Journal of Geoscience and Environment Protection, 2014, 2, 9-14
Published Online July 2014 in SciRes. http://www.scirp.org/journal/gep
http://dx.doi.org/10.4236/gep.2014.24002
How to cite this paper: Nwoko, C. O. (2014). Effect of Arbuscular Mycorrhizal (AM) Fungi on the Physiological Performance
of Phaseolus vulgaris Grown under Crude Oil Contaminated Soil. Journal of Geoscience and Environment Protection, 2, 9-14.
http://dx.doi.org/10.4236/gep.2014.24002
Effect of Arbuscular Mycorrhizal (AM) Fungi
on the Physiological Performance of
Phaseolus vulgaris Grown under Crude Oil
Contaminated Soil
Chris O. Nwoko
Department of Environmental Technology, Federal University of Technology, Owerri, Imo State, Nigeria
Received March 2014
Abstract
An experiment was conducted to assess the influence of arbuscular mycorrhizal (AM) fungi on the
performance of Phaseolus vulgaris under crude oil contaminated soil. P. vulgaris was grown on
soil under 2%, 4% and 8% (v/w) crude oil contamination. The experimental units were biostimu-
lated with 2 g NPK fertilizer pot−1 and were inoculated with 12 g AM inoculum pot−1. Non inocu-
lated pots served as control. The results showed that AM inoculated pots recorded higher and sig-
nificantly (P < 0.05) different dry matter yields and chlorophyll content than non AM inoculated
pots. Residual total petroleum hydrocarbon (TPH) increased as percent crude oil contamination
increased. Total petroleum hydrocarbon decomposition and removal was higher on pots inocu-
lated with AM than non inoculated pots. With AM colonization, physiological characteristics of P.
vulgaris and TPH decomposition improved. This is evinced by the linear regression analysis be-
tween colonization and TPH (R2 = 0.77).
Keywords
Arbuscular Mycorrhizae, Crude Oil Decomposition, Phytoremediation
1. Introduction
Crude oil contamination of agricultural soil often put severe stress to soil health and productivity. Record shows
that crude oil spillage on arable land has been on the increase since the 20th century when global production
doubled (Onosode, 2003). In the Niger delta region of Nigeria alone, about 1.8 million barrels of crude oil have
been lost to the environment from 1976 to 1996 (DPR, 1999). Soil, under this condition, is much constrained to
deliver the ecosystem goods and services. These constraints include soil moisture stress, low nutrient capital,
and high phosphorus (P) fixation, low levels of soil organic matter, reduced soil aeration, poor soil permeability,
bulk density and loss of soil biodiversity. The challenge for the next 50 years is to double food production in a
more sustainable approach that will ensure public health and safety.

C. O. Nwoko
10
Bioremediation is a low cost approach towards soil restoration. It involves the use of natural processes to
contain, reduce and degrade contaminants. Various soil characteristics are essential to achieve comprehensive
bioremediation of contaminated soil (Nwoko & Ogunyemi, 2010). Soil physical, chemical and biological prop-
erties are important in developing a biodegradation potential for contaminated soil (Rogers et al., 1993).
The fungi that are probably most abundant in agricultural soils are arbuscular mycorrhizal (AM) fungi (phy-
lum: Glomeromycota). They account for 5% - 50% of the biomass of soil microbes (Olsson et al., 1999). Pools
of organic carbon such as glomalin produced by AM fungi may even exceed soil microbial biomass by a factor
of 10 - 20 (Rillig et al., 2001). The external mycelium attains as much as 3% of root weight (Jakobsen & Ro-
sendahl, 1990). Mycorrhizal fungi contribute to soil structure by 1) growth of external hyphae into the soil to
create a skeletal structure that holds soil particles together; 2) creation by external hyphae of conditions that are
conducive for the formation of micro-aggregates; 3) enmeshment of microaggregates by external hyphae and
roots to form macroaggregates; and 4) directly tapping carbon resources of the plant to the soils (Miller & Ja-
strow, 2000). This direct access will influence the formation of soil aggregates, because soil carbon is crucial to
form organic materials necessary to cement soil particles. Arbuscular mycorrhiza is an important microflora in
the rhizosphere of plants and thus improve overall microbial activity in the root zone. Gao et al. (2011) observed
that optimized microbiota in mycorrhizal association was responsible for PAH degradation in AM phytoremedi-
ation. Wu et al. (2011) suggested that the hyphae and extraradical mycelium of AM fungi could play important
roles in the uptake and translocation of phenanthrene (PHE) and pyrene (PYR) in plants.
