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Role of phosphatase-producing fungi on the growth and nutrition of clusterbean (Cyamopsis tetragonoloba (L.) Taub.)
Journal of Arid Environments (1995)29:331-337
Role of phosphatase-producing fungi on the growth and nutrition of clusterbean ( Cyamopsis tetragonoloba (L.) Taub.)
J. C. Tarafdar, A. V. Rao & Praveen-Kumar Central Arid Zone Research Institute, Jodphur 342003, Rajasthan, India (Received 12 August 1993, accepted 4 September 1993) The efficiency of different phosphatase-producing fungi (PPF) in arid soils (loamy sand) was studied in pot experiments in order to evaluate the effect of these fungi on the enhancement of rhizosphere enzyme activities, the uptake of different nutrients, dry matter production and grain yield of the clusterbean. A significant increase in phosphatase (acid and alkaline), dehydrogenase and nitrogenase activities was observed. Nodulation and nitrogenase activity were most affected upon inoculation. Inoculation significantly improved the dry matter production, grain yield, number and dry weight of nodules and decreased shoot:root ratio. In general, there was a significant enhancement in the concentration of N, P, Ca, Mg, Fe and Zn while no effect on Mn with the inoculation of any PPF on clusterbean. A significant improvement in K concentration due to inoculation of Aspergillus fumigatus and Cu concentration with the inoculation of Aspergillus rugulosus and Aspergillus terreus was noted. Aspergillus rugulosus was found to be the best amongst PPF tested followed by Aspergillus fumigatus. The experiment confirmed that PPF have a significant effect on growth and nutrient uptake in the clusterbean and indicated the importance of these fungi on clusterbean production in an arid soil.
Keywords: arid soil; clusterbean; nutrition; phosphatase-producing fungi
Introduction Clusterbean, a drought-tolerant legume crop, is cultivated extensively in arid and semiarid regions of India. But the grain yields of this crop in aridisols are low because of low and erratic rainfall distribution and poor soil conditions besides the low availability of various nutrients, especially phosphorus. In general, the crop yields could be improved in these regions by enhancing the P availability in the soil through the application of P-fertilizers (Aggarwal & Venkateswarlu, 1989), inoculation with suitable VAM (vesicular-arbuscular mycorrhiza) (Rao & Tarafdar, 1993), P-solubilizers (Gaur, 1990) or phosphataseproducing fungi (Tarafdar et al., 1992). Phosphatases in the soil, whether microbial or of plant origin, catalyse the release of plant available P from the organic P compounds present in the soil (Tarafdar & Claassen, 1988; Adams & Pate, 1992). The production and distribution of these enzymes in arid softs under different land-use patterns (Tarafdar et al., 1989) and the potential of different soil fungi in the production of phosphatase (Tarafdar et al., 1988) have been assessed. Though the role of P-solubilizers and VAM fungi in crop production has been emphasized (Tarafdar et al., 1991; Rao & Tarafdar, 0140-1963/95/030331 + 07 $08"00/0
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1993) there is no information on the usefulness of phosphatase-producing fungi (PPF) except that of the authors' work on mung bean (Tarafdar et al., 1992). Hence, an attempt has been made to examine the effect of different phosphatase-produclng fungi on growth and nutrition of clusterbean on a loamy sand soil in an arid environment.
Materials and methods The soil used in the present study was a loamy sand (Table 1) collected from fallow virgin land on the C.R. Farm, Jodhpur. No fertilizer had been applied for 4-5 years as this land was not under cultivation. The air-dried soil was sieved ( < 2 mm) and placed in earthern pots (30 cm in diameter), 10 kg per pot. Four fungal cultures of Aspergillus niger van Tieghem, A. fumigatus Fres., A. rugulosus T h o m . & Roper and A. terreus Thom. were obtained from the culture collection centre of this Institute and maintained on potato dextrose agar slants. The fungi were grown for 7 days at 28°C in 250 ml Erlenmeyer flasks containing 150 ml Czapeks-Dox broth after inoculation with 8 mm discs of 4-day-old agar cultures. The inoculation was prepared by blending the culture broth in a homogenizer and dilutin~ it so that final inoculum contained 103 fungal propagules (spores and hyphal bits) ml- . This homogenized culture was applied just after placing the seeds in planting holes (5 ml each). Control pots received the same amount of steam sterilized inoculum. Four clusterbean plants (cv HFG-75) were grown in each pot. The