Legume Res.

, 33 (1) : 114 - 118, 2010

AGRICULTURAL RESEARCH COMMUNICATION CENTRE

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STUDIES ON PHOSPHATASE ACTIVITY AND CLUSTERBEAN PRODUCTION AS INFLUENCED BY THE P MOBILIZING ORGANISM EMERICELLA RUGULOSA
B.K. Yadav* and J.C. Tarafdar
Central Arid Zone Research Institute, Jodhpur- 342003, India

ABSTRACT
Phosphorus is one of the major plant growth limiting nutrients although it is abundant in soils in both inorganic and organic forms. In soils the hydrolysis of organic P is predominantly mediated by the activity of phosphatases, such as acid and alkaline phosphatases and phytase released by plant roots and microorganisms. In order to evaluate the effect of Emericella rugulosa on soil enzyme activities and crop yield, an experiment was conducted under green house conditions and field (clusterbean as a test crop). A significantly higher enzyme activities and improvement in plant biomass, root length and P content was observed in sterilized soil mainly due to increased population of Emericella rugulosa. The test plants influenced acid phosphatase and phytase activity but resulted in no significant increase in alkaline phosphatase activity in the inoculated soil. The depletion of organic P was much higher than mineral and phytin P The microbial . contribution was significantly (p=0.05) higher than the plant contribution to the hydrolysis of the different P fractions. A significant (p=0.05) improvement in seed and straw yield and P concentration of seed and straw resulted from inoculation. The results suggested that Emericella rugulosa produces phosphatases and phytase, which mobilize P from native P sources and enhance the production of clusterbean in an arid soil.

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Key words: Clusterbean, Emericella rugulosa, Phytase, P-mobilization. INTRODUCTION Clusterbean (Cyamopsis tetragonoloba (L.) Taub.) is the most important multipurpose legume crop in arid and semi-arid regions of India. The grain yield of the crop in arid soils is low because of low and erratic rainfall distribution and poor soil conditions besides the low availability of various nutrients, especially phosphorus. About 20- 25% of total phosphorus in the arid soils of Western Rajasthan (India) is organic in nature and 68% organic phosphorus in the soil is present as phytin (Tarafdar and Gharu, 2005; Yadav and Tarafdar, 2007). In order to become available to plants, these P compounds must be hydrolyzed by phosphatases, which are of plant roots (Yadav and Tarafdar, 2001) and microbial origin (Yadav and Tarafdar, 2003). Yadav and Tarafdar (2003) reported that fungal isolates differed in their abilities to hydrolyze different organic P compounds. Tarafdar and Gharu (2005) and Yadav and Tarafdar (2007) demonstrated the improvement of plant available P by applying microbial inoculants containing phosphatase producing fungi, resulting in higher yields of pearl millet. However, no information was reported on the usefulness of phosphatase producing fungi on clusterbean. Hence, an attempt has been made to examine the effect of Emericella rugulosa on growth and nutrition of clusterbean under both green house and field conditions. MATERIAL AND METHODS The soil used under the pot and field study was loamy sand (hypothermic typic haplocambids) with pH-8.2, EC-0.18, total P- 1261 mg kg-1, organic P- 367 mg kg -1 , available P- 10 mg kg -1 . The experiment was carried out with four treatments: sterilized soil with plants, sterilized soil without plants, non-sterilized soil with plants and non-sterilized soil without plants. Soil was sterilized by autoclaving on

*Present Address : Deptt. of Agril. Chemistry & Soil Science, Rajasthan College of Agri. M.P .U.A.T., Udaipur. Email : bkyadav74@yahoo.co.in

