Microbiol. Res. (2001) 156, 145–149 http://www.urbanfischer.de/journals/microbiolres
Improvement in bioavailability of tricalcium phosphate to Cymbopogon martinii var. motia by rhizobacteria, AMF and Azospirillum inoculation Neelima Ratti1, S. Kumar2, H. N. Verma3, S. P. Gautam1 1 2 3
Dept. of Biological Sciences, Rani Durgavati Vishwavidyalaya, Jabalpur - 482001 Dept. of Microbiology and Plant Pathology, Central Institute of Medicinal and Aromatic Plants, Lucknow -226015 Dept. of Botany, University of Lucknow, Lucknow
Accepted: December 5, 2000
Abstract The interactive effects of phosphate solubilizing bacteria, N2 fixing bacteria and arbuscular mycorrhizal fungi (AMF) were studied in a low phosphate alkaline soil amended with tricalcium insoluble source of inorganic phosphate on the growth of an aromatic grass palmarosa (Cymbopogon martinii). The microbial inocula consisted of the AM fungus Glomus aggregatum, phosphate solubilizing rhizobacteria Bacillus polymyxa and N2 fixing bacteria Azospirillum brasilense. These rhizobacteria behaved as “mycorrhiza helper” and enhanced root colonization by G. aggregatum in presence of tricalcium phosphate at the rate of 200 mg kg–1 soil (P1 level). Dual inoculation of G. aggregatum and B. polymyxa yielded 21.5 g plant dry weight (biomass), while it was 21.7 g in B. polymyxa and A. brasilense inoculated plants as compared to 14.9 g of control at the same level. Phosphate content was maximum (0.167%) in the combined treatment of G. aggregatum, B. polymyxa and A. brasilense at P1 level, however acid phosphatase activity was recorded to be 4.75 µmol mg–1 min–1 in G. aggregatum, B. polymyxa and A. brasilense treatment at P0 level. This study indicates that all microbes inoculated together help in the uptake of tricalcium phosphate which is otherwise not used by the plants and their addition at 200 mg kg–1 of soil gave higher productivity to palmarosa plants. Key words: Azospirillum brasilense – Bacillus polymyxa – Glomus aggregatum – phosphatase activity – tricalcium phosphate
Corresponding author: S. P. Gautam e-mail:
[email protected] 0944-5013/01/156/02-145 $15.00/0
Introduction Arbuscular mycorrhizae increase the uptake of nutrients particularly P (Joner and Jakobsen 1994; Kahiluoto and Vestberg 1998; Ludwig Müller et al. 1997), Zn (Kothari et al. 1991) and N (George et al. 1992). Among the nutrients, P has been subjected to the largest number of studies because improved growth by AM colonization and rhizospheric phosphate solubilizing bacteria is most often correlated with higher P uptake by the plant (Bolan 1991; Toro et al. 1997). Phosphorus is one of the major plant nutrients required in optimum amount for proper plant growth. About 98% of Indian soils are inadequate in available P (Gaur 1987). Native soil phosphorus is organically bound and available in the form of phytin, or its derivatives, has low solubility and mobility, and thus is not accessible to plants. This organic P must be hydrolysed to inorganic P for utilization by plants which can be mediated by phosphatases secreted by AM fungi or phosphate solubilizing bacteria (Gaur and Rana 1990). Excess inorganic fertilization decreases the extent of fungal infection by AM fungi, so an optimum situation must exist where optimal benefits from such fungi and inorganic fertilizers are achieved. Thus tricalcium phosphate was added as an insoluble source of inorganic phosphate which does not increase the P level in the rhizosphere soil. The rhizospheric bacteria also interact well with AM fungi in P-deficient soils or soils amended with tricalcium phosphate. Interaction of AMF with other microbes like Rhizobium, Azospirillum and Trichoderma Microbiol. Res. 156 (2001) 2
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has also been reported to be beneficial for plant growth (Rani et al. 1999; Ratti and Janardhanan 1996). The present work was done on the interaction of AMF, phosphate solubilizing bacteria and Azospirillum with tricalcium phosphate as a source of phosphate on palmarosa. Palmarosa, the host plant, is an aromatic grass known for its geraniol containing oil, which has high demands in perfume industries.
