STUDIES ON THE ARBUSCULAR MYCORRHIZAL FUNGAL DIVERSITY OF SELECTED MEDICINAL PLANT SPECIES FROM KODIKUTHIMALA, MALAPPURAM, KERALA

The present investigation has brought out the AM fungal association in some plant species of Kodikuthimala, Malappuram district Kerala. Totally, 25 plant species belongs to 15 families were analyzed for arbuscular mycorrhizal association. The root samples of all collected plant species showed mycorrhizal infection. The percentage of colonization was varied with plant species and it ranges from 12 (Commelina benghalensis) to 79% (Sida rhombifolia). Maximum spore population was observed in Gloriosa superba (574/100g of soil) and minimum in Euphorbia hirta (143/10g of soil). Totally 26 AM fungal species belongs to 13 genera were found. Among this Glomus was most dominated. In most of the plants, spores of Rhizophagus fasciculatus are seen. Present study confirms the Arbuscular Mycorrhizal association in the collected plant species.


INTRODUCTION
Mycorrhizae, which are a key soil microbial component and known to play an important role in reclamation and revegetation of such, degraded ecosystems (1). They also detoxify certain soil toxins thereby enable seedlings to withstand extreme nutrient absorption capacity of plants (2,3). Over 80% of terrestrial plants are able to associate symbiotically which mycorrhizal fungi and this usually results in positive plant growth response (4). Mutual nutrient transfer between the fungus and plant provides the plant with phosphate and micronutrients such as copper and zinc and the fungus with carbon-based compounds. The most common form of symbiosis involves arbuscular mycorrhizal (AM) fungi, which form two major structural classes of mycorrhizae with different host plants. In AM fungi, arbuscules are considered the major site of nutrient transfer to the plant (5,6).
AM Fung can efficiency absorbs mineral nutrients from the soil and delivers them to their host plants in exchange for carbohydrates and it also enhance tolerance of or resistance to root pathogens. Vascular plants host a great variety of fungi. In additions to being susceptible to soil-borne pathogens, plant roots are also colonized by nonpathogenic or mutualistic fungi like arbuscular mycorrhizae (AM), ectomycorrhizae (EM) and dark septate endophytes (DSE). A vast majority of terrestrial plant species are mycorrhizal associations. The AM fungi are associated with most herbaceous plants and with various woody plant families, while the EM fungi are confined chiefly to a limited number of woody plant families. It is now evident that the mycorrhizal fungi have many significant functions in ecosystems (7). Therefore, the present study aims to Enumeration of the arbuscular mycorrhizal fungal species in the rhizosphere soil samples of the plant species in Kodikuthimala, Malapuram district, Kerala.

Study area
Kodikuthimala is located at 32 km from Malappuram at the Latitude: 10.9802 and Longitude: 76.2917. Kodikuthimala has a watch tower that is popular with tourists visiting this serene place because of the vantage point it offers. British hoisted their flag on this hilltop during survey, thus getting the name Kodikuthimala. This place is noted for its various kinds of medicinal plants and ever flowering springs (Fig. 1). This city has a tropical climate. During most months of the year, there is significant rainfall in Kodikuthimala. There is only a short dry season. The average annual temperature in Kodikuthimala is 27.7°c in a year and the average rainfall is 2500 mm (Table 1).

Sample collection
Totally 40 plant species belonging to the 28 families were collected from Kodikuthimala, Malappuram, Keralain the period of 2016. Root samples and rhizosphere soil samples of plant species growing in and around areas of Kodikuthimala were collected. The root and soil samples were transported to the laboratory immediately after collection.

Root samples
Root samples, 5-15 cm long, were collected from the plant species during 2016 to 2017. During collection, care was taken to ascertain individual plants for which roots could positively identified as belonging to a particular plant species. For identification and nomenclature of the plant species the following manual was used (8,9).

Soil samples
The rhizosphere soils, dug up to a depth of 10 cm, were collected from each plant species after removing the surface of the soil and litter covering. These samples were kept in sterilized bags and were transported to the laboratory immediately after collection for the examination of arbuscular mycorrhizal fungal spore isolation.

Soil pH
The pH of soil samples was determined (soil-water suspensions 1:5) with the help of pH meter (Elico).

Estimation of arbuscular mycorrhizal colonization
minutes at 25°C until bleached. They were rinsed thoroughly with water to remove the H2O2, acidified in dilute HCl and stained as described earlier. In some cases the modified method of Merryweather and Fitter (11) was followed where autoclaving and bleaching with H2O2, were omitted. In a few cases, direct observation of unstained, fresh and intact roots (12) was made.
Arbuscular mycorrhizal infection in the roots was assessed following the grid line-intersect method of Giovannetti and Mosse (13). The stained root pieces were spread out evenly on a square plastic Petridish (10.2 x 10 cm). A grid of lines was marked on the bottom of the dish to form 1 cm inch squares. Vertical and horizontal gridlines were scanned under a dissecting microscope and the presence of infection was recorded at each point where the roots intersected a line. Four sets of observation were made, recording 100, 200, 300 and all the root gridline intersects. Each of the three replicates records was made on a fresh rearrangement of the same root sample. In the laboratory, the roots were separated from the soil by wet sieving. The roots were washed with water and processed a fresh whenever possible. Otherwise the washed roots were fixed in formaldehyde-acetic acid-ethanol (FAA) solution (90:5:5 V/N) modified method of Phillips and Hayman (10). The soil sample was air dried and stored at 4°C until processed. Each soil samples was used for chemical analysis, spore counts and classification in to various types and multiplication, concentration and separation of AM fungal spore for identification.

