Amutha and Kokila, IJALS, Volume (7) Issue (2) May RESEARCH ARTICLE

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1 Effect of on symbiotic association of Glomus aggregatum an Arbuscular Mycorrhizal Fungus K. Amutha and V. Kokila Department of Biotechnology, Vels University, Pallavaram, Chennai, Tamilnadu, India amutharavi40@gmail.com Corresponding Author K. Amutha Department of Biotechnology, Vels University, Pallavaram, Chennai, Tamilnadu, India amutharavi40@gmail.com Article History Received on 10 February, 2014; Received in revised form 18 March, 2014; Accepted 02 May, 2014 Abstract Interactions between as biocontrol agent and Glomus aggregatum, an Arbuscular mycorrhizal fungus (AMF) were studied. In all the experiments, the association of Glomus aggregatum in Allium cepa with was studied. Mycorrhizal activity was assessed by calculating the percentage of association of AMF with Allium cepa. The symbiotic activity of Glomus aggregatum depends on the presence of. Extra and intracellular proteins of tested on the symbiotic association of Glomus aggregatum were analyzed. Keywords : Glomus aggregatum, Allium cepa, and Extracellular protein Introduction Arbuscular mycorrhizal fungi (AMF) can have a large influence on plant growth by transfer of nutrients and protection of plant against pathogens (Davis et al., 1986). AMF are able to colonize the plant roots and helps in taking up nutrients such as phosphorus from the soil. It has other roles such as soil aggregation, protection of plant against drought stress and soil pathogens and increasing diversity which can be reduced by AMF (Borowicz, 2000). The effectiveness of AMF in biocontrol are dependent on the AM fungal involved as well as the substrate and host plant (Allium cepa). Pseudomonas is a better biocontrol agent (Cook, 1993). Extra and intracellular proteins of were isolated and tested on symbiotic associations of Glomus aggregatum. Proteins of Pseudomonas were identified as a key determinant for root colonization (Yousef - Coronado et al., 2008). Soil microorganism can be used to decrease the input of pesticides, fertilizers and chemicals. Pseudomonas are reported to be good root colonizers with the host plant by the formation of the nodules during endosymbiosis and attachment of bacteria to AM fungal hyphae surfaces change in fungal growth (Sen et al., 1996). Beneficial interaction of plant microbe with AMF in the rhizosphere helps for the growth of the plant and makes the soil fertile. Materials and methods The mixture of roots and rhizosphere soils, clay soil at Maduravoyal near Chennai were collected. Spores were extracted from the collected soil samples by wet sieving and decanting method (Gerdemann Int. J. Adv. Lif. Sci., Available online on at www. ijals.com Page 236

[ The International Journal of Advanced Life Sciences (IJALS) and Nicolson, 1963). AMF spores were identified by Morton and Msiska (1988).

After 12 days, roots of Allium cepa were stained in tryphan blue. A percentage of mycorrhizal colonization in the roots was assessed by the gridline-intersect method (Giovannett and Mosse, 1980).

The Pseudomonas aeruginosa were identified based on physical characterization - Gram staining and the biochemical tests - Catalase test, Oxidase test, Methyl red test, Indole test, Nitrate reduction

Isolation of protein from Extracellular protein Extracellular proteins were isolated by Hirose et al. (2000) (Trichloroacetic acid [TCA] / Methanol) precipitation method.

2 [ The International Journal of Advanced Life Sciences (IJALS) and Nicolson, 1963). AMF spores were identified by Morton and Msiska (1988). The total spore count was calculated by counting the number of spores per kg of soil. The spores were multiplied by using trap culture with Allium cepa as host plant. After 12 days, roots of Allium cepa were stained in tryphan blue. A percentage of mycorrhizal colonization in the roots was assessed by the gridline-intersect method (Giovannett and Mosse, 1980). Isolation of from soil samples were isolated from soil samples by the serial dilution method. The Pseudomonas aeruginosa were identified based on physical characterization - Gram staining and the biochemical tests - Catalase test, Oxidase test, Methyl red test, Indole test, Nitrate reduction test, Citrate utilization test outlined in Bergey s manual of determinative bacteriology (Williams and Wilkins. 1994). Pseudomonas aeruginosa were grown in Pseudomonas agar base medium. Isolation of protein from Extracellular protein Extracellular proteins were isolated by Hirose et al. (2000) (Trichloroacetic acid [TCA] / Methanol) precipitation method. Intracellular protein Intracellular proteins of were isolated by the modified method of Jarvis et al. (2003). Protein profile of Glomus aggregatum associated with Allium cepa as host plant isolated extra and intracellular proteins were introduced to trap cultures of Glomus aggregatum. Two weeks after mycorrhizal and non-mycorrhizal protein profile were analysed by estimating proteins by Lowry et al. (1951) and by SDS page analyses (Laemmli et al., 1970). Table - 1. Colony Morphology, Physical and Biochemical characterization of bacterial isolates from soil samples S. NO Cultural Characteristics Bacterial isolates Small, Pigmented circular, Flat, Entire, dry colonies 1. Colony Morphology 2. Gram s Staining Gram negative 3. Motility Active Mobile 4. Catalase + 5. Oxidase + 6. Methyl Red - 7. Indole + 8. Citrate Test + 9. Nitrate Test + + Positive; Negative Fig. 1. Spores of Glomus aggregatum Fig. 2. isolates from soil samples Int. J. Adv. Lif. Sci., Available online on at www. ijals.com Page 237

