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Journal of Plant Nutrition
ISSN: 0190-4167 (Print) 1532-4087 (Online) Journal homepage: http://www.tandfonline.com/loi/lpla20
Response of a wild-type and modern cowpea
cultivars to arbuscular mycorrhizal inoculation in
sterilized and non-sterilized soil
Marjorie Bonareri Oruru, Ezekiel Mugendi Njeru, Remy Pasquet & Steve
Runo
To cite this article: Marjorie Bonareri Oruru, Ezekiel Mugendi Njeru, Remy Pasquet & Steve
Runo (2018) Response of a wild-type and modern cowpea cultivars to arbuscular mycorrhizal
inoculation in sterilized and non-sterilized soil, Journal of Plant Nutrition, 41:1, 90-101, DOI:
10.1080/01904167.2017.1381728
To link to this article:  https://doi.org/10.1080/01904167.2017.1381728
Accepted author version posted online: 21
Sep 2017.
Published online: 03 Nov 2017.
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Response of a wild-type and modern cowpea cultivars to arbuscular
mycorrhizal inoculation in sterilized and non-sterilized soil
Marjorie Bonareri Orurua,b, Ezekiel Mugendi Njeru a, Remy Pasquetb, and Steve Runoc
aDepartment of Microbiology, Kenyatta University, Nairobi, Kenya; bInternational Center for Insect Physiology and
Ecology, Kasarani, Nairobi, Kenya; cDepartment of Biochemistry and Biotechnology, Kenyatta University, Nairobi, Kenya
ARTICLE HISTORY
Received 16 May 2016
Accepted 28 November 2016
ABSTRACT
Cowpea is an important crop that serves as a legume and vegetable source to
many smallholder farmers in sub-Saharan Africa. Soil fertility is a significant
limitation to its production thus; inoculation with beneficial soil biota such as
arbuscular mycorrhizal fungi (AMF) could improve its performance. However,
plant–AMF interaction could vary based on crop cultivar hence affecting
overall crop production. The present study aimed at determining the effect
of AMF inoculation and soil sterilization on root colonization and growth of a
wild-type and three modern cowpea cultivars grown by smallholder farmers
in Kenya. Potted cowpea plants were inoculated with a commercial AMF
inoculum comprising of Rhizophagus irregularis, Funneliformis mosseae,
Glomus aggregatum and Glomus etunicatum and maintained in a greenhouse
for 40 days. After harvesting, mycorrhizal colonization, nodule number and
dry weight, root and shoot dry weights, nitrogen (N,) phosphorus (P) and
potassium (K) content were determined. Interestingly, the modern cultivars
showed significantly (p < 0.001) higher root colonization, nodulation, shoot P
and N compared to the wild-type cultivar. Moreover, a strong positive
correlation between AMF root colonization and shoot P (r2 D 0.73, 0.90, p <
0.001), AMF root colonization and shoot N (r2 D 0.78; 0.89, p < 0.001) was
observed in both sterilized and non-sterilized soil, respectively. Soil
sterilization affected root colonization and growth parameters with plants
grown in non-sterilized soil performing better than those grown in sterilized
soil. This study provides major evidence that modern cowpea cultivars are
still responsive to mycorrhizal inoculation suggesting that modern breeding
programs are not deleterious AMF symbiosis.
KEYWORDS
arbuscular mycorrhizal fungi;
cowpea cultivars; root
colonization; smallholder
farmers
Introduction
Cowpea is a multipurpose legume that serves as human food, livestock fodder, income source,
and is grown in the semi-arid regions of most continents worldwide (Singh and Ajeigbe 2003).
Approximately, 5.7 million tons of cowpea was produced across the globe annually on about
11.3 million hectares in 2013, with sub-Saharan Africa (SSA) accounting for 70% of the total
global production (FAO 2013). Low soil fertility is a key challenge facing cowpea production,
especially among smallholder farmers in SSA due to poor soil health management practices. This
has led to an average annual depletion rate of 22 kg of nitrogen (N), 2.5 kg of phosphorus (P)
and 15 kg of potassium (K) hectare¡1 of cultivated land in 37 African countries over the last
30 years (Koohafkan and Altieri 2010). The traditional method of overcoming nutrient depletion
CONTACT Ezekiel Mugendi Njeru njeruezek@gmail.com Department of Microbiology, Kenyatta University, P.O. Box 43844-
00100, Nairobi, Kenya.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lpla.
© 2018 Taylor & Francis Group, LLC
JOURNAL OF PLANT NUTRITION
2018, VOL. 41, NO. 1, 90–101
https://doi.org/10.1080/01904167.2017.1381728
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is the use of chemical fertilizers, which are unfortunately too costly for the majorly resource-poor
smallholder farmers. This necessitates the use of sustainable agricultural practices such as incor-
poration of beneficial soil biota that promote uptake of limited soil nutrients such as N and P by
plants.
