N, P Contribution and soil adaptability of four arbuscular mycorrhizal fungi

Abstract

A glasshouse study was conducted to investigate the symbiotic efficiency and soil adaptability of four AMF using glass-bead cultivation systems. The results showed that efficiency and adaptability of four fungi varied among three soils. Particularly, efficiency of BEG167 shifted from positive in Beijing soil to negative in Guangdong soil. Furthermore, BEG167 had high adaptability in all three soils. Intraspecific differences of BRG168 and BEG221 were found in efficiency and adaptability in three soils. Taking efficiency and adaptabilty into consideration, it was concluded that BEG167, BEG168 and BEG221 in Beijing soil, BEG151 in Hubei soil, and BEG151 and BEG168 in Guangdong soil were effective AMF for maize.

Keywords:

• Symbiotic efficiency
• Fungal biomass
• Arbuscular mycorrhizal fungi
• Soil type

Introduction

Previous studies have shown that individual species of arbuscular mycorrhizal fungi (AMF), and even fungal isolates in one species, differ in their ability to promote plant growth and nutritional status (Munkvold et al., 2004; Gai et al., 2006). The large inter- and intraspecific functional diversity makes it essential to screen the efficient AMF for particular host-plant species or soils. A lot of AMF or isolates had been isolated in past decades from different ecological conditions in China. Many AMF are efficient in the original soils for a particular host plant, but how they perform in exotic soils, i.e. their symbiotic efficiency and soil adaptability, is unclear. Some studies had indicated that the efficiency of AMF could be determined or modified by soil types (Clark & Zeto, 1996a,b; Klironomos, 2003), soil pH (Van Aarle et al., 2002), and amendments (Raghuwanshi & Upadhyay, 2004). Therefore, the evaluation of efficiency and adaptability of AM fungi in different exotic soils is necessary work for screening efficient AMF. Up to the present time, only a few documents dealing with inter- and intraspecific functional variation among AM fungi in native or exotic soils have been published (Clark & Zeto, 1996a; Klironomos, 2003). For AMF isolated from China, however, no data at all have been presented on the above issues.

Redecker et al. (1995) developed a new cultivation system to recover extraradical fungal biomass of AMF, using a glass bead cultivation system, which made it possible to explore directly the fungal responses to different soil conditions. Chen et al. (2001) modified the method for studies on nutrient and trace metal uptake by arbuscular mycorrhiza. In the present study, four AMF in association with maize seedlings were cultivated in three soils by glass bead cultivation. The objectives of this study were to investigate the efficiency and adaptability of four AMF in native or exotic soils, and to select out the efficient AMF for each soil.

Materials and methods

AMF, soils, and host plant

Four different AMF were used (Table I). The fungi BEG221 and BEG168 were different ecotypes of the species Glomus etunicatum. Fungal inocula were raised in Beijing (BEG167 and BEG168), Hubei (BEG221), and Guangdong (BEG151), respectively, using a pot culture technique with maize. Culture soil for each fungus is the same type as the one of three soils used in this experiment as well as the isolate sites of the fungus. All inocula consisted of culture substrate, infected root segments, spores, and hyphae. Three experimental soils were collected, respectively, by author and co-workers from Beijing, Hubei, and Guangdong in arable land (the 0–30 cm topsoil). Basic chemical properties are given in Table II. The soils were air-dried, ground, sieved with a 3 mm sieve, and sterilized at 121 °C for 2 h.

Table I. Information about arbuscular mycorrhizal isolates used in the experiment.

BEG^a number	Isolation region	Species	Soil pH of isolation site	Isolation agency
BEG151	Guangdong	G. geosporum	3.81	Hong Kong Polytechnic Univ.
BEG167	Xinjiang	G. mosseae	8.20	China Agric. Univ.
BEG168	Hebei	G. etunicatum	8.20	China Agric. Univ.
BEG221	Hubei	G. etunicatum	7.96	Hua Zhong Agric. Univ.
^aRegistered numbers in the European Bank of Glomales.

Table II. Origin sites, type, and basic properties of soils used.

Collection region	Type	pH	Available P (mg/kg)	Organic matter (mg/kg)	Longitude (E) and latitude (N)
Beijing	Cinnamon	8.16	10.22	10.8	116° 33’, 39° 07’
Hubei	Brunisolic	7.20	24.06	17.3	114° 21’, 30° 37’
Guangdong	Red	4.74	20.63	14.7	113° 15’, 23° 08’

Maize (Zea mays), cv. Nongda 108, was used as host plant. Before planting, similar seeds were surface-sterilized by immersing them in 10% hydrogen peroxide for 10 minutes, and rinsed with deionized water. Then seeds were placed on moist filter paper in plates to germinate in an incubator under darkness at 28 °C for planting.

