Sundar, S. K ., et al. / Journal of Pharmacy Research 2012,5(8),3990-3993 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Studies on the Synergestic Effect of AM Fungi and PGPRS on Growth and Phytochemical Properties of Medicinally Important Indigofera tinctoria L

Sundar, S. K., A. Palavesam# and B. Parthipan* Department of Microbiology, M. R. Government Arts College, Mannargudi, Thiruvarur District, Tamil Nadu, India # Centre for Marine Science and Technology, M. S. University, Rajakkamangalam, Tamil Nadu, India *P.G. Department of Botany and Research Centre, S. T. Hindu College, Nagercoil-629 002, Tamil Nadu, India Received on:09-05-2012; Revised on: 14-06-2012; Accepted on:22-07-2012

ABSTRACT The impact of different combinations of native dominant AM fungi Glomus aggregatum and PGPRs viz., Bacillus coagulans and Trichoderma viride on the growth, primary metabolites and phytochemical parameters of the medicinally important natural dye yielding plant Indigofera tinctoria was studied by subjecting them to pot culture study. Dry matter content of I. tinctoria was higher in G. aggregatum + B. coagulans treatment whereas plant height, protein and amino acid contents were higher in G. aggregatum coinoculated with B. coagulans and T. viride. Chlorophyll, carotenoid, lipid and phenol contents were higher in triple inoculation of I. tinctoria than the other treatments. The inoculation of efficient AM fungi in combination with the two PGPRs had positively influenced the growth and phytochemical constituents of I. tinctoria whereas B. coagulans and T. viride as individual inoculations did not show any significant results.

