STUDIES ON THE AM FUNGAL DIVERSITY OF SOME SIGNIFICANT ETHANO-MEDICINAL PLANTS OF KARULAI HILLS, MALAPPURAM DISTRICT, KERALA

The study was carried out to observe the AM fungal diversity in some important medicinal plant species of Karulai hills, Malappuram district, Kerala. The root samples of all the collected plant species showed mycorrhizal infection. The percentage of AM fungal colonization ranged from 17 to 87. The highest AM fungal infection was exhibited in Desmodium triflorum (87%) and lowest in phyllanthes amarus (17%). The maximum spore population was observed in Desmodium gangeticum (874/100g of soil) and minimum in Piper longum (171/100g of soil). Totally 13 genera of AM fungi were found to be associated with the rhizosphere soil samples. Among them AM fungal species isolated, the dominant species is Rhizophagus fasciculatus. Ethanobotanical study reveals that the Cholanaykans tribes of Karullai hills posses great knowledge about the use of various herbal medicines to cure different ailments and are also conscious about the loss of their traditional medicinal practices. They know about number of medicinal plants and their applications.


INRODUCTION
Limited availability of soil nitrogen and phosphorus is frequently a major factor limiting sustainable productivity of tropical tree plantations. The situation in developing countries like India, fertilizer could be applied only for a few cash crops and stable food crops such as rice and wheat and not for afforestation of waste lands. Hence, microbial technologies hold great promise in the operation of scientific forest nursery managements by inoculating containers with biofertilizers viz., nitrogen fixing organisms, phosphate solubilising organisms and mycorrhizae. Of these, inoculation of forest trees with mycorrhizal fungi could help the plants to scavenge sparingly available nutrients in soil including phosphorus and also provide protection against plant pathogens and drought Baltruschat and Schonbeck (1). Arbuscular mycorrhizal (AM) fungal symbiosis facilitates the survival, growth and establishment of plants in extreme habitats Asmelash (2). Many factors stimulate differential spore production by AM fungi in the rhizosphere, leads to seasonal fluctuation in AM fungal colonization and spore densities Koske (3), Gemma and Koske, (4). The most wide spread symbiosis amongst plants is mycorrhizal association which involves various root inhabiting fungi and feeder roots. Among the different type of mycorrhizal fungi, the AM fungi are widely distributed in most ecosystems and associated with many plant species.
The beneficial effect of AM fungi on plant growth has been highlighted by Rafiq (5) and by several researchers. It has been found that AM fungi contributed to increased rate of nutrient absorption especially phosphorus from soil, longevity of feeder roots, increased tolerance to drought, heavy metals, soil toxins, extremes of soil pH and high temperature. Many commercially important tree species like Acacia, Eucalyptus, Teak etc. are naturally colonized by AM fungi. It is well known that AM fungi protect plants against soil and rootborne pathogens Bagyaraj (6) thereby improving plant growth and vigor.
Microorganisms are present in great number on and near the feeder roots and they play vital roles in numerous physiological processes. These dynamic processes are medicated by association of microorganisms participating in saprophytic, pathogenic and symbiotic root activities. The major symbiotic associations on tree species are mycorrhizal fungi. AM fungi, play an important role in plant survival and in the community stability of vegetation in natural ecosystems. Mycorrhizal symbiosis plays a critical role in mineral nutrition of terrestrial plants. The mycorrhizal fungi are an important part of the soil microbial system because the prevalence of these associations on plants is so common under natural soil conditions. Plants also find innumerable uses in the human civilization since its conception. The plants *Correspondence: Mr. K. Muthuraj, Research Scholar, Department of Botany, Kongunadu Arts and Science College, Coimbatore-also find their use as medicine in human healthcare. Several traditional systems have evolved in the world, which use plants to cater to needs of healthcare and are still in practice around the world. The use of plants and natural products received a fillip when World Health Organization recognized plant and natural products based medicinal systems as alternative and complimentary. The use of medicinal plants for human healthcare is well documented in India, China, Egypt and Arab world Lalrinzuali (7).
The traditional systems of medicine prescribe drug as single plant products or a mixture of several plants depending on the disease, which are mainly administrated orally. The ethanobotanical and ethanomedicinal studies have great significance in the collection traditional knowledge, preparation of recorded data and in conservation of endangered medicinal plant species Prakash (8). The present work aims, to documentation of ethanobotanical importance of medicinal plants practiced by Cholanaikkan tribes and Enumeration of the arbuscular mycorrhizal fungal species in the rhizosphere soil samples of these plant species in Karulai, Malapuram district, Kerala.

