A0560106

IOSR Journal of Pharmacy and Biological Sciences (IOSR-JPBS)
e-ISSN: 2278-3008.Volume 5, Issue 6 (Mar. – Apr. 2013), PP 01-06
www.iosrjournals.org
www.iosrjournals.org 1 | Page
AM Fungal Status in Ketaka: Pandanus fascicularis From Coastal
Region of Konkan, Maharahshtra
*
Vishal R. Kamble, Lalji R. Kanoujiya, Harshal L. Rahate and
Dinesh G. Agre
[Mycorrhizal Research Laboratory, Dept. of Botany, Bhavan’s College Andheri (W), Mumbai (MS) India]
Abstract: Present paper deals with assessment of Arbuscular mycorrhizal fungi (AM) associated with different
populations of Pandanus fascicularis found in coastal region of Konkan Maharashtra. All samples of P.
fascicularis roots were colonized by AM fungi. The mean percentage of root length colonization ranged from
39% to 74%. Amongst the thirteen AM fungal morphospecies Kuklospora colombiana was the most widely
distributed species. Species richness of AM fungi ranged from 3 to 6. Based on spore density and relative
abundance, three species were dominant viz., Acaulospora bireticulata, A. scrobiculata, and K. colombiana.
Details of AM fungal status in P. fascicularis are discussed in present paper.
Keywords - Arbuscular Mycorrhizal Fungi, Pandanus fascicularis, Relative abundance, Species diversity,
Spore density.
I. INTRODUCTION
More than 500 species are known to occur in genus Pandanus, out of these 36 species have been
recorded in India [1]. The Ayurvedic drug Ketaka is Pandanus fascicularis Lam. (Synonyms: P. tectorius, P.
odoratissimus) belonging to the family Pandanaceae [1-2]. Vernacular names of this plant are: Sansktit- Ketaki;
Hindi-Keura, Kewda, Gagandhul; Tamil- Tazhai; Telugu-Mugali; Kannada-Tale Mara; English-Screw pine [3-
4]. It grows wildly in coastal regions of India and Andaman islands [5]. Its distribution is documented over
coastal Districts of Orissa, Andhra Pradesh, Tamil Nadu, and to some extent in parts of Uttar Pradesh [6]. In
Konkan region of Maharashtra its distribution is confined with backshore zone of coastal area especially at
Raigad, Ratnagiri and Sindhudurg District. The fragrant spadices of staminate inflorescence are more frequently
used by Indian women for hair dressings in Maharashtra. Kewda attar and water are used for flavoring various
foods, sweets, syrups and soft drinks. They are popular in north India, especially on festive occasions.
It is now well documented that, different plants are widely used in various traditional systems of
medicine like Ayurveda, Siddha and Unani to treat rheumatic fever, rheumatism and rheumatoid arthritis and P.
fascicularis is a good example of it which has proved its analgesic activity [5]. In Ayurveda Ketaka used for
headache, anorexia, indigestion, constipation, eye diseases, leprosy, hypoglycemic activity [7-8]. Recently
Madhavan et al. [9] suggested that P. fascicularis root possesses the potent antihyperglycemic activity which
may be due to the presence of phenolic compounds, tannins, and flavonoids. Ecologically, the P. fascicularis is
also important as its root system binds soil and checks soil erosion. On sandy soils along the sea coast, it fixes
sand dunes, which act as a barrier against encroachment of the sea and protect human settlements along the
coast line [10].
The cultivation practices, marketing and trading of Kewda has proved positive impact on the local
economy at Ganjam District, Orissa, India [6], where it constitutes the backbone of the local economy [10].
Although Maharashtra has a coastline of 720 Km which is composed with either sandy or rocky area, it has wide
scope for cultivation of P. fascicularis. However, for such practices extensive study of coastal region is
necessary which includes physical, geological and biological aspects like microbial association with coastal
flora. Although, beach ecosystems in India particularly from Goa have been studied for first two aspects and for
restoration of degraded coastal dunes very well [11-13], unfortunately such efforts have not made in
Maharashtra. The microbial association (especially mycorrhizal) in coastal vegetation has been always remained
unexplored in Maharashtra.
