Phosphate solubilizing fungi in a Scottish upland soil

Mycol. Res. 95 (9): 1090-1093 (1991)

1090

Prinled in Greal Brilail!

Phosphate solubilizing fungi in a Scottish upland soil

D. JONES,! B. F. 1. SMITH/ M. J. WILSON AND B. A. GOODMAN 2 Division of Soil and Soil Microbiological Research l and Analytical Divisiorz 2, The Macaulay Land Use Research [nstilule, Craigiebuckler, Aberdeen AB9 2Qj, UK

In acid soils the availability of phosphate to plants is generally limited because of its fixation by free oxides and hydroxides of aluminium and iron, and the formation of insoluble metal phosphates. Although solubilization of inorganic calcium phosphates is known to be mediated by micro-organisms through their production of organic acids these forms are only of significance in alkaline soils. We now present evidence for the solubilization and utilization of iron phosphates by two soil fungi, isolated from an upland soil. One of these fungi has been identified as Tolypocladium geodes and this is the first report of the observation of this species in Scottish soil. The other species is a sterile green fungus producing dematiaceous mycelium and chains of chlamydospores.

Phosphate in soil which is unavailable to plants is said to be fixed. A proportion of the soluble phosphorus added to soils in the form of fertilizers always becomes fixed in some way (Hesse, 1971) and the cause depends on the soil type generally. Thus in acid soils, phosphorus becomes fixed by free oxides and hydroxides of aluminium and iron whilst in alkaline soils it is fixed by calcium. Phosphate solubilizing micro-organisms include species which are potential organisms for development as inoculants for the efficient utilization of phosphate resources. Phosphate solubilization by such micro-organisms is effected by the production of organic acids such as malic, oxalic, succinic, fumaric and citric (Caur, 1987). In comprehensive studies on soil bacteria dissolving mineral phosphate fertilizers, Louw & Webley (1959a; 1959b) found that none of the hundred or so bacterial isolates from the root region of oat plants which solubilized either calcium carbonate or di-calcium phosphates showed dissolving ability on agar media containing Cafsa rock phosphates (calcium phosphate), variscite (AlP0 4 • 2H2 0), strengite (FeP0 4 • 2H 2 0) or taranakite (2K 2 0. 3Al 20 3 . 5P 2°5' 26H 2 0). More recently Caur (1987) has isolated strains of bacteria, including Pseudomonas striata, Pseudomonas ralhonis and Bacillus polymyxa which are effective in the weathering and solubilization of insoluble phosphates such as phosphatic rocks and aluminium and iron phosphates. However, there is a paucity of reports of fungi capable of solubilizing iron phosphate in soil and, in particular, hit! and upland soils where pH is low and phosphate is in short supply to plants. This note describes the isolation and identification of iron phosphate-solubilizing fungal species using a soil dilution and agar plate method. The brown podsolic soil (Sourhope association, Sourhope series) was collected from the upland MLURI Research Station

(Sourhope), which is situated near Kelso in the Borders Region of Scotland (National Crid Reference: NT862204). The mineral soil from the Bh horizon was located below a 10-15 em organic layer and supported a predominantly Fescue (Festuca ovirza)-Bent (Agrostis tenuis) sward. The soil (1 g wet weight = 0'5 g dry weight) was suspended in sterile distilled water and aliquots from the subsequent soil dilutions incorporated in modified Melin Norkrans (MMN) agar (Marx, 1969) but with glucose substituted for sucrose and with the addition of streptomycin (30 IJg/ml) and biotin (10 IJg/ml); this procedure follows the soil dilution and agar plate method describeq by Johnson el al. (1959). Ammonium phosphate and potassium dihydrogen phosphate in the MMN agar were replaced by ammonium sulphate and potassium chloride respectively. One cm 3 of a stock solution (2'5 % w Iv in 0'5 % w/ v gum arabic) of colloidal iron phosphate (Tennessee Valley Authority, Wilson Dam, Alabama) was added to each plaStic Petri dish containing 1 cm 3 of soil suspension and 10 cm 3 agar (kept at 50°C). The plates were well agitated to give dispersal of the soil suspension and iron phosphate prior to allowing the agar to solidify. Plates were incubated at 25° for up to 4 weeks. Significant clearing of the agar, which showed solubilization of phosphate, occurred around colonies of two fungal species only, one of which is identified as TolypocIadium geodes W. Cams (Cams, 1971). The other has not been identified and is referred to as green sterile fungus because of its colour and lack of production of spores, despite exposure to near uv irradiation (information from Dr C. Hall, CAB. International Mycological Institute, Kew). It produces sterile dematiaceous mycelium and chains of chlamydospores. The formation of clear zones around T. geodes is illustrated in Figs 1 and 2. Colonies of T. geodes developed in 4 out of 5 plates at the