Among the key groups of important soil microorganisms are arbuscular mycorrhizal fungi
(AMF) that belong to the phylum Glomeromycota and form symbiotic association with the roots
of over 80% of wild and cultivated plant species (Barrow et al. 2008). AMF depend on the host
plant for the supply of photosynthetic carbohydrates and in return deliver many agroecosystem
services including soil aggregation, nutrient uptake and carbon sequestration (Oruru and Njeru
2016) through an extensive extraradical hyphal network that spreads from colonized roots into
the soil. Additionally, AMF enhance plant resistance to biotic and abiotic stresses, as well as the
synthesis of important plant secondary metabolites, which contributes to the production of safe
and high-quality food (Giovannetti et al. 2012). These symbiotic fungi have also been found to
increase nodulation and atmospheric N fixation potential in legumes such as cowpea (Turk et al.
2008), since the fungus improves P uptake by the plant, which in turn would avail more energy
for N fixation by rhizobia. AMF may form tripartite symbiosis with legumes and rhizobia to
stimulate nodulation and plant growth (Xavier and Germida 2002). Other additional effects pro-
duced by this interaction include greater number and dry weight of nodules, enhanced symbiotic
N fixation and higher N content (Shockley et al. 2004). The beneficial effect of N2 fixation by
AMF colonization has been thought to be caused by increased P supply to the nodules by the
symbiotic fungal partner.
Differences in the way plants respond to AMF have been observed among not only plant spe-
cies, but also cultivar types. Zhu et al. (2001) assessed six wheat varieties and reported that the
percentage AMF colonization was lower in modern varieties compared to old varieties (Landra-
ces). Tarawaya (2003) also showed that improved, high yielding wheat cultivars responded less to
AMF compared to landrace cultivars. Similar findings were obtained by (Zhu et al. 2003) in bar-
ley, where an improved cultivar was less responsive compared to a landrace. While conducting a
study to compare the interaction of wild-type, old and modern tomato cultivars with AMF, Glo-
mus mosseae, Steinkellner et al. (2012), reported a relatively higher AMF colonization in modern
cultivars compared to the wild-type and old cultivars. Similar results were reported by Njeru
et al. (2013), where composite cross-maize populations developed through evolutionary breeding
had relatively a higher level of AMF colonization compared to modern hybrids. Since very few
studies have been done on the response of cowpea to AMF, there is a possibility that different
cultivars differing in their genetic makeup or improvement respond differently to AMF inocula-
tion. Moreover, to our knowledge no study has been conducted to compare how different cowpea
cultivars maintained by farmers in Kenya interact with AMF although the crop is an important
food crop to many smallholder farmers as both a grain legume and vegetable.
Here, we tested the hypotheses that the susceptibility of AMF colonization differs between the wild-
type and the cultivated cowpea cultivars and that AMF inoculation would enhance shoot nutrition and
other growth parameters. The specific aims of the study were (1) to compare the susceptibility of a
wild-type vs. three cultivated cowpea cultivars to AMF colonization, (2) to determine the effect of fun-
gal root colonization in the wild-type and the cultivated cultivars and compare their effect on different
growth parameters, and shoot nutrition and (3) to determine the effect of soil sterilization on AMF col-
onization and growth of different cowpea cultivars.
Materials and methods
Fungal inoculum
Myco Apply custom super concentrate powder, comprising of four mycorrhizal species, Funneliformis
mosseae, Glomus aggregatum, Glomus etunicatum and Rhizophagus irregularis, was used as the inocu-
lum. The super concentrate powder contains 220,200 mycorrhizal propagules gram¡1. This
JOURNAL OF PLANT NUTRITION 91
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commercial product was provided by Symyco, a joint venture between Symbiotic Sciences based in
New Delhi, India and Mycorrhizal Application Inc., based in Grants Pass, Oregon, USA.
Experimental soil
The soil used for the greenhouse experiment was obtained from four farms in Embu County, which lies
about 120 km north east of Nairobi, on the South Eastern side ofMount Kenya. Based on FAO soil classi-
fication, the soil used belongs to the phaeozems category, characterized by well-developed subsoil with a
blocky structure and richness in organic matter. Phaeozems are also porous, fertile soils that make excel-
lent farmland. Crop rotation and use of organic manure are management practices used by farmers in
the four farms. Agriculture is the main economy driver in this county with more than 70% of the popula-
tion accounting for smallholder farmers. Cowpea is among the major food crops produced in the county,
although its production is quite lower than staples such as maize. Substantial rainfall in received in the
county with the average annual precipitation being 1,206 mm. Ten sub-samples of about 900 g were col-
lected from each of the four farms by removing soil core from 5 to 20 cm depth using a soil auger. The
experimental soil characteristics were; pH (water) 5.20, organic carbon (C), 1.82%, N 0.15%, exchange-
able K 0.50 Cmolkg¡1, sodium (Na) trace Cmolkg¡1, calcium (Ca) 6.50 Cmolkg¡1, magnesium (Mg)
