Journal of Tropical Crop Science Vol. 5 No.

3, October 2018
www.j-tropical-crops.com

The Diversity of Arbuscular Mycorrhizal Fungi in the

Black Cumin Rhizosphere (Nigella sativa L.) in Cianjur,
West Java, Indonesia

Faisal Al AsadA, Ani Kurniawati*B, Sri Wilarso Budi RC, Didah Nur FaridahD
A
Graduate School of Bogor Agricultural University, Bogor, 16680, Indonesia
B
Department of Agronomy and Horticulture, Bogor Agricultural University, Bogor, 16680, Indonesia.
C
Department of Silviculture, Department of Forestry Bogor Agricultural University, Bogor 16680, Indonesia
D
Department of Science and Food Technology, Bogor Agricultural University, Bogor 16680, Indonesia

*Corresponding authors: ani_kurniawati@yahoo.co.id

Abstract immobile elements on the soil and increase uptake

and transport of highly mobile elements such as N
Arbuscular Mycorrhizal Fungi (AMF) is a type of (Marschner and Dell, 1994; Liu et al. (2000).
fungus that can form a symbiotic mutualism with
most plants. Some AMF can only be symbiotic with a AMF form symbiotic associations with the roots of
certain plant species. This research aims to determine more than 80% of terrestrial plants (Smith and Read
and obtain the genus AMF from black cumin (Nigella 2008). This broad spread of AMF indicates that the
sativa L.) accessions from America, Turkey, Hong effect of specific host plants on AMF activities is very
Kong, Slovenia, India, and Kuwait accessions which small (Smith and Read, 2008). Klironomos and John
had been grown in West Java, Indonesia. Three (2000) reported that AMF successfully colonized
samples from each accession, four replications each, 96% of the species of grass and horticulture plants.
were collected for examination. The results showed This means that around 4% AMF has a specific host
that six generas of AMF were found in the rhizosphere and are not able to colonize well on species of grass
of black cumin: Glomus, Gigaspora, Acaulospora, and horticulture plants. Therefore, AMF exploration
Scutellospora, Dentiscutata, and Entrophospora. The in a plant such as black cumin is of fundamental
genus Glomus was predominantly found in the Indian importance to understand the level of colonization
accession, i.e. 96.42 spores. and the number of AMF spores in this species.

Keywords: black cumin, diversity, fungi, exploration,

AMF Material and Methods
All accessions of black cumin were grown on
Introduction polybags with Andosol soil in Pacet, Cianjur, West
Java, Indonesia. There were six accessions of
A Mycorrhiza is a symbiotic association and a black cumin from America (N1), Turkey (N2), Hong
mutualistic relationship between a vascular host plant Kong (N3), Slovenia (N4), India (N5) and Kuwait
and fungi of the Glomeromycota phylum (Peterson (N6). Three soil samples were collected from each
et al., 2004). Mycorrhiza can promote nutrient accession and repeated four times so that there were
absorption by the plants; the host plants supplied 72 samples in total. The research consisted of five
the carbohydrates as a source of nutrients to the stages: soil sampling, AMF isolation, spore counting,
mycorrhiza (Gaur and Kaushik, 2011), resulting in AMF identification, and AMF colonization calculation.
a mutualistic association. The association of AMF All stages of the research were conducted from
with host plants has important roles in the growth October to November 2017.
and development of plants (Lekatompessy and
Sukiman, 2015). AMF can increase the absorption of Soil Sample Collection
phosphorus (P) (Chairani, 2007) and uptake of copper
(Cu), Manganese (Mn), ferum (Fe) (Sreenivasa et Soil samples of 300 g each were collected from the
al., 1993) through expansion of root absorption area depths of 5 to 20 cm from the soil surface at four
in the presence of external hyphae that grows and different points of the rhizosphere area. The samples
develops through the roots of the plants. In addition, were then stored in plastic bags and labelled. Twenty
mycorrhiza fungi can improve the absorption of gram of soil sample was used for AMF isolation. Root

