Abstract

Aims

Mycorrhizal fungi can re-distribute nutrients among plants through formation of underground common mycorrhizal networks and therefore may alter interspecific plant competition. However, the effect of ectomycorrhizal (EM) fungi on interspecific plant competition in subtropical forests is poorly understood. In this study, we investigated the effects of EM fungal identity and diversity on the outcome of interspecific competition of plant species in relation to different successional stages in a Chinese subtropical forest.

Materials and Methods

This study selected four woody plant species, i.e. a pioneer tree Pinus massoniana, a late-pioneer tree Quercus serrata, a mid- successional tree Cyclobalanopsis glauca and a late-successional tree Lithocarpus glaber in a Chinese subtropical forest. The outcomes of interspecific competition were investigated in the seedlings of three plant pairs, i.e. between Cy. glauca and Pin. massoniana, between Q. serrata and Pin. massoniana, and between Li. glaber and Q. serrata in a pot experiment. In the Cy. glaucaPin. massoniana combination, plants in monoculture and two-species mixture were uninoculated or inoculated with EM fungi Paxillus involutus, Pisolithus tinctorius, Cenococcum geophilum, Laccaria bicolor and a mixture of these four fungal species. In the Q. serrataPin. massoniana and Li. glaberQ. serrata combinations, plants in monocultures and two-species mixtures were uninoculated or inoculated with EM fungi Pis. tinctorius, Ce. geophilum, La. bicolor and a mixture of these three fungal species. EM root colonization rate and seedling biomass of each plant species were measured, and the outcomes of interspecific competition were estimated using competitive balance index after 6-month cultivation.

Important Findings

All EM fungal inoculation significantly promoted a competitive ability of the mid-successional tree Cy. glauca over the pioneer tree Pin. massoniana compared with the uninoculated control treatment, and the extent to which EM fungi affected the outcome of interspecific competition was dependent on EM fungal identity in the Cy. glauca and Pin. massoniana combination. EM fungal inoculation had no significant effect on the outcomes of interspecific competition between the late-pioneer tree Q. serrata and Pin. massoniana combination and between the late-successional tree Li. glaber and Q. serrata combination, compared with the uninoculated control treatment. However, amongst the EM fungal inoculation treatments the competitive ability of Q. serrata over Pin. massoniana was significantly higher in EM fungi Ce. geophilum and La. bicolor treatments than in Pis. tinctorius treatment. EM fungal diversity did not show a complementary effect on the outcomes of interspecific competition in all three plant pairs. This study demonstrated that the effect of EM fungi on the outcome of interspecific competition was dependent on the plant pairs tested in the subtropical forest ecosystem.

INTRODUCTION

Interspecific plant competition is defined as an interaction between species, brought about by a shared requirement for a resource in limited supply, and leading to a reduction of the some competing species’ performance such as survival, growth and reproduction (Begon et al. 1996). Therefore, interspecific plant competition can influence plant productivity and community composition (Guo et al. 2017; Lehman and Tilman 2000; Martorell and Freckleton 2014). In the mechanisms of plant competition, plant–soil feedback theory proposed that soil microorganisms may influence plant communities through increasing soil nutrient availability and/or mediating plant co-existence (Bennett et al. 2016; Bever et al. 1997, 2012; van der Heijden et al. 2008; van der Putten et al. 2013; Revillini et al. 2016). As important components of the soil microorganisms, mycorrhizal fungi form symbiotic relationships with >90% of terrestrial plant species (Smith and Read 2008). In the symbiont, plant supplies photosynthetic carbon for fungal growth; in return, fungi enhance host plant nutrient and water uptake and resistance to biotic and abiotic stresses (Smith and Read 2008). Therefore, mycorrhizal fungi can influence interspecific plant competition through formation of underground common mycorrhizal networks that re-distribute nutrients among plants (Booth 2004; He et al. 2003; van der Heijden et al. 1998).