Table 2: Protein content of Control and Test plant Roots Trap plants Protein

2 c T 1 1 T 2 T 3 Fig 3: Trap Culture Allium cepa C - Control; (b) T 1 - Glomus

treated with extracellular protein of (d)t 3 - Trap culture of Glomus aggregatum

cepa Trap culture of Glomus aggregatum treated with extracellular protein of Trap

Arbuscules (40x) ; ( b) Glomus aggregatum associated with Allium cepa root (100x)

Glomus aggregatum treated with intracellular protein of root (40x) Fig 4. e.

Results and Discussion AMF spore were collected from Maduravoyal near Chennai

test as outlined in the Bergey s manual of determinative bacteriology the

3 Table 2: Protein content of Control and Test plant Roots Trap plants Protein (mg/100ml) Control plant 1.2 c T 1 1 T 2 T 3 Fig 3: Trap Culture Allium cepa C - Control; (b) T 1 - Glomus aggregatum associated with Allium cepa (c) T 2 Trap culture of Glomus aggregatum treated with extracellular protein of (d)t 3 - Trap culture of Glomus aggregatum treated with intracellular protein of Glomus aggregatum associated with Allium cepa Trap culture of Glomus aggregatum treated with extracellular protein of Trap culture of Glomus aggregatum treated with intracellular protein of a b c d Fig 4. Glomus aggregatum associated with Allium cepa root stained in tryphan blue (a) Arbuscules (40x) ; ( b) Glomus aggregatum associated with Allium cepa root (100x) ; c) Glomus aggregatum treated with extracellular protein of root (40x); (d) Glomus aggregatum treated with intracellular protein of root (40x) Fig 4. e. Glomus aggregatum associated with Allium cepa root stained in tryphan blue e Results and Discussion AMF spore were collected from Maduravoyal near Chennai (per 1 kg of soil). Glomus aggregatum spores were identified (Fig. -1.) ( With reference to colonial morphology, physical characterization and biochemical test as outlined in the Bergey s manual of determinative bacteriology the bacteria were identified as and the results are presented in Table - 1 and Fig Glomus aggregatum trap cultures were developed in Glass house conditions (Fig. - 3). Tryphan blue stained arbuscules, vesicles, and hyphae were observed in trap Int. J. Adv. Lif. Sci., Available online on at www. ijals.com Page 238

100% 80% 60% 40% 20% 0% S.NO 1 S.NO 2 S.NO 3 S.NO 4 L1 L2 L3 L4 L5 kda 66 43 29 20 Fig. 5. Percentage of association of Glomus aggregatum with Allium cepa S.No 1- Control (b) S.

