Effects of different nitrogen sources on interactions between ammonia fungi and non-ammonia fungi

Abstract

Four early-phase (EP) and one late-phase (LP) ammonia fungi were co-cultured with three non-ammonia fungi on urea, ammonium, nitrite and nitrate agar media at different pH values (5.5, 7.0 and 9.0). The early stage EP species Amblyosporium botrytis and Ascobolus denudatus showed deadlock competition with the non-ammonia fungi Aspergillus niger and Penicillium citrinum on urea agar medium, and neutral intermingling or weak competing deadlock on ammonium, nitrite and nitrate agar media in all pH conditions. The late stage EP species, Coprinopsis phlyctidospora and Lyophyllum tylicolor, showed antagonistic deadlock and invaded the region containing mycelia of the non-ammonia fungi, P. citrinum and Trichoderma viride, on urea, nitrite and nitrate agar media in all pH conditions. The LP species, Hebeloma vinosophyllum, showed competing deadlock with Asp. niger and P. citrinum on urea medium and weak competition on nitrite and nitrate agar media at pH 9.0. These findings suggest that the successive occurrence of ammonia fungi is partly due to changes in the interactions between ammonia fungi and non-ammonia fungi based on changes in inorganic nitrogen.

Keywords:

• co-culture
• ectomycorrhizal fungi
• inorganic nitrogen
• pH, saprobic fungi, urea

Introduction

To understand the structure and function of fungal communities, it is important to understand the interactions between different types of fungi (Rayner and Boddy 1988; Christensen 1989). Ammonia fungi are defined as a chemo-ecological group of fungi that sequentially develop reproductive structures exclusively or relatively luxuriantly on soil after a sudden addition of ammonia, alkalis, or other nitrogenous materials that react directly as a base or indirectly as a base after decomposition (Sagara 1975).

As described in previous studies, when the forest floor was treated with a large amount of urea, the ammonium nitrogen concentration in the soil increased, thus increasing the pH, and then both gradually returned to original levels (Yamanaka 1995a–c; Suzuki 2000; Suzuki et al. 2002). In these situations, the occurrence of ammonia fungi generally proceeded as follows: anamorphic fungi and cup fungi in the Ascomycota occurred when the soil ammonium-nitrogen concentration decreased (associated with a decrease in pH from 8 to 7). These species that occur at an early stage of the early phase are hereafter referred to as early stage EP species. When the oxidation of ammonium nitrogen decreased the soil pH from 7 to 6, fungi in the Basidiomycota with smaller fruiting bodies became dominant. These species that occur at the late stage of early phase are hereafter referred to as late stage EP species. Further decreases in pH (to a level similar to that of the control at pH 3.5–6.0) were associated with the occurrence of fungi with larger fruiting bodies (Sagara 1992; Yamanaka 1995a–c; Suzuki 2000; Suzuki et al. 2002). These late-phase occurring species are hereafter referred to as LP species (Sagara 1992; Yamanaka 1995a–c; Suzuki 2000; Suzuki et al. 2002). The LP species disappeared within 2–3 years, when concentrations of nitrite- and nitrate-nitrogen declined to similar levels as those in control soil (Sagara 1992; Yamanaka 1995a–c). All EP species and several LP species are saprobic and most LP species are ectomycorrhizal (Sagara 1995; Sagara et al. 2008). Yamanaka (1995a–c) suggested that ammonium nitrogen was the major nitrogen source for vegetative growth of LP species in urea-treated soils, and it was gradually replaced by nitrate-nitrogen. Licyayo et al. (2007) suggested that the interactions among ammonia fungi were one of the main factors affecting their successive occurrence. However, little is known about the effects of interactions between ammonia and non-ammonia fungi on the successive occurrence of ammonia fungi. Therefore, to examine in detail the interactions between ammonia fungi and non-ammonia fungi, we co-cultured various fungi on agar media containing urea, ammonium-nitrogen, nitrite-nitrogen and nitrate-nitrogen, adjusted to several different pH values.

Materials and methods

Organisms

Table 1 shows the fungal species used in these experiments. The fungi included four saprobic ammonia fungi, one ectomycorrhizal ammonia fungus that is commonly found in Japan after field applications of urea, two common soil fungi (saprobic non-ammonia fungi) and one mycoparasite (a saprobic non-ammonia fungus).

Table 1. Fungal isolates used in these experiments

Fungal species	Isolate no.	Ecological group of fungi	Abbreviation	Successional phase	Nutritional mode
Ammonia fungi
Amblyosporium botrytis	CHU M006	Ammonia fungi	Amb	Early stage EP	Saprobic
Ascobolus denudatus	CHU M007	Ammonia fungi	Asd	Early stage EP	Saprobic
Coprinopsis phlyctidospora	NBRC 30478	Ammonia fungi	Cop	Late stage EP	Saprobic
Lyophyllum tylicolor	PH (B2)	Ammonia fungi	Lyt	Late stage EP	Saprobic
Hebeloma vinosphyllum	CHU Kiyosumi	Ammonia fungi	Hev	LP	Ectomycorrhizal
Non-ammonia fungi
Aspergillus niger	IFM 55890	Soil fungi	Asp		Saprobic
Penicillium citrinum	IFM 40616	Soil fungi	Pec		Saprobic
Trichoderma viride	IFM 40938	Soil fungi*	Trv		Saprobic
*Fungicolus fungi; EP: early phase; LP: late phase.

Medium preparation

The basal medium was composed of 22.22 g glucose, 0.33 g KH₂PO₄, 0.33 g MgSO₄·7H₂O, 0.11 g CaCl₂·H₂O, 0.33 mg ZnSO₄·7H₂O, 0.15 mg FeSO₄·7H₂O, 0.10 mg CuSO₄·5H₂O, 0.10 mg MnSO₄·5H₂O, 0.02 mg Na₂MoO₄·2H₂O, 0.50 mg thiamine hydrochloride, 0.10 mg nicotinic acid, and 1000 ml distilled water (Kitamoto et al. 1972). Urea, ammonium chloride, sodium nitrite, or sodium nitrate was added to the basal medium at concentrations ranging from 0.041 to 4.18 g N/l, according to the optimal concentrations for each ammonia fungus (Table 2). All four types of media were adjusted to three different pH levels (5.5, 7.0 and 9.0) with 1 M NaOH and 1 M HCl, to determine the effects of pH on interactions between fungi. The medium adjusted to pH 5.5 served as the control because the soil pH in most Japanese forests is between 3.6 and 6.0 (Sagara 1975; Yamanaka 1995a–c; Fukiharu et al. 1997; Sato and Suzuki 1997; Suzuki 2000; Suzuki et al 2002; He and Suzuki 2004). Hereafter, the basal media containing urea, ammonium chloride, sodium nitrite, and sodium nitrate are described as urea agar medium, ammonium agar medium, nitrite agar medium, and nitrate agar medium, respectively. For all media, double-strength medium was sterilized by filtration through a membrane filter (cellulose nitrate; 0.22 μm pore size; Advantec, Tokyo, Japan). Agar medium [30 g agar (Nacalai Tesque, Kyoto, Japan)/1000 ml distilled water] was sterilized for 15 min at 120°C and kept at 60°C. Then, an equal volume of the agar medium and each double-strength test medium was mixed, keeping the mixture at 60°C before dispensing 20-ml aliquots of the medium into sterilized Petri dishes (90 mm in diameter).

Table 2. Nitrogen concentrations used in co-culturing experiments

Ammonia fungi	Urea (g N/l)^a	NH4Cl (g N/l)^b
Early stage EP species
Amblyosporium botrytis	13.9	4.1
Ascobolus denudatus	1.3	0.13
Late stage EP species
Coprinopsis phlyctidospora	1.3	0.418
Lyophyllum tylicolor	1.3	0.418
LP species
Hebeloma vinosphyllum	0.46	0.041
^aOptimum concentration of urea for each ammonia fungus (Barua et al. 2012).
^bOptimum concentration of NH4Cl for each ammonia fungus (Licyayo 2007).
Note: Concentration of sodium nitrite or sodium nitrate used for each
ammonia fungus was equivalent to the nitrogen concentration of ammonium chloride (Licyayo 2007).

Co-culturing of ammonia fungi and non-ammonia fungi

Agar discs (4 mm diameter) were cut from the sub-peripheral region of actively growing mycelial colonies of each fungal isolate. The discs were then cultured at room temperature separately on urea, ammonium, nitrite or nitrate agar media. Two discs from different fungal species were inoculated 55 mm apart on the agar media with different pH (5.5, 7.0 and 9.0). Slow-growing fungi were inoculated onto the medium before the fast-growing fungi (Table 3), because slow-growing fungi can be completely suppressed by fast-growing fungi when they are inoculated at the same time (Shaw et al. 1995). The co-culture experiments included saprobic EP species and the three saprobic non-ammonia fungus species, and also the ectomycorrhizal LP species H. vinosophyllum, which was included because it is a facultative mycorrhizal fungus, i.e. it is somewhat saprobic (Suzuki 2009). Pairs of the same isolates were also prepared as a control. All procedures were conducted under aseptic conditions. Co-cultures were incubated at 20.0±0.5°C under light irradiation at ∼500 lux with a white light florescent lamp (Hitachi, Tokyo, Japan). All co-cultures had five replicates.

Table 3. Inoculation schedule of each fungus on different nitrogen agar media

	Day of inoculation of isolate B after that of isolate A cultured on agar medium containing
Paired isolate A/B	Urea	NH4Cl	NaNO2	NaNO3
Amb^a/	0	0	0	0
Asn	3	10	10	3
Pec	3	10	10	3
Trv	4	10	10	4
Asd/	0	0	0	0
Asn	4	10	4	5
Pec	0	5	0	0
Trv	5	10	5	5
Cop/	0		0	0
Asn	4		5	4
Pec	0		0	0
Trv	4		4	4
Lyt/	0		0	0
Asn	10		7	7
Pec	0		0	7
Trv	10		10	10
Hev/	0		0	0
Asn	12		12	12
Pec	5		5	9
Trv	15		15	15
Note: Blank means no co-cultured.