Experimental design

A special Plexiglas pot, called a three-compartment glass bead culture pot, was made. Its configuration was length×width×height=(5 + 3+5) cm×10 cm×15 cm. One side-compartment contained glass beads (0.8 kg/pot; diameter about 1.5–2 mm, carefully cleaned, and sterilized at 121 °C for 2 h), and the central and the other side-compartment were filled with sterile soil (410 g/pot and 700 g/pot). Maize was planted in the central compartment. The three compartments were separated by two layers of 30 m nylon nets to prevent root penetration but not to restrict growth of hyphae.

Mycorrhiza inoculum (70 g dry weight with 700 fungal spores) of each AM fungus was inoculated into the central compartment before sowing. The spore numbers of inoculated inoculum for four fungi all exceeded 700 per pot. Three noninoculation treatments were designed for each soil by inoculation with the equivalent autoclaved corresponding inoculum (being raised in the same soil) and 10 ml of filter solution of inoculum (water:inoculum w/w, 10:1). Four uniform pre-germinated seeds of maize were planted in the central compartment, which were later thinned to two plants after emergence. All treatments were replicated four times. The three compartments of the experimental set were watered with tap water regularly to keep suitable water content in substrates for growth of maize and fungal hyphae. All compartments were watered regularly to keep suitable growth conditions. The plants were kept in a glasshouse with diurnal temperature fluctuations within 20 °C –30 °C.

Harvest and determination

After 10 weeks’ growth, maize was harvested. The root samples were carefully washed under a stream of water to remove adhering soil, then were blotted dry, cut into segments about 1 cm long, and mixed thoroughly. About 0.5 g sub-sample roots were taken randomly to determine root colonization. The sub-samples were cleared with 10% KOH, and stained with Trypan blue in lactoglycerol (modified from Phillips & Haymann, 1970). Percentage root length colonized by AMF was assessed under a dissecting microscope using a gridline intersect method (Giovannetti & Mosse, 1980). The shoot samples were dried in an oven at 70 °C for 48 h then weighed. The samples were ground and dry-ashed in a muffle furnace at 300 °C for 3 h and at 550 °C for 5 h continuously. The ash was dissolved using 2% (v/v) nitric acid. Phosphorus was determined by the molybdovanadophosphate method (Kitson & Mellon, 1944). N was determined titrimetrically after steam distillation. The fungal biomass in the glass bead compartment was recovered by immersing the glass beads in a clean vessel with water, collected with a 25 µm mesh to remove nutrients and soil by a stream of water, and finally, adhering tiny impurities and glass beads were eradicated under a microscope with forceps. The biomass was rinsed with deionized water, dried in an oven at 70 °C for 48 h, and weighed. Data were tested by analysis of variance and means were compared by least significant difference (LSD) at the 5% level.

Results

Root colonization and fungal biomass of AMF

All AMF infected the maize root and formed mycorrhizae to different extents. The root colonization varied with AMF and soils (Table III). Relatively low root colonization was found on Guangdong soil compared with Beijing soil and Hubei soil. No infected root was found in maize seedlings of the controls. Fungal biomass of most fungi was recovered. Only BEG151 in Beijing soil, BEG168 and BEG221 in Hubei soil, and BEG221 in Guangdong soil produced no detectable fungal biomass.

Table III. Root colonization and fungal biomass of AM isolate on three soils (%).

	Beijing soil		Hubei soil		Guangdong soil
Isolate	Colonization (%)	Fungal biomass (mg/pot, DW)	Colonization (%)	Fungal biomass (mg/pot, DW)	Colonization (%)	Fungal biomass (mg/pot, DW)
CK	0.0c	0.00b	0.0c	0.00c	0.0e	0.00c
BEG151	34.6b	0.00b	27.6ab	1.71ab	19.7ab	1.19c
BEG167	55.6a	17.88a	25.8b	2.36a	15.7bc	4.76ab
BEG168	33.0b	1.68b	24.1b	0.00c	20.8a	5.46a
BEG221	31.6b	1.74b	36.5a	0.00c	12.5cd	0.00c
In the same column, values followed by different letters indicate significant differences at 5% level. CK = Control. DW = dry weight.

