Induction of salt stress tolerance in cowpea [Vigna

unguiculata (L.) Walp.]

by arbuscular mycorrhizal fungi
Hashem Abeer1,2, E.F. Abd_Allah*3, A.A. Alqarawi3 and Dilfuza Egamberdieva4
ABSTRACT
The aim of present study was to examine the effect of arbuscular mycorrhizal fungi (AMF)
on the growth, lipid peroxidation, antioxidant enzyme activity and some key physiobiochemical attributes in cowpea (Vigna unguiculata [L.] Walp.) subjected to salt stress.
Salt stress (200 mM NaCl) reduced growth, biomass, relative water content and
chlorophyll pigment content in cowpea leaves. AMF ameliorated the negative impact of
salinity on the growth parameters studied. The activity of antioxidant enzymes such as
superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), peroxidase
(POD) and glutathione reductase (GR) enhanced under salt stress and AMF inoculation
further enhanced their activity, thus strengthening the plants defense system. Proline
content increased in salt stressed plants as well as AMF-inoculated plants providing
efficient protection against salt stress. Besides this AMF also increased uptake of mineral
elements which have direct impact on the osmoregulation of the plants. The present
study shows that AMF possesses the potential to enhance salt tolerance of cowpea.
INTRODUCTION
Plants are sessile and are frequently encountered by variety of environmental stresses
resulting in altered growth and metabolism (Ahmad, 2010). Among the various
environmental constraints salinity has stern effects on plant growth and development
(Ahanger et al., 2014). All over the world, particularly in arid and semiarid regions salinity
is one of the important problems and is increasing day by day. Increased use of saline
water for irrigation purposes continuously adds to the salt affected soils thereby resulting
in conversion of arable land into salinized waste land. Around 5 to 7% of global land is
salt affected (Egamberdieva et al., 2015; Ruiz-Lozano et al., 2012). Increased salinity
induces osmotic as well as toxic effects and hence affects growth and other physiological
and biochemical processes including photosynthesis and ion homeostasis (Porcel et al.,
2012;
Hashem et al., 2015). Altered photosynthesis is associated with perturbed carbon and
nitrogen metabolism. In legumes salinity alters nitrogen fixation and hence growth as
well as yield (Hashem et al., 2014a; Egamberdieva et al., 2015). Exposure to high salt
concentrations triggers production of toxic reactive oxygen species (ROS) ultimately
resulting in oxidative stress. Excess accumulation of ROS like superoxide, hydroxyl, and
peroxide radicals is detrimental to normal metabolism and growth (Wu et al., 2014). ROS
accumulate in leaves and leads to oxidation of various cellular molecules including lipids,
proteins and chlorophylls. Hence peroxidation of membrane lipids, protein oxidation, and
damage to nucleic acids are the immediate effects of the excess ROS (Wu et al., 2014).
Different defense mechanisms are employed by plants to cope up with the stress induced
oxidative damage. Increased activities of antioxidant enzymes are one of the key defense
mechanisms which help plants to avert oxidative stress damage. Superoxide dismutase
(SOD), catalase (CAT), peroxidase (POD) and glutathione reductase (GR) are among the
various intricate antioxidant enzymes which carry scavenging of ROS so as to protect
cells from the oxidative damage. In addition to this another strategy adopted for
ameliorating the deleterious impact of salinity involves accumulation of compatible
organic osmolytes (Ahanger et al., 2014). Accumulation of free proline, sugars and amino
acids help plants in bringing osmoregulation so that water content of the cell can be
maintained. Accumulation of compatible solutes and ions while sequestration of
deleterious ions into vacuole or apoplast is an important trait, which help plants to
maintain growth and protect various metabolic pathways (Ahanger et al., 2014; Alqarawi
et al., 2014a;b; Hashem et al., 2014a;b).