Observations and measurements

We studied the interactions between five ammonia fungi and three non-ammonia fungi (Table 1) co-cultured on different kinds of nutrient agar media. We categorized the interactions observed in co-cultures based on the features observed at the contact zone (CZ) of the mycelial colonies and those observed at the opposite sides to contact zone (OCZ) of the mycelial colonies. The types of interactions were recorded after 25–30 days of incubation. We also measured mycelial expansion (radial growth) on each medium after 2 to 30 days of incubation. The ratio between the mycelial colony radius in the CZ direction to that in the OCZ direction indicated the degree of growth inhibition.

Assessment of colony interaction types

Mycelial colony interactions were described according to Rayner and Boddy (1988), with modifications. When the mycelia of both inoculated fungi completely intermingled with each other, this was defined as neutral intermingling (N) (Figure 2J–L). When co-cultured fungi first showed contact zone inhibition but then intermingled, this was defined as Ci(N). When either of the inoculated fungi invaded past the CZ into the mycelial region of the other fungi and the invasion stopped within a few centimetres, this was defined as competing deadlock (Dc) (Figure 4A–C). When a CZ formed between the mycelia of the two fungi and low mycelial density was observed behind the CZ, this was defined as antagonistic deadlock (Da) (Figure 2H). When a significant growth inhibition was observed before the mycelia of the two fungi came into contact, this was defined as inhibition deadlock (Di) (Figures 2E and 5A–C). When the mycelia of either fungus covered the colony of the other fungus on the nutrient agar plate, this was defined as covering (C).

Figure 2. Mycelial interactions between Ascobolus denudatus and non-ammonia fungi. Asc. denudatus /Asp. niger at pH 5.5 (A) urea, 12^th day; (B) NH₄-N, 12^th day; (C) NO₂-N, 9^th day; (D) NO₃-N, 9^th day. Asc. denudatus/P. citrinum at pH 5.5 (E) urea, 9^th day; (F) NH₄-N, 12^th day; (G) NO₂-N, 18^th day; (H) NO₃-N, 12^th day. Asc. denudatus/T. viride at pH 5.5 (I) urea, 9^th day; (J) NH₄-N, 19^th day; (K) NO₂-N, 18^th day; (L) NO₃-N, 12^th day.

Figure 4. Mycelial interactions between Lyophyllum tylicolor and non-ammonia fungi. L. tylicolor /Asp. niger at pH 5.5 (A) urea, 12^th day; (B) NO₂-N, 18^th day; (C) NO₃-N, 12^th day. L. tylicolor/P. citrinum at pH 5.5 (D) urea, 9^th day; (E) NO₂-N, 18^th day, (F) NO₃-N, 12^th day. L. tylicolor/T. viride at pH 5.5 (G) urea, 12^th day; (H) NO₂-N, 18^th day; (I) NO₃-N 12^th day.

Figure 5. Mycelial interactions between Hebeloma vinosophyllum and non-ammonia fungi. vinosophyllum /Asp. niger at pH 5.5 (A) urea, 12^th day; (B) NO₂-N, 18^th day; (C) NO₃-N, 12^th day. H. vinosophyllum/P. citrinum at pH 5.5 (D) urea, 9^th day; (E) NO₂-N, 18^th day; (F) NO₃-N, 12^th day. H. vinosophyllum/T. viride at pH 5.5 (G) urea, 12^th day; (H) NO₂-N, 18^th day; (I) NO₃-N 12^th day.

Results

Intra-species co-culturing on media containing different nitrogen sources

Co-culturing of the early stage EP species, Amblyosporium botrytis and Ascobolus denudatus, with the late stage EP species, Coprinopsis phlyctidospora, resulted in neutral intermingling on urea, nitrite and nitrate agar media (Tables 4–8). When the late stage EP species, Lyophyllum tylicolor, and the LP species, Hebeloma vinosophyllum, were co-cultured, they first showed contact zone inhibition and finally intermingled on urea, nitrite and nitrate agar media (Tables 4, 5, 7 and 8).

Table 4. Mycelial interactions between ammonia fungi and non-ammonia fungi on agar media containing different nitrogen sources at pH 5.5

	Mycelial interaction on agar medium containing
Paired inocula A/B	Urea		NH4Cl		NaNO2		NaNO3
Amb/Amb ^a	N		N		N		N
Asp/Asp	Ci(N)		Ci(N)		Ci(N)		N
Pec/Pec	Ci(N)		Ci(N)		Ci(N)		N
Trv/Trv	Ci(N)		N		N		N
Amb/
Asn	Dc		N, C				N, C
Pec	Dc				N, C		N, C
Trv	N, C*		N, C		N		N, C
Asd/Asd	N		N		N	N	N
Asp/Asp	Ci(N)		N		N	N	N
Pec/Pec	Ci(N)		Ci(N)		N	N	N
Trv/Trv	Ci(N)		N		N	N	N
Asd/
Asn	Dc, C		N, C		N, C		N, C
Pec	Di, C		Di, C		Di, C		Da, C
Trv	Di, C		N, C		N, C		N, C
Cop/Cop	N		X		N		N
Asp/Asp	Ci(N)		N		N		N
Pec/Pec	Ci(N)		Ci(N)		Ci(N)		Ci(N)
Trv/Trv	Ci(N)		N		N		N
Cop/
Asn	Di, C				Dc, C		Dc, C
Pec	Dc				Dc		Dc, C
Trv	Dc				Dc		Dc
Lyt/Lyt	Ci(N)				Ci(N)		Ci(N)
Asp/Asp	Ci(N)		N		N		N
Pec/Pec	Ci(N)		Ci(N)		Ci(N)		Ci(N)
Trv/Trv	Ci(N)		N		N		N
Lyt/
Asn	Dc, C				Dc		Dc, C
Pec	Dc				Dc		Dc
Trv	Dc, C				Dc, C		Dc, C
Hev/Hev	Ci(N)				Ci(N)		Ci(N)
Asp/Asp	Ci(N)		N		N		N
Pec/Pec	Ci(N)		Ci(N)		Ci(N)		Ci(N)
Trv/Trv	Ci(N)		N		N		N
Hev/
Asn	Di				Di		Di
Pec	Di				Di		Di
Trv	N, C				N, C		N, C
Da: deadlock antagonism; Dc: deadlock competition; Di: deadlock inhibition.
N: neutral intermingling; same interaction pattern observed at pH 7.0 and 9.0.
Ci(N): contact zone inhibition and finally neutral intermingling.
Blank: not co-cultured.
**C: mycelia covered entire surface of agar medium.
ND: Two fungi did not come into contact during 30 days of culture.

Table 5. Growth response of fungi interacting on urea agar medium at three different pH levels

