全 文 ：Journal of Forestry Research (2010) 21(2): 137−142
DOI 10.1007/s11676-010-0022-2
Mycorrhizal colonization and distribution of arbuscular mycorrhizal
fungi associated with Michelia champaca L. under plantation system in
northeast India
Das Panna • Kayang Highland
Received: 2009-07-09; Accepted: 2009-12-22
© Northeast Forestry University and Springer-Verlag Berlin Heidelberg 2010
Abstract: Arbuscular mycorrhizal fungi (AMF) and dark septate endo-
phyte (DSE) colonization were investigated in three different plantation
sites (Umdihar, Umsaw and Mawlein) of Meghalaya, northeast India.
Isolation and identification of the AMF spore were conducted to evaluate
the AMF diversity and host preference in terms of AMF species distribu-
tion and abundance in the plantation sites. Results showed that AMF
colonization was significantly higher than dark septate endophyte coloni-
zation (p>0.05). AMF and DSE colonization had a narrow range of colo-
nization, varying from 50.91%−58.95% and 1.84%−4.11%, respectively.
Spore density varied significantly in all the sites (p>0.05). Out of 29
species identified from 7 genera, the species from Glomus was found to
be highly abundant. Sorenson coefficient (Cs) ranged from 0.35−7.0.
Species richness varied from 2.0−2.9 in the sites. Total species richness
was significantly correlated with total relative abundance (p=0.001). The
distribution, abundance and principal component analysis plot suggest
that Glomus macrocarpum, G. multicaulis, G. constrictum and Acau-
lospora sp 1 were the most host preferred species which possibly may
favour the host with proper nutrient acquisition and growth.
Keywords: arbuscular mycorrhizal colonization; dark septate endophyte
colonization; Glomus; Michelia champaca
Introduction
Michelia champaca Linnaeus (Magnoliaceae) is famous for its
striking appearance with large, very aromatic yellow blossoms,
The online version is available at http://www.springerlink.com
Das Panna • Kayang Highland ( )
Microbial Ecology Laboratory, Department of Botany, North Eastern
Hill University, Shillong-793 022, India. Email: hkayang@yahoo.com
Das Panna
Microbiology Laboratory, Department of Botany, Tripura University,
Suryamaninagar- 799 130, Tripura, India
Responsible editor: Hu Yanbo
smooth trunk, and large ovate, glossy leaves. The species has
highly economic value for perfume and timber industries in India.
Flower buds of M. champaca are used in most of the herbal
preparations for several diseases and possess active constituents
(Jarald et al. 2008).
Mycorrhiza is widespread in natural ecosystems, and plays a
crucial role in the uptake of mineral nutrition of forest trees,
which is one of important nutrient acquiring mechanisms (Pate
1994). Arbuscular mycorrhizal fungi (AMF) form associations
with the majority of terrestrial plant species (Smith and Read
1997). AMF stimulate plant uptake of nutrients such as P, Zn, Cu,
and Fe in deficient soils and mycorrhizal hyphae can signifi-
cantly improve 15N, P, and K uptake (Chen and Zhao 2009). It
can play an important role in ecological system protection, resto-
ration, and reconstruction (Wu et al. 2009). Moreover, AMF are
now well practiced in the forestry management (Mridha and
Dhar 2007). They belong to four orders: Glomerales, Archaeo-
sporales, Paraglomales and Diversisporales in the division
Glomeromycota (Schubler et al. 2001). Another type of my-
corrhizal association is dark septate endophyte (DSE). It com-
prises of miscellaneous group of root inhabiting conidial sterile
ascomycetous fungi that colonize living plant roots without caus-
ing noticeable harmful effects to the host (Jumpponen 2001).
