madhabi (1.61), Vinayaka X


(0.73-0.89cm.) Madhabi (1.03), RT 103 x T 13 (0.83)

No of seeds per -25.25 to 22.31 23(0) T 13 x E 8 (22.31), T 13 x Phuletil 1 (20.08),


capsule (57.73-92.37) B67 x Madhabi (14.36)

500 -Seed weight -13.37 to 12.84 35(0) TC 25 x CST 785 (12.84), CST 785 x TMV 5
(1.15-1.62gm) (11.88), TMV5 x E 8 (11.73)

Oil content -13.57 to 11.59 11(0) PratapxT13(11.59%), Pratap x


(45.53-58.4%) Madhabi(11.54%)

Seed yield per plant -15.8 to 131.5 60(0) T13 x E8 (224.6), Pratap x RT 103 (131.5), BS
(2.64-7.82gm) 5-18-6 x T13 (119.1), T13 x Madhabi(118.5),
CST 785 x E8 (116.7),Pratap x T13(85.9%),
Pratap x Madhabi(68.1%)
Note: a-Figures within the parenthesis indicates significant Hb value (%) of promising crosses at P0.05 or P0.01
b- Consideration for significant Hb values (%) in negative direction is marked bold.
from -38.68 to -116.67%,-15.6 to -102.56%, -13.69 to -18.84%, -25.25 to -22.31% and -13.37 to -12.84%
respectively (Table 1). The top ranking crosses with high positive Hb value have been sorted out in respect of
each of these important yield contributing traits. CST 785 x E8 with first highest (102.56%) and second highest
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(108.51%) significant positive Hb value for capsule number and number of primary branches; had also shown to
be equivalent to third highest position in terms of significant heterobeltiosis for seed yield/plant (116.7%).
Similarly, Pratap x RT 103 exhibiting second highest Hb value for number of capsules/plant; had resulted
tremendously higher seed yield with second highest estimated increase over better parent (131.5%).Prajapatiet
al. (2006) identified a sesame hybrid TMV-3 x C1013 which manifested highest significant positive heterosis
for number of branches per plant (163.2%) and number of capsules per plant (26.0%) in a set of 10x10 diallel
crosses. Similarly, Krishnaiahet al. (2003) observed significant positive heterosis ranging from 4.4 to 80.2% for
capsules on main stem in 9 out of 28 crosses of a 8 x 8 half diallel design.
It is worth to note that only five crosses e.g. TC 25 x Madhabi (1.61%), Vinayaka X Madhabi (1.03%), RT
103 x T 13(0.83%), RT 103 x TMV 5(0.53%) and TC 25 x CST 785 (0.36%) exceeded over the better parent
(Table 1)for capsule breadth and these crosses may result increase in 500-seed weight due to bold seeds. For
instance, the cross combination TC 25 x CST 785 that had shown positive significant heterobeltiosis for capsule
breadth; also was shown to have maximum increase (12.84%) in 500-seed weight over the better parent (CST
785). In contrast, Krishnaiahet al. (2003) observed lower estimates of maximum positive heterosis (4.6% and
5.92%) for 1000-seed weight in sesame while, Saravannan&Nadarajan (2002) identified significant positive
heterosis as high as 35.3% in 4 out of 28 crosses in a 8 x 8 diallel mating design for the trait.
Sesame being an oilseed crop, identification of heterotic crosses for oil content and seed yield per se is of
special concern. In the present investigation, RT 103, TC 25, TMV 5 and CST 785 exhibited high oil content in
seed (54.0%) among the parents while, CST785 x Pratap, CST 785 x Madhabi, CST 785 x Phule Til 1,
Pratap x T13 and Pratap x Madhabi and RT 103 x T13 resulted high per se oil content ( 58%) (data not
shown). Hb values for oil content ranged from -13.57 to 11.59% (Table 1) as also reported by Prajapatiet al.
(2006). However, Navadiyaet al. (1995) observed lower estimates of heterobeltiosis for oil content. In the
present study, only eleven crosses revealed significant increase in oil content over better parent among
whichPratap x T13 (11.59%) and Pratap x Madhabi (11.54%) had shown maximum positive Hb value
(11.0%).
Heterosis breeding is one of the potential techniques to improve yields in sesame. All crosses except only six
crosses exhibited significant positive increase over the respective better parent for seed yield in different cross
combinations. Among these, T13 x E8 (6.95gm/plant, 224.6%) followed by Pratap x RT 103 (131.5%), BS 5-
18-6 x T13 (119.1%), T13 x Madhabi (118.5%), and CST 785 x E8 (116.7%) may be sorted out as best five
heterotic crosses for seed yield. To exploit commercially viable heterosis the new crosses are compared with
released varieties or desirable parent, so that the crosses with high heterotic potential could be identified. In this
context, T13 x E8 (224.6%) had shown yield advantage of 93% over the best high yielding parent CST 785(3.6
gm/plant). The increased seed yield in the above crosses can be attributed to increase in one or more than one
yield component. Further, this envisaged that there is enough scope for increase in seed yield in selected crosses
through selection of transgressivesegregants in F2 and follow up selfing generations. It is worth to note that the
above mentioned crosses (T13 x Madhabi, Pratap x T13 and Pratap x Madhabi) that exhibited around 6%
increase in oil content over their better parent; had also shown significant increase in seed yield (over better
parent) to the extent of 118.5%, 85.9% and 68.1% respectively. Thus, these crosses may pave the way for
isolation of valuable pedigree lines in advance selfing generations for higher seed yield and more oil content.
Singh (2002) studied heterosis for 7 characters in 21 crosses among 7 varieties and they recorded significant
positive heterosis as high as 96.3% for number of capsules per plant and 120.7% for seed yield. Similarly,
Saravannan&Nadarajan (2002) observed maximum positive heterosis of 87.85% and 140.1% for seed yield in a
set of 5 x 5 and 8 x 8 diallel crosses in sesame respectively. Prajapatiet al. (2010) identified a cross ABT23 x
ABT26 showing highest heterosis for seed yield per plant in 45 F1s of sesame resulting from 10 x 10 diallel.
Besides, Padmasundari& Kamala (2012) identified a cross X-79-1 X EC 351887 in a 5 x 5 half diallel mating
design which showed promising performance with heterosis and highly significant genetic gain in F2 and F3 for
seed yield and yield components.
In the present investigation, 66 possible crosses are grouped into 15 high x high, 36 high x low and 15 low x
low crosses on the basis of yield performance of the parental varieties among which 9, 15 and 4 crosses
recorded significantly higher seed yield respectively (data not shown) Thus, it seems that high x low cross
combinations can result significantly high yielding hybrids with high frequency. In this context, Busch et al.
(1974) reported that the high x high crosses produced the highest frequency of superior F 6 lines, but the high x

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low crosses produced lines, though in low frequency, that was higher yielding than the best lines from high x
high crosses. While the low x low crosses did not produce any high yielding line.In contrast, in the present
study, the hybrids resulting from low yielding parents, involving RT 103 with Pratap and T13, exhibited
substantial increase in yield with highly positive and significant heterobeltiosis. Such a situation could be
attributed to high inter-allelic interaction canceling the individual unfavourable effects of each
other.Knysh&Norik (1978), however, had shown that heterosis occurred most frequently when both the parents
had high general combining ability i.e. in high x high GCA crosses, and more rarely in high x moderate crosses.
But, the F1 hybrids of high x low and moderate x low GCA crosses were inferior to the better parent in yield
performance.

REFERENCES
Arriel NHC, Mauro AOD, Arriel EF, Trevisoli SHU, Costa MM, Brbaro IM & Muniz FRS (2007) Genetic
divergence in sesame based on morphological and agronomic traits. Crop Breeding & Applied
Biotechnology 7: 253261.
Busch RH, Janke JC &Frolberg RC (1974) Evaluation of crosses among high and low yielding parents of spring
wheat (Triticumaestivum L.) and bulk prediction of the performance.Crop Science 14: 4750.
Das PK (1990) A simple modified technique for selfing and hybridization in (Sesamum indicum L.).Andhra
Agriculture Journal37: 104106.
Dixit RK (1978) Combining ability in sesame. Indian Journal of Agricultural Science 48(6): 362364.
Knysh AI &Norik IM (1978) Selection of inter varietal hybrids of winter wheat in F2 and later generations on
the basis of heterosis in the F1. SelskkhzyaistvennayaBiologiya 13: 140142.
Krishnaiah G, Reddy KR &Sekhar MR (2003) Heterosis and combining ability in sesame (Sesamum indicum
L.). Journal of Oilseeds Research 20: 229233.
Mishra AK, Yadav LN, Tomar RKS & Raghu IS (1994) Heterosis and combining ability in genetical diverse
line in sesame. Sesame and Saflower Newsletter 9: 2129.
Navadiya LJ, Godhani PR &Fougat RS (1995)Heterosis studies in sesame (Sesamum indicum L.). Gujarat
Agriculture University Research Journal 20(2): 7377.
Nupur M, Bhat KV & Srivastava PS (2010) Variation in fatty acid composition in Indian germplasm of sesame.
Journal of the American Oil Chemists Society 87 (11): 12631269.
Padmasundari M & Kamala T (2012) Heterosis in Sesamum indicum L. Asian Journal of Agricultural Sciences
4(4): 287290.
Panse VG &Sukhatme PV (1985) Statistical methods for agricultural workers.Fourth Edition. pp. 48117.
Prajapati KP, Patel KM, Patel CJ &Thakker DA (2006) Heterosis breeding in sesame (Sesamum indicum
L.).Journal of Oilseeds Research 23(2): 292294.
Prajapati N, Patel NCG, Bhatt AB, Prajapati KP & Patel KM (2010) Heterosis in Sesame (Sesamum indicum
L.). International Journal of Agricultural Sciences 6(1): 9193.
Raghunaiah E, Ganga KishanA& Ansari NA (2008) Combining ability and heterosis for yield and yield
components in sesame (Sesamum indicum L.). Journal of Research ANGRAU 36 (2&3): 916.
Saravanan S &Nadarajan N (2002) Studies on heterosis in sesame (Sesamum indicum L.).Indian Journal of
Genetics 62: 271272.
Sasikumar B & S Sardana (1990) Heterosis for yield and yield components in sesame.Indian Journal of
Genetics 50(1): 8788.
Singh PK (2002) Heterosis and inbreeding depression in sesame (Sesamum indicum L.).Indian Journal of
Genetics 62: 169170.
Singh AK, Lal JP, Kumar H &Agrawal RK (2007) Heterosis in relation to combining ability for yield and its
components in sesame ( Sesamum indicum L.). Journal of Oilseeds Research 24(1): 5155.
Tumer JH (1953) A study of heterosis in upland cotton, combining ability and inbreeding effects.
AgronomyJournal 45: 487490.
Wynne JC, Emery & PW Rice (1970) Combining ability estimates in Arachis hypogea 1. II. Field performance
of F1 hybrids. Crop Science 10(6): 713714.
Young J &Virmani SS (199) Heterosis of rice over environments.Euphytica51: 8798.

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ISSN (P): 2349 9265
3(2): 325327, 2016

Short communication

Taxonomic and comparative report of marine centric diatom


Pseudosolenia calcar-avis (Bacillariophyceae) after tasman spirit
oil spill, the affected area of North Arabian sea
Asma Tabassum1*, Hina Saeed Baig2 and Aliya Rehman1
1
Department of Botany, University of Karachi, Karachi, Pakistan
2
National Institute of Oceanography, Karachi, Pakistan
*Corresponding Author: centricdiatomist@gmail.com [Accepted: 24 June 2016]

[Cite as: Tabassum A, Baig HS & Aliya R (2016) Taxonomic and comparative report of marine centric diatom
Pseudosolenia calcar-avis (Bacillariophyceae) after tasman spirit oil spill, the affected area of North Arabian
sea. Tropical Plant Research 3(2): 325327]
In present study brief account of Pseudosolenia calar-avis has been presented, samples were collected
during a survey that was carried out along the Northern Arabian Sea after 2003 Tasman Spirit Oil Spill. Present
findings were also compared with other local and regional studies.
Pseudosolenia calcar-avis is a very important and commonly occurring centric diatom which may dominate
the phytoplankton community structure (Sunesen & Sar 2007). This diatom was previously included in genus
Rhizosolenia but due to asymmetric valves, absence of otaria and coiled rimportula (Round et al. 1990) they
have latterly been transferred in genus Pseudosolenia (Karpinsky 2010). This genus is exclusively present in
warmer waters where as infrequent reports have also been observed from temperate zones (Yun & Lee 2011).
Moreover manifestations suggest that they may also be found in temperate to colder waters (Hernandez-Becerril
et al. 2010). Reports from Mediterranean region revealed that this species was found in nutrient poor conditions
of summer months (Pearce 1998). Previous records from North Arabian Sea showed that this species occurred
7.14% of the total biomass of centric diatoms (Tabassum & Saifullah 2012). In this study a brief account of
description of Pseudosolenia calcar-avis have been presented during a survey carried out along the Northern
Arabian Sea after an oil spill. Present findings have also been compared within the same area and other regions
as well.

Figure 1. Map showing Tasman spirit oil spill sampling site in Northern Arabian Sea.

www.tropicalplantresearch.com 325
Received: 13 March 2016 Published online: 30 June 2016
Tabassum et al. (2016) 3(2): 325327
.
Phytoplankton samples were collected from Sea view, Karachi harbor, part of Northern Arabian Sea
bordering Pakistan after an oil spill named Tasman Spirit Oil Spill (Fig. 1). Samples were then immediately
fixed in 10% formalin. Water assessment parameters including pH, water temperature and salinity were also
measured. For light microscopy samples were treated with nitric acid and HCl to remove organic matter and
rinsed with distilled water to desalinize and then cleaned material were observed using LABX N-400M.
Pseudosolenia calcar-avis (Schultze) Sundstrom, 1986: 95, figs 40-46, 247-257. (Fig. 2)
Rhizosolenia calcar-avis Schultze., Hasle & Syvertsen, 1997, p. 160, Plate 30 (158); Sunesen & Sar, 2007, p.
637-640, Fig. 68-81 (638); Hernandez-Becerril, 2010, pp. 100, Figs. 16-21(p. 98); Karpinsky, 2010, pp. 82, Fig.
1; Yun & Lee, 2011, pp. 307, Figs. 4 (A-H) (pp. 308); Tabassum & Saifullah, 2012, pp. 73, Fig. 38.
Cylindrical, elongated, large, solitary cells with apical axis ranges from 80108 m. Conical valves show
asymmetric appearance ends with curved claw like process; narrow contiguous area with sigmoid depression
runs from process towards the valve; bands scale like in 23 columns.
Apical axis: 80108 m
General Distribution: Buenos Aires coastal waters, Argentina (Sunesen & Sar 2007); Mexican Coast
(Hernandez-Becerril 2010); Korean Coastal waters (Yun & Lee 2011); Northern Arabian Sea (Tabassum &
Saifullah 2012).

Figure 2. Girdle view of Pseudosolenia calcar-avis


The study reveals the presence of marine planktonic diatom Pseudosolenia calcar-avis from the Northern
Arabian Sea that was affected by the hazardous incident of Tasman Spirit Oil Spill. Pseudosolenia calcar-avis
has been previously recorded from Northern Arabian Sea bordering Pakistan (Tabassum & Saifullah 2012) but
in present study a marked difference in term of diameter of the specimens have been observed (Table 1). It is
concluded that this discrepancy in morphometric observation is related to the disaster of Tasman Spirit Oil Spill,
as the crude oil contents has effect on metabolism and growth of diatoms (Huang et al. 2010) and large sized
specimens have been recorded after the spill, so the discrepancy in morphometric observations in present studies
is justified and reason is oil pollution.
Table 1. Comparision of morphometric data among Pseudosolenia calcar-avis of present study with the previous records.
Cupp Hendey Moazzam Hasle & Hernandez- Tabassum & Yun & Present
(1943) (1964) (1973) Syvertsen Becerril et al. Saifullah Lee study
(1997) (2010) (2010) (2011) (2005)
Region Pacific Atlantic North Tropical North Koren North
Ocean Ocean Arabian ---------- Mexican Arabian Coastal Arabian
Sea Pacific Sea waters Sea
Apical 6 m 35 m 10 m to 45 m to 29 m 25 m 9.3 m 80 m
axis to 53 m to70 m 50 m 190 m to 55 m to 87 m to 90 m to108 m

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.
REFERENCES:
Cupp EE (1943) Marine planktonic diatoms of the west coast of North America. Bulletins of the Scripps
Institution of Oceanography 5(1): pp. 238.
Hasle GR & Syvertsens EE (1997) Marine Diatoms (5-386). In: Tomas CR (ed) Identifying Marine
Phytoplankton. Academic Press, San Diego, California, pp. 1385.
Hendey NI (1964) An introductory account of the smaller algae of British coastal waters. Fishery Investigations
Series 1V, Part V. Bacillariophyceae (Diatoms). H.M. Stationery Office, pp. 317.
Hernandez-Becerril DU, Herrera-Hernandez P, Perez-Mendoza A & Ceballos-Corona JGA (2010) Marine
planktonic diatoms of the order Rhizosoleniales (Bacillariophyta) from the Tropical Mexican Pacific. Vie Et
Milieu-Life And Environment 60(2): 95107.
Huang YJ, Jiang ZB, Zeng JN, Chen QZ, Zhao YQ, Liao YB, Shou L & Xu XQ (2011) The chronic effects of
oil pollution on marine phytoplankton in a subtropical bay, China. Environmental Monitoring and
Assessment 176(14): 517530.
Karpinsky MG (2010) Pseudosolenia calcar-avis (Bacillariophyta, Centrophyceae) in the Caspian Sea. Russian
Journal of Biological Invasions 1(2): 8186.
Moazzam M (1973) Taxonomic and seasonal studies of planktonic centric diatoms from Manora channel
(Lower Harbour) Karachi, M.Sc. Thesis. Karachi University, Pakistan, 350 p.
Pearce RB, Kemp AES, Koizumi I, Pike J, Cramp A & Rowland SJ (1998) A Limina-scale, SEM-Based study of
a lake quaternary diatom-ooze Sapropel from the Mediterranean Ridge, site 971. Robertson AHF, Emeis
KC, Richter C & Camerlemghi A (eds). Proceedings of the Ocean Drilling Program, Scientific Results, Vol.
160.
Round FE, Crawford RM & Mann DG (1990) The Diatoms, Biology and Morphology of Genera. Cambridge
University Press, Cambridge, pp. 1747.
Sunesen I & Sar EA (2007) Marine diatoms from Buenos Aires coastal waters (Argentina) IV. Rhizosolenia s.
str. Neocalyptrella, Pseudosolenia, Proboscia. Phycologia 46(6): 628643.
Tabassum A & Saifullah SM (2012) Centric Diatoms from North Arabian Sea Shelf of Pakistan. LAP Lambert
Academic Publishing, pp. 1136.
Yun SM & Lee JH (2011) Morphology and distribution of some marine diatoms, family Rhizosoleniaceae,
genus Proboscia, Neocalyptrella, Pseudosolenia, Guinardia and Dactyliosolen in Korean coastal waters.
Algae 26(4): 299315.

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ISSN (P): 2349 9265
3(2): 328333, 2016

Research article

Modulation of cell-wall bound phenolics accumulation by


shikimate pathway in yeast extract elicited fragrant roots of
Hemidesmus indicus
Anish Kundu*# and Adinpunya Mitra
Natural Product Biotechnology Group, Agricultural and Food Engineering Department, Indian Institute of
Technology Kharagpur, Kharagpur - 721302, India
#
Present address: National Institute of Plant Genome Research, New Delhi - 110067, India
*Corresponding Author: biok.anish@gmail.com [Accepted: 24 June 2016]

Abstract: Plant cell walls contain various phenolic compounds, originated from phenylpropanoid
pathway. Hemidesmus indicus is a well-known tropical Indian medicinal plant, traditionally used
as herbal medicine for different ailments. Several reports have been published on its soluble
phenolic constituents so far. Recently, shikimate pathway modulated biosynthesis of 2-hydroxy-4-
mrthoxybenzaldehyde, a major soluble phenolic compound of H. indicus, has been demonstrated.
Here we have reported three major cell-wall bound phenolic compounds (4-hydroxybenzoic acid,
4-coumaric acid and ferulic acid) in the fragrant Hemidesmus indicus root. We described their
accumulation pattern in quantitative manner by high performance liquid chromatographic analysis
before and after elicitation with yeast extract and correlation of their accumulation with shikimate
pathway by inhibition with glyphosate treatment.
Keywords: Phenylpropanoid pathway - Glyphosate - Shikimic acid.

[Cite as: Kundu A & Mitra A (2016) Modulation of cell-wall bound phenolics accumulation by shikimate
pathway in yeast extract elicited fragrant roots of Hemidesmus indicus. Tropical Plant Research 3(2): 328333]

INTRODUCTION
Hemidesmus indicus, commonly known as Indian Sarsaparilla is an endemic medicinal plant of Indian
subcontinent that accumulates a fragrant compound, 2-hydroxy-4-methoxybenzaldehyde in the woody
rootstocks only as a constitutive secondary metabolite (Sreekumar et al. 2000).These fragrant roots are used as
flavoring agent in sherbets (sweet-drinks) mainly in south India. This phenolic aldehyde was detected in the
soluble fraction of the methanolic extract of the root part of H. indicus (Sircar et al. 2007a). Though, in Indian
traditional medicine system this plant drew attention since ancient era, limited information is available on the
biosynthesis of soluble as well as cell-wall bound phenolics in this plant (Chakraborty et al. 2008). In recent
past, we made some attempts to demonstrate the biosynthesis of the major soluble methoxybenzaldehyde in
elicitor treated H. indicus roots (Chakraborty et al. 2008, Kundu et al. 2012) and it was observed that elicited
roots accumulated enhanced amount of this methoxybenzaldehyde when compared with the untreated one. It
was also evident that, enzymes of shikimate and phenylpropanoid pathway were induced after elicitor treatment
that leads to overproduction of the soluble methoxybenzaldehyde and blocking of shikimate pathway resulted in
reduction of its accumulation (Kundu et al. 2012). This incident have raised a question if there is any significant
effect of shikimate pathway on the cell-wall bound phenolics accumulation in elicited H. indicus root, as lignin
and related cell-wall bound phenolic biosynthesis follow the phenylpropanoid pathway (Nair et al. 2004). In this
communication we report the accumulation pattern of three major phenolic compounds in cell wall bound
fraction of both elicited and non-elicited H. indicus root for the first time. These are 4-hydroxybenzoic acid (4-
HBA), trans-ferulic acid (t-fer A) and 4-coumaric acid (4-com A). All these three have significant importance
in pharmaceutical and industrial aspects. For example, 4-HBA can be used as preservatives in pharmaceuticals,
cosmetics, foods, and industrial products as well as its esters showed bioactivity against fungi and gram positive
bacteria (Aalto et al. 2006). On the other hand, 4-comA has strong antioxidant properties (Kannan et al. 2013)

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and bactericidal activity which act on bacteria by dual damage mechanism (Lou et al. 2012). It has been
reported that t-fer A could be used as antioxidant, antimicrobial, anti-inflammatory, anti-thrombosis, anti-
carcinogen, antiviral, agent with vasodilatory effect, antithrombotic, and inducing agent of the viability of
sperms (Ou & Kwok 2004, Kumar & Pruthi 2014). We also demonstrated a probable modulation of their
accumulation pattern via shikimate pathway where inhibiting shikimate pathway with glyphosate (an inhibitor
of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase enzyme) treatment decreased the accumulation of
these phenolic compounds but uplifted shikimic acid in significant manner. Surprisingly, inhibition of shikimate
pathway with glyphosate also resulted in decrement of shikimate dehydrogenase (SKDH) activity which might
be due to over accumulation of substrate and feed-back inhibition.

MATERIALS AND METHODS


Plant material
All root samples of H. indicus were from same ecotype (from experimental garden of IIT Kharagpur
campus, India) and more or less of same age. The plant was identified on the basis of reproductive and
vegetative morphology by Pranjit Sarma, a former professor of botany at the University of Burdwan, India.

Chemicals
All solvents and chemicals were of analytical or high performance liquid chromatography (HPLC) grade
unless specified otherwise. Glyphosate (N-(phosphonomethyl) glycine), 4-hydroxybenzoic acid (4-HBA), 4-
coumaric acid (4-com A) and trans ferulic acid (t-fer A) were purchased from Sigma-Aldrich Chemical Co. Ltd.
(New Delhi); The chemicals purchased were used as received. Deionized water was used in all the experiments
and obtained from Barnstead/Thermolyne Diamond NanopureTM water purification system (Dubuque, USA).

Preparation of elicitor and its application to excised roots


Yeast extract solution was prepared and used for elicitation according to a published method (Kundu et al.
2012).

Extraction of cell-wall bound phenolic compounds


Cell wall bound phenolic compounds were extracted according to a published method (Sircar et al. 2007b).

Detection of cell-wall bound phenolic compounds


Cell wall bound phenolic acids were analyzed in a Waters HPLC system (Milford, USA) in isocratic mode
according to the method developed by Sachan et al. (2004) for separation of hydroxycinnamates and
hydroxybenzoates.

Glyphosate treatment and shikimic acid quantification


A stock solution of 5 mM glyphosate was prepared in deionized water in sterile environment and added as
required volume in the yeast extract solution. Shikimic acid was quantified by isocratic method through
WatersRP-HPLC using RP-Hydro C18 column and a mobile phase containing trifluoroacetic acid (1 mM)
added water as solvent A (68%) and HPLC grade methanol as solvent B (32%). Injection volume was 50 l and
detection was done at 194 nm.

Cell free extract preparation and SKDH assay


Cell free extract was prepared as described by Kundu et al. (2012). SKDH assay was done according to a
published method (Daz et al. 2001) with the prepared cell free extract before and after elicitation, and after 18 h
of 1.5 mM glyphosate treatment upon the elicited roots.

RESULTS
Enhancement of cell-wall bound phenolics upon elicitation with yeast extract
In this study, the profile of major cell wall-bound phenolic acids were studied in H. indicus excised roots,
both before and after 18 h of yeast extract treatment, aiming at finding any quantitative and qualitative
differences upon elicitation. This 18 h time point was selected on the basis of the report by Kundu et al.
(2012), where maximum elicitation was observed in H. indicus root after 18 h of incubation in yeast extract
solution. Three phenolic compounds were identified through RP-HPLC analysis in both elicited and control root
samples. Those were 4-HBA, 4-com A and t-fer A (Fig. 1A, 1B). Identification of these phenolic acids were
confirmed by comparing their retention times and UV spectral properties with their authentic standards.
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Quantification of these three compounds was done by standard chromatographic quantification method with
authentic standard of each of three compounds. Comparison of HPLC chromatograms of both control and
elicited samples clearly showed the increment of the phenolic accumulation (data not shown). The contents of 4-
HBA, 4-comA and t-ferA in elicited roots were increased five, four and three-folds, respectively, than that of
controls (Fig. 2A).

Figure 1. Chromatographic analysis: A, RP-HPLC chromatograms of authentic standards of different phenolic compounds.
Peak identity: 1. 4-Hydroxybenzoic acid, 2.vanillic acid, 3.vanillin, 4. 4-hydroxybenzaldehyde, 5.4-coumaric acid, 6. trans-
ferulic acid; B, RP-HPLC chromatograms of cell-wall bound fractions. Peak identity: 1. 4-hydroxybenzoic acid, 2. 4-
coumaric acid, 3.ferulic acid. UV spectra are shown in inset.
Effect of glyphosate on cell-wall bound phenolics
Roots were harvested and subsequently analyzed to check the wall bound phenolic contents after glyphosate
treatment in three different concentrations (0.5 mM, 1 mM, and 1.5 mM) for 18 h along with elicitor. This time
point was selected HPLC analysis of wall bound fractions of each set revealed that 4-hydroxybenzoic acid
accumulation decreased about five times by 0.5 mM glyphosate but decrement was more or less same with
further increment of glyphosate in compare with the positive control (only yeast extract treated roots). In a
similar way 4-coumaric acid accumulation was diminished eight times but higher concentration of glyphosate
did not show further reduction. In case of ferulic acid a clear decrement was found with increasing glyphosate
concentrations. Specifically, after increment of glyphosate from 0.5 mM to 1 mM a drastic fall in ferulic acid
accumulation was found (3 folds) (Fig. 2B).
Accumulation of shikimic acid upon glyphosate treatment
After 18 h of treatment with 1.5 mM Glyphosate, shikimic acid content in the H. indicus root extract was
quantified through HPLC analysis to check whether the shikimic acid pool was changed or not. Detection of
shikimic acid was done by comparison with authentic standard, co-chromatography and spectral analysis. It was
observed that inhibition of EPSPS enzyme resulted in increment of shikimic acid accumulation about four folds
(Fig. 2C). Therefore, the maximum amount of shikimic acid quantified upon glyphosate treatment on elicited H.
indicus root was 6.170.4 mg g-1 fresh mass.
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Figure 2. Effect of glyphosate treatment on elicited H. indicus roots cell-wall bound phenolics, shikimic acid accumulation
and SKDH activity: A, Major cell-wall bound phenolic acids detected in excised roots of H. indicus. Key to histograms: A -
4-hydroxybenzoic acid, B - 4-coumaric acid and C - trans-ferulic acid. The black and grey bars denote contents of the above
phenolic acids in elicited and control roots, respectively, after 18 h of treatments. Deionized water was used for treatment of
root in case of control sets; B, Suppression of cell-wall bound phenolics after treatment with different concentrations of
glyphosate (0.5. 1.0 and 1.5 mM) for 18 h in elicited H. indicus root; C, Enhanced shikimic acid accumulation upon
glyphosate treatment for 18 h; D, Effect of elicitation and glyphosate treatment on SKDH activity; E, Schematic
representation of the co-relation of shikimate pathway with cell wall bound phenolics biosynthesis as well as the route of
glyphosates effect on their accumulation. Discontinuous arrows represent multiple enzymatic reactions. All values are mean
SD of three individual extractions.
Effect of glyphosate on SKDH activity
SKDH activity was found to be enhanced more than two folds upon 18 h of elicitation with yeast extract but
this enhancement suppressed a little when Glyphosate was used together with yeast extract (Fig. 2D). Effect of
Glyphosate on SKDH was investigated as this enzyme is one of the key enzymes of shikimate pathway.

DISCUSSION
Detection and quantification of phenolic compounds in cell-wall bound fraction have been done first time for
H. indicus root. Three compounds, 4-HBA, 4-com A and t-fer A were confirmedly picked out through HPLC
and UV-VIS spectral analysis. It was found that 4-HBA is the most abundant in total phenolic acid fraction of
cell-wall that supported the earlier reports (Parr et al. 1997, Kang et al. 2008).We have previously reported that
elicitation experiments with yeast extract increased the accumulation of the soluble phenolic content, which
distinctly suggested yeast extract as potent elicitor for this system (Kundu et al. 2012). In the present
investigation, increment of each individual phenolic acid was checked that clearly showed the rise in 4-HBA

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content about 10 folds and it was maximum among three major phenolic acids. An enhanced accumulation of 4-
HBA upon elicitor treatment supports the previous reports (Abd-El-Mawla & Beerhues 2002, Gaid et al. 2009).
Lignin and related wall bound phenolic acids were synthesized from the phenylpropanoid pathway (Nair et
al. 2004); thus, interrogation of the effect of glyphosate on cell-wall bound phenolic fraction was carried out. In
our previous report, we found that methoxybenzaldehyde accumulation was decreased upon blocking shikimate
pathway with glyphosate treatment (Kundu et al. 2012). Here, glyphosate supplementation in elicited root
exhibited reduced accumulation of cell-wall bound phenolics. Shikimic acid accumulation was studied as a
confirmation of blocking of shikimate pathway. It was very significant that shikimic acid accumulation
increased 4 folds (upto 6.1 0.4 mg g-1 fresh mass) upon glyphosate treatment which could make H. indicus root
as a useful bio-resource of shikimic acid, as shikimic acid has several medicinal importance (Bochkov et al.
2011).
Reduction in the cell-wall bound phenolics accumulation has confirmed the correlation between shikimate
pathway and the phenylpropanoid mediated biosynthesis of cell-wall bound phenolics. Channeling of the
product formation is directed from shikimate pathway to phenylpropanoid metabolism where a probable
bifurcation results in biosynthesis of both methoxybenzaldehyde and cell-wall bound phenolics; thus blocking
shikimate pathway by glyphosate showed effect on both soluble and cell-wall bound phenolic contents (Fig.
2E).

CONCLUSION
Being a very well-known Indian medicinal plant Hemidesmus indicus had been studied from decades to
evaluate its medicinal properties. Though several investigations were done on its medicinal properties but least
is known about its secondary metabolism. This work has demonstrated an idea of the accumulation of major
cell-wall bound phenolic constituents in H. indicus roots, which have been suggested to be modulated via
shikimate pathway. This finding indicates a platform of future investigation to identify the enzymes and genes,
which are involved in the co-ordinate modulation of both soluble and cell-wall bound phenolic contents as well
as the shikimate pathway-derived benzoates in H. indicus.

ACKNOWLEDGEMENTS
This work was supported in part by a research grant (no. 2008/37/7/BRNS to A. Mitra) from the Board of
Research in Nuclear Sciences (BRNS), Department of Atomic Energy, Government of India.

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ISSN (E): 2349 1183
ISSN (P): 2349 9265
3(2): 334340, 2016

Research article

Liquid organic fertilizers for growth enhancement of Abelmoschus


esculentus (L.) Moench and Alternanthera sessilis (L.) DC.
J.M.N.P. Jayasundara, R. Jayasekara* and R.M.C.S. Ratnayake
Department of Botany, Faculty of Science, University of Kelaniya, Kelaniya, Sri Lanka
*Corresponding Author: ranjith@kln.ac.lk [Accepted: 26 June 2016]

Abstract: In the present study the influence of foliar application of liquid organic fertilizers on the
growth performance of two popular vegetables Abelmoschus esculentus (L.) Moench and
Alternanthera sessilis (L.) DC. was investigated. Six liquid fertilizers (F1; poultry manure +
Tithonia diversifolia, F2; poultry manure + Gliricidia. sepium, F3; poultry manure + Leucaena
leucocephala, F4; fish waste + Tithonia diversifolia, F5; fish waste + Gliricidia sepium, F6; fish
waste + Leucaena leucocephala) were formulated using leaves of the above plants, animal wastes,
coconut husk ash and potable well water in weight proportion of 4: 2: 1: 60. Allowing six weeks
for the decomposition, the extracts were filtered and the nutrient contents were determined using
standard analytical procedures. The highest nutrient containing F1, F2 and F4 were selected for the
application on A. esculentus and A. sessilis. Individuals of the plants were separately sprayed with
undiluted fertilizers, as well as with 50% and 25% strengths of the liquid fertilizers diluted with
well water. Plants spayed with well water only and commercial liquid fertilizer were served as the
control and the standard respectively. The selected fertilizers significantly (p<0.05) increased the
growth and yield of both plant species. Compared with the control, F1 recorded the highest number
of fruits per plant of A. esculentus, while F2 produced the highest fresh weight biomass per A.
sessilis plant. These findings suggested that the foliar application of F1 and F2 can be used
effectively to improve crop productivity of A. esculentus and A. sessilis respectively. The
undiluted mixtures of F1, F2 and 25% diluted F4 were the best strengths for growth enhancement of
A. esculentus, whereas the undiluted F1, F2 and F4 may be considered as the best strengths for
growth improvement of A. sessilis.
Keywords: Okra - Sessile joyweed - Foliar application - Crop yield.

[Cite as: Jayasundara JMNP, Jayasekara R & Ratnayake RMCS (2016) Liquid organic fertilizers for growth
enhancement of Abelmoschus esculentus (L.) Moench and Alternanthera sessilis (L.) DC. Tropical Plant
Research 3(2): 334340]

INTRODUCTION
The indiscriminate use of synthetic chemical fertilizers in modern agriculture results in adverse effects on
the environment, pollute agricultural products and affects human health. Hence, fertilization must be done in a
proper way to protect the sustainability of the ecosystems. This suggests the need to search for new approaches
of fertilization which is inexpensive, easy-to-use, effective and environmental friendly. The basis of sustainable
agriculture is the use of locally produced and low cost biomass resources to rebuild and maintain the soil
productivity. Proper processing and recycling of organic wastes for agriculture can greatly reduce the
environmental pollution. And also it is a solution for the increasing cost of chemical and synthetic fertilizers.
Improved public health and conservation of resources are the additional benefits of organic fertilizers (Kurdali
& Shammaa 2010).
Soil application is the most common method to supply essential nutrients for the plants which can cause
harmful effects on soil microorganisms as well as on fertility of soil in general. In view of these reasons, an
alternative method was required to fulfill the nutrient requirements of plants. So, the research on foliar
fertilization was started (Fageria et al. 2009). Researchers later proved that the higher plants can also absorb
mineral nutrients when applied as foliar sprays in appropriate concentrations. Although it has some

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Received: 27 March 2016 Published online: 30 June 2016
Jayasundara et al. (2016) 3(2): 334340
.
disadvantages, under certain circumstances foliar application is most effective method to overcome plant
nutritional disorders. It is now known that the foliar fertilization can effectively eliminate the deficiency
symptoms within less time than soil fertilization (Fageria et al. 2009).
Abelmoschus esculentus (L.) Moench (Okra/Ladies finger), a member of the Family: Malvaceae is a
common vegetable as well as a plant used in indigenous medicine and Alternanthera sessilis (L.) DC.
(Amaranthaceae) is a prostrate herb and widely used as a leafy vegetable in most Asian countries. According to
the traditional practices of fertilization, a wide range of plant species can be used as a low cost source to
formulate environmental friendly liquid organic fertilizers (LOFs) to improve the growth and productivity of
crop/ornamental plants. There are large amount of scientific research which has directly investigated the impacts
of G. sepium and T. diversifolia as nutrient sources (Gunapala & Amarasiri 1989, Heenkende et al. 2011).
Akande et al. (2010) have stated that application of organic fertilizer; poultry manure, Gliricidia leaves and
inorganic fertilizer enhance the growth and development of A. esculentus compared to untreated controls. The
green biomass of T. diversifolia was recognized as a high nutrient-rich and effective nutrient source for low land
rice and maize (Akanbi et al. 2007). Because of poultry manures high nitrogen content, it has long been
recognized as one of the most desirable manures to be used in the fertilization of crops (Maraikar 1996). This
research study was an effort to formulate low cost, environmental friendly LOFs to enhance the growth and
yield of A. esculentus and A. sessilis particularly in home gardening and urban agriculture.

MATERIALS AND METHODS


Tithonia diversifolia (Asteraceae), Gliricidia sepium (Fabaceae) and Leucaena leucocephala (Fabaceae),
were selected, as they are known as nutrient-rich green manure used in traditional agriculture in Sri Lanka.
Poultry manure and fish waste (heads, fins, bones, gills and inner parts) were selected, as they are used and
valued as nutrient-rich animal wastes. Coconut husk ash was added mainly to supply potassium.
Formulation of different LOF combinations
To prepare different LOFs, the following combinations were selected: F1; poultry manure + T. diversifolia,
F2; poultry manure + G. sepium, F3; poultry manure + L. leucocephala, F4; Fish waste + T. diversifolia, F5; fish
waste + G. sepium, F6; fish waste + L. leucocephala. In each mixture, 360 g of fresh green leaves, 240 g of air
dried poultry manure or fresh fish waste and 100 g of coconut husk ash were mixed with 6.0 L of potable well
water in closed plastic containers stirred with a wooden stick and aerated for two hours daily to facilitate
decomposition. After six weeks of decomposition, undiluted extracts were filtered using a cotton cloth and
subjected to nutrient analysis.
Nutrient analysis of extracts
Total nitrogen (N) contents were determined by Kjedahl method (Bennabi et al. 2013) and phosphorus (P)
by molybdovanadate method as described by Possingham & Obbink (1971). For the determination of Potassium
(K), calcium (Ca), magnesium (Mg) and iron (Fe) contents, dry ashing was done at 500 C as described by
Enders and Lehmann (2012) and were determined by atomic absorption spectrophotometry (GBC Aventa
932B plus) at the Industrial Technology Institute, Colombo, Sri Lanka. The potable well water used for the
fertilizer formulation and dilution was also analyzed for the presence of heavy metals by graphite furnace
atomic absorption spectrophotometry (Model: Analytik Jena 400p). Based on the highest nutrient levels, the best
LOFs were selected for foliar application.
Application of selected LOFs on A. esculentus
Seeds of A. esculentus (Cultivar : Haritha) spaced at 90 cm between two plants were sown in rows according
to the recommendation given by Department of Agriculture in a completely randomized block design with five
replicates in the soil beds. The experimental site was located in Kurunegala district, Sri Lanka, which has mean
annual rainfall of 2000 ml, mean monthly temperature range from 3037C, and mean annual relative humidity
of 70%. Water was supplied every other day to maintain the moisture level of the soil. Five milliliters of LOF
was sprayed between 08.0009.00 h once a week for two-week period, and then 15 ml of LOF was sprayed once
a week during the next two weeks followed by spraying of 20 ml of LOF for the next four weeks on each plant
as the plant grow. The plants in control beds were sprayed only with same volumes of well water and the growth
parameters were compared with a standard treatment of a commercially available LOF Maxicrop which was
diluted with potable well water as per the instructions before spraying. When plants were three months old (ripe
fruits are available), shoot height, number of flowers, stem circumference, number of fruits, fruit weight and leaf

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area were measured to determine the best LOF for A. esculentus. Another experiment was carried out to
determine the best strength of the prepared LOFs for foliar application in which the selected LOFs were diluted
into 50% [F(50)] and 25% [F(25)] with potable well water for application on the plants, and the undiluted
extract [F(U)] was also applied similarly.
Application of selected LOFs on A. sessilis
Healthy leaf cuttings with 68 leaves of A. sessilis (Cultivar: Piliyandala) were transplanted in polybags
(height and diameter each of 20 cm) filled with 750 g of garden soil. The plants were watered every other day to
keep the soil moist. The polybags were laid out in a completely randomized block design with five replicates.
As described above for A. esculentus, five milliliters of F(U), F(50) and F(25) of each LOF was sprayed once a
week for a period of two months. The data were collected on length of the plant, number of branches, number of
internodes, plant fresh weight and leaf area after a growth period of six weeks. The control plants were sprayed
with potable well water and the growth parameters were compared with the standard treatment of Maxicrop.
Statistical analysis
Comparison of measured growth parameters over time with different treatments was performed by one way
analysis of variance followed by Tukeys pair wise comparison tests using MINITAB 16 statistical software.