		Growth response observed on urea agar medium with pH adjusted to
		pH 5.5			pH 7			pH 9
Interacting species	Day^a	OC zone^b (mm)	C zone^c (mm)	Inhibition^d ratio	OC zone^b (mm)	C zone^c (mm)	Inhibition^d ratio	OC zone^b (mm)	C zone^c (mm)	Inhibition^d ratio
Amb/Amb^e	12	21±0.8/20±0.7	20±0.5/20±0.8	0.9±0.01/1±0.02	22±0.8/21±0.7	20±0.8/20±0.6	0.9±0.02/1±0.01	18±0.8/17±0.7	16±0.8/16±0.7	0.8±0.02/1±0.03
Asn/Asn	6	20±0.7/21±0.5	22±0.8/21±0.6	0.9±0.02/1±0.01	26±0.6/25±0.4	25±0.4/25±0.6	0.9±0.01/1±0.03	26±0.7/26±0.6	26±0.8/25±0.5	1±0.03/0.9±0.02
Pec/Pec	15	20±0.8/20±0.7	22±0.5/21±0.7	0.9±0.01/0.9±0.02	24±0.8/22±0.6	22±0.6/23±0.5	0.9±0.03/0.9±0.02	30±0.8/32±0.6	30±0.8/31±0.3	1±0.8/0.10±0.01
Trv/Trv	15	12±0.8/10±0.6	12±0.7/11±0.6	0.9±0.02/0.9±0.03	12±0.7/10±0.8	12±0.8/11±0.4	0.1±0.01/0.9±0.02	12±0.8/10±0.3	12±0.4/11±0.8	1±0.8/0.1±0.0.2
Amb/	12	21±0.8	20± 0.6	0.9±0.01	22±0.3	20±0.9	0.9±0.01	22±0.1	20±0.3	0.9±0.01
Asn	6	20± 0.6	18±0.1	0.9±0.01	25±0.9	22±0.3	0.8±0.02	19±0.1	16±0.9	0.8±0.02
Amb/	12	20±0.8	17±0.1	0.8±0.02	20±0.8	18±0.3	0.8±0.02	16±0.3	14±0.9	0.8±0.02
Pec	9	20± 0.6	18±0.3	0.9±0.01	20±0.8	15±0.5	0.7±0.04	10±0.5	5±0.3	0.5±0.01
Amb/	12	21±0.8	21±0.3	1±.0.03	22±0.1	22±0.9	1±0.02	22±0.1	22±0.9	1±0.02
Trv	8	11±0.3	11±0.5	1±0.03	12±0.1	12±0.3	1±0.01	12±0.3	12±0.9	1±0.04
Asd/Asd	9	24±0.2/4±0.6	18±0.4/19±0.6	0.7±0.0.2/0.7±0.0.1	25±0.7/24±0.4	18±0.7/19±0.7	0.7±0.02/0.7±0.01	23±0.6/24±0.7	18±0.5/19±0.7	0.7±0.02/0.7±0.0.3
Asn/Asn	6	23±0.6/21±0.7	24±0.4/21±0.7	0.9±0.0.2/1±0.0.1	27±0.4/26±0.5	26±0.7/25±0.7	0.9±0.01/1±0.02	26±0.4/26±0.5	26±0.6/25±0.4	1±0.04/0.9±0.01
Pec/Pec	15	21±0.5/22±0.7	22±0.7/21±0.7	0.9±0.02/0.9±0.01	24±0.3/22±0.8	22±0.7/23±0.7	0.9±0.01/0.9±0.02	30±0.5/32±0.6	30±0.7/31±0.5	1±0.02/0.1±0.02
Trv/Trv	6	26±0.4/24±0.6	20±0.7/20±0.7	0.7±0.03/0.8±0.02	26±0.9/24±0.8	23±0.9/22±0.6	0.7±0.01/0.9±0.02	20±0.7/18±0.5	16±0.5/16±0.7	0.8±0.01/0.9±0.03
Asd/	9	14±0.5	13±0.3	0.9±0.01	15±0.9	13±0.1	0. 8±0.02	11±0.4±0.5±0.7	9±0.3	0.8±0.02
Asn	6	8±0.3	8±0.9	1±0.03	10±0.3	11±0.5	1.1±0.03	12±0.9	13±0.5	1±0.03
Asd/	9	21±0.8	27±0.1	1.2±0.01	21±0.8	25±0.9	1.1±0.05	22±0.1	18±0.3	0.8±0.02
Pec	6	7±0.3	6±.02	0.8±0.02	7±0.3	6±0.4	0.8±0.02	7±0.5	6±0.3	0.8±0.02
Asd/	9	15±0.5	12±0.3	0.8±0.02	15±0.5	12±0.3	0.8±0.02	9±0.3	6±0.5	0.6±0.04
Trv	3	15±0.9	14±0.5	0.9±0.01	16±0.1	15±0.6	0.9±0.01	14±0.3	13±0.9	0.9±0.01
Cop/Cop	9	19±0.8/19±0.9	18±0.6/18±0.8	1±0.01/1±0.01	20±0.8/21±0.8	19±0.7/20±0.9	0.7±0.02/1±0.01	18±0.8/18±0.9	19±0.9/18±0.3	1±0.02/1±0.01
Asn/Asn	6	23±0.6/21±0.7	24±0.4/21±0.7	0.9±0.02/1±0.01	27±0.4/26±0.5	26±0.7/25±0.7	0.9±0.01/1±0.02	26±0.4/26±0.5	26±0.6/25±0.4	1±0.04/0.9±0.01
Pec/Pec	15	21±0.5/22±0.7	22±0.7/21±0.7	0.9±0.02/0.9±0.01	24±0.3/22±0.8	22±0.7/23±0.7	0.9±0.01/0.9±0.02	30±0.5/32±0.6	30±0.7/31±0.5	1±0.02/0.1±0.02
Trv/Trv	6	26±0.4/24±0.6	20±0.7/20±0.7	0.7±0.03/0.8±0.02	26±0.9/24±0.8	23±0.9/22±0.6	0.7±0.01/0.9±0.02	20±0.7/18±0.5	16±0.5/16±0.7	0.8±0.01/0.9±0.03
Cop/	9	18±0.1	16±0.3	0.8±0.02	20±0.8	18±0.3	0.9±0.01	16±0.5	14±0.3	0.8±0.04
Asn	9	18±0.9	15±0.4	0.8±0.02	18±0.1	14±0.2	0.7±0.02	10±0.3	6±0.5	0.6±0.04
Cop/	12	18±0.1	16±0.3	0.8±0.02	20± 0.6	18±0.3	0.9±0.01	16±0.1	14±0.9	0.8±0.02
Pec	12	14±0.5	12±0.9	0.8±0.02	18±0.1	16±0.3	0.8±0.02	14±0.9	12±0.5	0.8±0.02
Cop/	12	15±0.3	15±0.5	1±0.04	20±0.6	20± 0.6	0.7±0.02	20± 0.6	20± 0.6	1±0.04
Trv	3	20±0.8	15±0.3	0.7±0.02	20±0.6	15±0.9	0.7±0.02	10±0.5	5±0.3	0.5±0.04
Lyt/Lyt	12	17±0.8/17±0.8	16±0.7/16±0.8	0.9±0.03/0.9±0.01	16±0.6/18±0.9	19±0.5/18±0.8	0.8±0.02/1±0.03	15±0.1/15±0.2	14±0.7/15±0.8	0.9±0.01/1±0.02
Asn/Asn	6	23±0.6/21±0.7	24±0.4/21±0.7	0.9±0.0.2/1±0.0.1	27±0.4/26±0.5	26±0.7/25±0.7	0.9±0.01/1±0.02	26±0.4/26±0.5	26±0.6/25±0.4	1±0.04/0.9±0.01
Pec/Pec	15	21±0.5/22±0.7	22±0.7/21±0.7	0.9±0.02/0.9±0.01	24±0.3/22±0.8	22±0.7/23±0.7	0.9±0.01/0.9±0.02	30±0.5/32±0.6	30±0.7/31±0.5	1±0.02/0.1±0.02
Trv/Trv	6	26±0.4/24±0.6	20±0.7/20±0.7	0.7±0.03/0.8±0.02	26±0.9/24±0.8	23±0.9/22±0.6	0.7±0.01/0.9±0.02	20±0.7/18±0.5	16±0.5/16±0.7	0.8±0.01/0.9±0.03
Lyt/	12	15±0.3	14±0.9	0.9±0.01	16±0.1	15±0.9	0.9±0.01	14±0.3	14±0.9	0.9±0.01
Asn	9	10±0.3	9±0.5	0.9±0.01	9±0.3	8±0.5	0.8±0.02	7±0.5	6±0.3	0.8±0.02
Lyt/	9	8±0.3	8±0.5	1±0.04	9±0.3	9±0.3	1±0.04	9±0.3	9±0.9	1±0.04
Pec	9	11±0.3	10±0.9	0.9±0.01	11±0.3	10±0.9	0.9±0.01	9±0.3	8±0.9	0.8±0.02
Lyt/	12	15±0.1	14±0.3	0.9±0.01	15±0.9	14±0.3	0.9±0.01	13±0.1	12±0.3	0.9±0.01
Trv	3	20±0.8	15±0.1	0.7±0.02	20±0.6	15±0.9	0.7±0.02	10±0.3	5±0.9	0.5±.004
Hev/Hev	18	20±0.4/20±0.8	19±0.3/19±0.8	0.9±0.01/0.9±0.02	21±0.5/20±0.4	20±0.8/19±0.6	0.9±0.01/0.9±0.01	18±0.8/18±0.7	17±0.8/17±0.8	0.9±0.02/0.9±0.01
Asn/Asn	9	24±0.6/25±0.6	19±0.8/18±0.6	0.7±0.02/0.7±0.01	29±0.7/28±0.5	24±0.6/23±0.8	0.8±0.02/0.8±0.02	29±0.7/28±0.6	23±0.7/23±0.6	0.7±0.03/0.8±0.02
Pec/Pec	15	20±0.8/18±0.5	20±0.6/20±0.5	0.9±0.03/1±0.01	18±0.4/18±0.6	17±0.8/18±0.7	0.9±0.02/1±0.03	18±0.6/18±0.8	17±0.8/18±0.5	0.9±0.02/1±0.02
Trv/Trv	6	24±0.9/25±0.8	18±0.4/20±0.8	0.9±0.02/0.9±0.02	25±0.3/24±0.8	20±0.4/22±0.8	0.9±0.01/0.9±0.02	20±0.8/18±0.7	16±0.7/16±0.8	0.9±0.01/0.9±0.01
Hev/	18	16±0.1	12±0.3	0.7±0.02	17±0.5	12±0.3	0.8±0.02	14±0.1	11±0.3	0.7±0.02
Asn	6	20±0.8	13±0.1	0.6±0.04	18±0.1	14±0.3	0.7±0.02	10±0.9	6±0.3	0.6±0.04
Hev/	18	20± 0.6	15±0.1	0.7±0.02	20±0.8	15±0.1	0.7±0.02	10±0.3	5±0.9	0.5±0.04
Pec	9	18±0.1	14±0.9	0.7±0.02	17±0.3	13±0.9	0.7±0.02	9±0.3	5±0.3	0.5±0.03
Hev/	15	10±0.1	10±0.3	1±0.04	10±0.9	10±0.3	1±0.04	10±0.9	10±0.9	1±0.04
Trv	3	20± 0.6	17±0.9	0.8±0.02	20±0.8	17±0.1	0.8±0.02	14±0.1	11±0.3	0.7±0.02
^aIncubation period for the observation.
^bOCZ: mycelium radius in the direction to the opposite side of the contact zone.
^cCZ: mycelium radius in the direction of the contact zone.
^dOCZ/CZ.
^eSee Table 1.
Means±SE (n = 5).