experiment was laid out in a simple randomized block design consisting of four inoculation treatments and one control with eight replications. The plants were grown in a net house with sunlight for 10 h day -1 and were watered on alternate days up to field capacity. The temperature in the net house during the experiment ranged from 25°C (night) to 35°C (day). Four pots from each treatment were harvested after 30 days for different enzyme analyses and to determine nodulation. The rest of the pots were harvested at crop maturity and analysed for different nutrients. The root system was carefully lifted with the nodules intact. Rhizosphere soil samples were collected by the standard procedure. Nitrogenase
Table 1. Some characteristics of the soil used in the study Parameters Soil classification Sand (%) Silt (%) Clay (%) pH (Soil :Water, 1 : 2-5) Electrical conductivity (dS m- 1) Organic matter (%) Total N (mg g-l) Total P (mg g-a) Olsen-P (mg kg- 1) Organic-P (mg kg-1) Available Fe (mg kg- 1) Available Mn (nag kg-1) Available Zn (mg kg -1) Available Cu (mg kg -1) Acid phosphatase activity (n Kat 100 g-1) Alkaline phosphatase activity (n Kat 100 g- 1dry soil) Dehydrogenase activity (n Kat g-1 dry soil VAM propagules (100 g- 1dry soil)
Quantity Camborthid 85"1 5"5 7"9 8"1 0"2 0"4 310 270 7"0 50"0 2"3 5"7 0"43 0"21 6"6 8"2 2"0 140
NUTRITION OF CLUSTERBEAN BY PPF
333
activity was assayed immediately by acetylene reduction with an Aimil-Nucon gas chromatograph (Rao & Venkateswarlu, 1987). Following the assay, the nodules were separated and counted. The plants were dried at 60°C to a constant weight, ground to a fine powder and subjected to a mixed di- or tri- acid digestion. Estimations of N , P, K, Ca, Mg and Na contents were made by following the standard methods (Jackson, 1967), after wet digestion, while Cu, Zn, Fe and Mn contents were determined with an atomic absorption spectrophotometer (Varian AA 1475). Phosphatases were assayed by the method of Tabatabai & Bremner (1969) using p N P P as a substrate and dehydrogenase activity was estimated by a modified method of Casida et al. (1964). All parameters were expressed on oven-dry basis. Standard errors of means were calculated and when appropriate, analysis of variance carried out and means of inoculated and non-inoculated treatments separated by the Scheffe' test for planned comparisons (Sokal & Rohlf, 1981).
Results
A significant increase in the activities of both acid and alkaline phosphatases and dehydrogenase was observed in the rhizosphere of clusterbean upon inoculation with different phosphatase-producing fungi (Table 2) and the percentage increase in activity varied among different fungi. The activities of dehydrogenase and acid phosphatase were at a maximum with Aspergillus terreus and with A. rugulosus for alkaline phosphatase. Inoculation had significantly improved the number and dry weight of nodules and nitrogenase activity. The nitrogenase activity was enhanced by 350% in plants inoculated with A. rugulosus. Dry matter production and grain yields were improved upon inoculation while there was a decrease in s h o o t : r o o t ratio indicating thereby the enhancement in the root growth (Table 3). In general the response of inoculation was maximum with A. rugulosus followed by A. fumigatus, A. terreus and A. niger. Significant improvement in the concentration of N, P, Ca and Mg was observed in the clusterbean plants upon inoculation with P P F (Table 4) besides the enhancement in the uptake of K with A. fumigatus and Na with A. terreus. In general, the contribution of mineral nutrients due to PPF inoculation on clusterbean was maximum with A. rugulosus followed by A. fumigatus, A. terreus seems to be more suited under saline condition. Inoculation with PPF in clusterbean increases significantly the concentration of Fe and Zn (Table 5). There was absolutely no effect on Mn concentration with inoculation of any of the P P F studied but the concentration of Cu was significantly increased with the inoculation ofA. rugulosus and A. terreus. Amongst the micro-nutrients studied Fe was the most affected by the inoculation of PPF.
Table
2. Effect of inoculation with phosphatase-producing fungi on rhizosphere enzyme activities in clusterbean
Treatment
Acid phosphataset
Alkaline phosphataset
Dehydrogenase:~
Nitrogenase§
7"5 8"1" 8"0" 8'4** 8"9***
8"6 9"0* 9"9"** 10"8"** 9"4**
2"4 3"3** 3"1" 5"3"** 5"9"**
0"4 1"0"** 1"3"** 1"4*** 0"7*
Uninoculated control AspergiUus niger A spergillusfumigatus AspergiUus rugulosus Aspergillus terreus
t n Kat 100g-1 soil;~:p Kat g-t soil; § l~moleC2H4h-1 plant-L * p < 5%;** p < 1%; *** p<0"1%.
J.C. TARAFDAR ET AL.
