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The effect of pH on the growth of saprotrophic and ectomycorrhizal ammonia fungi in vitro

     Forestry and Forest Products Research Institute, Ibaraki 305-8687, Japan

	ABSTRACT

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Some saprotrophic and ectomycorrhizal fungi produce reproductive structures, preferably in slightly alkaline to neutral forest soil. This research examines the growth of these "ammonia fungi" in liquid medium at various pH values. In the first experiment, the capacity of six buffers was examined to select appropriate buffers for stabilizing pH in the neutral-to-alkaline range by culture of three species of the ammonia fungi in media initially adjusted to pH 7, 8 or 9. The highest buffering capacity was shown in 2-(N-morpholino) ethanesulfonic acid (MES) at pH 7, and N, N-bis (2-hydroxyethyl) glycine (Bicine) and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) at pH 8 and 9. In the second experiment, the growth of 15 strains of both saprotrophic and ectomycorrhizal ammonia fungi was tested on the medium initially adjusted to pH 3, 4, 5, 6 or 7 with MES, or to pH 8 or 9 buffered with Bicine. Many of the saprotrophic species grew well at pH 7 or 8; the ectomycorrhizal species showed optimum growth at pH 5 or 6. The pH suitable for the in vitro growth of these fungi was correlated with the pH of forest soil where these fungi occur.

Key words: ammonia fungi, ecology, forest health, pH, physiology, vegetative growth

	INTRODUCTION

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"Ammonia fungi" (Sagara 1975) sporulate sequentially on soil after treatment of soil with urea, aqueous ammonia or nitrogenous compounds (N compounds) that release ammonia during decomposition and cause an alkaline condition in soil. These fungi also occur after the decomposition of bodies or feces of animals. Therefore, these organisms also can be called postputrefaction fungi (Sagara 1995). The occurrence of ammonia fungi is divided into early and late phases (Sagara 1995). In the early phase, about one month after the treatment with N compounds, anamorphic fungi (e.g., Amblyosporium), cup fungi (e.g., Peziza, Ascobolus) and smaller agarics (e.g., Tephrocybe, Coprinus) sporulate or fruit for short periods. In the late phase, larger agarics (e.g., Laccaria, Hebeloma) are observed for several years. Investigations on the properties of soil in the course of the sporulation of the ammonia fungi after the addition of urea show that the early-phase (EP) species sporulate or fruit on the soil in neutral-to-alkaline conditions caused by an increase in ammonium concentration. The late-phase (LP) species fruit on acidic soil resulting from the decrease of ammonium concentration and a temporary increase of nitrate (Yamanaka 1995). This indicates that the ammonia fungi might be divided into different groups by the pH values favorable for or tolerable to their growth, as well as by the nitrogen use (Yamanaka 1999) or the timing of their appearance after soil treatment with N compounds (Sagara 1995). Some EP species possess cellulolytic enzymes having optimal pH, between 6.8–9.0, and the cellulolytic enzymes of LP species show an optimal pH range between 5.5–6.8 (Enokibara et al 1993). The optimal pH for the growth of ammonia fungi, however, has not been determined.

Conventional culture media used for physiological studies of fungi (potato-dextrose agar, modified Melin-Norkans agar, Czapek-Dox agar) have low buffering capacities, and the effects of pH on fungal growth on such media are difficult to assess (Child et al 1973, Hung and Trappe 1983). Therefore, organic and inorganic acids, such as acetates, citrate, phthalate and phosphate, have been used as buffers to stabilize pH. Some of the acids, however, inhibit fungi growth (Giltrap and Lewis 1981, Hilger et al 1986, Inoue and Ichitani 1990). Other buffers, such as 2-(N-morpholino) ethanesulfonic acid (MES), piperazine-N, N'-bis (2-ethanesulfonic acid) (PIPES), which are thought to be inert physiologically (Good et al 1966), have been used to stabilize pH under acidic conditions, although these buffers stimulate growth of some ectomycorrhizal fungi (Giltrap and Lewis 1981, Hilger et al 1986).

