Genetic Modification of Rhizobacteria for Enhanced Organic Acid Secretion and Phosphate Solubilization.
Department of Biochemistry, Faculty
of Science, M. S. University of Baroda, Vadodara – 390 002. INDIA
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Genetic
modification of rhizobacteria has been employed for enhancing plant growth
promotion. Organic acid secretion by soil bacteria, which is required for
phosphate solubilization, is associated with primary metabolism and is an
effect of the physiology of the bacteria in natural conditions. Therefore,
genetic modification to incorporate phosphate solubilizing ability into
rhizobacteria involves proper choice of the organic acid based on it’s
efficacy in soils as well as target genes.
Phosphate
deficiency for plants is determined by the nature of soils. Alkaline vertisols
and acidic alfisols are the major cultivated soils in India. Alkaline vertisols
are calcarious soils which have high buffering capacity whereas alfisols have
low buffering capacity. Mineral and organic phosphates are present as complexes
of metal ions. In Vertisols, phosphate is bound to Ca+2, Al+3
and Fe+3 whereas in Alfisols, phosphate is bound to Fe+3
and Al+3. Ca complexes of phosphate get solubilized when the pH
drops below 5.0. Indian alkaline vertisols require high amount of organic acids
for decreasing the pH of these soils to less than 5.0 (Gyaneshwar et al.. 1998). Citric and
oxalic acids are required at about 10 mM to release P from Indian vertisols,
which is lowest as compared to other organic acids studied. On the other hand,
Indian alfisols do not show release of phosphate upon addition of organic acids
or by addition of phosphate solubilizing microorganisms (PSMs). Addition of CaP
rich rock phosphates (RP) to acidic alfisols also did not enhance availability
of phosphate indicating that these soils efficiently fixed soluble phosphate.
Addition of RP along with oxalic acids in alfisols, however, facilitated the
release of phosphate.
PSMs in
the rhizosphere are postulated to improve the phosphate status of plants.
However, field experiments of PSM inoculations have not yielded consistent
increase in P status of plants. Reasons for the decreased efficacy of PSMs in
field conditions could be a result of many complex processes. In order to get
an insight, it is necessary to determine the ability of PSMs to secrete organic
acids and release P from soils under simulated field conditions. Many PSMs
which efficiently solubilize hydroxyapatitie and rock phosphate in laboratory
conditions could not release phosphate from alkaline vertisols even when
provided with other nutrients (Gyaneshwar et al.. 1998). Buffering capacity of alkaline vertisols requires much
higher levels of organic acids for P release. Screening of PSMs by modification
of conventional medium by incorporation of buffer (e.g. Tris- Cl pH 8.0)
resulted in the isolation of. Enterobacter asburiae PSI3 which colonizes
the rhizosphere of Pigeon pea (Gyaneshwar et al., 1999).
Nature and
amount of carbon sources available in bulk soil as well as in the rhizosphere
vary from soil to soil as well as depending on the plant variety. PSMs which
could utilize a variety of carbon sources and can effectively solubilize
phosphate under the amounts of carbon source available in the rhizosphere could
be more efficient as phosphate
biofertilisers. E asburiae PSI3 has glucose dehydrogenase which can act on mono- and
–disaccharides. E. asburiae PSI3 shows phosphate solubilization
phenotype at 75mM concentrations on glucose, xylose, arabinose, galactose,
mannose, maltose or cellobiose but in a mixture of these seven carbon sources,
15mM concentration of each are
sufficient for phosphate solubilization.
We have
studied the effects of genetic modifications in the P-solubilization ability of
gluconic acid secreting E. asburiae PSI3. Presence of plasmids of
medium and high copy number in E. asburiae PSI3 resulted in the loss of
P-solubilization ability. E. asburiae PSI3 containing pACYC184 plasmid retained phosphate
solubilization phenotype but the plasmid was unstable in the absence antibiotic
selection. It is necessary thus to either carry out genetic modifications in a
plasmid vector which is low copy number and is stable or to incorporate the
genes in the genomes.
Rhizobacteria
can be classified into two different categories based on glucose assimilation
pathways (i) glycolytic pathway and (ii)
Entner-Doudorof (ED) pathway. Glycolytic pathway is present Enterics and
Bacillus whereas ED pathway is present in Pseudomonas, Rhizobium, Azospirillum. Effect of
over-expression of citrate synthase and phosphoenol pyruvate genes could be
different in these two different categories of rhizobacteria. We are
investigating the effect of incorporation of cs and ppc genes in E. asburiae PSI3 and Pseudomonas
fluorescens.
REFERENCES
1. Gyaneshwar
P., Naresh Kumar, G. and Parekh, L. J. (1998) Effect of
buffering on the phosphate-solubilizing ability of microorganisms. World J.
Microbiol. Biotechnol. 14, 669-673.
2.
Gyaneshwar, P., Parekh, L. J., Archana, G., Poole, P. S., Collins, M.
D., Hutson, R. A. and Naresh Kumar, G. (1999) Involvement of a
phosphate starvation inducible glucose dehydrogenase in soil phosphate
solubilization by Enterobacter asburiae. FEMS Microbiol. Letters 171, 223-229.