The effects of arbuscular mycorrhizal (AM) fungal identity and diversity on interspecific plant competition have been widely investigated in previous studies (e.g. Facelli et al. 2010, 2014; Rinaudo et al. 2010; Scheublin et al. 2007; Shi et al. 2016; Wagg et al. 2011). In contrast, a few studies have investigated the effect of ectomycorrhizal (EM) fungi on interspecific plant competition (Booth 2004; Pande et al. 2007; Pedersen et al. 1999; Perry et al. 1989). For example, Pedersen et al. (1999) found that EM fungus Pisolithus arhizus promoted the competitive ability of Pinus elliottii over a grass Panicum chamaelonche in the southeastern USA. Booth (2004) found that EM fungi increased the competitive ability of Pinus strobus over Acer rubrum, Betula allegheniensis and Tsuga canadensis in a mature, mixed hardwood-conifer forest of USA. Pande et al. (2007) demonstrated that EM fungi Russula vesca and Amanita hemibapha had different effects on interspecific competition between Quercus leucotrichophora and Pinus roxburghii in the Himalaya region of India. Furthermore, Perry et al. (1989) found that the outcome of interspecific competition between Pseudotsuga menziesii and Pinus usponderosa was generally decreased with increasing EM fungal diversity in southwestern Oregon of USA.

Previous studies of the effect of EM fungi on interspecific plant competition have been carried out in temperate forest ecosystems (Booth 2004; Pande et al. 2007; Pedersen et al. 1999; Perry et al. 1989). By contrast, there are higher diversities of wood plant species (Bruelheide et al. 2011; Bu et al. 2017; Chi et al. 2017; Sun et al. 2017; Wills et al. 2006) and EM fungal species (Gao et al. 2013, 2015; Schuldt et al. 2015) in subtropical and tropical forest ecosystems. Besides, previous studies have demonstrated the response and feedback of EM fungal community to primary and secondary succession of forest ecosystems (e.g. Gao et al. 2015; Ishida et al. 2008; Twieg et al. 2007). However, the role of EM fungi on tree species competition from different successional stages in the subtropical forests is still poorly understood.

To better understand the effects of EM fungal identity and diversity on the outcome of interspecific competition of plant species in relation to different forest successional stages in a Chinese subtropical forest, we conducted three pairs of interspecific competitions, i.e. between a mid-successional tree Cyclobalanopsis glauca and a pioneer tree Pinus massoniana, between a late-pioneer tree Quercus serrata and Pin. massoniana, and between a late-successional tree Lithocarpus glaber and Q. serrata. These four plant species were cultivated in monocultures and two-species mixtures uninoculated or inoculated with single EM fungal species and a mixture of these EM fungal species in a pot experiment. Our aims were to reveal whether (i) EM fungal identity had various effects on the outcome of plant interspecific competition, (ii) EM fungal diversity had a higher effect on the outcome of interspecific plant competition than single EM fungal species and (iii) the effect of EM fungi on interspecific plant competition was dependent on different plant pairs in the subtropical forest.

MATERIALS AND METHODS

Plants and EM fungal inocula

In this study, we selected four tree species: Pin. massoniana, Q. serrata, Cy. glauca and Li. glaber, which represent a pioneer, a late-pioneer, a mid-successional and a late-successional species in a subtropical forest in the Gutianshan National Nature Reserve (GNNR) in southeastern China (29°15′6″–29°15′21″N, 118°07′01″–118°07′24″E). These four plant species commonly coexist in different successional stages in this forest ecosystem (Bruelheide et al. 2011). The GNNR has an annual mean temperature of 15.38°C and annual mean precipitation of 1964mm. During 2011, seeds of these four plant species were collected from this forest and were stored in moist vermiculite at 4°C for 20 weeks until the experiment. The top soil (20cm deep) was collected from a forest in the GNNR, and sieved through a 1-cm mesh to remove large stones and root fragments. Soil chemical analysis showed a pH of 4.6, 43.6g kg−1 total carbon, 2.2g kg−1 total nitrogen (N), 0.1g kg−1 total phosphorus (P), 1.2mg kg−1 Mehlich-3 extractable P, and 34.9mg kg−1 available N. Subsequently, the soil was mixed with sand and vermiculite (1:1:1 by volume), autoclaved for 2h at 120°C and used as growing medium in the following experiment. Three weeks prior to the experiment, seeds were surface sterilized twice with 30% H2O2 for 20min and rinsed five times with distilled water, and then sown individually in nursery flats to germinate.