Table 6. Growth response of fungi interacting on ammonium agar medium at three different pH levels

		Growth response observed on ammonium agar medium pH adjusted to
		pH 5.5			pH 7			pH 9
Interacting species	Day^a	OC zone^b (mm)	C zone^c (mm)	Inhibition^d ratio	OC zone^b (mm)	C zone^c (mm)	Inhibition^d ratio	OC zone^b (mm)	C zone^c (mm)	Inhibition^d ratio
Amb/Amb^e	9	10±0.4/9±0.3	10±0.3/10±0.3	1±0.01/0.9±0.02	11±0.5/12±0.3	11±0.4/13±0.3	1±0.01/0.9±0.02	8±0.3/9±0.2	8±0.3/10±0.4	1±0.01/0.9±0.02
Asn/Asn	6	20±0.3/21±0.2	22±0.3/21±0.4	0.9±0.01/1±0.01	26±0.6/25±0.5	25±0.3/25±0.3	0.9±0.02/1±0.01	26±0.3/26±0.3	26±0.3/25±0.5	1±0.01/0.9±0.02
Pec/Pec	15	20±0.3/20±0.3	22±0.4/21±0.3	0.9±0.02/0.9±0.02	24±0.3/22±0.4	22±0.3/23±0.4	0.9±0.02/0.9±0.01	30±0.3/32±0.4	30±0.4/31±0.3	1±0.01/0.1±0.01
Trv/Trv	6	25±0.3/24±0.4	20±0.3/20±0.3	0.9±0.01/0.9±0.01	26±0.3/24±0.5	20±0.3/22±0.5	0.7±0.02/0.9±0.01	20±0.4/18±0.3	16±0.3/16±0.3	0.8±0.01/0.9±0.02
Amb/	9	9±0.3	9±0.2	1±0.04	10±0.2	9±0.3	1±0.04	9±0.3	9±0.3	0.8±0.03
Asn	9	20±0.5	22±0.4	1±0.04	17±0.4	17±0.4	1±0.03	17±0.2	15±0.4	0.8±0.03
Amb/	9	9±0.3	9±0.2	1±0.04	10±0.2	9±0.3	1±0.03	9±0.3	9±0.3	0.8±0.03
Pec	6	13±0.5	12±0.4	0.9±0.02	12±0.4	13±0.2	1±0.03	11±0.2	14±0.4	1.2±0.04
Amb/	9	11±0.2	11±0.2	0.9±0.03	12±0.2	13±0.4	0.6±0.05	12±0.4	13±0.2	0.7±0.03
Trv	7	13±0.2	12±0.4	0.9±0.02	16±05	15±0.4	0.9±0.03	16±0.4	16±0.4	1±0.04
Asd/Asd	9	24±0.3/24±0.3	18±0.3/19±0.5	0.7±0.03/0.7±0.03	25±0.3/24±0.2	18±0.3/19±0.4	0.7±0.03/0.7±0.03	23±0.3/24±0.5	18±0.5/19±0.3	0.7±0.03/0.7±0.03
Asn/Asn	6	20±0.3/21±0.4	22±0.4/21±0.3	0.9±0.01/1±0.02	26±0.4/25±0.3	25±0.3/25±0.3	0.9±0.01/1±0.02	26±0.3/26±0.3	26±0.4/25±0.3	1±0.02/0.9±0.02
Pec/Pec	15	21±0.4/22±0.3	22±0.3/21±0.4	0.9±0.01/0.9±0.01	24±0.2/22±0.4	22±0.3/23±0.4	0.9±0.01/0.9±0.02	30±0.3/32±0.4	30±0.3/31±0.4	1±0.01/0.1±0.03
Trv/Trv	6	26±0.3/24±0.6	20±0.3/20±0.3	0.7±0.03/0.8±0.02	26±0.3/24±0.4	23±0.5/22±0.3	0.7±0.02/0.9±0.02	20±0.3/18±0.5	16±0.3/16±0.3	0.8±002/0.9±0.02
Asd/	12	10±0.4	7±0.3	0.7±0.03	12±0.4	8±0.3	0.6±0.05	11±0.2	7±0.2	0.6±0.03
Asn	6	14±0.4	12±0.2	0.8±0.02	17±0.4	17±0.2	1±0.01	17±0.5	17±0.4	1±0.04
Asd/	12	10±0.2	7±0.3	0.7±0.02	12±0.4	8±0.3	0.6±0.05	11±0.2	7±0.3	0.6±0.03
Pec	6	14±0.4	8±0.2	0.5±0.02	12±0.2	10±0.4	0.8±0.05	12±0.4	11±0.2	0.9±0.04
Asd/	12	11±0.2	7±0.4	0.6±0.02	12±0.4	8±0.3	0.6±0.05	12±0.2	10±0.4	0.8±0.03
Trv	3	20±0.4	21±0.2	1±0.04	20±0.5	21±0.4	1±0.04	22±0.5	22±0.5	1±0.04
Cop/
Asn
Cop/
Pec
Cop/
Trv
Lyt/
Asn
Lyt/
Pec
Lyt/
Trv
Hev/
Asn
Hev/
Pec
Hev/
Trv
^aIncubation period for the observation.
^bOCZ: mycelium radius in the direction to the opposite side of the contact zone.
^cCZ: mycelium radius in the direction of the contact zone.
^dOCZ/CZ.
^e See Table 1.
Means±SE (n = 5).
Note: Blank means no co-cultured, since they did not grow on ammonium agar medium.

Table 7. Growth response of fungi interacting on nitrite agar medium at three different pH levels