Some species, such as Coprinus and Mortierella, have been observed in alkaline soil and had an optimal pH for in vitro growth in neutral-to-alkaline conditions (Warcup 1951, Fries 1956). The EP species of ammonia fungi also were observed only on neutral-to-alkaline soil (Sagara 1975, Yamanaka 1995), and these ammonia fungi are expected to favor alkaline conditions in pure culture. Therefore, the effect of alkaline media on growth of these fungi should be examined in a buffered system. However, the effect of buffers on the in vitro growth of fungi under alkaline conditions has been little studied (Inoue and Ichitani 1990). In this study, six buffers were tested for their buffering under neutral-to-alkaline conditions in which three species of ammonia fungi were cultured. Then, using the buffers with the highest buffering capacity, the optimal pH values for the growth of EP and LP species of ammonia fungi were determined.

	MATERIALS AND METHODS

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Fungal species – Six EP [Amblyosporium botrytis, Pseudombrophila petrakii (=P. deerata in Yamanaka (1999)), Peziza urinophila, Tephrocybe tesquorum, Coprinus echinosporus, C. phlyctidosporus] and three LP [Laccaria bicolor, Hebeloma vinosophyllum, H. radicosoides (=Hebeloma sp. in Yamanaka (1999))] were used. The designation of the species as EP or LP follows Sagara (1992). In addition, two saprotrophic species [Collybia dryophila, Marasmius pulcherripes] and three mycorrhizal species [Amanita rubescens, Lyophyllum semitale, Suillus luteus] were isolated from untreated forest soil and tested for comparison. The detailed information regarding the origin of these fungi has been reported elsewhere (Yamanaka 1999). The isolates were maintained on glucose-yeast extract agar medium or glucose-dry yeast agar medium (Yamanaka and Sagara 1990).

In Experiment 1, fungal growth and six buffers in culture media initially adjusted to pH 7, 8 and 9 was examined. In Experiment 2, the growth of the ammonia fungi in liquid medium containing the appropriate buffer selected in Experiment 1 was determined at pH 3 to 9.

Experiment 1 – Two EP species, P. urinophila, T. tesquorum, and one LP species, H. vinosophyllum, were used in this experiment. A liquid medium (YG) containing 0.2% yeast extract (Difco) and 1% glucose was prepared as a basal medium. Synthetic media are likely to form insoluble compounds from phosphate and essential cations (magnesium, potassium) present in the media under alkaline conditions (Harley 1934) and may inhibit fungal growth (Munro 1970). In a preliminary experiment, insoluble compounds did not form in YG under alkaline conditions, indicating that it is a suitable basal medium for this study. These six buffers were added to the basal medium at 50 mM: MES (pka, 6.15), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES, pKa 7.55), N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS, pKa 8.4), N, N-bis (2-hydroxyethyl) glycine (Bicine, pKa 8.35), N-cyclohexyl-2-aminoethanesulfonic acid (CHES, pKa 9.3) and N-cyclohexyl-3-aminopropanesulfonic acid (CAPS, pKa 10.4). All of these buffers were supplied from Dojin-kagaku Laboratory, Kumamoto, Japan. The medium pH was adjusted to 7, 8 or 9 with NaOH or HCl, except for the pH of CHES and CAPS, which was adjusted to only 8 or 9. Forty mL of the liquid medium were poured into a 100 mL flask. Glucose was added aseptically through a 0.22 µm Millipore filter to the medium cooled after autoclaving at 121 C for 20 min. Inoculum disks were cut with a 4 mm diam cork borer from the edge of actively growing colonies on plates of YG containing 1.5% agar, and one disk was placed into each flask. Five flasks were used in each treatment. The flasks were incubated at 23 C in the dark for 14 d. The mycelia that developed during incubation were transferred to preweighed pieces of aluminum foil. These were oven-dried at 80 C for 48 h. The pH value of the medium also was measured with a glass electrode pH meter (Mettler, Toledo 320) at the end of the experiment.

Experiment 2 – The pH of the medium was adjusted to 3, 4, 5, 6 or 7 with MES and to 8 or 9 with Bicine, and various species of ammonia fungi and nonammonia fungi were cultured in them for 14 d. The procedure of preparation of the medium, inoculation of the fungi, incubation and harvesting were the same as described above. In addition, the pH of the medium without fungal inoculation and with or without the buffer was measured, 1 and 14 d after autoclaving.

Statistical analysis – Five replicates were prepared for each treatment. The Kruskal-Wallis test was employed for analyses of differences among treatments (P < 0.05).

