Influence of Arbuscular Mycorrhizal Fungus on Growth and Nutrient Status in Chickpea (Cicer Arietinum L.) Plant


 Background: The greatest challenge of today’s agriculture is to feed the growing population and restore the natural resources. World over demand of crops in market is more than production. Indian crop production needs to be doubled and just to maintain the present precipitate consumption. . Excessive use of chemical fertilizers causes environmental pollution both at the manufacturing and application sites. It is therefore most necessary to reduce the dependence on chemical inputs in agriculture. This is possible only through eco friendly approaches of farming system. Besides other biotechnological interventions, the arbuscular mycorrhizal fungi could be used as bio inoculants for promotion of growth, development, quality and yield of vegetables that too under an Integrated Plant Growth substances management system. Mycorrhizal fungi are used in conventional agriculture to improve crop production and productivity.Methods: A pot culture was performed at Botanical Garden of School of Studies in Botany, Jiwaji University Gwalior to examine the effect of Glomus hoi on growth and certain biochemical parameters. Plants were raised in triplicates for different mycorrhizal species through the pot culture, containing sterile soil. Plastic pots of 18” × 12”size were used for this purpose. Pots were placed at a sunny place after the seed sowing. And after the seed germination, plants were irrigated as when requiredResults: After germination the inoculated plants along with their controls were be sampled at 30, 60 and 90 days. During sampling it was found that due to AMF symbiosis all growth and biochemical parameters like plant were increased in all AMF treated plants.


Introduction
Arbuscular Mycorrhizal Fungi are obligate symbiotic organisms which are living in association with the roots of most land plants. AMF produce wide network of extracellular mycelium of independent length, Spreading from host roots into the surrounding soils and establishing below ground interconnections among plants belonging to the same or to different taxa. AMF are bene cial soil symbionts establishing mutualistic association with the roots of 80% of Plant species and the large majority of food crops including cereals, Legumes vegetables and fruits (Pepe et al, 2018). Arbuscular mycorrhizal (AM) fungi (AMF), one of soil inhabiting fungi, can form the symbiotic association with roots of higher plants, called AM symbiosis. The symbiosis is characterized by increased water and nutrient uptake diverted from the soil to the fungal partner, and in return receives the Photosynthetic supply from the host plant to the AMF2. Studies showed that AMF strongly enhanced drought tolerance of host plants via varied mechanisms viz., direct absorption of water by mycorrhizal extra radical hyphae at the rate of 375-760 nL water/h, contributing up to 20% of total water absorbed by plant roots3, improvement in osmotic adjustments, enhancement in antioxidant pro le coupled with enhanced e ux of hydrogen peroxide into rhizosphere and facilitating the formation of soil water-stable aggregates by both glomalin and mycorrhizal hyphae (Liu C.Y et al,2018). AMF shows increase in Biomass and Higher Tolerance in Biotic and Abiotic Stress (Lanfranco L. et al, 2018). Arbuscular mycorrhizal fungi (AMF) are considered as a potential biotechnological tool for improving Phytostabilization e ciency and plant tolerance to heavy metal-contaminated soils. However, the mechanisms through which AMF help to alleviate metal toxicity in plants are still poorly understood (Yang Y et al, 2015). During mycorrhiza formation, the arbuscular mycorrhizal fungus undergoes several developmental stages. Spores germinate and arbuscular mycorrhizal fungi shows limited hyphal development in the absence of a host plant during a symbiotic stage and they switch to the pre symbiotic stage which is characterized by extensive hyphal branching in the presence of root exudates (Buee et al, 2000). The plants which forms symbiotic association with mycorrhiza shows increase in growth than non mycorrhizal plants. Arbuscular mycorrhizal fungi increases the growth of some plants like Japanese mint, Spilanthes calva, Withania somnifera, Cartanospermum austral, Cymbopogon martini, Phyllanthus amarus, Gloriosa superba and Pueraria tuberose (Gianinazzi et al, 2010). AMF helps host plant in reducing stress which is responsible for soil drought. In AMF association, the fungal mycelium helps host plant for water v absorption (Mathur N et al, 2007). In agriculture, the increased uptake of soil minerals by colonized plants means that it is possible to consider reducing substantially the application of fertilizers and pesticides, and at the same time obtain equivalent or even higher crop yields (Jakobsen I et al, 1992). The plants which show mycorrhizal association possess higher concentration of esterase, pectin and endo-polymethyl glacturonase activities than controlled plants (Garcia Romera et al, 1991). The roots of Arachis hypohaea forms symbiotic association with Glomus fasciculatum shows higher amount of amino acids and proteins (Krishna and Bagyaraj, 1983). Arbuscular mycorrhizal fungi enhance the production of secondary metabolites such as alkaloids and terpenoids (Su Y. Y and Guo L. D, 2007). AMF helps host plant in increasing in nitrogen xation and increase dry weight of above ground parts of host plant (Nasr and Diem, 1987).

