Species Diversity of Arbuscular Mycorrhizal Fungi (AMF) in the Rhizospheric Soil of Spilanthes acmella Murr.

Chhavi Mangla1, Aditya Kumar2* and Ashok Aggarwal3

1, 2 Department of Botany, Dayanand Post Graduate College, Hisar, Haryana-125001, INDIA

3 Department of Botany, Kurukshetra University, Kurukshetra, Haryana-136119, INDIA

* Correspondence: E-mail: addy.gupta84@gmail.com

(Received 14 Dec, 2018; Accepted 26 Dec, 2018; Published 29 Dec, 2018 )

ABSTRACT: Microbes are very important components of soil plant complex where they show a network of interactions affecting plant health, growth and development. In the present investigation, attempts were made to screen out arbuscular mycorrhizal fungal (AMF) species from rhizospheric soil of Spilanthes acmella- an important medicinal plant. A total of ten species of arbuscular mycorrhizal (AM) spores were screened out. AM fungi belonging to genus Glomus were dominant. Root samples also showed a wide range of variation in terms of AM root colonization. It was found that number of spores in the rhizosphere of plant was not related to the intensity of AM root colonization. Arbuscular mycorrhizal spore population varied significantly in S.acmella as shown by results .

Keywords: Medicinal plants; Arbuscular mycorrhizal fungi; Spilanthes acmella.

INTRODUCTION: Among all the natural resources, one, that provides its invaluable support to sustain human race, is plantation. Besides sustenance, the plants have been used as therapeutic aid for alleviating various human ailments from very earlier times1. The sustainable use of these plant resources through in- situ and/ or ex- situ conservation becomes imperative. The present wave of awareness around environment and natural products is an opportunity for planned sustainable utilization of our valuable forest and cultivation bioresources in the Indian Himalayan zone.

The mycorrhizal symbiotic association appears to have evolved with plants since the colonization of dry land by plant began as a survival mechanism for fungi and higher plants, thus allowing each to survive in the existing environment of low temperature, low soil fertility, periodic drought, diseases, extreme environments and other stress situations2.

Wang and Qiu (2006)3, concluded that the mycorrhizas are present in 92% of plant families (80% of species) and most of these plants are colonized by fungi of the phylum Glomeromycota, which form VAM (Vesicular Arbuscular Mycorrhiza) or AM (Arbuscular Mycorrhiza), the ancestral and predominant form.

Keeping in view all these important aspects, the present investigation was undertaken to find out the abundance and distribution of AM fungi with S.acmella.

Description of Selected Medicinal Plants:

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Spilanthes acmella

Common Name: Akarkara, Alakada

Family: Asteraceae

Parts used: Leaves, flower head and roots.

S.acmella is an indigenous herb, grown as an annual herb throughout the tropics. Plant is erect or ascending stout herb, more or less pubescent, some time hairy. Stem 30-60 cm, usually decumbent near the base. Leaves are opposite, petiolate, broady ovate, narrow at base, acute or obtuse at apex. Flower heads discoid or radiate, conical, solitary on long stalks. Flowers are white or yellow in colour, mostly bisexual. Achenes flattened, each enclosed in a scale.

A number of constituents have been isolated from S.acmella e.g. spilanthol isobutylamide and triterpenoids4,5. It has been found that the plant possess vasorelaxant and antioxidant activity as well as antimicrobial activity6. Different bioactive metabolites have also been reported from S.acmella 7,8.

Medicinal Importance: S.acmella is the well known ‘toothache plant’, also commonly used as a spice. The whole plant is claimed to possess medicinal properties. It has a long history of use as a folklore remedy e.g. for toothache, rheumatism and fever9. In pharmaceuticals, the plant has found applications as an antitoothache, formulation for pain relief, swelling and gum infections and mouthwashes. Moreover, plant extract is added to body and beauty care cosmetics as a fast acting muscle relaxant to accelerate repair of functional wrinkles. As a nutritional supplement, small amount of the plant extract have been used for taste improvement as a sweetner.

MATERIAL AND METHODS:

Study Site: The study was undertaken in the hills of Himachal Pradesh. Wide differences in geo-physical features accounts for considerable variation in climate and rainfall in different sub-regions of the state. Physio-geographically, the state is a part of Himalayan system.

Field Sampling: The plant grew under natural environmental conditions. The three healthy individuals of medicinal plant species were randomly selected for collection of rhizospheric soil and root samples during course of investigation. The samples of each plant were collected for further processing for the isolation of AM spores and studying mycorrhizal root colonization.

Estimation of AM root colonization: The mixed soil and roots samples of each plant species were packed in polyethylene bags, labeled and brought to the laboratory. The soil samples were air-dried at room temperature. Roots were washed to remove soil particles and processed by ‘Rapid Clearing and Staining Method’ 10. Assessment of root colonization was done by estimating total percentage of root colonization by root slide technique11.