RESULTS
Reference to table 1, the highest nitrogen (N), phosphorus (P), calcium (Ca) and iron (Fe) concentrations
were detected in the standard (Maxicrop), with the exception of potassium (K) and magnesium (Mg). Of all the
formulated LOFs, the highest N levels were observed in the F2 (1678.9 mgL-1) followed by F5 (1564.6 mgL-1).
The highest P (675.1 mgL-1) and K (1924.4 mgL-1) concentrations were observed in the F1 combination, whereas
the K content was significantly higher than that of K of the standard (1373.3 mgL-1). Although F4 contain the
highest P content, it is much lower than that of the standard (831.8 mgL-1 ). Of all the formulated LOFs, the
highest Mg (169.1 mgL-1), Ca (170.6 mgL-1) and Fe (137.3 mgL-1) levels were observed in the F1 mixture.
However, Mg concentration of F1 was considerably higher than that of the standard (134.8 mgL-1). The levels of
cadmium, lead, zinc and copper of the well water used were below the detection limits.
Table 1. The nutrient levels of the formulated liquid organic fertilizers (Results are means of duplicate values and are in
mg/L).
Fertilizer N P K Mg Ca Fe
F1 1465.3 675.1 1924.4 169.1 170.6 137.3
F2 1678.9 612.3 1120.9 108.5 157.5 124.0
F3 1265.5 573.3 1047.3 117.4 82.3 87.3
F4 1454.2 649.4 1834.5 151.9 149.3 121.7
F5 1564.6 548.8 1028.8 113.2 103.1 112.5
F6 1297.4 512.4 908.8 105.3 106.8 105.8
Standard 2876.4 831.8 1373.3 134.8 231.5 208.5
Control 87.5 99.4 237.3 58.8 61.2 21.5
Note: F1; poultry manure + T. diversifolia, F2; poultry manure + G. sepium, F3; poultry manure + L.
leucocephala, F4; Fish waste + T. diversifolia, F5; fish waste + G. sepium, F6; fish waste + L. leucocephala.
Foliar application of different LOFs significantly influenced the growth performance of A. esculentus
(P<0.05). Although the highest leaf area was observed with the standard, plants treated with F1 possessed the
highest values for all the other growth parameters, but were not significantly different from the standard (Table
2).
Table 2. Effect of selected liquid organic fertilizers on the growth performance of A. esculentus*.
Growth parameter F1(U) F2(U) F4(U) Standard Control
Shoot height (cm) 152.6 3.4a 121.2 4.8b 138.4 7.5ab 152.0 2.6a 90.4 6.6c
Number of flowers 43.0 1.8a 23.0 2.9b 18.0 1.5b 43.0 1.3a 15.0 5.3c
Stem circumference (cm) 5.3 0.9a 4.2 0.7b 4.7 0.8b 5.2 0.9a 2.7 0.5c
Number of fruits/plant 38.0 1.2a 21.0 1.3b 15.0 1.6b 40.0 1.6a 13.0 1.2c
Leaf area (cm2) 295.2 1.4b 306.2 1.5a 307.6 1.1a 309.2 1.2a 260.6 2.7c
Fruit weight (g) 47.7 2.1a 24.8 1.6c 27.1 1.9c 39.6 1.3b 24.8 1.3d
Note: * Values are means of five replicates standard error; Means with the same letter in a row are not
significantly different at p<0.05, F1; poultry manure + T. diversifolia, F2; poultry manure + G. sepium, F4;
Fish waste + T. diversifolia, F1 (U) - undiluted F1, F2 (U) - undiluted F2, F4 (U) - undiluted F4

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Table 3a, b, c. Effect of three strengths of the selected liquid organic fertilizers on the growth performance of A. esculentus*.
Shoot height Number of Stem Number of Leaf area Fruit
Treatment
(cm) flowers circumference (cm) fruits/plant (cm2) weight (g)
(a) Effect of three strengths of the F1 on the growth performance of A. esculentus
F1 (U) 152.6 3.3a 43.0 1.8a 5.3 0.9a 38.0 1.2a 295.2 1.3a 47.7 2.1a
b b b b a
F1 (50) 178.8 3.8 26.0 1.5 4.9 0.9 21.0 1.6 299.4 1.1 44.0 1.6b
a b b b a
F1 (25) 140.8 8.3 24.0 1.4 3.5 0.6 22.0 1.4 293.1 0.8 29.3 2.1c
(b) Effect of three strengths of the F2 on the growth performance of A. esculentus
F2 (U) 121.2 4.8b 23.0 2.9b 4.2 0.7c 21.0 1.3c 306.2 1.5b 24.8 1.6d
c bc c d c
F2 (50) 116.4 4.8 18.0 2.1 4.7 0.8 16.0 2.0 305.8 0.8 20.3 1.5de
c c c d c
F2 (25) 125.0 2.6 16.0 1.3 3.8 0.7 13.0 1.2 298.8 0.7 17.8 1.1e
(c) Effect of three strengths of the F4 on the growth performance of A. esculentus
F4 (U) 138.4 7.5d 18.0 1.5d 4.7 0.8d 15.0 1.62e 307.6 1.0d 27.1 1.9f
d d e e d
F4 (50) 136.2 7.5 24.0 2.8 3.2 0.5 23.0 1.8 308.1 1.1 17.7 1.7g
d d e e d
F4(25) 124.8 7.76 24 1.63 4.0 0.75 21.0 1.8 302.2 0.8 15.3 1.8g
Standard 152.0 2.6 43.0 1.3 5.2 0.9 40.0 1.6 309.2 1.2 39.6 1.3
Control 90.4 6.6 15.0 5.4 2.7 0.5 13.0 1.3 260.6 2.7 24.8 1.3
Note: *Values are means of five replicates standard error; Means with the same letter in a column are not significantly
different at p<0.05, F1; poultry manure + T. diversifolia, F2; poultry manure + G. sepium, F4; Fish waste + T. diversifolia,
In order to determine the best strength of F1 fertilizer, two dilutions were made with well water, and the
results obtained revealed that the treatments significantly affected the growth performance of A. esculentus
(P<0.05) (Table 3a). F1(U) treatment produced plants with significantly higher number of fruits, flowers, shoot
diameter and fruit weight compared with F1(50) and F1(25). The highest shoot diameter and leaf area values
were obtained with F1(50). Application of the three strengths of F2 also significantly influenced the growth of A.
esculentus. The standard maxicrop treatment had a significant influence on most of the growth parameters
except the shoot diameter (Table 3b). Application of F2(U) produced plants with the highest number of fruits,
fruit weight and number of flowers. The highest shoot diameter and shoot height were obtained with the
treatment of F2(50) and F2(25) respectively. The effects of the three strengths of F4 fertilizer on the growth
performance of A. esculentus are presented in table 3c. Among the three strengths, the highest values were
observed in the plants treated with the F4(U), and the results were significantly different from those obtained
with the standard treatment. According to the Tukeys pair wise comparison, the F4(U) values for the number of
fruits, shoot height, number of flowers, stem circumference and leaf area were not significantly different from
the F4(50) and F4(25) except the fruit weight.
Another experiment was conducted to determine the best LOF for the growth enhancement of A. sessilis. As
evident from table 4, F2(U) treatment recorded significantly higher values for plant length, leaf area, plant fresh
weight and number of branches. The number of internodes was not much affected with the fertilizer treatments.
Treatment with the three different strengths of F1 had a positive impact on plant length and plant fresh weight of
A. sessilis (Table 5a). However, significant differences in growth parameters were not observed with the
application of three different strengths of F1.
Table 4. Effect of selected liquid organic fertilizers and the standard on the growth performance of A. sessilis*
Growth parameter F1(U) F2(U) F4(U) Standard Control
length of plant (cm) 37.0 2.0b 45.4 2.5a 35.4 1.8b 41.4 1.5ab 26.2 1.1c
Number of internodes 7.0 0.5ab 8.0 0.5a 7.0 0.5ab 9.0 0.3a 5.0 0.5b
bc a bc b
Number of branches 9.0 0.4 13.0 0.7 8.0 0.8 9.0 0.6 7.0 0.4c
2 b a b a
Leaf area (cm ) 5.9 0.2 7.3 0.1 6.3 0.1 7.3 0.1 4.7 0.1c
b a b b
Plant fresh weight (g) 9.6 0.2 11.6 0.3 9.2 0.1 10.6 0.2 5.4 0.2c
Note: *Values are means of five replicates standard error; Means with the same letter in a row are not
significantly different at p<0.05. F1 (U) - undiluted F1, F2 (U) - undiluted F2, F4 (U) - undiluted F4
Shown in table 5(b) are the results obtained with three different strengths of the F2 fertilizer. As can be seen,
F2 recorded a significant effect on plant length, number of internodes, number of branches, fresh weight and
leaf area of A. sessilis (p<0.05). Among the three different strengths of F2, the highest values were observed
with the F2(U) treatment, where a significantly higher fresh weight was observed than that with the other two
strengths. The growth of A. sessilis enhanced in general as the strength of the F4 increased (Table 5c).
Application of F4(U) produced plants with the highest length, number of internodes and number of branches,
fresh weight and leaf area.
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Table 5a, b, c. Effect of three strengths of selected liquid organic fertilizers on the growth performance of A. sessilis*
length of plant Number of Number of Leaf area Plant fresh
Treatment
(cm) internodes branches (cm2) weight (g)
(a) Effect of three strengths of F1 on the growth performance of A. sessilis
F1 (U) 37.0 2.0a 7.0 0.5a 9.0 0.4a 5.9 0.2a 9.6 0.2b
a a a ab
F1 (50) 34.8 1.3 7.0 0.6 13.0 0.7 5.4 0.2 8.1 0.1ab
a a a b
F1 (25) 32.6 1.1 6.0 0.5 8.0 0.7 4.7 0.14 7.4 0.18b
(b) Effect of three strengths of F2 on the growth performance of A. sessilis
F2 (U) 45.4 2.5b 8.0 0.5b 13.0 0.7a 7.3 0.1c 11.6 0.3a
bc b b c
F2 (50) 39.8 1.8 7.0 0.3 10.0 1.0 5.5 1.2 9.9 0.2b
c b bc c
F2 (25) 37.0 2.2 7.0 0.6 9.0 0.7 6.3 0.3 6.8 0.2c
(c) Effect of three strengths of F4 on the growth performance of A. sessilis
F4 (U) 35.4 1.8d 7.0 0.5d 8.0 0.8d 6.3 0.1d 9.2 0.1b
d d d e
F4 (50) 31.8 1.4 6.0 0.5 8.0 0.5 5.6 0.1 7.6 0.1a
d d d e
F4(25) 30.0 1.5 7.0 0.4 7.0 0.7 5.2 0.1 6.5 0.2b
Standard 41.4 1.5 9.0 0.3 9.0 0.6 7.3 0.1 10.6 0.3
Control 26.2 1.2 5.0 0.5 7.0 0.4 4.7 0.1 5.4 0.2
Note: *Values are means of five replicates standard error; Means with the same letter in a column are not
significantly different at p<0.05, F1; poultry manure + T. diversifolia, F2; poultry manure + G. sepium, F4;
Fish waste + T. diversifolia

DISCUSSION
This study attempted to formulate liquid organic fertilizers (LOFs) using widely available terrestrial plants,
animal manure, fish waste and coconut husk ash and test the efficacy of the formulated LOFs on the growth
performance of A. esculentus and A. sessilis plants. The plant materials used to formulate the LOFs were T.
diversifolia, G. sepium and L. leucocephala leaves. T. diversifolia leaves were decomposed faster than the other
two types of leaves, due to the lower content of lignin (6.5%) and polyphenols (1.6%) in T. diversifolia leaves
(Olabode et al. 2007). The rate of decomposition of L. leucocephala leaves was very low, probably due to the
presence of high amounts of lignified tissues. Based on the nutrient levels present, F1, F2 and F4 LOFs were
selected as the best fertilizer mixtures for the foliar application. F1 and F4 recorded the highest K contents
probably due to the high K levels present in T. diversifilia leaves as recorded by Nagarajah & Amarasiri (1977).
The highest nitrogen contents were observed in the fertilizer mixtures which contained G. sepium leaves
confirming the findings of Renuka & Wijesundara (2013). The use of G. sepium, a member of the nitrogen
fixing plant family, Fabaceae as a green manure is well known. According to the results obtained for A.
esculentus, the highest number of fruits, shoot height, stem circumference, fruit weight and the number of
flowers were observed in the plants treated with F1, but the highest leaf area was observed in the F4 treated
plants due to the higher nitrogen content of the F4 fertilizer combination (Table 1). The higher K levels present
in the F1 fertilizer resulted in the increase in flower and fruit formation, suggesting the F1 combination to be the
best fertilizer to enhance the fruit production of A. esculentus. In agreement with our results, increase in plant
growth and fruit quality of A. esculentus by the application of liquid sea weed fertilizer has already been
observed by Zodape et al. (2008), with the application of F1 fertilizer. The current results are also in agreement
with the findings of Ademiluyi (2012), where the application of high amounts of T. diversifolia in to the soil
resulted in higher vegetative and reproductive growth, suggesting the presence of the required nutrients in
correct proportion in T. diversifolia leaves. Taking all growth parameters observed in to consideration, the F1(U)
was selected as the best performing fertilizer for A. esculentus. The values observed for most of the growth
parameters with F2 (U) were higher than the F2 (50) and F2 (25) treatments suggesting the F2 (U) to be the best
strength for the application on A. esculentus. Both the F4 (U) and F4 (50) were performed in a more or less
similar manner. Therefore in terms of cost effectiveness the F4 (50) fertilizer mixture could be recommended for
the growth enhancement of A. esculentus.
As recorded by Mahaliyana et al. (2016), 2N: P: 2K fertilizer ratio is the best combination for A. sessilis to
obtain optimum growth performance in agriculture. Confirming the results obtained by Mahaliyana et al.
(2016), the highest growth performance of A. sessilis was observed in this study with the application of F2
fertilizer containing N: P: K in a similar ratio. Therefore it can be concluded that F2 fertilizer to be the best LOF
for A. sessilis. The undiluted liquid fertilizer of F1, F2 and F4 was the best strength for growth enhancement of A.
sessilis.
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To formularize the general usage of the tested LOFs for A. esculentus and A. sessilis, further in-depth studies
are required as crop productivity is also dependent on agro climatic zones and microhabitats. Further studies
should also be conducted on the presence of any pathogenic microorganisms and sterilization of the prepared
LOFs using low cost technologies, e.g. steaming before foliar application.

CONCLUSION
Application of leaf-based liquid manures containing plant extracts in organic farming is both effective and
ecologically sustainable method of supplying additional nutrients to crop plants with an appropriate, low cost
level of technology to suit the circumstances of most families. The results obtained in this study are suggestive
of the possibility of using T. diversifolia, G. sepium, L. leucocephala, poultry manure, fish waste and coconut
husk ash in combination can be used for the effective formulation of LOFs that can be used as environmental-
friendly alternative to chemical fertilizers to increase the growth performance of A. esculentus and A. sessilis.
Out of the three fertilizer combinations tested, F1 proved to be the best LOF for A. esculentus, whereas F2 proved
to be the best LOF for A. sessilis. F1 and the standard Maxicrop showed more or less similar performance. The
undiluted F1, F2 and F4(50) may be used as the best strengths of eco-friendly natural organic fertilizers to
effectively increase the growth and yield of A. esculentus while the undiluted F1, F2 and F4 seems to be the best
strengths for A. sessilis. The findings of the present study indicate that the prepared fertilizers have a great
potential to enhance the growth and yield of A. esculentus and A. sessilis.

ACKNOWLEDGEMENTS
The financial assistant obtained through a research grant from the University of Kelaniya is gratefully
acknowledged. We wish to thank Mr. J.K.A.B. Wijegunasekara of the Industrial Technology Institute, Colombo,
for the support extended in some chemical analysis.

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ISSN (P): 2349 9265
3(2): 341343, 2016

Short communication

Mitteriella ziziphi (Ascomycota) on Zizyphus nummularia from the


Himachal Pradesh and its distribution extension in India
A. K. Gautam1* and S. Avasthi2
1
Faculty of Agriculture, Abhilashi University, Mandi - 175028, India
2
Department of Botany, Abhilashi Institute of Life Sciences, Mandi - 175008, India
*Corresponding Author: a2gautam2006@gmail.com [Accepted: 26 June 2016]
[Cite as: Gautam AK & Avasthi S (2016) Mitteriella ziziphi (Ascomycota) on Zizyphus nummularia from the
Himachal Pradesh and its distribution extension in India. Tropical Plant Research 3(2): 341343]
Mitteriella, an anamorphic fungus was proposed by Sydow & Mitter (1933). The genus is characterized by
short, simple, macronematous, mononematous conidiophores with polyblastic, integrated terminal, sympodial,
denticulate broad conidiogenous cells and solitary, simple, ellipsoidal to limoniform, black, 04-septate conidia
(Ellis 1971).
During the exploration of foliicolous fungi of Himachal Pradesh, a black mildew infection was noticed on
Zizyphus nummularia (Rhamnaceae). Critical microscopic examination of the fungus identified it as Mitteriella
ziziphi. A detailed literature survey and comparative analyses revealed that this black mildew fungus has been
recorded from Uttar Pradesh, Madhya Pradesh, Kerela and Andaman Islands previously. However, no reports
were found from other part of the country. Therefore, we present here a new record from Himachal Pradesh
which is new addition to the mycoflora of state and distribution extension in India. The detailed taxonomic
descriptions of the fungi are presented in this study.
The plant leaves showing infection with black superficial fungal colonies were collected from Berthin
(District Bilaspur) of Himachal Pradesh, India. Field notes were made regarding nature of colonies, nature of
infection, locality and altitude, etc. These infected leaves along with a host twigs and reproductive parts were
dried between sheets of blotting paper and preserve for further studies. Host plants were identified and
confirmed by matching the collections with herbarium and by consulting botanists. The specimen was deposited
at Faculty of Agriculture, Abhilashi University Mandi (AUMH), Himachal Pradesh, India for further reference.

Figure 1. Map of India showing distribution of M. ziziphina in various states and its
distribution extension. (Blue colour denotes existing reports; red colour denotes new addition
to the mycoflora of Himachal Pradesh and distribution extension in India)
The morphological examination of colonies was carried out with the help of hand lenses for colour and
texture. In the laboratory, the Nail polish technique was used to study the micro-morphological characters of the

www.tropicalplantresearch.com 341
Received: 20 March 2016 Published online: 30 June 2016
Gautam & Avasthi (2016) 3(2): 341343
.
fungi. Black colonies were scraped directly from infected host, mounted in 5% KOH solution and then replaced
by lactophenol to make the septa visible (Hosagoudar & Kapoor 1984). The microscopic observations were
made under oil immersion by standard light microscopy to note down characters of appressoriate mycelium,
conidiophores, conidia, thyriothecia, ascus and ascospores used to identify species. Camera Lucida drawings
were also prepared to support the final confirmation of fungi.
The black mildew fungus was collected on aerial parts of infected plants during the course of mycological
survey. The infection was found more during or after severe spell of cold and least on shady side as compare to
sunny side of the bush. Description and illustrations of the fungi along with a discussion on its taxonomy is
presented as here. Distribution of M. ziziphina in various states of India along with its distribution extension is
also provided in figure 1.
Mitteriella ziziphina Sydow, Ann. Mycol. 31(1/2): 95, 1933. (Figs. 23)
Colonies black, amphigenous, mostly epiphyllous, without spots, at first minute, then more or less effuse,
becoming confluent and occupying a major part of leaf or the entire leaf, stem and even fruits; if hyphophyllous
thin, scattered, up to 2 mm in diameter, found on the margin or close to the veins. Hyphae straight to substraight,
branching opposite to alternate at acute angles, loosely reticulate, cells 835 47 m. Appressoria unicellular,
globose, ovate, entire, alternate, unilateral, rarely arranged closely to form bicellular, 711 7.711 m.
Conidiophores produced lateral to the hyphae, micronematous, mononematous, 14.582 68 m, 23 septate;
conidia ellipsoidal, ovateellipsoidal, gradually but distinctly narrowing towards both ends, at first continuous, 2
4 septate by dividing the spore into unequal cells, dark to opaque brown, smooth, thick walled, 2335 1519
m.
Material examined: India, Himachal Pradesh, Berthin (Distt. Bilaspur), 673 meters (2,208 ft), on leaves of
Ziziphus nummularia (Rhamnaceae), October - December 2015, coll. Ajay K. Gautam.

Figure 2. Black mildew infection on leaves of Ziziphus nummularia.


Mitteriella is a hyphomyceteous fungi of family Dothideomycetes. It is among one of the four
hyphomycetous asexual states of Schiffnerula (Hosagoudar 2003). It is widely distributed worldwide including;
India, Pakistan, Sudan, Uganda, Zanbia (Ellis, 1971). In India, Mitteriella was reported only on different
Ziziphus species. It was reported as both anamorphic and telomorphic forms. Mitteriella ziziphina is the
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Gautam & Avasthi (2016) 3(2): 341343
.
anamorphic state of Schiffnerula ziziphi. Previously, this fungus has been found on Ziziphus jujuba, Z. anoplia,
Z. latifolia, Z. nummularia, Z. xylopyrus, from various localities of Uttar Pradesh, Madhya Pradesh, Kerela and
Andaman Islands (Tandon 1935, Sahni 1966, Hosagoudar 2011, Hosagoudar et al. 2011). A detailed
distribution of the M. ziziphina is depicted in figure 3. Some species of Schiffnerula produced Questieriella or
Mitteriella alone, or no asexual morphs (Hughes 1983).
Himachal Pradesh is a north Indian hilly state with variable environmental conditions. We observed that the
state shows extreme winters with dense fog which may be most favorable weather for growth and development
of Mitteriella ziziphina. However, this black mildew has been reported from various parts of India; but no report
is available from Himachal Pradesh. Therefore, we present here a new record from Himachal Pradesh which is
new addition to mycoflora of state and distribution extension in India.

Figure 3. Mitteriella ziziphina: A, Branched appressoriate mycelium; B, conidia.

ACKNOWLEDGEMENTS
Authors thank their respective organizations for providing every possible help to complete this work
successfully.

REFERENCES
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3(2): 344353, 2016

Review article

Herbal remedies in cure of tuberculosis prevalent among ethnic


communities in Central India
Rajiv Rai
Tropical Forest Research Institute (Indian Council of Forestry Research and Education),
Jabalpur, Rajasthan, India
*Corresponding Author: rai_rajiv_57@hotmail.com [Accepted: 26 June 2016]

Abstract: Since ancient times, tuberculosis (TB) is an age-old contagious disease and patients are
suffering from illnesses. India bears a disproportionately large burden of the world's tuberculosis
rates, as it resides to be the biggest health problem in India. One third of the worlds population is
infected with TB. Recently, an increasing concern has been observed because the causing
organism of this disease has become multi-drug resistant. The present study was carried out among
Gond, Korku and Bhatra tribe in Central India. Out of 700 respondents surveyed 443 respondents
(63.2%) of them were found to be suffering from disease. Gond tribes preferred leaf or root
powder of Muskdana (Abelmoschus esculentus); Korku tribes preferred leaf or root powder of
Adusa (Adhatoda vasica) and Bhatra tribes favoured whole plant of Van Tulsi (Ocimum basilium).
Out of diseased 443, 307 (69.3%) were found to be cured by herbal medicines in their region for a
period of five months. The paper concludes that WHO has thus, recognized these herbal remedies
and found, it will not be feasible to replace and probably not desirable to replace these herbal
home remedies with modern medicines.
Keywords: Tribals - Herbal - Traditional healers - Formulations.

[Cite as: Rai R (2016) Herbal remedies in cure of tuberculosis prevalent among ethnic communities in Central
India. Tropical Plant Research 3(2): 344353]

INTRODUCTION
Tuberculosis (TB), an infectious deadly disease of worldwide occurrence is caused by various species of
Mycobacterium, especially Mycobacterium tuberculosis and its treatment is one of the most severe challenges at
the global level (Grange & Zunla 2002). Globally, about 2 million people die with this disease and 9 million
become infected each year (WHO 2006). In different regions comprising of 141 countries of globe as African,
American, East Mediterranean, European, South East Asian and Western Pacific area new cases of tuberculosis
(TB) were estimated as 7.96 (6.311.1) million in year of 1997, (Dye et al. 1999). An estimated 1.87 million
people died of TB and the global case fatality rate was 23% but exceeded 50% in some African countries (Dye
et al. 1999). Global prevalence of MTB infection was 32% (1.86 billion). Eighty percent of all incident TB cases
were found in 22 countries, with more than half the cases occurring in 5 Southeast Asian countries. The global
burden of tuberculosis remains enormous, mainly because of poor control in Southeast Asia, sub-Saharan
Africa, and Eastern Europe, and because of high rates of M tuberculosis and HIV co infection in some African
countries (Dye et al. 1999).
Tuberculosis, exhibiting specific symptoms was published in consumption of lungs in terms of diagnosis,
symptoms, signs of accuracy as per studies observed by Richard Morton in Latin Language for the first time in
year 1689 in book Phthisilogia (Richard 1694, Keers 1982). In 1700, John Jacob Manget further described in
year 1700 form of disseminated tuberculosis as miliary (TB) which were linked with tiny tubercles as evident
by gross pathological examination to had innumerable millet seed in size and appearance, which he coined the
term miliary (TB) had been fatal form of dissemination from Mycobacterium tuberculosis (Manget 1700, Sakula
1982). Mycobacterium tuberculosis (TB) is a potential lethal disease if not diagnosed and treated early. It is this
form of tuberculosis which pose patients suffering from human immune deficiency syndrome (HIV/AIDS)
significant problem in treatment of infection. Mortality of this form has remained high, despite effective therapy

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being available. The infection is considered to be a childhood disease (Udwadia et al. 2012). In 1720, Benjamin
Marten after invention of narrowly viewed microscope conjectured that certain species of animalcula on lungs,
particularly being contagious cause infection TB. He also developed a new theory of consumption (Doetsch
1978). At the beginning of the 19th century in 1865, Jean Antoine Villemin through further advanced
demonstration of the transmissibility of Mycobacterium tuberculosis infection and the identification of the
tubercle bacillusas the etiologic agent presented his results suggesting that TB was a contagious disease (Mohan
& Sharma 2009, Rubin 1995). However, it was Robert Koch who announced the discovery of the tubercle
bacillus during the monthly evening meeting of the Berlin Physiological Society on 24th March 1882 (WHO
1994). On this day, after thousands of years, Mycobacterium tuberculosis, the organism causing TB finally
revealed itself to humans. Commemorating the centenary of this event, since 1982, 24th March is being
celebrated as World TB Day world over. Wilhelm Conrad Roentgens discovery of X-rays, facilitated
radiographic visualization of changes caused by TB in a living person. Thus, it was in the early years of 20 th
century that basic concepts related to etiological agent of TB, consequent pathological changes in humans and
detection of the organism became established. In Belfast it was found tuberculosis infection was caused due to
failure of pasteurization of milk (John & Debra 2011). It was further reported that antibiotic streptothricin was
too toxic for human beings. In 1942 Streptomycin antibiotic was isolated and developed from actinomycin C
and D, which developed an era of antibiotics and their role in the conquest of tuberculosis infection (Waksman
1964).
India has a large burden, of the worlds tuberculosis patients as this developing country can ill afford, with
an estimated economic loss of US$ 43 billion and 100 US $ annually lost directly due to disease (Udwadia et al.
2012, WHO 2013). Tuberculosis infection is in rise in India, hence its important to prevent spreading rapidly by
help of reputed physician than to follow complications (Udwadia et al. 2012, Sharma et al. 2012). It has claimed
its victim throughout the history. It reached epidemic portion in Europe and North America (Anon 1893, Daniel
2006). India is a high TB burden country contributing to 26 per cent of global TB burden (WHO 2006). In 2008,
nearly 2 million cases were reported in India and 2.76 lacs of deaths are reported every year of this disease
(WHO 2009). The WHO reports in 2012 states that, there were almost 9 million new cases in 2011 and 1.4
million TB deaths (WHO 2013). Tuberculosis disproportionately affects the poor as things like crowded living,
poor ventilation, malnutrition all makes individuals more susceptible. This is despite the availability of treatment
that will cure most cases of TB. WHO reports of 2012 states that, 9 million people worldwide became sick with
TB disease, most of whom (80%) live in one of the 22 high burden countries for TB (WHO 2009, WHO 2012,
WHO 2013).
Globally, tuberculosis (TB) still remains a major public health problem. Which is wide spread in Africa
region (Ethopia, Gambia, Congo, Liberia, Mali, Malwi, Nigeria, Keyna, Uganda, Republic of Tanzania,
Zimbabwe), American region (Brazil, Argentina, Colombia, Eucador, Jamica, Mexico, Parague, Bolvia) ,
Eastern Mediterranean (Afghanistan, Sonali, Iran, Jordan), European region (Spain, Portugal, Check Republic
Bulgaria, Romania Norway ,Switzerland), South East Asian region (India, Indonesia, Pakistan, Sri Lanka,
Nepal, Myanmar, Thailand, Maldives), Western pacific region (China, Malaysa, PapauNew Guinea, Vietnam,
Cambodia, Korea) (Dye et al. 1999).
Tuberculosis is a leading killer of people living with HIV (PLHIV).Tuberculosis is a respiratory disorder
which is passed to other people through coughing and sneezing over a period of time under unsanitary
conditions. The disease is caused by bacterium Mycobacterium tuberculosis. This bacterium is passed through
fine spray of water vapors expelled when a person coughs or sneezes, if proper ventilation not exists in the
system. Since ancient times, there have been references to TB or illnesses resembling TB from several parts of
the world from many civilizations. The earliest references to TB can be found in the language Samskritam
(Sanskrit). In the ancient Indian scriptures, The Vedas, TB was referred to as Yakshma (meaning wasting
disease). Description of a TB-like disease has been documented in ancient Chinese and Arabic literature. In
English literature, the word consumption (derived from the Latin word consumer) has also been used to
describe TB. The word tuberculosis appears to have been derived from the Latin word tubercula (meaning a
small lump) (Dubos & Dubos 1952), Waksman (1964). Many studies conducted had reported that Fracastorius
(14431553) believed that TB was contagious, whereas Thomas Willis (16211675) had documented in clinical
presentation of consumption in detail in his treatise Pthisiologica (Rubin 1995, Keers 1978, Sakula 1982).

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Richard Morton (16371698) has also described several pathological appearances of TB (Anand & McKay
2012, WHO 2006).
Tuberculosis is an age-old contagious disease in Bangladesh, which often leads to fatality if not treated
properly. Recently, there has been increasing concerns because the organism causing this disease has become
multi-drug resistant. Since this disease is prevalent in Bangladesh and is often treated with herbal medicines by
the traditional medicinal practitioners (Kavirajes), we undertook an ethnomedicinal survey of Kavirajes in Bogra
district, Bangladesh to gather information on medicinal plants used to treat this disease. Plants were collected
from the Kavirajes and identified at the Bangladesh National Herbarium. The collected information indicates
that the following plants (with family name in parenthesis) are used to treat tuberculosis: Adhatoda vasica
(Acanthaceae), Andrographis paniculata (Acanthaceae), Centella asiatica (Apiaceae), Catharanthus roseus
(Apocynaceae), Holarrhena antidysenterica (Apocynaceae), Colocasia esculenta (Araceae), Pistiastratiotes
(Araceae), Aloe vera (Asphodelaceae), Calendula officinalis (Asteraceae), Shorea robusta (Dipterocarpaceae),
Ricinus communis (Euphorbiaceae), Swertia chirata (Gentianaceae), Ocimum sanctum (Lamiaceae), Allium
sativum (Liliaceae), Hibiscus rosa-sinensis (Malvaceae), Swietenia mahagoni (Meliaceae), Tinospora cordifolia
(Menispermaceae), Eucalyptus globules (Myrtaceae), Piper longum (Piperaceae), Cymbopo goncitratus
(Poaceae), Zizyphus mauritiana (Rhamnaceae), Morinda citrifolia (Rubiaceae) and Vitis vinifera (Vitaceae)
(Rahman et al. 2009).
TB in the United States reflects the global reality. TB is one of the most common infectious diseases
worldwide (WHO 2013). While significant progress has been made toward the elimination of TB in the United
States, this disease remains an urgent public health problem in many other parts of the world. Tuberculosis was
first isolated in 1882 by German Physician named Robert Koch (18431910). Tuberculosis (TB) most
commonly affects lungs but can also affect any part of the body. A person can be infected by tuberculosis
bacillus, which proved to be ineffective as a cure but became important diagnostic tool for tuberculosis; if a
healthy persons inhales minute particles of infected sputum from the air. Bacteria get into air when a person who
has lungs infection coughs, sneezes, shouts or spits. People in the vicinity of infected air if breathe such air the
bacteria is inhaled and healthy one is infected with tuberculosis. This bacterium after being perceived in lungs of
healthy person can remain inactive (dormant) state for several years, without causing symptoms or spreading
infection. As and when the immune system of the infected person with dormant Tuberculosis bacteria is
weakened, the person exhibits the symptoms of infection (Sakula 1982).
As soon as the immune system gets weakened symptoms of horrible cough extending for a period of more
than 34 weeks followed with chest pain, blood or sputum when coughing are observed. In acute and sub-acute
cases patient gets fatigue, reduced weight, loss of appetite, high fever, chills and night sweatness is observed.
The diagnosis is further confirmed leading to skin test, chest X-rays, sputum analysis and PCR tests. To detect
genetic material of causing bacteria are performed to confirm laboratory analysis of diseases. According to an
estimate of WHO about 89 million people are infested throughout the globe and about three million people are
killed every year (WHO 2013).
The present study focuses on use of home an herbal remedies in use of plants and their parts as prevalent in
Central India in pockets of Gond, Korku and Bhatra tribes in states of Madhya Pradesh and Chhattisgarh.

MATERIALS AND METHODS


The study has been carried out in states of Madhya Pradesh and Chhattisgarh, states in India during the
period 20042006. The study was carried out among Gond tribes in Madhya Pradesh (viz. Banjari, Lakhnadon,
Dhuma, Bitarwada), Korku Tribe in Madhya Pradesh: (Kesla, Tawa Nagar, Sohagpur, Bankhedi) and Bhatra
tribe in Chhattisgarh: (Kondagaon, Kolebeda, Makdi & Keshkal) to study health status pertaining to
Tuberculosis. The Survey conducted with examination of 700 persons, who were tested for Tuberculosis
infestation, comprising of Gond, Korku and Bhatra tribes in districts covered Seoni, Hoshangabad and Bastar in
tribal localities has been listed in table 1. During the visits a rapport was made with a number of elderly people
of tribal communities and traditional herbal healers who were contacted to collect the information and
interviewed. The discussion revealed local name of species, plant part used, formulation and dosages of herbal
drug used by traditional healers and tribal communities. The specimens were collected, processed and identified
with help of flora. During the visits in tribal pockets tribal communities, traditional healers, of different age
group were selected for recording the information. The people were randomly selected with help of traditional
healers and tribal communities who were suffering from tuberculosis to avoid any biasness for selection. The
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information was recorded from 443 patients with open structured schedule and discussions were held with
patients to confirm and record symptoms occurred due to infestation of tuberculosis. The herbal therapy was
recorded as prevalent among 307 patients who informed they were benefitted on account of continuous
medication in last five months, along with information on plant part used and formulations. The pathological
tests of patients and reports were conducted with assistance of Regional Medical Research Centre who carries
health examinations for Tribes. The discussion revealed local name of species, plant part used, formulation and
dosages of herbal drug used by traditional healers and tribal communities. The specimens were collected,
processed and identified with help of flora.

Table Table 1. Distribution of patients suffering from Tuberculosis in tribal pockets of MP & CG.
No. of persons No. of persons No. of % of % of
State

A) District Localities surveyed in infected with persons infected cured


B) Tribe (Cluster of villages) Population ( N) Tuberculosis (n 1) cured (n 2) persons persons
1 .Banjari - Ramchor, Nagan 50 42 35 21.0
Madhya Pradesh

Deori, Mohagaon
A) Seoni 2. Lakhnadon - Kunda, 50 39 31 19.5
Dungar gaon, Dhansor
MP

B) Gond tribe 3. Dhuma 50 27 19 13.5


4. Bitarwada - Kohma, 50 25 20 12.5
Dangani, Ari
Sub-total 1 200 133 105 66.5 78.94
1. Kesla - Semli-khurd, 75 69 38 27.6
Borkheda, Kuorda
Madhya Pradesh

2. Tawa Nagar - Gomtipara, 75 40 23 16.0


A) Hoshanagbad
Morpani
MP

3. Sohagpur - Samanapur, 50 22 13 8.0


B) Korku tribe
Rainabani
4. Bankhedi - Mandakheda, 50 27 19 18.8
Fatehpur, Goghari
Sub-total 2 250 158 91 63.2 56.1
1. Kondagaon - Kalipara, 75 66 47 26.4
Chhattisgarh

Matipara, Sugampara
A) Bastar
2. Kolebeda - Khapdri, 50 22 13 8.0
CG

Kormel, Paroda
B) Bhatra tribe
3. Makdi - Pathari, Mohali 75 61 42 24.4
4. Keshkal 50 13 07 5.2
Sub-total 3 250 162 109 64.8 67.2
Cumulative status of patients 700 443 307 63.2 69.3
The discussion revealed local name of species, plant part used, formulation and dosages of herbal drug used
by traditional healers and tribal communities (Fig. 1). The specimens were collected, processed and identified
with help of flora. The information recorded in field were further screened in laboratory as per work pertaining
to Indian ethno-botany and plants recorded (Chopra et al. 1965, Chopra et. al. 1982, Nadkarni 1982, Kapur
1990, Jain 1981, 1991& 1996). The information as genus of plant species, local name, family, plant part used of
plant species, formulation in cure of ailments prevalent among tribal community have been tabulated in the
present investigation.

RESULTS
Out of 700 persons, tested and examined for infestation due to tuberculosis in tribal pockets of Central India.
The study revealed that 443 tribal person were infested with tuberculosis in 2 districts of Madhya Pradesh
(Seoni and Hoshanagbad district inhabited with Gond and Korku tribes respectively) and 1 district in
Chhattisgarh state. (Bastar district inhabited with Bhatra tribes). The status of health of tribals in relation to
Tuberculosis is shown in figure 2.
The study shows that in state of Madhya Pradesh Gond tribals were surveyed (n=200) to know the health
status in relation to tuberculosis. The survey revealed that 66.5% of tribals (n=133), were found to be infested
with tuberculosis in Seoni district in localities in clusters of villages at Banjari, Lakhnadon, Dhuma and

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Figure 1. A, Interviewing a traditional healer; B, Interviewing Korku Tribal community; C, A view of forest; D, A family of
Bhatra tribe in Chhattisgarh.

Figure 2. Status of tuberculosis in tribal pockets respondent surveyed, patients affected and patients cured.

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Biterwada the infestation was found to be varying from 12.521 %. Patients were found to be infested with
disease since last 12 years. These patients were supplemented with Leaf or Root Powder of Muskdana,
Abelmoschus esculentus (Linn.) Moech. The herb was collected during rainy season from forest, washed and
dried, powdered and stored in cool place. About 35 gms of powdered leaf / root depending upon its availability
was orally administered empty stomach 56 times a day, for a period of 56 months. The dose is even
recommended for a period of 23 months, even after cure of illness as bacteria may be in a dormant state (Table
2). The traditional healers during the investigation accompanied to home of some of the patients to record
information and their experience about the cure. It was observed during the visit at patients home they have
planted Muskdana, Abelmoschus esculentus in their home. The patients collect fresh leaves from species and
powder is used for cure of illness. The patients were monitored for health status by examination of Chest X-ray
and sputum test and record is maintained. After a period of five months of continuous medication about 78.94%
(n=105) of infected persons were found to be cured.
Table 2. Herbal medicine prevalent with plant parts, formulation and dose in cure of Tuberculosis in tribal pockets of Madhya Pradesh and
Chhattisgarh state.
Plant Species District Plant
Family Tribe Formulation Dosages
(Local Name) (State) part used
Abelmoschus esculentus Malvaceae Gond Seoni Leaf Powder 35 gms of leaf or root
(Linn.) Moech. (Madhya Pradesh)
or powder is orally
Root administered with warm
(Muskdana) water empty stomach 56
times a day, for a period of
56 months. The dose is
even recommended for 23
months after cure as bacteria
may be in dormant state
Adhatoda vasica Nees Acanthaceae Korku Hoshangabad Leaf Powder 45 gms of leaf or fruit
(Madhya Pradesh) or powder is orally
(Adusa) Fruit administered twice a day
first empty stomach before
lunch with warm water and
5 hours after meals and
before dinner for a period of
56 months
Ocimum basilium Linn. Lamiaceae Bhatra Bastar Whole Decoction 12 gms of leaves, twigs,
(Chhattisgarh) Plant flowers, fruits (whole plant)
(Van Tulsi) is collected and boiled with
200 ml of water for 1520
minutes. The plant is
mashed and juice is
extracted filtered and stored
in cool place. This decoction
is orally administered 56
times a day for period of 56
months
When we talk about the health status of Korku tribes who were screened for Tuberculosis (n=250) in
Hoshangabad district in localities in clusters of villages at Kesla, Tawa Nagar, Sohagpur and Bankhedi, the
infestation was found that 63.2 % of respondents (N=158) infested with tuberculosis to be varying from 827.6
%. These patients were supplemented with Leaf or Fruits of Adusa, Adhatoda vasica Nees. About 45 gms of
leaf or fruit powder was orally administered twice a day first empty stomach before lunch with warm water and
5 hours after meals and before dinner for a period of 56 months (n=91). After a period of five months of
continuous medication 56.1 % of respondent (n=91) were found to be cured with the supplement of herbal
medicine. The patients were monitored for health status by examination of Chest X-ray and sputum test and
record is maintained.
On the other hand the status of health of Bhatra tribes in Chhattisgarh state who were screened for
Tuberculosis (n=250) in Bastar district in localities of in clusters of villages in Kondagaon, Kolebeda, Makdi
and Keshkal, the infestation was found varying from 5.226.4 % (n=162). These patients were supplemented
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with whole plant of Van Tulsi, Ocimum basilium Linn. 12 gms of leaves, twigs, flowers, fruits (whole plant) is
collected and boiled with 200 ml. of water for 1520 minutes. The plant is then mashed and juice is extracted,
filtered and stored in cool place. This decoction is orally administered 56 times a day to patients for period of
56 months. The traditional healers during the investigation accompanied by researcher to home of some of the
patients to record information and their experience about the cure. It was observed during the visit at patients
home they have planted Van Tulsi in their home and extract fresh leaves, twigs etc for preparation of decoction.
The patients were monitored for heath status by examination of Chest X-ray and sputum test and record is
maintained. After a period of five months of continuous medication about 67.2% (n =109) of the infected
persons were found to be cured. The tribals cured with use of herbal drug have been shown in figure 3.

Figure 3. Patients cured in tribal pockets by use of herbal formulations.

DISCUSSION
Tuberculosis (TB) is a disease that has affected mankind from very ancient times. Anti-TB allopathic
medications have been prescribed to control symptoms of this disease but results into side effects like hepatitis,
hypersensitivity reactions, nausea, vomiting etc. The use of herbal medicine becoming popular due to toxicity
and side effects of allopathic medicines. Medicinal plants from Ayurveda (Indian traditional medicine system)
and from foreign origin have been successfully employed to treat tuberculosis (TB) (Sharma 1998). Globally,
tuberculosis (TB) still remains a major public health problem. In India there is a high incidence of tuberculosis
(TB) in country, contributing to 26 per cent of global TB burden (WHO 2006). Tuberculosis (TB) is the most
common cause of death due to a single infectious agent worldwide in adults. The World Health Organization
(WHO) in year 1993, took an unprecedented step and declared TB to be a global emergence (Sharma & Mohan
2004). Tuberculosis is a highly infectious disease with about one third of the worlds population including 40
per cent from India estimated to be infected it (Agarwal 2004). Tuberculosis (TB) is a bacterial infection caused
mainly by Mycobacterium tuberculosis (MTB). The development of paleopathology and paleo-epidemiology in
infectious diseases has proven the very ancient origin of this disease (Godreui et al. 2007). Tuberculosis (TB) is
principally a disease of poverty, with 95 per cent of cases 98 per cent of deaths occurring in developing
countries (Sharma & Mohan 2004). Each year an estimated eight million new cases and two million deaths
occur due to TB worldwide (Kishore et al. 2007).
Tuberculosis, still remains one of the largest on India's health and wellness scale. India as and when
compared with global scenario remains at the highest tuberculosis (TB) burden country in the world (WHO
2012). According estimates of World Health Organization incidence of TB in India is 2.2 million cases rising to
2.6 million cases out of a global incidence of estimates as 8.7 million cases (WHO 2012). Where as in Canada,
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the reports are for about 1,600 new cases of TB every year (WHO 2013, Anand & McKay 2012), which does not
largely sum up, even closely, to the amount India suffers in India. Tuberculosis (TB) is principally a disease of
poverty, with 95 per cent of cases and 98 per cent of deaths occurring in developing countries (Sharma & Mohan
2004, Godreuil et al. 2007). Each year an estimated eight million new cases and two million deaths occur due to
TB worldwide (Kishore et al. 2007).
Tuberculosis is the biggest health issue around India, but what makes is worse is the newly and recently
discovered global phenomenon of drugs available, known as Totally Drug-Resistant Tuberculosis (TDR-TB
drug-resistant). The multi-drug resistant (MDR-TB - drug-resistant) drugs had been recommended, and
gradually in the market Extensive drug resistant of tuberculosis (XDR-TB) are available, but are very costly to
economically poor families. Gradually, the lowest but most dangerous and strongest of them all has situated
itself in India as TDR-TB. Emergence of multi-drug resistant (MDR) and extensively-drug resistant (XDR)
strains of Mycobacterium tuberculosis has further complicated the problem of tuberculosis (TB) control.
Medicinal plants offer a hope for developing alternate medicines for the treatment of TB. The studies on herbal
plants were done to evaluate in vitro anti-tubercular activity of five medicinal plants viz., Acalypha indica,
Adhatoda vasica, Allium cepa, Allium sativum and Aloe vera (Gupta et al. 2010). The ethnic communities in
Central India are living in most hostile conditions at hill tops, down hills ,where there are no approach roads,
community hospitals, compelled to live in lonely places and are not so financially viable, so as to purchase
costly drugs in remedial measures for tuberculosis and are only dependent on native flora for their health care.
Many of these have good knowledge on flora, their distribution pattern, pant part and formulation and they pass
such valuable herbal remedies from one generation to another through oral communication. Such remedies are
quite effective and cheap in comparison to drugs marketed in township. Tuberculosis (TB) is a disease that has
affected mankind from very ancient times. Anti-TB allopathic medications have been prescribed to control
symptoms of this disease but results into side effects like hepatitis, hypersensitivity reactions, nausea, vomiting
etc. The use of herbal medicine becoming popular due to toxicity and side effects of allopathic medicines. About
48 Medicinal plants from Ayurveda (Indian traditional medicine system) and from foreign origin have been have
been explained which have the potential of anti-tubercular activity from various sources in the literature and
successfully employed to treat tuberculosis (TB) (Arya 2011).
Medicinal plants based traditional systems of medicines are playing important role in providing health care
to large section of population, especially in developing countries. Interest in them and utilization of herbal
products produced based on them is increasing in developed countries also. It is a well-known fact that
Traditional Systems of medicines always played important role in meeting the global health care needs. They are
continuing to do so at present and shall play major role in future also. The system of medicines which are
considered to be Indian in origin or the systems of medicine, which have come to India from outside and got
assimilated in to Indian culture are known as Indian Systems of Medicine (Prasad 2002). India is one of the few
countries in the world which has unique wealth of medicinal plants and vast traditional knowledge of use of
herbal medicine for cure of various diseases (Gupta & Tandon 2004, Sharma 1998).
Medicinal plants based traditional systems of medicines are playing important role in providing health care
to large section of population, especially in developing countries. Interest in them and utilization of herbal
products produced based on them is increasing in developed countries also. It is a well-known fact that
Traditional Systems of medicines always played important role in meeting the global health care needs. They are
continuing to do so at present and shall play major role in future also. The system of medicines which are
considered to be Indian in origin or the systems of medicine, which have come to India from outside and got
assimilated in to Indian culture are known as Indian Systems of Medicine (Prasad 2002).
India is one of the few countries in the world which has unique wealth of medicinal plants and vast
traditional knowledge of use of herbal medicine for cure of various diseases (Gupta & Tandon 2004, Sharma
1998). India has a large burden of the world's tuberculosis (TB), one that this developing country can ill afford,
with an estimated economic loss of US $43 billion and 100 million lost annually directly due to this disease
(Udwadia et al. 2011). Treatment in India is on the rise just as the disease itself is on the rise. To prevent
spreading TB, it's important to get treatment quickly and to follow it through to completion by your doctor. This
can stop transmission of the bacteria and the appearance of antibiotic-resistant strains. It is a knowingly fact that
bacterial infections require antibiotics for treatment and prevention, thus, commonly you will see that patients
diagnosed with tuberculosis have certain pills and antibiotics carried around with them.

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In all the three tribal pockets a large number of traditional healers have pre-dominance in tribal pockets in the
states of Madhya Pradesh and Chhattisgarh. These traditional healers collect medicinal plants and their parts,
prepare formulations and supplement to their patients in health care of ailment. This indigenous information is
passed from one generation to another and is in practice since last several hundreds of centuries.