This present research examined the influence of arbuscular mycorrhizal fungi in the remediation of crude oil
contaminated soil under African bean (Phaseolus vulgaris) grown pot experiment.
2. Materials and Methods
2.1. Soil Microcosm Experiment
Soil (Table 1) was spiked with 2%, 4% and 8% (v/wt) of crude oil (Nigerian bonny light) and inoculated with
12 g of mycorrhizal inoculum (16 spores∙g−1 Glomus mosseae), in addition to soil resident microbes and was
thoroughly mixed. Non inoculated pots were steam sterilized at 121˚C for 2 h using the autoclave and this
served as control. Four Seeds of African bean (Phaseolus vulgaris L.) were planted per pot and thinned to two
after germination. These were laid out in a simple randomized block design and replicated thrice. The pots were
biostimulated by adding 2 g of NPK fertilizer pot−1. The moisture content was routinely monitored and main-
tained at 50% water holding capacity (WHC) and average room temperature of (25˚C ± 1˚C). The experiment
was left for 10 weeks in a screen house.
2.2. Analytical Methods
Soils were randomly collected from each experimental unit, homogenized, crushed and dried in the dark at room
temperature under a fume hood. The soil physicochemical properties were determined using methods generally
applied in soil chemistry laboratories. pH by a potentiometric method in 1:2.5 (w/v) soil water ratio. Organic
carbon content was determined by wet oxidation. Total petroleum hydrocarbon (TPH) was determined using a
modified EPA 8015 technique.
2.3. Mycorrhizal Colonization
To assess AM fungi colonisation, the fresh fine root sub-samples were cut into approximately 1 cm pieces,
Table 1. Initial soil characte ristics.
Property Soil Analytical methods
Porosity (%) 51.2 ± 2.1 Brandom (1986)
Org. C (%) 0.62 ± 0.01 Wet oxidation
Total N (%) 0.41 ± 0.02 Micro kjeldhal
pH (1:2) H2O 5.4 ± 1.2 pH meter
Org. C = organic carbon.

C. O. Nwoko
11
heated in a pressure pan at 120˚C in 10% KOH and stained using an adaptation of Phillips and Hayman (1970)
protocol including a longer incubation in 2% HCl (Oliveira et al., 2001). Stained root samples were examined
microscopically to assess the percentage of mycorrhizal colonisation using the grid-line intersect method (Gi-
ovannetti & Mosse, 1980).
To estimate the percentage of mycorrhizal colonization (x), intensity of infection (I) and arbuscular develop-
ment (A) in the infected region of the roots were estimated in P. vuglaris root samples.
2.4. Plant Analysis
Dry weight of roots and shoots were determined by drying at 70˚C for 24 hrs. Chlorophyll content of plants was
measured according to Harbon, (1984). The moisture content of plant tissues was determined as, an aliquot of
plant sample was weighed, dried at 105˚C for 24 h, and weighed again; the difference gave the percent moisture.
Data obtained were statistically analysed using Minitab software version 16.
3. Results and Discussion
The growth and development of Phaseolus vulgaris as influenced by crude oil contamination and AM inocula-
tion is shown in Table 2. Mycorrhizal inoculation generally influenced dry matter yield. Arbuscular mycorrhizal
inoculated pots recorded higher and significantly different (P < 0.05) yields compared to non AM inoculated
pots. At 2% crude oil contamination, dry matter yield on AM was more than non AM inoculated pots. Percent
crude oil contamination did not significantly affect dry matter yield on the average, low crude oil contamination
(2%) had higher yields than 8%.
Chlorophyll content of P. vulgaris was significantly affected by the crude oil contamination and mycorrhizal
inoculation. Shoot chlorophyll content decreased as percentage crude oil contamination increased. Mycorrhizal
inoculation significantly enhanced chlorophyll content of P. vulgaris irrespective of the level of crude oil con-
tamination (Table 2).