EM fungal species Paxillus involutus, Pisolithus tinctorius and Cenococcum geophilum were obtained from the culture collection of Inner Mongolia Agricultural University, China, and Laccaria bicolor was obtained from UFZ-Helmholtz Centre for Environmental Research, Germany. These four EM fungi can easily grow in artificial medium and were commonly used in previous pot experiments (Christophe et al. 2010; Kipfer et al. 2012; Sell et al. 2005). Each EM fungal species was grown in liquid culture with modified Marx-Melin-Norkrans medium (Marx 1969): KH2PO4 (0.5g L−1), (NH4)2HPO4 (0.25g L−1), CaCl2 (0.05g L−1), NaCl (0.025g L−1), MgSO4·7H2O (0.15g L−1), thiamine hydrochloride (100 μg L−1), FeCl3·6H2O (0.03g L−1), glucose (10g L−1) and malt extract (3g L−1). To propagate the inoculum, the cultures were mixed with a blender every 3–4 weeks and transferred into new Erlenmeyer flasks containing sterile growing medium. The inoculum was homogenized in a grinder immediately before application.

Experimental design

A pot experiment was carried out at a greenhouse for 6 months from 20 April to 20 October 2012. The experiment was laid out according to a randomized complete block design. In the Cy. glaucaPin. massoniana combination, plants in monoculture and two-species mixture were uninoculated or inoculated with Pax. involutus, Pis. tinctorius, Ce. geophilum, La. bicolor, and a 1:1:1:1 mixture of these four fungal species. In the Q. serrataPin. massoniana and Li. glaberQ. serrata combinations, plants in monocultures and two-species mixtures were uninoculated or inoculated with Pis. tinctorius, Ce. geophilum, La. bicolor, and a 1:1:1 mixture of these three fungal species; because the inoculum of fungus Pax. involutus was not enough, it was not used in these two plant combinations. There were four replicates for each treatment (Q. serrata monoculture was used twice in the two combinations), resulting in a total of 172 pots (13cm in diameter and 15cm in height, containing 1.15kg autoclaved mixed substrate). In each pot, we added 100ml of inoculum (ca. 2g fungal dry weight) for each single EM fungal treatment, 25ml of inoculum for each EM fungal species to give a total of 100ml of inoculum for the four EM fungal species mix (1:1:1:1) treatment or 33.3ml of inoculum for each EM fungal species to give a total of 100ml of inoculum for the three EM fungal species mix (1:1:1) treatment, and 100ml sterilized fungal inoculum for the uninoculated control treatment. In all cases the fungal inoculum was mixed throughout the substrate. Subsequently, 4 (monoculture) or 2 + 2 (mixture) plant seedlings were transplanted in each pot (an initial average height of 3.5cm for Pin. massoniana, 4.0cm for Q. serrata, and 5.0cm for Cy. glauca and Li. glaber). Pots were watered daily with tap water and received 10ml per week of a full-strength Hoagland nutrient solution (Hoagland and Arnon 1950) for the first month. Seedlings that did not survive were replaced up to 2 weeks after initial planting. Plants were grown in a glasshouse under natural light, in which the temperatures varied from 18°C during the night to 33°C in the day, respectively. The position of both the blocks and pots was randomized weekly.

Harvest

Plant seedlings were harvested after 6 months of growth, as previous studies (Scheublin et al. 2007; Wagg et al. 2011). Shoots (including stems and leaves) were harvested for each plant species by cutting at ground level, and roots were washed free from substrate. The roots of each species were cut into 1cm pieces and divided into two subsamples, and fresh mass was determined on both. Subsamples of roots and shoots of each plant species were oven-dried at 85°C for 72h, and weighed. The other subsample of roots was used for EM colonization observation, and its dry mass was calculated by multiplying the fresh mass by the dry to fresh mass ratio of the oven-dried root subsamples. The sum of the dry mass of both roots and shoots gave the total biomass for each plant species. The EM root colonization rate was determined according to the formula: EM root colonization rate (%) = ER/(ER + NR) × 100, where ER and NR denote the number of EM and non-EM roots, respectively (Choi et al. 2005).