The coarse structure of the root typified by the order Magnoli-
ales are especially dependent on AMF for mineral uptake (Baylis
1975). Moreover, occurrence of DSE and Paris type of AMF
colonization in M. champaca was reported earlier (Muthukumar
et al. 2006). However, there is no detailed study of AMF diver-
sity of this extremely important multipurpose tree. Composition
of indigenous AMF spores in the plantation sites may be helpful
in indicating the preference of AMF spores in inoculation pro-
gram for seedling production in nursery. Therefore, the study
was undertaken to evaluate (1) the status of AMF and DSE colo-
nization in the three plantation sites, (2) the biodiversity of AMF
in the plantation sites, and (3) whether some level of host prefer-
ence in terms of frequency and abundance exist among AMF
species in the plantation sites.
RESEARCH PAPER
Journal of Forestry Research (2010) 21(2): 137−142
138
Materials and methods
Study sites and sampling
Three plantation sites were selected in Ribhoi District of Megha-
laya, Northeast India. They were located in Umdihar
(N25º51.156; E91º52.699’; with an elevation of 544.0 m.a.s.l.),
Umsaw (N25º49.051; E91º52.642; 553.5 m.a.s.l.), and Mawlein
(N25º42.642; E91º53.55; 828.5 m.a.s.l.), respectively. The plan-
tations in Umdihar and Mawlein are privately managed sites,
while the plantation in Umsaw is under State Forestry Depart-
ment, Meghalaya.
The plant individuals with circumference breast height (CBH)
>5 cm were considered for sampling. Tree height (H) was meas-
ured with the help of a Clinometer (Suunto pm-5/1520). From
each sampling site, five trees were randomly selected with sam-
pling points approximately 5 m apart. M. champaca from the
order Magnoliales is distinguishable by its coarse root structure
(Baylis 1975) and characteristic interesting aroma. The rhizos-
pheric soil and roots at depths of 0−20 cm around each tree, at
four different points for each plant were collected. Combined
samples of approximately 500 g of soil per plant were placed in
3-kg polythene bags, labeled and transported for further analysis
in the laboratory.
Root processing
Electron microscopy of the root sample was carried out to con-
firm the arbuscular mycorrhizal association in the root. The
transverse sections of fresh root samples were fixed in 3% glu-
taraldehyde for 24 h at 4°C. The root sections were washed
thrice in 0.1 M sodium cacodylate buffer. The roots were then
dehydrated with acetone series. Final dry treatment was given
with the tetra methyl silane. The specimens were mounted in
brass stubs. Coating of the root sections with gold were done for
final examination in the microscope. The root segments were
examined under scanning electron microscope (Jeol, JSM 6360).
To determine percent root colonization, the root samples were
washed in tap water, processed and stained with black Faber
Castell stamp pad ink (Das and Kayang 2008). Root segments of
approximately 1-cm long stained samples were mounted on
slides in lactoglycerol and examined for mycorrhizal structures
under light microscope (Olympus 41209) to investigate different
colonization patterns (the structures of hyphal, arbuscular, ve-
sicular, and dark septate hyphal). The estimation of AMF and
DSE colonization were done by magnified intersection method
(McGonigle et al. 1990).
Spore analysis
The spores were extracted by modified wet sieving and decant-
ing method (Muthukumar et al. 2006). The isolated spores were
picked up with needle in polyvinyl alcohol-lactoglycerol under a
dissecting microscope (Koske and Tessier 1983) and also in
mixed polyvinyl alcohol-lactoglycerol: Meltzer’s reagent (1: 1, v:
v) for identification. The complete and broken spores were ex-
amined using a compound microscope, Olympus. Taxonomic
identification of spores to species level was based on sporocarpic
size, colour, and ornamentation and wall characteristics by
matching original descriptions (http://www.invam.caf.wvu.edu &
http://www.lrz-muenchen.de/~schuessler/amphylo). The photog-
raphy of the root segments colonized by fungi and spores of
AMF were done with the help of Leica EC 3 camera attached in
Leica DM 1000 microscope (Switzerland). Spore density (SD),
relative abundance (RA), isolation frequency (IF), species rich-
ness (SR), evenness (E), Simpson’s diversity index (D), Shan-
non-Wiener index of diversity (H) and Sorenson’s coefficient
(Cs) were calculated (Zhao and Zhao 2007).