CONCLUSION
Globally, tuberculosis (TB) still remains a major public health problem. India is a high TB burden country
contributing to 26 per cent of global TB burden. However, herbal home Remedies have been found to be very
effective in health care of tuberculosis a respiratory disorder which had spread throughout the globe and kills a
large number of patients in acute and sub-acute cases every year. Hence WHO has also now recently recognized
herbal medicines feasible to replace and probably desirable to replace herbal home remedies with modern
medicines where so ever applicable.

ACKNOWLEDGEMENTS
The author is thankful to Traditional healers (Vaidraj) who have shared the valuable information and in
sample collection from patients to study efficacy and for systematic documentation of information in states of
Madhya Pradesh and Chhattisgarh in pockets of Gond, Korku and Bhatra tribes. The author is also thankful to
Director, Tropical Forest Research Institute Jabalpur for providing facilities for conducting the study. The author
acknowledges support for examination of samples by Regional Medical Research Centre helpful for studies.

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Research article

Nitella flagelliformis A. Br. and Chara braunii Gm. - two new


records of Charophytes from fresh water bodies in Hooghly
district, West Bengal, India
Nilu Halder and Sankar Narayan Sinha*
Department of Botany, University of Kalyani, Kalyani-741235, West Bengal, India
*Corresponding Author: sinhasn62@yahoo.co.in [Accepted: 27 June 2016]

Abstract: The present paper was dealt with morpho-taxonomic descriptions of two Charophycean
algal species such as Nitella flagelliformis and Chara braunii, from one each of genera Nitella and
Chara of the family Characeae under the order Charales with different valuable information like
habitats, altitude of collection sites, phenologies, limnological notes, threats and significances for
the first time from Hooghly district, West Bengal, India. Both the species were collected from
freshwater aquatic ecosystems. They were found as waterlogged or submerged conditions and
attached with bottom muddy soil in water bodies by rhizoids (anchorage organ). The physico-
chemical parameters that favoured their distribution and growth in water bodies were recorded and
found to be congenial. The analysis of water revealed some important characteristics like slight
alkaline pH, lower level of essential nutrient contents (nitrate-nitrogen, phosphate) and salinity;
moderate total alkalinity and, higher chemical oxygen demand at the time of their collections from
freshwater bodies. Out of these two Charophycean algal species, Nitella flagelliformis is a new
report of occurrence from the state of West Bengal, India.
Keywords: Taxonomic description - Characeae - Hooghly district - West Bengal.

[Cite as: Halder N & Sinha SN (2016) Nitella flagelliformis A. Br. and Chara braunii Gm. two new records of
Charophytes from fresh water bodies in Hooghly district, West Bengal, India. Tropical Plant Research 3(2):
354360]

INTRODUCTION
Geographically, the state of West Bengal is located between 2138' to 2710' N latitude and 8550' to 8950'
E longitude and one of the most biodiversity rich states of phycoflora in India. Hooghly district (2001'2330'
N and 8730'8030'E ) covers a total area of about 3137.55 km2 and occupies different types of water resources
such as rivers, lakes, ponds, moats and flood plain wetlands ( Halder & Sinha 2013a, b). This district possesses a
typical tropical monsoon and thus, supports the magnificent reserve of plant resources including algae. The
monthly temperature of this district varied between 14C and 35C and, the average annual rainfall is 1500 mm
during monsoon (Halder & Sinha 2015a, b).
Charophytes are also known as "Stoneworts" (James & Jones 2016). They are submerged, heterotrichous
macroalgae (Schubert & Blindow 2003). Their plant bodies are grass-green, branched, differentiated into nodes
and internodes; bear two types of branchesbranches of limited and unlimited growths. They are very common
in temporary and permanent aquatic ecosystems (Caisov & Gabka 2009) like ponds, lakes, man-made
reservoirs, canals, swampy lands, water logged rice fields and rivers. Both the male (globule) and female
(nucule) sex organs are complicated and well protected by a sterile jacket. The zygotes of Charophycean
members are very resistant to dry and cold conditions that might be due to development of sporopollenin layer
surrounding the zygotes. From cytological point of view, presence of Golgi bodies and network of microtubules,
the formation of phragmoplasts and cell plates during late cytokinesis are the key characters of Charophytes
(Bennici 2008). Few species of Chara L. are bioindicator of water quality (Gudrun et al. 1996). Presently,
Charophytes are of great interests regarding biological control of pest management because of their pesticidal
properties that affect the central nervous system of insects (Sherby et al. 1986). Ecologically, Charophytes are

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Received: 19 February 2016 Published online: 30 June 2016
Halder & Sinha (2016) 3(2): 354360
.
distinct because they release some chemical substances which causing a strong and characteristic smell and,
suggested inhibition of phytoplankton growth due to allelopathic effects (Van den Berg et al. 1998, Schubert &
Blindow 2003). In the line of monophyletic origin of embryophytes, Charophyceae is considered as an ancestor
of land plants/bryophytes. Considerable evidences support this hypothesis (Graham 1993, Renzaglia et al. 2000,
Blackwell 2003, Qiu et al. 2006). The occurrence, distribution, species composition and productivity of algae
are regulated by several extrinsic forces of environments including physical and chemical delimiting factors and
by interactions among the species (Chambers & Prepas 1990, Dawes 1998). Hence, some limnological
parameters like temperature, pH, dissolved oxygen (DO), nitrate-nitrogen (NO3-N), phosphate (PO43-), chemical
oxygen demand (COD), total alkalinity, total suspended solids (TSS), salinity and turbidity of water were
assessed to find out correlations between environmental factors and algae.
In Bengal, some earlier reports were available on taxonomy of Charophytes by the investigations of Griffith
(1849), Agarkar & Kundu (1937), Kundu (1929, 19341935, 1937, 1941, 1959), Chaterjee (1975, 1979a), Pal &
Santra (1987) and Mandal & Ray (2004). As far as the karyotype is concerned, the work of cyto-taxonomy on
Charophytes by Chaterjee (1976, 1979b), Ray & Chaterjee (1987, 1994), Mukhopadhyay (1995) enriched our
knowledge of this interesting plant group. As, there was no comprehensive report of Charophytes from this
district the present study was undertaken. The main objective of the present work was to explore the occurrences
and diversities of Charophytes from this state.

MATERIALS AND METHODS


Samples of macroscopic algae were picked up by hands from different places viz. Khamargachi (23.05N,
88.43E), Tribeni (22.99N, 88.40E), Dumurdaha (23.03N, 88.43E), Dwarkeshwar river at Arambag
(22.53N, 87.47E) and Chinsurah (22.90N, 88.39E ) of Hooghly district, West Bengal, India. Fresh algal
samples were brought to laboratory in glass containers and polythene packets. For species identification of algae
upto species level detailed taxonomic study was made by examining these specimens under Olympus
microscope (Model-CH20i) for identification of species. Preservation was done in 4% formalin and the voucher
specimens were deposited in the Departmental Herbarium of University of Kalyani, Nadia, West Bengal.
Identifications of the taxa were done through authentic literatures (Imahori 1954, Pal et al. 1962, Wood &
Imahori 196465, Pal & Santra 1987, Subramainian 2002). The physicochemical characteristics of water bodies
were analyzed as described earlier (Halder & Sinha 2015b).

RESULTS AND DISCUSSIONS


A total number of two algal taxa namely Nitella flagelliformis A. Br. and Chara braunii Gm. belonging to
the family Characeae under the order Charales of the class Chlorophyceae were described morpho-
taxonomically for the first time from Hooghly district of West Bengal, India. Currently accepted name had been
provided with its author/s name. Asterisk mark (*) indicated new report from West Bengal.
Morphotaxonomic Enumeration
*1. Nitella flagelliformis A. Braun in Hookers J. Bot. 1: 294, 1849; Braun in Abh. Kon. Akad. Wiss. Berlin.10,
1847; Mukherji in Proc. 19th Indian Sci. Congr. Bangalore. 328, 1932. (Figs. 1, 3A, B)
Thallus greyish-green, dioecious; grows on sandy mud as submerged condition; stem rather slender, 384.0 to
600.0 m thick; lower internodes as long as branchlets, upper ones shorter; sterile branchlets long similar to the
fertile branchlets; fertile branchlets 57 in a whorl, 45 times furcated; primary rays half as long as the entire
branchlets; secondary rays 56 of which 34 again furcated into 23 quinary rays; dactyls of sterile and fertile
brachlets similar and 24, unequal in 35 tertiary rays; 23 of these again furcated into 34 quaternary rays;
some of the quaternary rays sometimes again furcated and length 2-celled; ultimate cell usually conical but
sometimes allantoid; male and female (globule & nucule) reproductive structures sessile, solitary and globule
lies above the nucule; globule spherical, 320.0 to 380.0 m in diameter; nucule 400.0 to 450.0 m long
(including coronula), 340.0 to 360.0 m broad; spiral cells showing 8-9 convolutions; coronula 45.0 to 58.0 m
high and 106.0 m broad at the base; oospores dark brown, ellipsoid, 310.0 to 340.0 m long, 250.0 to 280.0
m broad with 68 prominent sharp flange ridges and imperfectly reticulated.
Habitat: Canal water at Khamargachi in Hooghly district, West Bengal, India . 14 m (above sea level).
Collection No: NH 1045; Dated: 10.03.11.
Phenology: Winter to early summer (DecemberMarch)
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Limnological note: The limnological data after analysis of water which was collected from the above spot was
given. Water temperature: 21C; pH: 7.2; NO3-N: 0.15 mgl-1; PO43-: 0.20 mgl-1; DO: 7.2 mgl-1; COD: 160.0 mgl-
1
; Total alkalinity: 128.0 mgl-1; TSS: 136.0 mgl-1; salinity: 12.0 mgl-1; turbidity: 16.6 NTU.
Threat: Disturbances by cattle grazing and anthropogenic activities of human beings were found responsible for
the death and disappearance of this algal flora during taxonomic investigation.
Significance: It acts as primary producer and a part of aquatic food chain in freshwater ecosystem. Further the
alga provides shelter of zooplanktons.

Figure 1. , Nitella flagelliformis A. Br.: A plant body; B, Branchlet; C, Dactyl tips; D, Sex organs; E, Globule; F, Nucule;
G, Coronula with apex.
2. Chara braunii Gm. in Flor. Badens. Alsat. (suppl.) 646, 1826; Pal, Kundu, Sundaralingam & Venkataraman
in Charophyta 89. figs. 200203, 1962; Moore in Charophytes Great Britain and Ireland 80, figs.12 a-d, 1986
(Figs. 2, 3C, D).
Thallus grass-green, monoecious, grows on sandy mud as submerged condition; stem stout, 400.0 to 620.0
m in diameter; plant body ecorticated entirely; internodes a little longer or as long as the branchlets; branchlets
long; 911 in number with 45 segments each, lower segments elongated than the upper; stipulodes 910 in a
single whorl, alternating with the branchlets and elongated; 300.0 to 470.0 m long, 50.0 to 70.0 m broad at
base with acute apex; bract cells 34 with 23 small bracteoles at each node; terminal node with 23 bracts
forming a corona like termination; bracts 250.0 to 400.0 m long and 60.0 to 80.0 m wide but bracts of
terminal node upto 150.0 m long and 60.0 m broad; lower 2-3 nodes (except the base of the branchlet whorl)
with globule and nucule; both reproductive organs (globule & nucule) solitary, situated at the same node and
globule lies below the nucule; globule 250.0 to 310.0 m in diameter, nucule 460.0 to 520.0 m long excluding
coronula and 360.0 to 500.0 m broad at the middle region; spiral cells showing 89 convolutions; coronula

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.
ovoid-conical with rounded apex,140.0-180.0 m high and 182.0230.0 m broad at base; oospore black,
ellipsoid-cylindrical, 550.0 to 580.0 m long and 360.0 to 410.0 m (-550.0 m) broad with 89 ridges.
Habitat: Dwarkeshwar river, pond water at Chinsurah and Tribeni and, moat water at Dumurdaha. 14.2 m
(above sea level zone).
Collection No: NH 817, NH 1048; Dated: 03.01.11, 25.03.11.
Phenology: Winter to early summer (DecemberMarch)
Limnological note: The limnological parameters after analysis of water which was collected from a pond at
Dumurdaha had been depicted. Water temperature: 20.5C; pH: 7.3; NO3-N: 0.3 mgl-1; PO43-: 0.42 mgl-1; DO:
7.0 mgl-1; COD: 180.0 mgl-1; Total alkalinity: 112.0 mgl-1; TSS: 142.0 mgl-1; salinity: 12.0 mgl-1; turbidity: 15.0
NTU.
Threat: Same as mentioned for Nitella flagelliformis.
Significance: It provides shelter of zooplanktons, acts as primary producer and a constituent of aquatic food
chain in water body.

Figure 2. Chara braunii Gm.: A, Chara braunii plant body; B, Branchlet; C, Dactyl tips; D, Showing branchlets &
stipulodes; E, Sex organs; F, Globule; G, Nucule.
Members of Charophytes occurred in both freshwater and brackish water habitats (Krause 1997) but they are
mostly freshwater and occasionally grown in brackish water (Blindow 2000). In the present investigation, the
species of Charophytes had been only collected from freshwater bodies. Environmental factors such as water
temperature, light, salinity and altitude exert a immense impact on the distribution of Charophytes (Peechaty et
al. 2004, Gbka et al. 2007, Boszke & Bocig 2008). This opinion was found to be true in this study. Water
temperature below 25C and moderate intensity of light due to grown in winter months, low level of salinity

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because of freshwater and low altitude (not more than 20 m elevation) had been recorded during their
collections time form the above sites. When total phosphorus and turbidity values were lower and the impact of
regional environmental influences decreased, the dominance of Charophytes was noticed in aquatic bodies
(Rosqvist 2010). Here, more or less same finding was noticed because authors also recorded low level of
phosphate concentrations (0.200.42 mgl-1) and turbidity values (15.0 NTU16.6 NTU) from the sites. It was
noticeable that both the species occurred in slight alkaline water (7.27.3). The limnological study also revealed
a higher COD values (160.0 180.0 mgl-1) which is an indication of stressed ecosystem. Moreover, the species
were preferred to grow in winter probably lower temperature was one of the most important controlling factors
to persist them in water bodies.

Figure 3. Nitella flagelliformis: A, plant body; B, globule and Chara braunii: C, plant body; D, sex organs (globule &
nucule).

ACKNOWLEDGEMENTS
The author is grateful to University of Kalyani, Nadia, West Bengal, India for pursuing this work. The
author is also grateful to Dr. R. K. Gupta, Botanical Survey of India, Howrah for his co-operations.

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Short communication

Rediscovery of Amorphophallus margaritifer (Roxb.) Kunth


(Araceae) from Bangladesh
Md Sharif Hossain Sourav1* and Ronald Halder2
1
Department of Environmental Science and Management, North South University, Bashundhara, Dhaka, Bangladesh
2
Baikal Teal Production, Dhaka, Bangladesh
*Corresponding Author: nature.sourav@gmail.com [Accepted: 27 June 2016]
[Cite as: Sourav MSH & Halder R (2016) Rediscovery of Amorphophallus margaritifer (Roxb.) Kunth
(Araceae) from Bangladesh. Tropical Plant Research 3(2): 361363]
Five species of genus Amorphophallus (Araceae) are recorded from Bangladesh, namely Amorphophallus
bulbifer (Roxb.) Bl., A. longituberosus (Engl.) Engl. & Gehrm., A. margaritifer (Roxb.) Kunth, A. napalensis
(Wall.) Bogn. & Mayo, and A. paeoniifolius (Dennst.) Nicholson. Among of the five species, A. margaritifer
was first collected from Dhaka in 1893 by Sir J.D. Hooker (Siddiqui et al. 2007). The species seems to be rare in
Bangladesh and recommendations for immediate identification of the habitat preference is needed for
undertaking ex-situ and in-situ conservation measures (Siddiqui et al. 2007). This paper reports the rediscovery
of A. margaritifer after more than 120 years from Bangladesh.
On 13th June 2009, the second author while watching the grassland bird at Godagari Upazila of Rajshahi
district of Bangladesh has photographed an unusual Amorphophallus plants with mature spadix (Fig. 1C)
which growing in swampy place associated with tall grass. After five years gap, on 27 th June 2014, the first
author was conducted filed visit to explore the plant habitat and he has found five individuals of
Amorphophallus growing in the same location of Godagari (Fig. 2) beside an agriculture land associated with
grass, wild herbs, shrubs and climbers. Among the five single species plants, one plant was at flowering stage
and another one just post-flowering with a dried spadix and partially damaged spathe. Remaining individuals
were in vegetative stage with lamina in its natural habitat (Fig. 1). One plant was collected to prepare voucher
specimen. Later, the pictures and specimen were studied by Amorphophallus experts and were identified as A.
margaritifer. The specimens of the same are deposited at Bangladesh National Herbarium (DACB).The detailed
description and illustration of the species based on herbarium material is below.
Amorphophallus margaritifer (Roxb.) Kunth, Enum. Pl. 2: 34, 1837. (Fig. 1)
Tuber is nearly globose, 36 cm diam with roots. Immature offsets present but differ in size and associated
with mature tuber, each 46 mm diam. and around 813 mm long. Skin is muddy pale brown to yellowish
brown. Tuber found around 1020 cm down from the surface substrate soil. Petiole is soft and smooth, 1040
cm long, green with numerous narrowly elongated black-margined pale green stripes. Lamina is 2560 cm
diam., dark green and smooth. Peduncle is soft, 2060 cm long, 24 cm diam., colour dark green with some
brownish spot. Spathe is broadly ovate or broadly triangular, 1018 cm long, 1420 cm broad, tip acute, pale
greenish outside. Spadix is not fully open if not mature and longer than the spathe at mature stage, 1220 cm
long with, greenish; female zone 1.13.0 cm long, 918 mm diam.; male zone elongate-conoidal, 69 cm long,
1.01.5 cm diam. at base. Female flowers: each 56 mm high, ovary pale green; style very short, 0.51.0 mm
long, ca. 1 mm diam., colour as like ovary; stigma yellowish. Male flowers: each 23 mm high, 1.42.1 mm
broad, pale yellow. Terrestrial seasonal herb, survive around 78 months only. These characteristics observed
both nature and horticulture. Inundation and high precipitation is risk to survive in nature. Over watering is also
harmful.
Flowering & Fruiting: June to July.
Specimen Examined: BANGLADESH, Rajshahi, Godagari, 27.06.2014, M.S. H. Sourav 01 (DACB).
Habitat: Amorphophallus margaritifer was found in moist and shady places, and mixed with undergrowth and
grass vegetation adjacent to agriculture land and associated with wild shrubs (Ficus hispida, Lippia alaba);
herbs (Alocasia macrorrhizos, Amorphophallus paeoniifolius, Boerhavia diffusa, Chrozophora rottleri,
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Received: 02 March 2016 Published online: 30 June 2016
Sourav & Halder (2016) 3(2): 361363
.

Figure 1. Amorphophallus margaritifer: A, Illustrations (a, Tuber with leaf; b, A mature peduncle with inflorescence; c,
Inflorescence showing spadix, spathe removed and male and female flower zone; d, Female flower (L.S.); e, Single female
flower; f, Male flower); B. A mature peduncle with lamina in natural habitat; C, Spadix at mature stage; D, Inflorescence
showing spadix. (Illustrations by: MSH Sourav; Photographs by: MSH Sourav & Ronald Halder)
Coloccasia esculenta, Commelina benghalensis, Croton bonplandianus, Cyanoglossum sp., Cyanotis cristata,
Cyperus sp., Digera muricata, Eclipta laba, Euphorbia hirta, Hedyotis sp., Imperata cylindrica, Leucas
lavandulifolia, Lindernia sp., Mitracarpus hirtus, Parthenium hysterophorous, Phyllanthus virgatus, Physalis
minima, Saccharum spontaneum, Solanum villosum, Uraria picta etc.) and climbers (Cayrata trifolia and
Mikania cordata). The Godagari Upazila is within the Barind tract, which is mostly higher in altitude compare
to other floodplains. These areas are normally free from floods except some lower patches inundated to shallow
depth. The soil texture of the higher part is mainly loam, but lower part is clay loam.
Distribution: Genus Amorphophallus Blume ex Decne. (Araceae) is distributed in tropical Africa, Madagascar,
tropical and subtropical Asia, the Malay Archipelago, Melanesia and Australasia (Mayo et al. 1997). Perusal of
the related literature revealed that the species distribution encompasses Indian states of Maharashtra, Madhya
Pradesh, Uttar Pradesh, Rajasthan, Bihar, West Bengal, Sikkim and Assam (Jaleel et al. 2011), Myanmar
(Govaerts & Frodin 2002) and Dhaka of Bangladesh (Siddiqui et al. 2007).
Uses: The green and soft peduncle are cooked and eaten as a vegetable, informed by local peoples.
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Population status and conservation measures: A total of six field visits were conducted in 2014 and 2015 to
observe the current habitat and to find more plants on other locations adjacent to its current habitat, but failed to
find any plant from newly surveyed location. Local peoples were interviewed to know details of this plant. The
people explained that the species was not so much available even in the past and known as locally Shakrol. The
frequency of occurrence of the species was found to be very low at its current habitat and likely to be very rare
in other part of Godagari area. Further exploration is needed to determine the population status of the species
within the Barind Tracts. The species was found in a very vulnerable location with a high chance of conversion
to agriculture land. Habitat destruction and conversion of land for agriculture are therefore major threats to this
species. In-situ conservation step has been taken through local people's involvement at its current site. In 2015,
one plant was collected for ex situ conservation and to note the plant growth in horticulture.

Figure 2. Current habitat location of Amorphophallus margaritifer at Godagari, Rajshahi, Bangladesh.


ACKNOWLEDGEMENTS
The authors expressed their deep love and gratitude to Professor Dwijen Sarma for his great encouragement
to conduct study on flora of Bangladesh. Thanks also to Nidhan Singh; Surajit Koley, J.M. Garg for their
suggestion and identification help and finally Mohammad Abdur Rashid for information on Barind area and for
the habitat location map.

REFERENCE
Govaerts R & Frodin DG (2002) World Checklist and Bibliography of Araceae (and Acoraceae). The Board of
Trustees of the Royal Botanic Gardens, Kew, 560 p.
Hetterscheid WLA & de Sarker D (1996) Notes on the Genus Amorphophallus (Araceae) - 7. Amorphophallus
(Plesmonium) margaritifer (Roxb.) Kunth in Profile. Aroideana 19: 132138.
Jaleel VA, Sivadasan M, Alfarhan AH, Thomas J & Alatar AA (2011) Revision of Amorphophallus Blume ex
Decne. sect. Rhaphiophallus (Schott) Engl. (Araceae) in India. Bangladesh Journal of Plant Taxonomy 18(1): 126.
Mayo SJ, Bogner J & Boyce PC (1997) Amorphophallus. In: Eleanor Catherine. The genera of Araceae. Royal
Botanic Gardens, Kew, pp. 235239.
Siddiqui KU, Islam MA, Ahmed ZU, Begum ZNT, Hassan MA, Khondker M, Rahman MM, Kabir SMH, Ahmad M,
Ahmed ATA, Rahman AKA & Haque EU (eds) (2007) Encyclopedia of Flora and Fauna of Bangladesh. Vol. 11.
Angiosperms: Monocotyledons (Agavaceae-Najadaceae). Asiatic Society of Bangladesh, Dhaka, pp. 399.

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Research article

Similarity and difference of species among various plant communities


across grassland vegetation of north-eastern Uttar Pradesh
Sumit Srivastava and R. P. Shukla*
Plant Ecology Laboratory, Department of Botany, D.D.U. Gorakhpur University, Gorakhpur,
Uttar Pradesh, India
*Corresponding Author: drrpshukla@rediffmail.com [Accepted: 27 June 2016]

Abstract: The grassy landscape as a mosaic of grassland patches presented 287 species in the
sampled quadrats which represented 177 genera under 53 families. The species composition of
different sites varied significantly and the species richness increased with the heterogeneity of
communities. Some communities were found to be homogenous possibly due to similar ecological
conditions. The highly disturbed communities showed reduced dominance and sprouting of many
exclusive species in the communities. A few species, common to most of the concrete
communities were Aneilema nudiflora, Cynodon dactylon, Evolvulus nummularis, Desmodium
triflorum, Lindernia decussata, L. ciliata and Rungia repens. These species also showed high
presence and constance values indicating their wide ecological amplitude to cover various
microhabitats. The cluster analysis also showed distinct patterns of presence for various grassland
communities. Few communities showed more similarity among themselves, suggesting quite
similar structural and floristic patterns, probably due to better survival of common species. The
heterogeneity of communities may be created by habitat degradation, climate and soil conditions,
overgrazing, trampling and soil erosion in an area. Few species, which could not survive the
severity of these factors, added heterogeneity to the grassy landscape.
Keywords: Grassland communities - Habitat factors - Synthetic characters - Fidelity - Similarity
indices.

[Cite as: Srivastava S & Shukla RP (2016) Similarity and difference of species among various plant
communities across grassland vegetation of north-eastern Uttar Pradesh. Tropical Plant Research 3(2): 364
369]

INTRODUCTION
Plant community is a dynamic entity of vegetation (Cowles 1901). A community may also be defined as
aggregation of living organism having mutual relationship among themselves and to their environment (Oosting
1948). The structure of any plant community has been conventionally assessed through various
phytosociological attributes (Braun-Blanquet 1932, Mueller-Dombois & Ellenberg 1974). The structure and
composition of plant community can be described on the basis of several quantitative and qualitative
characteristics. Synthetic characters provide a picture of organization and characteristics of an abstract
community or landscape.
Braun-Blanquet & Pavillard (19221928) recognized five fidelity classes. For the understanding of the
degree of association between species and of similarity between stands, some mathematical treatments in the
form of similarity indices were given by Sorenson (1948). Further summarization of data was made possible
through ordination (Bray & Curtis 1957, Whittaker 1975) and vegetation mapping (Kuchler & McCormick
1965). In comparing species composition of two or more assemblages or communities, similarity or
dissimilarity indices provide quantitative bases of assessment (Magurran 2004). The degree of similarity or
dissimilarity between any two concrete communities may be shown by cluster analysis (Chahouki 2012). The
present study focuses on the synthetic characters like presence, constance and fidelity status of species and on
the degree of similarity among various communities. The multilateral relationship of plants and environmental
variables within different concrete communities has also been analyzed.

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Srivastava & Shukla (2016) 3(2): 364369
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MATERIALS AND METHOD
Study area
The Terai region is a belt of marshy grasslands, savannahs, and forests located south of the Siwalik foothills
and north of the Indo-Gangetic Plain. The region is sandwitched between the Bhabhar tract of the sub-
Himalayas and the main Gangetic Plain. The plains of north-eastern Uttar Pradesh cover 14 districts and occupy
45,760 km2 areas. The climax vegetation is forest which now remains only in patches (Bajpai et al. 2012). Most
of the area has been cleared for agriculture and the abandoned and vacant lands are subjected to recurrent
disturbances in the form of grazing, trampling, fire. The grassland vegetation characterises the vegetational
landscape which is traversed by many rivers, rivulets and nullahs and also possess a number of lakes and ponds.
The regional plain slopes gently from northwest to southeast. The landscape presents a mosaic of plant
communities with varying amount of grasses and forbs of contrasting life-forms. The grassland presents a stage
of arrested succession under the influence of recurrent biotic disturbances. The composition of various grassland
communities appears to vary in relation to the types of disturbance, soil types and soil moisture regimes.

Figure 1. Map showing the study sites and sampling locations (131).
Climate and Soil
The climate of the region is typically tropical monsoonal with 3 distinct seasons, viz. summer (March to mid-
June), monsoon (mid-June to mid-October) and winter (mid-October to February). Average annual rainfall is
about 1704mm for the entire study region, with 91% occurring during the wet summer. The numbers of rainy
days per annum is 513.2 and mean relative humidity is about 87% in the morning and 74% in the evening. The
eastern Terai plains receive more rainfall over a longer period and possess much richer plant biodiversity as
compared to the western and southern districts of the state. Mean maximum temperatures during wet summer,
winter and dry summer seasons are 23.3, 23.71 and 36.23oC and mean minimum temperatures are 23.9, 10.34
and 21.28oC, respectively (Indian Metrological Department 20122014). The soil of the region is part of the
trans-Sarju Plains and comprises Gangetic alluvium brought down by rivers like Ghaghara, Rapti, Rohin and
Gandak from the Himalayas in the north. The texture is sandy loam and pH is near neutral. In the northern area,
there are a few elevated mounds, locally called dhus, which have brown sandy soil and range in sizes from a few
hundred to five thousand square meters.
Vegetation
The growing season spans from mid-June to mid-September, when most species flower and set seeds. The
general grassland vegetation of Terai of north-eastern Uttar Pradesh is interspersed with patches of forest, old

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fields, open pasture, upland mounds (dhus), lowlands, orchards, playgrounds and human settlements. The
important tall grasses include the species of Saccharum, Phragmites, Arundo, Themeda and Erianthus etc. Some
short grasses like the species of Imperata, Andropogon and Aristida are also there. Several upland areas are
covered with forests dominated by sal (Shorea robusta). The belt also contains riverside tropical deciduous
forest commonly comprising the species of Mallotus, Syzygium, Bombax, Trewia and Garuga.
Methods
We started this study in June 2011 with a general survey of the vegetation and habitat conditions over a vast
stretch of grassy landscape of Terai of north-eastern Uttar Pradesh, encompassing more than 11 districts and
covering about 128,076 ha of a total 36,015 km2 geographical area (Fig.1). Finally, 31 locations, showing
marked differences in habitat conditions, were selected and sampled during August 2011 to March 2014.
Twenty quadrats each of 50 cm 50 cm size were randomly laid down at each locations. Thus, a total of 620
quadrats were sampled across the regional grassy landscape. The locations with quite similar species
composition and habitat conditions were pooled into 22 distinct communities or habitat types (Srivastava et al.
2015). The synthetic characters were derived treating each sub-community as concrete community and the
composite community as an abstract community.
The number of concrete communities in which a particular species occurred was expressed as percentage of
total concrete communities to derive the presence value of that species. Since 20 quadrats each of 50 cm 50
cm were sampled per concrete community, a total sampled area per concrete community was 5m2 based on
which the constance of different species was determined. The species were grouped into five different fidelity
classes as per their faithfulness or restriction of occurrences to various communities. Thus the presence,
constance and fidelity classes of species were derived and the similarity indices between any two concrete
communities were determined (Oosting 1956).
The degree of similarity between any two communities has been expressed mathematically on the basis of
quantitative characters i.e. frequency. The index of similarity (Is) between any two communities was estimated
by Sorensons (1948).

Where,
C = sum of common species between any two communities
A = Sum of all the species of community A.
B = Sum of all the species of community B.
Data analysis
The data was analyzed by using PAST (Paleontological Statistics software) Version 2.17 (Hammer et al.
2001, Bajpai et al. 2015). The presence/absence values were used to calculate the degree of similarity among
various communities using cluster analysis in PAST following the BrayCurtis method.

RESULT
Presence, Constance and Fidelity:
Distribution of number of species falling under five different presence classes are shown in figure 2A. The
highest number of species (175) was noticed in presence class A and least number in class E. 199 of the total
species, fell under constance class A followed by 48 species in class B, 21 species in class C and 14 species in
class D and 5 species in class E. The most dominant species namely Desmodium triflorum, Evolvulus
nummularis, Lindernia deccusata, Oldenlandia corymbosa and Rungia repens represented constance class E
across the grassy landscape community (Fig. 2B). The grouping of species according to the degree to which a
species is restricted to a particular concrete community has been represented in the form of five fidelity classes
(Fig. 2C). It is evident from the figure that the maximum number of species generally found in fidelity class 2
that represent generalist species and approximately similar number of species observed within fidelity classes 1
and 4. The least number of species were observed in fidelity class 5 which explain their exclusives status and
indicator value.
Cluster analysis:
The commonness of species that exist within various plant communities are represented in figure 3. The
highest similarity was (70%) between community O and R followed by communities F and H (67%), E and V
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(63%), R and U (61%) and Q and T (61.5%). The least similarity (0.22%) was observed between community J
and C.

Figure 2: Number of species of grassland community: A, Presence classes; B, Constance classes; C, Fidelity classes.

Figure 3. A bray-Curtis presence/absence based similarity indices between any two of the 22 communities occurring under
different ecological set ups as determined by 2 light regimes (O= open; PS= partial shade), 3 moisture regimes (LM= low
moisture; AM= average moisture; HM= high moisture), 3 disturbance regimes (HD= high disturbance; MD= moderate
disturbance; LD= low disturbance) and 6 textures of soil (SS= sandy soil; LS= loamy soil; CS= clayey soil; CLS= clayey
loam soil; SLS= sandy loam soil; GS= gravelly soil).

DISCUSSION
The relatively low number of shared species among the communities is not surprising because of the
irregular and heterogeneous nature of the environment (Swaine 1996) within the communities due to natural as
well as anthropogenic disturbance (Mwavu 2007). Some communities were found to be homogenous possibly
due to similar ecological conditions. These ecologically similar communities created habitats for similar
composition of herbaceous plants (Durrani 2010). A few species, common to most of the concrete communities
were Aneilema nudiflora, Cynodon dactylon, Evolvulus nummularis, Desmodium triflorum, Lindernia
decussata, L. ciliata and Rungia repens. These species also showed high presence and constance values
indicating their wide ecological amplitude to cover various microhabitats (Oosting 1956). A maximum number
of species falling under constance classes A and B, indicates considerable floristic heterogeneity and presence of
ephemeral adventives in the landscape.
The characteristic species is an important concept of community classification (Whittaker 1962, Westhoff &
van der Maarel 1973). They include species which preferably occur in a single community (character species) or
in a few communities (differential species). The concept of characteristic species has been associated with
fidelity, which is a measure of species concentration in a community (Szafer & Pawowski 1927). The much
greater number of exclusive species may be attributed to conditions in terms of disturbance and resource. Most
of the exclusive species had erect habit with foliage crown which may be related to low level of disturbances in
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the form of grazing, clipping and trampling. Regular clipping inhibited them to grow at various site. The highly
disturbed communities showed reduced dominance and sprouting of many exclusive species in the communities.
Few communities had more exclusive species, possibly due to the removal of disturbance-vulnerable species.
Similar observation was made by Overbeck et al. (2005). Few exclusive species like Alternanthera pungens,
Baccopa monnieri, Basella alba, Chrysanthellum indicum, Crotolaria calycina, Cyperus niveus, Evolvulus
alsinoides, Heliotropium ovalifolium, Indigofera linnaei, Ipomea aquatica, Ludwigia adscendens, Oxystelma
secamone, Perotis indica, Spermacoce pusilla and Tribulus terrestris fell under fidelity class 5. These species
are site-specific and encountered in the habitat conditioned by certain level of a few ecological factors.
Ecological amplitude of a species is the capacity of growing and reproducing within a definite range of
environmental conditions (Good 1974). Few other species- Alysicarpus bupleurifolius, Astercantha longifolia,
Cynoglossum lanceolatum, Elaphantopus scaber, Ficus heterophylla, Hemarthria compressa, Ionidium
suffruticosum, Lindernia antipoda, Ludwigia octovalis, Sphenoclea zeylanica and Zephyranthes citrina were
habitat specific and were found in only one community. They occurred very rarely in any other communities.
Although cluster analysis showed a distinct pattern for grassland communities, patterns among each
clustering group were not uniform. Few communities showed more similarity among themselves both in cluster
as well as in ordination analysis, suggesting a structural and floristic pattern, probably due to the survival of
common species (Mller et al. 2007). Degree of similarity between plant communities allows combining them
into an association of plant species. According to Muller- Dumbois & Ellenberg (1974) and Chao et al. (2006;
2008), communities having less than 65% similarities are regarded as dissimilar. The highest index of similarity
was reported in four communities (O & R and F & H). These communities showed more than 65% of similarity.
The lowest similarity occurred in two communities (C and J). These communities which have low similarity
value were also composed of a large number of annuals. These results were in accordance with those of Shah et
al. (1991).

CONCLUSION
A few species, common to most of the concrete communities showed high presence and constance values
indicating their wide ecological amplitude to cover various microhabitats. Most of the exclusive species had
erect habit which indicated low level of disturbance in the form of grazing, clipping and trampling. Several
exclusive species showed efficient sprouting. Only few communities showed significant number of exclusive
species. Few exclusive species were habitat specific and were found in only one community. Although cluster
analysis showed distinct patterns for grassland communities, patterns among each clustering group were not
uniform. Few communities also showed high similarity among themselves.

ACKNOWLEDGEMENTS
The authors are thankful to the then Head Department of Botany, D.D.U. Gorakhpur University, Gorakhpur,
for providing access and freedom to use departmental herbarium and other related facilities.

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Review article

A Mini Review on Tissue Culture Responses of Some auspicious


Wheat (Triticum aestivum L.) cultivars in different Media, Salts
and Growth hormones
Muhammad Bilal1*, Farah Ahmad2, Qandeela Nigar1, Quratul-ain Haidery2,
Qurat-ul-ain1, Abdul Hanan3, Shoaib Liaqat4 and Rana Imtiaz Ahmed4
1
Department of Plant Breeding and Genetics, PMAS Arid Agriculture University, Rawalpindi, Pakistan
2
Department of Biochemistry, PMAS Arid Agriculture University, Rawalpindi, Pakistan
3
Department of Agriculture, Bahauddin Zakariya University, Multan, Pakistan
4
Cotton Research Station, Multan, Pakistan
*Corresponding Author: m_bilal788@hotmail.com [Accepted: 28 June 2016]

Abstract: Wheat (Triticum aestivium) is grown all over the world as a cereal crop. Diverse biotic
and abiotic stresses seriously affect the wheat growth, which results in low yield. Plant tissue
culture is a technique used for in vitro regeneration of plants. Standardization for callus induction
and regeneration is of vital importance to deploy the transformation aim full in wheat but Poor
tissue culture performance, which is another obstacle in transformation of wheat genotypes.
Therefore, to address this obstacle this review depicts various works of various scientist. They
conducted many researches to evaluate response of different wheat genotypes to callus induction
and plant regeneration. Therefore, the information generated through this review could be utilized
to overcome poor tissue culture performance in wheat.
Keywords: Wheat - Tissue culture - Regeneration - Transformation - Callus.

[Cite as: Bilal M, Ahmad Q, Nigar Q, Haidery Q, Qurat-ul-ain, Hanan A, Liaqat S & Ahmed RI (2016) A Mini
Review on Tissue Culture Responses of Some auspicious Wheat (Triticum aestivum L.) cultivars in different
Media, Salts and Growth hormones. Tropical Plant Research 3(2): 370374]

INTRODUCTION
Tissue culture is an important tool for last few decades for various crop improvements. Many scientists used
novel genes by many techniques to create genetic variability for crop genetic improvement. La Rue (1949)
narrated about the very first culture from the origin maize endosperm, cereal callus culture. This is the reason
behind in vitro culturing of wheat, rye and rice. Mendoza & Kaeppler (2002) described the tissue culture as very
fine technique for cereals to genetically engineered. The genetic engineering of these cereals is fully dependent
on the techniques of tissue culture. Cilar et al. (2006) utilized mature form of the embryo among 5 different
species of the Triticum, that give rise to two mutually different media of MS like Murashige and Skoog (MS)
media that is Naphthaleneacetic acid (NAA) free or 2, 4-D. In the results of callus induction, its percentage
difference occurred depicted over two different media. The Yakar of Triticum aestivium depicted highest
amount of regenerative capacity in both media. Kiziltan cultivar (Triticum durumthe) depicted the maximum
regeneration capability in 4-D media and MS +2 but comparing the results with cultivar Yilmaz that showed the
maximum capacity for regeneration in the media MS+NAA. It suggests that on the induction of callus strong
effects of genotypes seen clearly. From these results, it concluded that various wheat species showed different
behavior in diverse media.
Redway et al. (1990) is his investigation used coconut milk in the media and eight hexaploid fined lines of
wheats immature embryo. The outcome depicted a huge difference in the progression of shoot and primordial
in the used wheat lines. Moreover, it was narrated that the induction of the callus is dependent on the genotype.
Bouiamrine et al. (2012) observed that response of seven different durum wheat genotypes were recorded with
the help of various immature and mature embryos for checking regeneration and the callus induction. Here five
different characteristics were measured i.e. zygotic germination, percentage of induced calli, callogenesis,
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number of plantlets per regenerated callus and callus growth. Genotype influenced the induction of callus and
the source of the explants. Mature embryo give comparatively low yield of the callus induction percentage as
compared with those embryos that were immature. Therefore, immature embryos produced by in vitro could be
select but albino immature plant showed excellent regeneration capacity so selection of albino plant would be
fruitful. Sarker et al. (2007) reported the use of immature embryos for the regeneration of the plants and
embryogenesis at the somatic stages. He moreover described that the presence of parameters like L asparagine,
casein hydrolysate and 2, 4-D, L- Proline, L asparagine can cause momentous effects on the callus formation
and embryogenesis at the somatic stages. Dagustu (2008) used four different segregating lines of wheat and five
Iranian cultivars of wheat. Different media including cold, induction medium were used for the placement of the
anthers of these cultivars and were subjected to the gamma radiations that resulted in giving differential
response i.e. higher in one genotype as compared with others cultivars. Anther culturing is usually inhibited by
the use of gamma radiations that caused a difference due to different concentrations of the radiations in the
different genotypes. Here the result also showed that the anther culture is dependent on genotype.
Machii et al. (1998) examined varying concentrations of the NaCl in medium culture to observe the result of
28 different durum wheat cultivars on salt tolerance via in vitro technique. Immature embryo, subjected to
experimentation; fresh weight growth of the cells of callus and frequency of induction of the callus was
measured. For checking the salt tolerance, different traits like relative necrosis percentage of callus and fresh
weight growth was examined. All the cultivars were significant different. The forms of callus derived from
Dipper-6 and PI40100 showed more salt tolerance compared with in vitro conditions of other cultivars.
Mahmood et al. (2012) used Triticum durum cultivar and Triticum aestivium cultivar for the induction of callus.
The composition of MS media used was 2, 4-D, 1KT, and 3 % sucrose. For the regeneration purpose, a basal
salt of MS without any hormones was utilized. Therefore, variety of variations showed by both the cultivars.
Regeneration was not initiated by the medium with 2, 4-D. but for some of the cultivars of wheat
Naphthaleneacetic acid (NAA) and KT were most effective regarding the formation of different organs. That
showed that for regeneration the medium and genotypes are dependent. Explants frequency of regeneration can
be increased with the help of different growth hormones. Hassan et al. (2009) used six diverse wheat genotypes
and compared the results of callus induction with the process of regeneration in media of MS in varying
concentrations of the carbohydrate sorbitol the varying concentrations of the sorbitol, different genotypes thus
show different results. Shimada & Makino (1975) noticed the maximum value of frequency of the induction of
the callus, which had concentration of sorbitol in increasing amount from 020 g.L-1. in contrast to that, Szakacs
et al. (1988) noticed the minimum value of frequency of the induction of the callus. Therefore, from the above
results, it has been concluded that with the increased in sorbitol concentration, there were chances of the
increase in frequency of the regeneration but time duration is lowered. Guo et al. (1982) narrated the same
outcomes. Bahieldin et al. (2000) reported that in the regeneration process, Dicamba has huge importance.
Three spring wheat genotypes had some immature embryos, which were used for checking the response of this
dicamba on the rate of calli regeneration and shoot formation. The lower concentration of the plant dicamba
give reciprocal effect on the shoot of per callus as the process of regeneration was totally genotype dependent.

RESPONSE OF CALLUS CULTURE IN SALTS


Akhtar et al. (2007) used MS media along with different concentrations of chloride salts for wheat
genotypes. Then the frequency of the regeneration of these different varieties was observed under sodium
chloride and calcium chloride stress that showed considerably differences in diverse wheat genotypes. Among
these four, LU-26 genotypes were declared most resistant genotypes. Abdullah et al. (2012) showed the salinity
effects on both without salt or with salt on callus regeneration capacity. Those genotypes with indole acetic acid
(IAA) and kinetin were mixed with the different concentrations of NaCl. By the addition of the salt low
generation of shoots number and low rate of callus regeneration was observed. Moreover, that callus which was
induced usually in the presence of the salt NaCl gives a rate of regeneration at 48% along with number of shoots
of as per callus that were generated of two shoots. This rate is relatively higher as compared to those that have
low amount of salt or no amount of saltinity at 32% along with number of shoots of as per callus that were
generated of 1.6 shoots. Regeneration from callus induced in the presence of salt can screen the cells within a
callus capable of regenerating plantlets. Salinity affects the rate of growth and the growth rate of relatively fresh
weight. If the salinity increases, the reduction in the rate of relatively fresh weight occurs. The results that were
seen of rate of relatively fresh weight were as after four weeks as: 0.1831 g, 0.1884 g and 0.1894 g for AS-2002,
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Punjab- 76 and Chenab-70 respectively. Chen (1986) also reported a reduced rate of relatively fresh weight.
Karadimova & Djambova (1993) recorded that with the progressing concentration of NaCl apparently necrosis
and brownish color was caused that resulted in the lowering of the growth of the callus. Arzani & Mirodjagh
(1999) described the similar experience related to the diverse durum wheat genotypes.