6.00 Cmolkg¡1, cation exchange capacity (CEC) 13.40 Cmolkg¡1and available P (CAL) 14.40 mg kg¡1.
Experimental design
The experiment had three factors (soil sterilization, cowpea genotype and mycorrhizal inoculation) fac-
torial in Randomized Complete Block Design with four replicates. The greenhouse experiment was
repeated in space for comparison and validity purposes. Cowpea genotypes included three cultivars
(Katumani 80, KenKunde 1 and Kunde Mboga) commonly grown by smallholder farmers in Kenya
and one wild-type cultivar. Katumani 80 and KenKunde 1 are dual-purpose genotypes suitable for
both vegetable and grain production, while Kunde Mboga is a local vegetable that plays a vital role in
restoration of soil fertility. The wild-type cowpea was accession SP 219, which belongs to Vigna ungui-
culata (L.) Walp. subsp. unguiculata var. spontanea (Schweinf.) Pasquet. The accession comes from
the South Coast of Kenya.
Planting and maintenance of greenhouse bioassays
Sterilized soil and sand (silica) were mixed (1:1 by volume) and mycorrhizal inoculation done by mix-
ing the inoculum with soil at a depth of 3–6 cm prior to sowing as per the manufacturer’s instruction.
All the pots were also supplied with a filtrate obtained by sieving an aliquot of non-sterilized soil
through a 40-mm sieve to provide the substrate with an equivalent soil microbiota (Njeru et al. 2014).
Two cowpea seeds were planted per pot, 4 inches apart and each seed approximately 1–2 inches deep.
The soil was watered immediately and then regularly after seed germination.
Plant sampling and analyses
The plants were sampled 40 days after planting and gently washed under running tap water to preserve
the root system. The roots, shoots and nodules were then separated. About 100 pieces of root segments
from each treatment were thoroughly washed, placed in falcon tubes and then cleared using 10% potas-
sium hydroxide (KOH) placed in 80
C water bath for 10–15 min, neutralized in 2% aqueous hydro-
chloric acid (HCl), and stained with 0.05% trypan blue in lactic acid. Root colonization was assessed
under a dissecting microscope at £25 or £40 magnification by the gridline intersect method
(Giovannetti and Mosse 1980).
The nodules detached from the root system were then counted, followed by dry weight of nodules,
roots and shoots obtained after oven drying at 65
C to a constant weight. To extract plant biomass,
0.025 g of oven dried, milled plant tissue was weighed and placed in a clean and dry digestion specimen
92 M. B. ORURU ET AL.
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tubes prior to ashing them in the muffle furnace for 41/2 hr and then allowed to cool. 0.5 ml of the
digestion mixture of acids and hydrogen peroxide was added and placed on the hot plate to evaporate
to dryness. 25 ml of 0.05 ammonium chloride (NH4Cl) was added to re-dissolve the digested sample
and allowed to stand for 5 hr after cooling. Shoot total N content was determined by Kjedahl method,
which involves the transformation of organic N to ammonium ions (Bremner 1982). The shoot P con-
tent was determined by colorimetric method, while readily exchangeable, water soluble K was deter-
mined in neutral NH4 acetate extractant following sulfuric/perchloric acid digestion (Page et al. 1982).
Data analyses
All data were tested for homogeneity of variance by Bartlett test before analyses. The % data were arc-
sine (xx) transformed wherever necessary to fulfill the assumptions of analysis of variance (ANOVA).
The data reported in tables and graphs were back transformed. Data were analyzed by two-way
ANOVA as a completely randomized block design. Pearson correlation coefficient was used to deter-
mine the relationship between shoot dry weight, N, P and K and mycorrhizal colonization. Wherever
applicable, post hoc test was performed using Tukey’s HSD test (p < 0.05). All statistical analyses were
performed with the SPSS (version 20.0 software).
Results
Effect of AMF inoculation, cowpea genotype and soil sterilization on root colonization
and growth parameters
Two way ANOVA data showed that root colonization was significantly affected by both the genotype
(F D 9.56, p <0.001) and AMF inoculation (F D 231.75, p < 0.001) with modern cultivars having
higher root colonization compared to the wild-type cultivar (Table 1). Moreover, significant differences
in AMF colonization were observed within the modern cultivars. Katumani 80 and Kunde Mboga had
higher level root AMF colonization compared to KenKunde 1, while non-inoculated plants were not
colonized. Plants grown in non-sterilized soil had significantly higher (F D 52.77, p < 0.001)
Table 1. ANOVA results for the effects of soil sterilization, cowpea genotype and AMF inoculation, and their interaction on root mycor-
rhizal colonization, nodule number, nodule dry weight, root dry weight and shoot dry weight of cowpea. The mean standard errors are
presented in parentheses.