Soil analysis
The soil samples were air dried after analysis of pH and moisture
content. They were cleaned, ground, sieved with a 2-mm sieve,
stored at 4°C, and processed for further soil analysis. Soil texture
was analyzed using sodium hexametaphosphate method (Allen et
al. 1974). For moisture content (%), 10 g sub sample of soil was
oven dried and weight was determined. Measurement of the soil
pH was done using microprocessor-based pocket pH tester 2
(Eutech Instruments). Available phosphorus of soil was deter-
mined following molybdenum-blue method (Allen et al. 1974).
The soil organic carbon was estimated using colorimetric method
(Anderson and Ingram 1993).
Data analysis
Standard errors of means were calculated. Analysis of variance
(ANOVA) was carried out and the means were separated by
Tukey test. Pearson correlation coefficients were computed be-
tween soil physico-chemical properties, mycorrhizal colonization,
CBH and H of tree species and between relative abundance and
species richness of AMF species. Principal component analysis
(PCA) was used to determine variation in AMF abundance. PCA
analysis was done with the help of software, PAST (Hammer et
al. 2001).
Results
Tree and soil characteristics
The circumference breast height and height of M. champaca in
the plantations of Umdihar were 169.92±2.53 cm and
2293.33±7.87 cm, respectively, 35.2±0.9 cm and 1486.92±4.72
cm in Umsaw, and 32.4±2.02 cm and 947.14±3.09 cm in
Mawlein, respectively. The soil physical and chemical properties
were presented in Table 1.
Mycorrhizal colonization
Scanning electron microscopy (SEM) reveals arbuscular my-
corrhizal structures likely arbuscules and hyphal coils (Fig. 1a &
b). The light microscopy also reveals various structures of dark
Journal of Forestry Research (2010) 21(2): 137−142
139
septate endophyte, including arbuscules, vesicles, hyphal coils
and hyphae of AMF (Fig. 1c-f). The mycorrhizal structural colo-
nization was presented in Table 2. Paris type of AMF morphol-
ogy existed in the roots. There was no significant difference in
total AMF and DSE colonization between the sites. However,
AMF colonization was significantly higher (p>0.05) than DSE
(Fig. 2). Spore density differed statistically in all the sites
(p>0.05). No significant correlation was found between my-
corrhizal colonization, soil properties, CBH and H (p>0.05).
Table 1. Physical and chemical properties of soil collected from the
three different sites of Michelia champaca
Texture (%) Sites
Moisture
(%) Silt Sand Clay
pH
Organic
carbon
(%)
Available
P (µg·g-1)
Umdihar 15.3±0.5 12.20 83.77 4.03 6.13±0.09 0.63±0.023 166.67±3.33
Umsaw 15.5±1.4 14.21 69.61 16.19 6.13±0.03 0.59±0.012 163.33±3.33
Mawlein 27.2±0.4 19.52 75.19 5.29 6.07±0.12 0.57±0.005 176.67±3.33
Fig. 1 (a-f) Mycorrhizal structure in the roots of M. champaca. (a & b)
Scanning electron microscopy of transverse section of the roots showed the
structures of hyphal coils (hc) and arbuscules (ar). (c-f) Light microscopy of
the root segments showed the structures of hyphal coils, arbuscules, vesicles
(v) and dark septate hyphae (dsh). Bar scale = 100 µm
Table 2. Mycorrhizal structural colonization in the roots of Michelia
champaca
Sites Arbuscules Vesicles Hyphae
Dark septate
hyphae
Spore density /
50 g soil
Umdihar 9.91±1.23b 4.24±0.87b 52.49±2.84c 4.11±1.11b 537.66±65.83 d
Umsaw 21.99±1.57a 8.46±0.95b 58.95±2.01c 1.84±0.97b 1939.66±58.37f
Mawlein 17.42±1.31a 9.30±1.14b 50.91±2.84c 1.99±0.40b 1163.00±177.88g
Tukey test showing different alphabetical letters varies significantly (p > 0.05)
Fig. 2 Mycorrhizal colonization in the roots of M. champaca from
three sites
Arbuscular mycorrhizal spore distribution
Acaulospora, Ambispora, Entrophospora, Gigaspora, Glomus,
Pacispora, and Paraglomus were extracted from three plantation
sites of M. champaca (Fig. 3). A total 29 species were identified
from all the soil samples (Table 3).
