GB2142933A - Method of culture - Google Patents

Abstract

It has been a problem that the beneficial VA mycorrhizal fungi respond very poorly to attempts to culture them non-biotrophically (in vitro), in the absence of the plant roots) which normally serve as the fungal host. It has now been found, however, that these fungi can be cultured on a growth medium containing an inorganic sulphur compound, preferably in a reduced form, containing a sulphur atom of valency less than 6. Potassium sulphite is particularly effective.

Description

SPECIFICATION Method of culture of VA mycorrhizal fungi This invention is in the field of soil microbiology and relates to a method of culture of VA (vesicular-arbuscular) mycorrhizal fungi.

VA mycorrhizal fungi are beneficial fungi which infect the feeding roots of plants and stimulate uptake of phosphorus from the soil. Hyphae of the fungi grow outwardly from the root well beyond the phosphate depletion zone, (the zone from which the available phosphate has already been consumed by the plant). The fungi have been grown well only in association with live host roots. Thus, inocula of the fungi for use in infecting plants have been produced only on a small scale from the roots of previously inoculated plants, see e.g. UK Patent 2043688B (National Research Development Corporation).

From a commercial viewpoint, the inoculum must be free from pathogens (both fungal and nematode) and of uniform quality. This is difficult to achieve in practice and the quality control requires rigorous, time-consuming, monitoring and testing of inocula. Therefore, axenic culture (i.e. "pure" culture, free of contamination by other biological species) of these has become a subject of interest to some commercial firms who foresee the possibilities of producing highquality inoculum under controlled conditions.

When mycorrhizal roots are chopped up and placed in water or on agar medium, fungal hyphae begin to regenerate from the cut ends. The new growth is from the intercellular hyphae and it occurs as readily from the proximal as from the distal end of the root. By providing boiled hemp seeds as an intermediate substrate for growth, J T Barrett, Recent Advances in Botany 2, 1725 (1961) was able to isolate a mucoraceous fungus from soil-grown roots which could be cultured on hemp seed extract agar with or without malt extract. Although not giving experimental details, he stated that this fungus could reinvade a test plant giving typical VA mycorrhizal infections, which is the criterion of successful culture. J W Gerdemann, Annual Review of Phytopathology, 6, 397 (1968) was unable to repeat this observation using the same isolates or others obtained in a similar way.

Surface-sterilised resting spores have generally been used to try to initiate cultures of VA mycorrhizal fungi. The approach has been to study the effect of various media or individual nutrients on the growth of hyphae from germinating spores. This presupposes that the nutrient requirements of the fungus when it grows non-symbiotically without the spore will be the same as those of the germ tubes.

J W Gerdemann, Mycologia 47, 619 (1955), looked at the effect of several media on a VA mycorrhizal fungus of the Gigaspora species and found that only water containing pieces of hemp seed or non-sterile organic matter promoted hyphal extension. Typical fungal media such as potato dextrose agar, cornmeal agar and malt extract did not support growth.

The effect of nutrients on the growth of hyphae from G. mosseae has also been examined by B Mosse, Transactions of the British Mycological Society 42, 273 (1959) and by the same author in the Annual Report of the Rothamsted Experimental Station, Harpenden, Herts, for 1969, Part I, 97(1970). Of the carbon sources tested, several organic acids promoted growth, in particular, oxaloacetic, pyruvic, acetic, citric and tartaric but carbohydrates such as maltose, cellulose, sucrose and glucose had an inhibitory effect on germ tube growth. Trehalose and mannitol, sugars which are commonly found in fungi, had no effect. No nitrogen source was found which radically improved growth. In another series of experiments using soil extract as a basic medium, the effect of several sulphur-containing amino acids, amino acid mixtures and proteins were tested.Of these only bovine serum albumin promoted hyphal growth.

A recent review of unsuccessful attempts to grow VA mycorrhizal fungi non-biotrophically on artificial media is provided by J O Siqueira, D H Hubbell and N C Schenck, Mycologia, 74, 952-959 (1982). These authors concluded: "Although the literature is contradictory, certain generalisations can be made: spore germination is inhibited by excess soluble salts, but trace amounts are required for germination and germ tube growth. Smail amounts of vitamins and some organic acids appear to stimulate the biosynthetic capacity of germinated spores.

Carbohydrates are not a good energy source for VA mycorrhizal fungi. Manipulation of the foregoing factors may permit culture of VA mycorrhizal fungi in the absence of host roots".

These authors' own work reported in their paper showed that CaH2PO4 (20 mg/litre) produced some slight increase in germination and germ tube growth. Potassium salts alone had no significant effect. Thiamine increased germination slightly or germ tube growth by a factor of 2 at various different concentrations.