Plant associated with microorganisms are of two general types, those forming a
symbiotic relationship with the plant and those that are free-living in the soil and root
(Hameed et al., 2014; Ahanger et al., 2014). Arbuscular mycorrhizal fungi (AMF) are
ubiquitous and have proved to posses the potential to improve soil structure and plant
growth under normal as well as stressful environments (Tang et al., 2009; Navarro et al.,
2013). Under salt stress conditions arbuscular mycorrhizal fungi (AMF) are believed to act
as essential bio-ameliorators of stress and hence helping plants in alleviating stress
induced damage (Ahanger et al., 2014). AMF colonization has been reported to enhance
plant growth and vigour (Ahanger et al., 2014; Alqarawi et
al., 2014a; Hashem et al., 2014b). AMF colonization brings morphological, nutritional and
physiological changes and has also been reported to enhance resistance of plants to
abiotic stresses (Hameed et al., 2014). AMF modifies root architecture so that roots
explore much water and nutrients (Aroca et al., 2013; Ahanger et al., 2014; Wu et al.,
2014).
Mycorrhizal inoculation not only affects root morphology but also physiological status in
host plants (Alqarawi et al., 2014a; Hashem et al., 2014a). For example, citrus plants
colonization with AM fungi have large leaf area and higher phosphorous content and
photosynthesis as compared to non colonized ones (Alqarawi et al., 2014b). Cowpea
(Vigna unguiculata L.) belongs to family Fabaceae, also known as black eye pea, is an
important legume grown as cash crop throughout the world. Cowpea is rich in protein
and is normally cultivated for green pods and dry beans (Hadi et al., 2012). Increasing
salinity affects its growth and hence causes a considerable decline in its yield. Uptake
and compartmentation of deleterious ions or understandings about the tolerance
mechanisms is an important aspect which is of prime interest for enhancing tolerance in
crops. Present study was aimed to study the impact of salt stress on growth,
physiological and biochemical attributes of cowpea and role of AMF in mitigating the salt
induced damage in plant changes caused by salt stress.
MATERIALS AND METHODS
Experimental design, soil and mycorrhizal fungi: The seeds of cowpea (Vigna
unguiculata [L.] Walp) var. Qaha1 were obtained from Agricultural Research Center, Giza,
Egypt. The seeds were surface sterilized with sodium hypochlorite (0.5%) for 3 min,
washed thoroughly with distilled water before germination on blotter. Hoaglands solution
(Hoagland and Arnon, 1950) supplemented with NaCl to get concentration of 200 mM/ L
was used for irrigation. The experiment was carried out in sandy soil collected from
Derab Agricultural Research Station, Riyadh, SA. The soil used throughout this
investigation was sandy with the following properties (%): sand (74.3); moisture content,
4.23; organic carbon, 0.17; total nitrogen, 0.007; (EC) = 7.12 dS m 1; and pH 7.8. The soil
was autoclaved for 3 h at 121oC, cooled and then divided among plastic pots (1 kg
capacity). The mycorrhizal fungi used in the present study were Funneliformis mosseae
(syn. Glomus mosseae),
Rhizophagus intraradices (syn. Glomus intraradices) and Claroideoglomus etunicatum
(syn. Glomus etunicatum), which were isolated previously from salt march habitat in
Alserw, Dakahlia, Egypt (Alqarawi et al., 2014a).
Production of mycorrhizal inoculums: The selected mycorrhizal fungi were
inoculated singly with maize (Zea mays) plants using autoclaved soil (clay:sand, 1:1,
w/w) as a host plant for three generations in pot cultures in a greenhouse for propagation
of fungal spores. The growth conditions were 25oC day/ 20oC night temperatures, 65%
relative humidity, 16/8 h light/dark photo period cycle with a photosynthetic photon flux
density of (750 mol m-2 S-1). Hoaglands solution without phosphorus source (free
phosphorus) used for irrigation to enhance mycorrhizal colonization. Spores of
mycorrhizal fungi were extracted from the trap cultures to develop pure cultures of
mycorrhizal fungi. Fungal inoculums potential was determined by the most probable
numbers method (Alexander, 1982), and each trap culture contained ~10.2 x 10 3
propagules per pot (1 Kg capacity). Fungal inoculums consisted of spores, hyphae and
colonized root fragments with mycorrhizal fungi. The mycorrhizal inoculum was added to

the experimental soil as 10 g of trap soil culture (approx. 100 spores/g trap soil, M =
80%)/ pot (1Kg). Non-mycorrhizal soil was used as reference.
Pot experiment: The pot experiment was carried out in complete randomized block
design with five replications. Healthy similar germinating seeds were selected for the
experiments. The germinating seeds were grown at 27 2 oC with 18 h light (750 mol
m-2 S-1) and 6 h dark photo-cycle, and RH of 70-75 % for eight weeks after transplantation.
At the end of pot experiment, the plants were harvested carefully, washed in distilled
water, separated into shoots and roots and analyzed for fresh and dry weight. The
samples were dried at 70oC for 48 h and dry weight was recorded. Fresh leaf samples
were used for estimation of photosynthetic pigments, membrane stability index, leaf
water content, malondialdehyde contents and enzymes activity. Fresh root samples were
used for mycorrhizal studies.
Determination of arbuscular mycorrhizal colonization: At end of the experiment,
the mycorrhizal spores were extracted from the experimental soil of each treatment by
wet sieving and decanting method as described by Daniels and Skipper (1982) and
modified by Utobo et al., (2011). The roots were washed carefully in ice-cold water (4 oC)
to remove the adhering soil followed by cleaning with KOH (10%) and subsequently
staining with trypan blue in lactophenol as described by Phillips and Hayman (1970)