RESPONSE OF CALLUS CULTURE IN GROWTH HORMONES


Farooq et al. (2004) showed the results of the responses of three different wheat genotypes i.e. Inqilab-91,
Bakhtawar-92 and Punjab-96 subjected to different media combinations. Bakhtawar-92 was considered best
suitable genotype for the induction of the callus. This had the rate of regeneration frequency as 40 %. This result
was for the media with BAP 2.5 mg.l-1 and IAA 0.1 mg.l-1 as compared to the Inqilab-91 and Punjab-96 that
showed the rate of regeneration frequency as 33 % and 25% respectively on the specified medium having BAP
0.5 mg.l-1 and IAA 0.1 mg.l-1 finally he gave results by concluding that the media having 2, 4-D shows
maximum rate of regeneration frequency of callus. Shah et al. (2003) reported the utilization of various growth
regulators required for the are also used in wheat for the induction of the callus and also in many other cereals
like Kinetin, BAP, IAA and 2, 4-D. Bouiamrine et al. (2013) placed immature embryo of four cultivars of
durum wheat media with varying concentrations of the ABA along with 2, 4-D 2 mg.l-1 and then the ability of
the regeneration was observed under the treatment of absicic acid. Results declared that the increase in ABA
concentration inside the medium, cause reduction in callus growth. Hence lowest concentrations of the ABA
cause increase in the frequency of the callus regeneration. Soliman et al. (2013) used six durum wheat
genotypes. This experiment was done to check the PEG response on calli culture necrosis, regeneration and
water content of growth. The result helped in the conclusion that the process of regeneration and parameters of
callus growth were gradually decreased in the specific media with increased PEG concentration.
Various effects of ascorbic acid were reported by Fazelienasab et al. (2004) on five different Iranian bread
wheat cultivars. He used varying concentrations in media of MS of abscisic acid (ABA) along with sucrose as
source. Huge difference was seen in callus induction among these five diverse cultivars and with the
enhancement of ABA concentration reduction in callus induction was observed. However, when the conditions
were controlled, optimized results were seen with glowing colors in callus but in the same way higher
concentrations of the ABA, the color of the callus is lowered. The conclusion was then made about the
interaction of the ABA and the five cultivars that was highly important. Gaspar et al. (1996), Mzouri & Amssa
(2002) and Carman et al. (1987) reported the influence about the callus induction in cereals by cytokinens and
auxin along with the morphogenesis. Wheat has mature embryos that were used by Mendoza & Kaeppler
(2001). They also used four types of auxins that were Propionic acid, Picloram, Dicamba and 2, 4-D for
checking the sugar and auxins effects on the callus induction and the ability to regenerate. They also compared
sucrose and maltose effects on the callus induction under all the optimized and sterilized conditions. Propionic
acid is the only acid that does not has good effect on the callus induction while all other forms of auxins are
considered beneficial. Sugar effect on the callus induction is usually depending on the treatment of auxins.
Combination of 2, 4-D and maltose caused enhancement in the regeneration frequency. However, when 2, 4-D
was used in combination with that of Dicamba, they lowered the frequency of regeneration. The Picloram use
also induced the callus induction and the frequency of the regeneration.
Fahmy et al. (2012) used two media in his findings that induced the callus i.e. CIM1, CIM2 and five
different media for regeneration i.e. WRM1, WRM2, WRM3, WRM4 and WRM5 have varying response on the
wheat cultivars of various Egyptian alite was also clearly checked. From his experimentations in Green house,
he made an opaque conclusion that medium WRM2 showed the greater shoot number per callus that was
cultured. Haliloglu (2006) reported in his study that 6-Benzyl Aminopurine, Kinetin and Indole-3-Acetic acid
have a remarkable high role in the plantlets regeneration.
Salt stress was applied to the calli of the genotypes of ten different wheat plants. These were further used for
the development and regeneration of plant in the MS media with no auxin e.g. 2, 4-D. The result obtained via
this experiment was in accordance with Varshney et al. (1991) and Hussain et al. (2009) in which explants of
immature embryos of triticum aestivium was used. They observed that plants were easily regenerated on that
MS media without hormone. Farshadfar et al. (2012) utilized wheat immature embryo for checking twenty
bread wheat genotypes response in-vitr of callus induction and the process of regeneration under drought stress.
They revealed thats the variations was present among genotypes for callus chlorosis percentage, callus water
content and relative growth rate of callus usually at drought level. Drought stress caused by Polyethylene glycol
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(PEG) was compared at different concentrations and various effects were observed at varying stress levels in in
vitro tolerance (INTOL), callus relative growth (CRG), callus growth rate (CGR), percentage of callus chlorosis
(PCCH) and percentage of callus water content (PCWC), callus relative growth rate (CRGR), and percentage of
callus water content (PCWC) and these effects were lowered with the increased drought effect.

CONCLUSION
Wheat (Triticum aestivum L.) is consideredas most vital cereal crop andplant breeders took interest to boost
up its productivity. Since several years, numerous efforts have been made to boost up its productivity under
various conditions through numerous techniques especially tissue culture. In this review, we focused on
responses of diverse wheat genotypes under different Media, salts and growth hormones. This comprehensive
review showed tremendous work of many scientists in field of tissue culture of wheat to overcome poor tissue
culture technique and transformation of wheat cultivars. So there is a need of time explore various techniques in
tissue culture to develop genotype having better performance.

ACKNOWLEDGEMENTS
The authors would like to express their special thanks and grateful to acknowledge the Rashid Mehmmod
Rana for their assistance, guidance, and all kind of support for the successful completion of this review article.

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ISSN (E): 2349 1183
ISSN (P): 2349 9265
3(2): 375379, 2016

Research article

Toxic level of Ni and its combination with micronutrient (B and


Cu) on antioxidative enzymes and synthesis of sugar and protein
in brinjal (Solanum melongena L.) metabolism
Shiv Shankar Yadav, Manoj Soni and Y. K. Sharma*
Laboratory of Environment Science, Department of Botany, Lucknow University, Lucknow-226007, India
*Corresponding Author: shivluresearch@gmail.com [Accepted: 28 June 2016]

Abstract: Brinjal (Solanum melongena) var NBH-774 were grown in pots at ambient temperature
20 30C. The 200 M, 500 M, 1000 M of nickel, boron (150 M, 300 M) and copper test
solutions (50 M, 100 M) were prepared in pure distilled water after quantification of nickel,
boron and copper as per percent availability in NiSO4, HBO3, CuSO4. Pure distilled water was
used as control. The 250 ml of test solutions were supplied daily in each pot, which was enhanced
to 500 ml having same concentration of nutrients at the time of flowering stage. Increased activity
of catalase (antioxidative enzymes) was recorded on 35th day of Ni treatment from 2001000 M,
whereas on 70th day of Ni treatment the catalase activity was increased up to 500 M of Ni, While
the recovery treatments using B and Cu reduced the activity on 70th day of treatment as compared
to lone Ni. The activity of peroxidase (an antioxidative enzymes) on 35th and 70th days of Ni
supply in leaves of brinjal showed increased activity except 1000 M of Ni on 70th day of
treatment. Eventually reduced activity of peroxidase was observed in recovery treatment using B
and Cu as compared to respective lone Ni supply (500 M and 1000 M). Excess Ni supply (200
M to 1000 M) significantly decreased the protein in brinjal leaves at 35th and 70th day of
treatment. Whereas, 500 and 1000 M of Ni in combination with each concentration of B (150
M, 300 M) and Cu (50 M, 100 M) improved protein content in leaves. Total sugar was
decreased with an increase in Ni supply from 200 M to 1000 M. The reduced sugar in 500 M
and 1000 M of Ni was improved in recovery treatment on both the time points of analysis.
Keywords: Nickel - Catalase - Peroxidase - Protein - Sugar.

[Cite as: Yadav SS, Soni M & Sharma YK (2016) Toxic level of Ni and its combination with micronutrient (B
and Cu) on antioxidative enzymes and synthesis of sugar and protein in brinjal (Solanum melongena L.)
metabolism. Tropical Plant Research 3(2): 375379]

INTRODUCTION
Heavy metal pollution is a worldwide problem with serious environmental consequences. Amongst heavy
metals, nickel (Ni) is an essential micronutrient for plant growth. Trace elements are necessary for normal
metabolic functions in plants, but higher concentrations of these metals are toxic and may severely interfere with
many physiological and biochemical processes of plants (Seregin & Kozhevnikova 2006, Chen et al. 2009).
Heavy metal affects plants in two ways. First, it alters reaction rates and influences the kinetic properties of
enzymes leading to changes in plant metabolism. Second, excessive heavy metals lead to oxidant stress. During
the period of metal treatment, plants develop different resistance mechanisms to avoid or tolerate metal stress,
including the changes of lipid composition, the profiles of isozymes and enzyme activity, sugar or amino acid
contents, and the level of soluble proteins and gene expressions. These adaptations entail qualitative and/or
quantitative metabolic changes that often provide a competitive advantage, and affect plant survival
(Schtzendbel & Polle 2002). The threshold concentration of nickel that leads to toxicity strongly depends on
the type of plant under investigation, because plants differ drastically in their ability to deal with nickel toxicity
(Manusadzianas et al. 2002).
The major environmental factors that affect heavy metal uptake by plants are the soil acidity, its cation
exchange capacity, the content of organic substance and lime, moisture potential, granulometric composition

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Yadav et al. (2016) 3(2): 375379
.
and concentration of macro- and micro- nutrients. The effect of these factors on the uptake of many heavy
metals are mostly nonspecific (Merkusheva et al. 2001, Kukier et al. 2004). The presence of Cu, Zn
significantly declined the uptake of nickel in Thlaspi montanum (Boyd et al. 1998). For instance the
concentration of one element may affect the level of accumulation of other metals or may modify the toxic
effects of other elements (Beckett & Davis 1978).They also stated that the nickel-zinc interaction and nickel-
copper interactions were ineffective in reducing the nickel toxicity, that is varying concentrations of zinc or
copper neither reduced nor increased the effect of nickel. Moreover copper and boron are micro- nutrients and
their deficiency causes wilting, melanism, white twisted tips, reduction in panicle formation, infertile pollen
grains (Shkolnik 1981). While boron deficiency causes chlorosis and browning of young leaves, death of
growing point and inhibition of multiplication of cells.( Bergmann & Cumakov 1977). Therefore, the
investigations were carried out to explore the nickel toxicity and to quantify the toxic responses of excess nickel
in terms of antioxidative enzymes, protein and sugar content. Whereas attempts were also made to find out the
effect of interaction of nickel with boron and copper in brinjal (Solanum melongena).

MATERIAL AND METHODS


Brinjal (Solanum melongena L.) var NBH-774 were grown in pots at ambient temperature 20 30C. The 20
seed were sown in prepared pots and five were maintained later. The 200 M, 500 M, 1000 M of nickel, (150
M, 300 M) boron and (50 M, 100 M) copper solutions were prepared in pure distilled water after
quantification of nickel, boron and copper as per percent availability in NiSO4, HBO3, CuSO4. Pure distilled
water was used as control for the study in triplicate. The 250 ml of treatments were supplied daily in each pot,
which was enhanced to 500 ml at the time of flowering stage. The activity of enzymes (Catalase, peroxidase),
total protein and total sugar was analyzed on 35th and 70th day of treatment supply.
Catalase and peroxidase activities were determined by the methods of Bisht (1989) and Luck (1963). Total
protein was estimated by the method of Lowry et al. (1951) and total sugar by the method of Dubais et al.
(1956). The data observed in the experiment, were statistically analyzed for the calculation of standard error
(S.E). Studentt test was administered for testing the hypothesis with the help of computer software sigma stat
2.0 programme.

RESULTS AND DISCUSSION

Figure 1. Effect of Ni on brinjal: A, Catalase activity; B, Peroxidase activity; C, Total protein; D, Sugar content. (1. Control.
2. 200 M Ni. 3. 500 M Ni. 4. 1000 M Ni. 5. Ni + B 500 +150 M. 6. 500 + 300 M. 7. 1000 +150 M. 8.1000+ 300
M. 9. Ni + Cu 500 + 50 M. 10. 500 +100 M. 11.1000+ 50 M. 12. 1000+ 100 M)
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Yadav et al. (2016) 3(2): 375379
.
Analysis of catalase activity on 35 and 70 days of treatment is show in table 1 and Fig 1A. Increase activity
of catalase was recorded on 35 day of Ni treatment from 200 M to 1000 M, whereas on 70 days of Ni
treatment the catalase activity was increased up to 500 M of Ni. While the recovery treatment reduce the
activity except the combination of 1000 M Ni with B (150 M, 300 M) and Cu (50 M, 100 M) on 70 days
of treatment as compare to lone concentration. However the activity of catalase was marked more on 35 days as
compare to 70 days of metal treatment.

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Yadav et al. (2016) 3(2): 375379
.
The activity of peroxidase both on 35 and 70 days of Ni supply in leaves of brinjal show increased activity
except 1000 M of Ni on 70 days of treatment. Eventually reduce activity was observed in recovery treatment
as compare to respective lone concentration of 500 M and 1000 M Ni supply (Table 1 and & Fig. 1B). Excess
Ni supply (200 M to 1000 M) significantly decreased the protein in brinjal leaves at 35 and 70 days of
treatment. Whereas 500 and 1000 M of Ni in combination with each concentration of B (150 M, 300 M)
and Cu (50 M, 100 M) improve protein in brinjal leaves (Table 1 & Fig. 1C). Total sugar was decreased with
an increase in Ni supply from 200 M to 1000 M (Table 1 & Fig. 1D). The reduce sugar in 500 M and 1000
M of Ni were improve in recovery treatment on both days of analysis.
Increase activity of catalase and peroxidase was recorded on 35th and 70th day of Ni treatment from lower to
higher concentration. Catalase, which catalyses conversion of hydrogen peroxide into water and oxygen, is the
major H2O2-scavenging enzyme in all aerobic organisms (Willekens et al. 1995). Yan et al. (2008) reported that
Ni treatment resulted in a significant increase in catalase activities of plant, while the results of MadhavaRao &
Sresty (2000) showed that the activity decreased significantly in pigeon pea seedlings grown at higher Ni levels.
These results suggested that catalase activities in plant tissues are correlated with the tested Ni concentrations.
The present results suggested that the catalase activities are remarkably increased in plant tissue under excessive
Ni stress, and these results are in agreement with the previous results. Accumulating evidence indicates that
catalase plays an important role in the protection against oxidative damage by breaking down hydrogen peroxide
(Mittler 2002). Induction of peroxidase activity after Ni treatment of plants was reported previously (Gomes-
Junior et al. 2006). Enhancement of peroxidase activity under metal stress was explained by its role in building
up physical barrier against toxic metals entering the cell as well as in scavenging H2O2 (Passardi et al. 2005).
Therefore, this peroxidase serves as a parameter of metabolism activity against nickel toxicity.
Total proteins and sugar were significantly decreased by increasing Ni concentration in brinjal at both times
of observation. The decrease in protein content may be cause by enhanced protein degradation as a result of
increase protease activity under stress conditions (Palma et al. 2002). Sridhar et al. (2005) conducted
experiment on wheat genotypes and found that total sugar decrease in all developing grains. Reduction in total
sugar content induced by heavy metal treatments may be due to its inhibitory effect on photosynthetic activities,
photosynthetic pigment concentrations, as well as on the activity of ribulose diphosphate carboxylase leading to
decrease in all sugar fractions (Stibrova et al. 1986).
In the recovery experiment the toxic effect of Ni were overcome by using boron and copper. The interaction
of boron with other metals is not very much studied but it is antagonistic or synergistic with Mo and Fe and
possible antagonism with Cr. Lou et al. (1991) found that Ni concentration and uptake was markedly reduced by
copper application. The presence of Cu, Zn significantly declined the uptake of nickel in Thlaspi montanum
(Boyd et al. 1998). Furthermore Cu involved in numerous physiological functions as a component of several
enzymes, mainly those which participate in electron flow, catalyze redox reactions in mitochondria and
chloroplasts (Hansch & Mendel 2009).

CONCLUSIONS
The result of the research showed that nickel treatment caused oxidative damage in brinjal as well as
synthesis of protein and sugar. Although use of boron and copper can help the plants to get rid of this inhibition
up to certain limit.

ACKNOWLEDGEMENTS
The authors are grateful to University Grant Commission, New Delhi, Grant sanction No. F.No. 31-
160/2005(SR) for providing funding for the work. Shiv Shankar Yadav is grateful for the fellowship under the
project.

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Research article

Heavy metal contaminated acidic soil: A source of useful fungi


Hruda Ranjan Sahoo, Madhuchhanda Sahoo, Mayeetreyee Baboo and Nibha Gupta*
Plant Pathology and Microbiology Division, Regional Plant Resource Centre,
Bhubaneswar-751015, Odisha, India
*Corresponding Author: nguc2003@yahoo.co.in [Accepted: 12 July 2016]

Abstract: The incidence of fungi endowed with exploitable potential in acidic laterite soil was
studied. The botanic garden located nearby to Chandaka forest of Odisha was considered the site
for collection of soil sample. Among the 16 fungal isolate found in the soil4 belonged to
Aspergillus sp., 1 belonged to Cladosporium sp., 6 belonged to Penicillium sp. while 5 were
Sterile mycelium. All the sixteen fungi were characterized morphologically and screened for
extracellular production of enzymes, organic acids and antagonistic behaviour. It is observed that
most of the fungi are amylase, lipase, L-asparaginase producer while few of them werepositive for
xylanase production. Antagonistic behaviour of sterile mycelium 7 exhibited its wide spectrum
activity against fungi tested. It is evident that soil having acidic pH and contaminated with heavy
metal is inhabited with useful fungi.
Keywords: Soil fungi - Heavy metal contamination - Antagonism.

[Cite as: Sahoo HR, Sahoo M, Baboo M & Gupta N (2016) Heavy metal contaminated acidic soil: A source of
useful fungi. Tropical Plant Research 3(2): 380383]

INTRODUCTION
Soil is a conglomerate system which harbours almost all major groups of fungi. Fungi dominate in soil in
terms of biomass but vary in number and kind with increasing depth of soil (Nagamani et al. 2006). They occur
in soil either in mycelial state or reproductive stage. Hyphae, hyphal fragments and sporulating fungi are also
present in soil (Nagamani et al. 2006). They occupy a variety of ecological habitats because of their immense
adaptability capacity; hence can be isolated even from unusual habitats such as salterns, peat soil, petroleum
polluted soils, inner tissues of plants and animals (Rajagopal et al. 2010). Fungi are relatively important than
bacteria in organic soils with low pH. They are known to be primary decomposers and recycle stored energy and
nutrients of the organic matter.Soil fungi are most studied group of fungi, and soil genera such as Acremonium,
Aspergillus, Fusarium and Penicillium can alsosynthesize a wide range of useful bioactive compounds. More
than 30% of isolated metabolites from fungi are found from Aspergillus and Penicillium (Brdy 2005). These
microorganisms mediate soil processes such as nutrient mobilization and mineralization, soil organic matter
decomposition, phosphate solubilization and nitrogen fixation, nitrification, denitrification and sulphur reduction
(Khan et al. 2007).
The industrial revolution and anthropogenic activities alleviated the metal concentration in soil (Yan-de et
al. 2007, Oves et al. 2012). Presence of excessive load of heavy metals in soil due to deposition of heavy metals
leads to heavy metal stress which results in pollution of soil. The excessive metals in soil also affect soil
properties and fertility making unsuitable for agricultural activities. Metal accumulation also results in reduced
microbial population (Wani & Khan 2013). However certain micro-organisms can survive in this type of abiotic
stress conditions. There are reports that metal tolerant micro-organisms have useful activity which help in
growth promotion and have positive effects onplants (Selvakumar et al. 2012, Oves et al. 2013, Sahu et al.
2014, Vyas & Gupta 2014). These microbes are also good source of industrial enzymes, organic acids and
antibiotics (Khan et al. 2009, Bello et al. 2016).
The present study was aimed to analyse the incidence of fungi in the soil having heavy metals and acidic pH.
Further, the characterization for their extracellular activity like enzyme production and mineral solubilisation
was the prime objective in order to search for the exploitable potential of these fungi.

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Sahoo et al. (2016) 3(2): 380383
.
MATERIALS AND METHODS
The red laterite soil was collected from the botanic garden of Regional Plant Resource Centre, Bhubaneswar
and treated for soil analysis (Jackson 1958). Serial dilution, plate culture and slide culture techniques (Mehrotra
& Aneja 1990) was used for the isolation of fungi and their morphologic characterization and tentative
identification. Colony characteristics of all the organisms were noted which includes the front and back
appearance of the organism on medium plate, growth pattern, texture, elevation and margin of the fungal colony
after 5 days of incubation at 25C. All the fungal cultures were characterized for their extracellular enzymatic
activity, phosphate solubilisation capacity, IAA production and antagonistic activity (Sahoo et al. 2014,
Rajoriya et al. 2014).

RESULTS AND DISCUSSION


In the present study, soil used for the analysis of incidence of fungi was highly acidic (pH 5.66.0). Mineral
analysis of the soil was confirmed the presence of Fe (32 mg.kg-1), Mn (11 mg.kg-1), Cu (1 mg.kg-1) and Zinc
(0.3 mg.kg-1) along with N, P, K in the ratio of 9:1:4. It was possible to isolate almost 16 numbers of fungi
characterized morphologically, identified at the generic level and categorised into Aspergillus, Penicillium,
Cladosporium and Sterile mycelium. All the 16 organisms were different in their colony morphology and vary
from each other in front and back appearance on medium plate, growth, colony characteristics such as margin,
texture and elevation (Table 1).
Table 1. Colony characteristics of fungi isolated from acidic laterite soil.
S.N. Organism Front Colour Reversecolour Growth Form Margin Texture Elevation
1 Aspergillus sp. 11 Brown To Dark Brown ++ Irregular Entire Velvety Flat
Pale Yellow
2 Aspergillus sp. 12 Black Pale Yellow ++++ Irregular Filliform Powdery Raised
3 Aspergillus sp. 13 Dark Green Pale White ++++ Irregular Filliform Powdery Raised
4 Aspergillus sp. 14 Black Pale Yellow ++++ Irregular Filliform Powdery Flat
5 Cladosporium sp. Black Black With ++ Irregular Entire Velvety Flat
White Margin
6 Penicillium sp. 5 Grey Black
++ Irregular Undulate Velvety Raised
7 Penicillium sp. 6 Olive Green Pale Yellow
++++ Irregular Filliform Velvety Flat
8 Penicillium sp. 7 Grey Pale Orange
+++ Irregular Undulate Slight Cottony Flat
9 Penicillium sp. 8 Light Sap Pale White
++++ Irregular Undulate Powdery With Flat
Green Slight Cottony
10 Penicillium sp. 9 Olive Green Pale White +++ Irregular Undulate Slight Cottony Raised
11 Penicillium sp. 10 Brown To Dark Brown +++ Irregular Undulate Slight Cottony Raised
Pale Yellow
12 Sterile mycelium sp. 3 Cream Cream ++ Irregular Undulate Slightly Cottony Flat
13 Sterile mycelium sp. 4 Black With Brown + Irregular Entire Slighty Cottony Flat
White Border
14 Sterile mycelium sp. 5 Blackish With Black To ++++ Filamentous Filliform Cottony Raised
Whitish Brown
Cottony
15 Sterile mycelium sp. 6 Olive Green Pale Yellow ++++ Circular Entire Slight Cottony Flat
To Yellow
16 Sterile mycelium sp. 7 White White ++++ Circular Filliform Cottony Raised
Note: (+++++), Maximum growth; (++++), Good growth; (+++), Moderate growth; (++), Less growth; (+), Least growth.
Occurrence of various fungi and their mineral solubilising potential in mine environment has been
documented very well (Gupta et al. 2007). It is important to note that most of the fungi isolated from the heavy
metal contaminated soil are found to be phosphate solubilising nature in the present study.Most of them were L-
asparaginase, protease and lipase producers and few among them were producing xylanase, amylase and
cellulase when screened through plate culture in specific medium (Table 2). Co-inoculation plate test performed
for the evaluation of antagonistic behaviour of these fungi and exhibited the antifungal potential of sterile

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mycelium. This fungus had inhibitory activity towards the growth of 5 Aspergillus and 2 Penicillium sp. among
the 16 test fungi used for the experiment (Table 3).
Table 2. Extracellular enzyme, organic acid production and phosphate solubilisation activity of fungal isolates.
Organic Phosphate
Organism Amylase Cellulase Iaa L- Asparginase Lipase Protease Xylanase
Acid Solubilisation
Aspergillus sp. 11 +++ ++ - - - ++++ + ++ +
Aspergillus sp. 12 + - - ++++ ++ + - +++ -
Aspergillus sp. 13 - ++++ - ++ - +++ - - +++
Aspergillus sp. 14 - - - + ++ - - +++ -
Cladosporium sp. + - - + +++ ++ - - -
Penicillium sp. 5 - - - + + - - - -
Penicillium sp. 6 - - - + + ++ + - -
Penicillium sp. 7 - - - + +++ - + ++ -
Penicillium sp. 8 - - - + ++ - + - -
Penicillium sp. 9 + - - - - - + + -
Penicillium sp. 10 + - - + ++ + + ++ -
Sterile mycelium sp. 3 - - - - - - ++ +++ -
Sterile mycelium sp. 4 - +++ - ++ - - ++ + -
Sterile mycelium sp. 5 ++ - - - - ++++ ++ ++ -
Sterile mycelium sp. 6 - - - ++ ++ - ++ - -
Sterile mycelium sp. 7 + +++ - ++ - +++ +++ - -
Note: (+++++), Highest activity; (++++), High activity; (+++), Good activity; (++), Medium activity; (+), Low activity; (-),
No activity.

Table 3. Antagonistic properties of fungal isolates.


Soil
A1 A 2 A 3 A 4 A 5 A 6 A 7 A 8 A 9 A 10 P 1 P 2 P 3 P 4 S 1 S 2
fungi
A 11 - - - - - - - - - - - - - - - -
A 12 - - - - - - - - - - - - - - - -
A 13 - - + + - - + - - + - - - - - -
A 14 - - - - - - - - - - - - - - - -
Cl sp. - - - - - - - - - - - - - - - -
P5 - - - - - - - - - - - - - - - -
P6 - - - - - - - - - - - - - - - -
P7 - - - - - - - - - - - - - - - -
P8 - - - - - - - - - - - - - - - -
P9 - - - - - - - - - - - - - - - -
P 10 - - - - - - - - - - - - - - - -
S3 +
S4 - - - - - - - - - - - - - - - -
S5 - - - - - - - - - - - - - - - -
S6 + - - - - - - - - + - - - - + -
S7 + - - + - + - - + + - + + - - -
Note: +, Inhibitory activity; -, No effect on growth.
(A 1, Aspergillus sp. 1; A 2, Aspergillus sp. 2; A 3, Aspergillus sp. 3; A 4, Aspergillus sp. 4; A 5, Aspergillus
sp. 5; A 6, Aspergillus sp. 6; A 7, Aspergillus sp. 7; A 8, Aspergillus sp. 8; A 9, Aspergillus sp. 9; A 10,
Aspergillus sp. 10; A 11, Aspergillus sp. 11; A 12, Aspergillus sp. 12; A 13, Aspergillus sp. 13; A 14,
Aspergillus sp. 14; Cl sp., Cladosporium sp.; P 1, Penicillium sp. 1; P 2, Pencilllium sp. 2; P 3, Penicilllium
sp. 3; P 4, Penicillium sp. 4; P 5, Penicillium sp. 5; P 6, Penicillium sp. 6; P 7, Penicillium sp. 7; P 8,
Penicillium sp. 8; P 9, Penicillium sp. 9; P 10, Penicillium sp. 10; S 1, Sterile mycelium 1; S 2, Sterile
mycellium 2; S 3, Sterile mycellium 3; S 4, Sterile mycellium 4; S 5, Sterile mycellium 5; S 6, Sterile
mycellium 6; S 7, Sterile mycellium 7).
It is confirmed that fungi present in the soil of acidic nature contaminated with heavy metal content are
endowed with some useful and exploitable potential of some industrial product like enzymes, organic acids etc.
Observation on their phosphate solubilising potential also makes them agriculturally important besides their
industrial potential. The negative antagonist behaviour for other fungi is an aided advantage to make these fungi
agriculturally more useful as bio fertiliser development. Hence, it is inferred that this acidic laterite soil is
endowed with a good number of fungal strains having exploitable potential. Further systematic research on
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enzyme extraction, purification and its utility, as well as their impact on growth and development of
agriculturally important crops and other plants of economic importance is required to reach any conclusion.

ACKNOWLEDGEMENT
The financial assistance obtained through Forest and Environment Department, Govt. of Odisha (State Plan
Project 2014-15) and INSPIRE programme (No. DST/INSPIRE Fellowship/2013/506) DST, Govt. of India is
gratefully acknowledged.

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Plant Research 3(1): 112119.
Brdy J (2005) Bioactive microbial metabolites: a personal view. Journal of Antibiotics 58: 126.
Gupta N, Sabat J & Parida R (2007) Solubilization of tricalcium phosphate and rock phosphate by microbes
isolated from chromite, iron and manganese mines. Acta Botanica Croatica 66: 197204.
Jackson ML (1958) Soil Chemical Analysis. Prentice Hall Inc. Englowoodcliff, New Jersey, USA.
Khan MS, Zaidi A & Wani P (2007) Role of phosphate solubilizing microorganisms in sustainable agriculture-
A review. Agronomy for Sustainable Development 27: 2943.
Khan MS, Zaidi A, Wani P & Oves M (2009) Role of plant growth promoting rhizobacteria in the remediation
of contaminated soils. Environmental Chemistry Letters 7: 19.
Mehrotra RS & Aneja KR (1990) An introduction to Mycology, Wiley eastern limited, New Delhi.
Nagamani A, Kunwar IK & Manoharachary C (2006) Handbook of Soil Fungi. IK International Pvt. Ltd., New
Delhi.
Oves M, Khan MS & Zaidi A (2013) Chromium reducing and plant growth promoting novel strain
Pseudomonas aeruginosa OSG41 enhance chickpea growth in chromium amended soils. European Journal
of Soil Biology 56: 7283.
Oves M, Khan MS, Zaidi A & Ahmad E (2012) Soil contamination, nutritive value and human health risk
assessment of heavy metals: an overview. In: Zaidi A, Wani PA & Khan MS (eds) Toxicity of heavy metals
to legumes and bioremediation. Springer, Wien, pp. 128.
Rajagopal K, Kalavathy S, Kokila S, Karthikeyan S, Kathiravan G, Prasad R & Balasubraminan P (2010)
Diversity of Fungal Endophytes in Few Medicinal Herbs of South India. Asian Journal of Experimental
Biological Sciences 1(2): 415418.
Rajoriya A, Panda A & Gupta N (2014) Comparative evaluation of nutritional, biochemical and enzymatic
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Sahoo HR, Sahoo P & Gupta N (2014) Extracellular enzymatic potential and antimicrobial activity of
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3(2): 384389, 2016

Research article

Diversity and distribution of lichens from the monuments of


Gwalior division, Madhya Pradesh with special reference to rock
porosity and lichen growth
Vindhyeshwari Uppadhyay1,2, Komal Kumar Ingle1,2, Suman Trivedi2 and
Dalip Kumar Upreti1*
1
Lichenology Laboratory, CSIR-National Botanical Research Institute, Lucknow-226001, India
2
Department of Botany, Motilal Vigyan Mahavidyalaya, Bhopal-462026, India
*Corresponding Author: upretidk@rediffmail.com [Accepted: 15 July 2016]

Abstract: The present study reports the diversity and distribution of lichens growing on historical
monuments of Gwalior division. Result revealed the occurrence of 28 lichen species belonging to
16 genera and 9 families. The members of the lichen family Physciaceae, Teloschistaceae and
Verrucariaceae dominate the lichen diversity on monuments as represented by 5 species each of
the families, followed by members of Peltulaceae and Lecanoraceae. Among the different growth
forms, crustose exhibits their luxuriant growth, followed by squamulose and foliose on various
monuments. Substrate preference for lichen colonization is apparent by occurrence of maximum
diversity of lichens represented by 27 species on sandstone, followed by concrete, igneous granite,
calcareous and clay represented by 7, 6, 5 and 2 species respectively. The rock porosity was
calculated to measure water holding capacity and the correlation between rock porosity and lichen
growth were studied which shows that the squamulose growth form lichen Endocarpon rosettum
and Endocarpon subrosettum with thick medullary zone, grows on rocks having maximum water
holding capacity of 43% each followed by Phylliscum indicum and Endocarpon nanum growing
on rocks with 23% and 16.5% water holding capacities respectively.
Keywords: Lichen distribution - Monuments - Gwalior division - Rock porosity.

[Cite as: Uppadhyay V, Ingle KK, Trivedi S& Upreti DK (2016) Diversity and distribution of lichens from the
monuments of Gwalior division, Madhya Pradesh with special reference to rock porosity and lichen growth.
Tropical Plant Research 3(2): 384389]

INTRODUCTION
Lichens are composite organisms formed by the symbiotic association of algae and fungi. Owing to their
desiccation resistant property, survival under extreme temperature and nutrient accumulating efficacy, lichens
occur in a wide range of habitats (Kumar & Kumar 1999, Kumar et al.2014). Lichens have an ability to grow on
any substrate of perennial nature. In nature they play important role in soil formation and grow as pioneer
organism. The role of lichens as biological weathering agents in the development of soils was formerly
considered in a geological context only, but recent researches have shown that these organisms are capable of
biodeteriorating stone substrata within a relatively short time-scale. Biodeterioration of stone monuments may
be classified into three categories: biophysical, biochemical and aesthetic deterioration (Allsopp & Seal 1986,
Caneva et al. 1991). The characteristics of lichens and their deterioration on stone have been studied by several
authors worldwide (Paine et al. 1933, Jones & Wilson 1985, Giacobini et al. 1985; Sabbioni & Monte del 1987,
Seaward 1988). James et al. (1977) observed numerous distinct lichen communities on hard limestone from
British Islets.
The distribution of lichens growing on historic monuments of different regions of India was enumerated in
number of studies (Ayub 2005, Singh & Upreti 1991, Chatterjee et al. 1995, Singh et al. 1999, Saxena et al.
2004). Although, some work exploring the lichen diversity on the monuments of central India has already been
done (Jain 2001, Upreti 2002, Upreti et al. 2004, Bajpai et al. 2008), but one of the monument rich Gwalior has

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Received: 22 May 2016 Published online: 31 August 2016
Uppadhyay et al. (2016) 3(2): 384389
.
not been explored well. Thus, the present study was conducted to know the diversity and distribution of lichens
growing on the monuments of Gwalior division.

MATERIALS AND METHODS


Study area
The Gwalior division is an administrative subdivision of Madhya Pradesh state in Central India. It includes
districts of Ashoknagar, Datia, Guna, Gwalior and Shivpuri. The Gwalior division is a home to a number of
forts, castles, temples and historical and religious monuments. The monuments in the study are having varied
construction material such as calcareous, clay, igneous granite and sandstone. Some monuments were
constructed with concrete as well. Being alkaline nature of most of the construction materials it provides an
excellent substratum for a large number of lichens to colonize together with other group of plants.
Sample collection and Identification
The study is based on the collection of lichens from more than 25 Monuments situated in five districts of
Gwalior division, Madhya Pradesh (Table 1). More than 200 samples were collected during August 2012 to
October 2014. The sample collections were made from the abandoned monuments and nearby rocks to avoid the
destruction of protected monuments.
Table 1. Localities survey for collection of lichens.
S.No. District Monuments and nearby area
1 Ashoknagar Kila kothi, Chanderi fort; Khuni Darwaja, Chanderi fort;, Kati Ghati; Digambar Jain
mandir; Jageshwari Mata mandir; Near Graveyard, Chanderi; Thubon village
2 Datia Pancham kavi ki pahadi; Chiviya for ki mata; Veer Singh palace; Karam Sagar dam; Dong
Karera village; Udnu ki pahadi; 5 Km from Datia towards Shivpuri
3 Guna Bajrang garh fort; Gader ki Gufa; Beesbhuji temple
4 Gwalior Chaturbhuj temple, Gwalior fort; Gujri mahal, Gwalior fort; Tansen ki dargah; Devkho;
Gupteshwar temple
5 Shivpuri Narwar fort; Survaya fort; Atal Sagar dam; Bhadaiya kund;10 Km from Shivpuri to Jhansi
highway
Though Gwalior division has rich diversity of a number of historical monuments, however Jain (2001)
enumerated few lichens and algae from a single monument of the district. Thus the present study is aimed to
investigate the lichen diversity on most of the monuments of the Gwalior division comprising of five districts.
The information regarding correlation between lichen diversity and the rock types will be significant for
conservators of the monuments to adopt appropriate conservational and management strategies.
The samples were identified morphologically, anatomically and chemically. The external morphology of the
thallus was observed under a stereo-zoom microscope. The anatomical structures were studied under a
compound microscope. For chemical spot tests, the usual reagents K (5% potassium hydroxide), C (aqueous
solution of Calcium hypochlorite), and PD (para-phenylenediamine) were used. Thin Layer Chromatography
was performed in solvent system C following Orange et al. (2001). The samples were identified up to their
species level and authenticated following literature of Awasthi (1991, 2007). The identified specimens are
preserved in herbarium of CSIR- National Botanical Research Institute (LWG).
Determination of porosity of rocks
The known volume (13 gm) of monumental rock samples are taken in glass bowl. This bowl already
contains distilled water 100 cc volume. These rock saturated by capillary action of water at room temperature
for 24 hours, till a thin film of water on the top surface of the rock was consequently seen. This saturated sample
was weight and dried in an oven at 105C for 24 hours, till constant weight was established. Then the rock
samples are removed and weight. Water holding capacity was calculated as wet minus dry weight and specified
as percentage water content related to dry weight.

RESULTS AND DISCUSSION


The study revealed occurrence of 28 species belonging to 16 genera and 10 families from the Gwalior
division (Table 2). The crust forming lichens exhibit their dominance in the area represented by 17 species
followed by squamulose, foliose and leprose growth forms with 8, 2 and 1 species respectively. The monument

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in Ashoknagar district exhibit the maximum dominance of lichens represented by 27 species followed by Guna
and Shivpuri with 14 and 7 species respectively, while Datia and Gwalior district represented by 6 species each.
Out of the 10 families of lichens, members of family Physciaceae and Teloschistaceae exhibit their dominance
followed by the members of families Peltulaceae, Lichinaceae and Lecanoraceae (Table 2). The most common
lichen genera growing on different monuments are Caloplaca, Peltula, Amandinea and Endocarpon.
Table 2. Distribution of lichens on various monuments of Gwalior division.
Localities in study sites
Lichen taxa GF Substratum
1 2 3 4 5
Lecanoraceae
1 Lecanora cenisia Ach. C 1 Sandstone
2 Lecanora pseudistera Nyl. C 1 Sandstone
3 Lecidella stigmatea (Ach.) Hertel & Leuckert C 1 Sandstone
Lichinaceae
4 Anema decipiens (A. Massal.) Forssell C 1 Sandstone, Igneous granite
5 Phylliscum indicum Upreti S 3 2 2 4 1 Sandstone, Calcareous, Concrete
Parmeliaceae
6 Parmotrema praesorediosum (Nyl.) Hale F 1 1 Sandstone, Concrete
Peltulaceae
7 Peltula euploca (Ach.) Poelt in Pisut S 1 6 1 4 Sandstone, Igneous granite,
Concrete
8 Peltula obscurans (Nyl.) Gyeln. S 1 1 1 Sandstone, Igneous granite
9 Peltula patellata (Bagl.) Swinscow & Krog S 3 2 1 1 3 Sandstone, Igneous granite,
Concrete
10 Peltula placodizans (Zahlbr.) Wetmore S 2 5 1 2 3 Sandstone, Clay, Igneous
granite, Concrete
Physciaceae
11 Amandinea Montana (H. Magn.) Marbach C 1 Sandstone
12 Amandinea subduplicata (Vain.) Marbach C 2 Sandstone
13 Amandinea submontana Marbach C 2 Sandstone
14 Buellia indica S.R. Singh & D.D. Awasthi C 1 Sandstone
15 Dirinaria confluens (Fr.) D.D. Awasthi F 2 1 Sandstone
Ramalinaceae
16 Bacidia arnoldiana Krb C 1 Sandstone
Stereocaulaceae
17 Lepraria lobificans Nyl. L 1 1 Sandstone
Teloschistaceae
18 Caloplaca cupulifera (Vain.) Zahlbr. C 2 Sandstone
19 Caloplaca pseudopoliotera Y. Joshi & Upreti C 5 Sandstone
20 Caloplaca subpoliotera Y. Joshi &Upreti C 1 2 1 1 Sandstone, Igneous granite
21 Caloplaca subsoluta (Nyl.) Zahlbr. C 3 1 Sandstone, Calcareous
22 Caloplaca tropica Y. Joshi &Upreti C 2 Sandstone
Thelenellaceae
23 Thelenella luridella (Nyl.) H. Mayrhofer C 2 Sandstone
Verrucariaceae
24 Endocarpon nanum Ajay Singh &Upreti S 3 1 Sandstone, Calcareous
25 Endocarpon rosettum Ajay Singh &Upreti S 2 1 Sandstone, Calcareous
26 Endocarpon subrosettum Ajay Singh &Upreti S 1 1 3 1 Sandstone, Calcareous, Clay,
Concrete
27 Staurothele fissa (Taylor) Zwackh C 1 2 1 Sandstone, Concrete
28 Verrucaria coerulea (Ramond) C 4 1 Sandstone
Note: GF, Growth forms; 1, Ashoknagar; 2, Datia; 3, Guna; 4, Gwalior; 5, Shivpuri.
The lichens were uniformly found growing on different rock types including Calcareous, Clay, Concrete,
Igneous granite and Sandstone. The sandstone is the major rock type used as a construction material for the
monuments throughout the division exhibit growth of most of the lichens The correlation between rock porosity
and the type of lichen growth have been studied which determined by water holding capacity method. Since
water holding capacity is directly proportional to the porosity of rock, which controls the availability of water
and hence influences the growth of lichens on monuments. The correlation between water uptake capacity of

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Uppadhyay et al. (2016) 3(2): 384389
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rocks and the type of lichen growth in Bhimbetka rock shelter have been studied by Upreti et al. (2006) and
suggested that the leprose lichens with thin medullary crust over rock have less water intake capacity, whereas
crustose lichens colonize on rocks having moderate water holding capacity. Among the foliose lichens, the taxa
with thin thallus grow on rocks having maximum water holding capacity.
In the present study among the different growth forms, squamulose lichen Endocarpon rosettum and
Endocarpon subrosettum with thick medullary zone, exhibit the maximum water holding capacity of 43% each
followed by Phylliscum indicum and Endocarpon nanum with 23% and 16.5% respectively. Caloplaca
subsoluta and Caloplaca tropica, crustose lichens have the maximum water holding capacity of 28% each
followed by species of lichen genus Amandinea, Bacidia, Buellia and Lecidella with 10% each. The foliose
lichen Dirinaria confluens and Parmotrema praesorediosum with thick thallus showed 10% water holding
capacity. The only leprose lichen found in the area Lepraria lobificans with a thin medullary crust spreading
over rocks have the lowest water holding capacity of 5%. Some common lichens of the study area have also
been shown in figure 1.

Figure 1. Some common lichen species reported from the study area: A, Endocarpon subrosettum Ajay Singh & Upreti; B,
Peltula euploca (Ach.) Poelt in Pisut; C, Peltula obscurans (Nyl.) Gyeln.; D, Peltula patellata (Bagl.) Swinscow & Krog; E,
Peltula placodizans (Zahlbr.) Wetmore; F, Phylliscum indicum Upreti. [Scale bars: AF = 2 mm]

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It is clear from the study that the anatomical characters play vital role in determining the water holding
capacity. Within different type of rocks, Calcareous rock showed the maximum water holding capacity ranging
from 1043% followed by Concrete and Sandstone with the range between 535% and 328% respectively.
Concrete rocks exhibit 35% and Clay showed range between 323% water holding capacity while the Igneous
granite exhibit the range between 510% (Fig. 2). The chemical composition and physical form of the rock also
play role in the water holding capacity. As calcareous rocks are rich in calcium salts, which has a tendency to
absorb moisture and retain it for longer period of time, while igneous rock solidified magma has little or no
tendency to absorb moisture. The non-porous rocks and the rocks with large pores have very less water holding
capacity as in both types the water absorption rate is very low. However, the rocks with smaller pores have high
water holding capacity because of its capillary action and lesser surface exposure of water to the air.

Figure 2. Graph shows water holding capacity of lichen species, correlated with different types of rock substratum.
In dry conditions lichen metabolism reduces water and nutritional needs to the minimum, and as a
consequence lichens are able to withstand xeric conditions for prolonged periods. Bare and exposed monument
surfaces therefore provide ideal, competition-free situations for lichen invasion and establishment as pioneers of
the bio-succession. Besides, many micro-environmental or microclimatic conditions develop in mosaic habitats
on account of conditions that create their own modified versions of the general lichen-flora supported by
prevailing macroclimate of the area. The present study will be helpful in conducting future bio-monitoring
studies and to the conservators for adopting conservation practices for the monuments in Gwalior division.

ACKNOWLEDGEMENT
We are grateful to the Director, CSIR-National Botanical Research Institute, Lucknow for proving
laboratory facilities.

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Ayub A (2005) Lichen flora of some major historical monuments & buildings of Uttar Pradesh. Ph.D. Thesis.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
3(2): 390395, 2016

Research article

Evaluation of in-vitro cytotoxic and thrombolytic activity of


methanolic extract of Xanthium indicum L.
Muhammad Sazzad Hossain1, Md. Hossan Sakib1*, Mohammad Shahin Alam1,
Ahsan Ullah2, Sadequr Rahman3 and Limon Kanti Shill2
1
Department of Pharmacy, University of Science and technology Chittagong, Bangladesh
2
Department of Pharmacy, International Islamic University Chittagong, Bangladesh
3
Department of Public Health, North South University, Dhaka, Bangladesh
*Corresponding Author: sakibiiucph@gmail.com [Accepted: 16 July 2016]

Abstract: The present study was designed to investigate the cytotoxic and thrombolytic activity of
methanolic extract of fruits of Xanthium indicum. Methanolic extract of Xanthium indicum, was
used to evaluate its cytotoxicity in Brine shrimp lethality bioassay where vincristine sulphate was
used as standard drug. Thrombolytic effect of the fraction was investigated in clot lysis
experiment. In Brine shrimp lethality bioassay, LC50 value of the extract was 30.90 g.ml-1 and
vincristine sulphate served as the positive control showed LC50 value 10.51 g.ml-1. The extract
exerted 48.47% lysis of the blood clot in thrombolytic activity test while 48.83% and 13.82% lysis
were obtained for positive control (streptokinase) and negative control respectively. Compared to
vincristine sulphate. It is evident that the methanolic extract of fruits of Xanthium indicum was
cytotoxic. So, the extract possessed considerable thrombolytic activity. Which compounds is
responsible for the present pharmacological actions and to know their mechanism of action,
extensive pharmacological and phytochemical experiments are essential.
Keywords: Xanthium indicum - Cytotoxic - Thrombolytic - Vincristine - Streptokinase.