The soil chemical characteristic as influenced by the crude oil contamination is presented in Ta ble 3. Soil pH
was not significantly affected by all the treatments. Percent crude oil contamination did not affect percentage
moisture of the residual soil. The overall effect of crude oil contamination on soil organic carbon indicated low-
est (0.68%) organic carbon at 8% crude oil, 0.90% at 4% crude oil and the highest (1.16%) at 2% crude oil con-
Table 2. Effect of AM on the performance of P. vulgaris on various crude oil contamination level.
Parameter
Crude oil concentration
2% 4% 8%
AM nonAM AM nonAM AM nonAM
Dry weight (g) 35.4 ± 0.87a 31.4 ± 1.61ab 33.3 ± 0.95a 27.67 ± 1.58b 34.6 ± 2.62a 16.4 ± 0.55c
Chlorophyll (µg/g) 230 ± 15.39a 170 ± 5.8b 140.6 ± 75.86c 94.07 ± 8d 121.6 ± 10.0c 72.67 ± 6.8d
AM = arbuscular mycorrhiza. Rows bearing the same letters are not significantly different.
Table 3. Residual soil chemical characteristics as influenced by AM under P. vulgaris grown pot experiment.
Parameter
Crude oil contamination
2% 4% 8%
AM nonAM AM nonAM AM nonAM
pH 5.7 ± 0.1a 5.8 ± 0.05a 5.7 ± 0.05a 5.7 ± 0.05a 5.8 ± 0.11a 5.63 ± 0.2a
Org. C (%) 1.16a 0.98b 0.84ab 0.90ab 0.91ab 0.68c
TPH (mg/g) 2.23 ± 0.21e 4.1 ± 0.05c 2.96 ± 0.208d 5.66 ± 0.11b 4.6 ± 0.26c 7.13 ± 0.21a
%moisture 57.2 ± 0.72a 40.6 ± 0.5c 52.9 ± 1.2b 36.6 ± 0.8d 53.3 ± 1.05b 32.2 ± 0.52e
Org. C = Organic carbon, TPH = total petroleum hydrocarbon, AM = arbuscular mycorrhizal Rows bearing the same letters are not significantly dif-
ferent.

C. O. Nwoko
12
tamination (Table 3). Residual TPH concentration increased as percent crude oil contamination increased in the
test soil. Arbuscular mycorrhizal inoculation significantly affected the decomposition of crude oil in this expe-
riment. This is evidenced in the decrease in TPH concentration when compared to non-AM inoculated pots at
different crude oil contaminations (Table 3). The overall assessment of impact of percent crude oil soil conta-
mination showed no significant difference on all parameters at different levels of contamination (Table 4).
The arbuscular mycorrhizal colonization of P. vulgaris roots is shown in Table 5. The crude oil contamina-
tion significantly affected the level of AM colonization, development and severity of infection in this experi-
ment. The levels of root colonization by G. moseae are expressed in three ways: 1) frequency of root segments
(X%) reflecting the proportion of roots colonized with mycorrhizal fungi. 2) Intensity of mycorrhizal coloniza-
tion in root tissues (I%). 3) The rate of arbuscular formation in root segments (A%) reflecting the potentiality of
exchange with the symbiosis. From the result, the root segmentation, intensity of colonization and arbuscular
formation decreased as crude oil contamination increased (Table 5).
The correlation matrix between AM infection on soil and plant characteristics is shown in Table 6. There is
positive correlation (P > 0.05) between percentage organic carbon and arbuscular mycorrhizal development and
infection in the crude oil contaminated soil. Dry matter yield and chlorophyll content of P. vulgaris had signifi-
cant positive correlation with mycorrhizal colonization and intensity of infection. Thus, with AM colonization,
the physiological characteristics of P. vulgaris were greatly improved. Total petroleum hydrocarbon concentra-
Table 4. Overall assessment of crude oil contamination on P. vulgaris and soil characteristics as influenced by arbuscular
mycorrhizal inoculation.