		Growth response observed on nitrite agar medium with pH adjusted to
		pH 5.5			pH 7			pH 9
Interacting species	Day^a	OC zone^b (mm)	C zone^c (mm)	Inhibition^d ratio	OC zone^b (mm)	C zone^c (mm)	Inhibition^d ratio	OC zone^b (mm)	C zone^c (mm)	Inhibition^d ratio
Amb/Amb^e	9	0	0	0	12±0.8/10±0.9	11±0.7/10±0.9	0.9±0.02/1±0.01	12±0.8/11±0.7	11±0.5/10±0.9	0.9±0.01/0.8±0.02
Asn/Asn	6	7±0.7/7±0.8	7±0.8/7±0.9	1±0.01/1±0.02	8±0.7/8±0.7	14±0.8/14±0.7	0.9±0.02/0.8±0.02	16±0.9/15±0.8	14±0.6/15±0.8	0.9±0.01/0.9±0.02
Pec/Pec	15	10±0.8/11±0.9	9±0.6/9±.0.7	0.9±0.03/0.9±0.02	12±0.9/11±0.8	10±0.7/9±0.8	0.9±0.02/0.9±0.01	10±0.7/11±0.9	9±0.7/9±0.9	0.9±0.03/0.9±0.02
Trv/Trv	4	12±0.7/10±0.9	12±0.8/11±0.9	0.9±0.01/0.9±0.01	12±0.7/10±0.9	10±0.5/10±0.7	0.1±0.01/0.9±0.01	11±0.4/10±0.9	8±0.7/9±0.9	0.9±0.01/0.9±0.01
Amb/	9	0	0	0	11±0.5	8±0.5	0.7±0.01	22±0.8	30±0.7	1.3±0.06
Asn	6	12±0.9	11±0.4	0.9±0.01	10±0.7	10±0.6	1±0.06	11±0.4	10±0.6	0.9±0.01
Amb/	9	0	0	0	11±0.5	8±0.6	0.7±0.01	22±0.7	30±0.6	1.3±0.07
Pec	6	4±0.1	4±0.4	1.1±0.06	4±0.1	4±0.1	1±0.06	3±0.1	3±0.1	1±0.06
Amb/	12	0	0	0	23±0.8	20±0.4	0.9±0.01	22±0.8	30±0.7	1.3±0.07
Trv	9	14±0.9	14±0.4	1±0.07	13±0.5	12±0.6	0.9±0.01	13±0.4	12±0.6	0.9±0.01
Asd/Asd	18	25±0.9	22±0.9	0.8±0.02	27±0.9	24±0.9	0.9±0.01	25±0.9	20±0.9	0.8±.0.02
Asn/Asn	9	15±0.9/14±0.8	14±0.8/14±0.9	0.9±0.02/0.9±0.01	29±0.6/28±0.9	22±0.8/23±0.9	0.7±0.01/0.8±0.02	29±02/28±0.4	22±0.9/23±0.2	0.7±0.02/0.6±0.02
Pec/Pec	15	10±0.6/11±0.8	9±0.8/9±0.7	0.9±0.01/0.9±0.02	12±0.8/11±0.8	10±0.5/9±0.6	0.9±0.01/0.9±0.02	10±0.9/11±0.8	9±0.9/9±0.9	0.9±0.02/0.7±0.01
Trv/Trv	4	26±0.8/27±0.5	15±0.9/15±0.5	0.6±0.1/0.6±0.02	22±0.2/20±0.4	13±0.9/13±0.7	0.5±0.02/0.6±0.01	20±0.9/20±0.9	13±0.9/13±0.8	0.6±0.01/0.6±0.01
Asd/	12	14±0.9	13±0.8	0.9±0.07	15±0.6	13±0.4	0.8±0.07	11±0.4	9±0.6	0.8±0.01
Asn	6	24±0.8	20±0.9	0.8±0.07	22±0.7	18±0.8	0.8±0.05	20±0.8	9±0.6	0.4±0.07
Asd/	12	20±0.8	15±0.5	0.9±0.07	27±0.6	24±0.8	0.9±0.07	22±0.8	15±0.6	0.7±0.01
Pec	9	13±0.5	10±0.5	0.7±0.07	11±0.6	9±0.4	0.8±0.05	6±0.1	5±0.1	0.8±0.07
Asd/	12	25±0.7	23±0.9	0.9±0.07	20±0.7	12±0.5	0.6±0.07	25±0.8	14±0.6	0.5±0.07
Trv	3	24±0.7	18±0.5	0.7±0.07	25±0.5	11±0.5	0.5±0.05	21±0.4	19±0.6	0.9±0.01
Cop/Cop	18	17±0.9/16±0.5	17±0.9/16±0.5	1±0.02/0.8±0.02	22±0.9/20±0.5	21±0.9/20±0.5	0.5±0.01/0.8±0.02	25±0.9/24±0.5	21±0.9/22±0.5	0.8±0.01/0.8±0.02
Asn/Asn	9	16±0.9/14±0.8	14±0.7/14±0.9	0.9±0.01/0.9±0.02	29±0.9/28±0.3	22±0.9/23±0.8	0.7±0.01/0.8±0.02	29±0.9/28±0.8	22±0.9/23±0.8	0.7±0.01/0.8±0.02
Pec/Pec	15	10±0.9/11±0.6	9±0.5/9±0.7	0.9±0.02/0.9±0.03	12±0.9/11±0.4	10±0.8/11±0.6	0.9±0.04/0.9±0.02	10±0.6/11±0.9	9±0.4/9±0.9	0.9±0.01/0.9±0.02
Trv/Trv	4	27±0.9/30±0.5	13±0.9/13±0.7	0.4±0.02/0.4±0.01	24±0.9/26±0.9	13±0.8/13±0.9	0.5±0.01/0.5±0.02	20±0.9/20±0.6	13±0.9/13±0.4	0.6±0.01/0.6±0.01
Cop/	9	18±0.5	16±0.9	0.8±0.02	20±0.7	18±0.7	0.9±0.05	16±0.7	14±0.6	0.8±0.01
Asn	6	6±0.5	5±0.1	0.9±0.02	6±0.1	5±0.6	0.8±0.05	5±0.1	5±0.1	1±0.06
Cop/	18	16±0.9	16±0.5	1±0.06	19±0.6	17±0.5	0.9±0.04	25±0.8	24±0.7	0.9±0.06
Pec	6	9±0.5	7±0.1	0.8±0.05	9±0.1	7±0.1	0.8±0.03	5±0.1	5±0.1	1±0.07
Cop/	18	16±0.4	16±0.5	1±0.06	20±0.6	14±0.5	0.7±0,05	27±0.8	27±0.6	1±0.06
Trv	6	17±0.4	10±0.4	0.6±0.05	25±0.8	12±0.6	0.5±0.05	18±0.7	5±0.1	0.3±0.07
Lyt/Lyt	18	18±0.9/18±0.9	17±0.9/17±0.9	1±0.01/09±0.02	20±0.9/21±0.9	21±0.9/20±0.9	0.5±0.02/09±0.01	20±0.9/24±0.9	21±0.9/22±0.9	0.8±0.02/1±0.04
Asn/Asn	9	16±0.9/14±0.8	14±0.7/14±0.9	0.9±0.01/0.9±0.02	29±0.9/28±0.3	22±0.9/23±0.8	0.7±0.01/0.8±0.02	29±0.9/28±0.8	22±0.9/23±0.8	0.7±0.01/0.8±0.02
Pec/Pec	15	10±0.9/11±0.6	9±0.5/9±0.7	0.9±0.02/0.9±0.03	12±0.9/11±0.4	10±0.8±0.6	0.9±0.04/0.9±0.01	10±0.6/11±0.9	9±0.4/9±0.9	0.9±0.01/0.9±0.02
Trv/Trv	4	27±0.9/30±0.5	13±0.9/13±0.7	0.4±0.02/0.4±0.01	24±0.9/26±0.9	13±0.8/13±0.9	0.5±0.01/0.5±0.02	20±0.9/20±0.6	13±0.9/13±0.4	0.6±0.01/0.6±0.01
Lyt/	6	20±0.7	17±0.4	0.8±0.05	14±0.7	13±0.6	0.9±0.03	16±0.7	9±0.6	0.6±0.07
Asn	18	22±0.7	20±0.9	0.9±0.06	24±0.9	11±0.5	0.4±0.02	19±0.9	17±0.7	0.9±0.06
Lyt/	9	24±0.7	14±0.9	0.6±0.02	14±0.8	13±0.6	0.9±0.06	21±0.8	14±0.9	0.7±0.06
Pec	27	13±0.8	11±0.6	0.8±0.02	19±0.8	17±0.6	0.9±0.07	19±0.9	15±0.8	0.8±0.07
Lyt/	12	15±0.8	13±0.6	0.8±0.02	17±0.6	15±0.6	0.8±0.02	17±0.6	14±0.9	0.7±0.07
Trv	6	22±0.9	14±0.9	0.6±0.02	22±0.9	17±0.5	0.7±0.02	20±0.9	9±0.6	0.4±0.07
Hev/Hev	18	20±0.8/20±0.7	18±0.9/18±0.8	0.9±0.01/0.9±0.02	21±0.8/20±0.9	20±0.8/19±0.9	0.9±0.01/0.9±0.02	18±0.9/18±0.5	17±0.9/17±0.8	0.9±0.03/0.9±0.01
Asn/Asn	9	15±0.7/14±0.9	14±0.7/14±0.9	0.9±0.01/0.9±0.03	29±0.9/28±0.5	22±0.9/23±0.7	0.7±0.02/0.8±0.03	29±0.9/28±0.8	22±0.9/23±0.8	0.7±0.02/0.8±0.02
Pec/Pec	15	10±0.8/11±0.9	9±0.7/9±0.8	0.9±0.03/0.9±0.02	12±0.8/11±0.9	10±0.9/9±0.7	0.9±0.03/0.9±0.03	10±0.8/11±0.9	9±0.9/9±0.7	0.9±0.03/0.9±0.02
Trv/Trv	4	27±0.9/30±0.9	13±0.9/13±0.8	0.4±0.01/0.4±0.02	24±0.8/26±0.7	13±0.9/13±0.6	0.5±0.02/0.5±0.01	20±0.9/20±0.9	13±0.7/13±0.9	0.6±0.02/0.6±0.02
Hev/	6	14±0.7	10±0.7	0.7±0.02	12±0.6	8±0.6	0.7±0.03	12±0.6	10±0.9	0.8±0.07
Asn	18	13±0.7	11±0.8	0.8±0.02	18±0.7	15±0.7	0.8±0.02	24±0.9	21±0.8	0.8±0.07
Hev/	6	23±0.9	13±0.7	0.6±0.03	14±0.9	9±0.6	0.6±0.02	21±0.8	13±0.6	0.6±0.07
Pec	6	10±0.8	10±0.9	1±0.03	12±0.9	8±0.6	0.6±0.03	14±0.9	12±0.9	0.8±0.07
Hev/	18	13±0.7	11±0.6	0.8±0.02	15±0.8	15±0.6	1±0.02	15±0.6	15±0.9	1±0.07
Trv	6	23±0.9	19±0.8	0.8±0.04	24±0.6	22±0.7	0.9±0.02	24±0.9	22±0.8	0.9±0.07
^aIncubation period for the observation.
^bOCZ: mycelium radius in the direction to the opposite side of the contact zone (mm).
^cCZ: mycelium radius in the direction of the contact zone.
^dOCZ/CZ.
^eSee Table 1.
Means±SE (n = 5).

Table 8. Growth response of fungi interacting on nitrate agar medium at three different pH levels