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3 alternate days at 1.05 kg cm-2 pressure and 105oC for 2 h. Each pot received 5 kg sterilized or nonsterilized soils. Clusterbean seeds (cv, RGC- 936) were inoculated by the method as described by Yadav and Tarafdar (2007). Twelve pots were used for each treatment with three pots harvested at each times. Ten seeds of clusterbean were sown in each pot and five plants were maintained after germination. Water was added at 50% water holding capacity (WHC) of the soil and watered every second day. The pots were completely randomized and repositioned weekly to minimize any effect of uneven environmental factors. The sampling was done after 7, 14, 21 and 28 days. Clusterbean (cv, RGC -936) was cultivated under rainfed condition during Kharif 2004 at Central Research Farm, CAZRI, Jodhpur, to evaluate the performance of Emericella rugulosa under field condition. A randomized block design with three replications was used. There were two treatments; with inoculation and without inoculation. The plot size of each replicate was 5 4 m. Clusterbean seeds were inoculated with Emericella rugulosa before sowing in inoculated treatment. Crops were grown under rainfed condition. Four plants of each replicate with intact roots were carefully freed from soil at 4, 5, 6, 7 and 8 weeks after germination. We harvested the crops after maturity (9 weeks). After each harvest, roots were thoroughly washed, separated from the soil and the root lengths were measured using a modified line-intersect method of Tennant (1975). Acid and alkaline phosphatases were assayed by adopting the standard procedure of Tabatabai and Bremner(1969) using acetate buffer (pH 5.4) and sodium tetraborteNaOH buffer (pH 9.4), respectively. Phytase activity was assayed by measuring the amount of inorganic phosphate (Pi) released by hydrolysis of sodium
Plant age (days) Acid Phosphatase ( EU x 10-4) Days after inoculation SS 7 14 21 28 LSD (p=0.05) 0.25 0.30 0.39 0.42 0.03 SS(P) 0.35 0.41 0.50 0.52 0.02 NSS NSS(P) 0.13 0.15 0.17 0.21 0.02 0.18 0.21 0.24 0.31 0.02 SS 0.43 0.58 0.81 0.94 0.12

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phytate (1mM) in 100M sodium acetate buffer (pH 4.5) and incubated at 37oC for 1h (Ames, 1966). Mineral, total and organic P was estimated as described by Seeling and Jungk (1996).The phytin P was estimated by extraction of phytate with 15% CCl3-COOH (trichloro-acetic acid) as described by Mega (1982). The microbial contribution to P hydrolysis was defined as the mineral, organic and phytin P depleted from the pots due to inoculation of Emericella rugulosa in pots without plants. The plant contribution was defined as the additional depletion of different forms of unavailable P after introduction of plants to inoculated pots. The pH (1:2), EC (1:2), partical size distribution and available P (Olsen s) using standard methods (Jackson, 1967).The P-content of the plant was determined using the vanadomolybdo phosphoric acid yellow colour method (Kiston and Mellon, 1944). The data were subjected to analysis of variance as described by (Panse and Sukhatme, 2000). RESULTS AND DISCUSSION The increase in acid phosphatase activity varies from 11.0 to 19.0 fold in sterilized soil and 0.5 to 1.4 fold in non-sterilized soil during the growth period of four weeks (Table 1). Introduction of plants in soil influences acid phosphatase activity, which was 32-49% higher under non-sterilized soil condition and 21-40% higher under sterilized soil condition. The increase in alkaline phosphatase activity was 53% more in sterilized soil due to inoculation of Emericella rugulosa compared to non-sterilized soil (Table 1). Plant introduction does not significantly influence alkaline phosphatase activity under both sterilized and non-sterilized condition. But under non sterilized soil condition more (14.4%) activity was noticed after two weeks of crop growth. Increase in phytase activity was 4.4 to 9.6 times more with Emericella rugulosa inoculation after four weeks
Phytase ( EU x 10-4) Days after inoculation SS 7.59 10.93 13.16 15.19 1.92 SS(P) 10.59 13.15 16.51 18.52 1.96 NSS NSS(P) 6.68 9.43 8.36 12.51 10.67 13.72 12.8 15.45 1.58 1.19

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Table 1: Enzyme activities after inoculation of Emericella rugulosa under different treatments.
Alkaline Phosphatase ( EU x 10-4) Days after inoculation SS(P) 0.45 0.59 0.90 0.98 0.07 NSS NSS(P) 0.26 0.38 0.50 0.61 0.09 0.31 0.44 0.68 0.75 0.06

SS: sterilized soil; NS: non-sterilized soil; SS (P): sterilized soil with plant; NS (P): non-sterilized soil with plant

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Table 2: Effect of inoculation of Emericella rugulosa to hydrolyze different unavailable P fractions.