% crude oil pH %moisture TPH (mg/g) Org. C (%) Dry weight (g) Chlorophyll (µg/g)
2% 5.68 ± 0.02a 49.7 ± 13.6a 2.48 ± 1.3a 0.89 ± 0.28a 31.9 ± 5.7a 206.7 ± 44.5a
4% 5.8 ± 0.17a 48.7 ± 12.4a 3.78 ± 1.6a 0.81 ± 0.06a 30.4 ± 5.1a 124.0 ± 44.3a
8% 5.83 ± 0.2a 50.2 ± 14.9a 4.3 ± 0.5a 0.76 ± 0.19a 28.7 ± 6.3a 121.4 ± 44.3a
P < 0.05 ns ns ns Ns ns ns
Table 5. Percentage of AM colonization on the root of P. vulgaris as influenced by crude oil contamination and soil particle
size.
Colonization Crude oil
2% 4% 8%
X% 87a 66.3b 49.3c
I% 30.6a 26.3ab 22.6b
A% 41.0a 30.3b 20.3c
X% = Frequency of mycorrhizal root segments, I% = intensity of mycorrhizal infection, A% = rate of arbuscular development. Rows bearing the
same letters are not significantly different.
Table 6. Correlation matrix of AM infection on soil and plant characteristics.
Measurement X% I% A%
Org. C (%) 0.44
(0.2)
0.54
(0.13)
0.210
(0.58)
TPH (mg/g) −0.89
(0.001***)
−0.92
(0.00***)
−0.684
(0.04**)
Dry weight (g) 0.734
(0.02**)
0.86
(0.003**)
0.317
(0.406)
Chlorophyll (µg/g) 0.876
(0.002**)
0.831
(0.006**)
0.80
(0.01**)
X = percentage mycorrhizal colonization, I% = intensity of AM infection, A% = arbuscular development.

C. O. Nwoko
13
tion showed significant negative correlation with the mycorrhizal colonization (P = 0.001), intensity of infection
(P = 0.0001), arbuscular development (P = 0.04). At higher degree of AM infection and severity, the crude oil
degradation and removal was enhanced. This is evinced by the linear regression analysis between TPH and co-
lonization. There was strong negative relationship between the AM root colonization of P. vulgaris and residual
TPH concentration (R2 = 0.77). Soil porosity measures the total volume of pore spaces and this negatively in-
fluenced soil residual TPH concentration (R2 = 0.77, P = 0.002).
The overall significant dry matter yield and chlorophyll content observed in mycorrhizal inoculated pots may
be attributed to improved nutrient acquisition, water relations, pollutant tolerance and sequestration potentials of
AM infected roots of P. vulgaris. One mechanism that may be involved is the oxidation of contaminant by acti-
vated oxygen species and concomitant enhancement of oxidoreductases to protect the plant from oxidative stress.
Satzer et al., (1999) noted enhanced levels of hydrogen peroxide in AM roots as well as enhanced levels of pe-
roxidase activity in mycorrhizal roots and the rhizosphere which may lead to enhanced oxidation of crude oil
around AM colonized root (Criquet et al., 2000). One peculiarity of crude oil polluted soil that may be overcome
by AM plant is the hydrophobicity and resulting limitations in uptake of water dissolved inorganic nutrients
(Leyval & Binet, 1998). There are good reasons to believe that mycorrhizal infection of roots of tropical plant
species induces tolerance against abiotic and biotic stresses.
Decomposition of crude oil was significantly improved in the mycorrhizal inoculated pots as evidenced in the
concentration of residual total petroleum hydrocarbon in this experiment. This important finding could be ex-
plained in the light of root physiology modification by AM that tends to increase enzyme activity level and root
exudation which directly stimulates crude oil degradation. Indirect mechanisms rely on root surface properties or
rhizosphere soil properties that act on crude oil availability through adsorption and co-metabolism (Jones &
Leyval, 2003).
The levels of root colonization by AM were decreased with increasing concentration of crude oil in soil. Of
note in this study is the behaviour of mycorrhizal activity in the contaminated soil. For example, the intensity of
colonization in the root tissues and rate of arbuscular formation in root segments showed significant positive and
negative correlations with dry matter yield, chlorophyll content and residual TPH, respectively. This observation
reflects enhanced degradation of crude oil and symbiotic activity of AM fungi with P. vulgaris plant. Houng-
nandan et al., (2000) concluded that farmers’ management practices that allow a buildup of AM fungal inocu-
lums would alleviate P-deficiency and hence increase N-fixation which will ultimately increase physiological
development of the plant species. Similar interactions between AM fungi and rhizobia have been demonstrated
for soybean (Glycine max) in low-P soils of the savanna in Nigeria (Nwoko & Sanginga, 1999; Sanginga et al.,
1999). AM fungal and rhizobial responses might show positive feedback. Rhizobial inoculation increased AM
colonization in soybean (Sanginga et al., 2000) and mucuna (Houngnandan et al., 2001). P. vulgaris, a legu-
minous plant, may have played significant role in nitrogen fixation that further improved rhizodegradation of the
crude oil contaminant.