		Growth response observed on nitrate agar medium with pH adjusted to
		pH 5.5			pH 7			pH 9
Interacting species	Day^a	OC zone^b (mm)	C zone^c (mm)	Inhibition^d ratio	OC zone^b (mm)	C zone ^c (mm)	Inhibition^d ratio	OC zone^b (mm)	C zone^c (mm)	Inhibition^d ratio
Amb/Amb^e	9	15±0.4/14±0.5	14±0.3/14±0.5	0.9±0.01/1±0.02	20±0.5/20±0.7	21±0.4/20±0.5	1±0.02/1±0.03	20±0.5/20±0.6	21±0.4/20±0.2	1±0.01/1±0.02
Asn/Asn	9	20±0.6/21±0.4	22±0.4/21±0.6	0.9±0.01/1±0.02	26±0.5/25±0.6	25±0.6/25±0.5	0.9±0.01/1±0.03	26±0.5/26±0.4	26±0.3/25±0.5	1±0.02/0.9±0.01
Pec/Pec	15	14±0.5/15±0.6	14±0.6/14±0.5	1±0.02/0.9±0.01	15±0.4/15±0.5	16±0.7/14±0.8	1±0.02/0.9±0.01	14±0.6/15±0.5	14±0.4/14±0.6	1±0.02/0.9±0.03
Trv/Trv	9	20±0.5/22±0.3	19±0.5/20±0.6	0.9±0.02/0.9±0.03	22±0.6/24±0.5	20±0.5/22±0.7	0.9±0.03/0.9±0.02	20±0.3/18±0.5	16±0.3/16±0.5	0.8±0.01/0.9±0.03
Amb/	9	20±0.1	16±0.5	0.8±0.02	27±0.1	21±0.5	0.7±0.03	25±0.1	22±0.5	0.8±0.02
Asn	9	28±0.5	33±0.1	1±0.04	17±0.5	17±0.2	1±0.04	17±0.5	15±0.2	0.8±0.03
Amb/	9	20±0.1	16±0.1	0.8±0.02	27±0.2	21±0.5	0.7±0.02	25±0.2	22±0.3	0.8±0.02
Pec	6	20±0.5	20±0.3	1±0.03	8±0.3	7±0.2	0.8±0.03	11±0.4	14±0.2	1.2±0.05
Amb/	9	20±0.1	14±0.5	0.7±0.05	20±0.3	12±0.5	0.6±0.02	25±0.4	18±0.3	0.7±0.03
Trv	7	28±0.5	30±0.4	1±0.04	16±0.5	15±0.3	0.9±0.04	16±0.3	16±0.5	1±0.04
Asd/Asd	9	24±0.03/24±0.5	18±0.5/19±0.4	0.7±0.01/0.7±0.01	25±0.4/24±0.5	18±0.5/19±0.4	0.7±0.5/0.7±0.3	23±0.5/24±0.4	18±0.3/19±0.5	0.7±0.01/0.7±0.02
Asn/Asn	9	22±0.04/24±0.6	18±0.5/17±0.4	0.9±0.01/0.7±0.02	29±0.6/28±0.4	22±0.5/23±0.6	0.7±0.5/0.8±0.4	29±0.5/28±0.3	22±0.5/23±0.6	0.7±0.01/0.8±0.02
Pec/Pec	15	20±0.5/20±0.4	22±0.6/21±0.5	0.9±0.02/0.9±0.01	24±0.5/22±0.3	22±0.6/23±0.4	0.9±0.4/0.9±0.3	30±0.4/32±0.5	30±0.3/31±0.4	1±0.03/0.10±0.01
Trv/Trv	6	24±0.2/24±0.3	18±0.7/20±0.5	0.9±0.02/0.9±0.01	25±0.3/24±0.5	20±0.5/22±0.5	0.8±0.5/0.9±0.4	20±0.5/18±0.4	16±0.3/16±0.4	0.8±0.03/0.9±0.02
Asd/	9	14±0.1	13±0.5	0.9±0.05	15±0.3	13±0.2	0.8±0.02	11±0.5	9±0.3	0.8±0.02
Asn	6	14±0.5	12±0.1	0.8±0.03	17±0.2	17±0.5	1±0.04	17±0.2	17±0.3	1±0.04
Asd/	12	21±0.5	9±0.3	0.4±0.07	15±0.3	13±0.4	0.8±0.03	22±0.3	15±0.5	0.7±0.02
Pec	6	8±0.3	14±0.1	1.7±0.04	10±0.5	10±0.3	1±0.04	10±0.4	6±0.1	0.6±0.03
Asd/	12	26±0.5	30±0.3	1±0.04	22±0.3	15±0.4	0.7±0.03	23±0.2	19±0.3	0.8±0.03
Trv	3	20±0.3	26±0.1	1.3±0.5	20±0.4	10±0.3	0.5±0.05	21±0.3	16±0.1	0.7±0.04
Cop/Cop	9	19±0.4/19±0.6	18±0.4/18±0.5	1±0.01/1±0.02	20±0.03/21±0.01	19±0.4/20±0.5	0.7±0.02/1±0.01	21±0.4/21±0.5	20±0.3/20±0.5	0.9±0.02/0.9±0.01
Asn/Asn	9	20±0.5/21±0.4	21±0.6/21±0.4	1±0.01/1±0.03	20±0.02/20±0.03	19±0.6/18±0.4	0.9±0.03/0.9±0.02	18±0.6/18±0.6	17±0.6/17±0.4	0.9±0.02/0.9±0.01
Pec/Pec	15	21±0.3/22±0.4	22±0.3/21±0.5	0.9±0.02/0.9±0.03	23±0.02/23±0.01	21±0.1/23±0.3	0.9±0.02/0.9±0.02	20±0.4/19±0.5	20±0.5/18±0.3	1±0.02/0.9±0.01
Trv/Trv	6	20±0.3/18±0.5	16±0.5/16±0.4	0.8±0.01/0.9±0.03	26±0.04/24±0.02	20±0.4/21±0.3	0.7±0.01/0.9±0.03	26±0.5/24±0.6	20±0.3/20±0.5	0.7±0.02/0.8±0.03
Asn	6	11±0.3	12±0.1	1±0.5	12±0.3	11±0.4	0.9±0.02	12±0.2	10±0.3	0.8±0.03
Cop/	12	24±0.5	20±0.3	0.8±0.04	24±0.5	22±0.3	0.9±0.02	17±0.1	17±0.1	1±0.04
Pec	6	10±0.3	10±0.5	1±0.5	10±0.3	8±0.5	0.8±0.03	8±0.5	7±0.3	0.8±0.02
Cop/	12	23±0.3	15±0.1	0.6±0.04	24±0.4	18±0.3	0.7±0.04	25±0.1	24±0.5	0.9±0.04
Trv	6	26±0.5	14±0.3	0.5±0.5	22±0.2	17±0.5	0.7±0.03	20±0.2	9±0.1	0.4±0.03
Lyt/Lyt	12	20±0.4/21±0.5	19±0.3/19±0.5	0.9±0.01/0.9±0.03	22±0.2/22±0.1	21±0.5/20±0.4	0.9±0.01/0.9±0.02	19±0.5/19±0.3	18±0.4/18±0.5	0.9±0.01/0.9±0.02
Asn/Asn	9	20±0.3/21±0.5	21±0.5/21±0.6	1±0.02/1±0.01	20±0.3/20±0.2	19±0.3/18±0.4	0.9±0.03/0.9±0.01	18±0.3/18±0.4	17±0.6/17±0.3	0.9±0.03/0.9±0.01
Pec/Pec	15	21±0.4/22±0.4	22±0.2/21±0.5	0.9±0.5/0.9±0.02	23±0.2/23±0.4	21±0.3/23±0.5	0.9±0.02/0.9±0.03	20±0.4/19±0.5	20±0.2/18±0.5	1±0.03/0.9±0.03
Trv/Trv	6	20±0.3/18±0.5	16±0.4/16±0.3	0.8±0.03/0.9±0.04	26±0.1/24±0.5	20±0.5/21±0.2	0.7±0.02/0.9±0.01	26±0.6/24±0.5	20±0.5/20±0.5	0.7±0.01/0.8±0.03
Lyt/	12	15±0.3	13±0.1	0.8±0.3	17±0.3	15±0.3	0.8±0.03	17±0.3	14±0.5	0.7±0.05
Asn	9	15±0.5	15±0.3	0.8±0.02	22±0.1	21±0.5	0.9±0.04	19±0.1	16±0.5	0.8±0.02
Lyt/	12	24±0.1	20±0.3	0.7±0.02	25±0.1	23±0.3	0.8±0.05	24±0.5	22±0.3	0.8±0.03
Pec	18	13±0.5	11±0.2	0.8±0.02	15±0.3	15±0.4	1±0.04	15±0.3	15±0.4	1±0.04
Lyt/	12	15±0.4	13±0.3	0.8±0.02	17±0.5	15±0.1	0.8±0.02	17±0.5	14±0.3	0.7±0.02
Trv	6	22±0.1	14±0.2	0.6±0.04	22±0.3	17±0.1	0.7±0.05	20±0.3	9±0.5	0.4±0.01
Hev/Hev	18	20±0.5/20±0.5	18±0.5/18±0.5	0.9±0.01/0.9±0.01	21±0.5/20±0.4	20±0.4/19±0.5	0.9±0.01/0.9±0.03	18±0.02/18±0.3	17±0.5/17±0.5	0.9±0.01/0.9±0.02
Asn/Asn	18	24±0.4/25±0.5	19±0.4/18±0.5	0.7±0.01/0.7±0.02	29±0.4/28±0.5	24±0.5/23±0.4	0.8±0.02/0.8±0.01	29±0.5/28±0.4	23±0.5/23±0.4	0.7±0.02/0.8±0.01
Pec/Pec	15	20±0.3/18±0.4	20±0.5/20±0.5	0.9±0.02/1±0.02	18±0.5/18±0.5	17±0.3/18±0.5	0.9±0.01/1±0.02	18±0.5/18±0.5	17±0.5/16±0.3	0.9±0.01/0.9±0.01
Trv/Trv	6	24±0.5/25±0.3	18±0.5/20±0.4	0.9±0.02/0.9±0.01	25±0.4/24±0.5	20±0.4/22±0.5	0.9±0.01/0.9±0.02	20±0.5/18±0.4	16±0.4/16±0.5	0.9±0.02/0.9±0.02
Hev/	18	10±0.3	8±0.4	0.8±0.02	16±0.4	12±0.1	0.7±0.02	14±0.4	12±0.2	0.8±0.02
Asn	9	19±0.4	9±0.5	0.4±0.01	17±0.3	11±0.4	0.6±0.01	18±0.3	16±0.3	0.9±0.03
Hev/	18	12±0.1	10±0.5	0.8±0.02	18±0.4	12±0.1	0.6±0.03	14±0.3	12±0.5	0.8±0.02
Pec	6	10±0.5	10±0.3	1±0.04	12±0.5	8±0.3	0.6±0.02	14±0.1	12±0.3	0.8±0.03
Hev/	18	13±0.1	11±0.5	0.8±0.02	15±0.2	15±0.1	1±0.04	15±0.4	15±0.5	1±0.05
Trv	6	23±0.5	19±0.1	0.8±0.02	24±0.3	22±0.5	0.9±0.02	24±0.3	22±0.1	0.9±0.03
^aIncubation period for the observation.
^bOCZ: mycelium radius in the direction to the opposite side of the contact zone.
^cCZ: mycelium radius in the direction of the contact zone.
^dOCZ/CZ.
^eSee Table 1.
Means±SE (n = 5).

When the non-ammonia fungi, Aspergillus niger, Penicillium citrinum and Trichoderma viride, were co-cultured, they first showed contact zone inhibition and finally intermingled on urea, ammonium, nitrite and nitrate agar media in all pH conditions (Tables 4–8). The ratio between of mycelial colony radius in the CZ direction to that in the OCZ direction showed growth inhibition even the same species (Tables 5–8).

Inter-species co-culturing on media containing different nitrogen sources

The late stage EP species, C. phlyctidospora and L. tylicolor, and the LP species, H. vinosophyllum, grew poorly on ammonium agar medium. Therefore, we did not co-culture these species with non-ammonia fungi in the following experiments (Table 4).