It is confirmed that, mycorrhizal colonization benefits soil rehabilitation and erosion control by
stimulating soil aggregation [14-15]. The extramatrical mycelium of Arbuscular Mycorrhizal fungi (AM) during
symbiosis with the plant also provides a large surface area on colonized root and thus help in maximum
absorption of orthophosphate from bulk soil [16]. Hence in present paper it was thought worthwhile to assess
the AM fungal diversity associated with different populations of P. fascicularis which is established as very
dense vegetation in Konkan region of Maharashtra.

AM Fungal Status in Ketaka: Pandanus fascicularis From Coastal Region of Konkan, Maharahshtra
II. Material And Methods
2.1 Sample collection:
Roots and rhizosphere soil samples of different populations of P. fascicularis found in coastal region of
Konkan Maharashtra were collected during 2010-2012. Study area comprises five sites belonging to three
Districts viz., Raigad: Revdanda beach; Ratnagiri: Murud beach Dapoli, Ganpatipule beach; Sindhudurg: Munge
beach and Tambaldeg beach respectively. At each of the field sites, 5 soil core samples per tree were taken at a
depth of 5-30 cm using a soil digger and spade. Approximately 1 kg total of rhizosphere soil from each site was
collected. Soil samples were mixed into composite samples and root samples were removed from the soil and
preserved in 70% ethanol for each tree.
2.2 AM fungal root colonization assessment:
In the laboratory, roots of each plant from every location were made free from sand and soil-debris by
washing, after clearing and staining technique of Phillips and Hayman [17]. Roots fixed in 70% ethanol were
cleared in 10% (w/v) KOH solution and autoclaved at 121°C and 15 lb/inch2
for 15 minutes. Then, roots were
washed with distilled water to remove KOH, stained with 0.05% cotton blue dye for overnight. They were
observed under a binocular microscope using lactophenol to evaluate mycorrhizal colonization [18]. Fifty
stained roots (each about 1 cm in length) were assessed for colonization percentage using the intercept method
[19] under a Olympus compound microscope.
2.3 AM fungal spore isolation and species identification:
After root removal, the sand samples from each location were combined to obtain a single sample per
location. AM fungal spores occurring in the rhizosphere soil samples were extracted directly by using the wet
sieving and decanting method of Gerdemann and Nicolson [20]. 100 g of each soil sample was suspended in 500
ml of water and stirred for 10 mins. Sieve sizes of 250, 210, 120, 75 and 45 μm, were used for spore collection.
The spores retained on each sieve were transferred to filter paper and subsequently examined under a Magnus
Stereomicroscope at a magnification of up to 400X and identified based on spore morphology. Each spore
morphotype was mounted in polyvinyl-lactoglycerol (PVLG) and PVLG mixed with Melzers reagent in 1:1
(v/v) ratio [21]. Identification of AM fungal species was based on original descriptions of Schenck and Pérez
[22] and also using current species descriptions and identification manuals (International Culture Collection of
Vesicular and Arbuscular Endomycorrhizal Fungi [http://
invam.caf.wvu.edu/Myc_Info/Taxonomy/species.htm]).
2.4 AM fungi species status:
2.4.1 Spore density (SD) is the number of spores in 100 g soil. Relative abundance (RA) was defined as the
percentage of spore numbers of a species divided by the total spores observed [23]. The frequency isolation (IF)
of each AM fungal species was calculated by the percentage of the number of the samples in which the species
or genus was observed. The dominant AM fungal species according to relative abundance (RA > 6%) and
spore density in 100 g soil (spore density higher than 40 spores) and species richness were determined for each
sampling site.
2.4.2 Diversity index and concentration of Dominance AM fungal diversity was evaluated using the
Shannon-Weiner diversity index which has two main components, evenness and number of species [24]. The
Shannon–Weiner index (H΄) was calculated according to the formula H΄ = -Σ(ni/N) log2 (ni/N), where ni
represents individuals of a species and N represents the total number of species. Concentration of dominance (C)
was also measured by the Simpson’s index [25] using the formula C = Σ(ni/N)2
, where ni and N are the same as
for Shannon–Weiner diversity index.