334
Table 3. Effect of inoculation with phosphatase-producing fungi on shoot: root ratio, nodule number and
dry weight, dry matter production and grain yield in clusterbean
Treatment
Shoot : root ratio
No. of nodules (plant- 1)
Dry weight of nodules (mg plant- 1)
Dry matter yield (g plant- 1)
Grain yield (g plant- 1)
9'2 8"8* 8"7* 7"6*** 8"2**
14"2 22"5*** 30"7*** 34"0*** 25"0***
21"2 61"2"** 57"5*** 71"2"** 40"0***
20"8 24"1" 26"5*** 27"2*** 24"4**
5"4 6"3* 6"9** 7"1"** 6"3*
Uninoculated control Asperg~llusniger Aspergillusfumigatus AspergiUusrugulosus Aspergillusterreus * p<5%;
** p < 1%; *** p < 0 " l % .
Table 4. Effect of inoculation of phosphatase-producing fungi on mineral uptake by clusterbean Treatment Uninoculated control Aspergillusniger Aspergillusfumigatus Aspergillusrugulosus AspergiUus terreus
Nt
Pt
Kt
Ca~
Mg~
Na~
19"0 26"1"* 29"7*** 31"5"** 24"9*
1'8 2"1" 2"2* 2"2* 2" 1"
3"8 4"2 4"7* 4"2 4'2
86 111"* 101" 108"* 114"**
216 244* 320*** 288*** 241"
125 129 137 106 96*
t Concentration, m g g - 1; :]: Concentration m g k g - 1. * p < 5 % ; ** p < 1%; *** p < 0'1%.
Table 5. Effect of inoculation of clusterbean with phosphatase-producing fungi on uptake of some trace elements (concentration mg kg- ) Treatment Uninoculated control Aspergillusniger Aspergillusfumigatus AspergiUusrugulosus Aspergillusterreus * p<5%;
Fe
Mn
Cu
Zn
3"4 4"9*** 5"9*** 4"4*** 5"1"**
5"0 5'0 5"5 4"9 5'2
8"0 7'2* 7"5 9"9* 9"8*
6"2 8"I* 8"2* 8"7** 8"0*
** p < 1%; *** p < O ' l % .
Discussion Activities of both the phosphatases are higher in the rhizosphere of inoculated plants compared with the uninoculated plants (Table 2). This increase can be attributed to the higher n u m b e r of fungi in the rhizosphere and increased growth of plant roots (Tarafdar & Claassen, 1988; Tarafdar et al., 1992). This is in accordance with that of Greaves & Webley (1965) who observed a specific stimulation of phosphatase-producing microorganisms by root exudates. The m a x i m u m increase in activity was noticed with A. rugulosus and A. terreus which were found to be more efficient in pure culture compared to others (Tarafdar & Claassen, 1988). The enhanced phosphatase activity may help the plants to use organic P sources (Tarafdar & Claassen, 1988). Sharpley (1985) also concluded that plants can use organic P fractions more efficiently when the soil root interface is enriched with phosphatases. The significant increase in dehydrogenase activity is a clear indication
NUTRITION OF CLUSTERBEAN BY PPF
335
of microbial build up. It is well established that dehydrogenase activity is an indicator of total microbial activity in soils and a significant and positive correlation of phosphatase activity with microbial populations in an arid soil has been reported (Tarafdar et al., 1989). The increased nitrogenase activity in the inoculated plants might have been due to an increase in nodule number and dry weight (Table 3) and by improved P nutrition (Table 4). A similar observation was noticed in our earlier study (Tarafdar et al., 1992) with inoculation by PPF in mung bean. Similarly N concentration and total nitrogen uptake increased considerably due to the improvement in symbiotic N2 fixation. AzconAguilar & Barea (1978) attributed higher N concentration and contents in lucerne plants to an increase in Nz fixation in plants inoculated with phosphatase-solubitizing bacteria. Further, it is well established that if the P nutrition of plants is improved, either through fertilization or biological means, symbiotic N2-fixation and the plant N contents are improved. The higher levels of dry matter production and grain yield upon inoculation with PPF (Table 3) are mainly due to higher enzyme activities in the rhizosphere and better nutrient availability besides the production of the plant growth regulators by PPF. Strzelczyk et al. (1989) reported that plant growth regulators such as ethylene and auxin, gibberellin and cytokinin-tike substances were produced by non-mycorrhizal fungi associated with the roots of forest trees. The inoculation with PPF led to a decrease in shoot : root ratio (W/W) indicating an increase in root biomass and this may be due to stimulation in the formation of lateral roots (Schonwitz & Ziegler, 1989) and also root hairs (Martin et al., 1989). As rhizosphere micro-organisms are known to influence root length (Martin et al., 1989; Schonwitz & Ziegler, 1989), it is possible that conditions in the clusterbean rhizosphere were favourable for