Extraction, Quantification and Identification of AM fungal Spores: Rhizospheric soil samples were wet sieved for AM spores using the technique of Gerdemann and Nicolson (1963)12. The quantification of AM spores was done by Grid Line Intersect Method 13. The spores were identified by using keys of Walker (1986), Schenk and Perez (1990), Kumar et al. (2009) 14,15,16.

RESULTS AND DISCUSSION: Basic knowledge of the fungal diversity associated with medicinal and aromatic plants may be useful for the conservation point of view. AM fungi are ecologically important root symbionts of most terrestrial plants. Root samples of the plant species showed a wide range of variation in terms of AM root colonization. The mycorrhizal structures present in the roots included mycelium, vesicles and arbuscules. Mycelia of various types like Y-shaped, H-shaped, coiled and parallel mycelia were reported in the roots of different plants. Vesicles of different shapes were observed. In some roots, vesicles were present in chain, in groups of two and more.

The species diversity and natural occurrence of AM spores isolated from the rhizosphere of studied medicinal plants is depicted in Table I. A total of ten AM fungal taxa were identified in the soil samples. AM fungi belonging to genus Glomus were dominant followed by Gigaspora and Acaulospora. A large number of spores were isolated and variation in spore population was observed in current study. A total of seven species of Glomus, two species of Gigaspora and one species of Acaulospora were isolated (Figure 1). The high spore number in the rhizosphere soils of studied medicinal plants supported the conclusion of Wang et al. (2004)17 in that the host species apparently had direct effects on spore density and colonization of AM fungi. The features favoring the higher population may either be the conductive to edaphic conditions for sporulation like low nutrient status, high aeration and optimum moisture or the undisturbed conditions of the soils which allowed sufficient time for the buildup of mycorrhizal spores18,19. Spore population is affected by a wide range of soil, climatic, fungal and host factors20.

The present results are in conformity with the findings of several workers who reported the dominance of the Glomus in various natural ecosystems21. Manoharachary et al. (2008) 22 screened thirty five AM fungi associated with sixteen medicinal and aromatic plants belonging to family Apocyanaceae and reported Glomus to be most dominant genera represented by twenty one species followed by Acaulospora with seven species. The involvement of intercellular or intracellular mycorrhizal associations or association of more than one mycorrhizal fungus with single host plant species might be attributed to their physiological, ecological and genetical variability23. The sporulation pattern of Glomus might bring about the dominance of the taxon. Spores of Glomus are grown in cluster and sporulate more frequently than others. Twenty three species of AM fungi, which belong to genus Glomus were reported from some medicinal plants of Himachal Pradesh16. The soil and plant types were found to be more or less important factors contributing to such an existence of Glomus. Wang et al. (2008)24 has reported the species of Glomus are the most widespread and abundant, followed by Acaulospora in agricultural soils of Sichuan Province of mainland China. More species of Glomus than that of Acaulospora was recognized by different workers while they worked on various natural ecosystems25,26.

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Figure 1: Number of AM species associated with Spilanthes acmella.

Table 1 : Natural occurrence of AM spores with medicinal plants.

Sr.No.

Name of Plant

AM Fungi

1.

Spilanthes acmella

A. laevis Gerdemann & Trappe

Glomus aggregatum Schenck & Smith emend. Koske

G. fasciculatum (Thaxter) Gerdemann & Trappe emend. Walker & Koske

G. geosporum (Nicolson & Gerdemann) Walker

G. minutum Blaszk, Tadych & Madej

G. mosseae (Nicolson & Gerdemann) Gerdemann & Trappe

G. indica Mano, Sharat & Adholeya

G. segmentatum Trappe, Spooner & Ivory

Gigaspora albida Schenck & Smith

G. gigantea (Nicol. & Gerd.) Gerd. & Trappe


CONCLUSION: Much of the data presented in our work elucidates patterns of spore distribution and their identification. This type of study could be the beginning of further research pursuits that will utilize such symbiotic fungi to manipulate the host in different ways. More studies are being emphasized to select the suitable indigenous AM fungal strains for the establishment and management of natural ecosystem such as medicinal plants and to make the people conscious about the role of mycorrhiza as a tool to maintain and manage the herbal flora of the state by environmental friendly approach.

ACKNOWLEDGEMENTS: The authors (CM & AK) are grateful to Kurukshetra University, Kurukshetra to carry out the work.

REFERENCES:

1. Sullivan, K. and Shealy, C. N. (1997) Complete natural home remedies. Elements Books Limited, Shafterburg, UK.

2. Manoharachary, C., Adholeya, A. and Kunwar, I. K. (2009) Mycorrhiza: some glimpses, Myco. News, 20(4), 2-6.

3. Wang, B. and Qiu, Y. L. (2006) Phylogenetic distribution and evolution of mycorrhizas in land plants, Mycorrhiza, 16(5), 299-363.

4. Ramsewak, R. S., Erickson, A. J. and Nair, M. G. (1999) Bioactive N- isobutylamides from the flower buds of Spilanthes acmella, Phytochemistry, 51, 729- 732.