III. Results And Discussion
All samples of P. fascicularis roots were colonized by AM fungi (Fig. 1a-f). The mean percentage of
root length colonization ranged from 39% in Pf3 to 74% in Pf2 (P < 0.05), and generally exceeded 45% (Table
1). Significant differences in percentage of root colonization occurred between sampling sites (P < 0.05). In
addition to regular components of AM fungi other fungal endophytes (ofe) were also recorded in Pf1, Pf2 and
Pf5. Whereas, chlamydospore (s) were observed in Pf1. Thus, P. fascicularis appears to be readily colonized by
AM fungi under a coastal soil conditions. Recently Kamble and Mulani, [26] reported Arum-type of AMF
colonization in the roots of P. fascicularis from a sandy beach at Murud Dapoli in Maharashtra. As a part of its
extension, in present work we have investigated AM fungal species associated with those samples which were
recently collected by Kamble and Mulani, [26].

In present work, total, 1275 AM fungal spores were derived using wet sieving and decanting technique
rhizosphere soil samples of P. fascicularis from five study sites (Pf1-Pf5). Spore density in samples ranged from
39 to 890 spores 100 g-1
soil (Table 1). Maximum spore density was observed in Pf 1 (890±7.4) and minimum
in CM1 (39±2.7). There was a significant difference (P < 0.05) in spore density between many of the sites
(Table 1).
Thirteen morphospecies of AM fungi were identified using spore characteristics. Species richness of
AM fungi ranged from 3 to 6 (average 4.4). Species were distributed as follows: 3 species from Pf3, 4 from Pf1
& Pf4, 5 from Pf2, and 6 species from Pf1 (Table 2). Acaulospora and Kuklospora occurred most frequently and
overall, were the most prevalent, containing 5 and 1 species, respectively. There were 2 species in Glomus, 1
species in Ambispora, Diversispora, Entrophospora and Scutellospora across the 5 sampling sites (Table 2).
In present study, Kuklospora colombiana was the most widely distributed species. It was found in 4
out of 5 sampling sites, namely Pf1, Pf2, Pf3 and Pf4 (80% IF). The next most widely distributed taxon was
Acaulospora scrobiculata which appeared in 3 sites, Pf1, Pf2 and Pf3 (60% IF). Acaulospora bireticulata and
Glomus etunicatum both were appeared in 2 sites, viz., Pf4 & Pf5 (40% IF). Out of thirteen, 8 species were only
found at one site (20% IF), for example, Acaulospora laevis in Pf3, Acaulospora spp 1, Acaulospora spp 2 &
Ambispora callosa in Pf3, Diversispora versiformis in Pf4, Entrophospora baltica in Pf1, Glomus
heterosporum in Pf2 and Scutellospora spp in Pf2 (Table 2). Although, Scutellospora projecturata was also
appeared in 3 sites, Pf1, Pf2 and Pf5 (60% IF), its relative abundance (0.549 RA) was comparatively low (Table
3). In present study AM fungal species viz., Acaulospora callosa, Entrophospora baltica, Kuklospora
colombiana and Scutellospora projecturata have been first time recorded for any other known coastal sand dune
plant from Maharashtra. Thus it makes new addition of these four AM fungal species for Maharashtra with
reference to P. fascicularis.
Based on spore density and relative abundance, three species were dominant (> 40 spores 100 g-1soil,
RA ≥ 6%); Acaulospora bireticulata (112 spores, 8.8%), Acaulospora scrobiculata (467 spores, 36.6%), and
Kuklospora colombiana (429 spores, 33.6%). Although, Diversispora versiformis and Entrophospora baltica
were encountered with more than 40 spores 100 g-1
soil, however relative abundance was 4.862 and 5.019
respectively (RA ≤ 6%). Hence, these two species were considered as non-dominant (Table 3). The
morphological characteristics of AM fungal spores recovered during study are illustrated in Fig. 2.
Species diversity was calculated using two indices. The Shannon–Weiner diversity index ranged from
0.915 to 1.471. The highest occurred in Pf5 (H΄ = 1.471) and the lowest in Pf1 (H΄ = 0.915). Similarly, the
Simpson’s index ranged from 0.209 to 0.575, the highest was in Pf5 and the lowest was in Pf1 (Table 1).