Key words: Glomus aggregatum, PGPRs, I. tinctoria, Biomass, Phytochemical constituents. INTRODUCTION India has about 18,000 species of angiosperms, of which about 3,000 species Seed pelleting with the plant growth promoting microorganisms, B. are considered as important sources of medicinal and aromatic chemical coagulans and T. viride were done before sowing. The seeds were spread on compounds1. The commercial medicinal plant growers started using chemical a polythene paper and the liquid based inoculums of both the PGPRs were fertilizers to increase the yield. Of late, the traditional medical practitioners sprinkled over the seeds and the inoculum coated seeds were then air dried and growers realized the rising toxicity in plant based medicines due to before sowing. The mass multiplied soil and root based inoculum of AM manuring with chemical fertilizes2.3. AM fungi are known to improve plant fungi were applied 3 cm below the pot culture soil as thin layer at the rate growth in different ways like increased phosphorus uptake, increase in of 5.0 g/pot7. biomass of plants etc4,5. In recent years, use of artificially produced inoculums of mycorrhizal fungi and other PGPRs for field, horticulture and of late, Experimental set up medicinal plants have increased due to their multifarious role in plant growth The experiment was carried out in random block design with five replicates. and yield and resistance to climatic and edaphic stresses, pathogens and Eight treatments consisting of various combinations of native AM fungi (G. pests6. I. tinctoria L., an economically important plant of the family aggregatum) and PGPRs and uninoculated control were maintained in pot Leguminosae and subfamily Papilionaceae (Fabaceae) is exploited in culture. traditional medicine and fabric dyeing process. The whole plant extract of I. tinctoria is used in the treatment of epilepsy and other nervous disorders Estimation of AM fungal colonization and enumeration of AM spores and the plant is also used as a natural dye. Considering the usefulness of the Roots collected from native dominant AM fungi and PGPRS inoculated and plant, native AM fungi G. aggregatum of I. tinctoria along with plant growth uninoculated I. tinctoria L. were fixed immediately in FAA after washing in promoting rhizomicroorganisms viz., B. coagulans and T. viride were applied running tap water8. The fixed root segments were cleared in 2.5% KOH at in combination to I. tinctoria plants in pot culture to study their synergistic 90º C for 30 minutes. They were then washed in distilled water and acidified influence on the growth and various biochemical parameters of the plant. with 5 N hydrochloric acid for 2-3 minutes. The roots were stained with 0.05% Tryphan blue in lactophenol for 15-30 minutes and the excess stain MATERIALS AND METHODS was removed with clear lactophenol9. The percentage of root colonization of cleared and stained root segments were estimated10. Selection of planting material Seeds of I. tinctoria plants were soaked in 5 % sodium chloride, the floating The native dominant AM fungal spores were isolated using wet sieving and seeds were discarded and viable seeds were used for further sowing in pots decanting method by Gerdemann and Nicolson11. Taxonomic identification of size 22 cm x 20 cm filled with sand and soil (1:3). Plant growth promoting of spores to species level was based on spore size, color, ornamentation and rhizomicroorganisms, Bacillus coagulans (MTCC 2499) and Trichoderma wall characteristics12. viride (MTCC 793), used in the present study were procured from Institute of Microbial Type Culture Collection and Gene Bank, IMTECH, Estimation of plant biomass and plant height Chandigarh. The bacterial and fungal cultures were maintained in nutrient The dry matter content of the AM fungi and PGRPs inoculated and agar and potato dextrose agar medium respectively for further