5. Mukharya, D. K. and Ansari, A. H. (1987) Olean-12-en-3-O-beta-D galactophyranosyl (1?4) –o- alpha-L- rhamnopyranoside: A new triterpenoidal saponin from the roots of Spilanthes acmella Murr, Indian J. Chem. B., 26, 86.

6. Wongsawatkul, O., Prachayasittikul, S., Piacham, T., Isarankura- Na- Ayudhya, C., Nantasenamat, C. and Prachayasittikul, V. (2008) Vasorelaxant and antioxidant activities of Spilanthes acmella (Murr.), Int. J. Mol. Sci., 9, 2724-2744.

7. Kumar, A., Mangla, C. and Aggarwal, A. (2015) The influence of mycorrhizal interactions on some physiological parameters of Spilanthes acmella Murr. plant, J. Biol. Chem. Chron., 1(1), 37-40.

8. Prachayasittikul, S., Suphapong, S., Warachartcheewan, A., Lawung, R., Ruchirawat, S. and Prachayasittikul, V. (2009) Bioactive metabolites from Spilanthes acmella Murr, Molecules, 14, 850-867.

9. Kirtikar, K. R. and Basu, B. D. (1987) Spilanthes acmella Murr. Indian Medicinal Plants (2nd edn). International Book Distributor, Dehradun.

10. Philips, J. M. and Hayman, D. S. (1970) Improved procedures for clearing roots and staining parasitic and VAM fungi for rapid assessment of infection, Trans. Brit. Mycol. Soc., 55, 158-161.

11. Giovannetti, M. and Mosse, B. (1980) An evaluation of techniques to measure vesicular- arbuscular infection in roots, New Phytol., 84, 489-500.

12. Gerdemann, J. W., and Nicolson, Y. H. (1963) Spores of mycorrhizaeEndogone species extracted from soil by wet seiving and decanting, Trans. Brit. Mycol. Soc., 46, 235-244.

13. Adholeya, A. and Gaur, A. (1994) Estimation of VAMF spore in soil, Myco. News, 6(1), 10-11

14. Walker, C. (1986) Taxonomic concepts in the Endogonaceae.II: A fifth morphological wall type in endogonaceous spores. Mycotaxon, 25, 95-105.

15. Schenck, N.C. and Perez, Y. (1990). Manual for identification of VA mycorrhizal (VAM) fungi. Univ of Forida, Synergistic Pub. Florida, USA, 241 p.

16. Kumar, A., Kaushish, S. and Aggarwal, A. (2009) Species diversity of the genus Glomus associated with some important medicinal plants. J. Indian Bot. Soc., 88(2), 80-86.

17. Wang, F. Y., Liu, R. J., Lin, X. G. and Zhou, J. M. (2004) Arbuscular mycorrhizal status of wild plants in saline-alkaline soils of the yellow river delta, Mycorrhiza, 14, 133-137.

18. Chulan, A. and Omar, M. (1991) Incidence of VAM spores in some Malaysian soils, Pertanika, 14, 133-137.

19. Kumar, A., Mangla, C., Aggarwal, A. and Srivastava, V. (2014) Rhizospheric effect of endophytic mycorrhiza and Trichoderma viride on physiological parameters of Mentha spicata Linn, Asian J. of Adv. Basic Sci. 2(1), 99-104.

20. Howeler, R.H., Sieverding, E. and Saif, S. (1987) Practical aspects of mycorrhizal technology in some tropical crops and pastures, Plant Soil, 100, 249-283.

21. Leal, P. L., Sturmer, S. L. and Siqueira, J. O. (2009). Occurrence and diversity of arbuscular mycorrhizal fungi in trap cultures from soils under different land use systems in the Amazon, Brazil. Braz, J. Microbiol., 40, 111-121.

22. Manoharachary, C., Swarupa Rani, S. and Kunwar, I. K. (2008) Arbuscular mycorrhizal fungi associated with some plants of apocyanaceae, J. Mycol. Pl. Pathol., 38(1), 91-92.

23. Sharma, S. K., Sharma, G. D. and Mishra, R. R. (1986) Status of mycorrhizae in subtropical forest ecosystems of Meghalaya, Indian For., 129, 371-376.

24. Wang, Y. Y., Vestberg, M., Walker, C., Hurme, T., Zhang, X. and Lindstrom, L. (2008) Diversity and infectivity of arbuscular mycorrhizal fungi in agricultural soils of the Sichuan Province of mainland China, Mycorrhiza, 18, 59-68.

25. Zhang, Y., Guo, L.D. and Liu, R.J. (2003) Diversity and ecology of arbuscular mycorrhizal fungi in Dujiangyan, Acta Phytoecol. Sinica , 27, 537-544.

26. Kumar, A., Mangla, C. and Aggarwal, A. (2017) Arbuscular mycorrhizal fungal association in some medicinal plant species in the hills of Himachal Pradesh, India,J. Biol. Chem. Chron.,3(2), 01-04.