Statistical analyses

The mycorrhizal responsiveness of each plant species in monoculture or mixture was calculated according to Janos (2007): responsiveness = M − NC, where M is the mean biomass of each plant species in the EM fungal inoculation treatments (all single fungal species and the mixture of the three or four fungal species), and NC is the mean biomass of each plant species in the uninoculated control treatment. A two-way analysis of variance (ANOVA) was used to test the effects of plant competition, EM fungi and their interaction on the mean of total biomass of each plant species per pot. Differences in EM root colonization rate and total biomass of each plant species among different treatments were tested using post hoc Tukey’s Honest Significant Difference (HSD) test. All data were tested for normality and homogeneity of variance before ANOVA. Of these data, the total biomass of Cy. glauca and Pin. massoniana and the EM root colonization rate of Pin. massoniana in the Cy. glaucaPin. massoniana combination, and the EM root colonization rate of Q. serrata in the Li. glaberQ. serrata combination were not normally distributed before and after transformation, and in this case a Dunnett’s T3 post hoc test was applied using the original data. We used the competitive balance index (Cb) to reflect the outcome of interspecific competition between two plant species through comparing the plant biomass between monoculture and two-species mixture. Cb was calculated according to Wilson (1988) as Cb = ln(Yij/Yii)/(Yji/Yjj) where Yii and Yij are the biomass of plant species i (Cy. glauca, Q. serrata or Li. glaber) in monoculture and mixture, and Yjj and Yji are the biomass of plant species j (Pin. massoniana or Q. serrata) in monoculture and mixture in the three combinations, respectively. A positive Cb value indicates that species i has higher competitive ability than species j, whilst a negative value indicates the opposite. Differences in the Cb values amongst treatments were examined using a one-way ANOVA followed by a Tukey’s HSD test. SPSS version 20.0 was used for all statistical analyses, and the significance level was at P < 0.05.

RESULTS

EM root colonization

Uninoculated plants became colonized by EM fungi, ranged from 7.0% to 15.6% probably due to airborne contamination (Table 1). The four plant species exhibited differences in EM root colonization rates among EM fungal inoculation treatments (Table 1). In the Cy. glaucaPin. massoniana combination, the root colonization rate of Cy. glauca was 11.5–28.5% in monoculture and 11.3–27.8% when grown with Pin. massoniana, and the root colonization rate of Pin. massoniana was 12.8–32.6% in monoculture and 14.4–28.4% when grown with Cy. glauca. In the Q. serrataPin. massoniana combination, the root colonization rate of Q. serrata was 17.5–29.5% in monoculture and 10.3–22.3% when grown with Pin. massoniana, and the root colonization rate of Pin. massoniana was 19.0–25.6% in monoculture and 12.5–30.5% when grown with Q. serrata. In the Li. glaberQ. serrata combination, the root colonization rate of Li. glaber was 16.8 – 27.5% in monoculture and 14.8–23.5% when grown with Q. serrata, and the root colonization rate of Q. serrata was 17.5–29.5% in monoculture and 13.8–23.5% when grown with Li. glaber.

Table 1:

ectomycorrhizal root colonization rate (%) of plant species in monocultures and two-species mixtures

