Efficiency of Tricalcium Phosphate Solubilization by Two Different Endophytic Penicillium sp. Isolated from Tea (Camellia sinensis L.).

Transcription

1 Available online at European Journal of Experimental Biology, 2012, 2 (4): ISSN: CODEN (USA): EJEBAU Efficiency of Tricalcium hosphate Solubilization by Two Different Endophytic enicillium sp. Isolated from Tea (Camellia sinensis L.). Ratul Nath 1, G. D. Sharma 1 and Madhumita Barooah* 2 1 Dept. of Life Science & Bioinformatics, Assam University, Silchar, Assam. 2 Dept. of Agricultural Biotechnology, Assam Agricultural University, Assam. ABSTRACT Microorganisms present within the intercellular spaces of plant parts, enhancing plant growth and development, are referred as endophytes. They are the endosymbionts without showing any visible symptoms of their presence. In this paper we compared the tri calcium phosphate solubilizing activity of two different endophytic enicillium species isolated from tea leaves. Both the isolates showed remarkable phosphate solubilizing activity up to eight days with an increase of acidity of the medium. Tri-calcium phosphate solubilizing activity of species1 ranged between 39.22±1.17 to 86.10±1.20µg/ml while that of species 2 varied between 32.57±1.41 to 84.25±1.5 µg/ml with in a span of 2 to 10 days. The of the medium decreased from 6.25±0.26 to 3.22±0.22 and 6.22±0.45 to 3.54±0. 35 for species1 and species2 respectively within the same peroid. The isolates showed good phosphate solubilizing ability and thus have potential to be used as biofertilizer agents. Key words: Endophytic microorganisms, hosphate solubilizer, Biofertilizer. INTRODUCTION hosphorus is an important mineral nutrient required for proper growth and development of plants. Deficiency of phosphorus is one of the important chemical factors restricting plant growth. hosphorus contributes remarkably to photosynthesis, energy and sugar production, nucleic acid synthesis, and also promotes N 2 fixation in legumes [1]. Though soil constitutes 0.5% phosphorus, only a minute amount is available for plant absorption, others remain as insoluble salts and cannot be absorbed by plants [2]. Besides this, a large portion of chemical fertilizers with high phosphorus content applied to soil is immobilized rapidly and becomes unavailable to plants [3]. Chemical fertilizers with readily available, when applied to soil with acidic are immediately converted to insoluble salts which comprises 95-99% of total phosphorus [4]. To minimize the phosphorus deficiency, phosphate fertilizers are repeatedly applied to agricultural fields. This leads to the loss of soil fertility and disturbs the normal microbial flora of soil and consequently reduces crop yield. The insoluble salts in the soil may be in the form of tri calcium phosphate, ferric phosphate, aluminium phosphate [5]. Some microorganisms associated with different pant rhizosphere are able to solubilise inorganic insoluble salts. seudomonas and Bacillus are two important genera of soil bacteria with promising activity of phosphate solubilisation [6]. Some strains of Rhizobium are also reported for their high phosphate solubilising activity [7]. Filamentous fungi are frequently used in laboratories to solubilise insoluble phosphate salts. enicillium and Aspergillus are two important genera frequently used for phosphate solubilization [8, 9, 10]. Ability of solubilization of inorganic insoluble phosphate salts by different microorganisms depends on their ability to produce and release organic acids to their respective environments. These organic acids 1354

