Solubilization of phosphates and micronutrients by the plant-growth-promoting and biocontrol fungus trichoderma harzianum rifai 1295-22

Abstract

We investigated the capability of the plant-growth-promoting and biocontrol fungus Trichoderma harzianum Rifai 1295-22 (T-22) to solubilize in vitro some insoluble or sparingly soluble minerals via three possible mechanisms: acidification of the medium, production of chelating metabolites, and redox activity. T-22 was able to solubilize MnO2, metallic zinc, and rock phosphate (mostly calcium phosphate) in a liquid sucrose-yeast extract medium, as determined by inductively coupled plasma emission spectroscopy. Acidification was not the major mechanism of solubilization since the pH of cultures never fell below 5.0 and in cultures containing MnO2 the pH rose from 6.8 to 7.4. Organic acids were not detected by high-performance thin-layer chromatography in the culture filtrates. Fe2O3, MnO2, Zn, and rock phosphate were also solubilized by cell-free culture filtrates. The chelating activity of T-22 culture filtrates was determined by a method based on measurement of the equilibrium concentration of the chrome azurol S complex in the presence of other chelating substances. A size exclusion chromatographic separation of the components of the culture filtrates indicated the presence of a complexed form of Fe but no chelation of Mn. In liquid culture, T. harzianum T-22 also produced diffusible metabolites capable of reducing Fe(III) and Cu(II), as determined by the formation of Fe(II)-Na2-bathophenanthrolinedisulfonic acid and Cu(I)-Na2-2, 9-dimethyl-4,7-diphenyl-1,10-phenanthrolinedisulfonic acid complexes. This is the first report of the ability of a Trichoderma strain to solubilize insoluble or sparingly soluble minerals. This activity may explain, at least partially, the ability of T-22 to increase plant growth. Solubilization of metal oxides by Trichoderma involves both chelation and reduction. Both of these mechanisms also play a role in biocontrol of plant pathogens, and they may be part of a multiple-component action exerted by T-22 to achieve effective biocontrol under a variety of environmental conditions.

FIG. 1

Concentrations of Fe, Cu, Mn, and Zn in T-22 cultures (●) and in control flasks (○) of an SY medium supplemented with CuO, Fe₂O₃, MnO₂, and metallic zinc, respectively. The dashed line shows the pH of the cultures. Error bars indicate standard deviations of three replicate determinations.

FIG. 2

Solubilization of rock phosphate. Concentrations of P and Ca in the SY medium in the presence (+) or absence (−) of T-22 are shown. Error bars indicate standard deviations of three replicate determinations.

FIG. 3

Solubilization of CuO, Fe₂O₃, MnO₂, metallic zinc (Zn), and rock phosphate (Rp) by cell-free culture filtrates of T-22 grown in SY medium (SYT) in comparison with noninoculated medium filtrates (SY). Filtrates were either untreated, autoclaved (121°C for 20 min), or digested with protease K (50 μg/ml for 4 h at 37°C). Inoculated SY medium not supplemented with any of the above minerals provided the basic levels of the various elements in culture filtrates. Error bars indicate standard deviations of three replicate determinations.

FIG. 4

GPC of the culture filtrates of T-22 grown on SY medium (◊) in the presence of Fe₂O₃ (top) or MnO₂ (bottom). The exit of free Fe²⁺ or Mn²⁺ ions occurred at an elution volume greater than 50 ml (○). When the filtrate SYFe was applied along with 0.1 mM FeSO₄ · 7 H₂O (■), an increase in the height of two peaks at 25 and 40 ml (arrows) occurred as a result of the complexed state of the Fe ions.

FIG. 5

Fe(III) and Cu(II) reduction by T-22 culture filtrates. The amount of Fe(II)-BPDS or Cu(I)-BCDS was determined spectrophotometrically by the increase of the absorbance (540 and 492 nm, respectively) after 4 h of incubation at 37°C (for more details, see the text).