Other unsuccessful attempts to obtain pure cultures on agar of mycorrhizal fungi of the En do gone (since re-named Glomus) species have been reported by R M Macdonald and J R Spokes, Annual Report of Rothamsted Experimental Station for 1978, 234 (1979) and by C M Hepper, Annual Report of Rothamsted Experimental Station for 1979, 1 89 (1980).

With G. caledonium it was found to substantiaily improve hyphal growth from spores by incorporating peptone, yeast extract, thiamine and lima bean agar into a basic medium, C M Hepper, Soil biology and biochemistry 11, 269 (1979). Peptone at 1-5 g/litre was the most stimulatory.

When cystine, glycine and lysine were incorporated in a medium together they gave a 5-fold increase in hyphal growth of G. caledonium compared to that on water agar, see C M Hepper and I Jakobsen, Soil Biology and Biochemistry, 15, 55-58 (1983).

It has now surprisingly been found that VA mycorrhizal fungi can be cultured, nonbiotrophically, (in vitro, in the absence of plant root host) on a growth medium containing an inorganic sulphur compound, desirably in a "reduced" form. The maximum valency of sulphur (in all normal compounds) is 6 and the sulphur compounds used in this invention desirably contain a sulphur atom of valency of less than 6, preferably a valency of 4. Spores of Glomus caledonium grown on agar containing potassium sulphite have shown hyphal growth of over 16 times the corresponding control not containing an inorganic sulphur compound and the hyphae so produced have been sub-cultured non-biotrophically to produce some further hyphal growth in the absence of a parent spore.

An important feature of the invention consists in a method of culturing a VA mycorrhizal fungus, non-biotrophically, which method comprises culturing the fungus, preferably aerobically, on a growth medium containing an inorganic sulphur compound, preferably one containing a sulphur atom of valence less than 6, most preferably of valence 4. Preferably the growth medium also contains amino acid(s). A carbon source can be included, but currently it seems best to avoid using a sugar.

The sulphur compound employed is preferably one containing an oxo-acid anion, especially suiphite: (S valency = 4)

disuiphite or metabisulphite: (S valencies = 6, 4)

thiosulphate: (S valencies = 6, 2)

dithionite: (S valencies = 4, 4)

disulphate: (S valencies t 6, 6)

or sulphate: (S valency = 6)

Sulphate and disulphate are less preferred.

When the sulphur compound contains two or more S-atoms, at least one of them preferably has a valence of less than 6. This is tantamount to saying that their average valence is less than 6, but it is preferred to express the preference in terms of individual atoms because the 4-valent state of an individual atom, as in disulphite, appears preferred over an average valence of 4 resulting from one atom being 6-valent and another 2-valent as in thiosulphate.

The sulphur compound can also be a sulphide, for example K2S.

Indications are that potassium salts give very much better results than sodium salts, which appear to inhibit growth in some way and are therefore less preferred. Any other sensible cation (compatible with culture of mycorrhizal fungi) can be used.

Potassium sulphite is the most preferred salt and is preferably freshly prepared (to minimise oxidation by air to the sulphate) and filter-sterilised.

The method of culture works well for Glomus caledonium and is expected to be suitable for any of the usual VA mycorrhizal fungi, for example Glomus mosseae, Glomus fasciculatus and other Glomus spp, and Gigaspora spp, Acaulospora spp and Sclerocystis spp. Spores, sporocarps and hyphae can be used to initiate culture.

The growth medium is conveniently agar-based, although it is contemplated that growth will also be obtained on appropriate liquid media. While growth has been produced without a carbon source in the medium, it is expected that improved results will be obtained by including one. Indications are that a non-sugar carbon source would be preferable to a sugar. Otherwise, the medium may contain any of the usual growth factors beneficial to fungal culture, for example yeast extract, peptone or thiamine and amino acid(s) such as cystine, glycine and lysine.

The concentration of sulphur compound in the medium is preferably in the range 1 to 500 mg/litre, more preferably 1 to 50 mg/litre, expressed as elemental sulphur. Generally stated, it is expected that at least 1 mg S/litre is likely to be required to impart satisfactory growth.

The pH of the medium will usually be in the range 4 to 9 and will depend on the species. A pH used in biotrophic culture will generally be appropriate. For Glomus caledonium the pH will normally be from 6 to 8, preferably 6.5 to 7.5. The temperature of culture will normally be around ambient, say 1 5-35'C, but higher or lower temperatures, e.g. from 5"C upwards or 45"C downwards would be expected to give some degree of growth.

The culture conditions are normally aerobic, although sub-aerobic quantities of oxygen may be usable under some conditions. The culture is not necessarily carried out on stationary plates, e.g. submerged shake culture would be possible.