2 decrease soil or any medial [9]), providing the facility to exchange the metal part of insoluble phosphates to potassium or sodium, resulting the formation of soluble phosphate salts. Inoculation of phosphate solubilising microorganisms to soil is a reliable technique for increasing soluble in soil [11]. revious studies have revealed that inoculation of phosphate solubilising microorganisms viz., enicillium, Enterobacter in soil significantly promote the growth and yield of several important crops like wheat [12], onion [13], alfalfa [14] and soybean [7]. Endophytic microorganisms are those microbes present within the intercellular spaces of plant parts without showing any visible symptoms of their presence and hence are less detrimental to their hosts [15, 16]. These endophytic microorganisms may enhance plant growth in various dimensions, for example, secretion of plant growth hormones e.g. indole acetic acid [17], via phosphate solubilization [18], production of siderophores [19], by enhancing hyphal growth and micorrhizal colonization [20] and by supplying biologically fixed nitrogen [21]. In addition endophytic microbes supply essential vitamins to plants [23]. Camellia sinensis, a major commercial crop of Assam grows well in acidic soil. Being commercial crop, planters apply high inputs of chemical fertilizers including phosphate fertilizers. Unfortunately, acidic soil condition renders most of the phosphate fertilizers unavailable due to immobilization leading to inefficient fertilizer uptake. Being an inherent part of plant system endophytic microbes may promote better utilization of phosphate fertilizers without detrimental effects on the plant. MATERIALS AND METHODS Isolation of endophytic fungi from tea leaves Thoroughly washed fresh tea leaves were subjected to surface sterilization by keeping it in 70% ethanol and then into 0.05% HgCl 2 solution for 3 and 2 mins respectively. Repeated washing with sterilized distilled water was done to remove the surface sterilizing agents. Tissue homogenate was prepared by crushing them with the help of autoclaved mortar and pastel, suspended in sterilized distilled water for serial dilution and spreaded on DA plates. lates were incubated at 28 0 C for one week [23]. Screening of the isolates for hosphate solubilization Fungal spores were inoculated to ikovskaya s agar medium and inoculated at 28 0 C for proper hyphal growth. Clear zones around the colonies indicated the capacity of phosphate solubilization [24]. Quantitative estimation of tri calcium phosphate solubilization Quantitative estimation of phosphate solubilization in broth culture was performed according to the procedure of Jackson (1973) [25]. One ml of fungal spore suspension was inoculated to 50 ml of ikovskya s broth in 150 ml conical flasks. Cultures were incubated at 28 0 C for eight days in a shaking incubator. Two ml of two, four, six and eight day s old cultures were centrifuged at 10,000 rpm for 10 mins. One ml of this supernatant was mixed with 10 ml of chloromolybdic acid and the volume was adjusted to 40 ml with distilled water. To this, 1 ml of chlorostannous acid was added and the volume was made 50 ml with distilled water. Absorbance of the developing blue colour was measured at 600 nm wave length with UV- VIS spectrophotometer (CECIL CE 7250). The amount of soluble phosphate was calculated from standard curve of KH 2 O 4. Estimation of culture eriodic estimation of culture was performed by using digital meter (EUTECH pc 510). Table 1: solubilization and change of. Isolases enicillium sp. 1 enicillium sp. 2 available in µg/ml and 2 days 4 days 6 days 8 days 10 days 39.22± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.37 Values are mean ± SD of five replicas. RESULTS This paper reports the efficiency of two endosymbiotic enicillium species isolated from tea leaves for their phosphate solubilizing activity. As shown in table 1, solubilization increased from day 2 to day 8 with a value 1355

3 ranging from 39.22±1.17 to 86.10±1.20 µg/ml for species 1 and between 32.57±1.4 to 84.25±1.5 µg/ml for species 2 and began to decrease slowly. Decrease of of the medium was noticed up to 8 days ranging from 6.25±0.26 to 3.22±0.22 for species 1 and from 6.22±0.45 to 3.54±0.35 for species 2 with an increase of soluble. After 8 days of incubation, of the medium was noticed to be increased slowly with a decrease of soluble. Fig: Trends in phosphate solubilization and change of by enicillium sp. 1. Fig: Trends in phosphate solubilization and change of by enicillium sp. 2. DISCUSSION Among different microbial groups, fungi have been reported to be more efficient phosphate solubilizer in comparison to bacteria [26]. This may be due to their ability of easy penetration through soil and other substrates 1356

4 and secretion of different chemicals responsible for solubilization. Out of different endophytic fungal isolates currently under investigation in our laboratory, two different enicillium species showed significant zone of phosphate solubilization on solid ikovskaya s medium supplemented with insoluble TC. eriodic estimation of available in the medium revealed the potential of the isolates in releasing from insoluble phosphate sources. Results of the present experiment revealed that the decrease in the of the medium that ranged between 6.25±0.26 to 3.22±0.22 was associated with increased amount of available that showed a value between 32.57±1.4 to 86.10±1.20 µg/ml in the medium. Several earlier studies have reported tat solubilizing activity is determined by the production of organic acids, which chelate cations through their carboxylic acid group and convert it into soluble form [27]. Results of the present study is supported by the observation of Gaur (1990)[24] on seudomonas striata, where he observed gradual increase of available and acidity of the medium up to certain period and then drop of available level with an increase of. roduction of organic acids by phosphate solubilizing microbes has been well documented (6, 27, 28, 29). Among them, gluconic acid is regarded as the most frequent agent of mineral phosphate solubilization. It is reported as the principal organic acid produced by seudomonas sp. [6], Erwinia sp [28] and in some enicillium sp.[29]. Ketogluteric acid is also reported as one of the major organic acids present in different phosphate solubilizing microbes [30, 31]. Besides these two organic acids, glycolic, malonic, oxalic and succinic acids were also identified among phosphate solubilizers [6, 32]. Gaind and Gaur (1989) [33] reported that the decrease in available after a period might be due to the deficiency in nutrients in the culture medium or refixation of solubilized with metal ions present in the broth.. It is also reported that drop of is not the sole determining factor of phosphate solubilization, besides it, solubilization is highly dependent on the nature of organic acids secreted by microbial cells [34]. roduction of gluconic acid in different enicillium sp. including. rugulosum,. notatum and. chrysogenum was found to very common [29]. Cause of increase of of the medium after a certain period can be explained as the catabolic activity of fungi on organic acids as reported earlier in. chrysogenum [35]. In presence of sucrose (disaccharide) as the sole source of carbon, enicellium sp. secrete both citric acid and gluconic acid for phosphate solubilization [36]. From the results of the experiment as well as from earlier studies it is observed that production of organic acid is the major mechanism of solubilization by phosphate solubilising fungi. CONCLUSION Biofertilizers have acquired importance during the recent years as an important input of the organic agriculture. In the light of the above statement, isolation of plant growth promoting microorganisms from novel sources has importance for development of efficient biofertilizers agents. Application of phosphate solubilizing microbes as sole or in combination of other biofertilizer expected enhance proper nutrient uptake by the crop plants, reduce the over application of phosphate fertilizers. This in turn will open a new vista in organic agriculture. Acknowledgement Authors are thankful to all the members of Department of Agricultural Biotechnology, Assam Agricultural University, Jorhat, Assam and Department of Life sciences and Bioinformatics, Assam University, Silchar, Assam, for their cooperation and suggestions. Authors are also grateful to DST India for providing the INSIRE fellowship to the scholar. REFERENCES [1] K. Saber, L. Nahla, D. Ahmed, A. Chedly, Agron. Sust. Develop, 2005, 25, [2] H. Rodriguez, R. Fraga, Biotechnol. Adv. 1999, 17, [3] A. H. Goldstein, Am. J. Altern. Agricult. 1986, 1, [4] D.S. Hayman, In: Soil Microbiology (Ed. Walker, N.). Butterworth Scientific ublications, London and Boston, 1975, [5] W.L. Lindsay,.L.G. Vlek, S.H. Chien, In: Dixon JB, Weed SB, eds. Soil environment. 2nd ed. Madison (WI): Soil Science Society of America. 1989, [6]. Illmer, and F. Schinner, Soil Biol. Biochem. 1992, 24, [7] M.H. Abd-Alla, Letters of Applied Microbiol. 1994, 18, [8] S.A. Omar, World Jour. of Microbiol. Biotechnol. 1998, 14, [9] S. Seshadri, S. Ignaimuthu, C. Lakshminarasimhan, Chem. Engg. Comm. 2004, 191, [10] S.A. Wakelin, R.A. Warren,.R. Harvey and M.H. Ryder, Biol. Fertil. Soils. 2004, 40, [11] I. Reyes, L. Bernier, and H. Antoun, Microb. Ecol. 2002, 44, [12] M.A Whitelaw, T.J. Harden and G.L. Bender, Austral. Jour. Soil. Resch. 1997, 35, [13] N. Vassilev, M. Toro, M. Vassileva, R. Azcón and J.M. Barea, Bioresource Technol. 1997, 61, [14] R. Rodríguez, N. Vassilev, and R. Azcón, Appl. Soil. Ecol. 1999, 11,