%MC NN
NDW
(mg plant¡1)
RDW
(mg plant¡1)
SDW
(mg plant¡1)
Soil
Non-sterilized 30.34 (1.99)a 21.15 (0.94)a 13.86 (1.24)a 162.06 (10.19)a 917.98 (57.62)a
Sterilized 18.47 (3.29)b 19.38 (1.18)b 10.01 (1.23)b 145.27 (10.51)b 803.71 (58.71)b
Cowpea genotype
K 29.22 (4.65)a 22.31 (1.10)a 14.13 (1.96)a 188.55 (14.27)a 1048.24 (78.80)a
KM 27.41 (4.46)a 20.31 (1.41)ab 12.98 (2.00)ab 166.76 (16.20)b 921.55 (90.68)b
KK 22.95 (3.88)ab 21.00 (1.76)a 11.65 (1.77)b 143.04 (12.56)c 829.59 (77.04)c
Wild 17.45 (3.06)b 17.44 (1.59)b 8.98 (1.26)c 116.31 (9.36)d 644.00 (51.83)d
AMF inoculation
M 37.17 (1.27)a 24.84 (0.63)a 18.24 (0.71)a 202.5 (6.83)a 1141.13 (36.43)a
NM 11.34 (2.20)b 15.69 (0.77)b 5.62 (0.45)b 104.83 (4.28)b 580.56 (24.13)b
p values of the main factors and interaction
Soil <0.001 0.0294 <0.001 <0.001 <0.001
Genotype <0.001 0.0006 <0.001 <0.001 <0.001
AMF In <0.001 <0.001 <0.001 <0.001 <0.001
Genotype£ AMF In 0.3592 0.0023 <0.001 <0.001 <0.001
Values followed by the same letter in a column within each treatment are not significantly different at p < 0.05 (Tukey’s HSD test)
%MC: Mycorrhizal colonization percentage, NN: Nodule number, NDW:Nodule dry weight, RDW:Root dry weight, SDW:Shoot dry
weight, K-Katumani 80, KM:Kunde Mboga, KK:KenKunde 1, M:Mycorrhizal, NM:Non-mycorrhizal AMF In: Arbuscular mycorrhizal fungi
inoculation.
JOURNAL OF PLANT NUTRITION 93
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percentage colonization compared to those grown in sterilized soil. The interaction between genotype
£ AMF was not significant.
Cowpea genotype significantly affected nodule number (F D 6.69, p D 0.0006), where Katumani 80
had the highest mean nodule number, while the wild-type had the lowest nodule number (Table 1).
Mycorrhizal plants had a higher nodule number (F D 132.13, p < 0.001) compared with the non-inoc-
ulated plants. Moreover, soil sterilization has a significant effect in the number of nodules (F D 5.00,
p D 0.0294) with plants grown in non-sterilized soil having higher nodule number compared to those
grown in sterilized soil. Additionally, there was a significant (F D 5.46, p D 0.023) genotype £ AMF
interaction on nodule number (Table 1). The highest increase in nodulation after AMF inoculation
was observed in Kunde Mboga (69.1%) followed by KenKunde 1 (68.9%), while Kunde Mboga and
Katumani 80 had the same percentage increase (63.4%) (Table 2).
Correspondingly, the nodule dry weight was significantly affected by the genotype (F D 28.99,
p D <0.001), where Katumani 80 had the highest nodule dry weight and the wild-type having the low-
est weight (Table 1). AMF inoculation (F D 930.58, p < 0.001) and soil sterilization (F D 87.23,
p D <0.001) similarly affected nodule dry weight, with AMF inoculation and unsterilized soil produc-
ing higher nodule dry weight compared non-inoculated plants and sterilized soils. Besides, a significant
genotype£ AMF interaction (FD 9.32, p< 0.001) on the nodule dry weight was observed. In this case,
AMF inoculated Kunde Mboga (52.03%) showed the highest increase in nodule dry weight followed by
KenKunde 1 (48.4%), while the wild-type and Katumani 80 showed a similar same percentage increase
(43.4%) after inoculation (Table 2).
The root and shoot dry weights varied significantly (F D 111.85, p < 0.001) in all the cowpea culti-
vars with Katumani 80 having the highest root and shoot dry weights, while the wild-type had the low-
est root and shoot dry weights per plant. Mycorrhizal plants had significantly (F D 1105.94, p < 0.001)
higher root and shoot dry weights than non-mycorrhizal plants. Soil sterilization also had a significant
(F D 32.72, p < 0.001) effect with plants grown in non-sterilized soil having higher weights compared
to those grown in sterilized soil. The effect of genotype £ AMF interaction on the root and shoot dry
weights was significant (F D 14.54, p < 0.001). The highest increase in root and shoot dry weights after
inoculation was observed in Kunde Mboga (57.1% and 58.1%), while Katumani 80 had the lowest
increase (45.5% and 47.2%) (Table 2).