Materials and methods Experimental design and biological material. Palmarosa plants (Cymbopogon martinii) were raised from surface sterilized seeds (0.5% sodium hypochlorite for 10 minutes) after subsequent washing with sterilized distilled water, in pots of 6 inches size filled with soil from the farm of Central Institute of Medicinal and Aromatic Plants (CIMAP). The soil was sterilized by autoclaving at 121 °C for 2 h on three consecutive days. After emergence of seedlings, they were thinned to three plants per pot. Phosphorus was supplied as tricalcium phosphate [Ca5(PO4)3OH], an insoluble inorganic source of P at the doses of 0 mg kg–1 soil, 200 mg kg–1 soil and 375 mg kg–1 soil in 24 pots each. Every phosphate dose had eight treatments. 1) control; 2) AMF only ; 3) Bacillus polymyxa only ; 4) Azospirillum brasilense only ; 5) AMF + Bacillus polymyxa + A. brasilense; 6) AMF + Bacillus polymyxa; 7) AMF + Azospirillum brasilense; and 8) Bacillus polymyxa + Azospirillum brasilense. Growth conditions and harvest. The mycorrhizal fungus Glomus aggregatum propagated on palmarosa in the greenhouse for eight weeks was used for mycorrhizal treatment. Infected root fragments and rhizospheric soil containing 20 spores g–1 soil were used. Ten g of mycorrhizal inoculum was placed in each pot 2 cm below the surface of the soil. In controls AMF was not inoculated. Sterile water was used for watering. The plants were harvested after 90 days and roots were carefully cleaned under tap water. Visual quantification of root colonization and number of spores. The root samples were cleared using 10% KOH for 1 hour at 90 °C and stained with trypan blue in lactoglycerol as described by Phillips and Hayman (1970). The percentage of infected root length was evaluated by the grid line intersect method of Giovannetti and Mosse (1980). Spores were isolated from the soil samples by wet sieving and decanting method (Gerdemann and Nicolson 1963). 300 g soil was suspended in 1 l of water, shaken vigorously; at the settlement of soil particles the suspension was decanted together with the spores through an arrangement of different mesh sizes (500, 250, 100 and 50 µm). The 146
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materials of the sieves were collected in a beaker. Dry weight of root and shoot was determined after drying at 60°C for 72 h. Analysis of phosphatase activity and P content. The P contents of shoots and roots were determined by the vanadomolybdo-phosphoric yellow colour method in nitric acid (Jackson 1973). Assay of acid and alkaline phosphatase activity was done by the modified method of Bergmeyer (1974). Activity of phosphatases is proportional to the amount of p-nitrophenol formed which gives a yellow colour with alkali. Tissue homogenates were prepared in ice-cold saline and centrifuged at 5000 g at 0°C for 15 minutes. For estimation of alkaline phosphatase activity and acid phosphatase activity the supernatant was incubated for 30 minutes with 1.0 ml of alkaline buffer substrate solution and acid buffer substrate, respectively. The reactions were stopped by adding excess alkali. Phosphatase activity was expressed as µmol mg–1 f wt min–1. The Phosphate solubilizing rhizosphere bacterium Bacillus polymyxa and the N2 fixing bacterium Azospirillum brasilense were cultured in nutrient broth (Beef extract 1.0 g, Yeast extract 2.0 g, Peptone 5.0 g, NaCl 5.0 g in 1 l, pH 6.8) and 2 ml per seedling were applied to the root surface. The bacterial suspension contained 108 CFU ml–1 adjusted by optical density. Statistics. Data were processed by variance analysis (ANOVA). Statistical significant differences were tested by the critical difference (C. D.) test at 1 and 5% level of probability.