use. uninoculated Indigofera species removed 90 days after sowing were AM fungi and PGPRs inoculation determined by drying the sample in an oven at 60ºC till constant weight was obtained. Plants were collected from each treatment and the height was measured from the bottom of the root to the tip of the plant and expressed *Corresponding author. in cm. B. Parthipan Estimation of plant macronutrients P.G. Department of Botany and Research The plant macronutrients namely nitrogen (N), phosphorus (P) and Centre, S. T. Hindu College, potassium (K) were estimated according to Piper13, Jackson14 and Jones Nagercoil-629 002, Tamil Nadu, India and Isaac15 respectively. Journal of Pharmacy Research Vol.5 Issue 8.August 2012 3990-3993 Sundar, S. K ., et al. / Journal of Pharmacy Research 2012,5(8),3990-3993 Plant pigments reported that the combined inoculation of G. mosseae and T. harzianum had The plant pigments, chlorophyll and carotenoids were extracted using the highest AM colonization and spore number. This indicates the efficacy acetone and estimated according to Witham et al.16 and Goodwin17. of inoculated efficient native AM fungi against AM fungi present in the rhizosphere soil for better colonization. Biochemical constituents The protein content was estimated by the procedure of Lowry et al.18 and In the present study the plant biomass and plant height of I. tinctoria were the total free amino acids according to Troll and Canon19. The estimation of influenced significantly (P < 0.05) by AM fungi and PGPRs inoculation. total phenols in plants was carried out by the protocol put forth by Farkas Earanna et al.31 also obtained maximum plant height and dry matter and Kiraly20. The total lipid was estimated with dry plant sample according production in Phyllanthus amarus with the inoculation of G. mosseae with to Sato and Murata21. either B. coagulans or T. harzianum. The maximum height and dry weight were found in plants with both AM fungi and PGPRs treatment (Table -1). The protocol for the estimation of alkaloids was proposed by Harborne22. The positive effects of the bioinoculants on the plant parameters may be The estimation of flavonoids was done according to Bohm and Kocipai attributed to the synergistic relation between them. Abyazan23. Obadoni and Ochuko24 proposed the method for the estimation of saponins. The protein and amino acid contents of I. tinctoria were higher in triple inoculation of the plants with G. aggregatum, B. coagulans and T. viride RESULTS AND DISCUSSION (1.47 mg/g plant) followed by dual inoculation with AM fungi and B. The role of AM fungi in improving plant growth is also well documented25,26. coagulans (Figure – 2). Krishna and Bagyaraj32 found that protein and Mycorrhizal fungi enhancing the number and activity of beneficial soil amino acid content increased in G. fasciculatum inoculated Arachis hypogeae organisms like nitrogen fixers and phosphate solubilizers with consequential roots. A three fold higher protein content was found in mycorrhizal than in beneficial effect on plant growth has also been reported27, 28. non-mycorrhizal red clover roots and in tobacco and onion33. In the present study, the AM fungal colonization and spore numbers in the Figure 2 - Effect of native AM fungi and PGPRs on the protein and rhizosphere soils were higher in single, dual and triple combinations of amino acid contents of I. tinctoria native dominant AM fungi G.aggregatum and PGPRs inoculated to the test plants. G.aggregatum was reported as the dominant AM fungi among the fifteen species of mycorrhizal fungi recorded in the rhizosphere soil of I.tinctoria surveyed in the terminal western Ghat regions of Kanyakumari district, TamilNadu by the same authors29. The highest colonization (88.09%) and spore number (428/100 g soil) were recorded in G. aggregatum + B. coagulans treated plants (Figure – 1). Anusuya and Senthilkumar30

