Effects of arbuscular mycorrhizal fungi on wheat

Int. J. Agri. & Agri. R.

International Journal of Agronomy and Agricultural Research (IJAAR) ISSN: 2223-7054 (Print) 2225-3610 (Online) http://www.innspub.net Vol. 7, No. 5, p. 30-42, 2015 OPEN ACCESS

RESEARCH PAPER

Effects of arbuscular mycorrhizal fungi on wheat growth, physiology, nutrition and cadmium uptake under increasing cadmium stress Sadia Kanwal*, Asma Bano, Riffat Naseem Malik Environmental Biology and Ecotoxicology Laboratory, Department of Environmental Sciences, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad PO 45320, Pakistan Article published on November 21, 2015 Key words: Arbuscular mycorrhizal fungi, Wheat, Cadmium toxicity, nutrient contents, Antioxidant enzymes.

Abstract A pot culture experiment was carried out to study the alterations in growth, biochemical activities and cadmium (Cd) uptake by wheat (Triticum aestivum) inoculated with or without arbuscular mycorrhizal (AM) fungi in sterilized soil with addition of different Cd levels (0, 100, 300, 600 mg.kg_1). In Mycorrhizal (M) plants, root colonization rates were significantly lower with the addition of high Cd concentration (600 mg.kg _1). AM inoculation increased shoot and root biomass at 100 mg kg_1 Cd addition but cause a reduction at 300 and 600mg.kg-1. Shoot and root Cd concentrations in mycorrhizal (M) plants were lower at all levels (0, 100, 300 and 600 mg.kg-1) and Cd accumulation and uptake efficiency were lower in M plants. AM inoculation improved shoot and root P nutrition at all Cd levels. In addition, mycorrhization also cause to improved shoot nutrients uptake (N, P, K, Ca, Mg, Na), chlorophyll, carotene, protein and sugar contents as compared to NM plants. Cd toxicity induced proline accumulation and significant reduction of antioxidant enzyme activities (SOD, POD, CAT, APX) were observed in NM plants however proline contents were lower in M except the higher Cd concentration (600 mg.kg-1). The results support the view that AMF can improve the capability of reactive oxygen species (ROS) and reduce Cd concentration in plants to protect wheat (Triticum aestivum L.) from Cd stress. Hence, AM fungi in combination with wheat is suitable for reduction of Cd toxicity and also shows a potential role in phytostabilization of soil moderately polluted with Cd. * Corresponding

Author: Sadia Kanwal  [email protected]

Kanwal et al. Page 30

Int. J. Agri. & Agri. R. Introduction

and Read, 1997). Several studies reported the mutual

Heavy metal contamination is a major environmental

symbiosis between AM fungi (AMF) and roots of

problem in the world (Davis, 2003). Cadmium (Cd) is

terrestrial plants by increasing plant biomass and

a non-essential element and highly toxic to humans,

uptake of immobile nutrients such as P, Zn Cu (Sheng

animals and plants. It disrupts the metabolic

et al., 2009) and reducing metal toxicity to plants by

processes of both plants and animals and is

decreasing

considered as one of the most phytotoxic heavy metal

translocation and shoot HM concentrations (Smith

pollutants (Aravind and Prasad, 2003). Cd enters into

and Read, 1997). AMF secreted several compounds

the

and

that cause to precipitate the metals in polyphosphate

anthropogenic sources. The natural sources include

granules present in soil, adsorping metals to fungal

weathering of rocks, forest fires and volcanic

cell walls and chelation of Cd inside the fungus (Gaur

eruptions. The anthropogenic sources that cause to

and Adholeya, 2004). However, the effect of AMF on

increase the natural limit of Cd towards toxic include

metal uptake in plants is still in controversy. Previous

industrial

in

reports suggested uptake of metals by AMF with

agriculture that has a huge quantity of Cd which

decreased or increased translocation to shoots or

directly transferred to food chain and poses hazards

sometimes even no uptake effects were studied (Gao

for both human and animal health (Schutzendubel et

et al., 2010).

environment

waste

through

release,

both

use

of

natural

fertilizers

root

to

shoot

heavy

metal

(HM)

al., 2002). In most of the plant species, Cd is readily translocated towards roots and accumulated in leaves

Wheat is one of the oldest and most widely cultivated

(Lopez-Millan et al., 2009).

crops. According to FAO reports, in 2007 world production of wheat was 607 million tons making it

High levels of Cd cause to generate toxic free radicals

the third most produced cereal after maize and rice.

i.e reactive oxygen species (ROS) produce oxidative

Cd is considered as one of the most toxic heavy metal

stress in plants (Dixit et al., 2001). These toxic free

causing serious problems in crops (Prasad, 1995). The

radicals (ROS) react with biomolecules like lipids,

toxic effects of Cd have been widely studied in

proteins,

lipid

different plant species is known to reduce or inhibit

peroxidation, harm to cell membranes and inhibit

plant growth and development (Shahabivand et al.,

enzymatic activity resulting in disruption of cell

2012). AMF can exert positive effects on terrestrial

functioning. On the other hand, plants has been

plants under Cd contamination (Shahabivand et al.,

developed an antioxidant defense mechanism which

2012; Medina et al., 2010a).

pigments,

nucleic

acid

cause

stimulate the functioning of antioxidative enzymes and fight against these toxic radicals to protect plants

