Effects of indigenous and introduced arbuscular mycorrhizal fungi on the growth of Allium fistulosum under field conditions

ABSTRACT

Enhanced phosphorus (P) uptake from the soil and increased plant growth related to arbuscular mycorrhizal (AM) fungi in pot culture, using sterilized soil, are well-known phenomena. However, these enhancements are not widely observed under field conditions because field sterilization is difficult. The aim of this study was to investigate the effects of AM fungi on P uptake and the growth of Allium fistulosum in non-fumigated and fumigated fields, under different levels of P availability. Plants were inoculated with the AM fungus Glomus R-10 and grown in fumigated soil. For the uninoculated treatment, a sterilized inoculum was applied directly. The field was fumigated using dazomet. Superphosphate was applied to the field at the rates of 0 (P0) or 500 (P500) kg P₂O₅ ha⁻¹. The inoculated and uninoculated plants were transplanted into the fields and sampled three times to measure AM fungal colonization, shoot P concentration, and shoot dry weight of the plants. At the transplanting stage, AM fungal colonization was observed in the inoculated plants (>70%) but not in the uninoculated plants. At the third sampling, irrespective of P treatment, AM fungal colonization was observed both in the uninoculated and inoculated plants in the non-fumigated field, and there was no difference in shoot P content and shoot dry weight between the inoculated and uninoculated plants. AM fungal colonization in the fumigated field was higher in the inoculated than uninoculated plants, irrespective of P treatment; shoot P content and shoot dry weight were both higher in the inoculated plants than in the uninoculated plants with P0. These results suggest that the responses of A. fistulosum to AM fungal inoculation under the low-P and fumigated conditions are similar to those observed in sterilized pot culture conditions.

KEYWORDS:

• Colonization
• dazomet
• phosphorus
• sterilization
• Welsh onion

1. Introduction

Phosphorus (P) fertilizer production is estimated to peak around 2030 because rock phosphate may be depleted within the next 50–100 years (Cordell et al. 2009). The concentration of inorganic orthophosphate in the soil is usually very low in most ecosystems, which frequently limits plant growth. Furthermore, a large fraction of P in the soil is present in plant-unavailable forms, mostly organic P and sparingly soluble inorganic P. Excessive P fertilizer application is a common practice in horticulture (Mishima et al. 2010; Reijneveld et al. 2010) and has resulted in the eutrophication of rivers, lakes, and marshes (Maguire et al. 2005). Rock phosphate depletion and environmental eutrophication must be addressed by reducing P fertilizer application to a level required by agricultural crops in one cropping cycle (Tawaraya et al. 2012).

AM fungi associate with 80% of terrestrial plants, promoting P uptake and the growth of their hosts (Smith and Read 2008). Enhanced P uptake and growth of host plants by AM fungal inoculation has been demonstrated under sterilized-(pot) culture conditions (i.e., in the absence of indigenous AM fungi) (Plenchette et al. 1983). Under low-P conditions, AM fungal inoculation improves plant P uptake and growth (Arias et al., 1991; Schweiger et al. 1995) but generally no differences. In contrast, no differences in P uptake and growth are observed between uninoculated and inoculated plants under high-P conditions (Smith and Read 2008). Pot experiments are particularly useful to demonstrate the enhancement of P uptake and growth of host plants by AM fungal inoculation, but not an increase in the yield of host plants. Consequently, field experiments are necessary to understand the effects of AM fungal inoculation on crop yield. However, soil sterilization is difficult under field conditions.

Several studies showed that, under field conditions, AM fungal inoculation increased olive (Olea europaea) (Estaun et al. 2003) and tomato (Solanum lycopersicum) (Al-Karaki 2006) yield compared to uninoculated plants. AM fungal colonization of uninoculated plants by indigenous AM fungi was relatively low (26% for olive and 6–18% for tomato), whereas AM fungal colonization of the inoculated plants was relatively high (76–81% for olive and 24–49% for tomato). In contrast, onion (Allium cepa) yield did not differ between inoculated and uninoculated plants, which presented comparable AM fungal colonization (70–80%) (Sorensen et al. 2008). These results indicate that high levels of colonization of indigenous AM fungi in uninoculated plants mask the effect of AM fungal inoculation on crop yield.

