Field and Greenhouse Trials Performed with Phosphate Solubilizing Bacteria and Fungi
E-mail: Antoun@rsvs.ulaval.ca
__________________
Soils
microorganisms are involved in a range of processes that affect P
transformation and thus influence the subsequent availability of phosphate to
plant roots (Richardson, 2001).
Free-living phosphate
solubilizing microorganisms (PSM) are always present in soils. The populations
of inorganic P-solubilizing microorganisms are sometimes very low, less than 102
cfu g-1 of soil as observed in a soil in Northern Spain (Peix et
al.,
2001). In four Quebec soils the number of root free PSM ranged from 2.5-to 3 x
106 cfu g-1 of soil and they represented from 26-to 46%
of the total soil microflora (Chabot et al., 1993). As observed with
other soil microbes the number of PSM is more important in the rhizosphere than
in non-rhizosphere soil (Kucey et al., 1989), and the number of
phosphate solubilizing bacteria is more important than that of fungi (Kucey,
1983). However, inoculation studies aimed are improving P nutrition in plants
involved bacteria and fungi, and commercially in Western Canada the phosphate
inoculant JumpStart (PhilomBios, Saskatoon, Sask.) sold for wheat, canola,
mustard and other legumes contain an isolate of Penicillium bilaii (http://www.philombios.ca/).
Rhizobacteria, are bacteria that aggressively colonize plant roots.
Plant growth promoting rhizobacteria (PGPR) are a very small portion of
rhizobacteria (2-5%) that promote plant growth (Antoun and Kloepper,
2001). PGPR use one or more of
direct or indirect mechanisms of action to improve plant growth and health.
These mechanisms can probably be active simultaneously or sequentially at
different stages of plant growth. P-solubilization, biological nitrogen
fixation, improvement of other plant nutrients uptake and phytohormone
production like indole-3-acetic acid are some examples of mechanisms that
directly influence plant growth. Some PGPR have the enzyme
1-aminocyclopropane-1-carboxylate (ACC) deaminase, which hydrolyses ACC, the
immediate precursor of ethylene in plants (Glick et al., 1995). By
lowering ethylene concentration in seedlings and thus its inhibitory effect,
these PGPR stimulate seedlings root length (Glick et al., 1999). Biological control of plant pathogens
and deleterious microbes, through the production of antibiotics, lytic enzymes,
hydrogen cyanide, and siderophores or through competition for nutrients and
space can improve significantly plant health and promote growth as evidenced by
increases in seedling emergence, vigor and yield (Antoun and Kloepper, 2001).
Induction of the systemic resistance against many pathogens, insect and
nematodes (Ramamoorthy et al., 2001; Zehnder et al., 2001) is also a
recent indirect mechanism of action of PGPR. All these traits that can be
present in PGPR, illustrate how it is complex and difficult to associate the
promotion of plant growth with P solubilization, and they explain in part the
reason of obtaining better responses from plant inoculated with a mixture of
PGPR. Furthermore, approximately two-thirds
of all land plants form the arbuscular mycorrhizal (AM) type of association
(Hodge, 2000). PGPR can promote mycorrhizal functioning. Recently for example,
Villegas and Fortin (2001) showed an interesting specific synergistic
interaction between the P solubilizing bacterium Pseudomonas aeruginosa and the AM fungus Glomus
intraradices. Transformed mycorrhizal carrot roots or G. intraradices external mycelium
and Pseudomonas aeruginosa solubilized more P from the sparingly soluble
tricalcium phosphate when combined than when each alone. No synergistic effect
was observed with the other two P-solubilizing bacteria tested (Pseudomonas
putida and Serratia plymuthica) showing the specificity of this interaction.
With the aim of developing a phosphate inoculant that will allow the use
of the different sparingly soluble P inorganic forms present in many Quebec
soils, and reduce the amounts of P fertilizers used with corn and lettuce, 69
bacteria and 23 fungi able to solubilize tricalcium phosphate were first
isolated from four different soils. Many organisms lost their P-solubilizing
ability after subculturing on media containing sparingly soluble P, and finally
31 bacteria and 14 fungi were retained. Among them, 10 bacteria and 3 fungi
produced large clarification halo zones on media containing Ca-P and exhibited
good growth on media containing Al-P or Fe-P as sole P sources. A maize inoculation trial with these
PSM was performed under greenhouse conditions in a silty clay loam soil (pH
6.00; organic matter 4.60%; available P and K 198 and 663 Kg ha-1).
