Arbuscular Mycorrhizal Fungal Diversity and Root Colonization in Pisum sativum

Abstract

Arbuscular mycorrhizal fungi (AMF) are soil fungi which form a mutualistic symbiosis with the roots of plants and enhanced uptake of immobile nutrients from the soil. The present study was carried out to study the association of arbuscular mycorrhizal fungi (AMF) with roots and rhizosphere of pea (Pisum sativum). A total of 17 AMF fungi belonging to 5 genera 17 species were isolated and identified from the rhizosphere soil. The dominant genus was Glomus (6 species), followed by Acaulospora (5 species); Boletus, Gigaspora (3 species), Scutellospora (2 species) and Sclerocystis represented by single species. Microscopic analyses of root samples revealed a variable degree of colonization by AM fungi. The different microscopic characters like size, colour, details of the wall layers and the nature of their subtending hyphae were also investigated to during this study.

Full text

Aishwarya et al., Biological Forum –An International Journal 14(1): 1626-1632(2022) 1626
ISSN No. (Print): 0975-1130
ISSN No. (Online): 2249-3239
Arbuscular Mycorrhizal Fungal Diversity and Root Colonization in Pisum sativum
Aishwarya1, Manjula1, Payal1, Shivani Kaundal2, Ravinder Kumar3, Ritika Singh4, Shubhi Avasthi5
and Ajay K. Gautam1*
1Plant Pathology Laboratory, School of Agriculture,
Abhilashi University Mandi (Himachal Pradesh), India.
2Microbiology and Crop Physiology Laboratory,
School of Agriculture, Abhilashi University Mandi (Himachal Pradesh), India.
3Soil Science Laboratory, School of Agriculture,
Abhilashi University Mandi (Himachal Pradesh), India.
4Plant Breeding and Genetics Laboratory, School of Agriculture,
Abhilashi University Mandi (Himachal Pradesh), India.
5School of Studies in Botany, Jiwaji University Gwalior (Madhya Pradesh) India.
(Corresponding author: Ajay K. Gautam*)
((Received 13 December 2021, Accepted 19 February, 2022)
(Published by Research Trend, Website: www.researchtrend.net)
ABSTRACT: Arbuscular mycorrhizal fungi (AMF) are soil fungi which form a mutualistic symbiosis
with the roots of plants and enhanced uptake of immobile nutrients from the soil. The present study was
carried out to study the association of arbuscular mycorrhizal fungi (AMF) with roots and rhizosphere of
pea (Pisum sativum). A total of 17 AMF fungi belonging to 5 genera 17 species were isolated and identified
from the rhizosphere soil. The dominant genus was Glomus (6 species), followed by Acaulospora (5
species); Boletus, Gigaspora (3 species), Scutellospora (2 species) and Sclerocystis represented by single
species. Microscopic analyses of root samples revealed a variable degree of colonization by AM fungi. The
different microscopic characters like size, colour, details of the wall layers and the nature of their
subtending hyphae were also investigated to during this study.
Keywords: AM fungi, pea, root colonization, Mid-hill conditions, Himachal Pradesh.
INTRODUCTION
Pea (Pisum sativum L.) is a common leguminous crop
belonging to the family “Fabaceae. “Faba” comes from
Latin word which simply means “beans”. It is third
most important pulse crop commonly grown worldwide
over six-million-hectare area. The major pea
producing countries of the world are Germany, Italy,
China and Canada followed by India, Australia, and the
United States. France, Canada and Australia are major
exporters of pea as they utilize over two million
hectares of land area for pea cultivation. As per FAO
Stat. (2014), pea occupies 4th position (10.53%) in area
under cultivation and 5th position in total production
(6.96%). Like other countries of the world, India also
occupies a key position in pea production. Uttar
Pradesh is the major pea growing state of India,
produces about 49 % of total pea produced. In addition,
Madhya Pradesh, Bihar and Maharashtra are also the
major pea growing states of the country. Himachal
Pradesh, a hilly state of India occupies 5th position in
pea production with total production of 294.96
thousand metric tons per year.