In previous studies, fungicides have been used to investigate the effect of AM fungal inoculation on P uptake and growth of host plants. Fungicide application decreased indigenous AM fungal colonization in Vulpia ciliata (Carey et al. 1992), Plantago lanceolata (Gange and West 1994), and Hyacinthoides non-scripta (bluebell) (Merryweather and Fitter 1996). Dazomet, which is widely used for soil fumigation, releases methyl isothiocyanate which is toxic to soil microorganisms. Thingstrup et al. (1998) showed that fumigation with dazomet reduced indigenous AM fungal colonization from 48% to 13% in flax (Linum usitatissimum) in a field. Hartnett and Wilson (2002) concluded that inhibiting AM fungi by fungicide application was the best approach to assess the roles and function of AM fungi in large-scale field experiments.

Only a few studies have analyzed AM fungal colonization and the growth of plants inoculated with AM fungi in fumigated and non-fumigated fields. Wininger et al. (2003) showed increased yield in four cultivars of chive (Allium schoenoprasum) inoculated with AM fungi, compared to uninoculated plants. However, as AM fungal colonization of these plants was not reported, it is unclear the extent to which the fumigation decreased AM fungal colonization and what effect this had on the growth of the plants. Additionally, although the yield of inoculated plants increased in the fumigated field, the yield tended to decrease in two of the varieties compared to the uninoculated plants in the non-fumigated field. However, the level of AM fungal colonization required to promote plant growth is unknown.

In this study, we aimed to investigate the effect of AM fungal inoculation on P uptake and growth of Welsh onion (Allium fistulosum L.) in non-fumigated and fumigated fields under two levels of P availability; in addition, we investigated whether AM fungal inoculation of fumigated fields is necessary for the recovery of the AM fungal population and for crop growth.

2. Materials and methods

2.1. Experimental design

An experimental field in the Faculty of Agriculture, Yamagata University (38° 44′ 14.7′′ N 139° 49′ 44.6′′ E), located in Tsuruoka city, Yamagata Prefecture, Japan, was used for the study. The soil chemical characteristics of the field before soil fumigation and fertilization were: pH (H₂O), 5.41; available P (Truog 1930), 300.4 mg kg⁻¹; P absorption coefficient, 558.75; cation exchange capacity, 15.42 cmol_c kg⁻¹; exchangeable Ca, 965.2 mg kg⁻¹; exchangeable Mg, 190.4 mg kg⁻¹; exchangeable K, 132.2 mg kg⁻¹; and total N, 1.3 g kg⁻¹. To avoid cross-contamination of AM fungi, the field was divided into two areas, one of which was fumigated. Dazomet (3,5-dimethyl-1,3,5-thiadiazinane-2-thione, Basamid®, Agro-Kanesho Co. Ltd., Tokyo, Japan) was manually applied to the soil surface at the rate of 2000 kg ha⁻¹, following the manufacturer’s protocol. The area was plowed to a depth of 15 cm and covered with polyethylene film for 1 week. After fumigation, the polyethylene film was removed, and the area was plowed twice every second week. Care was taken to ensure that dazomet was not applied to the area assigned for non-fumigation treatment. Ammonium sulfate and potassium sulfate were applied to both areas at the rates of 200 kg N ha⁻¹ and 190 kg K ha⁻¹, respectively. Superphosphate was applied at the rates of 0 (P0) and 500 (P500) kg P₂O₅ ha⁻¹. The fertilizers were broadcasted and incorporated manually into the top 10 cm of the soil. Available phosphate (Truog-P) of the P0 and P500 treatments was 111 ± 4 and 487 ± 70 mg P₂O₅ kg⁻¹, respectively. The plots in each area were arranged in a randomized complete block design with two AM fungal inoculation treatments (inoculated and uninoculated) and two soil P levels (P0 and P500) with four replications, resulting in a total of 16 plots. Each plot measured 2 × 2 m (4 m²) and had two 2 m rows.

2.2. Inoculation and nursery growth

Andosol was collected from a field in Tsuruoka city, Yamagata Prefecture, Japan. The soil was air-dried and sieved to <5 mm particles. Andosol and commercial Akadama soil (a subsoil of Andosol) were mixed (1:1 w/w), steam-sterilized twice at 80°C for 45 min and used in the nursery. Ammonium sulfate and potassium sulfate were added to the soil at the rates of 1.00 g N and 0.83 g K kg⁻¹ soil, respectively. Superphosphate was added to the inoculated soil at the rate of 1.00 g P₂O₅ kg⁻¹, and to the uninoculated soil at the rate of 2.0 g P₂O₅ kg⁻¹ to adjust the growth of the uninoculated plants to the inoculated plants. Soil pH (H₂O) was adjusted to 5.8 by addition of lime. An inoculum of AM fungi (Idemitsu Kosan, Tokyo, Japan) consisting of spores, extraradical hyphae, and colonized roots of Glomus R-10, was mixed with the soil at the rate of 50 g kg⁻¹ soil. Control plants were inoculated with the same amount of sterilized (121°C, 15 min) inoculum. Paper pots (220 plants, each in 26 × 26 × 38 mm, Nippon Beet Sugar, Tokyo, Japan) were placed in rearing trays (280 × 580 × 30 mm each), and each tray was filled with 4 kg of either the inoculated or uninoculated soil. Welsh onion (A. fistulosum L. cv. Motokura) seeds were sown in the paper pots on 27 April 2015. In all, 2640 inoculated or uninoculated paper pots were prepared. The seedlings were grown in a glasshouse at Yamagata University (38° 44ʹ 12.3′′ N 139° 49ʹ 42.4′′ E), Tsuruoka city, Japan, for 49 and 83 days for the non-fumigated and fumigated fields, respectively, and irrigated with tap water every second day.