Each value is an average of five replicates.

T0 - Control; T1 - G. aggregatum; T2 - T. viride; T3 - B. coagulans; T4 - B. coagulans + T. viride; T5 - G. aggregatum + B. coagulans; T6 - G. aggregatum + T. viride; T7 - G. aggregatum + B. coagulans + T. viride

In the present study, the dual inoculation of G. aggregatum with B. coagulans or T. viride and triple inoculation with G. aggregatum, B. coagulans and T. viride had enhanced the plant macronutrient contents of I. tinctoria when compared to the other treatments (Figure -3). This also upholds the observation made earlier in Phyllanthus amarus30. Under appropriate conditions, mycorrhizal plants are credited with 3-5 times Figure 1. Effect of native AM fungi and PGPRs on root colonisation and higher rate of phosphorus uptake per unit root length than non-mycorrhizal 34 spore number in the roots and rhizosphere soils of Indigofera tinctoria plants of the same dependent species .

Table - 1. Effect of native AM fungi and PGPRs on the plant biomass, plant height, pigments, The present study results indicate that the total phenol and total lipid contents of I. tinctoria inoculations of G. aggregatum along with B. coagulans and T. viride enhanced the total Treatments Dry weight Plant height Total Carotene Total Lipid Total Phenols (g/ plant) (cm/plant) chlorophyll (mg-1 g (µg/g (µg/ g chlorophyll (0.96 mg/g plant) and carotenoid (mg-1 g plant) plant) dry plant) fresh plant) (0.492 mg/g plant) contents than the uninoculated plants (Table -1). This increase Control 4.02 45.06 0.572 0.301 104.20 133.60 might be due to increase in the cytokinin content, Glomus aggregatum 6.54* 60.07* 0.857 0.443* 122.30* 157.10* which prevents chlorophyll degradation and Trichoderma viride 5.64 53.10 0.663 0.351 108.60 142.70 increases ion transport35. Similar results also Bacillus coagulans 5.86* 55.20* 0.806 0.387 112.10 139.50 have been obtained in Gloriosa superba by B. coagulans + T. viride 6.11* 56.55* 0.719 0.407 112.90 145.30* Elango36. G. aggregatum +B. coagulans 7.31* 72.39* 0.909 0.471* 139.20* 158.30* G. aggregatum +T. viride 7.03* 66.06* 0.87 0.455* 126.50* 161.80* G. aggregatum + 7.23* 74.20* 0.96 0.492* 145.70* 166.30* The maximum levels of total phenol and total B. coagulans +T.viride lipids were observed in I. tinctoria inoculated SEM 1.01 4.52 - 0.056 4.90 5.80 with G. aggregatum, B. coagulans and T. viride CD at 5% level 1.96 9.85 - 0.114 9.84 11.61 (145.70 µg/g dry plant and 166.30 µg/g fresh Each value is an average of five replicates.*Values indicate significance over control. Journal of Pharmacy Research Vol.5 Issue 8.August 2012 3990-3993 Sundar, S. K ., et al. / Journal of Pharmacy Research 2012,5(8),3990-3993 Figure 3 – Macronutrient content of I. tinctoria plants as influenced by In the present investigation, I. tinctoria plants inoculated with the native native AM fungi and PGPRs dominant AM fungi G. aggregatum along with PGPRs B. coagulans and T. viride showed significantly higher plant growth, dry matter content,

180 biochemical constituents such as protein, amino acids, lipids, pigments and the content of secondary metabolites than the uninoculated plants. 160

