2.10.7
SOLUBILIZATION OF PHOSPHATES AND MICRONUTRIENTS BY THE GROWTH-PROMOTING AND BIOCONTROL FUNGUS TRICHODERMA HARZIANUM RIFAI STRAIN 1295-22
C ALTOMARE1, WA NORVELL2, T BJÍRKMAN 3 and GE HARMAN3

1Istituto Tossine e Micotossine da Parassiti Vegetali, CNR, Bari, 70125, Italy; 2US Plant, Soil and Nutrition Laboratory, USDA-ARS, Ithaca, NY 14853, USA; 3Departments of Horticultural Sciences and Plant Pathology, Cornell University, Geneva, NY 14456, USA

Background and objectives
The nutritional state of plants greatly influences their susceptibility to plant pathogens. The correlation between availability of nutrients (especially micronutrients) to plants and incidence of some major plant diseases has long been known [1]. Members of the genus Trichoderma have been used for biological control of plant diseases. A phenomenon often associated with the use of biocontrol strains of Trichoderma is the increase of plant growth, even in the absence of any detectable disease and in sterile soil. We investigated the capability of Trichoderma harzianum Rifai strain 1295-22 (T-22), an effective biocontrol agent of several soilborne plant diseases, including Pythium, Rhizoctonia and Fusarium root rots, to solubilize in vitro some insoluble or sparingly soluble minerals. The solubilization of nutrients was studied with regard to three possible mechanisms, i.e. acidification of the medium, production of chelating metabolites, and redox activity.

Results and conclusions
Cultures of T-22 on a liquid sucrose-yeast extract medium supplemented with insoluble or sparingly soluble minerals were sampled daily over 5 ;days of growth. The concentrations of Cu, Fe, Mn, Zn, Ca and Fe in culture filtrates were quantified by inductively-coupled plasma emission spectroscopy (ICP). T-22 was able to solubilize MnO2, Zn metal, and rock phosphate (mostly calcium phosphate) as determined by the increase of Ca concentration in the medium, (while the P concentration decreased owing to the high efficiency of T-22 in the uptake of this element). In cultures supplemented also with rock phosphate, the Fe content significantly increased up to fivefold (0.1 ;mg/ml) the level in the control flasks. In all cultures the pH values never fell below 5.0, and in cultures containing MnO2 the pH increased from 6.8 to 7.4. Therefore, acidification does not appear to have a major role in solubilization. In addition, Fe2O3, MnO2, Zn metal and rock phosphate were all solubilized by cell-free culture filtrates.

The chelating activity of T-22 culture filtrates was assayed by the Cu-CAS (chrome azurol S) method [2]. Chelating activity was first detected in 3-day-old culture filtrates and its level did not change in the course of further growth of the culture. As the Cu-CAS assay indicated only that chelating metabolites were produced in culture, but did not provide any information about the actual state (free or chelated) of metal ions in the culture filtrates, we carried out the gel-permeation chromatographic separation of the components of the culture filtrates. In a preliminary experiment, this method proved to be suitable to separate free Fe2+ ions from EDTA chelated Fe3+. The chromatogram of the filtrates from cultures supplemented with MnO2 showed one single exit peak of Mn at the elution time of free ions (Mn2+), while the chromatogram of the filtrates from cultures supplemented with Fe2O3 showed two additional peaks, suggesting a complexed state of Fe ions. In liquid culture, T. ;harzianum strain T-22 also produced diffusible metabolites capable of reducing Fe(III) and Cu(II), as determined by the formation of Fe(II)-BPDS and Cu(I)-BCDS complexes [3]. As far as we know, this is the first report of the capability of a i>Trichoderma strain to solubilize insoluble or sparingly soluble minerals. The Trichoderma system for solubilizing nutrients involves both chelation and reduction. Both of these mechanisms are known to play a role in biocontrol of plant pathogens [4,5] and might be part of a multiple component action exerted by T-22 in order to achieve an effective biocontrol under a variety of environmental conditions.

References
1. Graham RD, Webb MJ, 1991. In: Welch RM, eds. Micronutrients in Agriculture. Madison: Soil Science Society of America, pp. 329-370.
2. Shenker M, Hadar Y, Chen Y, 1995. American Journal of the Soil Science of America 59, 1612-1618.
3. Norvell WA, Welch RM, Adams ML, Kochian LV, 1993. Plant and Soil 155/156, 123-126.
4. Baker R, Elad Y, Sneh B, 1986. In: Swinburne TR, ed. Iron Siderophores and Plant Diseases. Ney York: Plenum Publishing Corp., pp. 77-84.
5. Huber DM, McCay-Buis TS, 1993. Plant Disease 77, 437-447.