Plant combination Plant species Culture Ectomycorrhizal treatments 
UI Pi Pt Cg Lb Mix 
Cy. glaucaPin. massoniana Cy. glauca Monoculture 15.0±1.1bc 28.5±2.2a 25.5±1.6a 21.3±2.4abc 11.5±1.6c 23.8±3.1ab 
Mixture 11.8±1.2c 26.0±1.4a 27.8±2.8a 25.0±2.1ab 11.3±0.8c 22.3±3.0ab 
Pin. massoniana Monoculture 9.9±3.1c 32.6±3.2abc 29.4±2.2a 18.1±1.1abc 12.8±1.0c 28.0±2.2a 
Mixture 15.6±1.2abc 21.1±3.1abc 28.4±4.6abc 23.6±2.5abc 14.4±0.7abc 23.6±1.5a 
Q. serrataPin. massoniana Q. serrata Monoculture 7.5±1.0d 29.0±2.3a 17.5±2.6bcd 26.0±3.2ab 29.5±2.0a 
Mixture 9.8±2.3cd 22.3±1.7ab 21.3±1.5ab 10.3±1.7cd 18.0±1.8bc 
Pin. massoniana Monoculture 11.7±0.7cd 21.3±2.4abc 25.6±1.6ab 19.0±2.8bcd 24.5±3.3ab 
Mixture 9.8±0.9d 30.5±2.1a 18.8±2.2bcd 12.5±2.1cd 20.0±2.0bcd 
Li. glaberQ. serrata Li. glaber Monoculture 8.0±1.2c 17.3±1.7abc 23.8±1.3ab 16.8±2.7bc 27.5±3.2a 
Mixture 7.0±1.2c 14.8±1.7bc 23.5±1.7ab 19.3±1.9ab 23.0±3.5ab 
Q. serrata Monoculture 7.5±1.0d 29.0±2.3a 17.5±2.6bcd 26.0±3.2ab 29.5±2.0a 
Mixture 10.5±1.8d 21.3±1.8abc 23.5±1.6abc 22.3±3.2abc 13.8±1.3cd 
Plant combination Plant species Culture Ectomycorrhizal treatments 
UI Pi Pt Cg Lb Mix 
Cy. glaucaPin. massoniana Cy. glauca Monoculture 15.0±1.1bc 28.5±2.2a 25.5±1.6a 21.3±2.4abc 11.5±1.6c 23.8±3.1ab 
Mixture 11.8±1.2c 26.0±1.4a 27.8±2.8a 25.0±2.1ab 11.3±0.8c 22.3±3.0ab 
Pin. massoniana Monoculture 9.9±3.1c 32.6±3.2abc 29.4±2.2a 18.1±1.1abc 12.8±1.0c 28.0±2.2a 
Mixture 15.6±1.2abc 21.1±3.1abc 28.4±4.6abc 23.6±2.5abc 14.4±0.7abc 23.6±1.5a 
Q. serrataPin. massoniana Q. serrata Monoculture 7.5±1.0d 29.0±2.3a 17.5±2.6bcd 26.0±3.2ab 29.5±2.0a 
Mixture 9.8±2.3cd 22.3±1.7ab 21.3±1.5ab 10.3±1.7cd 18.0±1.8bc 
Pin. massoniana Monoculture 11.7±0.7cd 21.3±2.4abc 25.6±1.6ab 19.0±2.8bcd 24.5±3.3ab 
Mixture 9.8±0.9d 30.5±2.1a 18.8±2.2bcd 12.5±2.1cd 20.0±2.0bcd 
Li. glaberQ. serrata Li. glaber Monoculture 8.0±1.2c 17.3±1.7abc 23.8±1.3ab 16.8±2.7bc 27.5±3.2a 
Mixture 7.0±1.2c 14.8±1.7bc 23.5±1.7ab 19.3±1.9ab 23.0±3.5ab 
Q. serrata Monoculture 7.5±1.0d 29.0±2.3a 17.5±2.6bcd 26.0±3.2ab 29.5±2.0a 
Mixture 10.5±1.8d 21.3±1.8abc 23.5±1.6abc 22.3±3.2abc 13.8±1.3cd 

Abbreviations: Cy. glauca = Cyclobalanopsis glauca, Pin. massoniana = Pinus massoniana, Q. serrata = Quercus serrata, Li. glaber = Lithocarpus glaber, UI = uninoculated control, Pi = Paxillus involutus (N, not used in the treatment), Pt = Pisolithus tinctorius, Cg = Cenococcum geophilum, Lb = Laccaria bicolour, Mix = mixture of the three (Pis. tinctorius, Ce. geophilum and La. bicolor) or four fungal species. Means (n = 4 ± SE) shared the same letter are not significantly different according to Tukey’s HSD or Dunnett’s T3 post hoc test at P < 0.05.

Plant biomass

Two-way ANOVA results showed that plant competition, EM fungi and their interaction had significant effects on the total biomass of all plant species, apart from the effect of their interaction on Pin. massoniana in the Q. serrataPin. massoniana combination and on Li. glaber in the Li. glaberQ. serrata combination (Table 2). In the Cy. glaucaPin. massoniana combination, the total biomass of Cy. glauca was not significantly changed by any of the EM fungal inoculation treatments in monoculture, but was significantly increased by 119.0% by EM fungus Pax. involutus in mixture with Pin. massoniana, compared with the uninoculated control treatment (Fig. 1a). The Cy. glauca responsiveness to EM fungi was −0.07g in monoculture and 0.37g in mixture with Pin. massoniana. Compared with the uninoculated control treatment, the total biomass of Pin. massoniana was significantly increased by 156.5–287.3% by Pax. involutus, Pis. tinctorius and four fungal mix treatments in monoculture, but was not significantly changed by any of the EM fungal treatments when grown with Cy. glauca (Fig. 1a). The Pin. massoniana responsiveness to EM fungi was 0.41g in monoculture and 0.11g in mixture with Cy. glauca. In addition, the total biomass of Cy. glauca was significantly higher in monoculture than when grown with Pin. massoniana in the uninoculated control, Pax. involutus and four fungal mix treatments, while the total biomass of Pin. massoniana was significantly higher in monoculture than in mixture in Pax. involutus, Pis. tinctorius and four fungal mix treatments (Fig. 1a).