Interactions between ammonia fungi and non-ammonia fungi on urea agar medium

The early stage EP species, Am. botrytis showed antagonistic deadlock with the non-ammonia fungi, Asp. niger (Tables 3 and 4; Figure 1A) and P. citrinum (Tables 4 and 5; Figure 1E), but neutral intermingling with another non-ammonia fungus, T. viride (Tables 4 and 5; Figure 1I). T. viride grew poorly on urea agar medium containing the concentration of urea that was optimal for Am. botrytis (Table 5; Figure 1I; Barua et al. 2012.). Another early stage EP species, Asc. denudatus, showed antagonistic deadlock with non-ammonia fungi in all pH conditions (Tables 4 and 5; Figures 2A,E,I, 3A,E,I and 4A,E,I). After 30 days of incubation, the mycelium of Am. botrytis was slightly overgrown by those of Asp. niger and P. citrinum (Table 4; Figure 1A,E), whereas the mycelium of Asc. denudatus was covered gradually by those of all tested non-ammonia fungi in all pH conditions (Table 4). When Am. botrytis was co-cultured with non-ammonia fungi, it formed abundant conidiophores at pH 5.5 and pH 7.0 but few at pH 9.0 (Figure 1A). Asp. niger and P. citrinum formed abundant conidiophores after their mycelia contacted those of Am. botrytis at pH 5.5 and pH 7.0, but formed a few conidiophores at pH 9.0. Asc. denudatus did not form fruiting bodies in intra-species and inter-species co-cultures, whereas Asp. niger and P. citrinum showed poor conidiophore formation when co-cultured with Asc. denudatus in all pH conditions (Figures 2A and 3A). T. viride did not form conidiophores when co-cultured with Am. botrytis and Asc. denudatus in all pH conditions (Figures 3G and 4G).

Figure 3. Mycelial interactions between Coprinopsis phlyctidospora and non-ammonia fungi. C. phlyctidospora /Asp. niger at pH 5.5 (A) urea, 9^th day; (B) NO₂-N, 18^th day; (C) NO₃-N, 12^th day. C. phlyctidospora/P. citrinum at pH 5.5 (D) urea, 12^th day; (E) NO₂-N, 18^th day; (F) NO₃-N, 12^th day. C. phlyctidospora/T. viride at pH 5.5 (G) urea, 12^th day; (H) NO₂-N, 18^th day; (I) NO₃-N 12^th day.

Figure 1. Mycelial interactions between Amblyosporium botrytis and non-ammonia fungi. Am. botrytis/As. niger at pH 5.5 (A) urea, 12^th day; (B) NH₄-N, 12^th day; (C) NO₂-N, 9^th day; (D) NO₃-N, 9^th day. Am. botrytis/P. citrinum at pH 5.5 (E) urea, 12^th day; (F) (NH₄-N, 12^th day; (G) NO₂-N, 9^th day, (H) NO₃-N 9^th day. Am. botrytis/T. viride at pH 5.5 (I) urea, 12^th day; (J) NH₄-N, 9^th day; (K) (NO₂-N, 9^th day; (L) NO₂-N, 9^th day.

The late stage EP species C. phlyctidospora and L. tylicolor showed antagonistic deadlocks with non-ammonia fungi (Tables 4 and 5; Figures 2A,E,I, 3A,E,I and 4A,E,I). The mycelium of C. phlyctidospora was not covered by those of P. citrinum and T. viride even 30 days of incubation, but covered by Asp. niger, at any pH conditions (Tables 4 and 5; Figure 3D,G). The mycelium of L. tylicolor was covered gradually by those of the non-ammonia fungi. C. phlyctidospora and L. tylicolor did not form fruiting bodies during intra-species and inter-species co-culturing, whereas Asp. niger showed poor conidiophore formation when co-cultured with C. phlyctidospora (Figures 2A and 3A). P. citrinum formed conidiophores when co-cultured with L. tylicolor in all pH conditions (Figures 3G and 4E). T. viride did not form conidiophores when co-cultured with C. phlyctidospora and L. tylicolor in all pH conditions (Figures 3G and 4G).

The LP species H. vinosophyllum showed competing deadlock with Asp. niger and P. citrinum, and neutral intermingling with T. viride in all pH conditions (Tables 4 and 5; Figure 5A,E,I). The mycelium of H. vinosophyllum was rapidly covered by that of T. viride within 3 days after the two species came into contact. However, the mycelium of H. vinosophyllum was not covered by those of Asp. niger and P. citrinum even after 30 days of incubation in all pH conditions (Tables 4 and 5). H. vinosophyllum did not form fruiting bodies during intra-species and inter-species co-culturing, whereas Asp. niger and P. citrinum formed abundant conidiophores when co-cultured with H. vinosophyllum at pH 5.5 and 7.0, and but a few at pH 9.0. Mycelium of T. viride covered that of H. vinosophyllum and then formed conidiophores in all pH conditions (Table 4).

Interactions between ammonia fungi and non-ammonia fungi on ammonium medium

The early stage EP species Am. botrytis showed neutral intermingling with non-ammonia fungi (Table 4). Another early stage EP species, Asc. denudatus, also showed neutral intermingling with Asp. niger and T. viride, but inhibition deadlock with P. citrinum in all pH conditions (Table 4; Figure 2A,E,H). After 30 days of incubation, the mycelia of Am. botrytis and Asc. denudatus were covered by those of non-ammonia fungi (Table 4). Am. botrytis showed poor conidiophore formation when co-cultured with Asp. niger and T. viride at pH 5.5 and pH 7.0, but not at pH 9.0 (Figure 1B). Am. botrytis did not form conidiophores when co-cultured with P. citrinum in all pH conditions (Figure 1E). Asc. denudatus did not form fruiting bodies during intra-species and inter-species co-culturing. All of the tested non-ammonia fungi formed abundant co-nidiophores when co-cultured with Am. botrytis and Asc. denudatus in all pH conditions.

Interactions between ammonia fungi and non-ammonia fungi on nitrite medium

The early stage EP species, Am. botrytis grew poorly and showed neutral intermingling with non-ammonia fungi at pH 7.0 and pH 9.0. Am. botrytis started to grow 18 days after inoculation on medium at pH 5.5 (Tables 3 and 7). Another early stage EP species, Asc. denudatus, showed weak antagonistic deadlock with non-ammonia fungi in all pH conditions (Tables 4 and 7; Figure 2C,G,K). After 30 days of incubation, the mycelia of Am. botrytis and Asc. denudatus were covered by those of non-ammonia fungi (Table 4). Am. botrytis showed poor conidiophore formation when co-cultured with Asp. niger at pH 5.5 (Figure 1A) but no conidiophore formation at pH 7.0 and 9.0. Am. botrytis did not form conidiophores with P. citrinum and T. viride at any pH conditions. Asc. denudatus did not form fruiting bodies during intra-species and inter-species co-culturing.

The late stage EP species, C. phlyctidospora and L. tylicolor, showed weak antagonistic deadlock with non-ammonia fungi (Tables 4 and 7; Figures 2C,G,K, 3C,G,H, and 4C,G,H). The mycelium of C. phlyctidospora was slightly covered by that of Asp. niger, but not by those of P. citrinum and T. viride (Table 4; Figure 3C,G). The mycelium of L. tylicolor was gradually covered by those of Asp. niger and T. viride, but not by that of P. citrinum in all pH conditions (Table 4; Figure 4C,G). C. phlyctidospora and L. tylicolor did not form fruiting bodies during intra-species and inter-species co-culturing. The non-ammonia fungus T. viride showed poor conidiophore formation when co-cultured with C. phlyctidospora and L. tylicolor, and no conidiophore formation of non-ammonia fungi Asp. niger and P. citrinum when co-cultured with C. phlyctidospora and L. tylicolor in all pH conditions (Figure 3C).

The LP species H. vinosophyllum showed competition deadlock when co-cultured with Asp. niger and P. citrinum at pH 5.5 and 7.0, and neutral intermingling at pH 9.0 (Figure 5C,F). H. vinosophyllum showed neutral intermingling with T. viride in all pH conditions (Figure 5I). T. viride formed conidiophores surrounding the mycelium of H. vinosophyllum after its mycelium was covered by that of T. viride (Table 4). The mycelium of H. vinosohpyllum was covered by those of Asp. niger and P. citrinum in all pH conditions (Table 4; Figure 5C,G). When co-cultured with H. vinosophyllum, the non-ammonia fungus, Asp. niger formed conidiophores poorly at pH 5.5 (Figure 5B) and pH 7.0, but formed abundant conidiophores at pH 9.0. The non-ammonia fungus, P. citrinum, showed poor conidiophore formation when co-cultured with H. vinosophyllum at pH 5.5 (Figure 5F), and formed no conidiophore when cultured at pH 7.0 and 9.0. The non-ammonia fungus, T. viride, did not form conidiophores when co-cultured with H. vinosophyllum at any pH (Figure 5G).

Interactions between ammonia fungi and non-ammonia fungi on nitrate medium

The early stage EP species Am. botrytis grew weakly and showed neutral intermingling with the non-ammonia fungi, Asp. niger, P. citrinum and T. viride (Tables 4 and 8; Figure 1C,G,H). Another early stage EP species, Asc. denudatus, showed antagonistic deadlock with Asp. niger and T. viride in all pH conditions (Figure 1D). Asc. denudatus showed strong competing deadlock with P. citrinum and was not completely invaded (Figure 1H,L). The mycelia of Am. botrytis and Asc. denudatus were gradually covered by those of non-ammonia fungi in all pH conditions (Figures 1D,H,L and 2D,H,L). Am. botrytis showed poor conidiophore formation when co-cultured with P. citrinum, and did not form conidiophores with Asp. niger and T. viride in all pH conditions (Figure 1H).