Effect of AMF inoculation, cowpea genotype and soil sterilization on cowpea shoot nutrition
The effect of cowpea genotype on the level of shoot N was statistically significant (F D 220.08, p <
0.001) with Katumani 80 having the highest percentage of shoot N and the wild-type having the lowest
(Table 3). Mycorrhizal plants had significantly (F D 2418.61, p < 0.001) higher level of shoot total N
than the non-inoculated plants (Table 3). Similarly, soil sterilization significantly (F D 104.23, p <
0.001) affected shoot N content with plants grown in non-sterilized soil having a higher N content
compared to those grown in sterilized soil. Additionally, genotype £ AMF interaction showed
Table 2. The effect of genotype £ AMF inoculation interaction on mycorrhizal colonization, nodule number, nodule dry weight, root
dry weight and shoot dry weight of cowpea with the mean standard errors shown in parentheses.
Cowpea genotype AMF In NN NDW RDW SDW
K M 21.25 (1.15)a 24.22 (4.79)a 242.21 (4.79)a 1344.46 (26.09)a
NM 7.13 (1.07)d 13.48 (5.19)c 134.88 (5.19)c 752.01 (29.33)cd
KM M 20.25 (1.09)ab 22.74 (6.11)ab 227.44 (6.11)ab 1261.03 (33.05)ab
NM 5.71 (0.89)de 10.68 (5.95)cd 106.08 (5.95)cd 582.08 (34.99)d
KK M 18.14 (0.94)b 18.97 (6.01)b 189.68 (6.01)b 1123.46 (12.36)b
NM 5.16 (0.77)de 9.65 (4.57)d 96.49 (4.57)d 535.73 (24.72)de
Wild M 13.48 (0.74)c 15.07 (3.21)bc 150.74 (3.21)bc 835.58 (15.49)c
NM 4.49 (0.72)e 8.19 (5.13)d 81.89 (5.13)d 452.43 (28.02)e
Values followed by the same letter in a column within each treatment are not significantly different at p < 0.05 (Tukey’s HSD test).
NN: Nodule number, NDW:Nodule dry weight, RDW:Root dry weight, SDW:Shoot dry weight, K:Katumani 80, KM: Kunde Mboga, KK:
KenKunde 1, M:Mycorrhizal, NM:Non-mycorrhizal, AMF In: Arbuscular mycorrhizal fungi inoculation.
94 M. B. ORURU ET AL.
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comparable results (F D 30.01, p < 0.001), where the highest increase in percentage N after inoculation
was observed in Kunde Mboga (51.2%) followed by KenKunde 1 (50.1%) then the wild-type (43.4%).
Katumani 80 had the lowest increase (42.1%) in percentage N (Table 4).
The level of shoot P was significantly affected by the genotype (F D 428.14, p < 0.001) with Katu-
mani 80 cultivar having the highest shoot P content, while the wild-type cultivar had the lowest shoot
P level. Also, the effect of AMF inoculation on shoot P was significant (F D 2969.74, p < 0.001), with
mycorrhizal inoculated plants having a higher shoot P content compared to the non-inoculated ones.
Soil sterilization’s effect was significant (F D 160.02, p < 0.001) with the plants grown in non-sterilized
soil having higher P content compared to those grown in sterilized soil. Besides, a significant genotype
£ AMF interaction (F D 37.59, p < 0.001) in shoot P was observed where inoculated Kunde Mboga
showed the highest increase in shoot P (45.3%), while Katumani 80 and the wild-type showed the low-
est increase (35.5%) (Table 4).
Shoot K differed significantly (F D 10.22, p < 0.001) in all the cowpea cultivars with Kunde Mboga
and the wild-type having the highest level of shoot K. However, AMF inoculation did not significantly
affect (F D 3.26, p D 0.0762) shoot K content. Nonetheless, soil sterilization had a significant effect
(FD 78.04, p< 0.001) with plants grown in sterilized soil having a higher content of shoot K compared
to those grown in non-sterilized soil. The effect of genotype £ AMF interaction on shoot K was signifi-
cant (F D 2.78, p D 0.0493) with the highest increase in shoot K after inoculation observed in
Table 3. Means and p values from ANOVA of soil sterilization, cowpea genotype, AMF inoculation, and interactions on shoot N, K and
P nutrition of cowpea. The mean standard errors are presented in parentheses.