Table 3. Relative abundance and isolation frequency of arbuscular
mycorrhizal fungi from the three plantation sites.
Relative abundance (%)
AMF Species
Species
abbrevia-
tion
Umdihar Umsaw Mawlein
Isolation
frequency
(%)
Acaulospora sp 1 A1 27.91 0.46 5.39 100.00
A. bireticulata Ab 0.20 - - 33.33
A. rehmii Ar - 0.26 0.29 66.67
A. foveata Af 0.40 0.07 - 66.67
A. lacunose Al 10.04 - - 33.33
A. tuberculata At - 0.13 2.04 66.67
A. cavernata Ac - - 0.29 33.33
Ambispora sp 1 Am1 - - 0.15 33.33
Entrophospora colom-
biana
Ec - 0.13 - 33.33
Gigaspora sp 1 Gi1 - 0.07 1.02 66.67
Glomus sp 1 G1 0.4 0.40 - 66.67
Glomus sp 2 G2 - - 0.44 33.33
Glomus sp 3 G3 23.49 - - 33.33
G. aggregatum Gag 0.20 - - 33.33
G. ambisporum Gam 6.02 - - 33.33
G. aureum Gau - 0.07 0.15 66.67
G. constrictum Gcon 9.04 9.26 13.83 100.00
G. fuegianum Gfu - - 0.15 33.33
G. glomeratum Gglo - 2.67 1.6 66.67
G. intraradices Gin - 0.07 1.31 66.67
G. macrocarpum Gmac 13.05 45.11 32.02 100.00
G. microaggregatum Gmic - - 0.58 33.33
G. mosseae Gmos 0.40 0.13 - 66.67
G. multicaulis Gm 8.43 32.79 26.49 100.00
G. taiwanense Gtaw - 2.80 0.44 66.67
G. tortuosum Gto - 5.93 10.77 66.67
Pacispora boliviana Pb - - 1.60 33.33
P. chimonobambusae Pc 0.20 0.07 0.58 100.00
Paraglomus occultum Po 0.20 - 0.87 66.67
Total 100 100 100
Journal of Forestry Research (2010) 21(2): 137−142
140
Out of seven genera, four were isolated from Umdihar, five
from Umsaw, and six from Mawlein. Glomus macrocarpum, G.
multicaulis, G. constrictum, Acaulospora sp 1 and Pacispora
chimonobambusae were present in all the sites. Among all the 29
species, there were five species with the isolation frequency of
100%, 12 species with the frequency of 66.67%, and the rest 12
species having the frequency of 33.33%. Acaulospora sp 1 was
relatively more abundant in Umdihar than other species, and G.
macrocarpum was comparatively abundant than other species in
Umsaw and Mawlein. Ambispora sp 1, Entrophospora colombi-
ana and G. fuegianum were lower in relative abundance. Signifi-
cant positive correlation (p = 0.001) was found between relative
abundance and species richness of AMF spores (Fig. 4). Glomus
exhibited high relative abundance and high species richness,
whereas Entrophospora and Ambispora were the lowest in terms
of abundance and species richness (Table 4). In addition, species
richness increased with the increase in relative abundance as
depicted in the plot (Fig. 4). The highest species richness was
observed in the plantations of Mawlein (Table 5). Sorenson coef-
ficient varied between the sites. Moreover, high Cs was observed
between nearest sites and the lowest value between distant sites.
The dissimilar number of AMF species between Umdihar X
Umsaw, Umsaw X Mawlein and Umdihar X Mawlein were 15,
11 and 22, respectively. The similar number of species between
Umdihar X Umsaw, Umsaw X Mawlein and Umdihar X
Mawlein were eight, 13 and six, respectively (Fig. 5).