Plant age (Weeks) PC* 4 3.6 0.3 (15.0) 5 14.0 1.2 (22.2) 6
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Depletion of total unavailable P (mg kg-1) Mineral P MC** 20.4 1.2 (85.0) 49.9 3.2 (77.8) 47.5 4.1 (64.7) 55.9 4.7 (62.2) 58.8 5.2 (58.4) PC 12.8 1.0 (27.2) 18.2 1.1 (31.9) 28.9 1.4 (40.0) 35.2 1.8 (41.9) 45.9 2.5 (48.4) Organic P MC 33.4 2.1 (72.3) 38.8 2.7 (68.1) 43.2 2.9 (60.0) 48.8 3.4 (58.1) 49.0 4.1 (51.6) PC 4.7 0.7 (17.8) 11.9 0.9 (34.3) 19.5 1.2 (43.0) 26.6 1.5 (49.9) 33.4 1.9 (55.9) Phytin P MC 21.7 1.2 (82.2) 22.8 1.7 (65.7) 25.9 2.2 (57.0) 26.9 2.2 (50.1) 26.4 2.8 (44.1)

25.9 2.0 (35.3)

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33.9 2.5 (37.8)

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41.8 3.1 (41.6 )

*Plant contribution ** Microbial contribution; Figure in parenthesis denotes the per cent of total mineral/organic/phytin-P depleted.

under sterilized soil condition, whereas only 3.3-7.1 times higher under non-sterilized soil condition (Table 1). Clusterbean rhizosphere helps in increase in phytase activity, which was 18-40% under sterilized soil condition and 18-50% under non-sterilized soil condition. Plants and microorganisms (Tarafdar, 1989)) can release enzymes, therefore under pot microbial build up due to root exudates resulted in higher enzyme activity (Table 1). The introduction of plant does not automatically increase alkaline phosphatase activity, as microorganisms may be the only contributor of alkaline phosphatase in soil (Tabatabai and Bremner, 1969) Enhanced secretion of Phytase (Li et al., 1997) by plant roots and rhizosphere microorganisms (Rao et al., 1990) may contribute to inorganic P acquisition through the hydrolysis of organic esters in the rhizosphere. Crop age increased plant contribution but decreased microbial contribution to hydrolyze

different unavailable P fractions (Table 2). In general, microbial contribution varies between 44-85%, whereas only 15-56 % plant contribution was observed. At crop harvest grain yield of clusterbean was increased by 26% and straw yield by 42% due to inoculation of Emericella rugulosa. The microbial contribution was much higher than the plant contribution to the hydrolysis of different unavailable P fractions (Table 2). In addition to the cleavage of the C-O-P bond by microbial phosphatases and phytases, the microorganisms may also produce organic acids such as malate, citrate, oxalate, which may also help in the release of Pi (Jones, 1998). Enhanced secretion of phosphatases and Phytase (Tarafdar, 1989; Yadav, and Tarafdar, 2004) by plant roots and rhizosphere microorganisms (Tarafdar, 1989) may contribute to Pi acquisition through the hydrolysis of organic P esters in the rhizosphere. Richardson et al., (2001) reported the potential of