%N K (ppm) P (ppm)
Soil
Non-sterilized 2.77 (0.17)a 2599.81 (27.61)b 1195.28 (65.91)a
Sterilized 2.42 (0.18)b 2859.09 (24.27)a 1057.47 (64.20)b
Cowpea genotype
K 3.16 (0.24)a 2705.06 (44.10)ab 1396.00 (65.58)a
KM 2.77 (0.27)b 2804.19 (49.74)a 1189.69 (95.48)b
KK 2.51 (0.23)c 2606.94 (61.20)b 1060.69 (81.91)c
Wild 1.95 (0.16)d 2801.63 (11.95)a 859.13 (54.24)d
AMF inoculation
M 3.44 (0.11)a 2702.94 (29.09)a 1423.22 (45.42)a
NM 1.76 (0.07)b 2755.97 (39.29)a 829.53 (31.99)b
P values of the main factors and interaction
Soil <0.001 <0.001 <0.001
Genotype <0.001 <0.001 <0.001
AMF In <0.001 0.0762 <0.001
Genotype x AMF In <0.001 0.0493 <0.001
Values followed by the same letter in a column within each treatment are not significantly different at p < 0.05 (Tukey’s HSD test). K:
Katumani 80, KM:Kunde Mboga, KK:KenKunde 1, M:Mycorrhizal, NM:Non-mycorrhizal (control), AMF In: Arbuscular mycorrhizal fungi
inoculation.
Table 4. The effect of genotype£ AMF inoculation interaction on shoot N, K and P nutrition of cowpea. The mean standard errors are
presented in parentheses.
Cowpea genotype AMF In %N P (ppm) K (ppm)
K M 4.04 (0.072)a 1714.25 (29.74)a 2723.75 (51.90)ab
NM 2.28 (0.10)cd 1077.75 (39.53)d 2686.38 (74.44)b
KM M 3.79 (0.10)ab 1545.75 (41.34)b 2802.75 (64.10)a
NM 1.79 (0.11)d 833.63 (33.76)de 2805.63 (80.58)a
KK M 3.39 (0.04)b 1372.75 (20.11)c 2513.25 (29.71)c
NM 1.63 (0.08)d 748.63 (22.94)e 2700.63 (112.54)ab
Wild M 2.53 (0.05)c 1060.13 (9.89)d 2772.00 (1.67)ab
NM 1.37 (0.09)e 658.13 (31.10)e 2831.25 (18.93)a
Values followed by the same letter in a column within each treatment are not significantly different at p < 0.05 (Tukey’s HSD test).
K:Katumani 80, KM:Kunde Mboga, KK:KenKunde 1, M:Mycorrhizal, NM:Non-mycorrhizal, AMF In: Arbuscular mycorrhizal fungi
inoculation.
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Katumani 80 (5.3%). On the contrary, marginal decrease in shoot K content was observed in the other
three cultivars (Table 4).
There was a strong positive correlation between root AMF colonization and shoot N in both steril-
ized and non-sterilized soil (r2 D 0.78, p < 0.001 and 0.89, p < 0.001, respectively) (Figure 1). An
increase in percentage AMF corresponded to increased levels of shoot N and vice versa. Similarly, there
was a strong positive correlation between root AMF colonization and shoot P nutrition (r2 D 0.73, p <
0.001 and 0.90, p < 0.001 in sterilized and non-sterilized soil, respectively) (Figure 2). A high percent-
age of AMF colonization corresponded to high levels of shoot P. On the contrary, the correlation
between root AMF colonization and shoot K content was not significant.
Discussion
AMF responsiveness of cowpea genotypes
In this work, modern cowpea cultivars were more susceptible to AMF inoculation compared to the
wild type that accordingly resulted to better root colonization and growth in the modern cultivars.
Such findings are consistent to previous studies that have shown variation in AMF colonization among
different plant genotypes (Zhu et al. 2001; Tawaraya 2003). In an extensive comparison of several lines
of maize, modern hybrids showed a significantly higher percentage mycorrhizal colonization than
Figure 1. Relationship between AMF root colonization and shoot N (%) in sterilized soil ( ) (r2 D 0.78, y D 0.09x C 0.02) and non
sterilized soil ( ) (r2 D 0.89, y D 0.08x C 0.30) following AMF inoculation.
Figure 2. Relationship between AMF root colonization and shoot P (ppm) in sterilized soil ( ) (r2 D 0.73, y D 39.88x C 73.64) and
non sterilized soil ( ) (r2 D 0.90, yD 31.53x C 236.00) following AMF inoculation.
96 M. B. ORURU ET AL.
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older landraces and inbred lines (An et al. 2010). A study of mutants of soybean and Lotus japonicas
revealed accelerated AMF colonization and increased arbuscule formation compared to wild-type
plants (Solaiman and Senoo 2005).
In contrast, other studies have shown higher AMF colonization in wild-types. For instance, studies
conducted by (Y€ucel et al. 2009), led to the finding that old wheat cultivars relied more on mycorrhizal
symbiosis than the modern wheat cultivars. Hence, old wheat cultivars had higher AMF colonization
and infection than the modern cultivars. Besides, Steinkellner et al. (2012) demonstrated varying sus-
ceptibility of tomato cultivars to AMF colonization although such differences were not linked to culti-
var age.