Study area
Karulai village is located in Nilambur, Malappuram district, Kerala, India. It is situated 10 km away from the sub-district headquarters Nilambur and 48 km away from district headquarters Malappuram. Total extent of Karulai range is 26560.76 hecters which is notified under two reserve notifications viz., Amarambalam Reserve and Karimpuzha reserve. Karulai is the "Gods' own village" in Kerala state with green forest (Fig. 1). The average annual temperature in Karulai is 27.7°C in a year and the average rainfall is 2500 mm (Table-1). Karimpuzha is the largest tributary of Chaliyar River, Kerala, India. It is very near to Nilambur. Karimpuzha originates from Western slopes between Mukuthi peak and Avanlanche Dam in Nilgiri district of Tamil Nadu.

Sample collection
Totally 45 plant species belonging to the 31 families were collected from Karulai during the September, 2016 -March 2017. Root samples and rhizosphere soil samples of plant species growing in and around areas of Karulai 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 all three seasons of 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 Gamble(9) Nair and Henry (10).

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).

Sample preservation
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 (11). 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 (12) 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 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 (13) was followed where autoclaving and bleaching with H2O2, were omitted. In a few cases, direct observation of unstained, fresh and intact roots Arias (11) was made.
Arbuscular mycorrhizal infection in the roots was assessed following the grid line-intersect method of Giovannetti and Mosse (14). 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.
The percentage of AM infection was calculated using the formula: When sufficient root pieces are not available, the slide method Giovannetti and Mosse 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 Gerdemann and Nicolson (15). 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 (16) and Redecker (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.

Ethnobotanical study
Frequent field trips were conducted in the tribal villages located at Karulai hills, during the study period (2016-2017). Initial field trips were utilized to know about the land and people. As the tribal's are mostly illiterate, no structural questionnaire approach was used. Ethno medicinal data were collected through conversation with beneficiaries, traditional healers and elder people. During the interviews, local names, useful plant parts, method of preparation and dosage were recorded. Subsequent field trips were conducted in different season in the same localities for confirming the data collected and also for gathering, additional medicinal information. The medicinal plant species were collected from wild and also from the tribal peoples homestead gardens for herbarium preparation. The method of gathering information was the same as suggested by Jain (18).

Cholanaikkan tribes
The Cholanaikkans are an ethnic group and primarily inhabit the southern Kerala state, especially silent valley national park. The Cholanaikkan traditionally reside Karulai and Chunkathara forest ranges near Nilambur, Malappuram district. Until the 1960s, they were leading a secluded life with very limited contact with mainstream urban society. Since then, the Cholanaikkans traditional lifestyle has been altered. They currently have a 16% literacy rate. The Cholanaikkan call themselves as 'Malanaikan' or 'Sholanaikan'. They are called Cholanaikan because they inhabit the interior forests. 'Chola' or 'sholas' means deep ever green forests. And 'naikan' means king. The Cholanaikkan numbered 360 indivduals in the 1991 but only 191 members today. They are found widely scattered in the forest ranges. They subsist on food-gathering, hunting and minor forest produce collection. Their language is a mixture of Kannada, Tamil and Malayalam. They use rice as their staple food, also use wild tubers, roots, seeds, fruits, and meat.  Juice taken from crushed plant parts is applied for eye itching and other eye problems The juice from the heated leaves of the plant is applied in to ear for earache.

As a vegetable
The ripe seed can be eaten after cooking

7.
Cassia auriculata L. Diabetes Grind the dried bark, flowers, leaves and fruits in qualities boil with water. It is used to treat diabetes.

Eye diseases
The extract of the plant is useful for eye infection and irritation.