After 3 weeks of growth, inoculation with the PSM tested did not produce any
significant effect at the P<0.05 level on maize shoots dry matter yield or
on shoots total P content. However, a 5 and 6% increases in dry matter yields
were observed with strains 22c of Enterobacter sp. and 24 of Pseudomonas sp. (significant
at P<0.11 and P<0.15 levels). The total P content of maize inoculated
with strains 22a and 22c of Enterobacter sp. and Pseudomonas sp. 24 was 8 to 11% higher than that of the
inoculated control (significant at the P<0.13 and P<0.10 levels). The
highest non-significant increases in maize shoots dry matter yield and total P
content (4%) were observed with the fungus Rhizopus sp. 68. Field inoculation trials of maize were
performed with bacteria 22a, 22c and 24 and the fungus isolate 68 in a silty
clay loam soil (pH 5.47; organic matter 4.99%; available P and K 222 and 347 Kg
ha-1). After 60 days of growth the height of maize plant inoculated
with all bacteria tested (22a, 22c or 24) was significantly (P<0.01) higher
as compared to the uninoculated control.
After 108 days growth, inoculation with bacteria causes 14 to 23 %
increases of maize shoot dry matter yield. However, only the 23 % increase observed
with strain 22c was significant at the P<0.01 level. Although maize plants
inoculated with the bacteria contained 7 to 20 % more total P these value were
not statistically significant. Under the field conditions used the fungus 68
did not show any significant effect.
The results obtained in these two assays suggest that a 3 weeks
inoculation trial under greenhouse conditions in non-sterile soils is probably
a short period. With inoculated maize grown in the field the absence of
significant effects on P concentration or plant total P suggest that traits
other than P-solubilization might be involved. In fact all the bacterial
strains tested are IAA producers and like fungus 68 they also produce
siderophores. Under similar field conditions lettuce inoculated with bacteria
22a and 24 and with fungus 68 produced more fresh matter yields (18%,
P<0.01; 14%, P<0.01 and 11%, P<0.03 respectively) as compared to
uninoculated controls after 48 days of growth.
Strains from the genera Pseudomonas, Bacillus and Rhizobium are among the most
powerful P solubilizers (Rodriguez and Fraga, 1999). We focused our interest on rhizobia, because these bacteria
well known for their beneficial symbiotic atmospheric nitrogen fixing symbiosis
with legumes, have an excellent potential to be used as PGPR with non
legumes (Antoun et al., 1998). In a test
performed with 266 strains belonging to different genera and species, 54% of
the strains were able to solubilize dicalcium phosphate. The highest P
solubization activity was obtained with two strains P31 and R1 of Rhizobium
leguminosarum bv. phaseoli. The two selected rhizobia were superior root colonizers
of maize and lettuce as compared to other P-solubilizing bacteria (Chabot et
al.,
1996 b). These rhizobia were
included in maize and lettuce field inoculation trials performed in 3 sites
varying in their available P content from poorly to very fertile soils (Chabot et
al.,
1996a). The P-solubilization effect seems to be the most important mechanism of
plant growth promotion in moderately to fertile soils, and was less effective
in poor soils. Mutants altered in their P-solubilization activities
(solubilizing significantly less P than the wild type) were used to determine
the importance of this trait in strain R1 (Chabot et al., 1998). The
results obtained confirmed that growth promotion by P-solubilization is less
effective in poor soils as observed under field conditions.
In order to study the mechanisms of actions involved in the mineral
phosphate solubilizing (Mps) activity by a tropical Penicillium rugulosum (Mps+) isolated from a
Venezuelan soil, two unique UV induced mutants were produced (Reyes et al. 1999 b): a mutant
with altered (Mps-) or amplified (Mps++) activity. The
use of these mutants allowed the identification of three possible P
solubilization mechanisms in P. rugulosum: production of gluconic acid,
of citric acid and the H+ pump (Reyes et al., 1999a). These
mechanisms are influenced by the N, P and C sources present in the culture
media. These mutants were also used to study the solubilization of phosphate
rocks and minerals (Reyes et al. 2001) and in maize inoculation assays (Reyes et
al.
2002). All P. rugulosum strains were able to stimulate the growth of maize
plants as indicated by 3.6 to 28.6% increases in dry matter yields. In the
presence of rock phosphate, P-uptake by maize plants inoculated with the two
mutants Mps++ and Mps- was not always in agreement with
their P-solubilizing phenotypes.
Results on plant inoculation with PSM found in the literature will also
be presented and discussed.
Antoun, H., and
Kloepper, J.W. 2001. Plant Growth promoting rhizobacteria. Encyclopedia of
Genetics. Brenner, S., and Miller, J.F. (Eds in chief) p. 1477-1480. Academic
Press.
Antoun, H.,
Beauchamp, C.J., Goussard, N., Chabot, R., and Lalande, R. 1998. Potential of Rhizobium and Bradyrhizobium
species
as plant growth promoting rhizobacteria on non-legumes: effect on radishes (Raphanus
sativus L.). Plant & Soil 204, 57-67.