4 100% 80% 60% 40% 20% 0% S.NO 1 S.NO 2 S.NO 3 S.NO 4 L1 L2 L3 L4 L5 kda Fig. 5. Percentage of association of Glomus aggregatum with Allium cepa S.No 1- Control (b) S.No 2- Glomus aggregatum associated with Allium cepa root (c) S.No 3 - Trap culture of Glomus aggregatum treated with extracellular protein of (d) S.No 4 - Trap culture of Glomus aggregatum treated with intracellular protein of Pseudomonas aeruginosa. [roots of Allium cepa (Fig. - 4) and the percentage of Glomus aggregatum association was observed (Fig. - 5). Proteins isolated from extra and intracellular proteins of treated with Glomus aggregatum associated plants. Protein concentration were calculated and expressed in mg/100 ml and the results are shown in Table 2 and Fig.- 6. In the control root, the protein band on SDS PAGE gel found to be 58kDa. Similarly the size of the protein from Glomus aggregatum associated roots, the proteins from extra and intracellular treated proteins of Pseudomonas aeruginosa were 58 kda, 64 kda and 41 kda respectively. In this study, Glomus aggrgatum has shown very good association with extracellular proteins of. Pseudomonas produce an antifungal compounds, but it is not to be active against AM fungi (Burla et al., 1996). Combinations of AMF associated with bacteria are essential living components of the soil microbiota (Dharma Parkash Bharadwaj al., 2008). This shows a mutalistic relationship between Pseudomonas and Glomus aggrgatum. et Fig. 6. Protein profiling of Control and Test Plant Roots by SDS-PAGE L1 - Control Plant Root Protein of Allium cepa ; L2 - Glomus aggregatum associated with Allium cepa; L3 - Proteins from treated with extracellular protein of ; L4- Protein Marker e) L5 - Proteins from treated with intracellular protein of. Acknowledgement This research was supported by Vels University Research grant. References Bharadwaj, D.P., Lundquist, P.O. and Alstrom, S Arbuscular mycorrhizal fungus sporeassociated bacteria affect mycorrhizal colonization, Plant growth and Potato pathogens. Soil Biol Biochem., 40 : Borowicz, V.A Do Arbuscular Mycorrhizal Fungi Alter Plant Pathogenic Relations? Ecology., 82: Burla, M., Goverde, M., Schwinn, F.J. and Wiemken, A Influence of biocontrol organisms on root pathogenic fungi and on plant symbiotic microorganisms Rhizobium Phaseoli and Glomus mosseae. J. Plant. Dis. Protect., 103: Cook, R.J Making greater use of introduced microorganisms for biological control of pathogens. Annu Rev Phytopathol., 31: Int. J. Adv. Lif. Sci., Available online on at www. ijals.com Page 239

Davis, R.E., Mayo, K. and Motto, J. 1986. Stimulation of germination of spores of Glomus versiforme by spore-associated bacteria. Mycologia., 78 : 426-431. Gerdemann, J.W. and Nicolson, T.H.1963.

5 Davis, R.E., Mayo, K. and Motto, J Stimulation of germination of spores of Glomus versiforme by spore-associated bacteria. Mycologia., 78 : Gerdemann, J.W. and Nicolson, T.H Spores of mycorrhizal Endogone species extracted from soil by Wet sieving and decanting. Trans. Brit. Mycol. Soc., 46 : Giovannett, M. and Mosse, B An evaluation of techniques of measuring vesicular arbuscular mycorrhizal infection in roots. New phytol., 84 : Hirose, I., Sano, K., Shioda, I., Kumano, I., Nakamura, K and Yamane, K Proteome analysis of Bacillus Subtilis extracellular proteins: a two dimensional electrophoretic study. Microbiology., 146 : Jarvis, B.W Epicentre Forum. 10 (3), 4. Laemmli, U.K Clevage of structural proteins during the assembly the head of the bacteriophage T4. Nature., 227 : Lowry, O.H., Roserough, N.J., Farr, A.L and Randbell, R.J Protein measurement with the Folin Phenol reagent. J.Biol Chem.,193 : Morton, J.B. and Msiska, Z Phylogenic from genetic and morphological characters do not support a revision of Gigasporaceae (Glomero - mycota) into four families and five genera. Mycorrhiza., 20 : Sen, R., Nurmiaho Lassila., Haahtela, K. and Korhonen, K Specifity and mode of primary attachment of Pseudomonas fluorescens strains to the cell walls of ectomycorrhizal fungi. New phytol., 145 : Williams and Wilkins th Edition. Bergey s Manual of determinative bacteriology. Yousef- Coronado, F., Travieso, M.L and Espinosa- Urgel, M Different overlapping mechanisms for colonization of abiotic and plant surfaces by pseudomonas putida. FEMS Microbiol. Lett., 288 : Corresponding Author : K. Amutha, Department of Biotechnology, Vels University, Pallavaram, Chennai, Tamilnadu, India, amutharavi40@gmail.com, 2014, IJALS. All Rights Reserved. Int. J. Adv. Lif. Sci., Available online on at www. ijals.com Page 240

Wantira Ranabuht Department of Botany, Faculty of Science Chulalongkorn University

EFFECTS OF ARBUSCULAR MYCORRHIZAL FUNGI ON GROWTH AND PRODUCTIVITY OF LETTUCE Wantira Ranabuht Department of Botany, Faculty of Science Chulalongkorn University Lettuce Lettuce : Lactuca sativa L. Family