Evaluation of AM infection
The root samples were cleared and stained in tryphan blue with a modified version of the Phillips and Hayman's (10) method. Roots were cut in to 1-2 pieces, heated at 90°C in 10% KOH for about 1 hour. For thicker and older roots, the duration was increased. The root segments were rinsed in water and acidified with dilute HCl. The root pieces were stained 0.05% tryphan blue in lacto phenol for 5 minutes and the excess stain was removed with clear lacto phenol.
The pigmented roots were heated at 90°C in 10% KOH for 2 hours, washed with fresh 10% KOH and immersed in an alkaline solution of H2O2 for 30 When sufficient root pieces are not available, the slide method Giovannetti and Mosse (1980) was followed. Root pieces, 1 cm long, were selected at random from a stained sample and mounted on microscope slide groups of 10. Presence of infection was recorded in each of the 10 pieces and present infection was calculated. To observe hyphae, vesicles and arbuscles under light microscope, the root pieces were mounted on glass slides either temporarily in lacto phenol. The cover slip was pressed gently to make the roots flattened and sealed with DPX medium.

Isolation of arbuscular mycorrhizal spores from the soil samples
Spores were recovered from the soil samples by the wet-sieving and decanting method (14). From each soil sample, 100 g of soil was taken and mixed with 1:1 of warm water in a large beaker until all the aggregates dispersed to leave a uniform suspension. Heavier particles were allowed to settle down. To remove organic matter and roots, the suspension was decanted through a 710 µm sieve. The suspension that passed through 710 µm was decanted 425 µm, 250 µm, 150 µm, 75 µm and 45 µm sieves consecutively. The residues in the respective sieve were collected in petridishes with about 10-20 mL water observed under a dissecting microscope for AM fungal spores. The total spore count was calculated by counting the spores. Then the spores were separated using a glass pipette and segregated. The spore were mounted on clear glass slides using lacto phenol or polyvinyl alcohol lacto phenol (PVL), covered with cover slips and sealed with DPX medium.

Identification of AM fungi
Based upon microscopic characters, the AM fungal spores were identified. For identification and nomenclature, keys of the following manual authors were used: Raman and Mohankumar (15), Schenk and Perez (16) and Redecker et al., (17). Classification on based on color, size, shape, surface, structure, general nature of the spore contents and hyphal attachment. Photomicrographs were taken with the help of a Magnus Olympus Microscope.

RESULTS
AM fungal infection and spore population of 40 plant species belongs to 28 families present in the Table 2 to 4 and pH of the rhizosphere soil samples present in the Table 3 was 4 to 5.8. In the present study, totally 14 AM fungal species belongs to 7 genre were identified. Where the Glomus (4) was dominate genus followed by Gigaspora (3),Acaulospora (2), Ambispora (2), Claroideoglomus (1), Rhizophagus (1) and Scutellospora (1). Moreover the Rhizophagus fasciculatus was the most frequently abundant species in the study area (Table 4). H-hyphae, A-Arbuscules, V-Vescicle, + -Present, --Absent  The total number of 40 plant species belongs to 28 families were examined for AM fungal spore populations and colonization (Table 3 and 4). Of these, maximum spore population was recorded in the plant species of Gloriosa superba (574/100g of soil) belongs to the family Liliaceae and minimum spore population was noticed in the plant species of Euphorbia hirta belongs to Euphorbiaceae. The highest AM fungal infection was found in the roots of Sida rhombifloia (79%) belongs to Malvaceae and minimum infection was occurred in the plant species Commelina benghalensis (12%) belongs to Commelinaceae. In the present study, all the plants were examined form the study area have significantly influenced by AM fungal. Where, the plants were successfully surveyed by these fungal through their contribution in the plant community.

DISCUSSION
Vesicular-arbuscular mycorrhizal (VAM) association with plants is widely distributed and it is geographically ubiquitous.
In the present investigation all tree species were found to have mycorrhizal association. Microscopic observation of root segments revealed the presence of AM fungal structures ramified by extra-matrical hyphae and intracellular infestation of angular thick-walled hyphae. AM fungi have a potential importance in the recovery of disturbed lands and can be used in wasteland or semi-arid land could be improved by incorporating AM fungi. The variation in the intensity of root colonization and sporulation due to varieties and AM fungi recorded in the present study must be on the basis of host-symbiont specificity. In the present investigation, there was a change in AM spore number and infection in all the plant species. Others have also reported similar changes in different constituents of microbial population (18,19). Priya (20) showed that the activity of soil mycorrhizal population was greatly affected by soil pH, temperature and moisture.
In the present study the Cyperaceae family Kyllinga alba not infected by Arbuscular mycorrhizal infection. In contrast Cyperus conglomerathus, Cyperus rotundus both the species were found to be mycorrhizal (21). These findings are quite in line with the findings of Muthukumar and Udaiyan (22), Harikumar (23), Silva et al., (24). The probability of mycorrhizal colonization increases with the increase of soil pH because the availability of nutrients decreased with increasing pH (25). Chaudhry et al. (21) find out the AM fungal infection in the Poaceae members, particularly Cympopogon jwarancusa in an aromatic grass showed highest number of AMF species. The present study also revealed that the Poaceae member Chrysopogon zizanoides showed 56% of AM fungal infection. Most of the plant species in tropical rain forests and the members of Leguminosae sub families Papilonaceae and Mimosaceae form AM symbiosis (26). The same result was obtained in the present findings.
The present investigation of the AM fungal diversity in this study area, the tractability and ecological importance of mycorrhizal systems makes them ideal models to test and develop biodiversity in this study area. Consequently, recent studies have focused on the different functions of AM Fungal and their roles in ecosystem functioning. Hence, there is a new need of ecological concepts in AM Fungal community to increasing productivity and fitness of plants in ecosystems.