[Cite as: Hossain MS, Sakib MH, Alam MS, Ullah A, Rahman S & Shill LK (2016) Evaluation of In-Vitro
Cytotoxic and Thrombolytic Activity of Methanolic Extract of Xanthium indicum L. Tropical Plant Research
3(2): 390395]

INTRODUCTION
Due to the fact ancient times plants have served to be a natural source of treatments and therapies like
aspirin, quinine, and coffee. Today, scientists are using these renewable resources to generate a new generation
of therapeutic solutions. Plants improved by making use of biotechnology can produce the essential healthy
proteins for innovative treatments for diseases like cancer, HIV, heart disease, diabetes, Alzheimer's sickness,
kidney disease, Crohn's disease, cystic fibrosis, many sclerosis, spinal cord injuries, Hepatitis G, chronic
obstructive pulmonary disorder (COPD), morbid obesity, arthritis and iron deficiency. The by using natural
products with therapeutic properties is usually as ancient as human civilization and, for an extended time,
mineral, plant and animal products were the leading sources of drugs. Vincristine in addition to vinblastine from
Catharanthus roseus, atropine by Atropa belladonna and morphine and codeine by Papaver somniferum. It is
estimated that 60% connected with anti-tumor and anti-infectious drugs already out there or under clinical trial
are connected with natural origin (Rates 2001). The vast flavor these cannot yet be synthesized economically
and are also still obtained from wild or developed plants. Natural compounds can be cause compounds, allowing
the design and lucid planning of new drugs, biomimetic synthesis development along with the discovery of new
therapeutic properties not yet assigned to known compounds (Hamburger & Hostettmann et al. 1991). In
addition, compounds like muscarine, physostigmine, cannabinoids, yohimbine, forskolin, colchicine in addition
to phorbol esters, all obtained from facilities, are important tools used in medicinal, physiological and
biochemical studies (Archana et al. 2011). Cytotoxicity is the products being toxic to cells. Cells exposed to a
cytotoxic compound can respond in numerous ways. The cells may undergo necrosis, in which they lose
membrane integrity and die rapidly on account of cell lysis; they can stop rising and dividing; or they can
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Received: 10 April 2016 Published online: 31 August 2016
Hossain et al. (2016) 3(2): 390395
.
initialize a genetic program of controlled cell phone death, termed apoptosis. Cells undergoing necrosis
commonly exhibit rapid swelling, lose membrane sincerity, shut down metabolism, and release their contents
into your environment upon lysis. Apoptosis is seen as a well-defined cytological and molecular event,
including a big difference in the refractive index of this cell, cytoplasmic shrinkage, nuclear condensation, in
addition to cleavage of DNA. Cytotoxicity assays utilized widely in drug discovery research to help predict
which lead compounds might have safety concerns in humans before significant time and expense are incurred
in their development. Other researchers study mechanisms of cytotoxicity as a way to gain a better
understanding of the normal and abnormal biological processes that control cell growth, division, and death
(Patel et al. 2009).
Thrombolysis could be the breakdown (lysis) of blood vessels clots by pharmacological means. It is
colloquially called elot busting for this reason. It functions by stimulating fibrinolysis by plasmin through
infusion regarding analogs of tissue plasminogen activator (tPA), the particular protein that normally activates
plasmin. Thrombolytic therapy is the usage of drugs to break up or break up blood clots, which are the main
reason behind both heart attacks and stroke. Thrombolytic medications are approved for your immediate
treatment of stroke and also heart attack. The most widely used drug for thrombolytic therapy is muscle
plasminogen activator (tPA), but other drugs can do a similar thing (Kawsar et al. 2011).
Xanthium indicum L. yield Herbs perennial, monoecism or perhaps dioeciously, 2550 cm tall. Stems
climbing or erect, simple or branched, crispately pubescent. Results in alternate; nanophyllscordate or ovate, 3
10 mm; stipules lanceolate-linear, 570.50.8 mm, glabrous, without cystoliths; petiole 15 mm; Female
inflorescences individual, 46 mm in diam.; peduncle los angeles. 1 mm; receptacle nearly orbicular, 35 mm
inside diam.; bracts triangular; bracteoles lanceolate-linear. Men flowers 4-merous (Harnischfeger 2000).

MATERIALS & METHODS


Collection of Plant Components
The leaves of Xanthium indicum L. were accumulated from Chittagong local forest area; the leaves regarding
Xanthium indicum were collected at their totally mature form. After cleaning, the results in were taken and
splitting the peal, next air dried for 8 days, and kept in an oven at 45C with 72 hours. 250 gm of dehydrated
powder was cold extracted with Methanol. Dried powder soaked with methanol for 1 week. Then filtered to take
the targeted extract, extract containing beaker was added to the water bath (at 4045C) to evaporate the solvent
from your extract (Prasad 2007).
Preparation of Extraction
The extract is prepared by cold extraction process. In this process the coarse powder was submerged in
ethanol (95%) since ethanol is the most common solvent for extracting most of the constituents present in herbal
materials. Amber glass bottle were used for this purpose, which were kept at room temperature and allowed to
stand for 7 days with occasional shaking and stirring. When the solvent became concentrated the contents were
first decanted by using cotton and then filtered through Whatmann No.1 filter paper. The filtrate so obtained was
then concentrated to dryness through the evaporation of solvent using rotary evaporator. Finally we got the
concentrated semi-solid extract. The concentrated were then used as crude extract of respective test experiments.
In our present investigation, we used methanolic extract for cytotoxic and thrombolytic activity (Mackeen et al.
2000)
In-vitro Cytotoxic Review
Brine shrimp lethality bioassay is widespread in the bioassay for the bioactive chemical substances. Here
simple zoological organism (Artemia salina) was used to be a convenient monitor for the screening. The dried
cyst on the brine shrimp were collected from an aquarium shop (Chittagong, Bangladesh) in addition to hatched
in artificial seawater (3.8% NaCl alternative) with strong aeration for 24 hours day/dark cycles to mature shrimp
termed nauplii. The cytotoxicity assay was conducted on brine shrimp naupli using Meyer procedure (Sarkar &
Farooque 2004).
Materials
Artemia salina Leach (brine shrimp ova), Sea salt non ionized NaCl, Modest tank with perforated dividing
dam to hatch this shrimp, Lamp to attract the nauplii, Pipette (1 ml in addition to 5 ml), Micropipette (110
minuscule liter), Glass vials (5 ml), Magnifier, Test sample for experimental plants (Sarkar & Farooque 2004).
Hatching connected with Brine Shrimp Eggs
Artemia salina Leach (brine shrimp eggs) collected on the pet shop was used as this test organism. Simulated
sea water was consumed in the small tank and the shrimp ova (1.5 g.mL-1) were included in one side of the tank
and this also side was covered. The shrimps were permitted to one side of tank and that side was covered. The

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shrimp were allowed for 2 days to hatch and mature seeing that nauplii (larvae). Constant oxygen supply was
executed during the hatching time. The hatched shrimps were attracted to the lamp opposed to this of the
divided tank through this perforated dam. These nauplii were taken due to this bioassay (Islam et al. 2002.)
Preparation of the Simulated Beach Water
38 grams sea salt seemed to be weighted accurately, dissolved in 1 liter of sterilized distilled water then
filtered to get clear solution. The ph on the sea water was maintained between 8.08.5 applying 1n na0h solution
(mosaddik et al. 2003).
Preparation connected with Sample Solution
At first take 19 ml distilled mineral water in beaker add 1 ml DMSO (dimethyl sulfoxide) so prepares stock
solution. Clean test pipes were taken. These test tubes were for different concentration (one test tube for every
single concentration) of test samples. 5 mg methanolic extracts of Xanthium indicum were being accurately
weighed and dissolved in 4 ml stock options solution. Thus a concentration of 1000 g.ml-1 was obtained which
used for extract solution. Then taking 1ml get solution from beaker & add 9 ml stock options solution In vials
thus prepared remaining extract solution. From this extract alternative 12.5 g.ml-1, 25 g.ml-1, 50 g.ml-1, 100
g.ml-1, 200 g.ml-1 and 400 g.ml-1 were consumed in ten test tubes respectively and tweaked volume 5 ml sea
water. Eventually 20 nauplii are then applied with each test tube (Mosaddik et al. 2003)
Getting ready of Control group
Control groups are used in cytotoxicity review to validate the test method and be sure that the results
obtained are only a result of the activity of the test agent along with the effects of the other possible variables are
nullified. Usually two types connected with control groups are used-i) Constructive control lii) Negative control
(Robin et al. 1989)
Getting ready of Positive Control group
Positive control in cytotoxicity study is usually a widely accepted cytotoxic agent and a result of the test
agent is compared while using the result obtained for the positive management. In the present study, vincristine
sulphate was used for the reason that positive control. 3 mg of vincristine sulphate seemed to be dissolved in 1.
8 ml of distilled water to have a concentration of 5 mg.ml-1. This seemed to be used as stock solution of
vincristine sulphate. Through a micropipette 400, 200, 100, 50, 25 and 12.5 l on the stock solution were
transferred in 6 unique vials. NaCl solution (brine water) was included in each vial making the volume nearly 5
ml. The final concentration of vincristine sulphate from the vials became 400 g.ml-1, 200 g.ml-1, 100 g.ml-1,
50 g.ml, 25 g.ml-1 in addition to 12.5 g.ml-1 respectively. The experiment was repeated triple (Robin et al.
1989)
Preparation of negative control
100 l connected with distilled water, DMSO and ethanol was added to all of the three remarked glass vials
containing 5 ml connected with simulated sea water and 20 shrimp nauplii make use of as control groups. If the
brine shrimp nauplii in these vials show an immediate mortality rate, then the test is regarded as in valid as the
nauplii died caused by some reason other than the cytotoxicity on the samples (Robin et al. 1989)
Application of Brine shrimp Naupli
Through the Pasteur pipette 20 living nauplii were added to all of the vials containing 5 ml of simulated
beach water. A magnifying glass was for convenient count of nauplii. If the counting on the 20 nauplii was not
be doable accurately (Robin et al. 1989).
Counting of your Naupli
After 24 hours, the vials are observed using a magnifying glass and the sheer numbers of survival nauplii in
each vial ended up being counted and recorded. From this details, the percentage of mortality of nauplii was
calculated each concentration of the sample. The median lethal concentration (LC50) of your test samples was
obtained by a plot of percentage of your shrimps killed against the logarithm of your sample concentration
(Robin et al. 1989).
In-Vitro Thrombolytic Analysis
Thrombolysis is the breakdown (lysis) with blood clots by pharmacological means. It is colloquially
categorized as clot busting for this reason. It operates stimulating fibrinolysis by plasmin through infusion with
analogs of tissue plasminogen activator (tPA), a protein that normally activates plasmin.
Groundwork of Extract Solution for Thrombolytic Examine
10 mg of the extract appeared to be suspended in 10ml distilled water and shaken vigorously for a vortex
mixer. Then the suspension was kept overnight and decanted to eradicate the soluble supernatant, which was
filtered through the filter paper (Whatman No. 1). The best was then ready for in vitro review of clot lysis
activity (Mackeen et al. 2000)

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Groundwork of Streptokinase (SK) Resolution
To the commercially available lyophilized SK vial (PolaminWerk GmbH, Herdecke, Germany) of just one,
500,000 I.U., 5 ml sterile and clean distilled water was added and compounded properly. This suspension was
used for a stock from which 100 l (31,000 I.U) was used in in vitro thrombolysis (Mackeen et al. 2000).
Specimen with Thrombolytic Test
3ml blood was drawn from healthy human volunteers without using history of oral contraceptive or
anticoagulant therapy (with a protocol approved by the Institutional Strength Committee of Central India
Institute with Medical Sciences, Nagpur). 500 l of blood was transferred to every single ten previously
weighed alpine tubes to create clots (Mackeen et al. 2000).
Test Procedure for Thrombolytic examines
Experiments for clot lyses were toted as reported earlier (Mackeen et al. 2000). Venous blood drawn from
healthy volunteers was transferred within pre-weighed sterile Epen drop tube (500l/tube) plus incubated at
37C for 45 a matter of minutes. After clot formation, serum was wholly removed (aspirated out without
troublesome the clot formed). Each tube having clot was again weighed to look for the clot weight (Clot weight
= excess fat of clot containing tube - excess fat of tube alone). Each Epen drop tube containing clog was
properly labeled and 100 l of plant extract was added onto the tubes. All the tubes ended up being then
incubated at 37C for 95 minutes and observed for clot lysis. Just after incubation, fluid obtained was removed
and tubes were again weighed to see the difference in weight after clog disruption. Difference obtained in
weight utilized before and after clot lysis appeared to be expressed as percentage. Thrombolytic Activity with
methanol extract of Xanthium indicum clot lysis. Streptokinase and water were used for a positive and negative
(non-thrombolytic) regulate respectively. The experiment was repeated several times a day with the blood
samples of several volunteers.

RESULTS
Brine Shrimp Lethality Bioassay
Brine shrimp lethality results of the fraction of Xanthium indicum L. leaves is shown in figure 1 and LC50
calculated value is recorded in table 1. The fraction showed potential cytotoxic activity with LC50 value of 13.56
g.ml-1. Vincristin sulphate served as the positive control for this brine shrimp lethality assay and its LC50 value
was 10.51 g.ml-1.
Table 1. Cytotoxic activity of Xanthium indicum.
Conc Nauplii No.of naupli death Log C % of Mortality LC50 g/ml
12.5 20 9 1.096 35
25 20 13 1.39794 45
50 20 14 1.69897 80
100 20 15 2 85 13.561
200 20 18 2.3010 90
400 20 20 2.60205 100

Figure 1. Determination of LC50 value for fraction of Xanthium indicum leaves from linear correlation between log
concentrations versus percentage of mortality.
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Thrombolytic Activity
The methanolic extract of Xanthium indicum leaves is exerted 27.81% lysis of the blood clot in thrombolytic
activity test while 48.83% were obtained for positive control (streptokinase) and 13.82% were obtained for
negative control respectively which showed in table 2. So, the extract possessed considerable thrombolytic
activity (Fig. 2).
Table 2. Thrombolytic Activity of Xanthium indicum.
Exract Positive control Negetive control
27.806% 48.83% 13.82%

Figure 2. Methanolic extract of Xanthium indicum thrombolytic effect compared with standard (Streptokinase) and negative
control.

DISCUSSION
Plant-derived medicines contain a long history of usage for the prevention together with treatment involving
human diseases. Today, many pharmaceuticals currently approved when using the Food and Drug
Administration (FDA) get hold of origins to plant sources. A number of plants source especially several leaves
and vegetables are literally studied for their supplements having anticoagulant, antiplatelet and fibrinolytic
activity and the way to find evidence that consuming such food brings about deterrence of coronary events and
cerebrovascular event. Some of them plant products are generally modified further with recombinant technology
to make them more effective and site distinct. In some of our thrombolytic assay, the comparison of positive
control using negative deal with clearly demonstrated that clot dissolution won't occur when water was added
over the clot. When compared with the clog lysis percentage obtained through SK alongside water, an extremely
significant thrombolytic task was detected after treating the clots using C.arborea, chloroform percentage. Cell
floor bound plasminogen is easily activated to be able to plasmin, which could lead to fibrinolysis. Microbial
plasminogen activator: staphylokinase, streptokinase, be cofactor molecules that help with exosite formation and
increase the substrate presentation on the enzyme. Staphylokinase activates plasminogen that will melt clots,
also destroys the extracellular matrix alongside fibrin fibers that hold cells jointly.
Toxicity of plant materials is a serious concern to scientists and dieticians and thus cytotoxic assay was
conducted in this study to think about the toxicity profile of the plant extracts across the Brine Shrimp Lethality
(LC50, all day together with) test. Lagarto demonstrated a fantastic connection (r2 = 0. 91) relating into the
LC50 of the brine shrimp lethality make certainly the acute oral toxicity assay throughout mice. Influenced by
that correlation, brine shrimp lethality LC50< 10 g.ml-1 (LD50 involving 100 and 1000 mg.kg-1) is certainly as
the cut off value involving cytotoxicity. Depending on measured LC50 values in the extracts no one was found
severely lethal or toxic to build processed as pharmaceutical products in thrombolytic employs. Yet, the
extremely significant effect of Xanthium indicum demonstrates it to locate the best thrombolytic component for
even more processing.

CONCLUSION
The results of the present study led us to the inference that the plant extract possess modest cytotoxicity and
decreased level of thrombolytic properties. In the work we tried to give our maximum effort to fulfill the task
successfully and in the proper way of work. The work is done within the collaboration of all the teachers and we
tried to take each data carefully.
Since the extract is reported to contain a range of compounds, it is difficult to describe these observed
activities to any specific group of compounds. Hence, further studies are suggested to be undertaken to pin point
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Hossain et al. (2016) 3(2): 390395
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the exact compound(s) and to better understand the mechanism of such actions of Xanthium indicum
scientifically. The median lethal concentration (LC50) of Xanthium indicum was 13.56 g.ml-1. The thrombolytic
effect of Xanthium indicum was 27.81% at 10 mg in 10 ml conc.

ACKNOWLEDGEMENTS
Authors are grateful to Team of Pharmacy, International Islamic University Chittagong for giving all
facilities to use whole project. Authors are also fortunate to Dr. Shaikh Bokhtear Uddin, Link Professor,
Department of Botany, and College or university of Chittagong for identifying plants. Authors also
acknowledge the volunteer blood donors for cooperation from the project. The total research work seemed to be
supported by Department of Pharmacy, Overseas Islamic University Chittagong, Bangladesh. This project was
an inclusive element of academic degree of M. Pharm scholar.

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Immuno-Modulator. International Journal of Pharmacology 7: 198205.
Patel S, Gheewala N, Suthar A & Shah A (2009) In vitro Cytotoxicity Activity of SolanumNigrum Extract
Against Helacell Line and Vero Cell Line. International Journal of Pharmacy and Pharmaceutical Sciences
1(Suppl. 1): 3846.
Kawsar MH, Sikder MAA, Rana MS, Nimmi I & Rashid MA (2011) Studies of Thrombolytic, Antioxidant and
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Prasad S, Kashyap RS, Deopujari JY, Purohit HJ, Taori GM & Daginawala HF (2007) Effect of Fagonia
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Antimicrobial, antioxidant, antitumour -promoting and cytotoxic activities of different plant part extracts of
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Sarkar S & Farooque MA (2004) Antimicrobial andcytotoxic activities of 2-aminobenzoic acid and 2-
aminophenol and their coordinationcomplexes with Magnesium (Mg-II). Pakistan Journal of Biological
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Islam MA, Sayeed MA, Khan GRMAM, Mosaddik MA & Bhuyan MSA (2002) Terpenes from bark of
Zanthoxylum budrunga and their cytotoxic activities. Revista Latinoamericana De Quimica 30(1): 2428.
Mosaddik MA & Haque ME (2003) Cytotoxicity and antimicrobial activity of goniothalamin isolated from
Bryonopsis laciniosa. Phytotherapy Research 17(10): 11551157.
Robin JM, Farnsworth NR & Neill DA (1989) Isolation of A Novel Cytotoxic Polyacetylene From A
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
3(2): 396407, 2016

Research article

Species richness and diversity along the altitudinal gradient in


Tungnath, the Himalayan benchmark site of HIMADRI
Zubair A. Malik* # and Mohan C. Nautiyal
High Altitude Plant Physiology Research Centre (HAPPRC), HNB Garhwal University,
Srinagar (Garhwal) Uttarakhand-246174, India
#
Present address: Sheikh-ul-Aalam Memorial (SAM) Govt. Degree College, Budgam,
Jammu & Kashmir-191111, India
*Corresponding Author: malikmzubair081@gmail.com [Accepted: 18 July 2016]

Abstract: Alpine ecosystems are likely to show the effects of climate change earlier and more
clearly than other ecosystems. The main purpose of the recently launched research initiative,
HIMADRI (Himalayan Alpine Dynamics Research Initiative) is the long term monitoring of the
ecologically sensitive parameters at benchmark sites selected in various regions of Indian
Himalayan Region. The aim of the present study was characterization of one of the HIMADRI
benchmark sites (Tungnath, Western Himalaya) and to provide a baseline data about the species
richness and diversity along the altitudinal gradient for long monitoring. Four sites were selected
along an altitudinal gradient (32003600 m asl). A total of 52 plant species belonging to 40 genera
and 21 families were reported from the study area during the different seasons of year. Species
richness showed a non-significant positive correlation (r=0.53) with altitude but diversity showed
a slightly negative correlation (r=-0.05) with it. The reinvestigation of these sites in future (e.g.
after a decade or more) will help in understanding the effect of climate change in IHR in terms of
changes in species composition and diversity or in terms of species shifts from one vegetation zone
or/and ecotone to another.
Keywords: Alpine ecosystems - Climate change - Species migration - HIMADRI.

[Cite as: Malik ZA & Nautiyal MC (2016) Species richness and diversity along the altitudinal gradient in
Tungnath, the Himalayan benchmark site of HIMADRI. Tropical Plant Research 3(2): 396407]

INTRODUCTION
Altitude and climate are the two main factors that determine the prominent vegetation zones of the
mountains that are the most remarkable land forms on earth surface (Malik 2014). The area of mountains where
closed canopy forests end and give way to open vegetation is known as the alpine treeline ecotone. Alpine is
commonly used in a broad sense for the treeless areas above a low-temperature determined treeline in the high
reaches of mountains. The alpines are characterized by scanty rainfall, high wind velocity, low temperature,
high intensity of ultraviolet (UV) radiation, blizzards and snow storms (Nautiyal et al. 2004). The plants of this
zone show some adaptations to these conditions and are generally dwarfed, stunted, woolly or spiny, and
develop a mosaic patch of different forms (Walker et al. 1994). They possess an early growth initiation with a
short vegetative span ranging from several days to a few months (Bowman & Damm 2002). The community as
a whole usually exhibits seasonal fluctuations and its structure and composition are strongly influenced by the
extent to which periodic phenomena in the individuals are adjusted to each other (Kershaw 1973).
The distribution of plant species within alpine areas is often regulated by climate or climate-influenced
ecological factors. Therefore they are considered particularly sensitive to the influence of predicted climatic
change (Pauli et al. 2007). As a result, alpine ecosystems are likely to show the effects of climate change earlier
and more clearly than some other ecosystems (Grabherr 2000). Therefore, long term monitoring programs such
as the Global Observation Research Initiative in Alpine Environments (GLORIA) have been established
worldwide in different continents (www.gloria.ac.at). This program involves recording information about
composition of vascular species and soil temperatures according to a common protocol. This protocol can be

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Received: 08 April 2016 Published online: 31 August 2016
Malik & Nautiyal (2016) 3(2): 396407
.
used anywhere to examine patterns in species richness and diversity on the mountain peaks along altitudinal
gradients and for different aspects (Pauli et al. 2004).
Biological diversity includes the richness and evenness (relative abundance) of species amongst and within
living organisms and ecological complexes (Polyakov et al. 2008). Knowing the species diversity patterns and
the vegetation as a whole is fundamental for conservation of natural areas. Species richness and diversity are the
simple and easily interpretable indicators of biological diversity. These are ecologically sensitive parameters.
The number of species in a particular plant community varies markedly along the altitudinal range of its growth,
which depends on a set of complex factors that characterize the habitat of individual species (Malik et al. 2014).
Slobodkin & Sanders (1969) opine that community is a function of severity, variability and predictability of the
environment in which it develops. Therefore, diversity tends to increase as the environment becomes more
favourable and more predictable (Putman 1994).The factors such as soil nutrient content, slope, aspect and
altitude have been shown to exert an important control on species richness and diversity on a great variety of
ecosystems (Kharakwal et al. 2005).
The Indian Himalayan Region (IHR) occupies a special place in the mountain ecosystems of the world.
Himalayas, worlds youngest mountains with diverse vegetation are important locations for research into
ecology and biodiversity conservation (Pei 2001). But remoteness, difficult terrain, lack of resources and poor
infrastructure are some inherent difficulties that hamper the extent and quality of research in the region (Negi et
al. 2014). Thus there is a need for urgent attention from all concerned. Keeping in view all the aforesaid facts,
recently a multi-site research initiative, HIMADRI (Himalayan Alpine Dynamics Research Initiative) has been
launched that works on the protocol of GLORIA. This program, like GLORIA, involves recording information
about composition of vascular species and soil temperatures in the alpine regions of IHR. The aim of the present
study is to characterize the HIMADRI benchmark site (Tungnath) of Uttarakhand state of IHR on the basis of
species richness and diversity of plants along an altitudinal gradient.

MATERIALS AND METHODS


a) Study Area
The study area is being carried out in Tungnath area (N 3029'3030' and E 7912'7913') of Western
Himalaya, India. Tungnath forms a part of Kedarnath Wildlife Sanctuary (Fig. 1). It lies in the upper catchment
of the Alaknanda and the Mandakini Rivers, two major tributaries of the Ganges.

Figure 1. Map showing the location of study area (Tungnath).

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Geology, soil and climate
The rocks around Tungnath are mainly mylonitized gneisses, augen gneisses, schists and granites
constituting munsiari formation (Agarwala 1973). The weathering bedrocks, that provide the bulk of the loose
material in these mountains, are crystalline and metamorphic; with sedimentary deposits of Paleozoic age
(Gupta 1964). The soil texture is sandy loam, light grey to brown in colour and acidic in nature with a pH range
between 4 and 5 (Rai et al. 2012a). Meadows with deep soil cover are seen in northern aspects, while the
southern faces generally have large rock spurs and crevices and are either barren or have a few lithophytes. Four
distinct seasons are observed in the study area viz., short summer (MayJune), Monsoon (Julymid September)
and autumn (mid-SeptemberOctober) and long winter (NovemberApril). The snow cover lasts for about 45
months and melts during AprilMay that marks the arrival of favourable conditions for plant growth. The
growth period lasts for about 57 months only. Mean Annual temperature at the timberline ecotone (3300 m)
ranged between -8.91 (January) and +25.6C (May) with an average of 6.650.68C. Mean temperature of the
warmest month was 12.561.23C, in July (Fig. 2). Annual precipitation was 2410.5432.2 mm, of which
89.5%, recorded during JuneSeptember (Adhikari et al. 2011).

Figure 2. Mean air temperature, rainfall and snowfall at timberline ecotone in the study area during 2008-
2010 (Courtesy: Raiet al. 2012a).
Forest types and vegetation
According to Champion and Seth's (1968) classification the study area falls in sub-alpine forest and alpine
scrubs. The description of the vegetation types of the area is as under:
Sub-alpine forest: The sub alpine forest is formed by Abies spp., Betula utilis, Quercus semecarpifolia, Acer
spp., Sorbus sp. etc. Shrub layer is represented by Rhododendron campanulatum, R. barbatum, Viburnum
spp., Rosa sericea, Rubus niveus, Salix sp. etc. Herb layer is represented Trachydium roylei, Rumex
nepalensis, Persicaria wallichii, Thamnocalamus spathiflorus etc.
Timberline ecotone: Timberline in the study area ranges between 32503350 m which is formed by Betula utilis
and Abies spectabilis in the north to north-west facing slopes, while south to south west facing slopes
dominated by Q. semecarpifolia and R. arboreum (Rai et al. 2012b). In the steep rocky slopes timberline is
dominated by climatically modified dwarf and stunted individuals of R. arboreum, which grow very slowly
in harsh climatic conditions. Other major shrub species in this zone are Lonicera spp., Rubus niveus, Salix
denticulata, Rosa sericea, Berberis jaeschkeana and Viburnum grandiflorum, while in drier slopes and rocky
outcrops Cotoneaster microphylla and Juniperus indica. Cypripedium elegans, C. cordigerum, Neottia
pinetorum and Platanthera leptocaulon are some rare orchid taxa of the area which are mainly recorded at
timberline zone (Rai et al. 2012b). Beyond timberline ecotone other two species of Rhododendron viz., R.
anthopogon and R. lepidotum form the community and provide micro-habitat to several other herbaceous
species.
Krummholz layer: The 'krummholz' layer under the tree canopy is formed by Rhododendron campanulatum, a
dominant shrub species of this region.

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Alpine meadow and scrubs: The ground layer vegetation consists of cushionoid herb, grasses and sedges. The
herbaceous flora is represented by Ascomastylis elata, Anaphalis spp., Anemone spp., Bistorta spp., Carex
inanis, Danthonia cachemyriana, Gaultheris trichophylla, Trachydium roylei, Sibbaldia cuneata, Tanacetum
longifoliumetc.
b) Methodology
For the present study, four sites were selected along an altitudinal gradient (32003600 m asl). The details of
the selected sites are given in the table 1. Field expeditions were made to the selected sites during June, August
and October months of 2014 for vegetation analysis. At each altitudinal zone, a 3m 3m quadrat cluster was
marked permanently during the first visit in the month of June 2014. Each quadrat cluster consists of nine 1m
1m quadrats. Vegetation analysis was carried out thrice during 2014 in the four corner quadrats to study the
variation of species richness and diversity throughout the year.
Table 1. Characteristics of the study area.
Site Code Altitude (m) Vegetation Zone/Ecotone Geographic co-ordinates
TUN 1a 3236 Sub-Alpine N 30 29.723'; E 079 12.970'
TUN 1 3330 Timberline-Alpine Ecotone N 30 29.600'; E 079 12.985'
TUN 2a 3455 Ecotone between tree line and alpine zone N 30 29.449'; E 079 13.173'
TUN 2 3555 Upper Alpine Zone N 30 29.350' ; E 07913.242'
Species Richness was simply taken as a count of total number of species in a particular vegetation zone. The
index of diversity was calculated after Shannon & Wiener (1949). If pi is the proportion of individuals (from the
sample total) of species i.e. then diversity (H) is,

( )( )

Simpsons Index (D) was calculated following Simpson (1949). It measures the probability that two
individuals randomly selected from a sample will belong to the same species.
s
D Pi
2

i 1

Simpsons diversity index (SDI) was calculated by using following formula (Simpson 1949):
SDI=1-D
Where, D is the Simpsons index. SDI represents the probability that two individuals randomly
selected from a sample will belong to different species. Its value ranges between 0 and 1.
Equitability (Ep) or Evenness Index was calculated following Pielou (1975):

p
max
Where, = Shannon-Wiener Diversity Index; max lnS ; lnS = natural log of S.
Beta Diversity (-Div) was calculated as per following formula given by Whittaker (1972):
Sc
Div
S
Where, -Div = Beta Diversity; Sc is the total number of species occurring in a set of samples
counting each species only once whether or not it occur more than once and S is the average
number of species per individual sample.
Sorenson Similarity Index (IS) of similarity (in percentage) between forest sites was calculated following
Sorenson (1948):
2C
IS 100
A B
Where, IS= Sorenson Index of Similarity; C= Species common to both comparable sites; A=
Total number of species in site A; B= Total number of species in site B.

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RESULTS
A total of 52 plant species belonging to 40 genera and 21 families were reported from the study area during
the whole year (Table 2). Rosaceae with highest number of species (8) emerged as the dominant family,
followed by Asteraceae (7), Rananculaceae (5), and so on (Fig. 3). About 48% of the families were represented
by a single species.
Table 2. Occurrence of species during different seasons of the year. (+ is presence and is absence)
Species Jun Aug Oct Species Jun Aug Oct
Ascomastylis elata + + + Polygonatum verticillatum - + +
Anaphalis cuneifolia - + + Polygonum vaccinifolium - + -
Anaphalis royleana + + + Potentella atrosanguinea + - -
Anemone obustiloba + - - Potentella microphylla + + +
Anemone rivularis + - - Potentella polyphylla + + +
Bistorta amplexicaulis + + + Potentilla leuconata + - -
Bistorta macrophylla + + + Polygonatum vaccinifolium _ + +
Bupleurum longicaule - + + Primula denticula + + +
Carex inanis + + + Rananculus hirtulus + - -
Clematis montana + + + Rhododendron anthopogon + + +
Cyananthus lobatus - + - Rhododendron campanulatum + + +
Danthonia cachemyriana + + + Rubus niveus + + +
Euphraisa himailica - + - Salix lindleyana - + -
Gaultheria trichophylla + + + Salvia hians - + -
Gentiana pedicillata + - - Saussurea taraxacifolia + + +
Geranium wallichianum + + + Senecio graciliflorus - + -
Goodyera fusca - + - Sibbaldia cuneata + + +
Goodyera repens + + + Smilacina purpurea + + -
Jurinea macrocephala - + + Stachys melissifolia - + -
Myriactis javanica - + - Swertia ciliate - + +
Oxygraphis polypetalla + + + Swertia speciosa + + +
Parnassia laxmannii - + - Tanacetum longifolium + + +
Parnassia nubicola - + + Taraxacum officinale - + +
Parochaetus communis + + + Trachydium roylei + + +
Pedicularis pectinata + + - Viola biflora + + -
Plantago brachyphylla + + + Viola canescens + + -

8
8
7
7
Number of species

6
5
5
4
4
3 3
3
2 2 2 2 2 2
2
1 1 1 1 1 1 1 1
1

Families

Figure 3. Number of species in different plant families of the study area.


Species richness and diversity patterns
The number of species (SR) and diversity varied during different seasons of the year (Table 3). A total of 52
plant species were reported in the study area during different visits. Maximum species (46) were reported in the
month of August, followed by 34 in June and 32 in October (Table 2). In the month of June maximum number
of species (21) were reported from TUN 2a (ecotone between tree line and alpine zone, 3455 m asl) while
minimum species richness (18) was reported from TUN 1a (sub-alpine zone, 3236 m asl). During the month of
August, the SR varied from 21 (TUN 2a, 3455 m asl) to 24 (TUN 2, 3555 m asl), while during the month of
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October, SR varied from 12 (TUN 1a, 3236 m asl) to 21 (TUN 1, 3330 m asl). During all these seasons, the SR
did not follow any definite trend along the altitudinal gradient. SR first increased from lower altitude (sub
alpine) to mid altitude and then decreased (Fig. 4).
Table 3. Variation of ecological attributes in the study area during different seasons
June August October
Site Altitude H *
J SID -Div H J SID -Div H J SID -Div
TUN 1a 3236 m 1.09 0.37 0.27 0.59 2.63 0.85 0.65 0.67 1.23 0.49 0.38 0.66
TUN 1 3330 m 2.80 0.90 0.89 0.73 2.51 0.77 0.87 0.74 2.55 0.83 0.86 0.72
TUN 2a 3453 m 2.02 0.66 0.69 0.73 2.09 0.67 0.78 0.88 1.96 0.69 0.61 0.86
TUN 2 3475 m 1.77 0.59 0.67 0.80 1.89 0.59 0.62 0.66 1.45 0.50 0.74 0.77
Average 1.92 0.63 0.63 0.71 2.28 0.72 0.73 0.74 1.79 0.62 0.64 0.75
Note: *J= Species evenness, SDI= Simpsons Diversity Index, -Div= Beta Diversity.
Table 4. Diversity and dominance of plant species in TUN 1a during different seasons.
June, 2014 August, 2014 October, 2014
Species
*H *SI H SI H SI
Anemone rivularis -0.023 0.000 -0.076 0.0003 - -
Bistorta amplexicaulis -0.048 0.0002 -0.171 0.006 -0.105 0.002
Bistorta macrophylla - - -0.125 0.001 - -
Clematis montana -0.111 0.001 -0.075 0.0008 - -
Danthonia cahemyriana -0.060 0.0002 -0.088 0.001 -0.110 0.005
Geranium wallichianum -0.051 0.0001 -0.117 0.002 -0.146 0.002
Goodyera repens - - -0.074 0.0003 -0.036 0.0001
Myriactis javanica - - -0.185 0.004 - -
Parochaetus communis -0.025 0.000 -0.056 0.0002 - -
Polygonum vaccinifolium -0.032 0.0001 -0.087 0.0016 - -
Potentella atrosanguinea -0.079 0.0004 -0.274 0.019 -0.097 0.0007
Potentella polyphylla -0.050 0.0001 -0.127 0.005 -0.157 0.021
Potentilla leuconata -0.028 0.000 -0.088 0.0006 -0.042 0.000
Primula denticulata -0.025 0.000 -0.088 0.001 -0.052 0.0003
R. campanulatum -0.137 0.720 -0.253 0.311 -0.179 0.596
Rubus niveus -0.064 0.0005 -0.132 0.003 -0.111 0.001
Salvia hians - - - - -0.036 0.000
Senecio graciliflorus - - -0.160 0.003 - -
Smilacina purpurea -0.085 0.0005 -0.044 0.0002 - -
Stachys melissifolia - - -0.031 0.000 - -
Swertia speciosa -0.158 0.011 -0.190 0.018 -0.158 0.005
Viola biflora -0.021 0.000 -0.086 0.003 - -
Viola canescens -0.045 0.0001 -0.097 0.0013 - -
Note: *H= Shannon Wieners Diversity, SI= Simpsons Index.
30

25
24 24
22
Species Richness

21
21
20 20
20
18 18
17
15

12
10

0
3236 3330 3453 3475
Altitudinal Gradient
June August October

Figure 4. SR variation along the altitudinal gradient during different seasons.

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Table 5. Diversity and dominance of plant species in TUN 1 during different seasons.
June, 2014 August, 2014 October, 2014
Plant species
H SI* H SI H SI
Acomastylis elata -0.223 0.010 -0.214 0.008 -0.208 0.008
Anaphalis cuneifolia - - -0.011 0.000 -0.026 0.000
Anaphalis royleana -0.084 0.0006 -0.066 0.0004 -0.087 0.0008
Anemone obustiloba -0.002 0.000 - - - -
Anemone rivularis -0.017 0.000 - - - -
Bistorta macrophylla -0.241 0.012 -0.077 0.0005 -0.019 0.000
Bupleurum longicaule - - -0.013 0.000 -0.012 0.000
Carex inanis -0.133 0.002 -0.134 0.002 -0.151 0.002
Danthonia cachemyriana -0.176 0.004 -0.193 0.006 -0.185 0.005
Gaultheria trichophylla -0.197 0.007 -0.189 0.005 -0.213 0.01
Gentiana pedicillata -0.0002 0.000 - - - -
Geranium wallichianum - - -0.006 0.000 - -
Goodyera repens - - -0.006 0.000 -0.016 0.000
Oxygraphis polypetalla -0.142 0.002 -0.237 0.014 -0.203 0.008
Parnassia nubicola - - -0.004 0.000 - -
Pedicularis pectinata -0.07 0.0006 -0.018 0.000 - -
Plantago brachyphylla -0.243 0.013 -0.285 0.026 -0.238 0.012
Polygonum vaccinifolium -0.017 0.000 -0.011 0.000 -0.07 0.0006
Potentella polyphylla -0.09 0.001 - - -0.105 0.001
Potentilla atrosanguinea -0.08 0.001 -0.016 0.000 -0.031 0.000
Potentilla microphylla - - -0.007 0.000 - -
Potentilla polyphylla - - -0.111 0.0015 - -
Primula denticulata -0.017 0.000 -0.017 0.000 -0.036 0.000
Rhododendron campanulatum -0.289 0.024 -0.312 0.034 -0.258 0.015
Saussurea taraxacifolia -0.058 0.0004 -0.013 0.000 -0.022 0.000
Sibbaldia cuneata -0.239 0.014 -0.258 0.023 -0.232 0.013
Swertia ciliata - - -0.0002 0.000 -0.008 0.000
Taraxacum officinale -0.165 0.003 - - -0.095 0.0007
Trachydium roylei -0.273 0.029 -0.298 0.033 -0.329 0.073
Viola biflora -0.032 0.000 -0.012 0.000 - -
Note: *H= Shannon Wieners Diversity, SI= Simpsons Index.
TUN 1a (Sub-alpine Zone): SR ranged from 12 (October) to 22 (August). Shannon-Wieners diversity index
(H) varied from 1.09 (June) to 2.63 (August). Species evenness ranged from 0.37 (June) to 0.85 (August).
Simpsons Index of Diversity (SID) varied from 0.27 (June) to 0.65 (August) (Fig. 4). -diversity varied from
0.59 in June to 0.67 in August (Table 3). Details of Shannon-Wieners diversity and Simpsons index of
individual species of TUN 1a in different seasons is given in table 4.
TUN 1 (Timberline-Alpine Ecotone): SR ranged from 20 (June) to 24 (August). Diversity index (H) varied
from 2.51 (August) to 2.80 (June) (Fig. 4). Species evenness ranged from 0.77 (August) to 0.90 (June). SID
varied from 0.86 (Oct) to 0.89 (June). -diversity varied from 0.72 in October to 0.74 in August (Table 3).
Details of Shannon-Wieners diversity and Simpsons index of individual species of TUN 1in different seasons
is given in table 5.
TUN 2a (Treeline-Alpine Zone Ecotone): SR ranged from 17 (Oct) to 21 (June and August). Diversity index
(H) varied from 1.96 (October) to 2.09 (August). Species evenness ranged from 0.66 (June) to 0.69 (Oct). SID
varied from 0.61 (Oct) to 0.78 (August). -diversity varied from 0.73 in June to 0.88 in August (Table 3).
Details of Shannon-Wieners diversity and Simpsons index of individual species of TUN 2a in different
seasons is given in table 6.
TUN 2 (Upper Alpine Zone): SR ranged from 18 (Oct) to 24 (August). Diversity index (H) varied from 1.45
(Oct) to 1.89 (August). Species evenness ranged from 0.50 (October) to 0.59 (June & August). SID varied from
0.62 (August) to 0.74 (October). -diversity varied from 0.66 in August to 0.80 in June (Table 3). Details of

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Shannon-Wieners diversity and Simpsons index of individual species of TUN 2 in different seasons is given in
table 7.
Table 6. Diversity and dominance of plant species in TUN 2a during different seasons.
June, 2014 August, 2014 October, 2014
Plant Species
H SI H SI H SI
Acomastylis elata -0.239 0.018 -0.213 0.009 -0.213 0.011
Anaphalis cuneifolia - - -0.011 0.000 - -
Anaphalis royleana -0.111 0.001 -0.094 0.0006 -0.095 0.001
Bistorta amplexicaulis -0.053 0.0001 - - - -
Bistorta macrophylla -0.201 0.008 -0.121 0.001 -0.046 0.000
Carex inanis -0.076 0.0006 -0.156 0.004 -0.157 0.003
Danthonia cachemyriana -0.127 0.0051 -0.093 0.001 -0.101 0.001
Euphraisa himailica - - -0.006 0.000 - -
Gaultheria trichophylla -0.013 0.000 -0.052 0.0002 -0.139 0.002
Gentiana pedicillata -0.037 0.0001 - - - -
Jurinea macrocephala - - -0.076 0.0003 - -
Oxygraphis polypetalla -0.138 0.002 -0.277 0.024 -0.224 0.023
Parnassia laxmannii - - -0.016 0.000 - -
Parnassia nubicola - - -0.011 0.000 - -
Pedicularis pectinata -0.084 0.0005 -0.032 0.000 -0.043 0.0002
Plantago brachyphylla -0.125 0.002 -0.149 0.003 -0.121 0.002
Potentilla atrosanguinea -0.011 0.000 -0.018 0.000 - -
Potentilla leuconata - - -0.013 0.000 -0.015 0.000
Potentilla microphylla -0.040 0.000 - - - -
Potentilla polyphylla -0.058 0.0004 -0.072 0.0003 -0.060 0.0002
Rananculus hirtulus -0.009 0.000 - - - -
Saussurea taraxacifolia -0.097 0.001 -0.033 0.000 -0.065 0.0003
Sibbaldia cuneata -0.09 0.0006 -0.093 0.0009 -0.143 0.002
Swertia ciliata - - - - -0.007 0.000
Tanacetum longifolium -0.149 0.003 -0.153 0.005 -0.114 0.001
Taraxacum officinale -0.020 0.000 - - -0.077 0.0008
Trachydium roylei -0.321 0.265 -0.363 0.169 -0.339 0.209
Viola biflora -0.014 0.000 -0.0329 0.000 - -
Note: *H= Shannon Wieners Diversity, SI= Simpsons Index.
A t-test showed significant differences in diversity (t=6.12-8.20, df= 32-46, p=0.000) at 95% confidence
level along the altitudinal gradient. Similar significant differences are shown during different seasons of the year
(t= 5.47-11.18, df=33-46, p=0.000).SR showed a non-significant positive correlation (r= 0.53) with altitude but
diversity showed a slightly negative correlation (r= -0.05) with it. This means the species evenness decreased or
concentration of dominance increased with increase in altitude.
Table 7. Diversity and dominance of plant species in TUN 2 during different seasons.
June, 2014 August, 2014 October, 2014
Plant Species
H SI H SI H SI
Acomastylis elata -0.2071 0.012 -0.233 0.000 -0.240 0.018
Anaphalis cuneifolia - - -0.029 0.000 -0.022 0.000
Anaphalis royleana -0.104 0.0026 -0.049 0.0001 -0.057 0.0002
Bistorta amplexicaulis - - -0.027 0.000 - -
Bistorta macrophylla -0.099 0.001 -0.040 0.0001 -0.026 0.000
Bupleurum longicaule - - -0.0009 0.000 - -
Carex inanis -0.098 0.0013 -0.090 0.0009 -0.087 0.001
Cyananthus lobatus - - -0.366 0.109 - -
Danthonia cachemyriana -0.114 0.002 -0.126 0.002 -0.146 0.004
Euphraisa himailica - - -0.003 0.000 - -
Gentiana pedicillata -0.015 0.000 - - - -
Goodyera fusca - - - - -0.002 0.000
Jurinea macrocephala - - -0.021 0.000 - -
Oxygraphis polypetalla -0.14 0.005 -0.089 0.0009 -0.106 0.002
Parnassia nubicola - - -0.0313 0.000 - -
Pedicularis pectinata -0.02 0.000 -0.010 0.000 -0.0072 0.000
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Plantago brachyphylla -0.021 0.000 - - - -
Potentella microphylla - - -0.026 0.000 - -
Potentella polyphylla -0.014 0.000 -0.008 0.000 -0.031 0.000
Potentilla leuconata -0.017 0.000 - - -0.019 0.000
Rananculus hirtulus -0.02 0.000 - - - -
R. anthopogon -0.261 0.202 -0.198 0.090 -0.199 0.115
Salix lindleyana -0.122 0.002 -0.015 0.000 - -
Saussurea taraxacifolia -0.027 0.000 -0.008 0.000 -0.029 0.000
Sibbaldia cuneata -0.034 0.000 -0.080 0.0004 -0.032 0.000
Stachys melissifolia - - -0.015 0.000 - -
Swertia ciliata - - - - -0.020 0.000
Tanacetum longifolium -0.061 0.0002 -0.099 0.0012 -0.065 0.0008
Taraxacum officinale -0.045 0.0003 - - -0.037 0.0001
Trachydium roylei -0.327 0.099 -0.304 0.244 -0.320 0.249
Viola biflora -0.014 0.000 -0.019 0.000 - -
Note: *H= Shannon Wieners Diversity, SI= Simpsons Index.
Similarity Index: The similarity of any two communities depends on the number of species common to both of
them. Sorensons similarity index showed that the similarity varied not only among the sites but also with the
seasons (Table 8). Highest similarity index (87.17%) was found between the two upper sites viz., TUN 2 and
TUN 2a during the month of June, while lowest value for the similarity index (15.38 %) was recorded between
TUN 1a and TUN 2 during the same month (Table 8).
Table 8. Sorensons similarity index (%) for the studied sites during different seasons of the year.
June 2014
TUN 1 TUN 1a TUN 2 TUN 2a
TUN 1 100 - - -
TUN 1a 29.26 100 - -
TUN 2 71.42 15.38 100 -
TUN 2a 76.19 20.51 87.17 100
August 2014
TUN 1 TUN 1a TUN 2 TUN 2a
TUN 1 100 - - -
TUN 1a 33.33 100 - -
TUN 2 61.22 17.78 100 -
TUN 2a 72.34 69.77 86.36 100
October 2014
TUN 1 TUN 1a TUN 2 TUN 2a
TUN 1 100 - - -
TUN 1a 30.3 100 - -
TUN 2 61.53 20 100 -
TUN 2a 63.15 20.68 85.71 100

DISCUSSION
The alpine regions are particularly appropriate for a large-scale network to determine the effects of global
processes such as climate change (Pauli et al. 2004). The main purpose of the recently launched research
initiative, HIMADRI, is the long term monitoring of the ecologically sensitive parameters at benchmark sites
selected in various regions of IHR. The aim of the present study was the characterization of one of the
HIMADRI benchmark sites (Tungnath, Uttarakhand Himalaya) and to provide a baseline data about the species
richness and diversity along the altitudinal gradient for long monitoring. The reinvestigation of these sites in
future (e.g. after a decade or more) will help in understanding the effect of climate change in IHR in terms of
changes in species composition and diversity or in terms of species shifts from one vegetation zone or/and
ecotone to another. The average species diversity of 1.792.28 recorded in the present study is comparable to
the results of previous similar investigations in different Himalayan regions i.e. 2.394.63 in the Uttarakhand
Himalaya (Nautiyal & Gaur 1999), 1.44 to 2.48 from Sikkim Himalaya (Tambe & Rawat 2010), 1.022.17 in
Rama Valley Pakistan (Shaheen et al. 2011a) and 3.13 from Western Himalayan Alpine Pastures of Kashmir,
Pakistan (Shaheen et al. 2011 b). The Evenness (J) and Simpsons diversity index (SDI) varied from 0.62 to

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0.72 and 0.63 to 0.73, respectively. These values are comparable to some of the recent studies viz., Tambe &
Rawat 2010 (Sikkim Himalaya) and Shaheen et al.2011a (Pakistan Himalaya).
Maximum number of species was reported during the months of August and June because these are the
months of peak growth for most of the species of this region. Plant growth in alpine region starts with onset of
summer and snow melting. The growth activity increases with increase in temperature and moisture.
A gradual monotonic decrease in species richness with increasing altitude is considered a general pattern
(Stevens 1992). But in the present study, the SR did not follow this rule strictly. Neither, it followed any definite
trend along the altitudinal gradient. However, it showed a non-significant positive correlation (r= 0.53) with the
altitude. Rahbek (1995) made a critical review on species richness patterns in relation to altitude viewed that
approximately half of the studies detected a mild-peak in species richness. Grytnes & Vetaas (2002) have also
reviewed these aspects in Nepalese Himalaya. In the present study, SR first increased from lower altitude (sub
alpine) to mid altitude and then again decreased (Fig. 5). Similar types of results have been reported by
Pickering et al. (2008) and Shaheen et al. (2011a) from Australia and Pakistan respectively. Minimum SR was
reported from lowest site (TUN 1a, 3236 m asl) that forms the sub-alpine vegetation zone of the study area. This
region is very dense and dominated by Rhododendron campanulatum and Abies pindrow. Due to this dense
vegetation, ground flora is poorly represented. But beyond the tree line, the SR first increased in timberline-
alpine ecotone and then again decreased in treeline-alpine ecotone (Fig. 5). Alpine communities lack the
ultra-dominant tree cover which allows the herbs and grasses to flourish freely. Changes in altitude play an
important role in the composition of plant communities. Altitude itself represents a complex combination of
related climatic variables closely correlated with numerous other environmental properties viz., soil texture,
nutrients and substrate stability (Ramsay & Oxley 1997). Within one altitude the community composition is
affected by many co-factors like topography, aspect, slope and soil type. A negative correlation was observed
between diversity and altitude. Similar results were reported by Shaheen et al. (2011b) form alpine pastures of
Pakistan Himalaya. The similarity in species composition and community structure decreased with increasing
distance among sites, indicating high beta diversity in the area (Table 8).
Grazing activity is prevalent throughout the study area except for the sub alpine region. It is recommended
that grazing practices in these fragile alpine communities should be very limited and controlled. Himalayan
pastures have been a victim of severe human exploitation from centuries (Miller 1997). The degraded vegetation
is further not allowed to repair itself by harsh climatic conditions and very short growing period. It requires
immediate attention of monitoring authorities as well as to create awareness among locals about sustainable
utilization and conservation of alpine pastures to maintain ecosystem balance.