140 Productive and sustainable agriculture insists on agronomic practices such

N 120 as manuring at regular intervals, removal of weeds and management of pests P and diseases. In recent years, use of artificially produced inoculums of 100 K mycorrhizal fungi and other PGPRs for field, horticulture and of late, medicinal mg/plant 80 plants have increased due to their multifarious role in plant growth and yield and resistance to climatic and edaphic stresses, pathogens and pests42. 60 As the I. tinctoria plants surveyed in the present investigation has extensive 40 medicinal and dye yielding properties, application of chemical fertilizers is

20 normally not recommended, since it may have some implications on the secondary metabolite contents of the plants. In this context, application of 0 T0 T1 T2 T3 T4 T5 T6 T7 these beneficial microorganisms namely G. aggregatum, B. coagulans and Treatments T. viride will be safer and also will boost the growth and medicinal potential Each value is an average of five replicates. of the plant.

T0 - Control; T1 - G. aggregatum; T2 - T. viride; T3 - B. coagulans;

T4 - B. coagulans + T. viride; T5 - G. aggregatum + B. coagulans; REFERENCES

T6 - G. aggregatum + T. viride; T7 - G. aggregatum + B. coagulans + T. viride 1) Rajasekharan, P.E., 2001. Medicinal plant sector in India. In: plant respectively). The plants treated with G. aggregatum (either single or International Seminar on Medicinal Plants. The Voluntary Health, in combination with B. coagulans and T. viride) showed significantly higher Education and Rural Development Society, Chennai, pp. 23-31. (P < 0.05) lipid and phenol contents than the other two PGPR treatments 2) Anuradha, M. N., C. Kathiresan, M.M. Khan and A.A.Farooqui, and control plants (Table - 1). The increase in total phenols in AM inoculated 2001. Role of Biofertilizers in production of medicinal crops. plants could be attributed to the triggering of pathways of aromatic International Seminar on Medicinal plants. Quality biosynthesis37. The increase of total lipids in mycorrhizal plants could be standardization. The voluntary Health, Education and Rural attributed to the increase in neutral lipids, principally triacylglycerols that Development society, Chennai. pp. 65-69. are reportedly supplied to the plant roots by the fungal hyphae38. Increased 3) Abdul Malik, M. 2000. Association of VA-mycorrhizae with total phenols and total lipids were reported in mycorrhizal plants by some varieties of tobacco (Nicotiana tobacum L.) and its effect Hemalatha39 and Elango35 respectively. on their growth, nutrition and certain soil-borne diseases. Ph. D. Thesis, Bharathidasan University, Tiruchirapalli, P.104. The percentage alkaloids of I. tinctoria was high (1.45%) in G. aggregatum 4) Fattah, A. and M. Gamal, 2001. Measurement of the viability of treatment co-inoculated with B. coagulans and T. viride; whereas, it was arbuscular mycorrhizal fungi using three different strains, relation low in control plants (1.19%) (Figure -4). Plants inoculated with AM fungi to growth and metabolic activities of soybean plants. Microbiol. and PGPRs in combination showed significantly higher (P < 0.05) flavonoids Res., 156: 359-367. and saponins than the control plants. The B. coagulans + T. viride treatment 5) Javot, H., N. Pumplin and M. Harrison, 2007. Phosphate in the of I. tinctoria plants recorded significant (P < 0.05) flavonoid content than arbuscular mycorrhizal symbiosis: transport properties and the uninoculated plants while the treatment failed to show significant regulatory roles. Pl. Cell Enviro., 30: 310-312. increase (P < 0.05) in saponin content than the control plants. The increase 6) Raman, N. and A. Mahadevan, 1996. Mycorrhizal Research - a might be attributed to the production of hormones40 and the uptake of priority in Agriculture. In: Concepts in Mycorrhizal Research phosphorus and other nutrients by AM fungus26, 41. [K.G. Mukerji (ed.)], pp. 41-75. 7) Kumutha, K., S.P. Sundaram, and J. Sempavalam, 2006. Influence Figure 4 . Quantitative analysis of phytochemical constituents of na- of arbuscular mycorrhizal fungi and PGPR inoculation on growth tive AM fungi and PGPRs treated I. tinctoria and biochemical parameters of mulberry. Asian J. Microbiol. Biotech. Env. Sci., 8: 355-360. 8) Johansen, D.A. 1940. Plant micro technique. McGraw-Hill, New York. 9) Phillips, J.F. and Hayman, 1970. Improved procedures for clearing root parasitic and staining vesicular arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. Br. Mycol. Soc., 55: 158- 160. 10) McGonigle, T.P., M.H. Miller, D.G. Evans, G.L. Fairchild and J.A. Swan, 1990. A method which gives an objective measure of colonization of roots by vesicular-arbuscular mycorrhizal fungi. New Phytol., 115: 495-501. 11) Gerdemann, J.W. and T.H. Nicolson, 1963. Spores of mycorrhizal Endogone species extracted from soils by Wet-sieving and decanting. Trans. Br. Mycol. Soc., 46: 235-244. 12) Schenck, N.C. and Y. Perez, 1990. Manual for the identification of VA mycorrhizal fungi synergistic, Gainsville, INVAM, University of Florida, Gainesville, Florida, USA. Each value is an average of five replicates. 13) Piper, C.S. 1950. Soil and Plant Analysis, Inter. Sci. Pub. Inc., T - Control; T - G. aggregatum; T - T. viride; T - B. coagulans; T - B. New York, U.S.A., P. 368. 0 1 2 3 4 14) Jackson. M.L., 1973. Soil Chemical Analyses, Prentice Hall, New coagulans + T. viride; T5 - G. aggregatum + B. coagulans; T6 - G. aggregatum + T. viride; T - G. aggregatum + B. coagulans + T. viride Delhi, pp. 111-120. 7 15) Jones, J.B. and R.A. Isaac, 1969. Comparative elemental analy-