IV. Tables
Table 1. AM fungal root colonization, Spore density (SD), Shannon-Weiner index (H΄) and Simpson’s
index (D) in P. fascicularis at each sampling site.
Samples and Collection site AM colonization*1
(%) SD2
H΄ D
Raigad District
Pf1 [Revdanda beach :18°33'28"N 72°55'16"E] 50.33±1.9a 890±7.4 0.915 0.209
Ratnagiri District
Pf2 [Murud beach Dapoli: 17°45′32"N 73°11′8"
E]
74.66±1.3b 39±2.7 1.053 0.419
Pf3 [Ganpatipule beach:17°8'47"N 73°15'53"E] 39±1.2 c 74±2.6 1.084 0.555
Sindhudurg District
Pf4 [Munge beach 16°14'2"N 73°25'19"E] 51.66±1.8a 118±7.2 1.007 0.408
Pf5 Tambaldeg beach [16°16'56"N 73°24'31"E] 48.33%±1.9a 154±7.3 1.471 0.575
[*The same letter in the column indicates that there is no significant difference at α = 0.05; 1
mean±SD, n = 50;
2
mean±SD, n = 2]
Table 2. AM fungal Spore density (S) and relative abundance (RA) at each sample site of P. fascicularis.
AM fungal
Species
Sample site*
Pf1 Pf2 Pf3 Pf4 Pf5
S RA S RA S RA S RA S RA
Acaulospora 424 47.64 23 58.97 50 67.57 42 35.59 114 74.03
A. bireticulata - - - - - - 42 35.59 70 45.45

A. laevis - - - - 30 40.54 - - - -
A. scrobiculata 424 47.64 23 58.97 20 27.03 - - - -
Acaulospora spp 1 - - - - - - - - 32 20.78
Acaulospora spp 2 - - - - - - - - 12 7.79
Ambispora 0 0 0 0 0 0 0 0 23 14.93
A. callosa - - - - - - - - 23 14.93
Diversispora 0 0 0 0 0 0 62 52.54 0 0
D. versiformis - - - - - - 62 52.54 - -
Entrophospora 64 7.19 0 0 0 0 0 0 0 0
E. baltica 64 7.19 - - - - - - - -
Glomus 0 0 11 28.20 0 0 12 10.17 13 8.44
G. etunicatum - - - - - - 12 10.17 13 8.44
G. heterosporum - - 11 28.20 - - - - - -
Kuklospora 400 44.94 03 7.69 24 32.43 02 1.74 0 0
K. colombiana 400 44.94 03 7.69 24 32.43 02 1.74 - -
Scutellospora 02 0.22 02 5.12 0 0 0 0 04 2.60
S. projecturata 02 0.22 01 2.56 - - - - 04 2.60
Scutellospora spp - - 01 2.56 - - - - - -
Total spores 890 100 39 100 74 100 118 100 154 100
Species richness
(SR)
4 5 3 4 6
Table 3. Identified AM fungi with their spore density (S), isolation frequency (IF) and relative abundance
(RA) in P. fascicularis rhizosphere (dominant species are in bold).
AM fungal species S IF RA
Acaulospora bireticulata Rothwell & Trappe. 112 40 8.784
Acaulospora laevis Gerd &Trappe. 30 20 2.351
Acaulospora scrobiculata Trappe 467 60 36.626
Acaulospora spp 1 (Unidentified) 32 20 2.509
Acaulospora spp 2 (Unidentified) 12 20 0.941
Ambispora callosa (Sieverd.) C. Walker, Vestberg & A.
Schüssle
23 20 1.803
Diversispora versiformis (Karst.) Oehl, silva & Sieverd. 62 20 4.862
Entrophospora baltica Blaz., Madej & Tadych 64 20 5.019
Glomus etunicatum Becker & Gerd. 25 40 1.960
Glomus heterosporum Smith & Schenck 11 20 0.861
Kuklospora colombiana (Spain & Schenck) Ohel & Sieverd 429 80 33.646
Scutellospora projecturata Kramadibrata & Walker 07 60 0.549
Scutellospora spp (Unidentified) 01 20 0.078
Total: AM fungi 13 species 1275 - 100

V. Figures
Fig. 1(A-E): AM fungal root colonization in different samples (Pf1-Pf5 respectively) of P. fascicularis [A-
Arbuscule, H- Hyphae, hc -Hyphal coiling, Lm- Linear mycelium, Lv- Linear Vesicles, Ofe-Other fungal
endophyte, ofm- Other fungal mycelium, S- Chlamydospore, V- Vesicle].