ACKNOWLEDGEMENT
The work has been funded by Space Application Centre (SAC), ISRO Ahmadabad which is greatly
acknowledged.

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ISSN (E): 2349 1183
ISSN (P): 2349 9265
3(2): 408412, 2016

Review article

A concise review on Nerium oleander L. -


An important medicinal plant
Sankar Narayan Sinha* and Karabi Biswas
Microbiology Section, Department of Botany, University of Kalyani
*Corresponding Author: sinhasn62@yahoo.co.in [Accepted: 28 July 2016]

Abstract: Nerium oleander, commonly known as oleander, is an important medicinal plant in


Indian folk medicine. It is one of the best pharmacognostic devices available in now-a-days. The
modern as well as traditional uses make this plant much more valuable. Oleander is cultivated
recently in pots and hence, large scale propagation of plant material for commercial use has great
importance. This plant species also produces secondary metabolites such as alkaloids, flavonoids
and steroids which have pharmacological applications. The important pharmacological activities
are antibacterial, larvicidal, anticancer, antidiabetic activities. This review describes the evidence-
based information regarding pharmacological activity as well as phytochemicals of this plant.
Keywords: Medicinal plant - Secondary metabolites - Antibacterial activity - Phytochemicals.

[Cite as: Sinha SN & Biswas K (2016) A concise review on Nerium oleander L. - An important medicinal plant.
Tropical Plant Research 3(2): 408412]

INTRODUCTION
Nerium oleander L. is a small evergreen tree with 25 m in height and distributed in different geographical
and ecological places (Fig. 1). In Bengali the common name is Karabi. This plant is originated in the
Mediterranean region and Indo-Pakistan subcontinent (Patel 2010). Nerium oleander is a drought-tolerant plant
and belongs to the Family Apocynaceae. The leaves are 5 to 20 cm long, acuminate or acute, shortly petiolate,
narrow, with a coriaceus dark-green blade. Flowers are produced in terminal cluster about 5 cm in diameter with
five petals and different colours vary from lilac, salmon, carmine, deep to pale pink, purple, copper, apricot,
orange, white and yellow. The fruit consists of a narrow follicle of 7.5 to 17.5 cm long and opens to disperse
fluffy seeds. This plant can be propagated by seed (Pagen 1988) and shows great variability in seedling
populations.
Nerium oleander is widely grown as an ornamental plant in tropical, subtropical and temperate regions due
to its profuse flowering which are long lasting along with their moderate hardiness (Kingsbury 1964, Hardin &
Arena 1974). It is used for screens, hedging along highways, planting along beaches. It is able to form attractive
small trees by leaving just a few stems. In Northern regions it may be grown as an indoor or patio plant. Beside
these all, the plant showed antibacterial (Mostaqul et al. 1999, Derwic et al. 2010), antimicrobial (Hussain et al.
2004), anti-inflammatory, antinociceptive (Erdemoglu et al. 2003) and antitumor (Ali et al. 2010) activity.
Systematic position of this plant
Kingdom: Plantae
Division: Angiosperms
Class: Magnoliopsida
Subclass: Asteridae
Order: Gentianales
Family: Apocynaceae
Genus: Nerium L.
Species: oleander L.

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Received: 04 May 2016 Published online: 31 August 2016
Sinha & Biswas (2016) 3(2): 408412
.

Figure 1. Nerium oleander L. plant in flowering condition.

ETHNOMEDICINAL VALUE
Nerium oleander L. has been reported in ancient texts and folklore for many years. Literature survey shows
that most of its plant parts such as flowers, leaf juice, bark and latex leaves are used for the treatment of
microbial and fungal diseases. All parts of the plant are also used as therapeutic agent and have been used in
folklore to treat variety of ailments. The leaves and bark are used as expectorant, heart tonic, diuretic, emetic
and diaphoretic (Patel et al. 2010). Roots were boiled in water and helpful in skin complaints, herpes and also
ringworm infection. Very small doses of leave juice are used in snake and other venomous bites. Juice of young
leaves is effective in the cure of eye diseases. Root paste is administered in ulceration, haemorrhoids, various
types of cancer and leprosy (Ahmed et al. 2006, Sikkarwar et al. 2009, Vinayagam & Sudha 2011, Chauhan et
al. 2013). Oil obtained from the root is used in the treatment of leprosy and skin diseases (Saini 2010). For the
treatment of scabies and to reduce swellings, decoction of the leaves has been applied externally. The leaves and
the flowers are emetic, diaphoretic, cardiotonic, diuretic, expectorant and sternutatory (Asha & Chakraborthy
2010). N. oleander is used in the treatment of ulcers and also causes abortion (Hseini & Kahouadji 2007). In
some areas of Morocco, Africa the leaves are used in maceration and in external friction tale scabies, hair loss,
lice, diabetes and toothache (Lahsissene et al. 2009).
BIOLOGICAL ACTIVITIES
Antimicrobial activity
The roots of Nerium oleander possessed a new cardenolide, 12- hydroxy- 5- carda-8, 14, 16, 20 (22)
tetraenolide which showed antibacterial and digoxin-like cardiac activities (Huq et al. 1999). Hussain & Gorsi
(2011) showed that the ethanolic extract of the plant root and leaves was very effective against bacteria and
fungi. Tannu et al. (2011) noted the anti-microbial activity of stem extracts of Nerium oleander on wistar strain
albino rats against Pseudomonas aeruginosa and Bacillus subtilis. N. oleander showed effective against
Staphylococcus aureus as observed by Wong et al. (2013). According to Jude (2013) Nerium oleander, showed
the highest inhibitory zone against Klebsiella pneumoniae Proteus vulgaris, Salmonella typhi and Escherichia

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coli. As per the observation of Jeyachandran et al. (2010) the methanolic extract of Nerium oleander showed
maximum zone of inhibition (28 mm) against S. typhi. Swai et al. (2010) showed that B. subtilis was found to be
more sensitive than Gram negative bacteria. The ethanolic extract of Nerium oleander leaves showed highest
bactericidal activity at 900 mg.ml-1 concentration against Pseudomonas aeruginosa (Malik et al. 2015). The
ethanolic flower extracts of N. oleander showed anti-fungal activity in vitro against four important plant
pathogenic fungi viz.; Fusarium oxysporum, Alternaria alternata, Fusarium solani and Rizoctonia solani using
agar dilution bioassay. Oleander exhibited the best inhibition against F. oxysporum and F. solani (Hadizadeh et
al. 2009). Essential oil is obtained from the flowers of N. oleander and showed in vitro antibacterial activity
against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. It also showed a very effective
bactericidal activity with minimum inhibitory concentrations (MIC) ranging from 1.45 to 5.10 mg.ml-1 (Derwich
et al. 2010).
Larvicidal activity
The bark, stem, leaves, flowers and roots of Nerium oleander possess insecticidal and anti-feedant property
against Plutella xylostella (Gupta & Thorsteinson 1966, Jacobson 1975, Grainge et al. 1984). This plant has
been also reported for larvicidal activity against Aedes aegypti by Komalamisra et al. (2005). Insect growth
regulatory activity against Anopheles stephensi and Culex quinquefasciatus of this plant also recorded
(Pushpalatha et al. 1995). The aqueous leaf extract of Nerium oleander were exhibited ovicidal and larvicidal
properties (Kumar et al. 2012) and for ovicidal and adulticidal activity of this plant against Anopheles stephensi
was also recorded (Roni et al. 2013). Culex quinquefasciatus larval mortality was tested against Nerium
oleander crude hexane and aqueous flower extracts by Raveen et al. (2014). It was revealed that flower extract
with hexane possessed high larvicidal activity when compared to aqueous extract with LC50 values of 102.54
and 61.11 ppm after 24 and 48 hours.
Antidiabetic activity
Shikkarwar et al. (2009) studied antidiabetic activity of this plant. Neither exact biologically active
components responsible for anti-diabetic activity have not been reported nor, the exact mode of action was
reported previously. They found that single dose of ethanolic extract (300 mg.kg-1 bw) of Nerium indicum has
more significantly (P<0.01) reduced the blood glucose level as compared to seventh day of their study. The
chloroform extract (500 mg.kg-1 bw) showed significant reduction of blood glucose after one hour while the
ethanolic extract showed significant reduction after three hours. However, the water extract of this plant did not
able to reduce glucose level at sub-acute level. The effects of various solvent extracts on glucose tolerance level
in normal rats were done. After 30 minutes glucose administration, the blood glucose level peak increased
rapidly from the fasting value and then subsequently decreased. Glibenclamide treated group prevented glucose
induced hyperglycemia significantly at 30 min and 90 min (171.834.214 and 106.164.316) as compared to
control (167.832.301 and 146.832.960) respectively. Highest glucose tolerance in Nerium oleander extracts
was observed in chloroform extract (121.002.966) and lowest glucose tolerance was observed in aqueous
extract (155.33 3.018) in 90 minutes. The activity of Nerium oleander observed in chloroform extract showed
that the diabetic rats had lower body weights, high blood glucose level as compared to normal rats. Beside this
orally administered Nerium oleander chloroform extract and ethanolic extract significantly increased the body
weight and decreased blood glucose level in diabetic rats (Sikkarwar et al. 2009).
Anticancer activity
Ali and co-workers (2009) was able to extract essential oil from the flowers of the oleander. It showed
antitumor activity on the cell lines, Ehrlich Ascites Carcinoma (EAC). Pathak et al. (2000) used different
amount of Anvirzel (1.0 ng.ml-1 to 500 microgram.ml-1) or Oleandrin (0.01 ng.ml-1 to 50 microgram.ml-1) in
both continuously treated and pulse-treated/recovery Cell cultures. Both Oleandrin and Anvirzel were able to
induce cell killing in human cancer cells, but not in murine cancer cells.

PHYTOCHEMICALS
High quantity of polyphenols is present in the leaves of Nerium oleander as revealed by Siham et al. (2014).
Thus, different fractions of the phenolic compound were shown by HPLC analysis. The cinnamic acid was the
major component and other components were epicatechine, catechin and chlorogenic acid. The aqueous extract
of leaves of Nerium oleander yielded 2.3% of crude polysaccharide. Major of the fractions was pectic

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polysaccharide which is composed of arabinose, galacturonic acid, galactose, and rhamnose. Two new coumaryl
oxy-triterpenoids, neriucoumaric and isoneriucoumaric acids have been isolated from fresh leaves of Nerium
oleander. The preliminary phytochemical screening showed that the leaves of this plant possess carbohydrates,
flavonoids, alkaloids, steroids, cardiac glycosides and tannins (Yadav et al. 2013). Siddiqui et al. (2012)
reported oleanderocinoic acid, a pentacyclic triterpene, flavonoid glycosides,quercetin-5-O-[-L-
rhamnopyranosyl-(16)]--Dglucopyranoside and kaempferol-5-O-[-L-rhamnopyranosyl- (16)]--D-
glucopyranoside and a cardenolide, oleandigoside found in the leaves of Nerium oleander this compounds
showed The growth inhibitory and cytotoxic acrivites against MCF-7, human breast cancer cell lines using
sulforhodamine B assay. Two new compounds heptacosane-3-enyl-5- hydroxyhexanoate and 4-oxooctyl-2-
hydroxyundecanoate were isolated by Sharma et al. (2012) from the stems of Nerium oleander. Polysaccharide
fraction was isolated from the hot water extract of flowers of this plant using ethanol precipitation,
cetyltrimethylammoniumbromide (CTAB) complexing, gel permeation chromatography and anion exchange
chromatography. It has been found to contain L-rhamnose, L-galactose and D-galacturonic acid (Qun et al.
2010). Siddiqui et al. (1995) reported two novel cytotoxic pentacyclic triterpenoids ciskarenin (3--
hydroxyphenoxy-28-Z-p-coumaroyloxy-urs-12-en- 27-oic acid) and trans-karenin(3--hydroxy-28-E-
pcoumaroyloxy- urs-12-en-27-oic acid) from N. oleander leaves. A new labdane diterpene, oleanderoic acid and
a new triterpene, oleanderen were isolated from the fresh, leaves of Nerium oleander (Siddiqui et al. 1987).
Siddiqui et al. (1986) had been isolated Kaneroside and neriumoside,type of cardiac from the fresh, undried,
winter leaves of Nerium oleander and their structures evaluated as 3-O-(D-diginosyl)- 2- hydroxy -8, 14-
epoxy-5- carda-16: 17, 20: 22- dienolide and 3- O- (D- diginosyl)- 2, 14- dihydroxy-5- carda - 16: 17, 20:
22- dienolide, respectively by spectral and chemical studies.

CONCLUSION
From the review of the existing work it was concluded that N. oleander has been used in the treatment of
skin diseases, cancer, diabetes, inflammation ,CNS depression and also any other microbial infection. Larvicidal
activity also found. Various bioactive compounds isolated from different parts of this plants. So it is an utmost
of importance to explore its potential in the field of medicinal and pharmaceutical sciences for novel application.
As N .oleander is a popular remedy among the various ethnic groups, this plant is used in Ayurvedic and
traditional medicine. So further or more work is needed to investigate the therapeutic potential of this plant.

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ISSN (E): 2349 1183
ISSN (P): 2349 9265
3(2): 413427, 2016

Research article

Ethno-botanical survey of plant species used in traditional


medicine in Kinshasa city (Democratic Republic of the Congo)
Koto-te-Nyiwa Ngbolua1, Shetonde O. Mihigo2, Clment Inkoto Liyongo1, Masengo C.
Ashande3, Damien S.T. Tshibangu2, Ben Gbolo Zoawe1, Robijaona Baholy2,
Pierre Ruphin Fatiany4,5 and Pius T. Mpiana2,*
1
Dpartement de Biologie, Facult des Sciences, Universit de Kinshasa, B.P. 190, Kinshasa XI, RD Congo
2
Dpartement de Chimie, Facult des Sciences, Universit de Kinshasa, B.P. 190, Kinshasa XI, RD Congo
3
Comit scientifique pour la recherche, le dveloppement et la conservation de la biodiversit (CSB), Facult
des Sciences, Universit de Kinshasa, B.P. 190, Kinshasa XI, RD Congo
4
Dpartement de Gnie chimique, Ecole suprieure Polytechnique, B.P. 1500, Universit dAntananarivo, 101
Antananarivo, Madagascar
5
Institut Malgache de Recherches Appliques, Avarabohitra Itaosy lot AVB 77, B.P. 3833, 102 Antananarivo,
Madagascar
*Corresponding Author: ptmpiana@yahoo.fr [Accepted: 11 August 2016]

Abstract: An ethno-botanical survey was conducted among traditional healers and medicinal plant
vendors in Kinshasa city (DR Congo) in order to identify plant species used in traditional medicine
to treat common diseases, with the aim of documenting, preserving, and sustaining this valuable
traditional knowledge. Surveys were conducted from February to April 2014 among 50 medicinal
plant vendors, in five markets (Limete, Makala, Matete, Mont-Ngafula, and Ngaba). The education
level of the majority of informants was secondary school. The age of the informants ranged
between 20 and 68 years. Cited plant species were collected and identified at the herbarium of
the Faculty of Science, University of Kinshasa. Their ecological status was also determined. The
50 informants used 32 plant species (belonging to 22 families and 30 genera) in traditional
medicine in Kinshasa. Their herbal remedies were administered as aqueous decoctions against 38
different diseases. It was found that ligneous, savanna, phanerophyte, and pantropical-type plant
species were predominant both in numbers of species as well as in citations. Roots were the most
used plant part, and malaria and haemorrhoids were the most treated diseases.
Keywords: Traditional healers - Medicinal plant vendors - Sustainable management.

[Cite as: Ngbolua KN, Mihigo SO, Liyongo CI, Ashande MC, Tshibangu DST, Zoawe BG, Baholy R, Fatiany
PR & Mpiana PT (2016) Ethno-botanical survey of plant species used in traditional medicine in Kinshasa city
(Democratic Republic of the Congo). Tropical Plant Research 3(2): 413427]

INTRODUCTION
In Africa, over 80% of the population relies on medicinal plants for their primary healthcare (WHO 2002,
Ngbolua et al. 2011a,b, Fatiany et al. 2013, Fatiany et al. 2014a). There is limited access to modern medicine
and the facilities are poor (Kabena et al. 2015, Ngbolua et al., 2014b,c,d). The Democratic Republic of the
Congo (DRC) is one of the most plant diversity rich countries in Africa, and contains 47% plant diversity of the
African rain forests (Debroux et al. 2007). Although traditional medicines are used throughout the country and
across all cultures, only a small percentage of its flora has been subject to detailed phytochemical and
toxicological evaluations (Mihigo 2005, Mpiana et al. 2007, Ngbolua 2012).
Thus, and in order to document, preserve, and sustain this traditional and valuable knowledge; several
studies on the uses of traditional medicines in the country have been undertaken; in such provinces, tribes and
cities as Kisangani city (Wome 1985, Katemo et al. 2012), Bobangi city (Ilumbe 2006) and Bikoro city (Ilumbe
2010). Likewise, some studies have also been conducted in Kinshasa city on plant species used to treat
infectious and pathologies (like malaria and amibiasis) and for intimate female hygiene in selected markets
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Received: 20 May 2016 Published online: 31 August 2016
Ngbolua et al. (2016) 3(2): 413427
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(Ngbolua et al. 2014d, Kabena et al. 2015). In addition, recent rural population displacement due to war has
caused important changes in the demographic profile of Kinshasa city (by rural migration, the number of
Kinshasa inhabitant passed from 8 million to about 12 million people these fifteen last years), making it a
necessity to update the knowledge and uses of medicinal plants in this city by surveying markets no previously
included. It should be noted that in Kinshasa city as in other central Africa capitals, people occupy the
municipalities according to their ethnic origin. Thus, the present study would make it possible to identify plant
species not previously included in the former studies or to know the new use of the existing plant species.
Indeed, a same plant can be used for different uses in different parts of the country by others ethnic groups
(Ngbolua et al. 2011a, b).
The main objective of the present study was to make an updated inventory of plant species used in traditional
medicine in Kinshasa city, particularly in Limete, Makala, Matete, Mont-Ngafula, and Ngaba municipalities.
The specific objectives consisted of a botanical survey and identification of medicinal plants, their ecological
study, and a preliminary phytochemical screening of those plant species for which a higher informant consensus
factor was obtained.

MATERIALS AND METHODS


Study Area
Geographic location: Kinshasa is located between 418' and 425' S latitude, and between 1515' and 1522' E
longitude. The citys boundaries are the Bandundu province that covers the North and East parts, the Bas-Congo
province in South, and the Republic of Congo in the West. Its average altitude is 360 m above sea level.
Soils: According to Sys (1961), Kinshasa soils belong to the following classification. Order: Kaolisoils, Sub-
order: Hydro-xerokaolisoils, and the group of Arenoferrals.
Hydrography: Kinshasa is located along the Congo River, and its hydrology network comprises local and
allogenic rivers of which the most important are Ndjili, Nsele, and Mai-Ndombe.
Climate: In Kinshasa, the climate is warm, tropical humid, of the Aw4-type following Kppen classification
(Bultot 1954). Two seasons alternate in Kinshasa, the dry season (June to September) and the rainyseason
(October to May).
Vegetation: The original vegetation in Kinshasa consisted of forests, savannas, and the aquatic and semi-aquatic
valleys of the Congo River and Pool Malebo. Studies on the African vegetation by White (1979) and Troupin
(1966) have successively placed the DR Congo in the following sub-divisions: Guineo-Congolese region,
Congo-basin domain and the Congo-Zambezian transition Sector.
Ethnography: Kinshasa city is a town with a heterogeneous population; inhabitants originate from many
different ethnic groups. The 450 tribes present can be grouped into four predominant linguistic groups: Lingala,
Tshiluba, Swahili and Kikongo. However, the indigenous population of this city consists of Teke and Humbu
(Mokengo 2011).
Ethno-Botanical survey
Ethno-botanical information about the plant species reported in this study was obtained by interviewing
traditional healers and medicinal plant vendors in Kinshasa city (Bajpai et al. 2016). Surveys were conducted
from February to April 2014, in five markets (viz. Limete, Makala, Matete, Mont-Ngafula, and Ngaba). A total
of 50 traditional healers and medicinal plant vendors were interviewed,on a voluntary basis. Local language
Lingala was used during anthropological interviews.
The study followed principles laid out in the Declaration of Helsinki (Ngbolua et al. 2013, Ngbolua et al.
2014d, Fatiany et al. 2014b, Fatiany et al. 2015). The research was performed according to the principles laid
out in the Nagoya protocol (Coomb 2005, Buck 2011, Soares 2011). Required permission was obtained from the
Ministry of Environment of the DRC in order to collect plant samples and conduct non-commercial research on
Congolese medicinal plants.
The questionnaires were divided into three sections: (i) personal information (including name, age, sex, marital
status, studies level and profession) and reason of the recourse to the traditional medicine; (ii) vegetable material
(including plant vernacular and scientific names); (iii) traditional medicine practice (including knowledge of
diseases and symptoms, diagnosis of disease, plant used parts, state of the plant materials, modes of preparation
and conservation of recipes, route of the administration and dose of recipes, results of the treatments of diseases,
knowledge about toxic plants). Collected plants were identified using taxonomic keys and by comparison of

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voucher specimens with specimens deposited at the herbarium of the Institut National dEtudes et Recherches
Agronomiques (INERA), located at the Faculty of Science of the University of Kinshasa.

Figure 1. A, One of the authors (Clment Inkoto Liyongo) interviewing a medicinal plant vendor in a local market; B, Some
medicinal plant species in the local marked.
Data analyses
The reported medicinal plants species were classified on the basis of their ecological characteristics,
including morphological type, biological type, habitat type, and phytogeographic distribution. The following
parameters were used for additional data analyses: number of plant species, number of recipes, number of
citations, and number of informants, the informant consensus factor, usage value, confirmation indices and the
usage value agreement. The usage value agreement (UVAs) which is an index of evaluating the medicinal and
cultural value of plants was calculated using the following formula:

(1) [with (2) and (3)].
Where, UVs : usage value, Where, CIs : confirmation indices, Uis : number of usage of the
species s quoted by informant i, Ns : number of species, Ni : number of informants having
cited this species, Nt : total number of informants.
The usage value agreement is defined as the relative importance of each plant type known to be used as
herbal medicine. This index is useful in identifying plants with the highest use (most frequently mentioned) in
the treatment of a disease with a given informant consensus factor value (ICF) (Ilumbe et al. 2014). ICF
(ranging from 0 to 1) is used to deduce the homogeneity in the information on the use of a specific plant to treat
a certain disease. The informant consensus factor value was calculated using the following formula:
(4)
Where, Nc is the number of plant use reports (citation number) per each category (disease)
and Nps, number of plant species (taxa used).
A high value of ICF (close to 1) shows that a reduced number of plant species are quoted by a large number
of informants for a specific type of treatment; indicating the consistence of the use of this medicine (Alsarhan et
al. 2012).

RESULTS AND DISCUSSION


The 50 informants in the ethno-botanical survey used 32 plant species (belonging to 22 families and 30
genera) in traditional medicine in Kinshasa city/DRC.
Floristic Study
Medicinal plants reported during the course of this investigation were characterized for their morphological
type, biological type, habitat type and phytogeographic distribution (Fig. 25).
Morphological type
The morphological classification (Fig. 2) of medicinal plant species was carried out as previously reported
(Katemo et al. 2012, Ngbolua et al. 2013, Ngbolua et al. 2014d); they were classified as follows: trees, shrubs,
lianas, annual herbs, perennial herbs and under-shrubs.
The 32 reported medicinal plant species consist of mainly shrubs that account for 31% of the total. The next
important morphological type was made of perennial herbs (22%), followed by lianas and trees: 16% each,
under-shrubs (9%) and annual herbs (6%).
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Figure 2. Morphological types of the inventoried medicinal plant species.


The high number of shrubs in traditional medicine practices in Kinshasa can be justified by the fact that the
city is located in a savanna-type ecosystem. In addition, it has been argued that the increased use of savanna
plant species in traditional recipes may be due to them having a relatively higher content of potentially bioactive
metabolites (Bitsindou 1996).
Biological type
The biological types have been classified as follows: mesophanerophytes, microphanerophytes,
nanophanerophytes, lianescent phanerophytes, dressed therophytes, climbing herbs, bulbous geophytes or
climbing therophytes. Theinventoried plantspecies were found to belong to various biological types (Fig. 3),
including the microphanerophytes (28%), nanophanerophytes (22%), lianescentphanerophytes (16%),
mesophanerophytes (13%), climbing herbs (13%), dressed therophytes(3%), climbing therophytes (3%) and
bulbous geophytes (3%).
The predominance of phanerophytes among the reported plantspecies is a characteristic of tropical regions
and may also correlate to the ease with which their tissues have been claimed to synthesise bioactive secondary
metabolites (Betti et al. 2013, Ngbolua et al. 2014d). In addition, the perennial character of the reported species
supports their high availability and usage by the communities (Ilumbe et al. 2014).

Figure 3. Biological types of the inventoried medicinal plants. [McPh, microphanerophytes; Nph, nanophanerophytes; Lph,
lianescent phanerophytes; MsPh, mesophanerophytes; clH, climbing herbs; bG, bulbous geophytes; clTh, climbing
therophytes; dTh, dressed therophytes]
Habitat type
The biotopes of the inventoried plant species were classified as follows: forest, savanna (including wooded
grassland), ruderal habitat, farmland (including fallow land) and cultivated plants. About 47% of the reported
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plant species were found to be of forest type, while the cultivated plants-type, the savanna-type, the ruderal type
and the farmland type plants accounted for 31, 13, 6 and 3%, respectively (Fig. 4).

Figure 4. Biotope types of the inventoried medicinal plants.


Phytogeographic distribution
The phytogeographic distribution (Fig. 5) of species was recorded according to Central Africas
chorographic subdivisions (White 1979, Denys 1980, White 1983) as follows: Guinea-Congolese, Afro-tropical,
pan-tropical, Guinean, paleo-tropical and Afro-Malagasyplant species.

Figure 5. Phytogeographic distribution of the inventoried medicinal plants. [GC, Guinea-Congolese; At, Afro-tropical; Pan,
pan-tropical; Guin, Guinean; Pal, paleo-tropical; AMg, Afro-Malagasy plant species]
It was found that the reported medicinal plant species are widely distributed and belong to the following
distribution classes: pantropical (41%), Guinea-Congolese (37%), Afrotropical (9%), Paleo-tropical (6%),
Guinean and Afro-Malagasy: 3% each. The reported 32 medicinal plant species are presented in Appendix with
their scientific names, ecological characteristics, vernacular names, treated diseases, used plant parts, and the
dosage.

Plant species usage and other considerations


The number of plant species per treated disease, the number of citations, and the informant consensus factor
are given in table 1.
Table 1. Treated diseases and Informant consensus factors.
Number of Number of Informant
Treated diseases
plant species citations consensus factor
Anemia 1 1 0
Backache 7 9 0.25
Buruli ulcer 1 1 0
Cough 1 1 0
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Cyst 1 1 0
Demons 1 1 0
Diabetes 2 4 0.67
Diarrhorea 1 1 0
Fever 1 1 0
Fibroma/Fibroid 1 1 0
Gastritis 2 4 0.67
Gonorrhea 1 1 0
Guts 2 2 0
Haemorroids 10 12 0.81
Headache 2 4 0.67
Hepatitis 1 1 0
Hernia 2 2 0
High blood pressure 2 3 0.5
Infections 1 2 1
Kidney 1 2 1
Lack of appetite 1 1 0
Madness 1 1 0
Malaria 8 13 0.42
Myoma 2 2 0
Pain 1 1 0
Poison 1 1 0
Pruritus 1 1 0
Rheumatism 1 1 0
Sexualimpairment 2 3 0.5
Shock 1 1 0
Sinusitis 1 1 0
Skin diseases 1 1 0
Sore throat 1 2 1
Sterility 1 1 0
Tinea 2 2 0
Toothache 1 1 0
Typhoidfever 3 3 0
Yellow fever 3 3 0
Note: Informant consensus factor: 1 = 100% Consensus; 0.5-0.9 = High Consensus;
0.1-0.4: Weak Consensus; 0: Absence of consensus (Disagreement).
Out of 38 recorded diseases, malaria was the most cited as having been treated, and it was followed by
haemorrhoids and backache. These three diseases are treated using 33% of the reported/inventoried medicinal
plant species. Infection, sore throat and kidney complicationsare treated by all the informants (maximum
consensus). On the other hand, Annona senegalensis, Aframomum alboviolaceum and Mondia whitei, were cited
by every informant (high usage value agreement).This can indicate that these plants are probably the most
active. So, these three plant species were submitted to a preliminary phytochemical screening in order to have an
idea on their secondary metabolites profile. Qualitative analysis showed that the three plants species contain
saponins and leuco-anthocyanins but lack flavonoids. In addition, anthocyanins and quinones were absent in
Aframomum alboviolaceum and Annona senegalensis, respectively; but were present in Mondia whitei. These
secondary metabolites are known for their bioactivity. Indeed, saponins are well known for their haemolytic
inducing properties (Bruneton 1999). Anthocyanins were reported to possess antisickling activity while
quinones were reported to have antiplasmodial and cytotoxic activities in vitro (Fatiany et al. 2013, Mpiana et
al. 2008).
Diabetes, gastritis and headache also recorded a high (67%) consensus among the informants. Morinda
morindoides, Annona senegalensis, Garcinia kola, Aframomum alboviolaceum, Nicotiana tabacum, Mondia
whitei, and Sida rhombifolia were the reported plant species used in the treatment of these diseases. The
treatment of high blood pressure and sexual impairment recorded a moderate (50%) consensus; high blood
pressure was treated using Aframomum alboviolaceum and Sida rhombifolia, while sexual impairment was
treated by Oldenlandia herbacea and Heinsia crinita. Haemorroids, backache and malaria havent recorded a
maximum (100%) consensus between informants despite the fact that they were claimed to being treated with a
high number of medicinal plant species (10, 7 and 8 plant species respectively).
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Moreover, the recorded medicinal plant species were studied for their confirmation indices, the usage values
and the usage value agreements. Eighteen plant species were found to have higher ( 1.5) usage values, and
these are Morinda morindoides, Mondia whitei, Aframomum alboviolaceum, Alchornea cordifolia, Cogniauxia
podolaena, Crossopteryx febrifuga, Cymbopogon citratus, Nicotiana tabacum, Hyptis suaveolens, Capsicum
annuum, Harungana madagascariensis, Sarcocephalus latifolius, Gladiolus gregarius, Gongronema latifolium,
Annona senegalensis, Senna alata, Sida rhombifolia, Selaginella myosurus (Table 2). Indeed, il was reported
that medicinal plant species having an UV 1.5 has great cultural and medicinal values (Ilumbe et al. 2014).
Table 2. Reported plant species and their usage value agreements (UVAs).
N Plant species (Family) NR NI NC UV CI UVA
1 Aframomum alboviolaceum (Ridl.) K.Schum. (Zingiberaceae) 10 4 6 1.5 0.08 0.12
2 Aframomum melegueta (Roscoe) K.Schum. (Zingiberaceae) 5 3 2 0.67 0.06 0.04
3 Alchornea cordifolia (Schumach. &Thonn.) Mll. 2 1 2 2.0 0.02 0.02
Arg.(Euphorbiaceae)
4 Aloe buettneriA.Berger (Xanthorrhoeaceae) 1 1 1 1.0 0.02 0.02
5 Ananas comosus (L.) Merr. (Bromeliaceae) 1 1 1 1.0 0.02 0.02
6 Annona senegalensis Pers. (Annonaceae) 11 3 7 2.3 0.06 0.14
7 Capsicum annuum L.(Solanaceae) 2 1 2 2.0 0.02 0.02
8 Citrus limon (L.) Burm.f. (Rutaceae) 2 1 2 2.0 0.02 0.04
9 Cogniauxia podolaenaBaill. (Cucurbitaceae) 3 1 3 3.0 0.02 0.06
10 Crossopteryx febrifuga (Afzel.) Benth. (Rubiaceae) 2 1 2 2.0 0.02 0.04
11 Cymbopogon citrates (DC.) Stapf(Poaceae) 2 1 2 2.0 0.02 0.04
12 Erythrina abyssinica Lam. ex DC. (Leguminosae) 1 1 1 1.0 0.02 0.02
13 Garcinia kola Heckel (Clusiaceae) 6 3 3 1.0 0.06 0.06
14 Gladiolus gregariusWelw. ex Baker (Iridaceae) 2 1 2 2.0 0.02 0.04
15 Gongronema latifolium Benth. (Apocynaceae) 2 1 2 2.0 0.02 0.04
16 Harungana madagascariensis Lam. ex. Poir (Hypericaceae) 2 1 2 1.0 0.02 0.04
17 Heinsia crinita (Afzel.) G.Taylor (Rubiaceae) 1 1 1 1.0 0.02 0.02
18 Hyptis suaveolens (L.) Poit. (Lamiaceae) 3 1 3 3.0 0.02 0.06
19 Millettia eetveldeana (Micheli) Hauman (Leguminosae) 1 1 1 1.0 0.02 0.02
20 Mondia whitei (Hook.f.) Skeels (Apocynaceae) 9 4 7 0.8 0.08 0.14
21 Morinda morindoides (Baker) Milne-Redh. (Rubiaceae) 3 1 3 3.0 0.02 0.06
22 Nicotiana tabacum L.(Solanaceae) 2 1 2 2.0 0.02 0.04
23 Oldenlandia affinis (Roem. &Schult.) DC.(Rubiaceae) 1 1 1 1.0 0.02 0.02
24 Oryza sativa L. (Poaceae) 1 1 1 1.0 0.02 0.02
25 Persea americana Mill. (Lauraceae) 1 1 1 1.0 0.02 0.02
26 Piper nigrum L.(Piperaceae) 2 2 1 0.5 0.04 0.02
27 Quassia Africana (Baill.) Baill. (Simaroubaceae) 6 4 3 0.8 0.08 0.06
28 Sarcocephalus latifolius (Sm.) E.A. Bruce (Rubiaceae) 2 2 2 1.0 0.04 0.04
29 Securidaca longipedunculata Fresen. (Polygalaceae) 1 1 1 1.0 0.02 0.02
30 Selaginella myosurus (Sw.) Alston (Selaginellaceae) 2 1 2 2.0 0.02 0.04
31 Senna alata (L.) Roxb. (Leguminosae) 3 2 3 1.5 0.04 0.06
32 Sida rhombifoliaL. (Malvaceae) 4 1 4 4.0 0.02 0.08
Note: NR = Number of recipes, NI = Number of informants, NC = Number of citations, UV = Usage value, CI = Confirmation
indices, UVA = Usage value agreement.
Considering the confirmation indices, six plantspecies (Mondia whitei, Aframomum alboviolaceum, Quassia
africana, Aframomum melegueta, Garcinia kola and Annona senegalensis) seemed to be the most used, their
confirmation indices values being higher than 0.5. However, combining the usage values and the confirmation
indices, only three plant species (Aframomum alboviolaceum, Annona senegalensis and Mondia whitei) had the
highest usage value agreements (>0.1) and are the most used to treat/cure diseases.

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As for the informants, they were studied for their age, sex, marital status, profession, and education level. It
was found that they ranged between 2068 years (Fig. 6). Among the informants, females were the most
abundant (68%), while the majority (82%) of informants attended secondary school.

Figure 6. Age-based distribution of informants.


Informants having 3440 years of age were the most abundant; 38% of the informants were in this age class.
Other informants ages ranged between 4147 (16%), 4854 (14%), 5561 (12%), 2026 (10%), 6268 (6%)
and 2733 years (4%). It was also found that younger (2034 years: 14%) people used medicinal plants more
than the elders (6268 years: 6%). t-Student test confirmed that this difference was statistically different at a
probability threshold of 0.05 (p <0.05). Of the informants, 90% were married and 10% were single.
Regarding the professional activities of the informants, medicinal plant vendors (44%) and traditional
healers (44%) were the most common informants, followed by electricians (4%), house wives (4%), students
(2%), and aestheticians (4%) (Fig. 7).

Figure 7. Professional activities-based distribution of informants.


Moreover, 50% of the informants turned to traditional medicine exclusively, while 4% used the modern
medicine and 46% of them used both traditional and modern medicines. As for the composition of recipes, 64%
of the informants used a single plant while the remaining used a combination of at least two plant species for the
preparation of their recipes.

Figure 8. Used plant parts for the inventoried medicinal plant species.
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Various plant parts are used in recipes in traditional medicine practices in Kinshasa. Roots are the most used
plant part, accounting for 35%, followed by leaves (28%), fruits (10%), seeds (9%), barks (9%), stems (5%),
and bulbs (2%); while the whole plant was used in 2% of recipes (Fig. 8). It should be noted that any extensive
use of roots presents a serious threat to the sustainable preservation of biodiversity. The use of leaves may be
justified by the abundance of chemical groups they contain, and the fact that they have been claimed to be the
main synthesis site of secondary metabolites in plants (Dibong et al. 2011, Ngbolua et al. 2013).
Fifty-two percent of the informants use freshly collected plant materials while 16% use dried materials and
32% use both fresh and dried plant materials. For those informants who dry their plant material, 23% exposes
their samples to direct sun but over 75% of them dry their materials under shade.

Figure 9. Modes of preparation of recipes.


In addition, and as reported by several other investigators (Saoud et al. 2010, Katemo et al. 2012), water is
the used solvent; and decoction was found to be the preferred mode of preparation of recipes, accounting for
49%. This may be due to the fact that it has been reported that traditional healers believe that heat and steam
remove toxic substances from plant materials (Ngbolua 2012). Other modes of preparation of recipes include
maceration (23%), infusion (11%), mastication (11%) and instillation (6%) (Fig. 9).
Prior to and during the sales, the prepared recipes are packed through different means including bottles, bags
and other packaging tools. The conservation methods (some being also the packaging tools) were reported to be
shade (46%), sun light (4%), plastic bottles (34%), plastic bags (7%), glass bottles (5%), paper (3%) and other
unspecified means that account for about 1% (Fig. 10).

Figure 10. Modes of conservation of recipes.


The informants were also interviewed for their knowledge about the diagnosis of diseases and the results of
the treatments. They revealed that 74% of the diseases are diagnosed by the patients themselves, while medical
staff and patients family members were responsible for the diagnoses of 24% and 2%, respectively.
Concerning the results on the treatments of diseases (Fig. 11), the informants indicated that 44% of their
patients were healed, while 17% got improvement, 32% of the treatments failed and that side effects were
observed in about 7% of the cases.
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Figure 11. Results of the treatments of diseases.


Another important part of the investigation was the awareness of the existence of toxic plants among the
prescribers of traditional medicine. In this regard, 52% of the informants indicated that some of their plant
species were toxic and these consisted of Cogniauxia podolaena, Hyptis suaveolens and Quassia africana.
These plant species are being used in the management of such diseases as malaria, hernia, typhoid fever,
fibroma, myoma, cysts, haemorrhoids, rheumatism and shock. It is therefore important that people relying on
the above-mentioned plant species be made aware of the possible toxicity dangers associated with those plants.
Indeed, it was reported that the stem and root barks of Quassia africana were cytotoxic for MRC-5 cells with a
CC50< 10 g.ml-1 (Musuyu et al. 2012).