Journal of Pharmacy Research Vol.5 Issue 8.August 2012 3990-3993 Sundar, S. K ., et al. / Journal of Pharmacy Research 2012,5(8),3990-3993 ses of plant tissue by spark emission and atomic absorption of VAM fungi and Trichoderma on micropropagated Dianthus spectroscopy. Agronomy J., 61: 393–394. caryophyllus L.. Mycorrhiza News, 14:13-15. 16) Witham, F.H., D.F. Blaydes and R. M.Devlin, 1971. Experimen- 31) Earanna, N., D.J. Bagyaraj and A.A. Farooqi, 2003. Response of tal Plant Physiology. Van Nostrand, New York, pp. 245. Phyllanthus amarus to inoculation with Glomus fasciculatum 17) Goodwin, T.W., 1954. Carotenoids. In: Hand Books of Plant and plant growth promoting rhizomicroorganisms. Geobios, 30: Analysis [K. Peach and M. V. Traccy (eds.)], pp. 272-311. 183-187. 18) Lowry, O.H., N.J. Rose Brough, A.L. Farr and R.J. Randall, 1951. 32) Krishna, K.R. and D.J. Bagyaraj, 1983. Interaction between Glo- Protein estimation with folin-phenol reagent. J. Biol. Chem., 193: mus fasciculatum and Sclerotium rolfsii in peanut, Arachis hypo- 265-275. gea. Can. J. Bot., 61: 2349-2351. 19) Troll, W. and K. Canon, 1953. A modified phytometric Ninhy- 33) Dumas, E., V. Gianinazzi-Pearson and S. Gianinazzi, 1989. Pro- drin method for the analysis of amino-imino acids. J. Biol. Chem., duction of new soluble proteins during VA-endo-mycorrhiza for- 200: 803-811. mation. Agric. Ecosys. Environ., 29: 111-114. 20) Farkas, G.L. and Z. Kiraly, 1962. Role of phenolic compounds in 34) Schachtman D.P., R.J. Reid and S.M. Ayling, 1998. Phosphorus the physiology of plant disease and disease resistance. uptake by plants: from soil to cell. Pl. Physiol. 116: 447-453. Phytopathol., 2: 105-150. 35) Allen, M.F., W.K. Smith, Jr. T.S. Moore and M. Christensen, 21) Sato, N. and Murata, 1988. Membrane lipids. In: Methods in 1981. Comparative water relations and photosynthesis of myc- Enzymology L. [Packer and A.N. Glazer (eds.)], pp. 251-259. orrhizal and non-mycorrhizal Bouteoua gracilis H.B.K. New Phy- 22) Harborne, J.B., 1999. Phytochemical methods, 3rd edn. Chapman tol., 88: 683-693. and Hall, Ltd. London. pp: 86-112. 36) Elango, K.V., 2004. Studies on the effect of native AM fungi and 23) Bohm, B.A. and R. Kocipai Abyazan, 1994. Flavonoids and plant growth promoting rhizomicroorganisms on growth and pro- condensed tannin from leaves of Hawaiian Vaccinium vaticulatum ductivity of Gloriosa superba L.. Ph. D. Thesis, Bharathidasan and V. calycinium. Pacific Sci., 48: 458-463. University, Tiruchirapalli, India. 24) Obadoni. B.O. and P.O. Ochuko, 2001. Phytochemical studies 37) Mahadevan, A., 1991. Post infectional defence mechanisms, To- and comparative efficacy of the crude extracts of some Homostatic day and Tomorrow Publishers, New Delhi. plants in Edo and Delta States of Nigeria. Global J. Pure Appl. 38) Gasper, M.L., R. Pollero and M. Cabello, 1997. Variations in Sci., 8:203-208. lipid composition of alfalfa roots during colonization with 25) Lakshman, H.C., 1992. Development and response of vesicular arbuscular mycorrhizal fungus Glomus versiforme. Mycologia, arbuscular mycorrhizal fungi in Terminalia bellirica Roxb. J. Trop. 89: 37-42. For., 8: 179-182. 39) Hemalatha, M., 2002. Synergistic effect of VA-Mycorrhizae and 26) Krishna Naik, L., N. Jayasheela, A.A. Farooqui and D.J. Bagyaraj, Azospirillum on the growth and productivity of some medicinal 1998. Influence of Glomus fasciculatum, Bacillus megaterium plants. Ph.D. Thesis, Bharathidasan University, Tiruchirapalli, and phosphorus on growth and oil yield of Citronella java pp. 108. (Cymbopogon winterianus). J. Soil Biol. Ecol., 18: 17-22. 40) Allen, M.F., T.S. Jr. Moore and M. Christensen, 1980. Phyto- 27) Raj, J., D.J. Bagyaraj and A. Manjunath, 1981. Influence of soil hormone changes in Bouteloua gracilis infected by vesicular- inoculation with vesicular arbuscular mycorrhiza and a phos- arbuscular mycorrhizae –I.. Cytokinin increases in the host plant. phate dissolving bacterium on plant growth and P uptake. Soil Can. J. Bot., 58: 371-374. Biol. Biochem., 13: 105-108. 41) Sreenivasa, M.N. and D.J. Bagyaraj, 1988. Suitable level of zinc, 28) Bagyaraj, D.J., 1992. Vesicular-arbuscular mycorrhiza: Applica- copper and manganese for mass production of VAM fungi Glo- tion in Agriculture, In: Methods in Microbiology, 24: 359-373. mus fasciculatum. In: Proc. Ind. Acad. Sci. (Plant Sci.). 98: 135- 29) Sundar, S.K., A. Palavesam and B. Parthipan .2011. AM fungal 138. diversity in selected medicinal plants of Kanykumari District , 42) Raman, N. and A. Mahadevan, 1996. Mycorrhizal Research - a Tamil Nadu, India,51(3):259-265 priority in Agriculture. In: Concepts in Mycorrhizal Research 30) Anusuya, D. and K. Senthilkumar, 2003. Mutualistic symbiosis [K.G. Mukerji (ed.)], pp. 41-75.

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Journal of Pharmacy Research Vol.5 Issue 8.August 2012 3990-3993