Table 4. Species richness and relative abundance of arbuscular my-
corrhizal fungi associated with Michelia champaca
AMF species Species richness Relative abundance (%)
Glomus 9.7 89.14
Acaulospora 4.0 9.60
Pacispora 1.3 0.63
Gigaspora 0.7 0.26
Paraglomus 0.7 0.26
Ambispora 0.3 0.04
Entrophospora 0.3 0.07
Total 17 100
Table 5. Diversity index of arbuscular mycorrhizal fungi associ ated
with Michelia champaca
Sites
Species
richness
Shannon - Wiener
index of diversity (H)
Simpson’s index
of diversity (D)
Evenness
(E)
Umdihar 2.0 1.9 0.18 0.38
Umsaw 2.4 1.4 0.32 0.20
Mawlein 2.9 1.9 0.21 0.43
Diversity index reveals that H and E were high in Umdihar
and Mawlein, however, D was high in Umsaw (Table 5). PCA
plot showed the distribution of AMF species and host preference
(Fig. 6). The variability in the distribution existed between spe-
cies, where G. macrocarpum, G. multicaulis, G. constrictum and
Acaulospora sp 1 accounted for 78.57% of the total variation.
The other species were clumped together in one spot, indicating
no variation. PCA plot showed close relation of the four species
to the sites. G. macrocarpum and G. multicaulis were closely
related to Mawlein and Umsaw. Acaulospora sp 1 was closely
correlated with Umdihar, while G. constrictum was relatively
associated with Mawlein and Umsaw. Moreover, the entire three
axes showed that these four species were distributed in all the
sites.
Fig. 3 (a-u) Arbuscular mycorrhizal spores. (a) Acaulospora tuberculata
with cicatrix. Bar scale = 200 µm. (b) A. lacunosa with cicatrix and wall
layers. Bar scale = 150 µm. (c) A. rehmii with cicatrix. Bar scale = 100 µm. (d)
A. foveata. Bar scale = 150 µm. (e) A. cavernata with soporiferous saccule.
Bar scale = 200 µm. (f) A. bireticulata. Bar scale = 50 µm. (g) Glomus con-
strictum Bar scale = 150 µm. (h) G. mosseae. Bar scale = 200 µm. (i) G.
multicaulis. Bar scale = 150 µm. (j) Glomus sp 1. Bar scale = 100 µm. (k) G.
feugianum. Bar scale = 100 µm. (l) G. taiwanense. Bar scale = 200 µm. (m) G.
aureum. Bar scale = 100 µm. (n) G. macrocarpum. Bar scale = 100 µm. (o) G.
glomeratum. Bar scale = 50 µm. (p) G. microaggregatum. Bar scale = 250 µm.
(q) Paraglomus occultum. Bar scale = 100 µm. (r) Entrophospora colombiana.
Bar scale = 150 µm. (s) Ambispora sp 1. Bar scale = 100 µm. (t) Pacispora
chimonobambusae. Bar scale = 50 µm. (u) Unidentified Bar scale =100 µm.
Journal of Forestry Research (2010) 21(2): 137−142
141
Fig. 4 Relation between species richness and relative abundance of
arbuscular mycorrhizal fungi
Fig. 5 Sorenson coefficient (Cs) of arbuscular mycorrhizal fungi in
the three plantations
Fig. 6 Array of the relative abundance of AMF species distribution in
the three plantation sites, which were determined by PCA biplot. For
other abbreviations, see Tab. 3
Discussion
M. champaca was colonized by AMF and DSE in all the sites.
Presence of DSE and AMF confirms the findings of Muthuku-
mar et al. (2006). Mycorrhizal colonization recorded in the pre-
sent study was less than the earlier report (Muthukumar et al.
2006). Moreover, our results showed that the AMF colonization
was significantly higher than DSE colonization. This may be due
to the presence of coarse structure of the root, the characteristic
feature of Magnoliales, which favors mycorrhizal infection (Bay-
lis 1975). The results suggest that mycorrhizal colonization of M.
champaca in different plantations recorded in the investigation
had a narrow range i.e., intermediate AMF colonization
(50.91%−58.95%) and low in DSE (1.84%−4.1%).