Results Plant growth and oil content The shoot and root dry weight was significantly increased by inoculation with AM fungi irrespective of P addition (Table 1). The highest shoot dry weight was obtained in the G. aggregatum and B. polymyxa treatment at P1 level of phosphate where it was 17.8 g. In general for each treatment P1 level was better than P0 and P2 level. The shoot dry weight was increased by 64%, 34.4% and 16.3% on mycorrhizal inoculation at P0, P1 and P2 level as compared to noninoculated plants (Table 1). The dry root weight was also increased in mycorrhizal treatment by 32.4%, 118.8% and 97.6%, respectively as compared to control at P0, P1 and P2 levels of phosphate. The highest biomass was recorded in Bacillus polymyxa + A. brasilense at P1 level where it was 21.7 g 45.6% and 6.9% over control and G. aggregatum treatment, respectively at P1 level (Table 1). Total biomass of the treatment G. aggregatum + Bacillus polymyxa at P1 was 21.5 g which is next to the maximum 21.7 g (Table 1). Because of best results
Table 1. Effect of different treatments on growth, biomass, percent root colonization and spore number of palmarosa at three levels of tricalcium phosphate Treatments
Control Tr1 Tr2 Tr3 Tr4 Tr5 Tr6 Tr7
Height pot–1
Dry wt shoot
Dry wt root
Total dry wt
Spore no.
(cm)
(g pot–1)
(g pot–1)
(g pot–1)
(100 g–1 soil)
Root colonization (%)
P0
P1
P2
69.7 78.4 73.0 76.1 68.3 83.8 80.2 70.7
83.9 71.1 94.7 87.2 97.7 93.5 69.0 93.8 90.5 75.2 71.6 78.7 91.9 89.7 95.4 110.9
CD 5% 14.3 28.3 CD 1% 19.8 39.2
P0
P1
P2
P0
P1
P2
P0
P1
P2
P0
P1
P2
P0
P1
P2
7.5 12.3 13.9 10.3 13.0 16.0 10.9 9.5
12.5 16.8 13.8 15.5 12.5 17.8 16.3 15.5
8.6 10.0 13.0 17.1 12.2 14.5 13.0 12.5
2.38 3.15 3.23 3.63 1.96 3.87 3.7 3.23
1.6 3.50 4.27 3.23 3.12 3.68 2.52 6.15
2.49 4.92 4.37 3.1 2.37 3.15 3.42 2.2
9.1 15.5 17.2 13.9 14.9 19.9 14.6 12.7
14.9 20.3 18.0 18.7 15.6 21.5 18.9 21.7
11.1 14.9 17.4 20.2 14.5 17.7 16.4 14.7
– 337 – – 623 723 753 –
– 610 – – 823 740 797 –
– 530 – – 610 432 580 –
– 43.5 – – 51.5 73.5 75.2 –
– 78.7 – – 71.8 85.3 88.3 –
– 50.0 – – 46.8 27.0 30.3 –
21.5 0.83 0.95 0.92 0.24 0.43 0.31 29.8 1.15 1.32 1.28 0.34 0.59 0.43
0.79 0.99 1.1 31.1 31.4 41.8 4.17 8.92 8.0 1.11 1.38 1.47 47.1 47.6 63.5 6.32 13.5 12.1
Control = sterilized soil without any microbial inoculation; Tr1 = inoculation with G. aggregatum alone; Tr2 = inoculation with Bacillus polymyxa alone; Tr3 = inoculation with Azospirillum brasilense alone; Tr4 = inoculation with G. aggregatum + B. polymyxa + A. brasilense together; Tr5 = inoculation with G. aggregatum + B. polymyxa together; Tr6 = inoculation with G. aggregatum + A. brasilense together; Tr7 = inoculation with B. polymyxa + A. brasilense together. P0 = without tricalcium phosphate; P1 = 200 mg tricalcium phosphate/kg soil; P2 = 375 mg tricalcium phosphate/kg soil. CD = critical difference test at 5% and 1% probability level. Table 2. Effect of different treatments on phosphatase activity, percent P content of palmarosa at three levels and percent oil and geraniol content at P1 level of tricalcium phosphate. Treatments
Phosphatase activity (µm mg–1 fresh wt min–1) P0
Control Tr1 Tr2 Tr3 Tr4 Tr5 Tr6 Tr7 CD5% CD1%
P1
Percent P content P2
P0
P1
P2
Oil (%)
Geraniol (%)
P1
P1
Acid
Alk
Acid
Alk
Acid
Alk
Stem
Root
Stem
Root
Stem
Root
Leaf
Leaf
2.27 3.46 3.76 0.87 4.75 1.82 1.13 2.64
1.36 2.25 1.74 1.05 1.26 0.76 1.50 0.87
1.87 6.05 3.01 3.88 2.52 2.26 4.37 1.55
0.63 1.13 1.13 0.88 0.25 0.13 0.05 0.88
2.65 6.37 2.32 4.88 3.86 2.66 2.64 1.32
0.63 1.26 1.50 1.38 0.76 1.07 1.88 1.85
.085 .107 .145 .128 .042 .056 .078 .118
.019 .017 .025 .023 .037 .017 .044 .025
.086 .122 .106 .103 .167 .102 .104 .033
.034 .038 .053 .016 .094 .042 .027 .032