D. Jones, B. F. L. Smith, M.

J. Wilson and B. A. Goodman

1091 Table 1. Mean dry weight and phosphorus content (%) of mycelium of the asporogenous green fungus from MMN cultures with and without strengite after six weeks with soluble iron and after 12 weeks without soluble iron in media Culture with strengite -Fe inMMN

Culture without strengite +Fe inMMN

Mean dry wt 166'4 (mg) Phosphorus (%) n.d. n.d. = Not determined.

-Fe inMMN

+Fe inMMN 37

n.d.

Table 2. The pH change, phosphorus and iron content of filtrates from cultures of the green asporogenous fungus grown for 9 weeks at 25° in liquid MMN medium (with no soluble Fe). The data represent the mean of six replicate experiments

Culture with strengite

Culture without strengite

Fig. 1. Dilution agar plate incorporating soil suspension from

Sourhope soil and insoluble iron phosphate (the dilution factor was I: 20 000). Arrow shows fungal colony Tolypocladium geodes with clear zone, indicating solubilized phosphate.

Fig. 2. Agar plate with iron phosphate suspension inoculated at five points with 5 mm diam. 'plug' from culture of Tolypocladium geodes. Note clear zone (arrowed) around each plug (after 2 weeks at 25°). 1 :20 000 soil dilution but this organism did not occur on any of the five plates at the 1: 2000 dilution. No appreciable clearing of the agar occurred around any of the other fungal colonies or bacterial and actinomycete colonies on the plates. A modified Bunt & Rovira agar (Bunt & Rovira, 1955), with the addition of glucose (0'5 % w Iv), in which (NH4)zS04 replaced (NH 4)zHP0 4 and incorporating iron phosphate as sole source of phosphate, was used to selectively encourage bacterial growth but failed to give colonies with clear zones at soil dilution factors of 1: 20 000 and 1: 200 000 (2 and 3

Uninoculated Inoculated Uninoculated Inoculated Mycelium yield (mg dry wtml- 1) 4'6 Final pH Final P (ppm) 12 Final Fe (ppm) 0'8

1'0

2'2 2'7 13'3 17'5

5 9 <0'01

3'6

1'4 0'4

replicate agar dishes, respectively). Similarly, the same two media (modified Bunt & Rovira's and MMN) with ground Kola rock phosphate (calcium phosphate) incorporated as sole source of phosphate failed to give colonies of fungi, bacteria and actinomycetes with clear zones around their colonies. The same dilution factors and replicates were used. This does not mean that the soil micro-organisms, other than the two fungi, do not have the ability to solubilize the inorganic iron and calcium phosphates but rather, perhaps, that the method is not sufficiently sensitive to reveal them. Evidence of the ability of the sterile green fungus to actively solubilize iron phosphate in liquid media is given in Tables 1 and 2 and illustrated conclusively in Fig. 3. In Table 1 measurements of yield were performed with and without strengite (as sole source of phosphate) in the presence and absence of FeC1 3 . The dry weight of mycelial mats in flasks with strengite was twice that in control flasks without any source of phosphate. The percentage P, measured spectrophotometrically by the Murphy & Riley molybdenum blue method (Murphy & Riley, 1962) after fusion with sodium hydroxide (Smith & Bain, 1982), in the fungal mycelium from two flasks with strengite was about five times that in mycelium from flasks without strengite. Data in Table 2 confirms the ability of the sterile green fungus to actively solubilize strengite and also that little or no phosphate (by the above Murphy & Riley method) is present in the culture filtrate, indicating that it is incorporated into the fungal biomass. However, significant amounts of iron (detected spectrophotometrically by the orthophenanthroline method of Jeffrey, 1970) are present in the culture filtrates. 69-2