4. Conclusion
The physiological development of P. vulgaris under abiotic stress may be improved through soil biological im-
provement strategies such as arbuscular mycorrhizal inoculation. Arbuscular mycorrhizal tends to ameliorate
unfavourable conditions posed by crude oil contamination by enhanced production of oxidative enzymes and
overall improvement in the soil aggregation.
References
Brandom, T. M. (1986). Groundwater Occurrence, Development and Protection. Water Practice Manuals (p. 615). London:
Institute of Water Engineers and Scientists.
Criquet, S., Joner, J., Leglize, P., & Leyval, C. (2000). Effect of Anthracene and Mycorrhiza on the Activity of Oxidoreduc-
tases in the Roots and the Rhizosphere of Lucerrne (Medicago sativa). Biotechnology Letters, 22, 1733-1737.
http://dx.doi.org/10.1023/A:1005604719909
Directorate of Petroleum Resources (DPR) (1999). Nigeria: Environmental Guidelines and Standards for the Petroleum In-
dustry in Nigeria (EGAS) (Draft Revised Edition). Lagos, Nigeria: Department of Petroleum Resources.
Gao, Y., Li, Q., Ling, W., & Zhu, X. (2011). Arbuscular Mycorrhizal Phytoremediation of Soils Contaminated with Phe-
nanthrene and Pyren. Journal of Hazardous Materials, 185, 703-709. http://dx.doi.org/10.1016/j.jhazmat.2010.09.076

C. O. Nwoko
14
Giovannetti, M., & Mosse, B. (1980). An Evaluation of Techniques for Measuring Vesicular Arbuscular Mycorrhizal Infec-
tion in Roots. New Phytologist, 84, 489-500. http://dx.doi.org/10.1111/j.1469-8137.1980.tb04556.x
Harborne, B. (1984). Photochemical Methods. A Guide to Modern Techniques of Plant Analysis. London: Chapman & Hall
Press. http://dx.doi.org/10.1007/978-94-009-5570-7
Houngnandan, P., Sanginga, N., Okogun, A., Vanlauwe, B., Merckx, R., & Van Cleemput, O. (2001). Assessment of Soil
Factors Limiting Growth and Establishment of Mucuna in Farmers’ Fields in the Derived Savanna of the Benin Republic.
Biology and Fertility of Soils, 33, 416-422. http://dx.doi.org/10.1007/s003740100347
Houngnandan, P., Sanginga, N., Woomer, P., Vanlauwe, B., & Van Cleemput, O. (2000). Response of Mucuna pruriens to
Symbiotic Nitrogen Fixation by Rhizobia Following Inoculation in Farmers’ Fields in the Derived Savanna of Benin. Bi-
ology and Fertility of Soils, 30, 558-565. http://dx.doi.org/10.1007/s003740050036
Jakobsen, I., & Rosendahl, L. (1990). Carbon Flow into Soil and External Hyphae from Roots of Mycorrhizal Cucumber
Roots. New Phytologist, 115, 77-83. http://dx.doi.org/10.1111/j.1469-8137.1990.tb00924.x
Jones, E. J., & Ley val, C. (2003). Rhizosphere Gradients of Polycyclic Aromatic Hydrocarbon (PAH) Dissipation in Two
Industrial Soils and the Impact of Srbuscular Mycorrhiza. Environmental Science & Technology, 37, 2371-2375.