2.3. Transplanting and growth conditions

Forty Welsh onion seedlings were transplanted to each row (e.g., 80 plants per plot), on 11 June 2015, for the non-fumigated field and 16 July 2015, for the fumigated field. Monthly mean air temperature ranged from 10.8°C to 25.1°C, and total precipitation was 624.5 mm during the growth period. All plots were manually weeded as needed. The insecticide dinotefuran was applied to the seedlings at the transplanting stage, and nitenpyram was applied to the field once.

2.4. Sampling and sample analysis

Shoots and roots were sampled three times: 35 (first), 70 (second), and 105 (third) day after transplanting for the non-fumigated field; and 35 (first), 70 (second), and 111 (third) day after transplanting for the fumigated field. Four plants were randomly selected and sampled from each plot. The four plants were pooled and treated as one replicate. Each plant was washed with tap water and divided into shoot and root; fresh weights were recorded.

Shoot dry weight was determined after drying at 70°C for 2 days. Ground dried shoots were digested in a HNO₃-HClO₄-H₂SO₄ (5:2:1) solution. The P concentration in the digested solution was determined colorimetrically by the vanadomolybdate-yellow assay (Olsen and Sommers 1982).

Percentage AM fungal colonization was determined using the gridline intersect method (Giovannetti and Mosse 1980) after staining the roots with 500 mg L⁻¹ aniline blue solution (Tawaraya et al. 1998). Eighty root segments from each replicate were observed under a microscope (ECLIPSE 80i, Nikon, Tokyo, magnification 100×).

2.5. Statistical analysis

All the data are presented as means of four replicates, and error bars represent standard error. Two-way analysis of variance and Tukey’s honestly significant difference test (Tukey-HSD test) for inoculation × P level on AM fungal colonization, shoot P content, and shoot dry weight were performed with Kaleida Graph 4.0j (HULINKS, Tokyo, Japan) to identify significant differences (P < 0.05) between all treatments under the same fumigation treatment.

3. Results and discussion

3.1. Mycorrhizal colonization, shoot P uptake, and shoot growth of Welsh onion in the non-fumigated field

At the time of transplanting, 49 days after sowing, AM fungal colonization in the inoculated plants was 71% and no colonization was observed in the uninoculated plants (Table 1); shoot P content and shoot dry weight were higher in the uninoculated plants than those in the inoculated plants.

Table 1. Arbuscular mycorrhizal (AM) colonization, shoot P content, and shoot dry weight at the transplanting stage of Allium fistulosum with (+M) or without (−M) AM fungus Glomus R-10 inoculation, grown in unsterilized (−D) or sterilized (+ D) fields. Data are expressed as means ± standard error (S.E.; n = 4). Different letters indicate significant differences between uninoculated and inoculated plants, as determined by Tukey’s-honestly significant difference test (P < 0.05).

		AM colonization				Shoot P content				Shoot dry weight
Fumigation	Inoculation	(%)		S.E.		(mg plant^−1)		S.E.		(mg plant^−1)		S.E.
-D	-M	0	±	0		0.090	±	0.011	a	166	±	13	a
	+M	71	±	2		0.062	±	0.002	b	89	±	5	b
+D	-M	0	±	0		0.122	±	0.012	b	126	±	10	b
	+M	74	±	2		0.176	±	0.011	a	185	±	6	a

AM fungal inoculation increased the levels of AM fungal colonization at the first and second samplings, but not at the third (Table 2). Application of P did not affect AM fungal colonization, irrespective of the sampling stage. There was no difference in AM fungal colonization between the inoculated and uninoculated plants due to indigenous AM fungal colonization. Although soil available P reached 487 mg P₂O₅ kg⁻¹ because of heavy P application, indigenous AM fungal colonization of the uninoculated plants with P0 was comparable to that with the P500 treatment. This result indicates that the introduced and/or indigenous AM fungi can colonize under high P availability. Tawaraya et al. (2012) indicated that high levels of soil P did not decrease AM fungal colonization either by indigenous fungi or by inoculation with Glomus R-10 in Welsh onion.