126 Hafith Furqoni, Darda Efendi

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samples for AMF colonization analysis were taken and then immersed in 20% (w/v) KOH solution for
from four points of the rhizosphere and placed in 1 day until the roots turned white.
plastic containers containing 70% alcohol b. The solution was removed and the roots were
washed under running water, then immersed in a
AMF Isolation solution of 0.1 M HCl for 3 to 4 minutes without
washing
For AMF isolation, a modified method by Pacioni c. The roots were soaked in a trypan blue solution
(1992) and Brundrett et al. (1996) were used and (0.25 g in 475 mL lactic acid and 25 mL of aquadest)
described below: for 24 hours followed by the root washing
a. The soil sample was placed into the glass and d. Soak the roots in a de-staining solution of 25 mL
mixed with 1 L of water and stirred to get fine aquadest and 475 mL lactic acid for 24 hours
particles e. The roots were then cut into approximately 10 mm
b. Sieves of 500 μm, 125 μm, and 63 μm were length and lined on a glass slide then covered with
arranged and placed under running water. a cover slip; each slide has 10 pieces of roots.
c. The soil suspension was poured into a stratified f. Two samples were prepared from each
siever (size 500 μm, 125 μm, and 63 μm). This plant. Each sample was observed under the
step was repeated until the soil suspension compound microscope; fragmented root sections
became clear. characterized by hyphae, arbuscular or vesicles
d. Soil deposits of 125 μm and 65 μm were removed were photographed and recorded.
and transferred into centrifuge tubes and 60%
sugar solution was added with a ratio of 3:2, The percentage of root colonization was calculated
shaken and centrifuged for 3 minutes at 2500 rpm. using the formula developed by Brundrett et al.
e. The supernatant was poured into a plastic funnel (1996):
with millipore paper to filter the sample into the
glass bottles. The previously used milipore paper
was divided into eight gradients to facilitate
observation.
f. The filtered spores were then washed, cleaned Result and Discussion
with water from a spray bottle to prevent spore
lysis. The Diversity of AMF Genus
g. Spores that had been trapped in the millipore
paper were removed using a tweezer into a petri Six genera of AMF were found from the rhizosphere of
dish black cumin accessions (Table 1). Glomus is the most
h. Spore counting and genus identification were commonly discovered genus, with more than 96%,
observed under a compound microscope followed by Gigaspora, Acaulospora, Scutellospora,
(Olympus SZ261) under 40x magnification Dentiscutata, and Entrophospora (Table 1).

Spore Counting The presence of Glomus in each of black cumin

accession indicates that Glomus has a very wide
Spores were counted using a hand counter under the dispersal area. This is in accordance with Hartoyo
Olympus SZ61 Microscope under 40 x magnifications. et al. (2011) that Glomus has the widest distribution
The area of the millipore were divided into eight compared to any other AMF genus. The presence of
sections; spores were counted from the 1st to the Glomus species that are more than other genera is
8th section using a hand counter under a compound also the cause of frequent encounters of this genus
microscope; the number of spores from each section (INVAM 2018). The optimum pH of planting medium,
were added up to get the total spore count. which was neutral to alkaline, might have provided an
optimal environment for Glomus germination.
AMF Identification
The highest number of spores was found in Indian
Spores were identified based on their AMF morphology accession (96.42 spores). It is suspected that in
according to the method of INVAM (2018). Each general the Indian AMF accession is more active
sample consists of 20 g rhizospheric soil. compared to AMF of other accessions. The American
and Kuwait accessions were found in six genera
AMF Colonization Calculation of AMF, i.e. Glomus, Gigaspora, Acaulospora,
Scutellspora, Dentiscutata, and Entrophospora. The
Observation of AMF colonization was conducted number of spores found was still relatively low, i.e.
after cleaning and staining the roots. Root coloration 4.32 spores per g of sample. Growing black cumin
was scored using the method of Clapp et al. (1995) in polybags might be the one of the causes of the
described below: small AMF population. In addition, the often rainy
a. The roots were washed thoroughly using a sieve field conditions affect the increase in the number of

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Table 1. Quantity of AMF spores in black cumin rhizosphere

Accessions*
AMF Genera
N1 N2 N3 N4 N5 N6
Glomus 79.17 79.58 76.17 82.33 96.42 83.58
Gigaspora 2.42 1.08 1.08 1.42 1.33 1.08
Acaulospora 1.83 0.42 1.67 1.08 1.08 0.83
Scutellosprora 0.17 0 0 0 0 0.83
Dentiscutata 0.92 0.75 0.67 0.58 0.58 0.25
Entrophospora 0.17 0 0.08 0.17 0 0.17
*N1 = American accession, N2 = Turkey accession, N3 = Hong Kong accession, N4 = Slovenian accession, N5 =
Indian accession, and N6 = Kuwait accession

