Arbuscular Mycorrhizal and Endophytic ( DSE ) Association in the Dominant Grasses of Melghat Forest ( Phase-I ) , India

Crossref, Index Copernicus International (ICI), Directory of Research Journal Indexing (DRJI), Scientific Indexing Services (SIS), CiteFactor, Investigations on arbuscular mycorrhizal fungi (AMF) and dark septate endophytic (DSE) association in some of the dominant grasses from Melghat forest (phase I) of Satpura terrain India, was carried out to examine their existence and symbiotic relationships with the host plants. This forest area was not surveyed earlier by anybody to evaluate the AMF status diversity hence it was decided to survey the buffer and core area. Rhizosphere soil of each sampled grass was analyzed for the AM fungal structures in the roots to study percent root colonization by AMF and AMF spore density in rhizospheric soils of respective samples. The composite soil sample was prepared for each site and used for physicochemical analysis by standard methods. In the first phase of the project, forty-eight dominant grass species from twenty-one different sites were collected along with roots and rhizospheric soil to find out AMF and DSE status of grasses. Both types of fungal associations was found in almost all the grass species collected during studies. All of them were found colonized by AMF hyphae along with moderate to poor development of mycorrhizal structures in roots. DSE colonization was also found in maximum forty-three species of grasses. Physicochemical characterization of all the soil samples were performed to find out its correlation with AM percent colonization and spore count. Mean AMF percent colonization was in between 1.33 to 52.85 and DSE in with 0.00 to 18.97. Viable AM spore count was in between 0 to 98 per 100g of soil. Altogether four AMF genera with its thirty-nine-different species were isolated and identified. A database of indigenous AM species richness for Melghat forest has been generated to plan and design the future management practices for grasses establishment and development especially in burnt and overgrazed areas.


INTRODUCTION
Melghat forest is amongst the first nine tiger reserve declared and notified by the Indian government in 1974 as project tiger of Satpura terrain, spread in 2029 Km 2 area.The forest type is a southern tropical mixed dry deciduous with the dominance of finest teak (Tectona grandis Linn.f.).The forest is located at 21°26′45″N 77°11′50″E of northern part of Amravati district of Maharashtra state, India.

Map I: Study Area Map
Grasses are monocotyledonous flowering plants belonging to family Poaceae formerly known as Gramineae are distributed widely throughout the world.They are ubiquitously found in almost all type of habitats and are helpful for growth of all plant communities (Mitra and Mukherjee, 2005).They are used as forage for domestic and wild animals, birds and for soil conservation.The Grains of grasses are provided as staple food for human (Gould, 1968) which satisfy about 80% of human nutrient requirement (Stebbin, 1972).They also play an important medicinal role (Mitra and Mukherjee, 2005;Jeeva et al., 2006).Ecologically grasses are crucially important as their root system is very extensive and act as an effective soil binder (Skerman and Riveros, 1990); they consequently protect the erosion of banks of water tributaries (Saini et al., 2007).
Soil microbes are effectively responsible for the health and productivity of plants (Bloemberg and Lugtenberg, 2001), hence the high level of soil microbial community is highly responsible for maintenance of high diversity of plant species (Wardle et al., 2004).Bellgard (1993); Schwab and Reeves (1981) concluded that arbuscular mycorrhizal fungi (AMF) are mainly found in upper layer of soil and occur in high quantity in rhizosphere area and establish symbiotic association in almost all-natural ecosystems (Brundrett, 1991).On the global basis, mycorrhizae occur in 83% dicot and 79% of monocot plants and in all the gymnosperms (Wilcox 1991).AMF is associated with the roots of plant taxa and becoming an integral part of it, they occur in most of the ecosystems like dense rain forest, grasslands, sand dunes, savannas, woodlands, arid and semi-arid regions and are most common in different zones of the world and widely found in temperate, tropical and arctic regions of the earth forming broad ecological range.
Like AMF, Dark Septate Endophytes (DSE) which forms septate hyphae and microsclerotial structure in cortical region of roots are also capable to form mutual association (Jumpponen, 2001) and are common microbial community in tallgrass prairies (Mandyam and Jumpponen, 2005;2008) and highly compatible with grasses and act beneficial to plant health in drought condition.
Plantae Scientia, March 2019; Volume 01 Issue 06:87-98 In the perusal of literature very scanty information is available about AMF and DSE association with grasses.The overall assessment of Melghat forest flora and fauna cannot be completed unless information available about this area is complete in all the respect.Therefore, the present investigation was undertaken to examine AMF and DSE relationship with dominant grasses of forest along with its colonization and diversity status of AM species in the Melghat forest for the first time.