	RESULTS

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Experiment 1 – The pH values were lowered during the culture period in all media examined (Fig. 1). In many cases, the greatest drop in pH occurred when the medium had a higher initial pH. The pH of media initially adjusted to pH 7 was reduced slightly in the presence of MES or HEPES during the culture of all three isolates tested; TAPS had the weakest buffering capacity at an initial pH of 7. At an initial pH of 8 or 9, Bicine, HEPES, or TAPS had a relatively high buffering capacity compared to other treatments. Peziza urinophila grew better in media with a buffer than in media without a buffer at any initial pH, except for CAPS or CHES at pH 8 and CAPS at pH 9 (Fig. 1). The growth of T. tesquorum was better in media initially adjusted to pH 7 with a buffer, while this fungus in the media initially adjusted to pH 9 grew well without a buffer. The growth of H. vinosophyllum was reduced with Bicine at initial pH of 7 and with all buffers tested except for CAPS at initial pH of 9.

View larger version (37K): [in this window] [in a new window]   FIG. 1. Dry weight of Peziza urinophila, Tephrocybe tesquorum and Hebeloma vinosophyllum and the change in pH value in the medium containing six buffers. Values are means calculated from five replicates for each treatment. Columns with the same letter for each pH value of each species were not significantly different at P < 0.05 according to the Kruskal-Wallis test

The decline of pH of the media during the culture period was less than 1.0, except for the medium in which P. urinophila was cultured at an initial pH of 9 (Table II). In the medium adjusted initially to pH 9, the pH value declined 0.2–0.4, even when little or no fungal growth was observed. The pH of the medium with buffer declined with time even without fungi in liquid medium initially adjusted to pH 8 or 9.

	DISCUSSION

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MES has been used often as a buffer for the control of pH (Child et al 1973, Giltrap and Lewis 1981, Myers and Campbell 1985, Inoue and Ichitani 1990). This study also demonstrated a high buffering capacity for MES in the media initially adjusted to pH 3 to 7 during the culture of all species examined (Table II). MES has been considered to be physiologically inert (Good et al 1966), but this buffer recently has been reported to stimulate fungal growth. The growth of Laccaria laccata, Pisolithus tinctorius and Paxillus involutus was enhanced by this buffer on a solid medium (Hilger et al 1986). Giltrap and Lewis (1981) showed a stimulatory effect of MES on the growth of Rhizopogon roseolus and Suillus bovinus. Child et al (1973) reported the stimulation of this buffer on the sporulation of Chaetomium funicola. On the other hand, inhibitory effects of MES on the metabolism of fungi have been reported, viz. possible inhibition by MES on the induction or activity of invertase of R. roseolus and S. bovinus (Giltrap and Lewis 1981) and reduction in the use of organic phosphate by P. involutus (Hilger et al 1986).

Fungi generally grow well in acidic conditions (Dix and Webster 1995), but some species favor neutral to slightly alkaline conditions. Fries (1956) reported that Coprinus species (e.g., C. radiatus, C. micaceus, C. ephemerus) grew well at above pH 8; these species were collected from habitats with a high pH. El-Abyad and Webster (1968) also reported that some carbonicolous species grew well at pH 6.2 to 8.2, and that the highest percentage of germination in these species was obtained under alkaline conditions. The soil from which fruit bodies of these carbonicolous species were collected was alkaline to neutral after a fire (El-Abyad and Webster 1968).

In the case of the ammonia fungi, optimal pH for EP species ranged from pH 7 to 8, and for LP species was pH 5 to 6 (Table I). Therefore, the optimum pH for these fungi generally was correlated with the soil pH where they sporulated; EP species sporulated in neutral to slightly alkaline conditions, LP on acidic soil (Sagara 1992, Yamanaka 1995). Although favorable conditions for vegetative growth are not necessarily correlated with conditions required for fruit body formation, these findings suggest that the pH value of the habitat could be a determinant for the developmental pattern of these fungi.

Erland et al (1990) showed that mycorrhizal fungi possess a generally broader range of pH tolerance in symbiosis than in pure culture and emphasized the danger of extrapolating the results from pure culture studies to symbiotic systems. Typical LP species such as Hebeloma spp. and L. bicolor are mycorrhizal (Sagara 1995) and therefore might have the ability to tolerate alkaline conditions better as a symbiont with their host trees than as a saprophyte. In field observations, however, fine roots of trees in soil were damaged by urea treatment and did not spread again into the treated soil, while the EP species appeared in alkaline soil (Sagara 1975). Accordingly, LP species did not form mycorrhizal associations with plants at this stage.