Mycorrhiza is a non-disease-producing association in
which the fungus invades the root to absorb nutrients.
These fungi are found in a wide range of habitats
usually inside the roots ramify into the surrounding
bulk soil extending the root depletion zone around the
root system. They transport water and mineral nutrients
from the soil to the plant while the fungus is benefiting
from the carbon compounds provided by the host plant
(Warburton, 2005). This plant root association with
these fungi play a vital role in supporting plant’s health
by improving plant nutrition (Jacoby et al., 2017),
suppress pathogen outbreaks (Pieterse et al., 2014),
nutrient exchange and modulation of abiotic stress
tolerance (Cheng et al. 2019; Baum et al., 2015). They
mainly facilitate nutrient uptake, mainly phosphorus,
nitrogen (Campo et al., 2020) potassium, sulphur,
copper, zinc, calcium etc. (Avio et al. 2006; Fanaei et
al., 2015; Prasad et al., 2017; Wang et al., 2018; Liu et
al., 2018) and enhance the availability of nutrients as
well as their translocation (Rouphael et al., 2015).
Keeping in view the key benefits of association of
mycorrhiza fungi with plants roots, the present study
was carried out to investigate the diversity of AM fungi
Biological Forum –An International Journal 14(1): 1626-1632(2022)

Aishwarya et al., Biological Forum –An International Journal 14(1): 1626-1632(2022) 1627
associated with pea in different location of mid hill
conditions of Himachal Pradesh.
MATERIALS AND METHODS
A. Study area
The study areas selected for sample collections were
lies in the mid hill regions of district Mandi, Himachal
Pradesh. The areas selected for surveys and sample
collection were Chachyot, Naugroun, Ganai and Gohar
in Chail-Chowk areaand from Balh valley: Kummi,
Ratti, Surandi, Dadour, Gagal and Sakroha. The areas
are situated in mid hill conditions of District Mandi and
fall in second zone, Mid-hill zone of Himachal Pradesh.
Total five pea plants were randomly selected from each
study site for the collection of plants and soil samples
(Plate I). Soils up to the depth of 0-30cm were collected
in sterile polythene bags and carried to the laboratory
for further analyses.
Plate I: Map of study area.
B. Assessment of root colonization by
Arbuscular mycorrhizal fungi (AMF)
For staining of root to assess root colonization of
AMF, a method described by Phillips and Hayman
(1970) for roots and modified by Kormanik et al.
(1980) was employed. Freshly collected roots of pea
plants were washed thoroughly with tap water, cut it
into 1 cm length and cleared in 10% (w/v) KOH for 1
hour at 90°C, acidified with 1 % HCl and stained with
0.05% trypan blue overnight and then finally de-
stained with lactic acid- glycerin (1:1 by volume) at
room temperature. Slides were prepared and observed
under a compound microscope for any of structures
associated with mycorrhizal fungi viz., hyphae,
vesicles or arbuscules. Root colonization was
assessed by using following formula:
Total number of colonized root/tissues pieces
% colonisation Total number of root / tissue pieces examined
=×100
C. Isolation and Identification of AM Fungi
The isolation of AM fungal spores was carried out by
wet-sieving and decanting method (Gerdeman and
Nicolson 1963). The soil samples were carried to the
laboratory in polythene bags and stored in a refrigerator
at 4°C for isolation of AMF spores. Total 25g of soil
was mixed in 100 ml of water in a glass beaker and
stirred constantly with a glass rod to make a uniform
suspension. The suspension was left for five minutes so
that mycorrhizal debris floated on the top. The
suspension was passed through a set of sieves of
different sizes (240µm, 120µm, 100µm, 63µm, 30µm).
The final decanted suspension of sieving was passed
thorough what man filter paper. This process was
repeated 8-10 times to trap all spores of AM fungi. The
sieved material collected from sieves was observed
under stereomicroscope and the spores were isolated
using hypodermal needle. Spore population was
expressed in terms of number of spores per 25 gm of
dry soil.
To aid in the identification of AM fungi, the resting
spores were mounted in polyvinyl lactic acid and the
size, colour, details of the wall layers and the nature of
their subtending hyphae were recorded as per the
method suggested by (Phillips and Hayman 1970). The
AMF isolates were identified at least to species level.