However, the association between AMF colonization

from stess (Liet al., 2009). The antioxidative defense

and accumulation of toxic elements in crops is an area

mechanism consist of both enzymatic and non-

of considerable interest relating to both production of

enzymatic

enzymes

safe food and bioremediation strategies (Smith and

include superoxide dismutase (SOD), catalase (CAT),

Read, 2008). There is scarce information about the

guaiacol peroxidase (GPX), ascorbate peroxidase

effects of AMF on physiological and biochemical

(APX)

Non-

changes of cereal crops especially wheat under Cd

soluble

stress. Therefore, this study was carried out to

membrane associated antioxidants (e.g, a-tocopherol

examine the effects of AMF (combination of Glomus

and b-carotene) and water soluble reductants (e.g.,

species) on physiology, biochemical contents and

ascorbic acid and glutathione).

shoot and root Cd accumulation in Triticum aestivum

and

enzymatic

antioxidants.

glutathione antioxidants

Antioxidative

reductase include

(GR). lipid

under different Cd contamination levels. The role of arbuscular mycorrhizal fungi (AMF) for land remediation has been commonly studied (Smith


Int. J. Agri. & Agri. R. Materials and methods

fungal inoculum containing about 2500 spores per

Plant materials

pot. AMF inoculation was performed during the

Wheat seeds (T. aestivum) were obtained from the

transplantation process and was not provided in non-

Department of Crop Science, National Agriculture

mycorrhizal treatments.

Research Centre, Islamabad. Seeds were surface sterilized (10 min, 3% Chlorox) and gently washed by

Pot experiment and growth conditions

deionized water five times and germinated on sterile

A pot culture experiment was carried out under

wet filter paper (Xin Hua No.101, China) in Petri

growth chamber conditions consisted of a completely

dishes at 28oC for 48 hours. These were selected for

randomized design with six replicates. Each pot

uniformity before sowing. Five pre-germinated seeds

(10cm diameter and 12cm height) contained 2kg

were sown per pot and the plants were allowed to

growth medium plus 50g inoculum to mycorrhizal

grow for 8 weeks.

treatments, while the same amounts of growth medium were added to non-mycorrhizal treatments.

Preparation of soil

The treatments were either inoculation or non-

The experimental soil and sand were collected from

inoculation of the AM fungi and the addition of four

the top layer (0-20cm) in the vicinity of Quaid-i-Azam

Cd concentrations to the soil (0, 100, 300 and

University, Islamabad. The soil and sand were air-

600mg/kg). The experimental pots were placed in the

dried and sieved with a 2-mm diameter sieve for

growth chamber under conditions of 14 h of light, 10

analysis. The soil and sand were air-dried and sieved

h darkness, 28/20oC day/night temperature, relative

with a 2-mm diameter sieve for analysis. Soil was

humidity of 50-65% day. Water lost was replaced

chemically

characterized

Phosphorus (4.3 Calcium (34.45 Extractable

T.

daily by top watering with deionized water at 24th

T. Potassium (19.5

mgkg-1),

intervals during growth period to maintain the

Magnesium (42.50

mgkg-1),

moisture of the soil at about 60% until the end of the

nitrogen

mgkg-1),

experiment. Each pot was irrigated with long Ashton’s

mgkg-1),

mgkg-1),

nitrate

with

Extractable potassium (1.45

a

pH

(6.7),

(1.04

Extractable

nutrient solution (20ml) every week. Six pots per

phosphorus (1.53 mgkg-1), Zinc (1.50 mgkg-1), Nickel

treatment were used and plants were harvested after

(1.33 mgkg-1), Copper (30.3 mgkg-1), Cadmium (1.60

60 d for chemical analysis.Roots and shoots of the

mgkg-1),

mgkg-1),

harvested wheat samples were rinsed with tap water

mgkg-1)

to remove soil particles and then carefully washed

Iron (28.51

Chromium (4.25

mgkg-1),

mgkg-1),

mgkg-1)

Lead (1.6

and Manganese (10.4

respectively. The soil and sand were autoclavedsterilized

(121oC,

with deionized water.

2 h) in order to eliminate native AM

fungal propagules and other microorganisms. The soil

Plant measurements and analysis

was manually mixed with sand in ratio of 1:3 (v/v).

AM Root colonization

The mixture of soil and sand were used as growth

Root mycorrhizal colonization was estimated after

medium of plants. CdCl2 was added to the growth

clearing and staining (Koske and Gemma, 1989) using

medium as Cd stress at the concentrations of 100,

the grid-line intersect method (Giovannetti and

300 and 600 mg/kg respectively.

Mosse, 1980). The stained roots were then mounted on glass slides (5 pieces of root per slide) for

Fungal inoculum

examination with an eyepiece cross-hair. Colonization

The AMF used was the mixture of different Glomus

percentage of mycorrhiza was estimated for each

species with dry soil substrates obtained from the

sample by examination of one hundred 1cm long

AMF

pieces of roots.

collection

maintained

by

the

company

(Agrauxine) in France. Spores and dried sand-soil mixture (growth medium) were used in mycorrhizal

Plant biomass

inoculated treatments. Each pot received 50 g of AM

At harvest, roots and shoots were separated. Sub-


Int. J. Agri. & Agri. R. samples of fresh roots were taken to assess

Antioxidant enzymes

mycorrhizal colonization. Fresh weights of total roots

For enzyme analysis, fresh samples of leaves (300 mg

and

and

each) were ground in a chilled mortar and extracted

remaining roots were rinsed with tap water and then

with 3 ml of 100 mm potassium phosphate buffer (pH

with deionized water. Tissues were weighed after

7.5). The homogenate was centrifuged at 12,000 rpm

oven drying at 60°C for 72 h and then ground to