Genotypic variation to AMF responses may arise from differences in the degree of fine root develop-
ment (Lebron et al. 2012). In this case, the wild cowpea cultivar might allocate less photosynthate to
AMF, which might limit their ability to grow into the soil, and absorb nutrients. The differences could
also be a reflection of the inherent traits of the cultivars, the rhizosphere, or differences in soil nutrients’
availability. The plant genotype determines the effect of AMF by influencing AMF development and
consequently the AMF populations flourishing in the soil. Some AMF that are good colonizers on other
species may even be denied access in some species (Sanders et al. 2003) and fail to reproduce while
other crop varieties are highly compatible with AMF, resulting to improved crop nutrient and water-
use efficiency as demonstrated by (Singh et al. 2012) in durum wheat. The genetic differences in com-
patibility of durum wheat with AMF led to notable effects in straw biomass production, straw P, grain
P and straw K contents. This differential colonization accounts for the genotypic differences observed
with respect to parameters such as nodulation, dry matter and nutrition.
AMF colonization and cowpea growth
Shoot and root dry matter increased significantly across all the cowpea cultivars following inoculation
with AMF. Such findings suggest that mycorrhizal inoculation has a positive effect on plant height,
leaves and roots, which consequently results to increased dry weight. These results are in line with the
findings of (Sharif et al. 2009), which showed that the root and shoot dry matter of wheat increased
after inoculation with AMF. The dry matter differences between mycorrhizal and non-mycorrhizal
plants were due to beneficial effects derived from mycorrhizal association. Al-Karaki et al. (1998) stud-
ied the effects of AMF inoculation on two wheat genotypes and the findings revealed that AMF inocu-
lated genotypes had higher root and shoot dry matter than non-inoculated plants. Similar findings
were reported by (Jan et al. 2014), indicating a positive correlation between root dry weights and shoot
P in wheat samples inoculated with AMF, results that are consistent with our findings. According to
(Mandou et al. 2015), AMF inoculation increases the shoot and root dry matter of micropropagated
banana plantlets, which may enhance photosynthesis rate and nutrient uptake. It has been noted that
enhanced growth effects on mycorrhizal plants are due to improved water relations, resulting from
enhanced P nutrition.
In this study, AMF inoculation increased the nodule number and nodule dry weight in all the cow-
pea genotypes with mycorrhizal plants having a higher nodule number and dry weight compared to
the non-inoculated plants. This is in line with studies that have shown that different AMF species are
able to increase nodulation and N fixation. Tajini et al. (2012), found that common bean (Phaseolus
vulgaris L.) plants inoculated with Glomus intraradices and Rhizobium had higher number of nodules
and higher nodule dry weight than non-inoculated plants. This suggested that combined inoculation
of plants with both AMF and rhizobia increases the P use efficiency for symbiotic N fixation. Our
results are also consistent to those obtained by Goicoechea et al. (2004) on Anthylis cytisoides L., a
drought-tolerant legume that can form symbiosis with both mycorrhizal and rhizobial microsym-
bionts. The study demonstrated that AMF inoculation increased nodulation and N fixation, which in
turn improved N nutrition. Similarly, a study done by Huang et al. (2014), on white clover (Trifolium
repens) found a significant correlation between percentage AMF colonization and number of nodules.
Nonetheless, contrary findings were obtained in alfalfa (Catford et al. 2003), whereby colonization of
roots with AMF systematically inhibited further mycorrhization as well as nodule formation.
JOURNAL OF PLANT NUTRITION 97
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Larimer et al. (2014) investigated the synergistic effects of AMF and rhizobia on growth and nodula-
tion of a prairie legume, Armorpha canescens. Strong synergistic effects between both symbionts were
found on plant biomass production and nodulation, which were dependent on nutrient level. AMF
infection increased root nodule number and mass, while rhizobia inoculation decreased AMF hyphal
root colonization. It was further noted that the relative benefits of each combination of symbionts were
determined by the P level. P is the key nutrient utilized by legumes for nodulation process. The combi-
national effect of rhizobia and a mixture of AMF (Aculospora laevis, Glomus geosporum, Glomus mos-
seae and Scutellospora armeniaca) was a significant increase in nodulation, N fixation and growth of
Vicia faba under alkalinity stress (Abd-Alla et al. 2014). In our study, AMF inoculation resulted to
increased P uptake, and this could account for increased nodulation.
As expected, AMF inoculation increased shoot N and P across all the cowpea cultivars although this
was not reflected on K uptake. Interestingly, shoot nutrient content however varied significantly based
on the cultivar type, with wild-type recording the lowest shoot N and P content. Besides, we observed a
strong positive correlation between AMF colonization and shoot N and P content. These results are
consistent with those obtained by Yaseen et al. (2011), who found out that nutrient uptake in two cow-
pea varieties inoculated with AMF was higher than that in non-inoculated plants. Our results are also
in line with those of (Ghazala 2005; Singh and Gogoi 2012 and Sharma et al. 2013), who reported that
mycorrhizal plants had higher nutrient uptake compared to non-mycorrhizal plants.