Memory power Consumption of whole plant juice can improves memory
Intestinal worms Rhizome has been used to treat fever, rash, and intestinal 11. Clitoria ternatea L.

Head ache, Inflammation
Leaf juice is used as a nasal drops in headache. The leaf can be grind in to fine paste and applied any kind of inflammation. 12. Costus pictus D.Don. Diabetes Juice prepared from the leaves is used to treat diabetes. Crotalaria pallida Aiton.

Swelling
The poultice made of the root applied in painful swelling

Blood purifier Crushed tubers are mixed with milk is used as blood
The oil is used to reduce pain and inflammation associated with snake bite.

16.
Cyclea peltata (Lam.) Hook.f.&Thomson The crushed leaves are applied externally on wounds and skin problems. The whole plant is used to promoting

15.
women. Leaf paste is used to treat swelling. Latex is used to treat warts on face. Gliricidia sepium (Jacq.) Walp.
The leaves paste is used as a sedative and insecticides. It is used for the treatment of fracturesand wounds.
Their livelihood is totally dependent on the forest. The collection and selling of minor forest produce is the major source of income. The tribes, unlike any other tribes, under the leadership of the Mooppan (Elder) are willing to come out of the deep forest (Fig. 2).
Medicinal plants used in folk herbal remedies are prepared and administered in various forms in the Karulai hills. Majority of the plant remedies were prepared by decoction and juice. The paste was prepared by grinding the fresh or dried plant parts with oil or water. Powder was prepared by the grinding of shade dried parts. The most frequently used mode of remedy administration is oral ingestion, followed by tropical uses, nasal drops, face crams, hair cleaners, and bath. The most treated illness of the Karulai hills using a number of medicinal plants are grouped in to several disorders. We found the highest number of plant species are used against cold, followed by cough, diabetes, kidney stones, stomachache, swelling, headache, eye diseases, ageist intestinal worms, toothache, snake and scorpion bites, mosquito repellent, vomiting, jaundice and rheumatism ( Table 6). The present study noticed that, single disease can be cured with infusions of more than one plant. Similarly, the single plant can be utilized to cure more than one disease.

DISCUSSION
The arbuscular mycorrhizae are reported to be ubiquitous both geographically and ecologically Mosse (14). Seasonal fluctuations in moisture, temperature, p H and soil nutrient status show high and dramatic effects on arbuscular mycorrhizal spore population and percentage of root colonization. Soil physiological characters played an important role in distribution and density of mycorrhizal fungi. All the plant species 45 belongs to 31 families of rhizosphere soil samples observed the AM fungal spores. Among the AM fungal species Glomus is most common. All the plant species colonized by AM fungi. The plant species infected by hyphae, vesicles and arbuscules. Grasses they have evolved the fibrous root system or an alternative phosphate acquisition strategy which enables them to do without mycorrhiza.
In the present finding the Poaceae member Cymbopogon flexuosus infected by arbuscular mycorrhizae. The infection in the plant species has 38%. Mycorrizal association occurred naturally with many important forest trees. Ectomycorrhizae mostly occur in temperate forest whereas in tropics endomycorrizae are more common. The present finding is in agreement with the results obtained by seasonal workers.
Brundrett and Abbott (20) analyzed the most of the plant species in tropical rain forests and the members of Leguminosae and the subfamilies of Papilonaceae and Mimosaceae. The same results was obtained in the present investigation that the Leguminosae members of Abrus precatorius, clitoria pictus, crotalaria pallida infected by AM fungal infection. Arbuscular micorrhiza is most common in Angiosperms, Gymnosperms, Pteridophytes and Bryophytes. The association of AM fungi with all the plants studies confirms the ubiquitous nature of AM Hayman (11) although the extent of root infection and number of AM spores found in the rhizosphere were different.
In this investigation, the mycorrhizal colonization was vary this may be the host specificity. The major ecosystem function of mycorrhizae is to assist host plants in the acquisition of resources from soil. This study displays the different degrees of AM fungi in plant host specificity. Such as mycorrhizal symbioses play fundamental roles in shaping plant communities, terrestrial ecosystems and high value for sustainability of this ecosystem.