MATERIALS AND METHODS
Studies were conducted in the Melghat forest of Satpura hill ranges from Amravati district, (Maharashtra), India.The forest is dry deciduous dominated by one of the best teak of India.The forest has rich biodiversity of different important medicinal plants along with various species of grasses.The grasses are considered to be a very important integral part of forest ecosystem and arbuscular mycorrhizae are one of the key components for the development and sustenance of forest ecosystems.In the first phase of survey, twenty-one different locations were selected for sampling.The samples were collected between September to December (2014).The photographic collection of grasses and its herbarium is maintained in the laboratory.
The top 2-3cm of soil around each grass species were removed and rhizosphere soils along with root systems from 4-15cm depth were collected in polyethene bags, labelled and brought to the laboratory for further processing.The soil and root samples in triplicate were maintained in refrigerator at 4 0 C to find out physicochemical properties of soil, AMF spore count and taxonomic status of isolated AM spore.
Wet sieving and decanting technique of Gerdemann and Nicolson (1963) was used for the spore extraction and for spore count the method given by Gaur and Adholeya (1994) was followed.Only viable spores were counted and picked up by needle using Carl Zeiss semi DV 4 stereo zoom microscope.The mean spore density was expressed as spores in 100g soil.Permanent slides of these spores were prepared using polyvinyl alcohol.The detailed photography of these spores was done by Tucsen camera attached to Carl Zeiss Primostar Trinocular compound microscope under 10, 40 and 100x objectives.Morphotaxonomic identifications of extracted spores were carried out using the manuals of Schenck and Perez (1990) and INVAM.
All the permanent slides are deposited in Mycorrhizal biotechnology lab of Shri Shivaji Science College, Amravati

Data Analysis
The standard deviation of means was calculated.Pearson correlation coefficients were computed for soil physicochemical properties with AMF spore count and colonization.Ecological measures of diversity to describe AMF communities includes spore count, species richness, relative abundance, isolation frequency, Shannon-Winer index of diversity, Evenness and Simpson's index of dominance were used.

RESULTS
The soil physicochemical characteristics of the study sites of phase-I (buffer area) is presented in Table no 1.The soils were slightly acidic to alkaline in nature with pH ranged between 6.5 to7.8 and soil texture was mostly sandy clay loam.The range of other soil parameters such as EC ranged from 0.13 to 32.1 dsm -1 ; phosphorus (P) 1.31 to 786.5 kg Ha -1 whereas some sites were P-deficient; potassium(K) 192.64 to 2723.84 kg Ha-1; Nitrogen (N) 109.76 to 517.44 kg Ha -1 ; % Organic carbon 0.16 to 0.77.The micronutrients like Copper (Cu) ranged from 2.68 to 13.03 and Zinc (Zn) 0.52 to 4.23 ppm.
The site wise mean of AMF and DSE % colonization was found in between 1.33 to 52.85 and 0 to 18.97% respectively.The mean of spore count of AMF ranged from 0 to 98 per 100g of soil.
Intact viable AMF spores were isolated from all 21 sites of phase-I.In all 1430 AM spores were extracted and identified from all the sites from 100g soil of each site.As per morpho taxonomic characters 39 different AMF species belonging to 4 genera were recovered from different location (Table-2 and Photo Plate-II).Amongst 39 AM fungal species, Glomus represented by 29 different species, Acaulospora with 5 species, Gigaspora with 4 species and only one species of Scutellispora.Glomus found to be the most dominant genus with 74.35% species population followed by Acaulospora 12.82%, Gigaspora 10.25% and the least species that of Scutellispora with 2.56%.

Statistical Analysis of correlation studies
Correlation studies between soil physicochemical properties, % AMF colonization, % DSE colonization and mean spore count from all sites of phase-I is presented in Table No.3.
In phase-I, % AMF colonization was found to be positively correlated with Potassium, Copper and Zinc, likewise % DSE colonization was found to be positively correlated with EC, Organic carbon, Nitrogen but % AMF was found to be negatively correlated with pH, Phosphorus, Potassium, Copper and Zinc.AMF spore count was found to be positively correlated with pH and Potassium but negatively correlated to EC, organic carbon, nitrogen, Phosphorus, Copper, Zinc, % AMF and % DSE.
In the present investigation, AMF % root colonization were in between 1.  Chaudhry et al., (2006) was also isolated only 12 AMF species belonging to 3 genera from rhizospheric soils of perennial grasses from Cholistan desert, Pakistan.Thus, it can be said that the Melghat forest has more diversity of AMF species in compare to other studied area of the world and India.Anjum et al., (2006) in their correlation studies reported that % mycelia and vesicular infection and number of vesicles were negatively correlated with all root and shoot growth parameters.Similarly, it was also reported by Baylis (1974) and Janos (1980).

CONCLUSION
The present investigation, moderate to very good AMF association along with DSE in different grasses were traced out.It can also make emphasis on the fact that mycorrhizal and DSE symbiosis was controlled by various edaphic factors.Moderate to high level of AMF colonization was an indication of better hyphae and root contact amongst different grasses of monocot plant species for increased benefits of AMF symbiosis and better adaptation to soil.Though there was less number of viable AMF spores in the rhizosphere soil of grasses there was high biodiversity of AM fungal species in the Melghat forest in comparison to previous studies on grasses.
From the present studies, it can be concluded that AMF diversity was high in compared to other regions of world.
Glomus was recovered as the most dominant genus throughout the forest.An expanded AMF study from Melghat forest may be needed to determine relationships between AMF and DSE colonization.With the proper knowledge of mycorrhizal diversity in the rhizosphere of grasses, we can ensure better mycorrhizal inoculation programmes for sustainable availability of grass resources.
The selection of native dominant AMF, mass multiplication and its incorporation as biofertilizer is an important need for future technical development, restoration of burnt and destroyed grasses in the long-term sustenance of forest ecosystem in view to safeguarding the existence of wild animal life.