5 [15] D. Wilson, Oikos. 1993, 68: [16] K. Saikkonen, M. Helander and S.H. Faeth, In: lant microbiology, Fungal endophytes: hitchhikers of the green world. M. Gillings and A. Holmes, eds. Oxford: BIOS Scientific ublishers, 2004, [17] S. Lee, M. Flores-Encarnación, M. Contreras-Zentella, L. Garcia-Flores, J. E. Escamilla and C. Kennedy, Jour. Bacteriol. 2004, 186, [18] S. A. Wakelin, R. A. Warren,. R. Harvey and M. H. Ryder Biol. Fertil. Soils 40, [19] J.M. Costa and J. E. Loper, Mol. lant-microbe Interact. 1994, 7, [20] M.E. Will, D.M. Sylvia, Appl Environ Microbiol. 1990, 56, [21] E.K. James, V. M. Reis, F. L. Olivares, J. I. Baldani, J. Dobereiner, Jour. Exp. Bot. 1994, 45, [22] B. Rodelas, V. Salmeron, M. V. Martinez-Toledo, J. Gonzalez-Lepz, lant. Soil. 1993, 153, [23] N. Tejera, E. Ortega, R. Rodes and C. Jour. lant. hysi. 2006,163, [24] A. C. Gour, In: A.C. Gour, Eds. phosphate solubilizing micro-organisms as biofertilizers. Omega scientific publishers. New Delhi, 1990, [25] M. L, Jackson. Soil chemical analysis. rintice hall of India, New delhi, 1973, 392. [26] E. Nahas, World J. Microbiol. Biotechnol.1996, 12, [27] K. Kpomblekou and M. A. Tabatabai, Soil Science, 1994, 158, [28] T. S. Liu, L.Y. Lee, C.Y. Tai, C.h. Hung, Y. S Chang, J. H. wolfram, R. Rogers and A.H Goldstain, J. bacterial, 1992, 174, [29] K.Y. Kim, D. Jordan, and H.B Krishnan, FEMS Microbiol. Lett. 1997, 153, [30] A.K. halder, A.K. Mishra, Bhattacharyya and.k. Chakrabartty, J. Gen. Appl Microbiol. 1990, 36, [31] A.K. halder and.k. Chakrabartty, Folia Microbiol.1993, 38, [32] S. Banik and B.K. Dey, lant Soil. 1982, 69, [33] Gaind, S. and Gaur A.C. Curr. Sci. 1989, 58, [34] M.C Bardiya,. and A.C Gaur.. Indian J. Microbiol. 1972, 12, [35] J. Nielsen, C.L. Johansen and J. Villadsen, J. Biotechnol. 1994, 38, [36] I. Reyes, L. Bernier, R.R. Simard and H. Antoun, FEMS Microbiol. Ecolo. 1999, 28,