Chabot, R.,
Beauchamp, C.J., Kloepper, J.W., and Antoun, H. 1998. Effect of phosphorus on
root colonization and growth promotion of maize by bioluminescent mutants of
phosphate solubilizing Rhizobium leguminosarum biovar phaseoli. Soil
Biol. Biochem. 30, 1615-1618.
Chabot, R., Antoun,
H., and Cescas, P. 1996 a. Growth promotion of maize and lettuce by
phosphate-solubilizing Rhizobium leguminosarum bv. phaseoli. Plant &
Soil 184, 311-321.
Chabot, R., Antoun,
H., Kloepper, J.W., and Beauchamp, C.J. 1996 b. Root colonization of maize and
lettuce by bioluminescent Rhizobium leguminosarum biovar phaseoli. Appl. Environ.
Microbiol. 62, 2767-2772.
Chabot, R., Antoun,
H., and Cescas, P. 1993. Stimulation de la croissance du maïs et de la
laitue romaine par des microorganismes dissolvant le phosphore inorganique. Can. J.
Microbiol. 39, 941-947.
Glick, B.R.,
Patten, C.L., Holgin, G., and Penrose, D.M. 1999. Biochemical and Genetic
mechanisms used by plant growth promoting bacteria. Imperial College Press,
London, 267 p.
Glick, B.R.,
Karaturovic, D.M., and Newell, P.C. 1995. A novel procedure for rapid isolation
of plant growth promoting pseudomonas. Can. J. Microbiol. 41,533-536.
Hodge, A. 2000. Microbial ecology of the arbuscular mycorrhiza. FEMS Microbiol. Ecol. 32, 91-96.
Kucey, R.M.N., 1983. Phosphate-solubilizing
bacteria and fungi in various cultivated and virgin Alberta soils. Can. J.
Soil Sci. 63, 671-678.
Kucey, R.M.N.,
Janzen, H.H., and Leggett, M.E. 1989. Microbially mediated increases in
plant-available phosphorus. Ad.
Agronomy 42, 199-228.
Peix, A., Rivas-Boyero, A.A., Mateos, P.F.,
Rodriguez-Barrueco, C., Martinez-Molina, E., and Velazquez, E. 2001. Growth promotion of
chickpea and Barley by a phosphate solubilizing strain of Mesorhizobium
mediterraneum under growth chamber conditions. Soil Biol. Biochem. 33, 103-110.
Ramamoorthy, V.,
Viswanathan, R., Raguchander, T., Prakasam, V., and Samiyappan, R. 2001. Induction of systemic resistance by
plant growth promoting rhizobacteria in crop plants against pests and diseases.
Crop Prot. 20, 1-11.
Reyes, I., Bernier,
L., and Antoun, H. 2002. Rock phosphate solubilization and colonization of
maize rhizosphere by wild and genetically modified strains of Penicillium
rugulosum. Microbial Ecology (In Press).
Reyes, I.,
Baziramakenga, R., Bernier, L.,
and Antoun, H. 2001. Solubilization of phosphate rocks and minerals by a
wild type strain and two UV-induced mutants of Penicillium rugulosum. Soil Biol.
Biochem. 33, 1741-1747
Reyes, I., Bernier,
L., Simard, R.R., and Antoun, H. 1999 a. Effect of nitrogen source on the
solubilization of different inorganic phosphates by an isoalte of Penicillium
rugulosum and two UV-induced mutants. FEMS Microbiol. Ecol. 28, 281-290.
Reyes, I., Bernier,
L., Simard, R.R., Tanguay, P., and Antoun, H. 1999 b. Characteristics of
phosphate solubilization by an isolate of a tropical Penicillium rugulosum and two UV-induced
mutants. FEMS
Microbiol. Ecol. 28, 291-295.
Richardson, A.E.
2001. Prospects for using soil microorganisms to improve the acquisition of
phosphorus by plants. Aust.
J. Plant Physiol. 28, 8797-906.
Rodriguez, H., and Fraga, R. 1999. Phosphate
solubilizing bacteria and their role in plant growth promotion. Biotech. Adv. 17, 319-339.
Villegas, J., and
Fortin, J.A. 2001. Phosphorus solubilization and pH changes as a result of the interaction between soil
bacteria and arbuscular mycorrhizal fungi on a medium containing NH4+
as nitrogen source. Can. J. Bot. 79, 865-870.
Zehnder, G.W., Murphy, J.F., Sikora, E.J.,
and Kloepper, J.W. 2001. Application of rhizobacteria for induced resistance. Eur.
J. Plant Pathol. 107,
39-50