aspergilli, causing increased root dry matter. The observed changes in root growth due to PPF should have important implications for the acquisition of mineral nutrients, particularly of those with low mobility in the soil such as P and Zn. Greater phosphatase activity has been directly implicated in the acquisition of more P (Table 4) by plants (Dodd et al., 1987; Tarafdar & Jungk, 1987). A significant improvement in the uptake of Ca and Mg might be explained by the activity of aspergilli in synthesizing organic acids which can react with Ca and Mg containing minerals releasing them in larger quantities (Smith, 1954). A significant increase in K concentration only with the inoculation of Aspergillus fumigatus indicates the ability of this fungus reacting with K-containing minerals. It is also known that one of the most important properties of fungi is the ability to translocate plant nutrients especially N, P and K (Harley, 1959). The results (Table 5) clearly demonstrate the capacity of PPF to mobilize Zn and Fe from a loamy sand soil and make them available to the plants. The very significant effect on Fe is difficult to explain unless interactions with microbial siderophore production and corresponding increase in supply of ferrated siderophores to the roots is considered. Similar results obtained with VAM in maize (Kothari et al., 1991). They had observed that in non-mycorrhizal plants, at the interface between hyphal and root compartment, the soil was firmly aggregated, indicating high microbial activity and perhaps, also the production of siderophores. Reid et al. (1985) showed that E D D H A and Desferal increased the diffusion of Fe to the same extent and concluded that the microbial siderophores were important in plant Fe nutrition. There are differences between the four major phosphatase producing fungi in their ability to stimulate plant growth and nutrient uptake in clusterbean. Similar variations were noticed in our earlier study with mung bean (Tarafdar et al., 1992) and among VAM fungi isolates for soybean (Carting et al., 1979). These may be specific interaction between the plant species and the Aspergillus strain. The present results demonstrate the high capacity of phosphatase-producing fungi for nutrient uptake and transport to plants. However, the process of transferring nutrients from the fungus to the host needs to be investigated. The authors wish to thank Dr A. N. Lahiri, Head, Division of Arable Cropping System for his keen interest and helpful discussion.
336
J. C. TARAFDAR ET AL.
References Adams, M. A. & Pate, J. S. (1992). Availability of organic and inorganic forms of phosphorus to lupins (Lupinus spp.). Plant and Soil, 145:107-113. Aggarwal, R. K. & Venkateswarlu, J. (1989). Long term effect of manures and fertilizers on important cropping systems of arid region. Fertilizer News, 34: 67-70. Azcon-Aguilar, C. & Barea, J. M. (1978). Effects of interactions between different culture fractions of'Phosphobacteria' and Rhizobium on mycorrhizal infection, growth and nodulation of Medicago sativa. Canadian Journal of Microbiology, 24: 250-254. Carling, D. F., Brown, M. F. & Brown, R. A. (1979). Colonization rates and growth responses of soybean plants infected with vesicular-arbuscular mycorrhizal fungi. Canadian Journal of Botany, 57: 1769-1777. Casida, L. E., Klein, D. A. & Santoro, T. (1964). Soil dehydrogenase activity. Soil Science, 98: 373-376. Dodd, J. C., Burton, C. C., Burns, R. G. & Jeffries, P. (1987). Phosphatase activity associated with the roots and the rhizosphere of plants infected with vesicular-arbuscular mycorrhizal fungi. The New Phytologist, 107: 163-172. Gaur, A. C. (1990). Phosphate Solubilizing Microorganisms as Biofertilizers. New Delhi: Omega Scientific Publishers. 265 pp. Greaves, M. P. & Webley, D. N. (1965). A study of the breakdown of organic phosphates by microorganisms from the root region of certain pasture grasses. Journal of Applied Bacteriology, 28: 454-465. Harley, J. L. (1959). The Biology ofMycorrhiza (2nd Edn). London: Leonard Hill. 233 pp. Jackson, M. L. (1967). Soil Chemical Analysis. New Delhi: Prentice Hall. 498 pp. Kothari, S. K., Marschner, H. & R6mheld, V. (1991). Contribution of VA mycorrhizal hyphae in acquisition of phosphorus and zinc by maize grown in calcareous soil. Plant and Soil, 131: 177-185. Martin, P., Glatzle, A., Kolb, W., Ornay, H. & Schmidt, W. (1989). N2-fixing bacteria in the rhizosphere: quantification and hormonal effects on root