Under alkaline conditions, YG did not form insoluble compounds possibly from phosphate and cations. Yeast extract contains 3.27% phosphate (Difco laboratories 1996), corresponding to 0.065 g/L in YG. This value is lower than that found in culture media, such as modified Melin-Norkans medium (0.53 g/L) or Czapek-Dox medium (0.54 g/L) used in physiological studies. Yeast extract contains a variety of amino acids and vitamins (Difco laboratories 1996) and should support good growth of the ammonia fungi in spite of low phosphate content.

In the media adjusted initially to pH 9, the pH of the media declined 0.2–0.4 even when no or little fungal growth was observed (Tables I and II). Under alkaline conditions, carbon dioxide becomes soluble and carbonate ion in solution increased; this might result in a decrease of medium pH.

In addition to the tolerable pH range for the ammonia fungi, nitrogen metabolism of ammonia fungi has been examined for the successive occurrence of these fungi. EP and LP species showed different responses to a nitrogen source in pure culture, which is well correlated with the dominant form of inorganic nitrogen in soil during the occurrence of the ammonia fungi (Yamanaka 1995, 1999). Many EP species grew well on ammonium nitrogen as a sole N source, and LP species grew well on nitrate as well as on ammonium. Ammonium nitrogen in soil increased at an early phase after the treatment with urea, and nitrate nitrogen as well as ammonium nitrogen increased at the late phase. Dix and Webster (1995) pointed out that in the studies on coprophilous fungi the successional appearance of fruit bodies of each species is a result of a characteristic minimum time that the fungi require to form a fruit body and interspecific antagonism was an explanation of the cessation of fruiting in each species. The results of a previous study (Yamanaka 1999) and this study, however, showed that the successional appearance of the ammonia fungi from EP to LP species may be controlled by both pH and the form of inorganic nitrogen.

Dix and Webster (1995) pointed out that environmental H⁺ concentration has little direct effect on fungal metabolism due to the buffering system in hyphae but may influence the ionization of salts in solution and the permeability of the plasmalemma of the hyphae. Furthermore, enzyme activity is affected by H⁺ concentration. Enokibara et al (1993) examined the activity of cellulase at different pH values in some fungi, including the ammonia fungi. In their study, the favorable pH range for cellulase in EP species was pH 6.8 to 9.0, and LP species showed a comparatively weak cellulolytic activity at pH 5.5–6.8.

The results of this study have ecological implications. Growth of six out of 10 EP species at pH 4 (Table I) suggests that even in soil not treated with nitrogenous materials, these species may survive in the form of vegetative hyphae. Growth of Hebeloma spp. and L. bicolor, LP species, at pH 8 (Table I) also show that these species can develop mycelia during the early phase, although these species do not form their fruit bodies at this stage. In neutral to slightly alkaline conditions, LP species with such characteristics possibly infect root tips and fruit more easily than the fungi preferring acidic conditions, such as the nonammonia species used in this study. Spore germination of H. vinosophyllum is stimulated by ammonia (Suzuki 1978). Ammonia causes alkalinity in aqueous solutions, and Suzuki's (1978) results also suggested that mycelial growth could occur in soils at high pH.

Ammonia fungi have been observed after urea treatment of forest litter in various regions besides Japan, such as U.K. (Lehmann 1976), Finland, U.S.A. (Sagara 1992) and Taiwan (Wang and Sagara 1997). These observations indicate that this fungal group is present latently in forests with various types of vegetation and climate and is likely to play a role in nutrient dynamics under the conditions of a high ammonium concentration after decomposition of organic materials. This ubiquitous group of fungi, therefore, might be one of the indicators of a healthy forest ecosystem (Sagara and Hamada 1965).

	ACKNOWLEDGMENTS

 
I thank Dr C.Y. Li, U.S.D.A. Forest Service, U.S.A., for reading the manuscript, Emeritus Prof. N. Sagara, Graduate School of Human and Environmental Studies, Kyoto University, Ms. K. Akama and Mr. T. Akema, Forestry and Forest Products Research Institute, for kindly providing isolates of some of the fungi used in this study.

	FOOTNOTES

 
¹ E-Mail: yamanaka{at}ffpri.affrc.go.jp

Accepted for publication November 18, 2002.

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