Maize shoot biomass

Shoot growth response of maize seedlings to AMF inoculation varied with isolates and soils (Table IV). In Beijing soil, all isolates significantly improved shoot biomass of maize seedlings. In Hubei soil, BEG151, BEG168, and BEG221 had a positive effect on maize growth. However, maize shoot biomass was markedly promoted only by BEG168 in Guangdong soil. In particular, as for BEG167, it functioned positively in Beijing soil, and neutrally in Hubei soil, while functioning negatively in Guangdong soil in terms of shoot biomass contribution.

Table IV. Shoot biomass of maize inoculated with or without AM isolate on three soils (g/pot).

Isolate	Beijing soil	Hubei soil	Guangdong soil
CK	16.75c	21.37c	9.46c
BEG151	20.46a	25.01ab	10.26ab
BEG167	19.49a	20.71c	7.30d
BEG168	20.20a	23.18b	11.10a
BEG221	20.18a	27.41a	9.20c
In the same column, values followed by different letters indicate significant differences at 5% level. CK = Control.

N and P uptake of maize shoot

Compared with controls, all AMF in Beijing soil, BEG168 and BEG221 in Hubei soil, and BEG168 in Guangdong soil significantly promoted the P uptake of maize shoot (Table V). As for BEG167, it functioned positively in Beijing soil, and neutrally in Hubei soil, while functioning negatively in Guangdong soil in terms of shoot P contribution. BEG151, BEG168, and BEG221 in Beijing soil, BEG221 in Hubei soil, significantly increased shoot N uptake, while no AMF in Guangdong soil contributed significantly to shoot N uptake.

Table V. Shoot N and P uptake of maize inoculated with or without AM isolate on three soils (mg/pot).

	Beijing soil		Hubei soil		Guangdong soil
Isolate	N uptake	P uptake	N uptake	P uptake	N uptake	P uptake
CK	45.54c	10.03c	55.58bc	16.74d	37.71a	5.62b
BEG151	59.31a	22.44a	57.54b	23.37cd	32.38ab	5.95ab
BEG167	50.01bc	22.56a	50.67c	23.46cd	26.55b	4.51c
BEG168	55.39ab	23.20a	55.12bc	26.02bc	34.94a	6.65a
BEG221	58.49a	17.40b	65.62a	38.51a	33.18a	5.78ab
In the same column, values followed by different letters indicate significant differences at 5% level. CK = Control.

Discussion

In this experiment, all tested AMF formed mycorrhiza with maize seedlings in the respective soil. They colonized the root differently depending on fungal species and soils. All AMF in Guangdong soil with low pH presented relatively low root colonization, which agrees with some previous results (Clark & Zeto, 1996a; Coughlan et al., 2000). Published data showed that soil pH is an important factor for mycorrhizal formation (Van Aarle et al., 2002; Rohyadi et al., 2004). In terms of extraradical biomass, BEG168, BEG167, and BEG221 were well adapted to Beijing soil, and BEG167, BEG151, and BEG221 were more adapted to the Hubei soil. In the Guangdong soil, all fungi produced detectable fungal biomass except BEG221, which indicated that BEG167, BEG168, and BEG151 have an adaptability to sustain themselves in low-pH soil. BEG167 adapted strongly to all soils. No diseases occurred during the experiment, and few hyphae of other fungi were found in fungal biomass recovered, which ensured the accuracy of our results.

In terms of beneficial effects on maize seedling growth and N, P uptake, all tested fungi in Beijing soil, BEG151, BEG168, and BEG221 in Hubei soil, and BEG151 and BEG168 in Guangdong soil were efficient. The number of efficient fungi in the soils decreased as pH increased. In addition, some AMF were effective in increasing shoot N uptake of maize seedlings in three soils. BEG151, BEG168, and BEG221 in Beijing soil, as well as BEG221 in Hubei soil, significantly increased shoot N uptake. Taking efficiency and adaptability into consideration, it was concluded that BEG167, BEG168, and BEG221 in Beijing soil, BEG151 in Hubei soil, and BEG151 and BEG168 in Guangdong soil were effective fungi. The two ecotypes of Glomus etunicatum BEG168 and BEG221 presented large differences with respect to efficiency and adaptability in the soils.

The symbiotic status of arbuscular mycorrhizae ranges from mutualism to commensalism, even to parasitism (Johnson et al., 1997). Three typical positions along the continuum were presented in this experiment for BEG167, which functioned positively in Beijing soil, while having no effect in Hubei soil, and being highly negative in Guangdong soil. Obviously, in Guangdong soil, BEG167 was a parasitic symbiont, not mutualistic, for over-production of fungal biomass.

Acknowledgements

I would like to thank the mycorrhizal researchers in Hong Kong Polytechnic University, China Agricultural University and Hua Zhong Agricultural University for friendly provision of the AMF inoculants.