http://dx.doi.org/10.1021/es020196y
Leyval, C., & Binet, P. (1998). Effect of Polyaromatic Hydrocarbons (PAHs) and Arbuslar Mycorrhizal Colonization of
Plants. Journal of Environmental Quality, 27, 402-407. http://dx.doi.org/10.2134/jeq1998.00472425002700020022x
Miller, R. M., & Jastrow, J. D. (2000). Mycorrhizal Fungi Influence Soil Structure. In: Y. Kapulnik, & D. D. Douds, Eds.,
Arbuscular Mycorrhizas: Physiology and Function (pp. 3-18). Dordrecht: Kluwer Academic.
http://dx.doi.org/10.1007/978-94-017-0776-3_1
Nwoko, C. O., & Ogunyemi, S. (2010). Effect of Palm Oil Mill Effluent (POME) on Microbial Characteristics in a Humid
Tropical Soil under Laboratory Conditions. International Journal of Environmental Science and Development, 1, 308-314.
Nwoko, H., & Sanginga, N. (1999). Dependency of Promiscuous Soybean and Herbaceous Legumes on Arbuscular Mycorr-
hizal Fungi and Their Response to Bradyrhizobial Inoculation in Low P Soils. Applied Soil Ecology, 13, 251-258.
http://dx.doi.org/10.1016/S0929-1393(99)00038-4
Oliveira, R. S., Dodd, J. C., & Castro, P. M. L. (2001). The Mycorrhizal Status of Phragmites australis in Several Polluted
Soils and Sediments of an Industrialized Region of Northern Portugal. Mycorrhiza, 10, 241-247.
http://dx.doi.org/10.1007/s005720000087
Olsson, P. A., Thingstrup, I., Jakobsen, I., & Baath, E. (1999). Estimation of the Biomass of Arbuscular Mycorrhizal Fungi
in a Linseed Field. Soil Biology & Biochemistry, 31, 1879-1887. http://dx.doi.org/10.1016/S0038-0717(99)00119-4
Onosode, G. O. (2003).The Vision: Niger Delta Environmental Survey. In B. A. Chokor, Ed., Environmental Issues and
Challenges of the Niger Delta (pp. 78-98). The CIBN Press Ltd.
Phillips, J. M., & Hayman, D. S. (1970). Improved Procedures for Clearing and Staining Parasitic and Vesicular-Arbuscular
Mycorrhizal Fungi for Rapid Assessment of Infection. Transcriptions of the British Mycology Society, 55, 158-161.
http://dx.doi.org/10.1016/S0007-1536(70)80110-3
Rillig, M. C., Wright, S. F., Nichols, K. A., Schmidt, W. F., & Torn, M. S. (2001). Large Contribution of Arbuscular My-
corrhizal Fungi to Soil Carbon Pools in Tropical Forest Soils. Plant Soil, 233, 167-177.
http://dx.doi.org/10.1023/A:1010364221169
Rogers, J. A., Tedaldi, D. J., & Kavanaugh, M. C. (1993). A Screening Protocol for Bioremediation of Contaminated Soil.
Environmental Progress, 12, 146-149. http://dx.doi.org/10.1002/ep.670120213
Salzer, P., Corbiere, H., & Boller, T. (1999). Hydrogen Peroxide Accumulatiob in Medicago Truncatula Roots Colonized by
the Arbuscualr Mycorrhiza Forming Fungus Glomus intraradices. Planta, 208, 319-325.
http://dx.doi.org/10.1007/s004250050565
Sanginga, N., Carsky, R. J., & Dashiell, K. (1999). Arbuscular Mycorrhizal Fungi Respond to Rhizobial Inoculation and
Cropping Systems in Farmers’ Fields in the Guinea Savanna. Biology and Fertility of Soils, 30, 179-186.
http://dx.doi.org/10.1007/s003740050606
Sanginga, N., Lyasse, O., & Singh, B. B. (2000). Phosphorus Use Efficiency and Nitrogen Balance of Cowpea Breeding
Lines in a Low P Soil of the Derived Savanna Zone in West Africa. Plant and Soil, 220, 119-128.
http://dx.doi.org/10.1023/A:1004785720047
Wu, F. Y., Yu, X. Z., Wu, S. C., Lin, X. G., & Wong, M. H. (2011). Phenanthrene and Pyrene Uptake by Arbuscular My-
corrhizal Maize and Their Dissipation in Soil. Journal of Hazardous Materials, 187, 341-347.
http://dx.doi.org/10.1016/j.jhazmat.2011.01.024