The rhizosphere soil samples were expressed in term of
percentage occurrence as per the given formula:
Total no.of Spores of Individual AM fungi
%age of occurance Total no. of Spores of AM Fungi
=×100

Aishwarya et al., Biological Forum –An International Journal 14(1): 1626-1632(2022) 1628
RESULTS
A. Root colonization by AM Fungi
The analyses of root samples confirmed association of
Arbuscular mycorrhizal fungi (AMF) with Pea roots.
The root samples collected from all the locations
showed variable percentage of root colonization with
various mycorrhizal fungi. The highest root
colonization was observed with root samples plants
collected from Kummi (56.6%) followed by Surandhi
(53%) and Chachyot (42%) whereas, the range of root
colonization form Ratti, Sakroha, Chail-Chowk, Ganai,
Gagal, Dadour, Gohar was observed in the range of 34-
40.5%. The detailed results of root colonization of pea
plants with Arbuscular mycorrhizal (AM) fungi are
presented in Table 1.
Table 1: Arbuscular mycorrhizal AM fungi colonization (percentage) of root or tissue of pea plants.
Sampling
Sites
Samples
Number of roots
colonized
Number of root segments
examined
Rate of
colonization
(%age)
Average
(%age)
Ratti
Sample 1
3
6
50
44
Sample2
4
10
40
Sample 3
6
10
60
Sample 4
4
8
50
Sample 5
2
10
20
Surandhi
Sample 1
4
8
50
53
Sample2
4
8
50
Sample 3
6
10
60
Sample 4
2
8
25
Sample 5
8
10
80
Sakroha
Sample 1
4
8
50
39
Sample2
2
8
25
Sample 3
6
10
60
Sample 4
2
10
20
Sample 5
4
10
40
Gagal
Sample 1
2
8
25
34
Sample2
4
10
40
Sample 3
1
4
25
Sample 4
3
10
30
Sample 5
4
8
50
Dadour
Sample 1
2
10
20
41
Sample2
4
8
50
Sample 3
6
10
60
Sample 4
2
4
50
Sample 5
2
8
25
Chail-
Chowk
Sample 1
4
8
50
39
Sample2
2
8
25
Sample 3
6
10
60
Sample 4
2
10
20
Sample 5
4
10
40
Chachyot
Sample 1
3
10
30
42
Sample2
5
10
50
Sample 3
7
10
70
Sample 4
2
5
40
Sample 5
1
5
20
Ganai
Sample 1
4
10
40
34.5
Sample2
2
10
20
Sample 3
3
12
25
Sample 4
5
8
62.5
Sample 5
2
8
25
Gohar
Sample 1
5
10
50
40.5
Sample2
6
12
50
Sample 3
3
8
37.5
Sample 4
2
8
25
Sample 5
4
10
40
Kummi
Sample 1
4
10
40
56.5
Sample2
6
10
60
Sample 3
8
10
80
Sample 4
5
8
62.5
Sample 5
4
10
40

Aishwarya et al., Biological Forum –An International Journal 14(1): 1626-1632(2022) 1629
B. Association of AM Fungi
The soil samples collected from different study sites
were analysed for occurrence of Arbuscular
mycorrhizal (AM) fungal spores. The occurrence and
abundance of AM fungi from different soil samples was
calculated on 30th, 60th and 90th days after sowing of
pea plants. A variation in diversity of Am fungi was
observed with respect to collection sites however, no
significant difference was observed age of the plants
(30th, 60th and 90th days after sowing). The percentage
of occurrence of Arbuscular mycorrhizal (AM) fungi on
30th day was observed highest in Dadour (20.04%)
followed by Gohar (20%), Kummi (19.94) and
Surandhi (19.73%). Similarly, this percentage of
occurrence was observed maximum in Chail-chowk
(20%) followed by Chachyot (20%) and Kummi
(19.96%) and Gohar (18.86%)on 60th days after sowing
whereas, on 90th day it was observed highest in Ratti
(19.98%) and Chail-Chowk (19.98%) and then Sakroha
(19.96%) and (15.23%). The detailed results of
occurrence of Arbuscular Mycorrhizal fungi with pea
plants on 30th, 60th, 90th days after sowing are presented
in Table 2.