The late stage EP species C. phlyctidospora and L. tylicolor showed inhibition deadlock when co-cultured with non-ammonia fungi (Tables 4 and 8; Figures 3C,F,I and 4C,F,I). The mycelium of C. phlyctidospora was gradually covered by that of Asp. niger, but not by those of P. citrinum and T. viride (Table 4; Figure 3C,F,I). The mycelium of L. tylicolor was gradually covered by those of non-ammonia fungi in all pH conditions (Table 4; Figure 4C). C. phlyctidospora and L. tylicolor did not form fruiting bodies in intra-species and inter-species co-cultures. The non-ammonia fungus, T. viride, showed poor conidiophore formation when co-cultured with C. phlyctidospora and L. tylicolor. Non-ammonia fungi Asp. niger and P. citrinum did not form conidiophores when co-cultured with C. phlyctidospora and L. tylicolor in all pH conditions (Figure 3C,F,I).

The LP species H. vinosophyllum showed competing deadlock with Asp. niger and P. citrinum and neutral intermingling with T. viride (Table 4; Figure 5C,F,I). The mycelia of Asp. niger and P. citrinum covered those of H. vinosophyllum in all pH conditions (Table 4; Figure 5 H). Asp. niger formed abundant conidiophores when co-cultured with H. vinosophyllum at pH 9.0, but a few at pH 7.0 and 5.5 (Figure 5C). P. citrinum formed conidiophores poorly when co-cultured with H. vinosophyllum in all pH conditions (Figure 5F). T. viride formed conidiophores surrounding the mycelium of H. vinosophyllum after its mycelium covered that of H. vinosophyllum in all pH conditions (Table 4).

Discussion

Ammonia fungi occur sequentially on soil after a sudden addition of alkalis, urea, aqueous ammonia, or nitrogenous materials that release ammonia during decomposition and cause alkalinisation of the soil (Sagara 1975). In Castanopsis and Quercus-dominated mixed forest, addition of urea to soil (800 g/m²) increased the amount of ammonium-nitrogen to 30–40 mg N/g dry soil. This was associated with an increase in pH to 8–10. Subsequent oxidation of ammonium-nitrogen into nitrate-nitrogen via nitrite-nitrogen decreased the amount of ammonium nitrogen to levels similar to that of the control (0.05–0.28 mg N/g dry soil; pH ∼5.5) (Suzuki et al. 2002). The early stage EP species, Am. botrytis and Asc. denudatus and the late stage, C. phlyctidospora and L. tylicolor, became dominant when the pH increased from 6.6 to 9.4 due to an increase in the amount of ammonium-nitrogen (1.5–25 mg N/g dry soil) derived from hydrolysis of urea. During the occurrence of EP species, the amounts of nitrite-nitrogen and nitrate-nitrogen in urea-treated soil were 0.280–0.930 and 1.1–10 mg-N/g dry soil, respectively, although these amounts of nitrogen were less than that of ammonium-nitrogen. The LP species, Hebeloma spoliatum, occurred on urea-treated soil during a temporary increase in amounts of nitrite-nitrogen (0.080–0.230 mg-N/dry soil) and nitrate-nitrogen (0.93–1.2 mg-N/g dry), which was associated with a return of the pH to a level similar to that of the control (Suzuki et al. 2002). Similar patterns in the changes in the amounts of inorganic nitrogen and pH in urea-treated soil, and the successive occurrence of ammonia fungi, were reported after nitrogen disturbances in Pinus densiflora forest (Yamanaka 1995a,b), and in mixed forests dominated by Quercus serrata (Fukiharu et al. 1997) or Q. serrata and Abies firma (Suzuki 2000). The first flush of H. vinosophyllum occurs alongside the appearance of other Hebeloma spp. including H. spoliatum, and Alnicola lactariens and Laccaria bicolor on urea-treated forest floors (Sagara 1975; Yamanaka 1995a; Fukiharu and Horigome 1996; Suzuki 2000; Imamura and Yumoto 2004). At this time, the soil pH returns to a level similar to that of the control, although the soil contains more ammonium-nitrogen and more nitrate-nitrogen than the control (Yamanaka 1995b; Suzuki 2000). This suggests that the soil conditions suitable for the colonization and occurrence of H. vinosophyllum would be similar to those described above for H. spoliatum. Imamura et al. (2006) reported a rapid increase in the activity of ureases in urea-treated soil, especially in the O layer, and also reported that there were similar patterns of increases in the amount of ammonium-nitrogen among different vegetation types and among different urea application times.

In vitro, the mycelia of the late stage EP species and the LP species showed extremely sparse radial growth on agar media containing higher concentrations (0.041–0.41 g N/l) of ammonium chloride in all pH conditions (Table 6). Ellouze et al. (2009) also reported that ammonium chloride inhibited radial growth. Most saprobic EP species colonized vigorously on urea-containing media under weak alkaline to neutral conditions, and showed strong extracellular urease activities under neutral conditions (Barua et al. 2011). These observations suggested that EP species could decompose urea into ammonium-nitrogen, thereby altering the concentration of ammonium-nitrogen to a level suitable for their vegetative growth. In this present study, therefore, we evaluate the growth responses of fungi on urea agar medium instead of ammonium agar medium to avoid the influence of inhibitive effect of chloride ion.

The mycelia of the early stage EP species, Am. botrytis and Asc. denudatus, showed vigorous radial growth on urea agar medium and invaded into the territory of the non-ammonia fungi in all pH conditions (Table 5; Figures 1A and 2A). In contrast, Am. botrytis showed poor radial growth and was covered by mycelia of non-ammonia fungi on ammonium, nitrite, and nitrate agar media in all pH conditions (Tables 6–8; Figure 1B). On all of the tested media and in all pH conditions, Asc. denudatus showed vigorous radial growth, but did not invade into the territory of the non-ammonia fungi. The mycelium of Asc. denudatus was covered by mycelia of non-ammonia fungi on urea, ammonium, nitrite and nitrate agar media in all pH conditions (Tables 5–8; Figure 1A), except for P. citrinum, which did not invade into the territory of Asc. denudatus on urea and ammonium agar media. (Tables 5 and 6; Figure 1F). When co-cultured with non-ammonia fungi, Am. botrytis formed abundant conidiophores on urea agar medium in all pH conditions (Figure 1A).

These findings indicate that the early stage EP species would have an advantage over non-ammonia fungi to colonize in the presence of a large amount of ammonium-nitrogen, which is associated with neutral and weak alkaline conditions. This also suggests that period in which the early stage EP species, Am. botrytis and Asc. denudatus, occur would be shortened in the field as a result of a decrease in their abilities to colonize and form conidiophores when competing with non-ammonia fungi. Occurrence of the non-ammonia fungi is associated with increased concentrations of nitrate-nitrogen, which result from oxidation of ammonium-nitrogen after a nitrogen disturbance. In other words, nitrate-nitrogen would be a key factor for the disappearance of the early stage EP species. The mycelia of the late stage EP species, C. phlyctidospora and L. tylicolor, invaded into the territory of the non-ammonia fungi on urea agar medium (Tables 4 and 5; Figures 3A and 4A) and weakly invaded or intermingled on nitrite and nitrate agar media in all pH conditions (Tables 7 and 8; Figure 3B). In contrast, none of the other saprobic and ectomycorrhizal ammonia fungi evaluated showed such invasion abilities. The late stage EP species showed strong invasion abilities, and were able to compete against non-ammonia fungi, including mycoparasites. This was particularly evident in the presence of a large amount of ammonium-nitrogen. These strong competitive abilities would be an advantage for establishment of late stage EP species, and would allow them to retain their territory for a longer period than early stage EP species. This is because the late stage EP species can compete with non-ammonia fungi even under high concentrations of nitrite-nitrogen and nitrate-nitrogen. The mycelium of the LP species, H. vinosophyllum, did not invade into the territories of the non-ammonia fungi, Asp. niger, P. citrinum and T. viride, on urea, nitrite and nitrate agar media in any pH conditions (Tables 4–8; Figure 5A). The mycelium of H. vinosophyllum was not covered by those of Asp. niger and P. citrinum on urea agar medium, but was covered by their mycelia on nitrite and nitrate agar media in all pH conditions. The mycelium of H. vinosophyllum was slightly covered by that of T. viride on urea agar medium, but was completely covered with its mycelium on nitrite and nitrate agar media in all pH conditions (Table 4). These findings suggest that LP species would not be able to retain their territories and/or survive without mycorrhizal symbiosis. In other words, they rely on their mycorrhizal symbioses due to the drastic loss of their ability to compete with non-ammonia fungi when the ammonium-nitrogen is converted into nitrite-nitrogen and nitrate-nitrogen in the field.

Suzuki (1989) also proposed that the sequential occurrence of ammonia fungi results from changes in the ammonium-nitrogen concentration and pH. Yamanaka (1999, 2003) suggested that the successive change in the species composition of ammonia fungi from EP to LP species may be affected by changes in pH and the form of inorganic nitrogen. Suzuki (2006) and Licyayo et al. (2007) suggested that the successive occurrence of ammonia fungi was caused not only by the physiochemical soil characteristics of soil, such as pH and nitrogen concentration, but also by the interspecific interactions among ammonia fungi associated with changes in pH and nitrogen concentration.

In conclusion, the changes in the interactive abilities of ammonia fungi and non-ammonia fungi, namely the increasing in invasion abilities of non-ammonia fungi as the levels of ammonium-nitrogen, nitrite-nitrogen and nitrate-nitrogen decrease to similar levels to those of undisturbed soil, is an important factor in determining the occurrence and persistence of each ammonia fungus.

Acknowledgements

We sincerely thank Naohiko Sagara (Professor Emeritus, Kyoto University) and the Medical Mycology Research Center, Chiba University, for providing most of the fungal isolates used in this study.