.053 .045 .042 .101 .075 .073 .106 .112
.048 .016 .013 .018 .052 .057 .037 .068
3.67-02 1.20 0.58 0.97 1.43 1.18 1.43 0.78
74.3 95.64 94.83 87.99 92.69 92.91 92.18 91.82
.005 .007
.004 .006
.003 .004
.004 .006
.004 .005
.003 .004
8.23-02 0.11
.028 .039
.054 .074
.064 .089
.032 .046
.042 .059
.082 .115
–
Control = sterilized soil without any microbial inoculation; Tr1 = inoculation with G. aggregatum alone; Tr2 = inoculation with Bacillus polymyxa alone; Tr3 = inoculation with Azospirillum brasilense alone; Tr4 = inoculation with G. aggregatum + B. polymyxa + A. brasilense together; Tr5 = inoculation with G. aggregatum + B. polymyxa together; Tr6 = inoculation with G. aggregatum + A. brasilense together ; Tr7 = inoculation with B. polymyxa + A. brasilense together. P0 = without tricalcium phosphate; P1 = 200 mg tricalcium phosphate/kg soil; P2 = 375 mg tricalcium phosphate/kg soil. CD = critical difference test at 5% and 1% probability level.
at P1 phosphate level the oil content was analysed qualitatively and quantitatively. Oil was found significantly increased over control in all the treatments but it was maximum in G. aggregatum + Azospirillum brasilense and G. aggregatum + Bacillus polymyxa + Azospirillum
brasilense where it was 19.2% higher than in G. aggregatum treatment (Table 2). Geraniol content was highest (95.6%) in G. aggregatum treatment which is 28.7% more than at control. It was 94.8% in Bacillus polymyxa treatment (Table 2). Microbiol. Res. 156 (2001) 2
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Phosphatase activity and P content Acid phosphatase activity was higher than alkaline phosphatase activity irrespective of the addition of tricalcium phosphate in all treatments (Table 2). Acid phosphatase activity was 4.75 µmol mg–1 f wt min–1 in G. aggregatum + Bacillus polymyxa + Azospirillum brasilense treatment at P0 level where it was 109.3% and 37.3% higher than in control and G. aggregatum treatment, while it was 6.05 and 6.37 µmol mg–1 f wt min–1 at P1 and P2 level in G. aggregatum treatment (Table 2). It was 140.4% higher than control in G. aggregatum treatment at P2 level. Percent P content of the shoot was higher than in the root in all the treatments irrespective of P addition (Table 2). P content of the shoot was maximum (0.167%) in G. aggregatum + Bacillus polymyxa + Azospirillum brasilense treatment at P1 level. It was 94.2% and 36.9% higher than in control and G. aggregatum treatment of the same level (Table2). Next to the maximum P content was 0.145% in the Bacillus polymyxa treatment at P0 level and it was 70.6% and 35.5% higher than in control and G. aggregatum treatment of the same level, respectively (Table 2). AMF colonization and sporulation The addition of tricalcium phosphate and inoculation with Azospirillum brasilense increased root colonization by Glomus aggregatum at both P0 and P1 level by 72.9% and 12.2%, respectively (Table 1). Root colonization by G. aggregatum was increased in tricalcium phosphate treatment P1 as compared to P0 by 80.9%; however, at P2 level it was increased by 14.9% as compared to P0 (Table 1). The spore number/100 g soil were increased at the P1 and P2 levels by 81% and 57.3% in only G. aggregatum inoculation as compared to the P0 treatment. It was further increased by the microbial addition of Bacillus polymyxa and Azospirillum brasilense. The maximum increase was in the combined inoculation with G. aggregatum, B. polymyxa and A. brasilense where it was 34.9% more in the P1 treatment than in the G. aggregatum only treatment (Table 1). The addition of tricalcium phosphate at the recommended dose of P1 was best for mycorrhizal colonization and spore number as compared to the P0 and P2 treatments.