Table 2: Percentage occurrence of Arbuscular Mycorrhizal fungi on 30th, 60th, 90th days after sowing.
Sampling sites
Samples
%age of occurrence
Average (%age)
30th Day
60th Day
90th Day
30th Day
60th Day
90th Day
Ratti
Sample 1
7.14
9.67
11.11
19.99
19.96
19.98
Sample 2
17.85
19.35
19.44
Sample 3
28.57
22.58
22.22
Sample 4
25.01
25.80
25
Sample 5
21.42
22.58
22.22
Surandhi
Sample 1
18.75
19.04
19.23
19.73
19.94
19.96
Sample 2
18.75
14.28
15.38
Sample 3
12.05
19.04
19.23
Sample 4
37.05
33.33
30.76
Sample 5
12.05
14.28
15.38
Sakroha
Sample 1
22.22
19.04
19.23
19.99
19.94
19.96
Sample 2
11.11
19.04
19.23
Sample 3
22.22
14.28
15.38
Sample 4
11.11
33.33
30.76
Sample 5
33.33
14.28
15.38
Gagal
Sample 1
25.92
26.66
2.64
19.99
19.96
15.23
Sample 2
40.74
40
38.23
Sample 3
14.81
16.66
20.58
Sample 4
11.11
6.66
8.82
Sample 5
7.40
10
5.88
Dadour
Sample 1
30.01
25.80
28.12
20.04
19.96
19.96
Sample 2
10.01
12.90
15.62
Sample 3
20.01
22.58
25
Sample 4
23.33
19.35
21.87
Sample 5
16.66
19.35
9.37
Chail-Chowk
Sample 1
46.66
40
37.5
19.99
20
19.98
Sample 2
6.66
10
12.5
Sample 3
6.66
10
16.66
Sample 4
13.33
15
8.33
Sample 5
26.66
25
25
Chachyot
Sample 1
18.18
18.75
18.18
19.99
20
19.96
Sample 2
18.18
18.75
22.72
Sample 3
18.18
18.75
18.18
Sample 4
36.36
31.25
27.27
Sample 5
9.09
12.5
13.63
Ganai
Sample 1
21.73
23.07
22.58
19.99
19.94
19.96
Sample 2
13.04
7.69
9.67
Sample 3
30.43
30.76
29.03
Sample 4
8.69
11.53
12.90
Sample 5
26.08
26.92
25.80
Gohar
Sample 1
16.66
17.64
18.18
20
18.86
19.96
Sample 2
25.01
23.52
27.27
Sample 3
16.66
17.64
13.63
Sample 4
25.01
17.64
9.09
Sample 5
16.66
17.77
31.81
Kummi
Sample 1
15.90
17.77
17.30
19.99
19.96
19.94
Sample 2
20.45
22.22
21.15
Sample 3
13.63
24.44
23.07
Sample 4
22.72
15.55
15.38
Sample 5
16.66
20
23.07

Aishwarya et al., Biological Forum –An International Journal 14(1): 1626-1632(2022) 1630
C. Identification and Diversity assessment of
Arbuscular Mycorrhizal Fungi
Total five genera and 17 species of mycorrhizal fungi
were isolated from the rhizosphere of Pea (Pisum
sativum) from all samples collected from various
sampling sites of mid hill conditions of district Mandi,
Himachal Pradesh. The genus Glomus was isolated with
maximum 6 species followed by Acaulospora (05 sp.),
Gigaspora (03 sp.), Scutellospora (02 sp.) whiles
Sclerocystis with single species.The genus Glomus is
classified as a mycorrhizal type with a wide distribution
found in almost all ecosystems (Ibou et al., 2021; Lara-
Capistran et al., 2021; Sukmawati et al., 2021). While
the genera Scutellospora and Acaulospora have limited
distribution (Ibou et al., 2021; Lara-Capistran et al.,
2021; Sukmawati et al., 2021). The various microscopic
characteristics of AM fungi observed in present study
are summarized in Table 3, Plates II&III.