Table 2. Arbuscular mycorrhizal (AM) colonization, shoot P content, and shoot dry weight of Allium fistulosum with (+M) or without (−M) Glomus R-10 inoculation, grown in non-fumigated (−D) fields with (P500; 500 kg P₂O₅ ha⁻¹) or without (P0) phosphate fertilizer at the first, second, and third sampling. Data are expressed as means ± standard error (S.E.; n = 4) and analyzed by analysis of variance (ANOVA) and Tukey’s-honestly significant difference test. Asterisks and n.s. indicate significant differences by ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001) and not significant, respectively.

			AM colonization			Shoot P content			Shoot dry weight
Sampling	P level	Inoculation	(%)	±	S.E.	(mg plant^−1)	±	S.E.	(g plant^−1)	±	S.E.
First	P0	-M	76	±	6	2.73	±	0.51	0.812	±	0.181
	P0	+M	86	±	1	3.68	±	0.51	1.091	±	0.128
	P500	-M	58	±	10	2.80	±	0.24	0.817	±	0.136
	P500	+M	82	±	3	2.89	±	0.25	0.965	±	0.044
ANOVA
P value		Inoculation	0.018	*		0.219	n.s.		0.132	n.s.
		P level	0.113	n.s.		0.393	n.s.		0.654	n.s.
		Inoculation × P level	0.287	n.s.		0.303	n.s.		0.627	n.s.
Second	P0	-M	51	±	6	12.0	±	2.4	4.55	±	1.20
	P0	+M	68	±	5	16.5	±	1.7	5.54	±	0.41
	P500	-M	45	±	3	13.7	±	2.1	4.72	±	0.82
	P500	+M	66	±	6	16.7	±	1.7	5.26	±	0.47
ANOVA
P value		Inoculation	0.003	**		0.089	n.s.		0.353	n.s.
		P level	0.482	n.s.		0.651	n.s.		0.949	n.s.
		Inoculation × P level	0.637	n.s.		0.712	n.s.		0.782	n.s.
Third	P0	-M	72	±	4	26.3	±	2.5	10.20	±	1.15
	P0	+M	65	±	2	30.4	±	2.7	11.47	±	0.62
	P500	-M	70	±	7	40.9	±	5.6	13.37	±	1.95
	P500	+M	71	±	8	38.0	±	3.1	13.18	±	1.00
ANOVA
P value		Inoculation	0.633	n.s.		0.878	n.s.		0.682	n.s.
		P level	0.755	n.s.		0.011	*		0.081	n.s.
		Inoculation× P level	0.521	n.s.		0.354	n.s.		0.579	n.s.

The shoot P content and shoot dry weight of Welsh onion were not affected by AM fungal inoculation (Table 2). We applied large amounts of P fertilizer to the fields since soil P availability before fertilization (i.e., 300.4 mg kg⁻¹ Truog-P) was not sufficient for the growth of the Welsh onion (Tawaraya et al. 2012). However, P application did not affect AM fungal colonization, shoot P content, or shoot dry weight, except for shoot P content at the third sampling. There are two possible explanations for these results (1) introduced and indigenous AM fungi had a similar capacity to promote plant growth, or (2) the introduced fungus was replaced with indigenous AM fungi. A previous study showed that the growth of plants inoculated with indigenous AM fungi did not differ from plants inoculated with introduced AM fungi (Oliveira and Sanders 2000). Our results indicate that indigenous AM fungi colonized well in the Welsh onion (Table 2). However, in this study, we could not distinguish between the introduced and indigenous AM fungal colonization types, which is necessary to clarify the effects of indigenous AM fungi and introduced AM fungi on the growth of Welsh onion.

3.2. Mycorrhizal colonization, shoot P uptake, and shoot growth of Welsh onion in the fumigated field

At the time of transplanting, 83 days after sowing, AM fungal colonization in the inoculated plants was 74%, and no colonization was observed in the uninoculated plants (Table 1). Shoot P content and shoot dry weight were higher in the inoculated plants than in the uninoculated plants (Table 1). The inoculation increased AM fungal colonization of the plants irrespective of the sampling stage, in which P application did not affect AM fungal colonization (Table 3). Interactions between inoculation and P level were observed at the second sampling. At the first sampling, AM fungal colonization of the inoculated plants was higher than 80%, irrespective of P levels, and did not fall below 60% until the third sampling, whereas AM fungal colonization in the uninoculated plants was lower than 20% until the third sampling.