Discussion Bacillus polymyxa and Azospirillum brasilense promoted root colonization in mycorrhizal association, confirming previous findings of Azcon-Aguilar et al. (1986), Sreenivasa and Krishnaraj (1992) and Toro 148
Microbiol. Res. 156 (2001) 2
et al. (1997). Omar (1998) found that addition of three microorganisms with rock phosphate resulted in highest growth and P content of wheat plants, followed by inoculation with Glomus constrictum and Aspergillus niger together. Root colonization by G. aggregatum was enhanced in the phosphate amended soil at P1 level as compared to P0, but it was not increased at P2 (Table 1). The improved phosphate uptake and growth of inoculated plants over uninoculated plants, where tricalcium phosphate was added to the soil, must have resulted from the microbial inocula in some way increasing the accessibility of plant to this insoluble form of phosphate (Kahiluoto and Vestberg 1998). This effect did not appear to be directly attributable to phosphate solubilization, because the percentage of P in the plants did not increase with increasing concentration of tricalcium phosphate. However, increased content at P1 compared to P0 also confirms that the insoluble source of phosphate is utilized by palmarosa plants with the help of AM inoculants (Table 2). This uptake of tricalcium phosphate could be due to both, increase in surface area and phosphatase activity of the roots. Root phosphatase activity was enhanced at P1 level of phosphate as compared to P0 (Table 2). Similarly, Toro et al. (1997) reported an increase in biomass and N as well as P accumulation in plant tissues by dual inoculation of Glomus intraradices and Bacillus subtilis. In the present study plants which were not inoculated with AM fungi but received a bacterial inoculum had more P content in their shoots at P0 level (Table 2). However, when all test microbes were inoculated together they helped in the uptake of P from ticalcium phosphate amended soil at P1 level (Table 2). This indicated that test bacteria may solubilize the endogenous P sources and tricalcium phosphate more easily than AM fungi (Table 2). But in combined inoculation AM mycelium plays a role in taking up P ions released from tricalcium phosphate, thereby maintaining a low soluble P concentration in the discrete soil microhabitats where the tricalcium phosphate is attacked by phosphate solubilizing bacteria, thus favouring a continuous and sustained P release. The enhanced effect of combined inoculation of G. aggregatum, Bacillus polymyxa and Azospirillum brasilense on uptake of tricalcium phosphate cannot be exclusively attributed to higher phosphatase activity or hyphal uptake as root growth was also enhanced (Table 1). Increase in root growth will also have favourable effect on the uptake of tricalcium phosphate. Compared to the P0 level of phosphate root growth was much higher at P1 level with the inoculation of B. polymyxa and A. brasilense (Table 1).
Dry weights of plants inoculated only with bacteria increased steadily with tricalcium phosphate concentration (Table 1). Two possible explanations are (i) the bacteria might affect plant growth by hormones that they synthesize. Such substances could influence the early stages of plant growth (Brown 1974), and (ii) some solubilization of phosphate by the bacteria cannot be excluded especially as the bacteria produce acids. Dry weight of mature palmarosa plants is strongly related to the amount of root colonization with AMF. Similar linear relationships between dry weight of plant shoots and percentage colonization of roots was found by Thompson (1994) for linseed colonized by AMF.
Acknowledgements The authors are grateful to Dr. Sushil Kumar, Director CIMAP Lucknow for facilities provided by him. The first author thanks the Council of Scientific and Industrial Research for awarding Research Associateship, during the tenure of which the present investigation was carried out. Thanks are also due to Mr. Srikant Sharma, for his help in statistical analysis of the data.
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