Similarity of plant species usage


The convergence of the remedies used and the medicinal practices in different countries is a very significant
criterion in ethno-pharmacology. The frequency of citations by both traditional healers and literature is an
indication of the probable biological activity of the plant. If a plant species is employed as remedy by local
communities in different countries, this may be considered as a strong indication that the biological activity
could be effective (Ngbolua et al. 2011b).
Some of the 32 plant species have been reported by other authors to treat various ailments (Neuwinger 2000,
Karou et al. 2011, Betti et al. 2013). For example, seven of the 32 plant species were repeatedly cited by the
traditional healers in other central African countries, while others (eight plant species) have been scientifically
validated as anti-parasitic or antisickle cell disease in previous studies (Mpiana et al. 2007, Betti et al. 2013).
In addition, compared to former work (Ngbolua 2015), 17 out of 32 plant species in the present study were
not quoted in our previous study. A possible reason for this could be that the recent rural migration following
the war in the RDC influenced ethno-medical knowledge of Kinshasa city, whose population currently amounts
to approximately 12 million inhabitants. Another reason could be the possibly that the informants were not the
same in the two studies. To this end, the great interest of the population in traditional medicine for their primary
health care in the context of the demographic explosion in Kinshasa city and the fact that the roots are the most
used parts, conducting to the destruction of the plants, can represent an environmental risk for the forest
resources surrounding the city, because of the increasing demand for medicinal plants. Thus, the creation of
community based agro-forest plantations could constitute the solution for a sustainable management of the
ecosystems surrounding the city.

CONCLUSION
The aim of this ethno-botanical study was to determine plant species used in traditional medicine in
Kinshasa city (DRC). Results from this study have shown that herbal remedies are widely used in the city and
administered as aqueous decoctions against as many as 38 different diseases. It was found that ligneous,
savanna, phanerophytes, and pantropical-type plant species predominate both in numbers of species as well as in
citations. The education level of the majority of traditional healers and medicinal plant vendors was secondary
school. Their age ranged between 20 and 68 years. Roots were the most used plant parts and malaria and
haemorrhoids were the most treated diseases.

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This report may be used as data base and information source for researchers who follow the ethno-
pharmacology approach for their investigation of natural sources for bioactive secondary metabolites. Within
this context, it is recommended that detailed phytochemical and pharmacological studies be performed on the
most interesting species determined by the present study as these could lead to the development of active
substances against the cited diseases.

ACKNOWLEDGMENTS
The authors thank the Acadmie de Recherche et d'Enseignement suprieur (ARES), Belgium for
Research grants PAH ARES/UNIKIN 2015 offered to Koto -te- Nyiwa Ngbolua, Pius T. Mpiana and Damien
S.T. Tshibangu. The authors are also indebted to The World Academy of Sciences (TWAS) (Grant No. 15-156
RG/CHE/AF/AC_GFR3240287018) for providing financial assistance.

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Appendix: Ethno-botanical and ecological data of reported plant species.
Plant species
Vernacular names Treated diseases Used plant parts Dosage
(Ecological characters and voucher no.)
Aframomum alboviolaceum (Ridl.)K.Schum. glass 2x/day (Maceration filtrate); 4
Tondolo High blood pressure, malaria, headache, myoma, gastritis, pruritis Leaves, fruits, roots
(Perennial herb; clH; GC; Savanna; Dumont 149) drops/day (instillation) (5 days)
Aframomum melegueta (Roscoe) K.Schum 1 fruit/day; 1 glass 2x/day (maceration)
Mondongo Cough, sore throat, backache Fruits
(Perennial herb; clH; AMg; Savanna; Denis 4) filtrate
Alchornea cordifolia (Schumach. & Thonn.) Mll. Arg.
Mbunzi Mbunzi Haemorroids, fever, Leaves, roots 2 glasses/day (5 days)
(Shrub; McPh; At; Forest; Devred 265)
Aloe buettneri A. Berger 3-4glasses/day Maceration filtrate (7
Badinseke Tinea Leaves
(Shrub; Nph; GC; Savanna; -) days)
Ananas comosus (L.) Merr.
Langa (Ananas) Lack of appetite Fruits 1 fruit/day
(Perennial herb; Nph; Pan; Cultivated; -)
Annona senegalensis Pers
Lolo Haemorroids, infections, malaria, diabetes Roots 2 glasses/day (7 days)
(Shrub; McPh; GC; Savanna; A. Lonard 5635)
Capsicum annuum L.
Tuenga Madness, toothache Leaves 2 drops 2x/day (5 days)
(Undershrub; Nph; Pan; Cultivated; Breyne 2161)
Citrus limon (L.) Burm.f.
Kpepke (Citron) Fever, cough Fruits, leaves 2 glasses/day; 4 fruits/day
(Shrub; McPh; Pan; Cultivated; -)
Cogniauxia podolaena Baill.
Kisakamba Fibroma,myoma, cyst Roots glass 2x/day (5 days)
(Liana; Lph; GC; Forest; Devred 144)
Crossopteryx febrifuga (Afzel.) Benth.
Mapela ya zamba Haemorroids Leaves, roots 2 pumps/day, 1x/week
(Shrub; McPh; At; Savanna; Donis 1577)
Cymbopogon citrates (DC.) Stapf
Sinda Malaria, pain Leaves Tea-like drink (5 days)
(Perennial herb; clH; Pan; Cultivated; -)
Erythrina abyssinica Lam. ex DC
Mukadi pembe Haepatitis, Roots Small portion
(Shrub; McPh; GC; Forest; Eleanor Phillips 4222)
Garcinia kola Heckel
Ngadiadia Malaria, guts, diabetes Seeds 2-5 grams/day (3 days)
(Tree; MsPh; GC; Forest; A. Lonard 1848)
Gladiolus gregarious Welw. ex Baker
Litungulu ya zamba Haemorroids, backache Bulbs 1 Pump 2x/day (3 days)
(Perennial herb; bG; Pan; Cultivated; Schmitz 6013)
Gongronema latifoliumBenth
Lolango Haemorroids, poison Roots 1 Pump/day (3 days)
(Liana; Lph; Pan; Forest; Compere 1162)
Harungana madagascariensis Lam. ex Poir
Ntunu Hernia, yellow fever Roots 2 glasses/day (7 days)
(Tree; McPh; GC; Forest; Davio 37)
Heinsia crinita (Afzel.) G.Taylor
Kitamata Sexual impairment Roots 2 glasses/day (14 days)
(Shrub; McPh; Gui; Forest; Breyne 3264)
Hyptis suaveolens (L.) Poit. glass 2x/day (Maceration filtrate) (5
Musunda Haemorroids, rheumatism, shock Roots
(Shrub; Nph; Pan; Cultivated; Breyne 3655 ) days)
Millettia eetveldeana (Micheli) Hauman
Mbuengi Haemorroids Roots 2 glasses/day (5 days)
(Tree; MsPh; GC; Forest; Devred 364)
Mondia whitei (Hook.f.) Skeels
Kimbiolongo Kidney, gastritis, backache, guts Stem, roots 1 glass/day (decoction) (3 days)
(Liana; Lph; GC; Forest; Compere 773)
Morinda morindoides (Baker) Milne-Redh.
Kongo bololo Malaria, diabetes, typhoid fever Leaves glass 2x/day
(Liana; Lph; GC; Forest; Breyne 3384)
Nicotiana tabacum L.
Bulu ( Fumu) Headache, sinusitis Leaves 2 bowls/day
(Annual herb; Nph; Pan; Cultivated; -)

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Oldenlandia affinis (Roem. & Schult.) DC.
Ankoro Sexual impairment Roots Small portion
(Annual herb; clTh; Pan; Ruderal; Diankenda 156)
Oryza sativa L.
(Loso) Riz Diarrhoea Seeds 1 glasses/day
(Perennial herb; dTh; Pan; cultivated; -)
Persea americana Mill.
Avocatier Anemia Leaves 1 cup/goblet, 2x/day
(Tree; MsPh; Pan; Cultivated; Devred 1366)
Piper nigrum L.
Ketshu Haemorroids Seeds, stem 2 glasses/day (3 days)
(Liana; Lph; Paleo; Forest; Breyne 830)
Quassia Africana (Baill.) Baill.
Mumpesepesi Malaria, hernia, typhoid fever Leaves, roots 2 glasses/day (5 days)
(Shrub; MsPh; GC; Forest; Dechamps 8080)
Sarcocephalus latifolius(Sm.) E.A. Bruce
Kilolo,Kiloloki Kwango Haemorroids, backache, diabetes, infections Roots 2 glasses/day
(Shrub; McPh; At; Savanna. Devred 389)
Securidaca longipedunculata Fresen.
Nsunda Exteralhaemorroids Roots 2 glasses/day
(Tree; McPh; Paleo; Forest; Collens 60)
Selaginella myosurus (Sw.) Alston
Tosangango Demons, Buruli ulcer Leaves, stem glass, 2x/day
(Perennial herb; clH; GC; Forest; -)
Senna alata(L.) Roxb.
Jokote Tinea, (gonorrhea) Leaves 2 drops ( glass), 2x day (7 days)
(Undershrub; Nph; Pan; Ruderal; Breyne 2418)
Sida rhombifoliaL.
Lukuliande Gastritis, high blood pressure, lack of appetite, backache Leaves 2 glasses/day
(Undershrub; Nph; Pan; Cultivated; Devred 531)

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ISSN (E): 2349 1183
ISSN (P): 2349 9265
3(2): 428433, 2016

Research article

Comparative phytochemical analysis of Cuscuta reflexa Roxb.


Parasite grown on north India by GC-MS
Dhanendra Kumar Rai1*, Vibhu Sharma1, Krishan Pal1 and Rajan Kumar Gupta2
1
Dept. of Biotechnology, Shri Venkateshwara University, Gajraula, Uttar Pradesh, India
2
Dr.P.D.B.H. Govt. P.G. College Kotdwar, Pauri Garhwal, Uttarakhand, India
*Corresponding Author: dhanendra010187@gmail.com [Accepted: 13 August 2016]

Abstract: The present study was aimed to determine the phytochemicals present in Cuscuta
reflexa parasite grown on two different areas. The comparative GC-MS analysis of extract of
Cuscuta reflexa the plant grown on North India was performed. The extract of plant sample was
dissolved in 75 ml of methanol for 24 hrs. Then the filtrates were collected and evaporated under
liquid nitrogen. The GC-MS analysis was carried out using a Clarus 500 Perkin-Elmer (Auto
system XL) Gas Chromatograph equipped and coupled to a Mass detector Turbo mass gold-
Perking Elmer Turbomas 5.1 spectrometer with an Elite-1 (100% Dimethy1 ply siloxane), 30 m x
0.25 mm ID x 1 m df capillary column. The instrument was set to an initial temperature of
110C, and maintained at this temperature for 2 min. At the end of this period, the oven
temperature was raised up to 280C, at the rate of an increase of 5C min-1, and maintained for 9
min. Injection port temperature was ensured as 250C and Helium flow rate as 1 ml min-1. The
ionization voltage was 70eV. The samples were injected in split mode as 10:1. Mass spectral scan
range was set at 25400 mhz. The chemical constituents were identified by GC-MS. The result of
the GC-MS analysis of C. reflexa shows the presence nitrogen (13.56%), aromatic compound
(7.88%), fluro (28.40%), alkaloid (7.64%), silica (5.66%), phosphorus (16.31%) and chlorine
compounds (6.26%). In general the isolated compounds are reported to possess antimicrobial,
antitumor, anticarcinogenic and anti-inflammatory properties. This comparative study confirms
that phytochemicals present in Cuscuta reflexa parasite depends on nature of plants.
Keywords: Cuscuta reflexa - Phytochemistry - Parasitic Plants - GC-MS analysis.

[Cite as: Rai DK, Sharma V, Pal K & Gupta RK (2016) Comparative phytochemical analysis of Cuscuta reflexa
Roxb. Parasite grown on north India by GC-MS. Tropical Plant Research 3(2): 428433]

INTRODUCTION
Cuscuta reflexa Roxb. (Convolvulaceae) is an extensive climber parasite. It occurs throughout the plains of
India. It is more often called dodder in English. Traditional healers called in Hindi Akash bel in Tamil
Akashavalli. Other names include hell weed, devil's gut, and beggar weed, strangle tare, scald weed, dodder of
thyme, greater dodder, and lesser dodder. In Chinese, Cuscuta seeds are called tu si zi. It has no chlorophyll and
cannot make its own food by photosynthesis. Some research studies say that the plant has very low levels of
chlorophyll and can slightly photosynthesis. But other species of Cuscuta are entirely dependent on the host
plants for nutrition. The stem is thread like filaments it is begin to grow and attach themselves to nearby host
plants. The nature plants lives its entire life without attachment to the ground. It has long history of
ethnomedicinal use. Cuscuta is a genus of about 100170 species.
In North India Cuscuta reflexa is usually associated with parasitism in ornamental plants and its occurrence
in medicinal crops is unusual. This species is originally from India, is common over the Northern region of the
country from the state of Uttar Pradesh and Uttarakhand. Cuscuta reflexa Roxb. is a valuable medicinal herb
(Fig. 1). Stem of this plant is antibacterial and used externally to treat itch and internally in fever (Pal et al.
2006). It is useful in treatment of androgen induced alopecia (Pandit et al. 2008). It also gives anti-inflammatory
and anti-cancer activity (Suresh et al. 2011). The aqueous and alcoholic extract of C. reflexa has diuretic
property (Sharma et al. 2009). The crude water extract of C. reflexa also shows anti HIV activity (Mahmood et
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Received: 13 May 2016 Published online: 31 August 2016
Rai et al. (2016) 3(2): 428433
.
al. 1997). It is a parasitic plant completely dependent on host plant for food and nutrition. The organic matter is
transported from the phloem of the host to the parasite through the hostorium (Kumar et al. 2012). It is believed
that the parasitic herbs extract healthy and potential sap from host plant and if their host plant is medicinal plants
then these parasitic herbs how many similar properties to host plants. Cuscuta species feeding on commonly
used medicinal herbs are given special attention by traditional healers. Present work evaluates the comparative
study of phytochemical activity of extract of Cuscuta reflexa grown on Muzaffarnagar and Pantnagar, North
India.

MATERIAL AND METHOD


Collection of plant material
Cuscuta reflexa plant leaves were collected from village area of district Muzaffarnagar, Uttar Pradesh and
area of Pantnagar, Uttarakhand, India. The plants were authenticated by Dr. Anju Pal, Dept. of Horticulture,
G.B. Pant University of Agriculture and Technology, Uttarakhand, India.

Figure 1. Cuscuta reflexa Roxb. on his host Euphorbia tirucalli L.


Preparation of aqueous extract
Hundred grams each of dried leaves of C. reflexa collected from different location of North India were
macerated with 100 ml sterile distilled water in a blender for 10 min. The macerate was first filtered through
double layered muslin cloth and centrifuged at 4000 rpm for 30 min. The supernatant was filtered through
Whatman No.1 filter paper and heat sterilized at 120C for 30 min. The extracts were preserved aseptically in
brown bottles at 4C until further use.
Preparation of plant Solvent extracts
Soxhlet extraction will be the method used for plant extraction. A portion of dried leaves (100 g) of Cuscuta
reflexa was placed in a Soxhlet apparatus. Extraction was performed with 500 ml of an appropriate solvent
(Ethanol, Methanol, Chloroform) with increased polarity for 24 h at 950C temperature not exceeding the boiling
point of the solvent. The extract was filtered through a 45 m filter paper and concentrated under vacuum. In
this experiment three solvents were used: Ethanol, Chloroform and methanol. The resulting three solutions were
concentrated in vacuoum to dryness to give Ethanol (4 g), Chloroform extract (10 g) and methanol extract
MeOHE (12 g). The stock solutions were kept at 4C until further use.
Sample preparation
The extract of plant sample was dissolved in 75 ml of methanol for 24 hrs. Then the filtrates were collected
and evaporated under liquid nitrogen. The GC-MS analysis was carried out using a Clarus 500 Perkin-Elmer
(Auto system XL) Gas Chromatograph equipped and coupled to a Mass detector Turbo mass gold-Perking
Elmer Turbomas 5.1 spectrometer with an Elite-1 (100% Dimethy1 ply siloxane), 30 m x 0.25 mm ID x 1 m df

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Rai et al. (2016) 3(2): 428433
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capillary column. The instrument was set to an initial temperature of 110C, and maintained at this temperature
for 2 min. At the end of this period, the oven temperature was raised up to 280C, at the rate of an increase of
5C/min, and maintained for 9 min. Injection port temperature was ensured as 250C and Helium flow rate as 1
ml/min. The ionization voltage was 70eV. The samples were injected in split mode as 10:1. Mass spectral scan
range was set at 25-400 mhz. The chemical constituents were identified by GC-MS. The fragmentation patterns
of mass spectra were compared with those stored in the spectrometer database using National Institute of
Standards and Technology - Mass Spectral database (NIST-MS). The percentage of each component was
calculated from the relative peak area of each component in the chromatogram.

RESULTS
The phytochemical compounds present in the methanolic extract of Cuscuta reflexa were identified by GC-
MS analysis. The active principles with their retention time (RT), molecular formula (MF), molecular weight
(MW) and concentration (%) in the extracts of C. reflexa were presented. From Muzaffarnagar sample, totally
15 compounds were identified table 1. The prevailing compounds were lauric acid (2.46%), ester compound
(0.05%), alkanes (0.05), phenolic compound (0.08%), myristic acid (2.77%), plasticizer compound (4.15%),
palmitolic acid (2.27%), palmitic acid (13.97%), diterpene (2.31%), stearic acid (1.68%), mono unsaturated fatty
(5.19%), chlorine compound (2.16%), steroid (11.6%), alkaloid (1.78%), triterpenes (3.56%) and amino
compound (39.27%).
Table 1. Qualitative and quantitative determination of biochemical constituents of plants sample collected from Muzaffarnagar.
S.No. RT Name of the Molecular MW Peak Compound
Activity
Compound Formula Area (%) Nature
1 8.13 Tetradecane C14H30 198 0.05 Alkane No activity reported
2 9.07 Phenol, 3,5-bis (1,1- C14H22O 206 0.08 Phenolic Analgesic, Anesthetic,
dimethylethyl)- Compound Antioxidant, Antiseptic,
Antibacterial, Antiviral Cancer
preventive, Fungicide
3 10.50 Dodecanoic acid C12H24O2 200 2.46 Lauric acid Antipyretic, Antiinflammatory,
Analgesic, Antiseptic,
Pesticide, Cancer preventive,
Carminative
4 13.56 Tetradecanoic acid C14H28O2 228 2.77 Myristic acid Antioxidant, Cancer preventive,
Nematicide,
Hypocholesterolemic
5 14.84 1,2-Benzenedi C16H22O4 278 1.67 Plasticizer Antimicrobial, Antifouling
carboxylic acid, bis (2- Compound
methyl propyl) ester
6 16.30 Hexadecenoic acid, C16H30O2 254 2.27 Palmitolic Hypocholesterolemic
Z-11- Acid
7 16.77 n-Hexadecanoic acid C16H32O2 256 13.97 Palmitic acid Antioxidant, Flavor,
Hypocholesterolemic,
Nematicide, Pesticide,
Lubricant, Antiandrogenic,
Hemolytic,
5-Alpha reductase inhibitor
8 18.45 1-Hexadecanol, C17H36O 256 2.16 Alcoholic Antimicrobial
2-methyl- Compound
9 19.01 Phytol C20H40O 296 2.31 Diterpene Antimicrobial, Anticancer
Anti-inflammatory, Diuretic
10 19.50 Oleic Acid C18H34O2 282 5.19 Mono Antiinflammatory,
unsaturated Antiandrogenic,
fatty acid Cancer preventive,
Dermatitigenic,
Hypocholesterolemic,
11 22.29 Tris (1,3-dichloro C9H15Cl6O4P 428 2.16 Chlorine Antimicrobial
isopropyl) phosphae Compound
12 25.66 1,2Benzenedicarboxylic C24H38O4 390 4.15 Plasticizer Antimicrobial, Antifouling
acid, diisooctyl ester compoud

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13 30.53 Squalene C30H50 410 3.56 Triterpene Antibacterial, Antioxidant,
Antitumor, Cancer preventive,
Immunostimulant, Chemo
preventive,
14 32.59 Tetrazol-5-amine, N- C10H13N5O2 239 39.37 Amino Antimicrobial
(3,4- compoud
dimethoxybenzyl)-
15 34.19 Cholestan-3-one, C29H50O2 430 11.61 Steroid Antimicrobial, Antiarthritic
Cyclic1,2 ethanediyl Antiasthma, Anti-inflammatory
The mass spectrum and structures of these above mentioned compounds were shown in figure 2. They are
suggested to be the medicinally important compounds which can be used as antimicrobial, anti-inflammatory,
cancer preventive, antioxidant, antiviral, antidiabetic, antifouling and hepatoprotective agent.

Figure 2. GC-MS chromatogram of methanolic extracts of Cuscuta reflexa (Muzaffarnagar Sample).


The result of the GC-MS analysis of C. reflexa from Pantnagar plant samples is presented in the table 2. The
GC-MS chromatogram of these medicinal plant extracts was shown in the figure 3. Nearly 12 compounds were
indentified in the Pantnagar sample. They were nitrogen (13.56%), aromatic (7.88%), fluro (28.40%), alkaloid
(7.64%), silica (5.66%), phosphorus (16.31%) and chlorine compounds (6.26%). In general the isolated
compounds are reported to possess antimicrobial, antitumor, anticarcinogenic and anti-inflammatory properties.
Table 2. Qualitative and quantitative determination of biochemical constituents of plants sample collected from
Pantnagar.
S.No. RT Name of the compound Molecular Molecular Peak
Compound Nature
Formula Weight Area (%)
1 2.15 1,4 Bis*4 5 Bis C10 N4 532 28.40 Fluro Antimicrobial
(Trimethylfluromethyl) 1, F12 S4 compound
3- Dithiac
2 2.29 Dihydrofuranoartobilochro C25H22O7 434 10.28 Pigment No activity
men A
3 2.49 Tetra (Diphenylphosphinyl) C51H40P4 76 16.31 Phosphorus No activity
Allene compound
4 2.80 Methyl-2-hydroxy-2- C9H16O3 172 8.30 Acetate No activity
cyclohexylacetate compound
5 3.51 Methyl-6,8 dioxo- C20H17O5 351 7.68 Nitrogen No activity
2,C4Diphenyl- N compound
7-oxa-3-Azabicyclo(3
6 3.69 Isopropyl(P-metho C13H14O 214 11.62 Nitrogen No activity
xyphenyl)malononitrile N2 compound
7 4.18 Dimetilan C10H16O3 240 13.56 Nitrogen No activity
N4 compound
8 4.44 TMS-8,11-Di C30H54O4 562 5.66 Silica No activity
OHTetrahydrocannabinol Si3 compound
9 22.62 1,2 Di-Hydroxy C20H24O4 384 7.88 Aromatic No activity
anthroquinone DITMS Si2 compound
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10 4.71 Tetramethyl 5 (Hexachloro- C20H12O8 590 6.26 Chlorine Antimicrobial
2,4,6 Cl6 compound
Cycloheptatrien
11 5.18 N [1,2,2,2 tetrafluro-1- C6H9O2N 306 5.68 Fluro Antimicrobial
(Trifluromethyl)ethyl]SU 2F7S compound
12 5.82 Ditetrazolo1,5- A:5,1-C C4 H2 N8 162 7.64 Alkaloid Antimicrobial
Pyracine
Figure 3 shows mass spectrum and structures of these compounds, and also are suggested to be the
medicinally important compounds which can be used as antimicrobial, anti-inflammatory, cancer preventive,
antioxidant, antiviral, antidiabetic, antifouling and hepatoprotective agent.

Figure 3. GC-MS chromatogram of methanolic extracts of Cuscuta reflexa (Pantnagar Sample).

DISCUSSION
In the present study the GC-MS analysis of ethyl acetate extract of C. reflexa grown on North India .showed
the fifteen and three compounds respectively.Tetradecane ,Phenol,3,5-bis (1,1 dimethylethyle,1,2 Benzenedi
carboxile acid bis ( 2 methyle propyle ester), n Heaxadecenoic acid ,1 hexadecanol, 2 methyle, phytol, tetrazol
5- amine N (3,4- dimethoxybenzyl,cholestan 3- one cyclic 1,2 ethanediyle. Are present in Muzaffarnagar village
area sample. And 1,4 Bis 4,5 trimethylefluroment and other 11 types of compound nature are present in
Pantnagar area sample .tetramethyle and its derivatives are known to have bacterial inhibiting effect.

CONCLUSION
It is revealed from this study that C. reflexa from both the region is rich in secondary metabolites which
possess wide range of biological activities. Different compounds are present in C. reflexa on two different
region thus it is concluded that variation in phytochemicals in C. reflexa is different region dependent. Further
study need to be undertaken to investigate the biological activity and other phytochemicals present in C. reflexa
grown on North India.

ACKNOWLEDGEMENT
The authors thankful to Dr. Anju Pal Dept. of Horticulture, G.B.Pant University of Agriculture. And
Technology, Pantnagar, U.K, India and the Ex. Dean Dr. D.P Mishra and Director Dr. S.K Garg Deppartment of
Science and Humanities G.B. Pant University of Ag. & Technology, Pantnagar, U.K. And also thanks to Dr.
Krishan Pal Dept. of Biotechnology, Shri Venkateshwara University, Gajraula, and U.P. India for providing
necessary laboratory requirement, facilities to carry out this work and useful discussion and suggestion.
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REFRENCES
Kumar A, Rani S, Sagwal S & Niketa (2012) Recent review on plant molecular biology, phytophysiology,
phytochemistry and ethnopharmacology of Cuscuta reflexa Roxb. A wonderful parasitic plant. International
Research Journal of Pharmacy 3(7): 3038.
Mahmood N, Piacente S, Burke A, Khan AL & Pizza C (1997) Constituents of Cuscuta reflexa are viral Agents.
Antiviral Chemistry and Chemotherapy 8(1): 70.
Pal DK, Mandal M, Senthil Kumar GP & Padhiari A (2006) Antibacterial activity of Cuscuta reflexa stem and
Corchorus olitorious seed. Fitoterapia 77: 589591.
Pandit S, Chauhan NS & Dixit VK (2008) Effect of Cuscuta reflexa ROXB on androgen induces alopecia.
Journal of Cosmetic Dermatology 7: 199204.
Sharma S, Hullati KK, Prasanna SM, Kuppast IJ & Sharma P (2009) Comparative study of Cuscuta reflexa and
Cassytha filiformis in diuretic activity. Pharmacognosy Research1: 327330.
Suresh V, Sruthi V, Padmaja B & Asha VV (2011) In vitro anti inflammatory and anti cancer activities of
Cuscuta reflexa Roxb. Journal of Ethnopharmacology 134: 872877.

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ISSN (P): 2349 9265
3(2): 434439, 2016

Research article

New records of lichens from foothills of Kumaun Himalayas to


the lichen flora of Uttarakhand, India
Shiksha Mishra1*, Dalip Kumar Upreti2 and Anand Kumar Srivastava3
1
Botany Department, Sardar Bhagat Singh Government PG College, Rudrapur (Udham Singh Nagar),
Uttarakhand, India
2
Lichen Laboratory, CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow-226021, India
3
Government Post Graduate College, Dwarahat, Almora, Uttarakhand, India
*Corresponding Author: shikshamis@gmail.com [Accepted: 15 August 2016]

Abstract: The paper presents an enumeration of seven lichen species as addition to the lichen flora
of foothills of Kumaun Himalayas, Uttarakhand. The enumerated species belong to six genera and
six families. The explored area showed dominance of crustose lichens while only two foliose and a
single leprose species was recorded. A morpho-taxonomic note together with ecology and
distribution of all the recorded lichen species as new addition to lichen flora of Uttarakhand is
provided.
Keywords: Lichen - New addition - Foothills - Kumaun Himalayas - Uttarakhand.

[Cite as: Mishra S, Upreti DK & Srivastava AK (2016) New records of lichens from foothills of Kumaun
Himalayas to the lichen flora of Uttarakhand, India. Tropical Plant Research 3(2): 434439]

INTRODUCTION
Indias ranks fifth with greatest biodiversity in the world, which is about 10% of the 20,000 species of
lichens recorded in the world (Groombridge 1992). About 2305 lichen species (with additional 21 species
unpublished) under 305 genera and 74 families have been reported to occur on various substrata in tropical,
subtropical, temperate and alpine regions of India (Singh & Sinha 2010). The Himalayan region is represented
by about 1000 species of lichens out of which 747 species (Rai 2013) are known to occur in Uttarakhand state.
The topography of the state provide a wide altitudinal range from plain foothills to higher alpine region, hence
the state exhibit tropical type of climate in the lower Himalayan region and temperate to alpine type of climate
condition in higher Himalayas (Gupta et al. 2016a).
Kumaun region of Himalayas has been the reservoir of enormous natural resources of medicinal wealth.
Great altitudinal variations as well as variation in rainfall are the main factors for the rich lichen flora of
Kumaun. The intensity of rainfall becomes less and lesser from lower to higher altitude. Number of species is
directly proportional to the altitude within the area, lower the altitude lesser the number of species. Mishra &
Upreti (2015) carried out the floristic studies in the Kumaun Himalayan region and reported the occurrence of
630 species of lichens belonging to 134 genera and 44 families from the area. The Kumaun Himalayas
comprised of six district of Uttarakhand. Amongst all the six districts, Udham Singh Nagar having a subtropical
region, is situated at foothills of the Himalayas. The foothill region of Kumaun includes unique physiographic
ecosystem as bhabhar and terai (Gupta et al. 2016b). The terai region is water logged alluvial plain with gentle
South East Slope, deep and fertile moist loamy soil forming marshy land free from boulders and gravels. Being
situated in the foothills of the Himalayas, having industrialized areas and agriculture fields, the district exhibit
poor representation of lichens as compared to the region at higher altitude and only 28 species of lichen
belonging to 17 genera and 13 families are known from the area (Mishra et al. 2012).
Mishra et al. (2012, 2015) listed a small number of lichen species from the foothills of Kumaun Himalayas
in the lichen flora of Kumaun based on the few cursory collection of the area. During a recent intensive and
extensive field survey and collection of lichens more than 300 lichen specimens were collected and the
identification of the specimens resulted into the addition of seven more taxa to the flora of the state of
Uttarakhand.
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Received: 27 May 2016 Published online: 31 August 2016
Mishra et al. (2016) 3(2): 434439
.
MATERIALS & METHODS
This study is based on more than 300 specimens of lichens collected from different localities of Udham
Singh Nagar district (2853' 2923' N and 7845' 8008' E), located in the foothills of Kumaun Himalayas
(Fig. 1). The specimens were collected from different substrata such as barks and twigs from the forests and
mango orchards. The various trees (Eucalyptus, Mangifera indica, Mallotus philippensis, Murraya koengii,
Shorea robusta and Syzygium cumini) exhibited remarkable growth of lichens. The identification of the
specimens was done by studying their morphology, anatomy and chemistry. The morphology was studied using
a LaboMed Digi Zoom dissecting microscope, and the anatomical details were studied using a Leica TM DM
500 optical microscope. Colour tests were performed using KOH (K), calcium hypochlorite (C) and para-
phenylenediamine (P) solution. Secondary metabolites were identified by thin layer chromatography (TLC) as
described by Orange et al. (2001) and Elix et al. (1993). The chromatograms were done in solvent system A
(toluene: dioxane: acetic acid 180:60:8 ml) and solvent EA (Diethyl ether: Acetic acid: 200:2 ml). Specimens
were identified by comparing the morphological and the biochemical test results with the literature and
identification keys (Singh & Sinha 2010). The identified voucher specimens were deposited in the lichen
herbarium of CSIR-National Botanical Research Institute, Lucknow (LWG).

Figure 1. Collecting sites in foothills of Kumaun Himalayas.


RESULTS
The study enlisted seven lichen taxa as new addition to the lichen flora of foothills of the Kumaun
Himalayas. The recorded lichen taxa belong to the six genera viz. Anisomeridium, Amandinea, Graphis,
Hyperphyscia, Opegrapha, Lepraria and six families which are Calicaceae, Monoblastaceae, Graphidaceae,
Physciaceae, Rocellaceae and Stereocaulaceae. All the taxa are reported for the first time for Uttarakhand state
from the foothills of Kumaun Himalayas.
The study area exhibit dominance of crustose, foliose and leprose lichen growth forms, were found growing
on bark and twigs in the forests as well as mango orchards. Though, the area is not rich in lichen diversity but
exhibit diversity at generic as well as species level. A brief documentation of each species along with their
ecology and distribution is provided.
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Species enumeration
Caliciaceae
Amandinea subduplicata (Vain.) Marbach, Biblioth. Lichenol. 74: 101 (2000). (Fig. 2A)
Thallus corticolous or saxicolous, crustose, smooth or verrucose, 12 cm wide; esorediate; apothecia 0.40.9
mm wide, lecideine, immersed to adnate; disc black, plane to concave, epruinose; proper margin broad; proper
exciple thick; hymenium 80120 m thick, not markedly inspersed, but with a few scattered oil droplets in the
lower part; asci 8-spored; ascospores Buellia type, olive-green to olive-brown, ellipsoidal, 1723 79 m;
thallus K+ yellow, C, KC, PD+ pale yellow, UV; atranorin detected in TLC.
Distribution and ecology: The species is rare but found growing on bark as well as rocks. From India, it has
been reported earlier from the states of Madhya Pradesh, Rajasthan and Tamil Nadu. The species is also known
from Asia, North and South America.
Specimen examined: Udham Singh Nagar, Khatima, Khatima forests, on bark, 02.09.2015, S. Mishra & G. K.
Mishra 15027744 (LWG).
Graphidaceae
Graphis crebra Vain., Hedwigia 38:256. 1899. (Fig. 2B)
Thallus corticolous, crustose, corticate, dull to somewhat shiny, pale grey; ascomata lirellate, lirellae
erumpent with lateral thalline margin, short, unbranched to rarely once branched, straight to slightly curved;
labia entire; disc becoming exposed very early, with a distinct white pruina; excipulum laterally carbonized;
hymenium inspersed; ascus 8 spored, hyaline, 912 celled, 2933 89 mm; thallus K+ yellow orange, C,
KC, P+, norstictic acid in TLC.
Distribution and ecology: In the Neotropics, Graphis crebra is known from Guadeloupe, St. Helena and the
Galapagos. During field trip, it was found growing on bark, distributed in the forests of Khatima and Tanda of
Udham Singh Nagar district.
Specimens examined: Udham Singh Nagar, Khatima, Khatima forests, on twig of Shorea robusta, 02.09.2015,
S. Mishra & G. K. Mishra 15019917, 15019918 (LWG); Rudrapur, Tanda forests, on bark, 03.09.2015, S.
Mishra & G. K. Mishra 15027702 (LWG); on bark of Shorea robusta, 03.09.2015, S. Mishra & G. K. Mishra
15027710 (LWG); on twig, 03.09.2015, S. Mishra & G. K. Mishra 15027711 (LWG).
Monoblastiaceae
Anisomeridium albisedum (Nyl.) R. C. Harris, The Bryologist 90 (2): 163 (1987). (Fig. 2C)
Thallus corticolous, crustose, ecorticate, white, UV; perithecia solitary; ostiole apical; hamathecium
filaments thin, anastomosing above the asci; asci 8 spored; ascospores hyaline, 1 septate, ovoid, 915 45
m, not ornamented, septum mostly median.
Distribution and ecology: A neotropical species has been earlier reported from Maharashtra and
Mahabaleshwar in India. In the study area, the species is found growing on bark near base of tree trunk of
Eucalyptus tree.
Specimens examined: Udham Singh Nagar, Nanak Sagar, Sitarganj, Eucalyptus, 02.09.2015, S. Mishra & G.
K. Mishra 15027742, 15027742/A, 15 027742/B (LWG); Bajpur, way to Kaladhungi, on bark, 03.09.2015,
S. Mishra & G. K. Mishra 15027743 (LWG).
Physciaceae
Hyperphyscia adglutinata var. adglutinata (Flrke) H. Mayrhofer & Poelt, Herzogia 5: 62. 1979. (Fig. 2D)
Thallus corticolous, foliose, orbicular, small, 0.62.4 cm across, greenish grey to brownish, sorediate; lobes
minute, 0.20.4 mm wide, slightly pruinose, free from the substratum; lower surface pale, yellowish white to
grey white, rhizinate; rhizines dark grey with white tips, sorelia capitate, soredia granular, greenish medulla
white to greenish; apothecia absent; thallus K, C, KC, P; no chemicals detected in TLC.
Distribution and ecology: The species is cosmopolitan in distribution and found growing on bark and rarely on
rock. During field trip, it is collected from forests as well as mango orchards from various localities (Bajpur,
Gadarpur, Kichha, Khatima and Rudrapur) on different phorophytes. Earlier, in India, it is reported from
Himachal Pradesh and Jammu & Kashmir. Outside India, it is known from Australia, Bhutan, New Zealand and
Taiwan, Africa, Europe, North America.
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Mishra et al. (2016) 3(2): 434439
.
Specimens examined: Udham Singh Nagar, Bajpur, enroute to Kaladhngi, Murraya koengii, 03.09.2015, S.
Mishra & G. K. Mishra 15027791 (LWG); on twigs, 15027790/A (LWG); Bajpur, on bark Mallotus
philippensis, 03.09.2015, S. Mishra & G. K. Mishra 15027789 (LWG); Rudrapur, Tanda, 02.09.2015, S.
Mishra & G. K. Mishra 15027784 (LWG); Kichha, near Rudrapur, Magnifera indica, 02.09.2015, S. Mishra &
G. K. Mishra 15027742 (LWG); Khatima, Khatima forests, Shorea robusta young bark, 02.09.2015, S. Mishra
& G. K. Mishra 15027786, 15027786/A(LWG); Gadarpur, before Kelakhera, Magnifera indica, 03.09.2015,
S. Mishra & G. K. Mishra 15027742, 15027796 (LWG).

Figure 2. Thallus of newly added lichen taxa to the foothills of Kumaun Himalaya: A, Amandinea subduplicata (Vain.)
Marbach; B, Graphis crebra Vain.; C, Anisomeridium albisedum (Nyl.) R. C. Harris; D, Hyperphyscia adglutinata var.
adglutinata (Flrke) H. Mayrhofer & Poelt; E, Hyperphyscia adglutinata var. pyrithrocardia (Mll. Arg.) D. D. Awasthi; F,
Opegrapha varia Pers.; G, Lepraria coriensis (Hue) Sipman.
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Mishra et al. (2016) 3(2): 434439
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Hyperphyscia adglutinata var. pyrithrocardia (Mll. Arg.) D. D. Awasthi, Comp. Macrolich. India, Nepal &
Sri Lanka: 197. 2007. (Fig. 2E)
Thallus corticolous, foliose, 1.03.0 cm diam., often coalescing with neighbouring thallus, upper surface
whitish grey to brownish, darkening at margin, lobes free from substratum, 0.20.4 mm wide; sorelia capitate to
irregular, laminal, soredia farinose lower surface pale at margin, rhizinate, medulla white, sometimes orange and
K+ purple; apothecia absent; thallus K, C, KC, P; skyrin present in TLC.
Distribution and ecology: The species is widely distribution in the Udham Singh Nagar. It is collected from
forests of Bajpur, Khatima and Rudrapur on bark and twigs. Previously, it has been reported from Tamil Nadu
in India and outside India, recorded from U.S.A.
Specimens examined: Udham Singh Nagar, Rudrapur, Tanda, on bark 03.09.2015, S. Mishra & G. K. Mishra
15027795, 15 027795/A, 15027792 (LWG); Bajpur, on the way to Kaladhungi, on twig 03.09.2015, S.
Mishra & G. K. Mishra 15027793 (LWG); Khatima, Khatima forests, Shorea robusta young bark 03.09.2015,
S. Mishra & G. K. Mishra 15027794 (LWG).
Rocellaceae
Opegrapha varia Pers. Ann. Bot. (Usteri) 1:30. 1794. (Fig. 2F)
Thallus corticolous, crustose, ecorticate, greenish grey, sometimes inconspicuous; ascomata lirellate, lirellae
black, mostly simple, rarely furcated, 0.307 0.10.2 mm, disc slit like, epruinose; exciple brown black to
carbonized, convergent, thinning and continuous below hymenium, 1634 m thick; hymenium hyaline to
slightly yellowish, upto 38 m high; ascus 8 spored, clavate, 5771 1722 m; ascospores hyaline, 45
septate, middle locule slightly larger, fusiform with thick perispore, 3134 7110 m; thallus K, C, KC,
PD; no chemicals detected in TLC.
Distribution and ecology: The species is cosmopolitan and colonize on the bark of the tree. During the study, it
has been collected from Bajpur and has been earlier reported from plain areas of Andaman & Nicobar Islands,
Kerala and West Bengal. Outside India, it is reported from Australia, Fiji, Indonesia, Hongkong, Papua New
Guinea the Philippines and Africa.
Specimen examined: Udham Singh Nagar, Bajpur, enroute to Kaladhungi, on bark, 03.09.2015, S. Mishra & G.
K. Mishra 15027802 (LWG).
Stereocaulaceae
Lepraria coriensis (Hue) Sipman, Herzogia 17: 28. 2004. (Fig. 2G)
Thallus corticolous, crustose-leprose, yellowish grey, ecorticate, forms a thin to moderately thick, non-
areolate crust of powdery granules or, in part, to form irregular rosettes with lobed margins; marginal lobes
rounded, 0.52.0 mm wide, with raised marginal rim; surface smooth near the margin, with more or less
scattered granules in the centre; usnic acid and zeorin present in TLC, pannaric acid derivatives absent.
Distribution and ecology: The species is corticolous reported from Khatima. In India, it has been reported from
Karnataka while outside India it is widely distributed in Australia, Korea, Hong Kong and Taiwan, SE Asia,
extending to North Australia.
Specimens examined: Udham Singh Nagar, Khatima, Khatima forests, on bark 02.09.2015, S. Mishra & G. K.
Mishra 15027721 (LWG), 15027721/A (LWG).

ACKNOWLEDGEMENTS
The authors are grateful to Kumaun University, Nainital and Director, CSIR-National Botanical Research
Institute, Lucknow for providing the necessary laboratory facilities and numerous supports during the study.

References
Awasthi DD (1991) A key to the microlichens of India, Nepal and Sri Lanka. Bibliotheca Lichenologica 40: 1
337.
Elix JE & Ernst-Russel KD (1993) A catalogue of standardized thin layer chromatographic data and
biosynthetic relationships for lichen substances, 2nd edn. Australian National University, Canberra.
Groombridge B (1992) Global biodiversity: Status of the earths living resources. Chapman and Hall. James.

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Gupta N, Gupta V & Dwivedi SK (2016b) New addition to lichen flora of Uttar Pradesh, India. Tropical Plant
Research 3(1): 153156.
Gupta S, Rai H, Upreti DK, Sharma PK & Gupta RK (2016a) New addition to the Lichen flora of Uttarakhand,
India. Tropical Plant Research 3(1): 224229.
Mishra GK, Upreti DK, Nayaka S & Punetha N (2012) An enumeration of lichens from Udham Singh Nagar
district of Uttarakhand with Bacidia delicata as new record for India. Phytotaxonomy 12: 105108.
Mishra GK & Upreti DK (2015) Lichens Flora of Kumaun Himalaya. Lap Lambert Academic Publishing,
Deutschland, Germany.
Orange A, James PW & White FJ (2001) Microchemical Methods for the Identication of Lichens. British
Lichen Society, London.
Rai H (2013) Lichens of Uttarakhand: A checklist. Available from: https:// sites.google.com/site/himanshurai
india/checklist-ofuttarakhand (accessed: 20 July 2013).
Singh KP & Sinha GP (2010) Indian lichens: An annotated checklist. Botanical Survey of India. p. 1507.

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ISSN (E): 2349 1183
ISSN (P): 2349 9265
3(2): 440448, 2016

Research article

Characterization of iron Bacterium Gallionella ferruginea isolated


from the drinking water of the collector wells in
Northern Sri Lanka
Aberamy Sayanthan1, Ponipus T. J. Jashothan1, Suntharalingam Saravanan2
and Ranganathan Kapilan1*
1
Department of Botany, University of Jaffna, Jaffna, Sri Lanka
2
National Water Supply and Drainage Board, Jaffna, Sri Lanka
*Corresponding Author: ranganat@ualberta.ca [Accepted: 18 August 2016]

Abstract: The objective of the study was to isolate, identify and characterize the organism/s
responsible for the brownish slime colour and bad odour of the water from the collector wells at
Vallipuram area of the Northern Sri Lanka. The iron concentrations in the collector well samples
that are free of fecal coliform bacteria, varied from 0.093 to 0.307 mg.l-1 during the day time.
Based on the microscopic biochemical and molecular characterization, the bacterial strain isolated
from the collector wells was identified as Gallionella ferruginea. They are gram negative kidney-
shaped mycoplasmodial bodies found in clusters. The colonial growth was powdery, opaque and
flat in elevation. The biochemical characterization showed the positive interpretation for indole
and catalase tests while methyl red, citrate, Voges-Proskaeur, urease production, nitrate reduction,
tyrosine utilization, acetoin production and oxidase tests showed negative. The bacteria were
capable of fermenting glucose with the production of acid in anaerobic condition, but not in
aerobic condition thus confirmed as Gallionella ferruginea. This bacterial strain grew well in iron
added liquid media at temperatures between 2540oC and the optimum growth was observed at
35oC. Though Gallionella grew well at a broad range pH values between 6.010.0, the optimum
growth was obtained at neutral pH. Addition of NaCl in the iron added liquid media can inhibit the
growth and multiplication of Gallionella ferruginea.
Keywords: Brownish slime - Water quality - Iron bacteria - Gallionella ferruginea - Vallipuram.