Figure 2. The morphological characteristics of AM fungal spores recovered from different samples of P.
fascicularis: 2.1. Acaulospora bireticulata Rothwell & Trappe:. [a] Spore, [b] Enlarged view of spore wall
showing three layers (SWL), [c] Enlarged view of the surface ornamentation (i-iv); 2.2. Acaulospora laevis
Gerd &Trappe.; 2.3. Acaulospora scrobiculata Trappe; 2.4. Acaulospora spp 1 (Unidentified): [a] Spore
(s) and sporiferous sacule (sac); subtending hypha (h), [b-c] Enlarged part of spore showing spore walls (SWL1-
SWL3) and cicatrix (cix); 2.5. Acaulospora spp 2 (Unidentified): Spore (s) showing disintegrating sporiferous
sacule (sac) and subtending hypha (sh) in water mount [a-b] and in PVLG & Melzer’s reagent [c-d]; Enlarged
part of spore showing spore walls (SWL1-SWL3) [e]; 2.6. Ambispora callosa (Sieverd.) Walker, Vestberg &
Schüssle: Spores crushed in PVLG with Melzer’s reagent, showing the rust-coloured reaction of the outer wall
component, the yellow reaction of the contents exuded through the break, and the creasing resulting from the
pliable nature of the wall components. 2.7. Diversispora versiformis (Karst.) Oehl, Silva & Sieverd.: [a]
Spores, [b] Enlarged spore, [c] Spore with short, fragile subtending hypha (sh) that is principally continuous
with semi-persistent outermost spore wall layer (SWL1) but not with structural layer SWL2; 2.8.
Entrophospora baltica Blaz., Madej & Tadych: Mature spores with characteristic hyphal mantle (orn) and
round cicatrix (pc) formed after detaching sacule; 2.9. Glomus etunicatum Becker & Gerd. spore with
sloughing layer patchily stained with Melzer’s reagent; 2.10. Glomus heterosporum Smith & Schenck: Spore
mount in- water [a], PVLG [b], PVLG & Melzer’s reagent [c]; 2.11. Kuklospora colombiana Spain &

Schenck) Ohel& Sieverd.: Spore (s) showing sporiferous sacule (sac) and stalk; 2.12. Scutellospora
projecturata Kramadibrata and Walker.: [a] Spores, [b] Enlarged view of spore showing prominent
protuberances (p) of various shapes that give the species its name, and arrow headed bulbous base (b) and 2.13.
Scutellospora spp (Unidentified).
VI. CONCLUSION
Besides to aromatic and medicinal potential of Ayurvedic drug Ketaka: P. fascicularis, ecologically
this plant is also important as its root system binds soil and checks soil erosion, it fixes sand dunes, which act as
a barrier against encroachment of the sea and protect human settlements along the coastline line. Present paper
has provided sound data on AM fungal association and species diversity with P. fascicularis. Thus, an
establishment of AMF association with P. fascicularis apparently helps to strengthen the ecological efficacy in
relation with coastal region. However, for introducing the coastal eco-agriculture practices of P. fascicularis at
mostly underestimated regions in Konkan region of Maharashtra or so, application of this potential mycorrhiza
on large scale is the most effective method. For this purpose production of native AM fungal inoculum or
consortium on large scale is the need of near future and hence extension of work is required.
Acknowledgements
VRK is personally grateful to the Wild life warden of Sindhudurg District, Govt. of Maharashtra (Prof. Daptardar
Dept. of Botany Kelkar College Devagad), Prof. Ghalame and Dr. V. Masal, Dept. of Botany, Dapoli College, for their
assistance during field visits. Sincere thanks are also due to Dr. (Mrs) V.I. Katchi, Principal and (Dr. Mrs.) M. S.
Chemburkar (Vice Principal), BCA (W), Mumbai, for providing Laboratory facilities.
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