Phosphate Solubilization by Rhizobium Isolates from Crotalaria Species
Rhizobium species isolated from Crotalaria species (C. junceaC. laburnifoliaC. retusa and C. verrucosa) were tested for their ability to solubilize tricalcium phosphate (TCP). Among these four species, Rhizobium sp. isolated from Crotalaria retusa and C. verrucosa showed zone of TCP solubilization on Pikovskaya`s agar medium. Highest solubilization index (2.70) was observed in Rhizobium sp. from C. retusa. Effect of different carbon and nitrogen sources was also studied. Among the carbon sources, glucose was found to be best carbon source for TCP solubilization. Presence of highest concentration (3%) of glucose enhanced TCP solubilization. Among the nitrogen sources, ammonium sulphate was the best promoter for the process. Addition of low levels (0.5Âµg mL-1) of EDTA increased TCP solubilization.
Plant Growth Promoting Rhizobacteria (PGPR) are involved with host plants in
mutual interaction. They promote plant growth by the production of phytohormones,
solubilization of insoluble phosphates and biocontrol of host plant diseases
or improvement of plant nutritional status (Deshwal et
al., 2003). Rhizobia are the first group of bacteria, which are attributed
to the ability of PGPR to solubilize insoluble phosphates (Johri
et al., 2003). So far only a few reports on phosphate solubilizing
ability of Rhizobium strains (Halder et al.,
1990, 1991; Halder and Chakrabarty,
1993; Rivas et al., 2006; Daimon
et al., 2006).
Crotalaria is one of the largest genera of Fabaceae with more than 500
species commonly occurring in diverse climatological situations (Allen
and Allen, 1981). Some Crotalaria species are of great agronomic
interest, since they are used as used as green manure to improve soil fertility
or control nematode populations in infested soils (Sy et
al., 2001). Because of its importance the present study was taken up
to investigate phosphate solubilizing ability of Rhizobium isolates from
commonly occurring Crotalaria species (C. juncea L., C. laburnifolia
L., C. retusa L. and C. verrucosa L.) and the ability of these
isolates to solubilize tricalcium phosphate (TCP) in the presence of different
carbon sources, nitrogen sources and cell affecting agents was also studied.
MATERIALS AND METHODS
Organism and Phosphate Solubilization
Rhizobium species isolated from root nodules of C. juncea,
C. laburnifolia, C. retusa and C. verrucosa on Yeast Extract
Mannitol Agar (YEMA) medium. The identification of the isolates as Rhizobium
was confirmed by plant infection test (Vincent, 1970).
The phosphate solubilizing ability of the Rhizobium species was tested
on Pikovskayas agar (Pikovskaya, 1948) containing
tricalcium phosphate (TCP) as insoluble phosphate source. The formed halo zone
surrounding the colony revealed phosphate solubilization and was expressed in
solubilization index (Arun and Sridhar, 2005). The Rhizobium
species, which formed zone of solubilization on agar medium, was further tested
in Pikovskayas broth having initial pH 7.0.
After incubation the final pH and phosphorus (P) content of the medium were
also estimated in culture supernatant (Subba Rao, 1993).
Effect of Carbon and Nitrogen Sources
Effect of different carbon sources (1%) on phosphate solubilization was
tested by replacing glucose with 14 carbon compounds (mannitol, galactose, fructose,
rhamnose, arabinose, trehalose, inositol, raffinose, sucrose, maltose, mannose,
dulcitol, ribose and xylose) sterilized separately and added aseptically to
the medium before sterilization. The effect of different concentrations (1.0,
1.5, 2.0, 2.5 and 3.0%) of most effective carbon source was also studied. Effect
of different nitrogen sources (0.1%) on TCP solubilization was studied by replacing
ammonium sulphate with five different nitrogen sources (sodium nitrate, potassium
nitrate, calcium nitrate, asparagine and urea).
Effect of EDTA
The chelating effect of EDTA on TCP solubilization was tested by adding
different concentrations (0.5, 1.0, 2.0, 2.5 and 3.0 μg mL-1)
of EDTA to the medium.