development. Journal of Plant Nutrition and Soil Science, 152: 237-245. Rao, A. V. & Venkateswarlu, B. (1987). Nitrogen fi afion as influenced by water stress in selected crop legumes of the Indian arid zone. Arid Soil Research and Rehabilitation, 1: 89-96. Rao, A. V. & Tarafdar, J. C. (1993). Role of VAM fungi in nutrient uptake and growth of clusterbean in an arid soil. Arid Soil Research and Rehabilitation, 7: 275-280. Reid, R. K., Reid, C. P. P. & Szaniszlo, P. J. (1985). Effects of synthetic and microbiaUy produced chelates on the diffusion of iron and phosphorus to a stimulated root in soil. Biology and Fertility of Soils, 1: 45-52. Schonwitz, R. & Ziegler, H. (1989). Interaction of maize roots and rhizosphere microorganisms. Journal of Plant Nutrition and Soil Science, 152:217-222. Sharpley, A. N. (1985). Phosphorus cycling in unfertilized and fertilized agricultural soils. Soil Science Society of Arnerica Journal, 49:905-911. Smith, G. (1954). An Introduction to Industrial Mycology (4th Edn). London: Edward Arnold Ltd. 144 pp. Sokal, R. R. & Rohlf, F. J. (1981). Biometry ~ The Principles and Practice of Statistics in Biological Research(2nd Edn). New York: W. H. Freeman and Co. 859 pp. Strzelczyk, E., Pokojska, A., Kampert, M., Michalski, L. & Kowalski, S. (1989). Production of plant growth regulators by non-mycorrhizal fungi associated with the roots of forest trees. In: Vancura, V. & Kunc, F. (Eds.), Interrelationships between Microorganisms and Plants in Soil, pp. 213-222. Tabatabai, M. A. & Bremner, J. M. (1969). Use ofp-nitrophenyl phosphate for assay ofphosphatase activity. Soil Biology and Biochemistry, 1: 301-307. Tarafdar, J. C. & Jungk, A. (1987). Phosphatase activity in the rhizosphere and its relation to the depletion of soil organic phosphorus. Biology and Fertility of Soils, 3: 199-204. Tarafdar, J. C. & Claassen, N. (1988). Organic phosphorus compounds as a phosphorus source for higher plants through the activity of phosphatases produced by plant roots and microorganisms. Biology and Fertility of Soils, 5: 308-312. Tarafdar, J. C., Rao, A. V. & Bala, K. (1988). Production of phosphatases by fungi isolated from desert soils. Folia Microbiologia, 33: 453-457.
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Tarafdar, J. C., Bala, K. & Rao, A. V. (1989). Phosphatase activity and distribution of phosphorus in arid soil profiles under different land use pattern. Journal of Arid Environments, 16: 29-34. Tarafdar, J. C., Chattrath, R. & Bala, K. (1991). Role of phosphate solubilizing bacteria in pearl millet (Pennisetum americanum)rhizosphere as affected by genotypes in arid soils. Transactionsof Indian Society of Desert Technology, 16: 129-137. Tarafdar, J. C., Rao, A. V. & Praveen-Kumar, (1992). Effect of different phosphatase producing fungi on growth and nutrition of mung bean [Vigna radiata [L.] Wikzek] in arid soil. Biology and Fertility of Soils, 13: 35-58.
Role of phosphatase-producing fungi on the growth and nutrition of clusterbean ( Cyamopsis tetragonoloba (L.) Taub.)
J. C. Tarafdar, A. V. Rao & Praveen-Kumar Central Arid Zone Research Institute, Jodphur 342003, Rajasthan, India (Received 12 August 1993, accepted 4 September 1993) The efficiency of different phosphatase-producing fungi (PPF) in arid soils (loamy sand) was studied in pot experiments in order to evaluate the effect of these fungi on the enhancement of rhizosphere enzyme activities, the uptake of different nutrients, dry matter production and grain yield of the clusterbean. A significant increase in phosphatase (acid and alkaline), dehydrogenase and nitrogenase activities was observed. Nodulation and nitrogenase activity were most affected upon inoculation. Inoculation significantly improved the dry matter production, grain yield, number and dry weight of nodules and decreased shoot:root ratio. In general, there was a significant enhancement in the concentration of N, P, Ca, Mg, Fe and Zn while no effect on Mn with the inoculation of any PPF on clusterbean. A significant improvement in K concentration due to inoculation of Aspergillus fumigatus and Cu concentration with the inoculation of Aspergillus rugulosus and Aspergillus terreus was noted. Aspergillus rugulosus was found to be the best amongst PPF tested followed by Aspergillus fumigatus. The experiment confirmed that PPF have a significant effect on growth and nutrient uptake in the clusterbean and indicated the importance of these fungi on clusterbean production in an arid soil.