Phosphate dissolving fungi

1092

I I I

: x 100 I

I I I

B

Fig. 3. Solubilization of iron phosphate (strengite). Flask on right was

c

uninoculated.

Considerable difficulties were encountered in separating residual strengite from spores and mycelium of T. geodes, unlike the situation with the sterile green fungus which produced relatively few, but large, colonies in liquid media. Consequently, it was difficult. or impossible, to prove from mycelial dry weight determinations alone that T. geodes solubilized iron phosphate in liquid media. However, analysis of culture fluids did indicate a release of significant amounts of iron (but not phosphate) into the media - between 26 and 46 ppm from six replicate flasks. Because these fungi were active in dissolving iron phosphates but ineffective in the dissolution of calcium phosphate in agar plates, the fate of the iron with respect to the mycelium has also been investigated by using electron paramagnetic resonance (EPR) spectroscopy, a technique that is able to produce information on the chemical environments of a number of paramagnetic metal ions (Goodman & Cheshire, 1987). EPR spectra of freeze dried specimens of both fungi that had strengite in their growth media consist of two features (e.g. Fig. 4A) originating from Fe(III). These were essentially absent (i.e. < 1 % of the intensity of the weaker feature in Fig. 4A) in the spectra of the specimens that had not received any added iron. The feature with g = 2 arises from Fe(III) in a symmetrical environment and corresponds either to the free ion or to a mononuclear complex with a simple organic acid. In either case it must have come from a phase with a very low pH ( ~ 2) because of the strong tendency for Fe(III) to hydrolyse at higher pH values. The component with g ca 4'2 is typical of a range of Fe(III) chelates (e.g. Fe(III) EDTA), where the iron has an environment of rhombic symmetry. In the unidentified green sterile fungus, the iron forms were not uniformly distributed with the centre of the colony having a somewhat darker coloration than the periphery. EPR spectra of the darker regions (Fig. 4 B) showed an approximately 10fold increase in the g = 4'2 component and IO-fold decrease in the g = 2 component relative to the EPR spectra of the lighter regions. It appears, therefore, that uptake of iron occurs as a simple ion and that this is slowly incorporated into more complex chelates. It should be noted that the greater height of the g = 2 signal in Fig, 4 A does not necessarily mean that

D

100

200

300

400

Magnetic field (mT) Fig. 4. EPR spectra of (Al freeze dried specimen of the green sterile

fungus at room temperature, (B) as (A) but using the more denselycoloured inner regions of the samples, (C) as (B) but at 77 K, and (D) T. geodes in its natural state (not freeze-dried). All specimens were obtained from media to which strengite had been added as sole iron and phosphate sources. Spectra (A), (B) and (D) were recorded at a microwave frequency of 9'5 GHz whilst spectrum (e) was recorded at 9'1 GHz. Note g-values are obtained from the relationship hv = g[3B, where h is Planck's constant, [3 is the Bohr magneton, v is the microwave frequency and Bthe magnetic field. When v is in GHz and Bin Tesla, g = O'071449v/B. there is a greater amount of the free Fe(III) than the complex chelate because the g = 4'3 feature represents only a part of the spectrum of the latter; other transitions being highly anisotropic are not seen in the spectra of randomly oriented complexes such as occur in the present sample. At 77 K there was noticeable enhancement of the g = 4'2 component, relative to that with g = 2 (Fig. 4 C). Measurements were also performed on T. geodes in the undried state, because it was thought that the g = 2 signal may have arisen from the breakdown of complexes as a result of the lowering of pH during the freeze drying process. The EPR spectrum of the fungus from the solution with strengite (Fig. 4 D) again consisted of components with g = 2 and 4'2 as before. With this specimen, however, the g = 2 feature consisted of two components of different width. The narrower of these is similar to that from the freeze dried specimens, whilst the