No correlation was found between mycorrhizal colonization,
soil properties, CBH and H. It implies that AMF colonization
may be affected by the comprehensive interactions of several
factors, such as the factors inherent to the host plant, climatic and
edaphic factors, and effects of the soil community (Moreira et al.
2006).
The spore number varied significantly between the sites. This
might be due to production of AMF spores in the rhizosphere
vicinity of surrounding herbaceous species (Kruckelmann 1975).
Presence of herbaceous community in Umsaw and Mawlein were
observed but in Umdihar spore density and herbaceous commu-
nity were comparatively lower. The herbaceous community was
removed or not frequent in Umdihar, however, the spore density
could be disturbed as the site was located very near to national
highway No. 44. Moreover, in an undisturbed ecosystem, higher
spore population was quite natural as the number of AMF spores
and the population diversity were higher in native undisturbed
forests than the disturbed and replanted areas (Moreira-Souza et
al. 2003). Spore population is affected by a wide range of soil,
climatic, fungal and host factors (Anderson et al. 1983; Howeler
et al. 1987). Plant phenology and root production are closely
related to the patterns of spore production and spore quantity
(Brundrett 1991).
Out of seven genera, Glomus was the most widely distributed
genera, followed by Acaulospora and Pacispora. Glomus
sporulated abundantly regardless of the sites selected. Das and
Kayang (2009) also reported the dominance of the Glomus from
this region. They described the wider adaptation of the taxon in
varied soil conditions. The sporulation pattern of Glomus might
bring about the dominance of the taxon. Spores of Glomus grow
in cluster and sporulate more frequently while the spores of other
genera like Gigaspora sporulated singly (Dhar and Mridha 2006).
The altitudinal variation and distances of plantation sites could
play an important role in the similarities of AMF species. Higher
similarity coefficient (Cs) was found between the nearest sites
(Umsaw and Umdihar) with a relatively low altitude, and lower
between the distant sites (Umdihar and Mawlein) with a rela-
tively high altitude. In contrast, the results of An et al. (2008)
showed that highest similarity index was observed between the
most distant sites and the lowest value was observed between the
nearest sites.
In the present study, diversity attribute of M. champaca plants
such as H and E were higher in Mawlein and Umdihar than in
Umsaw. The variation in the diversity attribute can be substanti-
ated to the study of Allen and Boosalis (1983) where the diver-
sity of mycorrhizal fungi associated with the same plants was
found to vary.
Journal of Forestry Research (2010) 21(2): 137−142
142
G. macrocarpum, G. multicaulis, G. constrictum, Acaulospora
sp 1 and P. chimonobambusae were most frequently distributed
in the three sites, of which G. macrocarpum, G. multicaulis, G.
constrictum and Acaulospora sp 1 were relatively abundant.
However, P. chimonobambusae was less abundant than other
four species. PCA plot with relative abundance of AMF species
in all three sites showed that there was close relation between the
sites and the highly abundant species. The distribution and rela-
tive abundance indicate that these four species may favour M.
champaca.
Conclusion
Relative abundance, species distribution and PCA plot suggest
that G. macrocarpum, G. multicaulis, G. constrictum and Acau-
lospora sp 1 were most preferred by the host plants, which may
possibly favour host nutrition and growth. Furthermore, the in-
vestigation was emphasized to select the suitable indigenous
AMF species for sustainable management of tree plantations, and
to create consciousness among foresters and local folks about the
significance of mycorrhiza as a tool to maintain such ecosystem
environment friendly. In addition, this work provides a platform
for analysis the role of AMF and DSE in several tree species that
are used in northeast Indian forestry practice.
Acknowledgements
The authors are thankful to the Head, Department of Botany for
providing infrastructures. The first author is grateful to the Uni-
versity Grant Commission, New Delhi for financial support in
the form of fellowship. We are also thankful to Dr. Dibyendu
Adhikari for preparation of map.
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