Keywords: arid soil; clusterbean; nutrition; phosphatase-producing fungi
Introduction Clusterbean, a drought-tolerant legume crop, is cultivated extensively in arid and semiarid regions of India. But the grain yields of this crop in aridisols are low because of low and erratic rainfall distribution and poor soil conditions besides the low availability of various nutrients, especially phosphorus. In general, the crop yields could be improved in these regions by enhancing the P availability in the soil through the application of P-fertilizers (Aggarwal & Venkateswarlu, 1989), inoculation with suitable VAM (vesicular-arbuscular mycorrhiza) (Rao & Tarafdar, 1993), P-solubilizers (Gaur, 1990) or phosphataseproducing fungi (Tarafdar et al., 1992). Phosphatases in the soil, whether microbial or of plant origin, catalyse the release of plant available P from the organic P compounds present in the soil (Tarafdar & Claassen, 1988; Adams & Pate, 1992). The production and distribution of these enzymes in arid softs under different land-use patterns (Tarafdar et al., 1989) and the potential of different soil fungi in the production of phosphatase (Tarafdar et al., 1988) have been assessed. Though the role of P-solubilizers and VAM fungi in crop production has been emphasized (Tarafdar et al., 1991; Rao & Tarafdar, 0140-1963/95/030331 + 07 $08"00/0
~ 1995 AcademicPress Limited
J. c. TARAFDAR ET AL.
332
1993) there is no information on the usefulness of phosphatase-producing fungi (PPF) except that of the authors' work on mung bean (Tarafdar et al., 1992). Hence, an attempt has been made to examine the effect of different phosphatase-produclng fungi on growth and nutrition of clusterbean on a loamy sand soil in an arid environment.
Materials and methods The soil used in the present study was a loamy sand (Table 1) collected from fallow virgin land on the C.R. Farm, Jodhpur. No fertilizer had been applied for 4-5 years as this land was not under cultivation. The air-dried soil was sieved ( < 2 mm) and placed in earthern pots (30 cm in diameter), 10 kg per pot. Four fungal cultures of Aspergillus niger van Tieghem, A. fumigatus Fres., A. rugulosus T h o m . & Roper and A. terreus Thom. were obtained from the culture collection centre of this Institute and maintained on potato dextrose agar slants. The fungi were grown for 7 days at 28°C in 250 ml Erlenmeyer flasks containing 150 ml Czapeks-Dox broth after inoculation with 8 mm discs of 4-day-old agar cultures. The inoculation was prepared by blending the culture broth in a homogenizer and dilutin~ it so that final inoculum contained 103 fungal propagules (spores and hyphal bits) ml- . This homogenized culture was applied just after placing the seeds in planting holes (5 ml each). Control pots received the same amount of steam sterilized inoculum. Four clusterbean plants (cv HFG-75) were grown in each pot. The experiment was laid out in a simple randomized block design consisting of four inoculation treatments and one control with eight replications. The plants were grown in a net house with sunlight for 10 h day -1 and were watered on alternate days up to field capacity. The temperature in the net house during the experiment ranged from 25°C (night) to 35°C (day). Four pots from each treatment were harvested after 30 days for different enzyme analyses and to determine nodulation. The rest of the pots were harvested at crop maturity and analysed for different nutrients. The root system was carefully lifted with the nodules intact. Rhizosphere soil samples were collected by the standard procedure. Nitrogenase
Table 1. Some characteristics of the soil used in the study Parameters Soil classification Sand (%) Silt (%) Clay (%) pH (Soil :Water, 1 : 2-5) Electrical conductivity (dS m- 1) Organic matter (%) Total N (mg g-l) Total P (mg g-a) Olsen-P (mg kg- 1) Organic-P (mg kg-1) Available Fe (mg kg- 1) Available Mn (nag kg-1) Available Zn (mg kg -1) Available Cu (mg kg -1) Acid phosphatase activity (n Kat 100 g-1) Alkaline phosphatase activity (n Kat 100 g- 1dry soil) Dehydrogenase activity (n Kat g-1 dry soil VAM propagules (100 g- 1dry soil)
Quantity Camborthid 85"1 5"5 7"9 8"1 0"2 0"4 310 270 7"0 50"0 2"3 5"7 0"43 0"21 6"6 8"2 2"0 140
NUTRITION OF CLUSTERBEAN BY PPF
333
activity was assayed immediately by acetylene reduction with an Aimil-Nucon gas chromatograph (Rao & Venkateswarlu, 1987). Following the assay, the nodules were separated and counted. The plants were dried at 60°C to a constant weight, ground to a fine powder and subjected to a mixed di- or tri- acid digestion. Estimations of N , P, K, Ca, Mg and Na contents were made by following the standard methods (Jackson, 1967), after wet digestion, while Cu, Zn, Fe and Mn contents were determined with an atomic absorption spectrophotometer (Varian AA 1475). Phosphatases were assayed by the method of Tabatabai & Bremner (1969) using p N P P as a substrate and dehydrogenase activity was estimated by a modified method of Casida et al. (1964). All parameters were expressed on oven-dry basis. Standard errors of means were calculated and when appropriate, analysis of variance carried out and means of inoculated and non-inoculated treatments separated by the Scheffe' test for planned comparisons (Sokal & Rohlf, 1981).