Table 3: Types and density of Arbuscular Mycorrhizal fungi (AMF) spores.
Sr. No.
AM fungi
Identification parameters
Diameter
Wall width
Colour
Hypha
1.
Glomus fugianum
264 × 231µm
16µm
Dark brown to yellow
Absent
2.
Glomus macrocarpum
132 × 165µm
4µm
Light yellow & transparent
Present
3.
Glomus melanosporum
231 × 231μm
3µm
Dark black and brown inside
Present
4.
Glomus multicauli
297 × 297 μm
15µm
Brown and black
Absent
5.
Glomus spercum
40.5 × 98.5μm
10 μm
Dark yellow to black inside
Absent
6.
Glomus sp.
297 × 198 µm
10 µm
Dark yellow to brown inside.
Absent
7.
Acaulospora denticulata
191 × 165 μm
8.5 μm
Yellow to brown
Absent
8.
Acaulospora bireticulata
181.5 × 214.5 μm
15 μm
Dark brown
Absent
9.
Acaulospora rehmii
264 × 231 μm
16 μm
Yellow to brown
Absent
10.
Acaulospora dilatata
171.6 × 188.1μm
16 μm
Brown
Present
11.
Acaulospora undulata
181.5 × 214.5μm
10 μm
Brown to dark brown
Present
12.
Gigaspora albida
264 × 231µm
8 µm
Dark brown
Present
13.
Gigaspora margarita
184.5 × 132µm
13 μm
Yellow
Absent
14.
Gigaspora rosea
214.5 × 198µm
6 µm
Black
Present
15.
Scutellospora dipurpurscens
297 × 297 µm
6 μm.
Brown
Present
16.
Scutellospora minuta
397 × 297 µm
4 μm
Transparent shade
Present
17.
Sclerocystis sp.
330 × 330 µm
4 μm
Dark brown
Absent
The five genera and 17 species of mycorrhizal fungi
were isolated during the present study are as Glomus
fugianum, G. macrocarpum, G. melanosporum, G.
multicauli, G. spercum, Glomus sp., Acaulospora
denticulate, A. bireticulata, A. rehmii, A. dilatata, A.
undulate, Gigaspora albida, G. margarita, G. rosea,
Scutellospora dipurpurscens, S. minuta and Sclerocystis
sp. The isolated AM fungi showed a great degree of
variations in occurrence at different sampling sites. The
diversity and distribution of AM fungi at different
sampling sites is given in Table 4.
Table 4: The distribution of AM fungi in different sampling sites.
AM Fungi
Balh Valley
Chail –Chowk valley
Ku
Sa
Su
Da
Ra
Gag
Cc
Ch
Gan
Go
Ft 1
Ft 2
Glomus fugianum
+
+
+
-
-
-
+
+
-
+
-
-
Glomus macrocarpum
+
+
+
+
-
-
-
+
-
-
+
-
Glomus melanosporum
+
-
+
+
+
-
+
-
-
-
+
+
Glomus spercum
+
-
-
+
+
+
-
-
-
+
-
+
Glomus spp.
-
+
+
-
+
-
-
+
-
+
-
-
Acaulospora denticulate
+
-
+
-
-
-
+
-
+
-
+
-
Acaulospora bireticulata
+
+
-
-
-
-
-
+
-
-
-
-
Acaulospora rehmii
-
-
-
-
+
+
-
+
+
-
-
-
Acaulospora dilatata
-
-
-
-
+
-
-
-
-
-
+
-
Acaulospora undulate
+
-
-
+
-
-
+
+
+
+
-
+
Gigaspora albida
+
-
+
-
-
+
+
+
+
-
+
-
Gigaspora margarita
+
+
+
+
+
-
+
-
-
+
+
+
Gigaspora rosea
-
+
-
-
+
-
-
+
-
-
-
-
Scutellospora dipurpurscens
-
-
-
-
-
-
-
+
-
-
+
+
Scutellospora minuta
-
-
-
-
-
-
+
-
-
+
-
-
Sclerocystis spp.