[Cite as: Sayanthan A, Jashothan PTJ, Saravanan S & Kapilan R (2016) Characterization of iron Bacterium
Gallionella ferruginea isolated from the drinking water of the collector wells in Northern Sri Lanka. Tropical
Plant Research 3(2): 440448]

INTRODUCTION
Water is one of the principal components in determining the quality of our live and the living environment.
Although water covers about 70 percent of the earths total surface, only 0.3% of it can be utilized by humans
(Tiimubet et al. 2012). Jaffna Peninsula of Sri Lanka has a low percentage of surface water sources because of
its karstic nature and flat terrain (Meisler 1977, Dissanayake & Senaratne 1981, Senaratne & Dissanayake 1982,
Wijesekera et al. 2012, Hidayathulla & Karunaratna 2013). Group of aerobic bacteria that can utilize the
oxidation of ferrous or manganous ions as an essential element in their metabolic utility are defined as iron
bacteria. The resultant production of ferric acid or manganic salts within the cell or cell coating gives the
bacteria their typical brown coloration. Studies of pyrite deposits have shown them to contain a range of fossil
bacteria including representatives of two major groups of iron bacteria, Gallionella and Sphaerotilus (Schopf et
al. 1965). Iron can be deposited as filamentous amorphous gelatinous type of brown-reddish slime that
precipitates from water that contains iron. Iron deposits have become a serious issue in a variety of water sauces
including drippers and could lead to plugging of a low volume system. There is a class of bacteria that can
absorb ferrous ion and interchange it to a slimy matrix of bacterial bodies and convert into ferric ion. The
complication typically exists in well water areas where the earth water aquifers are formed mainly of sandy soils

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Received: 05 May 2016 Published online: 31 August 2016
Sayanthan et al. (2016) 3(2): 440448
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normally with a pH of down from 7.0 and in the absence of dissolved oxygen. These ground waters contain
ferrous ion (Fe2+) which is chemically reduced, completely water soluble and which serves as the starting raw
material for slime production. The dissolved ion may precipitate out of the water due to differences in pressure
and temperature, an increase in pH, electro conductivity or through the action of bacteria. The result is a sludge
or slime that may reduce system performance.
The encrustations of wells are caused by iron and manganese bacteria, various species of which exist in the
soil and could presumably enter a well during the initial boring operations or by seepage into the aquifer feeding
the well (Hasselbarth & Ludemann 1972). The existence and morphology of Gallionella ferruginea, one of the
important iron bacteria living in diverse aquatic environment, have been subjected for continuous discussion.
Litho autotrophic metabolism of these bacteria has been questioned (Jarrgensen 1989). Gallionella ferruginea
generally grows to a very low cell number, approximately 1 106 cells ml-l, in the appropriate liquid medium
(Hallbeck & Pedersen 1990). Iron bacteria of the well water react with the ferrous (Fe+2) during the oxidation
process and converts it to insoluble ferric (Fe+3). This Ferric ion is enveloped by the filamentous bacterial
colonies and forms the sticky iron slime gel that is responsible for clogging the water movement. The ferrous
ion concentration low as 0.150.22 ppm is considered as a potential hazard to drip systems (Ford 1982).
Between 0.21.5 ppm, of ferrous can cause clogging hazard in a moderate level. Concentrations above 1.5 ppm
can cause severe blocking (Bucks & Nakayama 1980). Water resources that contain concentrations higher than
0.5 ppm of iron, should be considered for immediate chemical treatment before using filter. Chlorination can
successfully control iron slime when iron concentrations were less than 3.5 ppm and the pH was below 6.5
(Nakayama & Bucks 1986). Ferrous ion in the water stream could be mixed with the wastewater that has an
organic load associated with it. Microbial corrosion is an electrochemical process where microorganisms are
able to initiate, facilitate or accelerate corrosion reaction without changing its electrochemical nature (Dexter et
al.1991, Iversen 2001).
Jaffna lagoon of the northern province of Sri Lanka is a shallow water body and has extensive mudflats, sea
grass beds and some mangroves. Most of the wells that are closer to sea are not used for public consumption
because of the salty in nature (Sayanthan et al. 2015, Kapilan 2015). It was decided to analyze its ground water
since the ground water is collected and purified by the addition of chemicals and this processed water is
distributed for public consumption. The occurrence of iron in geological strata of this Island is already well
established. Because of the limited sources available and the difficulty of monitoring the organisms, limited
attention has been paid to identify the organism and its role in the strata or aquifers in the change of water colour
and the bad odour is not well understood. Soil, water and mud could be a habitat for the growth of diverse iron
bacteria. Dissolved organic materials of the groundwater may be de-oxygenated by microbes feeding on that
dissolved organic material. In water-supply wells, iron bacteria derive the energy they need to live and multiply
by oxidizing the dissolved ferrous ion (Andrews et al. 2013). These iron bacteria causes problems in mining
operations, piped water and sewage systems and in open bodies of water. The colour change and brownish slime
formation would be always possible and the water becomes undrinkable and further growth would lead to
plugging of freshwater wells. The problem continued and the Iron bacteria also formed and the bad hydrogen
sulphide odour was observed. Sulfurous smell of rot or decay results from enzymatic conversion of
soil sulfates to volatile hydrogen sulfide as an alternative source of oxygen in anaerobic environmentsthat are
used to collect water for drinking purpose. In the collector wells from where the drinking water is supplied to
public, the major problems are, growths plugging the screens, coating of the pipes, impellers and motors,
reduction in the water flow rates reduced portability of the water and all these may lead to total plugging of the
well. However there is no recent scientific water analysis conducted for the collector wells to determine the
water quality and to identify the potential pollutants specially the iron bacteria in the northern province of Sri
Lanka so that some remedies for improvement could be possible. Vallipuram area that supplies water to the
public for consumption through the collector well system was selected as an ideal place of the above mentioned
situation. Therefore this study was aimed at determining the iron bacteria that is responsible for the bad odour
and brownish slimecolour of the water in the drinking water collector wells in the Vallipuram area of the
northern Sri Lanka.

MATERIALS AND METHODS


Study Area

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This study was conducted in the Vallipuram costal area between September and December in 2015.
Collector wells are located in the sand dunes. The location is rich in water and there are less hardness issues,
less population density in the area so that anthropogenic activity is also very less.
Sampling method
Samples were collected randomly from four collector wells. From each of the water sources samples were
collected in two positions one from top and other from bottom and used for the analysis. Standard precautionary
measures were adopted to minimize cross contamination of samples. Sterilized plastic containers and Durant
bottles were used for collecting water from collector wells. This was done carefully to avoid contact between the
containers and the walls of the wells, thus avoiding contamination of samples. Samples were labelled as Sample
01: collector well 04 water sample, Sample 02: observation well - water sample, Sample 03: collector well 02
water sample, Sample 04: collector well 03 slime and water sample, Sample 05: collector well 03 water sample
and Sample 06: collector well 01 water sample. The samples were carefully transported and stored at 4oC in the
laboratory refrigerators and later used for the physico-chemical analysis based on SLS and APHA guidelines
and microbiological and molecular biological studies.
Design of sampler
The sampler was made of stainless steel cylinder of 300 ml capacity. The upper end of the cylinder was
sealed but contained three holes through which the water can enter into the cylinder. A movable piston was set
up inside the cylinder and connected with the upper part of the sampler. Two long running threads were tied
each on the middle of the cylinder as well as the piston. The thread fixed to the piston was used to bring down
the sampler to a desired depth. Once the sampler reached to a desired depth the thread fixed to the piston was
released by tightening the other thread, which was fixed to the cylinder. After a few minutes again the thread
fixed to the piston was tightened, while releasing the other and the sampler was taken out from the well.
Optimization of growth conditions
The optimum temperature and NaCl concentration required for the growth of the bacterial isolate was
determined. For determination of optimum temperature, 0.1 ml of freshly prepared suspension of the bacterial
isolate was inoculated in 10 ml of iron added liquid media and incubated at different temperatures ranging from
065oC. After an incubation period of 72 hr, the growth was checked in the form of turbidity and by measuring
the OD at 600nm. Similar experiment was done with different concentrations of NaCl ranging from 050g.l-1
while incubated at 35oC in the iron added liquid media. Similar experiment was done with different pH values of
the media ranging from 4.012.0. Different buffer solutions were used to maintain the buffer of the liquid media
constant throughout the experiment.
Morphological and biochemical characterization of isolates
Colony morphology such as form, elevation, margin,opacity, diameter after 40 h growth (in mm), colour and
surface of the strain, were studied to identify the Genus of the isolated strain.Shape and arrangement of
endospore were observed under oil-immersion microscope after gram staining. Production of acid from different
carbohydrates such as glucose, xylose and mannose were tested. Production of urease, hemolysis of blood agar,
indole test, nitrate reduction test, decomposition of tyrosine, hydrolysis of starch, citrate utilization test and
Voges-Proskauer (VP) test were done on the isolated strain (Barrow & Feltham 1993, Fisher 1975,
Theivendrarajah 1990). Growth of the selected strain was tested at 5, 15, 25, 35, 40, 45, 50,55 and 60C at pH
9.0, and 100 rpm. The bacterial strain that was isolated from the water sample was subjected to gram staining
(Kaiser 2001) and motility test by hanging drop (Theivendrarajah 1990). Oxygen requirement test, test for
anaerobic growth, Catalase test, Oxidase test, Triple Sugar Iron Agar test and Lactose Fermentation test were
done on the isolated strain (Kapilan & Arasaratnam 2010, Theivendrarajah 1990).
Molecular Identification of bacterial isolate
The bacterial isolate was sent for identification by 16S rRNA sequencing at NCCS, India. The 16S rRNA
gene sequence of the isolate was aligned with reference 16S rRNA gene sequences of the European
Microbiological Laboratory (EMBL), GeneBank and the database of Japan using the BLAST algorithm
available online (http://blast.ncbi.nlm.nih.gov/).

RESULTS AND DISCUSSION

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Iron is found to be dissolved in water and whenbrought to the surface, it can form rust (Goonetilleke et al.
2015). Iron-reducing bacteria add iron to the water by attacking thepiping of the system. Removal of natural
ironsin the water requires special water treatment equipment (Hamilton 1985, Trivedy & Goel 1986).
Multiplication and spread of iron-reducing bacteria couldbe controlled by adequate chlorination.The most
common features that could be visible because of the colonization of iron bacteria in the water are appearance of
reddishwater, spotting, metallic taste of water and staining ofplumbing fixtures. These features are possible
when the concentration of iron in the water is more than 0.3 mg.l-1. The smells of most of the water ofthe
collector well is like irony. Irony smell may be due to thecorrosion of iron pipes.
Morphological characterization of isolates
Identification of genus of the isolated strain
To identify the species of the isolated strain, diverse guides (Chessbrough 1984, Theivendrarajah 1990) were
used.Tests on ground waters in Vallipuram area have been 95% positive for the presenceof iron bacteria and
microscopic examination of the surface growths has revealed the dominant types to be Crenothrix, Leptothrix,
Sphaerotilus and Gallionella. The cells possess a long spirally twisted stalk arising from centre of cell. The
bacterium is a kidney-shaped mycoplasmodial cell body. This body has a single elongated stalk that is made
from "numerous helically wound, uniquely mineralized fibrils outward from the convex side". These stalks are
generally covered in bacteriogenic iron oxide precipitate which gives it a redish-brown colour (Anderson &
Pedersen 2003). After 40 h of growth, the white colour colony with a diameter between 1.5 and 2.0 mm was
observed. Based on studies so farcarried out, it can be confirmed that the isolated strain belongs to the genus
Gallionella. Its species hasbeen identified, using the following experiments.
Morphological Characteristics
Gram staining showed gram negative kidney shaped (curved) and found in clusters (Fig. 1). But these
curvedcellshad a sheath on the outer surface which appeared lightly stained. The growth was powdery, opaque
and flat in elevation (Fig. 2). The bacterium Gallionella is generally a kidney-shaped mycoplasmodial cell body.
This body has a single elongated stalk that is made from numerous helically wound, uniquely mineralized fibrils
outward from the convex side (Anderson & Pedersen 2003, Halbach et al. 2001). Growth of the Gallionella sp.
was observed only in the iron agar media. When a loopful of the enriched broth added with iron was isolated on
the same solidified medium, a pale yellow coloured growth was observed on the plates after an incubation of 72
hours. Individual separate colonies were also obtained. Since only one type of growth was observed hence, it
could be concluded that it is possible to obtain the isolate as a pure culture. Since the iron agar media tend to dry
up very fast even after refrigeration, frequent sub-culturing was performed.

Figure 1. Microscopic view of bacterium Gallionella ferruginea growing on the iron agar media (Kidney shaped cells).
Determination of species of the isolated strain
When serum-water glucose medium, serumwater xylose medium and serum-water mannose medium were
inoculated with the strain and incubated at 37oC for 24 h, the colour changed to red with water glucose medium.
This indicated that the strain can produce acid from glucose but not from xylose and mannose. If the strain is a
urease-producer, the enzyme will hydrolyze urea by releasing ammonia and carbon dioxide. Ammonia released
will change the phenol red indicator to red pink colour due to the change in pH of the medium to alkaline
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conditions. Medium inoculated with strain did not show any colour change. This indicated that strain is not a
urease producer.When blood agar plate was inoculated with strain, around the bacterial colony a transparent
clear zone was not observed. This indicated that the strain has no capacity to break down the haem in
hemoglobin present in blood. When tryptophan is metabolized by an organism using the enzyme
tryptophanase,indole is produced and the presence of indole can be detected using Kovacs reagent. Kovacs
reagent is yellow in colour. It contains 4(p)-dimethylamino-benzaldehyde, which reacts with indole and
produces a red colour on the surface of the reagent. Light red or pink colour at the border of Kovacsindole
reagent and tryptophan solution (tryptone water) indicates the formation of indole (Theivendrarajah 1990).
When tryptone water medium was inoculated with the isolated strain and mixed with Kovacsindole reagent, red
colour ring was observed. This indicated that strain can utilize tryptophan and produce indole.

Figure 2. Colony morphology of the bacterium Gallionella ferruginea while growing on the iron agar media.
Reduction of nitrate to nitrite by an organism can be tested by incubating one loopful of inoculum in a broth
containing nitrate and after 4 h adding sulphanilic acid reagent to the broth. If nitrite is present the acid reagent
is diazotized and forms a pink-red compound with alpha napthalamine. The strain did not show any colour
change, indicating that it does not have the ability to produce nitrate reductase.If the organism decomposes
tyrosine crystals there should be a clear zone under and around the bacterial growth. When the tyrosine-agar
plates were inoculated with strain on the iron agar plate, no clear zone was observed around and under the
colonies. This indicated that the strain did not utilize tyrosine. If the organism produces starch hydrolyzing
enzymes it can hydrolyse starch into monosaccharides. To check whether the organism has utilized starch, after
the growth of the organism I2 has to be added. If there is blue colour formation, it indicates the production of
starch hydrolyzing enzyme (Theivendrarajah 1990). When starch-agar medium was inoculated with the strain
and I2/KI was added after 48 h of incubation, a blue colour was formed. This indicated that the strain did not
produce starch-hydrolyzing enzymes.
Bacteria that utilize citrate can extract nitrogen from the ammonium phosphate incorporated in the medium,
resulting in the production of ammonia, which combines with water to form NH4OH. These reactions in
combination produce an alkaline pH, resulting a colour change in the indicator from green to blue. If the
organism does not utilize citrate, the medium remains green in colour. When citrate phosphate broth was
inoculated with the strain, the medium remained green. This indicated that the bacterial strain did not utilize
citrate as the carbon source. If acetoin is produced by the organism, in the presence of sodium hydroxide
acetoine is oxidized to diacetyl. The diacetyl will give a pink compound with creatine. The isolated bacterial
strain was cultured in glucose-phosphate-peptone water and incubated at 37oC for 48 h. When creatine and
sodium hydroxide were added the culture and exposed to air, no pink colour was obtained. This indicated that
the strainhas no ability to produceacetoin by the fermentation of glucose.
Microscopic studies and Biochemical tests
Biochemical tests were carried out to confirm the genus of the strain and to identify the species. The
bacterial strain had shown good growth both under aerobic condition and anaerobic condition (in anaerobic jar).
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This indicated that the strain is mostly anaerobic. The strain could not produce O2 from H2O2. This showed that
the strain is not a catalase producer. If the bacterium oxidizes tetramethylp-phenylenediaminedihydrochloride, it
will turn purple, indicating that the organism can produce cytochrome oxidase. No change in colour indicates
that there is no production due to lack of cytochrome oxidase. The strain did not bring about a colour change.
Therefore, it does not produce cytochrome oxidase. The strain ferment lactose, sucrose and glucose. The strain
inoculated slants showed pink red butt and yellow slope. These results indicated that the bacterial strain
ferments glucose. When Mac Conkey agar medium was inoculated with the strain it did not change colour into
red. This indicated that strain does not ferment lactose.
Optimization of growth conditions
Table 1. Growth of bacterial strain at 72 hrs in iron added liquid culture media and different temperatures while shaking
at 100 rpm.
Temperature (oC) 0 5 15 25 30 35 40 45 55 65
OD (600nm) 0 0.026 0.569 0.953 1.122 1.297 0.988 0.674 0.192 0
Isolated bacterial strain was grown at varying temperatures, ranging from 5 to 55oC in liquid LB media. The
temperature range for growth of the isolated strain was 25 to 40oC. This bacterial strain grows at 35oC but no
growth at 0 and 65oC (Table 1). This bacterial strain grew well at temperatures between 2540oC and did not
grow at or below 0oC. At 55oC less growth (OD600 nm 0.192) was observed. Optimum growth of the bacteria
growing in the iron added liquid media, was observed at 35oC.When different concentrations of NaCl were
added to the iron added liquid growth media, the growth (OD at 600nm) of the strain was decreased
from1.264to 0.453at 42 h in the liquid media with NaCl concentration was increased from 0 g.l-1 to 10 g.l-
1
.Beyond 10g.L-1concentration of NaCl, the growth of the strain decreased. In the presence of 25g.L-1NaCl, no
growth (OD 600nm) was observed (Table 2). This bacterial strain grew well at pH valuesranging between 5.0
and 10.0 but it did not grow at pH values less than 5.0 and above 10.0.Optimum growth (OD 600nm= 1.383)
was obtained at pH 7.0 (Table 3).
Table 2. Growth of bacterial strain at 72 hrs in iron added liquid culture media containing different
concentrations of NaCl and 35C while shaking at 100 rpm.
NaCl (g.l-1) 0 1 2 4 6 8 10 25 50
OD (600nm) 1.264 1.036 0.956 0.854 0.825 0.694 0.453 0 0

Table 3. Growth of bacterial strain at 72 hrs in iron added liquid culture media at 35C and different pH values
while shaking at 100 rpm.
pH values 4 5 6 7 8 9 10 11 12
OD (600nm) 0 0.221 0.768 1.383 1.024 0.592 0.089 0 0
Final confirmation of species of identified strain Gallionella
Characteristics of the isolated bacterial strain were compared with other iron bacterial species. If the
character of strain is similar to the known species its score would be 1. If the character is not similar and
variable, it will not get any score. Total score was counted, divided by total characteristics and it was multiplied
by 100 and presented as a percentage. Based on these morphological findings and biochemical studies, the
isolated strain got the highest score of 95% showing similarities with Gallionella ferruginea. The strain showed
clear characteristics of Gallionella ferruginea than the other suspicious iron bacterial species. As the bacterial
strain got the highest score, it was identified as Gallionella ferruginea.
Molecular Identification of bacterial isolate
The alignment results using the BLAST algorithm showed that the 16S rRNA sequence was from a
bacterium Gallionella ferruginea. The most common water complaints are those of red water, laundry spotting,
metallic tastes, and staining of plumbing fixtures. These are usually due to the presence of iron above 0.3 mg.l-1.
The smells of most of the water of the collector well two is like irony. It may be due to the corrosion of iron
pipes collecting water in the Vallipuram area. The shortage of iron causes a disease called anaemia and
prolonged consumption of drinking water with high concentration of iron may be lead to liver disease called as
haermosiderosis. Industrialization without proper waste management system and excessive applications of
fertilizers and pesticides in agriculture are the two principle reasons of water pollution. Similar experiments
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have been performed worldwide to determine the water quality of diverse water sources (Health Canada 2007,
Kalwela & Savale 2012, Kapilan 2015, Perera et al. 2012, Tiimub et al. 2012).
Table 4. Brief summary of the bio chemical tests done on the water samples collected at different sources in the Vallipuram area.

Gallionella ferruginea is an iron-oxidizing chemolithotrophic bacteria that has been found in ground water.
These bacteria show a crucial role in fixing and oxidizing iron (Fe). It is a genus of stalked, ribbon-like bacteria
which employ iron in their metabolism, and cause staining, plugging and odour issues in well water system
(Anderson & Pedersen 2003). They cause dangerous problems in well water systems. Because it is challenging
to eradicate Gallionella ferruginea once they have entered well systems, prevention is the best safeguard. This
research is concerned primarily with the behaviour of iron in solution. Iron occurs in two oxidation states, the
divalent or ferrous form and the trivalent or ferric form. Iron in aqueous solution is subject to hydrolysis. The
iron hydroxides formed in these reactions. The ferric form, have very low solubility (Aiyesanmi et al. 2008).
The retention of iron in solution is consequently affected by the pH of the solution. In most natural waters, the
pH is not low enough to prevent hydroxides from forming, and under oxidizing conditions, practically all the
iron is precipitated as ferric hydroxide (Perera et al. 2012). Irons have a tendency to form complex ions with
inorganic and organic materials in solution state. These ions may be considerably more stable than the
noncomplex ion and more may remain in solution that might spoil the quality of the drinking water. The primary
source of iron is the water bearing strata (Maddison & Gagnon 1999, Perera et al. 2012). Corrosion of iron pipes
in a water distribution system can cause three different types of problems: 1. The pipe mass is lost through
oxidization to soluble iron species or iron-bearing scale. 2. The scale can accumulate as large tubercles that
increase head loss and decrease water capacity. 3. There will be a release of soluble or particulate iron
corrosion-by products into the good quality water. This will decrease the aesthetic quality of water and often
leads to yellowish or reddish water at the tap. Therefore corrosion of iron pipes has become as a serious issue in
the water industry.

CONCLUSION
Based on the microscopic, biochemical and culture studies, the isolated strain from thedrinking water
collector wells at Vallipuram area of the Northern Sri Lankawas identified as Gallionellaferruginea. This
bacteria was very active in growth at pH 7.0 and temperature 35oC in iron added liquid growth media. Reddish
brown slime formation and bad odour of the drinking water from the fresh water collector wells at Vallipuram
area could be prevented or minimized by the addition of NaClat very low concentration.
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ACKNOWLEDGEMENTS
The authors thank the funding and support provided by the University of Jaffna and the National water
supply and drainage Board, Jaffna, Sri Lanka. Special thanks to Selvaratnam, S and Thananjeyan, A. for their
timely help.

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ISSN (E): 2349 1183
ISSN (P): 2349 9265
3(2): 449459, 2016

Research article

Prediction of gene action and combining ability for yield and quality
traits in F1 and F2 generations of wheat (Triticum aestivum L.)
Saurabh Verma1, Ramanuj Maurya2* and Satyamvada Maurya3
1
Chandra Shekhar Azad Agriculture University, Kanpur, Uttar Pradesh, India
2
ICAR-Indian Institute of Pulses Research, Kalyanpur, Kanpur, Uttar Pradesh, India
3
Dr. A.P.J. Abdul Kalam Technical University, Lucknow, Uttar Pradesh, India
*Corresponding Author: rocksanuj918@gmail.com [Accepted: 19 August 2016]

Abstract: Combining ability analysis was studied in a 10 x 10 diallele set of bread to understand
the inheritance pattern of several yield and quality traits in bread wheat. Results showed that the
GCA variances were higher than SCA variances for plant height, spike length, duration of
reproductive phase, gluten content and grain yield per plant in both the generations. General
predictability ratio (GPR) did not reach near to unity for any of the traits in both the generations
except tryptophan content in F1 generation. On the basis of overall performance, parents K8962,
K9423, PBW343, GW373, HD2733 and Sonalika were best general combiners for grain yield and
other important characters. Among the cross significant and desirable SCA effects in order of
merit for yield and yield contributing traits were exhibited by GW373/K8962, K9107/K8027,
GW373/Sonalika, K8027/K8962, K8962/Sonalika, K9107/K9423, K8020/K9423, K9107/K9351,
K9107/K8962, K9107/Sonalika. These parents may be used for simultaneous improvement in
grain yield and quality attributes through an intermating population involving all possible
combinations among themselves.
Keywords: Triticum aestivum - GCA - SCA - Combining ability.

[Cite as: Verma S, Maurya R & Maurya S (2016) Prediction of gene action and combining ability for yield and
quality traits in F1 and F2 generations of wheat (Triticum aestivum L.). Tropical Plant Research 3(2): 449459]

INTRODUCTION
Wheat (Triticum aestivum L.) is the principal food grain of the world population belonging to the family
Gramineae are well known for their containing various phytochemicals that function as antioxidants (Ghose et
al. 2013) and its huge global consumption as human food. Wheat is the second most important staple food crops
(Joshi et al. 2007) next to rice, consumed by nearly 35% of the world population and providing 20% of the total
food calories (Desale & Mehta 2013). Wheat is a rich source of essential human diet ingredients like 70%
carbohydrates, 12% protein, 22% crude fibers, 2% fat, 12% water and 1.8% minerals. Yield and Quality traits
like grain protein-content (GPC) are primary importance in bread-wheat breeding programs. The success of any
crop improvement program depends mainly on a judicious selection of promising parents from gene pools, a
clear cut understanding of genetic mechanisms involved in the inheritance of characteristics which help the
breeders in deciding the most appropriate breeding procedure to enhance the genetic potentialities. The
identification of superior parents for hybridization is based on the ability of a particular line to combine with
other lines and produce desirable segregants/recombinants are important pre-requisite for efficient breeding
programme (Jaiswal et al. 2013). Hybridization is the most potent technique for breaking yield barriers and
evolving varieties having a built in high yield potential (Jaiswal et al. 2013). Singhet al. (2013) suggested that
heterosis play significant role in combining ability of parents and can be include in the breeding programme.
Diallel analysis provides a systematic approach for the identification of appropriate parents and crosses superior
to the traits investigated so far (Joshi et al. 2004).
The combining ability analysis is an important tool for the selection of desirable parents together with
information regarding nature and magnitude of controlling quantitative traits (Masood et al. 2014). The term
general combining ability (GCA) is associated with genes of additive effects, used to designate the average
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Received: 06 May 2016 Published online: 31 August 2016
Verma et al. (2016) 3(2): 449459
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performance of a line in hybrid combinations and specific combining ability (SCA) caused by dominance and
epistasis, to define those case in which certain combinations do relatively better or worse than expected on the
basis of the average performance of the lines involved. The diallel analysis is an efficient method for the study
of combining ability and gene action involved in the inheritance of traits. Kamaluddin et al. (2007) studies GCA
and SCA effects for yield and yield components in wheat. In bread wheat grain yield and grain protein content
are two very important traits (Groos et al. 2003). In a systematic breeding programme, selection of parents with
desirable characteristics having good general combining ability (GCA) and specific combining ability (SCA)
effects for yield and its trait components are essential (Gowda et al. 2012, Desale et al. 2014).The number of
parental lines can be tested in all possible combinations through diallele crosses (Hassan et al. 2007).
Therefore, knowledge about combining ability is important in selecting suitable parents for hybridization,
understanding of inheritance of quantitative traits and also in identifying the promising crosses is of paramount
importance in formulating an efficient breeding programme. Keeping in view, present investigation was carried
out to obtain more precise estimates of combining ability and genetic architecture for yield and quality traits in
bread wheat.

MATERIALS AND METHODS


Plant material
The material used for the present study was comprised of 10 varieties of hexaploid wheat (Triticun aestivum
L.), namely, K 9107, GW 373, PBW 343, HD 2733, K 8020, K 9423, K 9351, K 8027, K 8962 and Sonalika
were selected on the basis of their differences in maturity, plant height, grain yield and other important quality
traits at Section of Economic Botanist (Rabi Cereals), C.S. Azad University of Agriculture and Technology,
Kanpur. The details of genotypes and their pedigree are presented in table 1. These genotypes were used to
obtain hybrid combinations according to a diallel crossing scheme. In order to get F2 seeds of for final
experiment about 30 F1 seeds were raised in Wellington, Nilgiri Hills (Tamil Nadu) nursery during the off
season of summer in 2009.
Table 1. Pedigree details and some distinguishing features of the parents used in 10 diallel analysis in wheat.
SI No Parents Pedigree Diagnostic features
1 K 9107 K8107/K68 Single gene dwarf (d1), late in maturity, rusts tolerant, protein
content over 14%
2 GW373 HW2008/J505 Double gene dwarf (d2), resistant to rusts.
3 PBW 343 ND/VG1944//KAL//BB/3/ Double gene dwarf (d2), resistance to rusts, protein content over
YACO5/4/VEE5S 12%.
4 HD2733 ATTILA/3/TUI/CARC/ Double gene dwarf (d2), high yielding resistance to rusts
CHEN/CHT014/ATTILA
5 K 802 K. Sona/ HD 1982 Double gene dwarf (d2), early in maturity, resistance to rusts,
6 K 9423 HP 1633/ K.Sona/UP262 Double gene dwarf (d2), resistance to rusts and heat tolerant, high
protein content
7 K9351 K72/HD2160 Double gene dwarf (d2), medium in maturity, resistance to brown
rust
8 K8027 HD1696/ K852 Single gene dwarf (d1), resistant to rusts
9 K8962 K7401/ HD2160 Double gene dwarf (d2), early maturity, highly tolerant to all the
three rusts
10 Sonalika II54-388/AN/3/YT54/ Single gene dwarf (d1), tolerant to rusts
N10B//LR64
Experimental design
The experiment for the present investigation was carried out with 10 parents and their resulting F1 which
were grown in a triplicate randomized block design during crop years 20072008. The seeds were dibbled in
rows keeping within and between row distances at 10 and 25cm, respectively. Single row of 3 m length served
as an experimental unit. Three seeds per hill were sown to ensure the crop stand, which were thinned to single
seedling per site after germination. At maturity 5 randomly selected competitive plants in each test entry per
replication in F1 and F2 were tagged before flowering and observations were recorded for 14 traits: days of 75%
flowering, days of 75% maturity, duration of reproductive phase, number of ear bearing tillers/ plant, plant
height (cm), spike length (cm), ear density (cm), number of grains/spike, seed hardness (by O.S.K. 201 grain

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hardness Tester), protein content (by Biuret method in %), tryptophan content (%), phenol colour reaction
(score), gluten content (%), and grain yield/ plant (g).
Statistical analysis
The data of various traits were compiled and mean values of the replicated data were used for statistical
analysis using WINDOSTAT software, Hyderabad, India. The mean data of each replication were tested for
significance by the method of analysis of variance (ANOVA) suggested by Panse and Sukhatme (1967). The
breeding values of the parental lines have been evaluated by analyzing the combining ability estimates
according to Griffings (1956) for both F1 and F2 generations.

RESULTS
Analysis of variance (ANOVA)
Analysis of variance for combining ability revealed that the variances due to GCA and SCA were significant
for all the traits in both generations (Table 2). This indicated existence of genetic variability in the parental lines
and involvement of both additive and non-additive gene effects in the inheritance of these traits. The magnitude
of estimates of GCA variances were higher for plant height, spike length, duration of reproductive phase, gluten
content and grain yield per plant than the respective estimates of SCA variances in both the generations. General
predictability ratio (GPR) did not reach near to unity for any of the traits in both the generations except
tryptophan content in F1 generation indicated non-additive component played relatively greater role in the
inheritance of traits.
Table 2. Analysis of variance (ANOVA) for combining ability of 14 characters in a 10 parent-diallel cross in F1 and
F2 generations of wheat.
Source GCA SCA Error 2g 2s GPR
df 9 45 108
DF 75% F1 6.03** 8.57** 0.32 0.47 8.25 0.10
F2 9.35** 12.33** 4.11 2.44 8.22 0.05
PH F1 144.71** 126.11** 0.92 11.98 25.19 0.32
F2 130.11** 123.99** 0.75 10.78 23.24 0.31
NEBT F1 1.42** 1.34** 0.39 0.09 0.95 0.80
F2 0.91** 1.88** 0.40 0.04 1.48 0.26
SL F1 0.65** 0.41** 0.12 0.04 0.29 0.12
F2 0.40** 0.30** 0.10 0.03 0.20 0.13
DM F1 157.21** 215.23** 0.46 13.06 14.47 0.47
F2 153.13** 116.70** 0.51 12.72 16.19 0.44
DRP F1 5.86** 3.42** 0.36 0.46 3.06 0.13
F2 8.06** 2.85** 0.27 0.67 2.58 0.20
ED F1 49.33** 33.55** 2.44 3.91 31.11 0.12
F2 41.12** 43.52** 4.50 3.05 39.02 0.07
NG F1 24.21** 31.73** 0.51 1.97 31.22 0.05
F2 14.00** 40.00** 6.65 0.62 33.85 0.02
PC F1 0.28** 0.53** 0.06 0.01 0.47 0.03
F2 0.31** 0.42** 0.04 0.02 0.38 0.05
TC F1 0.08** 0.08** 0.008 0.06 0.07 1.77
F2 0.06** 0.06** 0.004 0.004 0.05 0.07
PCR F1 0.79** 1.14** 0.41 0.03 0.73 0.03
F2 1.09** 0.95** 0.41 0.05 0.54 0.08
GC F1 0.07** 0.03** 0.002 0.005 0.02 0.2
F2 0.66** 0.04** 0.005 0.05 0.03 0.62
SH F1 0.54** 0.68** 0.19 0.02 0.49 0.007
F2 0.23** 0.67** 0.14 0.007 0.53 0.013
GY F1 24.37** 12.90** 0.94 1.95 11.96 0.14
F2 21.00** 16.24** 1.21 1.64 15.03 0.098
Note: *Significant at 5% level; ** significant at 1% level
GCA-General combining ability variance, SCA-Specific combining ability variance, 2g-Estimates of GCA variance,
2S-Estimates of SCA variance, GPR - General predictability ratio.
Days to 75% flowering (DF 75%), Plant height (PH), Number of ear bearing tillers/plant (NEBT), Spike length (SL),
Days to maturity (DM), Duration of reproductive phase (DRP), Ear density (ED), Number of grains / spike (NGS),
Protein content (PC), Tryptophan content (TC), Phenol colour reaction (PCR), Gluten content (GC), Seed hardness
(SH), Grain yield/Plant (GY).
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General combining ability and performance of parents
Considering GCA effects of the parents for all the 14 characters in F1 and F2 progenies are presented in table
3. Lowest negative and significant values of GCA effects were considered to be desirable for number of days to
75% flowering, plant height, days to maturity, phenol colour reaction while for remaining characters positive
and significant GCA effects were taken into account for sorting out the desirable general combiners. In F1
generation, significant and desirable GCA effects were observed for days to 75% flowering in K8020, K9351
and K8962, for plant height in K9423, K9351 and K8962 for days to maturity in K8020, K9423, K9351 and
K8962, for phenol colour reaction in K8027. In F2 generation, significant and desirable GCA effects were found
for days to 75% flowering in K8962, days to maturity in K8020, K9423, K9351 and K8962, for plant height in
K9423, K9351 and K8962; for phenol colour reaction in K9107 and K8027.
Table 3. Estimates of GCA effects and corresponding mean performance of the parents for 14 characters in 10 parents of diallel cross in
F1and F2 generation of wheat.
Parent K9107 GW373 PBW343 HD2733 K8020 K9423 K9351 K8027 K8962 Sonalika SE(gi ) SE(gi - gj)
DF 75% F1 0.11 0.84** 0.79** 0.11 -0.38** 0.81** -0.76** 0.01 -1.37** 0.63** 0.17 0.24
F2 0.59 1.43* 0.99 0.51 -0.05 0.2 -1.06 -0.86 -1.30* 1.07 0.57 0.86
PH F1 -0.2 3.76** 3.25** 0.48 4.76** -6.86** -2.11** 1.13** -3.22** 2.25** 0.26 0.41
F2 0.44 3.61** 3.33** -0.12 5.16** -5.45** -2.10** 0.66* -4.09** 1.88** 0.24 0.37
NEBT F1 0.15 0.64** 0.82** -0.33 0 -0.24 -0.39** -0.33 0.2 0.39* 0.17 0.26
F2 0.07 0.43* 0.51** -0.26 -0.23 -0.35 -0.23 0.22 0.18 0.27 0.18 0.27
SL F1 -0.07 0.46** 0.62** -0.24* -0.31** -0.06 -0.04 0.26* 0.12 -0.12 0.1 0.14
F2 -0.07 0.40** 0.59** -0.20* -0.17 -0.04 -0.08 -0.04 0.22* -0.02 0.09 0.14
DM F1 2.40** 4.09** 3.17** 0.64** -1.50** -4.91** -5.66** 0.70** -1.30** 5.55** 0.19 0.28
F2 2.55** 4.21** 4.33** 0.15 -1.55** -4.73** -5.76** 0.73** -0.85** 5.33** 0.2 0.3
DRP F1 0.13 1.38** 1.12** -0.71** -0.81** 0.85** -0.50** -0.29 -0.29 0.23 0.17 0.25
F2 1.22** 1.30** 1.16** 0.2 -0.14 -1.22** 0.29 0.07 -1.02** -0.11 0.15 0.22
ED F1 1.72** 3.15** 2.95** 0.06 -0.81 0.12 -2.95** -0.38 -3.02** 2.11** 0.44 0.66
F2 -0.41 2.20** 2.32** 1.51* -0.83 0.5 -1.14 0.08 -3.94** 2.20** 0.6 0.9
NG F1 0.52* 0.89** 0.92** 0.95** -2.84** 0.58* 1.58** 0.34 -2.14** 0.13 0.2 0.3
F2 0.07 1.43* 1.61** 1.19 -2.29** 0.28 0.29 0.2 -1.11 -0.05 0.73 1.1
PC F1 -0.07 -0.06 -0.12 0.09 0.02 0.18* 0.18* -0.33** -0.02 0.13 0.07 0.1
F2 0.01 -0.20** 0.25** 0.09 0.02 0.19** 0.09 -0.35** 0.1 0.05 0.05 0.08
TC F1 -0.08* 0.16** 0.19** 0.09** 0.02 -0.07* 0.03 -0.08* -0.02 -0.05 0.03 0.04
F2 -0.01 0.09** 0.09** 0.09** 0.01 0.001 -0.02 -0.12** 0.05* -0.10** 0.02 0.03
PCR F1 -0.2 -0.08 -0.06 0.25 0.13 0.43* 0.01 -0.51* -0.05 0.01 0.18 0.27
F2 -0.41* -0.04 -0.05 0.14 0.17 0.65** -0.04 -0.38* -0.07 -0.01 0.18 0.27
GC F1 0.03** 0.07** 0.06** 0.05** -0.12** 0.07** -0.01 0.02 -0.16** 0.05** 0.01 0.02
F2 0.02 0.08** 0.09** 0.07** -0.11** 0.01 -0.01 0.03 -0.12** 0.04 0.02 0.03
SH F1 -0.05 -0.25* -0.27* 0.11 0.27 0.34* 0.11 -0.17 -0.08 -0.29* 0.12 0.18
F2 0.01 -0.28* -0.31* 0.08 0.01 0.24* 0.04 0.01 0.02 -1.13** 0.1 0.16
GY F1 0.85** 2.76** 2.32** 0.78** -1.05** 0.76** -1.03** -0.43 -1.61** 1.14** 0.26 0.41
F2 0.75* 2.19** 2.45** 0.81* -0.47 0.78* -0.84* 0.04 -2.62** 0.92* 0.31 0.47
Note: * Significant at 5% level; ** significant at 1% level.
Days to 75% flowering (DF 75%), Plant height (PH), Number of ear bearing tillers/plant (NOEBT), Spike length (SL), Days to
maturity (DM), Ear density (ED), Number of grains / spike (NOGS), Protein content (PC), Tryptophan content (TC), Phenol
colour reaction (PCR), Gluten content (GC), Seed hardness (SH), Grain yield (GY).
Specific combining ability effects
The specific combining ability (SCA) effects estimated for all the traits are presented in Appendix I. On the
basis of SCA effects and per se performance of the hybrids (Table 4), it was noted that the crosses were not
exactly in the same order of ranking. In the present findings, best combinations mostly involved high low and
low low general combiners for the characters under study high x low GCA effects played an important role in
the expression of positive and significant SCA effects. The cross combinations GW373/K8962, K8027/K8962
and K9423/K8962 in F1 generation and K9423/K8962 and K9107/K9423 in F2 generation involved one parent
with desirable and significant GCA effects and other with poor or negative effects.
Considering both the F1 and F2 generations simultaneously, for grain yield per plant GW373/K8962,
K9107/K8027 and GW373/Sonalika for protein content K8027/K8962, for tryptophan content K8962/Sonalika,
for gluten content K9107/K9423 and K8020/K9423, for seed hardness K9107/K9351, K9107/K8962 and
K9107/Sonalika were most desirable and common cross combinations based on SCA and per se performance.

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Table 4. Good specific combinations on the basis of Per Se performance and SCA effects for 14 Characters in a 10parents
diallel cross in wheat.
Cross combination Good specific combinations Common cross
Character with high Per Se combination based on sca
performance F1 F2 and Per Se performance
DF 75% GW373 X K8027 K8027 X K8962 PBW343 X K8020 K8027 X K8962
K9107 X HD2733 K8020 X K8962 K9107 X K8962
PH GW373 X PBW343 K9107 X K8027 K9107 X K9423 K9107 X K9423
K9107 X HD2733 K8020 X K9351 K8020 X K9351
NEBT GW373 X K8962 GW373 X K8962 PBW343 X K8027 K9107 X K8962
GW373 X Sonalika GW373 X Sonalika GW373 X K8962
SL K8020 X K9423 K8020 X K9423 K8027 X Sonalika K8027 X Sonalika
PBW343 X K8962 GW373 X K9423 PBW343 X K8020
DM PBW343 X K8962 PBW343 X K8962 K9423 X K8962 K9423 X K8962
PBW343 X Sonalika PBW343 X onalika K9423 X Sonalika K9423 X Sonalika
DRP K9107 X K8027 K9107 X K8027 K8020 X K9423 K9107 X K8027
GW373 X Sonalika GW373 X K8962 PBW343 X K9351
ED GW373 X Sonalika PBW343 X K8962 K8020 X K9423 GW373 X K8962
GW373 X K8027 GW373 X K8027 K9107 X K8027 GW373 X Sonalika
NG K8962 X Sonalika GW373 X K8027 K8020 X K9423 GW373 X K8027
K8027 X K8962 K8020 X K9423 K8027 X K8962 K8027 X K8962
PC GW373 X PBW343 K8020 X K8027 K8962 X Sonalika K8027 X K8962
HD2733 X K8962 GW373 X K8962 K8027 X K8962
TC K9351 X K8962 K8962 X Sonalika K8962 X Sonalika K8962 X Sonlika
K9351 X Sonalika K9351 X Sonalika K9107 X K8962
PCR K8020 X K9351 K8020 X K8027 K9423 X Sonalika
K8020 X K8027 K8020 X K9351 K9423 X K8962 -
GC K9107 X K8027 K9107 X K9423 GW373 X Sonalika K9107 X K9423
K9107 X K9423 K8020 X K9423 K8020 X K9423 K8020 X K9423
SH K9107 X K9351 K9107 X K9351 GW373 X K8020 K9107 X K9351
GW373 X K9423 K9107 X K8962 K9107 X K8962 K9107 X K8962
GY GW373 X K8962 GW373 X K8962 K8020 X K9423 GW373 X K8962
GW343 X HD2733 K9107 X K8027 K9107 X K8027 K9107 X K8027
Note: Days to 75% flowering (DF 75%), Plant height (PH), Number of ear bearing tillers/plant (NEBT), Spike
length (SL), Days to maturity (DM), Ear density (ED), Number of grains / spike (NOGS), Protein content (PC),
Tryptophan content (TC), Phenol colour reaction (PCR), Gluten content (GC), Seed hardness (SH), Grain
yield/Plant (GY)

DISCUSSION
The population of India is continuously growing and many times we face the problems of drought and heavy
rains which decrease the production of wheat. The systematic breeding with improved quality of seed grains can
fulfill the hungry problems in India. The development of wheat varieties possessing improved yield related
characters has been the major objective of wheat breeders. Availability of genetically based variation for these
traits viz. Plant height, Number of ear bearing tillers/plant, Spike length, Days to maturity, Duration of
reproductive phase, Ear density, Number of grains / spike, Protein content, Tryptophan content, Phenol colour
reaction, Gluten content, Seed hardness, Grain yield/Plant is a pre requisite for the selection of new cultivars.
Present wheat material was studied to generate information on general and specific combining ability for these
traits. Similar findings were also reported by Saeed et al. (2010) and Zahid et al. (2011). Combining ability
describes the breeding value of parental lines to produce hybrids (Romanus et al. 2008). The general combining
ability has been equated with additive gene action and specific combining ability with non-additive gene action
(Griffing 1956). A basic requirement in any effective hybridization programme is to identify superior genotypes
which could excel in their combining ability. General combining ability effects have successfully been used in
making the choice of the parents and also for the isolation of the germplasm base for further improvement. This
information on yield and other agronomic traits is important and greatly help in the proper classification of
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parental lines. Hassan et al. (2007) studied combining ability in the F1 generations of diallele for yield and yield
components in wheat found best general combiner for grain yield per plant and grain weight per spike. The
crosses showing high SCA effects with at least one good general combiner could produce desirable
transgressive segregants in subsequent generations. Nazir et al. (2005) studied combining ability analysis for
yield and yield contributing traits using 5 x 5 diallele cross of wheat show similar finding. The crosses involving
good general combiners and showing high SCA effects may be utilized for further breeding purposes.

CONCLUSION
In conclusion, it is clear the information on GCA effects should be supplemented by SCA effects and hybrid
performance of cross combinations to predict the gene action for yield and quality related traits in segregating
generations. The identified crosses hold great promise in improving the grain yield and quality attributes in
future breeding programme of bread wheat.

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