Results
A significant increase in the activities of both acid and alkaline phosphatases and dehydrogenase was observed in the rhizosphere of clusterbean upon inoculation with different phosphatase-producing fungi (Table 2) and the percentage increase in activity varied among different fungi. The activities of dehydrogenase and acid phosphatase were at a maximum with Aspergillus terreus and with A. rugulosus for alkaline phosphatase. Inoculation had significantly improved the number and dry weight of nodules and nitrogenase activity. The nitrogenase activity was enhanced by 350% in plants inoculated with A. rugulosus. Dry matter production and grain yields were improved upon inoculation while there was a decrease in s h o o t : r o o t ratio indicating thereby the enhancement in the root growth (Table 3). In general the response of inoculation was maximum with A. rugulosus followed by A. fumigatus, A. terreus and A. niger. Significant improvement in the concentration of N, P, Ca and Mg was observed in the clusterbean plants upon inoculation with P P F (Table 4) besides the enhancement in the uptake of K with A. fumigatus and Na with A. terreus. In general, the contribution of mineral nutrients due to PPF inoculation on clusterbean was maximum with A. rugulosus followed by A. fumigatus, A. terreus seems to be more suited under saline condition. Inoculation with PPF in clusterbean increases significantly the concentration of Fe and Zn (Table 5). There was absolutely no effect on Mn concentration with inoculation of any of the P P F studied but the concentration of Cu was significantly increased with the inoculation ofA. rugulosus and A. terreus. Amongst the micro-nutrients studied Fe was the most affected by the inoculation of PPF.
Table
2. Effect of inoculation with phosphatase-producing fungi on rhizosphere enzyme activities in clusterbean
Treatment
Acid phosphataset
Alkaline phosphataset
Dehydrogenase:~
Nitrogenase§
7"5 8"1" 8"0" 8'4** 8"9***
8"6 9"0* 9"9"** 10"8"** 9"4**
2"4 3"3** 3"1" 5"3"** 5"9"**
0"4 1"0"** 1"3"** 1"4*** 0"7*
Uninoculated control AspergiUus niger A spergillusfumigatus AspergiUus rugulosus Aspergillus terreus
t n Kat 100g-1 soil;~:p Kat g-t soil; § l~moleC2H4h-1 plant-L * p < 5%;** p < 1%; *** p<0"1%.
J.C. TARAFDAR ET AL.
334
Table 3. Effect of inoculation with phosphatase-producing fungi on shoot: root ratio, nodule number and
dry weight, dry matter production and grain yield in clusterbean
Treatment
Shoot : root ratio
No. of nodules (plant- 1)
Dry weight of nodules (mg plant- 1)
Dry matter yield (g plant- 1)
Grain yield (g plant- 1)
9'2 8"8* 8"7* 7"6*** 8"2**
14"2 22"5*** 30"7*** 34"0*** 25"0***
21"2 61"2"** 57"5*** 71"2"** 40"0***
20"8 24"1" 26"5*** 27"2*** 24"4**
5"4 6"3* 6"9** 7"1"** 6"3*
Uninoculated control Asperg~llusniger Aspergillusfumigatus AspergiUusrugulosus Aspergillusterreus * p<5%;
** p < 1%; *** p < 0 " l % .
Table 4. Effect of inoculation of phosphatase-producing fungi on mineral uptake by clusterbean Treatment Uninoculated control Aspergillusniger Aspergillusfumigatus Aspergillusrugulosus AspergiUus terreus
Nt
Pt
Kt
Ca~
Mg~
Na~
19"0 26"1"* 29"7*** 31"5"** 24"9*
1'8 2"1" 2"2* 2"2* 2" 1"
3"8 4"2 4"7* 4"2 4'2
86 111"* 101" 108"* 114"**
216 244* 320*** 288*** 241"
125 129 137 106 96*
t Concentration, m g g - 1; :]: Concentration m g k g - 1. * p < 5 % ; ** p < 1%; *** p < 0'1%.