The data were statistically analyzed by correlation coefficient and ANOVA
(two way classification technique), using Statistica software.
RESULTS AND DISCUSSION
The Rhizobium species isolated from C. juncea, C. laburnifolia,
C. retusa and C. verrucosa were fast growers as they produced
acid in YEM broth and colony diameter was greater than 2.0 mm. Among these species,
the Rhizobium species from C. retusa and C. verrucosa formed
clear zones around the colonies on Pikovskayas agar medium after 3 days
of incubation and it gradually increased up to 9 days. The colony diameter is
almost similar throughout the incubation period. Solubilization Index (SI) of
these species on solid media ranged between 2.4 and 2.7 (Table
1). Maximum SI was observed in Rhizobium sp. from C. retusa (2.7).
In liquid medium maximum solubilization was also recorded in this species (840
|| Solubilization of tricalcium phosphate by Rhizobium
species from Crotalaria retusa and C. verrucosa
|Solubilization Index (SI) = Diameter of the colony+Halo zone/Diameter
of the colony; *: Correlation coefficient (r); Between pH and P liberated
= -1; Between zone of solubilization and P liberated = 1
A fall in pH accompanied phosphate solubilization, due to production of organic
acids was observed up to 9 days, but there is sudden increase in pH after 9
days. This may be due to utilization of organic acids produced during phosphate
solubilization by the strains as reported earlier in Pseudomonas (Dave
and Patel, 1999). The data were statistically analyzed using correlation
coefficient and it was found that there is positive correlation between zone
of solubilization on agar medium and liberated P in broth and negative correlation
between liberated P and final pH of the medium.
Effect of different carbon sources (1%) on phosphate solubilizing activity
by the Rhizobium sp. revealed that the maximum solubilization occurred
in glucose containing medium (Table 2). That the glucose was
the best carbon source for phosphate solubilization was also reported earlier
in Bradyrhizobium sp. isolated from Cicer arietinum (Halder
et al., 1991). Maximum decrease in pH was recorded in glucose containing
medium. In other carbon sources little decrease in pH and no correlation between
acidic pH and P liberated were observed. The relative efficiency of Rhizobium
spp. on different carbon sources could be due to the organic acids secreted
by the isolates rather than the total acidity (Dave and
Patel, 1999). The data on effect of carbon sources on TCP solubilization
by Rhizobium spp. were statistically analyzed using analysis of variance
(two way classification technique) and it was found that variations due to both
carbon sources and Rhizobium spp. were found to be significant and the
Rhizobium spp. differ significantly with different carbon sources.
As the glucose at 1% concentration (as in Pikovskayas medium) supported maximum solubilization of TCP, effect of different concentrations of glucose was studied. Rhizobium sp. from C. retusa and C. verrucosa showed maximum solubilization at 3.0% concentration of glucose (Table 3). Linear increase in TCP solubilization was observed as the concentration of glucose increased. This may be due to greater acidity produced at 3.0% concentration of glucose, which is an important factor in phosphate solubilization. The variation in the effect of different concentrations of glucose is found to be statistically significant.
|| Effect of different carbon sources on TCP solubilization
|*: Significant at 1%
|| Effect of different concentrations of glucose on TCP solubilization
|*: Significant at 1%
|| Effect of nitrogen sources on TCP solubilization
|*: Significant at 1%
|| Effect of EDTA on TCP solubilization
|*: Significant at 1%
Effect of different nitrogen sources on TCP solubilization revealed that maximum
solubilization of TCP occurred in ammonium sulphate containing medium (Table
4) and reduced the pH of the medium. Further it was observed that the inorganic
nitrogen sources supported better solubilization of TCP than organic nitrogen
sources. This could be due to the production of inorganic acids by proton exchange
mechanism in presence of NH4+ cause accelerated phosphate
solubilization (Halder et al., 1991). Statistical
analysis showed that the effect of different nitrogen sources on TCP solubilization
was also found to be significant.