Materials And Methods
A pot culture was performed at the Botanical Garden of School of Studies in Botany, Jiwaji University Gwalior to study the symbiotic association between an arbuscular mycorrhizal fungus (Glomus hoi) and Chickpea plant (Cicer arietinum). Plants were raised in ve replicates for mycorrhizal species (Glomus hoi) as well as for non inoculated plants through the pot culture, containing sterile soil. Plastic pots of 18" × 12"size were used for this purpose. A total number of 40 pots were placed, 20 pots were kept as control and 20 pots were inoculated with mycorrhizal spores. In Each pot 3 plants were grown per plant. For procuring AMF free tissues, the seeds were surface sterilized by 4% hypochlorite solution before sowing. Pots were placed at a sunny place after the seed sowing. And after the seed germination, plants were irrigated as when required. After germination the inoculated plants along with their controls was sampled at 30, 60 and 90 days. During sampling plants will be thoroughly watched and were washed several times with the tap water followed by the jet of distilled water. The water adhered to the plant surface was removed by wrapping the harvested plants in blotting papers. Fractionation of the root and the shoot components was carefully done and the fresh weights of the root and the shoot were measured. The detailed methodology for various parameters of growth and biochemical analysis is described as below Root clearing and staining technique: Roots were thoroughly washed with the tap water, cleared in 10% KOH by autoclaving for 20-25 minutes at 121°C and 15lbs pressure. Cleared roots were rinsed with water 4-5 times then kept in HCl for 5 min. The HCl solution was decanted and left overnight in 0.05% tryphan blue stain as per the method of Philip and Hayman, (1970).

Estimation of root colonization:
Above treated roots were cut in 1 cm long segments, mounted on microscopic slides in Lacto glycerol solution. 10 such root segments were arranged properly on a glass slide and then covered with another glass slide. Slides were observed under compound microscope for mycorrhizal structures viz. hyphae, vesicles and arbuscules as per the method of Bierman and Linderman (1981).
Estimation of growth and development parameters: Estimation of Chlorophyll Content: For chlorophyll a, chlorophyll b and total chlorophyll the method of Arnon (1949) and Withman et al., (1971) were employed. The fully expanded fresh plant leaves from all the pots trials were collected in the polythene bags and transported to the laboratory. Weighted (0.5gm) fresh leaf material was homogenized and extracted thrice in chilled 80% acetone. The volume of the acetone extract was made up (10 ml) to a known one and the optical density was read at 645nm and 663nm wavelengths on a spectrophotometer Calculation: The concentration of the chlorophyll pigments was calculated using the following formula Estimation of biochemical parameters: The following methods were employed for certain biochemical analysis of the stem modi ed tissue.
Estimation of non reducing sugar by Nelson- Somogyi (1952).

Estimation of reducing sugars by Nelson-Somogyi (1952).
For ascertaining total carbohydrates by Anthrone method of Hedge and Hofreiter (1962) will be used.

Estimation of total phenol by the method of Mallick and Singh (1980)
Statistical Analysis Results on growth analysis and chemical components were analyzed by average ± standard error.        Abdel-Fattah and Asrar, (2012) observed that addition of AM fungi signi cantly increased growth parameters of wheat such as plant height as compared to non-mycorrhizal wheat plants. Our result shows that AMF being in the symbiotic relationship with crop plant enhances growth and biochemical parameters of the plants. In our experiment inoculation of AMF spores increased growth and developmental characters, chlorophyll content, carbohydrate, protein and several other biochemical parameters in chick pea. Based on our results we suggest that AMF must be explored at all levels to further investigate their role in nature as a bio-fertilizer for sustainable agricultural production.

Conclusion
Mycorrhiza is a bene cial type of fungi that grows in association with most plant roots. Mycorrhizae increase the root's ability to absorb nutrients and water from the soil by increasing the surface absorbing area of roots from 100 to 1,000 times. Mycorrhizae also release powerful enzymes that help dissolve nutrients such as organic nitrogen, phosphorous, and iron. Mycorrhiza plays a vital role in plant growth, disease protection and overall soil quality. There are seven types of mycorrhiza viz: arbuscular, ecto, ectendo, Arbutoid, monotropoid, ericoid and orchidaceous mycorrhizae. Arbuscular Mycorrhizal Fungi (AMF) constitutes a group of root obligate biotrophs that exchange mutual bene ts with about 80% of plants. They are considered natural bio fertilizers, since they aid in the acquisition of water and soil nutrients, also help host defense mechanism against pathogens in exchange for photosynthetic products. An increase in crop production is essential to meet the future food demand. Soil fertility and soil structure of agricultural systems is to be managed by effective use of fertilizers with increased pro tability and reduced harm to the environment. Microbial inoculants, including arbuscular mycorrhizal (AM) fungi for increasing the e cient use of fertilizers are potential components of such management. The process of re-establishing the natural level of AMF richness can represent a valid alternative to conventional fertilization practices, with a view to sustainable agriculture. The need for AMF as a biofertilizer, with a view to sustainable agriculture, is becoming increasingly urgent since the appropriate management of these symbiotic fungi could potentially decrease the use of agro chemicals.