Table 3. Arbuscular mycorrhizal (AM) colonization, shoot P content, and shoot dry weight of Allium fistulosum with (+M) or without (−M) Glomus R-10 inoculation, grown in fumigated (+D) fields with (P500; 500 kg P₂O₅ ha⁻¹) or without (P0) phosphate fertilizer at the first, second, and third sampling. Data are expressed as means ± standard error (S.E.; n = 4) and analyzed by analysis of variance (ANOVA) and Tukey’s-honestly significant difference (HSD) test. Different letters indicate significant differences between inoculated and uninoculated plants with P0 or P500 in the field, sampled at the same time and analyzed using Tukey’s-HSD test (P < 0.05). Asterisks and n.s. indicate significant differences by ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001) and not significant, respectively.

			AM colonization				Shoot P content				Shoot dry weight
Sampling	P level	Inoculation	(%)	±	S.E.		(mg plant^−1)	±	S.E.		(g plant^−1)	±	S.E.
First	P0	-M	5	±	3		1.50	±	0.23	b	0.631	±	0.072
	P0	+M	84	±	3		8.44	±	0.68	a	1.898	±	0.190
	P500	-M	19	±	8		3.19	±	0.75	b	1.083	±	0.230
	P500	+M	84	±	2		6.75	±	1.06	a	1.576	±	0.208
ANOVA
P value		Inoculation	<0.001	***			<0.001	***			<0.001	***
		P level	0.159	n.s.			0.998	n.s.			0.729	n.s.
		Inoculation × P level	0.173	n.s.			0.043	*			0.059	n.s.
Second	P0	-M	11	±	1	b	12.2	±	1.0		4.34	±	0.37	c
	P0	+M	77	±	4	a	30.0	±	2.0		9.77	±	0.49	a
	P500	-M	27	±	13	b	21.4	±	4.6		6.43	±	1.21	bc
	P500	+M	62	±	3	a	28.0	±	2.1		7.52	±	0.33	ab
ANOVA
P value		Inoculation	<0.001	***			<0.001	***			<0.001	***
		P level	0.971	n.s.			0.214	n.s.			0.905	n.s.
		Inoculation × P level	0.038	*			0.063	n.s.			0.009	**
Third	P0	-M	20	±	8		23.2	±	3.6		7.88	±	0.96	b
	P0	+M	77	±	8		55.5	±	2.4		15.73	±	0.72	a
	P500	-M	22	±	5		34.4	±	5.8		9.87	±	1.80	ab
	P500	+M	67	±	7		38.7	±	11.9		10.01	±	2.78	ab
ANOVA
P value		Inoculation	<0.001	***			0.023	*			0.042	*
		P level	0.588	n.s.			0.693	n.s.			0.311	n.s.
		Inoculation × P level	0.455	n.s.			0.068	n.s.			0.049	*

Irrespective of the sampling stage, inoculation affected shoot P content and dry weight of Welsh onion, while P application had no effect on these two parameters. Interactions between inoculation and P level for shoot dry weight were observed at the second and third sampling. Multiple comparison showed that there was an increase in shoot dry weight of the inoculated plants with P0 at the second and third sampling, but not those with P500 (Table 3). The root system of Allium spp. is thinner than those of other crop species (Greenwood et al. 1982), implicated in their higher mycorrhizal dependency (Tawaraya 2003). Consequently, the growth of these Allium species increased considerably with AM fungal inoculation under sterilized pot culture conditions (Hepper et al. 1988). These results indicate that AM fungal inoculation is important for plant growth when if population density of indigenous AM fungi is low, such as in a fumigated field. Wininger et al. (2003) demonstrated that AM fungal inoculation increased chive yield compared to uninoculated plants in a fumigated field. Our results support these findings and indicate that AM fungal inoculation increased P uptake and plant growth in the fumigated field, especially under low-P conditions.

4. Conclusion

During the growing season, both pathogenic and symbiotic fungi colonize plant roots under field conditions. To eliminate pathogenic fungi, fields are occasionally fumigated (Hartz et al. 1989). However, soil fumigation also decreases AM fungal colonization and thus their contribution to plant P nutrition (Zhao et al. 2016), leading to increased input of P fertilization (Kunishi et al. 1989). Our study suggests that AM fungal inoculation to fumigated field is an effective approach to maintain yield through recovery of AM fungal population.

Additional information

Funding

This work was supported by JST ACCEL [Grant Number: JPMJAC1403), Japan.