While it is preferred to pre-germinate the spores before contacting them with the growth medium, in situ germination has also been carried out successfully. Thus, using water agar and potassium sulphite, the spores germinated well and hyphal growth was stimulated.

The invention includes, of course, the VA mycorrhizal fungi obtained by the method of culture of the invention and inocula based on them.

The following Examples illustrate the invention.

EXAMPLE 1 Spores of Glomus caledonium were isolated from around the roots of stock plants of Nardus stricta, Allium cepa or Zea mays by wet-sieving. They were surfaced-sterilised by immersing them in a solution of 20 g/litre chioramine T containing 200 mg/litre streptomycin sulphate for 20 minutes, washed three times in sterile water and plated on to Difco Bacto agar (10 g/litre).

The spores were incubated at 23"-25"C and transferred individually to the plates of test medium when they had just germinated (i.e. when the germ tube length was less than the spore diameter). For each treatment there were six 25 ml agar plates each with 5 spores. These plates were incubated for 14 days at 23"-25"C and then the amount of growth from each spore was assessed, after staining with the dye acetic aniline blue, by counting the number of intersections which the hyphae made with a microscope eyepiece grid.

All test media were prepared using Difco Bacto agar (10 g/litre) and were checked for pH after sterilisation at 121 C for 15 minutes. All pHs were between 6.6 and 7.5 and did not differ by more than 0.5 unit in any experiment. The inorganic sulphur-containing compounds tested are listed in Table 1 below. Each compound was tested at the concentration given in Table 1 (equivalent to 1 2.3 mg S/litre) and also at one-tenth and one-hundredth this concentration.

TABLE 1 Inorganic sulphur-containing Concentration compounds tested Grade used* mg/litre K2SO3 GPR 60.5 Na2SO3. 7H20 AR 97.0 K2S205 AR 42.7 3K2S203.5H2O GPR 42.2 Na2S2035H2O AR 47.5 K2SO4 AR 66.6 Na2SO4.1OH2O AR 126.0 * GPR = General purpose reagent AR = Analar grade The results obtained are shown in the accompanying drawings (Figs. 1-4) in which hyphal growth is plotted on the ordinate and concentration in mg. sulphur/litre on the abscissa. Each compound was tested twice in separate experiments, with the exception of the sodium sulphate, and substantially the same results were obtained both times. It will be seen that the reduced forms of sulphur present in the sulphite, disulphite and thiosulphate all produce good fungal growth at the higher concentrations employed.Results with sodium salts were markedly poorer than for potassium salts, and the most oxidised form of sulphur, i.e. the sulphate, gave the poorest results of all the sulphur-oxygen anions tried.

EXAMPLE 2 Four sets of additional experiments were carried out in which were used (a) the agar of Example 1, (b) a different agar (Oxoid type L28) and (c) and (d) two agarose preparations. In each set of experiments the medium was used with and without potassium sulphite (60.5 mg/litre). Conditions were otherwise in accordance with Example 1. As Table 2 shows, hyphal growth from germinating spores was best when the Bacto agar of Example 1 was used.

TABLE 2 Mean number of Medium Additive intersections/spore 10 g/litre Difco Bacto agar None 23 " K2SO3 402 10 g/litre Oxoid agar None 18 K2S03 338 10 g/litre BDH agarose None 4 K2S03 163 5 g/litre Sigma agarose None 6 K2S03 76 EXAMPLE 3 Example 1 was repeated using different inorganic sulphur-containing compounds, each compound being again tested at a concentration equivalent to 1 2.3 mg S/litre and also at onetenth and one-hundredth this concentration. The compounds tested and the results obtained are shown in Table 3.

TABLE 3 Concentration of compound equivalent Compound Grade used to 12.3 mg. S/litre K2S Laboratory reagent 42.3 2 2 7 Laboratory reagent 48.8 Na2S204. x H20 Laboratory reagent 33.3 (x = an unknown amount of water of hydration; for the purposes of reckoning the equivalent in S, the water of hydration was ignored).

S content Mean number of intersections/spore (mg/litre K2 S K 2S207 Na 2S204 12.3 34* 52 223* 1.2 26 60* 8 0.12 35* 40 6 0 16 41 5 Significantly different from control at 5% level.

These experiments indicate that both K2S and K2S207 are stimulatory to hyphal growth. These salts were supplied in the laboratory reagent grade, which has a lower standard of purity than the GPR and AR reagents used in Example 1. It is quite possible therefore that the stimulation due to these salts is less than that observed with K2SO3 and K2S205 because of the presence of impurities which might be acting as inhibitors.