D. Jones, B. F. L Smith, M. J. Wilson and B. A Goodman broader component probably corresponds to a partially hydrolysed ion, possibly Fe(OH)2+. This observation suggests that the pH in this specimen is somewhat higher than that experienced during the freeze drying procedure, but nevertheless is still low « 3). Our preliminary data indicate that there are iron phosphate solubilizing fungi in the upland soil examined. It would also appear that this is the first report of Tolypocladium geodes in Scottish soils. It has been reported to be antagonistic to other soil fungi in Sweden (Lundgren, Baath & Soderstrom, 1978).

We thank Mrs. Angela Norrie for technical assistance and Mr Donald Duthie for X-ray diffraction characterization of the phosphates used. We are also grateful to Drs M. A J. Williams, G. Hall and B. C. Sutton, C.AB. International Mycological Institute (I.M.I.), Kew, Surrey for examining (and identifying T. geodes) the fungi described here and Mr Donald McPhail for his help in EPR determinations.

REFERENCES Bunt. j. S. & Rovira, A. D. (1955). Microbiological studies of some subantarctic soils. journal of Soil Science 6, 119-128. Gams. W. (1971). Tolypoc/adium. eine Hyphomycetengattung mit geschwollenen phialiden. Persoonia 6, 185-191.

(Received for publication 8 October 1990 and in revised form 15 january 1991)

1093 Gaur. A. C. (1987). Abstract in 8th International Symposium on Environmental Biogeochemistry, Nancy, France. Goodman. B. A. & Cheshire, M. V. (1987). Characterization of iron-fulvic acid complexes using Mossbauer and EPR spectroscopy. The Science of the Total Environment 62, 229-240. Hesse, P. R. (1971). A Textbook of Soil Chemical Analysis. London: john Murray. jeffery, P. G. (1970). Photometric determination of iron in silicate rocks by 1.1O-phenanthroline. In Chemical Methods of Rock Analysis. pp. 278-281. Oxford: Pergamon Press. Johnson, L. F., Curl, E. A., Bond, J. H & Fribourg. H A. (1959). Methods for studying soil micro-flora-plant disease relationships, Minneapolis, u.s.A.: Burgess. Louw, H. A. & Webley, D. M. (1959a). The bacteriology of the root region of the oat plant grown under controlled pot culture conditions. journal of Applied Bac/eriology 22, 216-226. Louw, H. A. & Webley, D. M. (1959b). A study of soil bacteria dissolving mineral phosphate fertilizers and related compounds. journal of Applied Bacteriology 22. 227-233. Lundgren, B" Baath, E. & Soderstrom, B. E. (1978). Antagonistic effects of Tolypocladium species. Transactions of the British Mycological Society 70, 305-307. Marx, D. H. (1969). The influence of ectotrophic mycorrhizal fungi on the resistance of pine roots to pathogenic infections. 1. Antagonism of mycorrhizal fungi to root pathogenic fungi and soil bacteria. Phytopathology 59, 153-163. Murphy, ). & Riley, J. P. (1962). A modified single solution method for the determination of phosphate in natural waters. Analitica Chimica Ac/a 27, 31-36. Smith, B. F. L. & Bain, D. C (1982). A sodium hydroxide fusion method for the determination of total phosphate in soils. Communications ill Soil and Plant Analysis 13, 185-190.