spores. Even though the average rainfall during the accessions of black cumin were infected by AMF
study was classified as low at 21.95 mm, but the high (Figure 3). This indicates an association between AMF
intensity of rain often occurred. According to Delvian and black cumin, and a successful symbiosis of AMF
(2003) the formation of spores is influenced by the with black cumin. One of the successes of infection
amount of rainfall and soil moisture fluctuations. can be seen from AMF colonization characterized by
the discovery of hyphae in the root tissue of plants
Differences in the number of spores among genera (Figure 2).
could possibly due to differences in exudates released
by each accession of black cumin. According to The highest colonization value in the accessions
Giovannetti et al. (1993) germination of spores and of India (N5) and Kuwait (N6) is 8.33% followed by
growth of hyphae are influenced by root exudates. Hong Kong 5.83% accession, 5% US, Turkey, and
Slovenia 3.33%. The percentage of infections found
AMF Colonization in the multiple accessions of black cumin is still low.
According to O’Connor et al. (2001) colonization
AMF colonization observation showed that all values of <10% is considered low, 10-30% is

133,408 μm 185,393 μm 65,611 μm

68,493 μm
137 μm
205,479 μm 3
1 2

376,392 μm

91,215μm 113,3995μm

95,890μm 110,730μm
4 5 6
384,704μm

98,639μm 334,244μm 76,404μm

98,179μm 77,272μm
7 8 9
351 138 μm
Figure 1. Glomus sp 1 (1), Glomus sp 2 (2), Glomus sp 3 (3), Glomus sp 4 (4), Gigaspora (5), Dentiscutata
(6), Acaulospora (7), Scutellospora (8), Entrophospora (9).

128 Faisal Al Asad, Ani Kurniawati, Sri Wilarso Budi R, Didah Nur Faridah
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moderate, whereas >30% is high. This low value of 2004). P availability in the rhizosphere is related to
colonization was possibly related to the fact that the soil pH. Acidic soil media conditions will increase
planting medium was less optimal for AMF growth. the value of AMF colonization (Singh, 2004). The
According to Chalimah et al. (2007) one of the successful utilization of AMF can be seen in the size
factors that determine spore germination and AMF of colonization. The higher the AMF colonization in the
colonization is the composition and pH of the planting host plant the higher symbiotic association between
medium. The planting media in this study was soil, AMF and the host plants.
manure, and charcoal husk (2: 1: 1 v/v) with a pH of
7.05. According to Goltapeh et al. (2008) the colony
growth will be optimal at pH 5.6. The increasing pH Conclusion
of the planting medium from 4.7, 5.6 to 6.4 increased
the values of AMF colonization (Nurlaeny et al., 1996; AMF can readily associate and form a symbiotic
Singh, 2004). relationship with various accessions of black cumin
grown in Indonesia. Six genera of AMF were found in
The condition of the planting medium that is the rhizosphere of black cumin: Glomus, Gigaspora,
suspected to have a high availability of P might have Acaulospora, Scutellospora, Dentiscutata, and
caused the less activity of the AMF. AMF activity will Entrophospora with the genus Glomus to be
be optimal for planting media with low P availability, predominant (96.42 spores) in black cumin Indian
whereas in planting media where P availability is accession.
high, AMF activity will not affect host plants (Swift,

 

Figure 2. AMF colonization of black cumin roots in the form of vesicular (A), arbuscular (B).

9 8,33 8,33
8
Percentage of colonization

7
5,83
6
5,00
5
4 3,33 3,33
3
2
1
0
American Slovenian Turkey Indian Hong Kuwait
Kong
Black cumin accession
Figure 3. Percentage of AMF colonization in the rhizosphere of six accessions of black cumin

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Acknowledgement Mycorrhizal Fungi In “Proceedings of the 8th