Other studies that have shown enhanced P nutrition following AMF inoculation have been done on
cowpea (Yaseen et al. 2011), maize (Antunes et al. 2009; Miransari 2011; Njeru et al. 2014), tomato
(Cavagnaro et al. 2006; Cavagnaro and Martin 2010; Abdel Latef and Chaoxing 2011) and cucumber
(Ortas 2010). Although P is critical for plant growth and makes up about 0.2% of dry mass, it is one of
the most difficult nutrients for plants to acquire (Habibzadeh 2015). It may be present in relatively
large amounts in soil, but much of it is poorly available because of the very low solubility of phosphates
of aluminum, iron and Ca, or very low mobility (Ryan et al. 2005). The increase in P uptake is one of
the most dramatic effects of mycorrhizal infection on the host plant (Bai et al. 2008), and this is because
mycorrhizal fungi have the ability to absorb phosphate from soil and transfer it to the host plant. Our
results are further supported by the statement that AMF improve uptake of immobile nutrients such as
P and Zn (Balakrishnan and Subramanian 2012). The extensive AMF hyphal network alters the physi-
ochemical properties of soil and directly or indirectly contributes to the release of phosphates from
inorganic complexes that have low solubility (Finlay 2008).
The AMF cytoplasm may serve as a host to bacterial endophytes, particularly, plant growth promot-
ing rhizobacteria. Although, there is a need to clarify the role of endophytes living within AMF spores,
some evidence has shown that they could be involved in nutrient exchange between the partners
(Varennes and Goss 2007). Tripartite symbiosis, which incorporates N fixation by rhizobia, could
explain why there was increased N uptake by AMF inoculated plants in the current study. Enhanced N
uptake through AMF symbiosis has been shown in maize (Zea mays L.) (Miransari 2011), melon plant
(Cucumis melo L.) (Martınez-Medina et al. 2011) and Long pepper (Piper longum L.) (Singh and Gogoi
2012). Nonetheless, contradictory results have been reported by Reynolds et al. (2005), who found that
AMF did not enhance N acquisition and growth of old-field perennials under low N supply.
Although limited studies have been done to investigate the role of AMF in K uptake, a few studies
have reported increased uptake of the nutrient. Studies done on tomato (Lycopersicon esculentum)
(Abdel Latef and Chaoxing 2011), maize (Zea mays) (Miransari 2011) and melon plant (Cucumis melo
L.). Martınez-Medina et al. (2011) showed that K uptake was enhanced following AMF inoculation.
These results are contrary to those from the current study where there was no significant relationship
between K uptake and AMF colonization.
Effect of soil sterilization on AMF colonization and growth
In the current study, soil sterilization had a significant effect on root AMF colonization and different
growth parameters. Plants grown in non-sterilized soil not only had higher colonization but they also
performed better than those grown in sterilized soil. In this case, the increased growth observed in
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unsterilized soil could be associated to AMF colonization since all the pots in had been provided with
equivalent soil microbiota except AMF.
These findings are consistent to those obtained by Farzaneh et al. (2009), which showed that AMF
colonization level was higher in chickpea that additional inoculation increased colonization in non-
sterilized soil. Without sterilization, the soil contained indigenous AMF populations which were able
to colonize chickpea, thereby enhancing its performance. This could explain why the cowpea plants
that were grown in non-sterilized soil in this study had a higher root colonization, nodulation and nod-
ule dry weight compared to those grown in sterilized soil. Our findings are contrary to those obtained
by Nelly and Heung-Kyu (2013), where soil sterilization increased the growth and survival of Kalopa-
nax septemlobus microplants during the acclimatization period.
Conclusion
In this study, AMF might improve cowpea production by enhancing the uptake of nutrients, particu-
larly P and N. AMF inoculation had a positive effect on different cowpea growth parameters including
nodule number, nodule dry weight and root and shoot dry weights. Thus, the study further revealed
that modern cultivars were more responsive to AMF inoculation since they had higher root coloniza-
tion, dry matter and shoot P and N than the wild-type cultivar. Although modern breeding programs
may suppress AMF colonization since plant cultivars are usually bred in nutrient-rich environment,
this was not the case in our study. Hence, there is the need to screen different cowpea cultivars for
AMF symbiosis, which should be extended to other crops. Considering the differences revealed in
terms of responses of different plant genotypes to AMF, as demonstrated in this and other studies,
future studies should elucidate appropriate genotype-AMF combinations in order to obtain the optimal
benefit from mycorrhizal symbiosis.
Acknowledgment
This work was supported by the International Centre for Insect Physiology and Ecology (ICIPE), Nairobi, Kenya and the
Department of Microbiology, Kenyatta University. The authors wish to thank Symyco Inc. and Mycorrhizal Applications,
Inc (Grants Pass, Oregon, U.S.) for providing with AMF inoculum.
ORCID
Ezekiel Mugendi Njeru http://orcid.org/0000-0002-9104-808X
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