EXAMPLE 4 If the effect of reduced inorganic sulphur compounds is due to a change in the oxidationreduction potential of the medium, then it was thought possible that the same effect might be obtained with a compound such as ascorbic acid. This was tested at a range of concentrations by the procedure of Example 1. The results in Table 4 below show that there was no stimulation of hyphal growth at any concentration of ascorbate tested; it was in fact inhibitory to fungal growth in the concentration range 0.1-10 g per litre.

TABLE 4 Mean number of Ascorbic acid concentration intersections per spore 10 2* 1 10* 0.1 5 0.01 16 0 16 * Significantly different from control at 5% level.

EXAMPLE 5 An experiment was carried out to check whether it is desirable to heat-sterilise the inorganic sulphur compounds with or without agar in order to produce a stimulatory effect. The following media were used: (i) water agar control (ii) K2SO3 was sterilised at 121 C together with the agar (iii) K2SO3 was sterilised at 121 C separately and mixed with molten agar (iv) K2SO3 was filter-sterilised through a 0.22 micrometre Millipore filter and mixed with molten agar.

The procedure was otherwise as in Example 1, the final concentration of K2SO3 in each case being 60.5 mg per litre, equivalent to 12.3 mg S per litre. The results are shown in Table 5.

TABLE 5 Mean number Medium intersections/spore Water agar (control) 18 K2S03 heat-sterilised with agar 130 K2S03 heat-sterilised separately 192 K2S03 filter-sterilised separately 189 The results from the three media containing potassium sulphite differed significantly from the control and the results from the media which were made by sterilising the potassium sulphite separately differed significantly from the result for the medium prepared by heat-sterilising the sulphite together with the agar.

EXAMPLE 6 The effects of inorganic sulphur-containing compounds described above have been observed on the growth of hyphae which are still attached to their parent spores. To obtain pure cultures of VA mycorrhizal fungi, it is necessary for the hyphae to grow independently of the spore, deriving all their nutrients from the medium. Spores of Glomus caledonium were germinated on water agar and then transferred to the fresh medium shown in Table 6 below, which includes potassium sulphite. The spores were grown in the manner of Example 1.

TABLE 6 Concentration Component mg/litre Yeast extract (Difco) 400 Peptone (Oxoid bacteriological) 1,000 Thiamine HC1 5 Glycine 556 Lysine 825 K2S03 60.6 CaCl2. 2H20 36.8 K2HPO4 11.2 Lima bean agar (Difco) 100 Difco Bacto agar 8,000 0 Note: the pH of this medium after sterilising at 121 C for 10 minutes was 6.8.

When the spores have been on this medium for 5-9 days and have produced a net of mycelium, small squares of agar, containing hyphae with at least one apical growing point, were cut from the plates and transferred to fresh medium of the same composition. Small pieces of boiled seed of Phaseofus lunatus were placed around each group of hyphae, because these are known to promote growth of hyphae from spores, see C M Hepper, Soil Biology and Biochemistry, 11, 269-277 (1979). The size of the pieces of hyphae which have been transferred was noted by making drawings. The extent and position of any new growth was then recorded. Most subcultures grew to some extent and approximately 50% of them increased their size by at least half as much again.

Since the medium did not include a carbon source for the fungus, an organic acid, oxaloacetic acid, which was found to be marginally better than other acids or sugars in earlier tests was added to the medium. A range of concentrations from 5 to 500 mg/litre was used and all concentrations gave better results than the unsupplemented medium. Approximately 85% of the subcultures increased their size by at least half as much again and some subcultures grew up to five times their initial size.

An attempt to transfer the new hyphae which grew from the subcultures on to fresh medium of identical composition produced only small amounts of growth. There was no growth at all from subcultures which were plated on to fresh agar with no added nutrients.

Claims (10)

1. A method of culturing a VA mycorrhizal fungus non-biotrophically, which method comprises culturing the fungus on a growth medium containing an inorganic sulphur compound.

2. A method according to Claim 1 wherein the sulphur compound is a reduced one, containing a sulphur atom of valence less than 6.

3. A method according to Claim 1 wherein the sulphur compound contains a sulphur atom of valence 4.

4. A method according to Claim 3, wherein the sulphur compound is a sulphite, disulphite or dithionite.

5. A method according to any preceding claim wherein the sulphur compound comprises a potassium salt.

6. A method according to any preceding claim wherein the concentration of the sulphur compound is at least 1 mg/litre reckoned as elemental sulphur.

7. A method according to any preceding claim wherein the species of VA mycorrhizal fungus is Glomus caledonium and the pH of the medium is from 6 to 8.

8. A method according to any preceding claim, wherein the growth medium is agar-based.

9. A method according to any preceding claim wherein the culture is aerobic.

10. A method according to any preceding claim, wherein spores or hyphae of the fungus are cultured.