Table 5. Effect of inoculation of clusterbean with phosphatase-producing fungi on uptake of some trace elements (concentration mg kg- ) Treatment Uninoculated control Aspergillusniger Aspergillusfumigatus AspergiUusrugulosus Aspergillusterreus * p<5%;
Fe
Mn
Cu
Zn
3"4 4"9*** 5"9*** 4"4*** 5"1"**
5"0 5'0 5"5 4"9 5'2
8"0 7'2* 7"5 9"9* 9"8*
6"2 8"I* 8"2* 8"7** 8"0*
** p < 1%; *** p < O ' l % .
Discussion Activities of both the phosphatases are higher in the rhizosphere of inoculated plants compared with the uninoculated plants (Table 2). This increase can be attributed to the higher n u m b e r of fungi in the rhizosphere and increased growth of plant roots (Tarafdar & Claassen, 1988; Tarafdar et al., 1992). This is in accordance with that of Greaves & Webley (1965) who observed a specific stimulation of phosphatase-producing microorganisms by root exudates. The m a x i m u m increase in activity was noticed with A. rugulosus and A. terreus which were found to be more efficient in pure culture compared to others (Tarafdar & Claassen, 1988). The enhanced phosphatase activity may help the plants to use organic P sources (Tarafdar & Claassen, 1988). Sharpley (1985) also concluded that plants can use organic P fractions more efficiently when the soil root interface is enriched with phosphatases. The significant increase in dehydrogenase activity is a clear indication
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335
of microbial build up. It is well established that dehydrogenase activity is an indicator of total microbial activity in soils and a significant and positive correlation of phosphatase activity with microbial populations in an arid soil has been reported (Tarafdar et al., 1989). The increased nitrogenase activity in the inoculated plants might have been due to an increase in nodule number and dry weight (Table 3) and by improved P nutrition (Table 4). A similar observation was noticed in our earlier study (Tarafdar et al., 1992) with inoculation by PPF in mung bean. Similarly N concentration and total nitrogen uptake increased considerably due to the improvement in symbiotic N2 fixation. AzconAguilar & Barea (1978) attributed higher N concentration and contents in lucerne plants to an increase in Nz fixation in plants inoculated with phosphatase-solubitizing bacteria. Further, it is well established that if the P nutrition of plants is improved, either through fertilization or biological means, symbiotic N2-fixation and the plant N contents are improved. The higher levels of dry matter production and grain yield upon inoculation with PPF (Table 3) are mainly due to higher enzyme activities in the rhizosphere and better nutrient availability besides the production of the plant growth regulators by PPF. Strzelczyk et al. (1989) reported that plant growth regulators such as ethylene and auxin, gibberellin and cytokinin-tike substances were produced by non-mycorrhizal fungi associated with the roots of forest trees. The inoculation with PPF led to a decrease in shoot : root ratio (W/W) indicating an increase in root biomass and this may be due to stimulation in the formation of lateral roots (Schonwitz & Ziegler, 1989) and also root hairs (Martin et al., 1989). As rhizosphere micro-organisms are known to influence root length (Martin et al., 1989; Schonwitz & Ziegler, 1989), it is possible that conditions in the clusterbean rhizosphere were favourable for aspergilli, causing increased root dry matter. The observed changes in root growth due to PPF should have important implications for the acquisition of mineral nutrients, particularly of those with low mobility in the soil such as P and Zn. Greater phosphatase activity has been directly implicated in the acquisition of more P (Table 4) by plants (Dodd et al., 1987; Tarafdar & Jungk, 1987). A significant improvement in the uptake of Ca and Mg might be explained by the activity of aspergilli in synthesizing organic acids which can react with Ca and Mg containing minerals releasing them in larger quantities (Smith, 1954). A significant increase in K concentration only with the inoculation of Aspergillus fumigatus indicates the ability of this fungus reacting with K-containing minerals. It is also known that one of the most important properties of fungi is the ability to translocate plant nutrients especially N, P and K (Harley, 1959). The results (Table 5) clearly demonstrate the capacity of PPF to mobilize Zn and Fe from a loamy sand soil and make them available to the plants. The very significant effect on Fe is difficult to explain unless interactions with microbial siderophore production and corresponding increase in supply of ferrated siderophores to the roots is considered. Similar results obtained with VAM in maize (Kothari et al., 1991). They had observed that in non-mycorrhizal plants, at the interface between hyphal and root compartment, the soil was firmly aggregated, indicating high microbial activity and perhaps, also the production of siderophores. Reid et al. (1985) showed that E D D H A and Desferal increased the diffusion of Fe to the same extent and concluded that the microbial siderophores were important in plant Fe nutrition. There are differences between the four major phosphatase producing fungi in their ability to stimulate plant growth and nutrient uptake in clusterbean. Similar variations were noticed in our earlier study with mung bean (Tarafdar et al., 1992) and among VAM fungi isolates for soybean (Carting et al., 1979). These may be specific interaction between the plant species and the Aspergillus strain. The present results demonstrate the high capacity of phosphatase-producing fungi for nutrient uptake and transport to plants. However, the process of transferring nutrients from the fungus to the host needs to be investigated. The authors wish to thank Dr A. N. Lahiri, Head, Division of Arable Cropping System for his keen interest and helpful discussion.
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