International Symposium on Microbial Ecology”
The authors thanked Lembaga Pengelola Dana (C.R. Bell, M. Brylinski, and P. Johnson-Green,
Pendidikan (LPDP) and Penelitian Terapan Unggulan eds.) pp. 845-851. Atlantic Canadian Society of
Perguruan Tinggi 2018 (PTUPT) for funding this Microbial Ecology.
research, and the manager of IPB Pasir Sarongge
Experimental Station for facilitating the study. Hartoyo, B., Ghulamahdi, M., Darusman, L.K., Aziz,
S.A., and Mansur, I. (2011). Keragaman
fungi mikoriza arbuskula (AMF) pada rizosfer
References tanaman pegagan (Centella asiatica L urban).
Jurnal Litri 1, 32-40
Brundrett MC, Bougher N, Dell B, Grove T, and
Malajczuk N. (1995). “Working with Mycorrhizas [INVAM]. (2018). International culture collection of
in Forestry and Agriculture”. Australian vesicular arbuscular Mychorizal Fungi http://
Centre for International Agricultural Research. invam.caf.wvu.edu [July 13, 2018]
Canberra. https://www.aciar.gov.au/node/7406
[July 13, 2018]. Lekatompessy, S.J.R. and Sukiman, H.I. (2015).
Peran mikroba dalam penyediaan bibt
Chairani H. (2008). Pertumbuhan dan kadar P akar berkualitas dalam menunjang penghijauan
kedelai bermikoriza pada perlakuan cekaman kota. Prosiding Seminar Nasional Masyarakat
alumunium dan kekeringan In “Proceding Biodiversitas Indonesia 1, 2000-2005
Pecepatan Sosialisasi Teknologi Mikoriza
untuk Mendukung Revitalisasi Pertanian, Liu, A., Hamel, C., Hamilton, R.I. and Smith, D.L.
Perkebunan, dan Kehutanan” (S.W. Budi, M. (2000). Mycorrhizae formation and nutrient
Turjaman, N.F. Mardatin, A.D. Nusantara, uptake of new corn (Zea mays L.) hybrids
O. Trisilawati, I.R. Sitepu, A.S. Wulandari, with extreme canopy and leaf architecture
M. Riniarti, L.Setyaningsih, eds.), pp. 72-75. asinfluenced by soil N and P levels. Plant Soil
Bogor, Indonesia. 221, 157–166

Chalimah, S., Muhadiono., Aznam Latifah., Haran Marschner, H. and Dell, B. (1994). Nutrient uptake in
Said., and Mathius, N.T. (2007). Propagation mycorrhizal symbiosis. Physiologia Plantarum
of Gigaspora sp. and Acaulospora by pot 159, 89–102.
culture in green house. Biodiversitas 4, 12-1
Nurlaeny., Marschner, H., and Goerge, E. (1996).
Clapp, J.P., Young, J.P.W., Merryweather, J., Effects of liming and mycorrhizal colonization
and Fitter, A. H. (1995). Diversity of fungal on soil phosphate depletion and phosphate
symbionts in arbuscular mycorrhizas from a uptake by maize (Zea mays L.) and soybean
natural community. New Phytologist 130, 259– (Glycine max L.) grown in two tropical acid
265. soils. Plant and Soil 181, 275-285

Delvian. (2003). “Keanekaragaman Cendawan O’Connor, P.J., Smith, S,E., and Smith, F.A. (2001)
Mikoriza Arbuskula (CMA) di Hutan Pantai Arbuscular mycorrhizal associations in the
dan Potensi Pemanfaatannya”. Thesis. Bogor southern Simpson Desert. Australian Journal
Agricultural University. Bogor. of Botany 49, 493–499

Gaur, S. and Kaushik, P. (2011). Biodiversity Pacioni, G. (1992). Wet sieving and decanting
of vesicular arbuscular mycorrhiza with techniques for the extraction of spores of VA
Catharanthus roseus, Ocimum spp. and mycorrhyzal fungi. Methods in Microbiology
Asparagus rocemosus in Uttarakhand State of 24, 317-322.
Indian Central Himalaya. International Journal
of Botany 7, 31-44 Peterson, R.L., Massicotte, H,B., and Melville, H.
(2004). “Mycorrhizas: Anatomy and Cell
Goltapeh, M., Danesh. Y.R., Prasad, R., and Varma, Biology”. 173 pp. CABI Publishing. Ottawa.
A. (2008). “Mycorrhizal Fungi: What We Know
and What Should We Know?” 3rd ed. Springer, Singh, S. (2004). Effect of soil pH on mycorrhiza in
Heidelberg. agricultural crops. Mycorrhizal News 16, 2-7

Klironomos, J.N. (2000) Host-Specificity and Smith, S.E. and Read, D. (2008). “Mycorrhizal
Functional Diversity among Arbuscular Symbiosis”. 3rd ed. 800 pp. Academic Press.
United Kingdom.

130 Faisal Al Asad, Ani Kurniawati, Sri Wilarso Budi R, Didah Nur Faridah
Journal of Tropical Crop Science Vol. 5 No. 3, October 2018
www.j-tropical-crops.com

Sreennivasa, M.N., Krishnaraj, P.U., Gangadhara,

G.A., and Manjunathalah, H.M. (1993).
Response of chilli to the inoculation of an
efficient vesicular-arbuscular mycorrhizal
fungus. Scientia Horticulturae 53, 45-52.

The Diversity of Arbuscular Mycorrhizal Fungi in the Black Cumin Rhizosphere .......... 131