-
-
-
-
-
-
-
+
-
+
-
-
Ku= Kummi, Sa= Sakroha, Su= Surandhi, Da= Dadour, Ra= Ratti, Gag= Gagal, Cc= Chail-Chowk,
Ch= Chachyot, Gan= Ganai, Go= Gohar, Ft1=Field trial=1, Ft2=Field trial 2.
DISCUSSION
Root colonization was checked first to observe the AM
fungi presence or absence in plant root samples. The
variation in percentage root colonization with various
AM fungi under natural conditions was observed in this
study. Variability in humidity, temperature, moisture
texture, pH of soil and available nutrients played an
important role in root colonization by AM fungi
(Herold et al., 2014; Bhardwaj and Chandra 2018; Liu
et al., 2016). The root colonization by different AM
fungi has already been studied on very wide scale
throughout the world. The plant like Asparagus sp.,

Aishwarya et al., Biological Forum –An International Journal 14(1): 1626-1632(2022) 1631
Smilax sp., Rhizophagus sp., Withania sp.,
Claroideoglomus sp. has been investigated for
association of AMF with plant roots (Thangavelu and
Raji 2016; Yaseen et al., 2016; Johny et al., 2021).
The AM fungi in the rhizosphere of pea plant revealed
the association of five genera and 17 species. However,
a great variability in the diversity of these fungi was
observed from soil samples collected from various
study sites. This variation could be due to the reason of
variation in physical and chemical properties of the soil
(Urcoviche et al., 2014; Liu et al., 2016; Abedi and
Esfandiari 2017) and seasonal periods and host plant
(Guyonnet et al., 2017). Being a commercial
agricultural crop, tillage as well as land use intensity
also affects the diversity and structure of arbuscular
mycorrhizal fungal communities (Jansa et al., 2002;
Oehl et al., 2004; Mathimaran et al., 2005). A study of
Krüger et al., (2012) also reported the isolation of
Glomus is the most diverse of the genus from
rhizosphere of some medicinal plants. Similarly,
Garampalli et al. (2012) also isolated Glomus as
predominant genus in the rhizosphere 46 medicinal
plants whereas, Bhat et al. (2014) also isolated it as
predominant genus in the rhizosphere Catharanthus
roseus. Several academics are working on identifying
certain mycorrhizal fungus and their role in
phytochemical production (Kumar et al., 2021).
CONCLUSION
In conclusion, the present study was focused mainly on
investigation of pea (Pisum sativum L.) roots and
rhizosphere soil samples for the association of
Arbuscular Mycorrhizal fungi. As pea is one of the
major commercial crop of mid hill regions of Himachal
Pradesh and the associations of AM Fungi in general
may be useful to improve soil microbial status and
overall performance of these plants. considering the
importance of pea as commercial crop and usefulness of
AM fungi, further studies should be focused on the
evaluation of dominant mycorrhizal fungi association
with agricultural crops and impact on plant growth and
metabolite production.
Plate II: Root colonization by AM fungi A) HCl solution; B) Fine roots acidified in HCl solution; C) & D) microscopic view of
AMF colonized roots.
Plate III: Different species of the genus Glomus isolated from rhizosphere of pea: A) Acaulospora denticulate, B) Acaulospora
bireticulata, C) Acaulospora rehmii, D) Acaulospora dilatata, E) Acaulospora undulate. F) Glomus fugianum, G) Glomus
macrocarpum, H) Glomus melanosporum, I) Glomus multicauli, J) Glomus spercum, K) Glomus sp., L) Scutellospora
dipurpurscens, M) Scutellospora minuta, N) Sclerocystis sp.
Acknowledgement. Authors are grateful to their
respective organizations for necessary laboratory
facilities and encouragement to carry out this research
work successfully.
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How to cite this article: Aishwarya, Manjula, Payal, Shivani Kaundal, Ravinder Kumar, Ritika Singh, Shubhi Avasthi and Ajay
K. Gautam (2022). Arbuscular Mycorrhizal Fungal Diversity and Root Colonization in Pisum sativum.Biological Forum –An
International Journal,14(1): 1626-1632.