Vol. 33, No. 2, 2010

inoculation with Emericella rugulosa. Plant age (days) Plant biomass ( mg plant-1) Control 7 14 21 28 LSD (p=0.05) SS 282 368 338 676 792 728 956 1353 1232 1925 2415 2130 55 53 68 22 44 67 107 Root length ( cm plant-1) 62 125 217 485 17 69 65 152 141 282 265 679 647 24 18 3 12 19 36 0.066 0.040 0.033 0.018 0.013 P content (mg g-1) SS 0.075 0.045 0.027 0.016 0.010 NSS LSD (p=0.05) Control SS NSS LSD (p=0.05) Control

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Table 3: Changes in plant biomass, root length and P content of clusterbean under sterilized and non-sterilized soils due to

NSS LSD (p=0.05) 0.057 0.040 0.027 0.018 0.008 0.008 0.004 0.003 0.002

SS: Sterilized soil; NSS: Non-sterilized soil

Table 4: Straw yield, Grain Yield and P concentration of Clusterbean after harvesting of the crop.
Treatment Un inoculated Inoculated LSD (p=0.05) Yield ( q ha-1) Straw 22.0 31.2 3.8 Grain 5.0 6.3 1.1 P concentration (mg g-1) Straw 6.09 6.85 0.14 Grain 11.38 12.56 1.05

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soil microorganisms to increase the availability of P from phytate both through phytase activity and perhaps by affecting the availability of phytate itself. However, they identified that the extent to which microorganisms activity release P from phytase in soils for its subsequent uptake by plant roots, remained to be determined. In this experiment the results clearly demonstrated the efficiency of Emericella rugulosa in releasing P from phytate (phytin-P) and other unavailable P sources. The result demonstrated significantly (p<0.01, n=6) higher plant biomass with crop age, which was more in sterilized soil. The plant biomass accumulation was 28% higher in sterilized soil and 17% higher in non-sterilized soil due to inoculation of Emericella rugulosa (Table 3).The increase in root length was significantly higher in sterilized soil than non-sterilized soil. In general, Emericella rugulosa helps to improve root length by 26% in sterilized soil and 18% in non-sterilized soil (Table 3).A significant improvement in P content was observed during the

growth period. There was 12% more P accumulation due to Emericella rugulosa after 4 weeks of plant growth (Table3). At crop harvest grain yield of clusterbean was increased by 26% and straw yield by 42% due to inoculation of Emericella rugulosa. A significant (p<0.01, n=6) improvement in plant P content (12%) and seed P content (10%) was also observed (Table 4). Microbial activity results in quantitative and qualitative alterations of root exudates composition due to the degradation of exudates compounds and the release of microbial metabolites (Neumann and Rmheld, 2000). Microbial activity is a central factor in the soil organic P cycle and affects the transformations of inorganic P (Kucey et al., 1989). Higher enzyme activity in soils indicated the potential of soil to affect the biochemical transformations necessary for the maintenance of soil fertility (Rao et al., 1990). The results clearly demonstrate the positive influence of Emericella rugulosa on clusterbean production apparently as a result of the increased release of phosphatases and phytase.

Ames, B.N. (1966). Method Enzymol. 8,115-118. Jackson, M.L. (1967). Soil Chemical Analysis. Prentice-Hall of India, Delhi. Jones, D.L (1998). Plant Soil 205, 25-44. Kiston, R.E. and Mellon, M.G. (1944). Ind. Eng. Chem. Anal. Ed. 2, 379-383. Kucey R.M.N. et al. (1989). Adv. Agron. 42,198-228. Li, M. et al. (1997). Soil. Sci. Plant Nutr. 43, 179-190. Mega, J.A. (1982). J. Agril. Food Chem. 30, 1-9. Neumann, G. and Rmheld. V. (2000) In: The rhizosphere, biochemistry and organic substances at the soil-plant interface (Ed. Pintor, R. et al.) Marcel Dekker Inc, p 41-93. Panse, V.G. and Sukhatme, P (2000). Statistical methods for agricultural workers. ICAR, New Delhi, 359p. .V.

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