The effect of different concentrations of EDTA (0.5, 1.0, 2.0, 2.5 and 3.0
μg mL-1) revealed that the addition of 0.5 μg mL-1
EDTA to the standard medium results in significant increase of TCP solubilization,
but increasing concentration of EDTA had little effect on TCP solubilization
(Table 5). This could be due to the chelation of Ca2+
produced during TCP solubilization as reported in Bradyrhizobium sp.
(Halder et al., 1991). The effect of EDTA on
TCP solubilization was found to be statistically significant.
Only a few reports on phosphate solubilization have been reported earlier (Halder
et al., 1990; 1991; Halder
and Chakrabarty, 1993; Rivas et al., 2006;
Daimon et al., 2006) and studies were confined
to only limited number of hosts and a single species of Rhizobium from
a single host. From this it may concluded that, in addition to symbiotic nitrogen
fixation, some species of Rhizobium can also involved in phosphate solubilization.
Furthermore, the capacity of phosphate solubilization by the Rhizobium
species can be exploited as PGPR.
Allen, O.N. and E.K. Allen, 1981.
The Leguminosae a Source Book of Characteristics, Uses and Nodulation. University of Wisconsin Press, Masison, WI
Arun, A.B. and K.R. Sridhar, 2005.
Growth tolerance of rhizobia isolated from Sand Dune legumes of Southwest coast of India. Eng. Life Sci., 5: 134-138.Direct Link |
Daimon, H., K. Nobuta, M. Ohe, J. Harada and Y. Nakayama, 2006.
Tricalcium phosphate solubilization by root nodule bacteria of Sesbania cannabina
and Crotalaria juncea
. Plant Prod. Sci., 9: 388-389.Direct Link |
Dave, A. and H.H. Patel, 1999.
Inorganic phosphate solubilizing soil pseudomonads. Indian J. Microbiol., 39: 161-164.
Deshwal, V.K., R.C. Dubey and D.K. Maheshwari, 2003.
Isolation of plant-growth promoting strains of Bradyrhizobium
sp.) with biocontrol potential against Macrophomina phaseolina
causing charcoal rot of peanut. Curr. Sci., 84: 443-448.Direct Link |
Halder, A.K., A.K. Mishra, P. Bhattacharya and P.K. Chakrabarty, 1990.
Solubilization of inorganic phosphate by Rhizobium
. Indian J. Microbiol., 30: 311-314.
Halder, A.K., A.K. Mishra, P. Bhattacharya and P.K. Chakrabarty, 1991.
Solubilization of inorganic phosphates by Bradyrhizobium
. Indian J. Exp. Biol., 29: 28-31.
Halder, A.K. and P.K. Chakrabarty, 1993.
Solubilization of inorganic phosphate by Rhizobium
. Folia Microbiol., 38: 325-330.Direct Link |
Johri, B.N., A. Sharma and J.S. Virdi, 2003.
Rhizobacterial diversity in India and its influence on soil and plant health. Adv. Biochem. Eng. Biotechnol., 84: 49-89.Direct Link |
Pikovskaya, R.I., 1948.
Mobilization of phosphorous in soil connection with the vital activity of some microbial species. Microbiologia, 17: 362-370.
Rivas, R., A. Peix, P.F. Mateos, M.E. Trujillo, E. Martinez-Molina and E. Velazquez, 2006.
Biodiversity of populations of phosphate solubilizing rhizobia that nodulates chickpea in different Spanish soils. Plant Soil, 287: 23-33.CrossRef | Direct Link |
Subba Rao, N.S., 1993.
Biofertilizers in Agriculture and Forestry. 3rd Edn., Oxford and IBH Publishing Co. Pvt. Ltd., Oxford, UK
Sy, A., E. Giraud, P. Jourand, N. Garcia and A. Willems et al
bacteria nodulate and fix nitrogen in symbiosis with legumes. J. Bacteriol., 183: 214-220.CrossRef | Direct Link |
Vincent, J.M., 1970.
A Manual for the Practical Study of Root-Nodule Bacteria. IBP Handbook No. 15, Blackwell Scientific Publications Ltd., Oxford, UK., ISBN-13: 9780632064106, Pages: 164