Table 2:

results (P values) of two-way ANOVA on the effects of plant competition, ectomycorrhizal fungi and their interaction on the total biomass of plant species

Plant combination Plant species Source of variation df Total biomass 
Cy. glaucaPin. massoniana Cy. glauca COMP 0.000 
EM fungi 0.025 
COMP × EM fungi 0.002 
Pin. massoniana COMP 0.000 
EM fungi 0.000 
COMP × EM fungi 0.000 
Q. serrata–Pin. massoniana Q. serrata COMP 0.000 
EM fungi 0.000 
COMP × EM fungi 0.007 
Pin. massoniana COMP 0.000 
EM fungi 0.027 
COMP × EM fungi 0.754 
Li. glaber–Q. serrata Li. glaber COMP 0.000 
EM fungi 0.001 
COMP × EM fungi 0.600 
Q. serrata COMP 0.000 
EM fungi 0.000 
COMP × EM fungi 0.002 
Plant combination Plant species Source of variation df Total biomass 
Cy. glaucaPin. massoniana Cy. glauca COMP 0.000 
EM fungi 0.025 
COMP × EM fungi 0.002 
Pin. massoniana COMP 0.000 
EM fungi 0.000 
COMP × EM fungi 0.000 
Q. serrata–Pin. massoniana Q. serrata COMP 0.000 
EM fungi 0.000 
COMP × EM fungi 0.007 
Pin. massoniana COMP 0.000 
EM fungi 0.027 
COMP × EM fungi 0.754 
Li. glaber–Q. serrata Li. glaber COMP 0.000 
EM fungi 0.001 
COMP × EM fungi 0.600 
Q. serrata COMP 0.000 
EM fungi 0.000 
COMP × EM fungi 0.002 

Abbreviations: Cy. glauca = Cyclobalanopsis glauca, Pin. massoniana = Pinus massoniana, Q. serrata = Quercus serrata, Li. glaber = Lithocarpus glaber, COMP = plant competition, EM = ectomycorrhizal, df = degrees of freedom.

Figure 1:

total biomass of plant species in monocultures and mixtures among different treatments in the Cyclobalanopsis glaucaPinus massoniana combination (a), Quercus serrataPinus massoniana combination (b) and Lithocarpus glaberQuercus serrata combination (c). UI = uninoculated control, Pi = Paxillus involutus, Pt = Pisolithus tinctorius, Cg = Cenococcum geophilum, Lb = Laccaria bicolor and Mix = mixture of the three (Pis. tinctorius, Ce. geophilum and La. bicolor) or four fungal species. Means (n = 4 ± SE) shared the same letter within a plant species are not significantly different according to Tukey’s HSD or Dunnett’s T3 post hoc test at P < 0.05.

Figure 1:

total biomass of plant species in monocultures and mixtures among different treatments in the Cyclobalanopsis glaucaPinus massoniana combination (a), Quercus serrataPinus massoniana combination (b) and Lithocarpus glaberQuercus serrata combination (c). UI = uninoculated control, Pi = Paxillus involutus, Pt = Pisolithus tinctorius, Cg = Cenococcum geophilum, Lb = Laccaria bicolor and Mix = mixture of the three (Pis. tinctorius, Ce. geophilum and La. bicolor) or four fungal species. Means (n = 4 ± SE) shared the same letter within a plant species are not significantly different according to Tukey’s HSD or Dunnett’s T3 post hoc test at P < 0.05.

In the Q. serrataPin. massoniana combination, compared with the uninoculated control treatment the total biomass of Q. serrata was significantly decreased by 43.8% as a result of treatment with La. bicolor in monoculture, and decreased by 27.4% in the presence of La. bicolor and by 12.4% in the presence of three fungal mix when grown with Pin. massoniana (Fig. 1b). The Q. serrata responsiveness to EM fungi was −0.06g in monoculture and −0.07g when grown with Pin. massoniana. Compared with the uninoculated control treatment, the total biomass of Pin. massoniana was not significantly changed by any of the EM fungal inoculation treatments either in monoculture or when grown with Q. serrata (Fig. 1b). The Pin. massoniana responsiveness to EM fungi was −0.02g in monoculture and −0.02g in mixture with Q. serrata. In addition, the total biomass of Q. serrata was significantly lower in monoculture than when grown with Pin. massoniana in the uninoculated control, Pis. tinctorius, Ce. geophilum and La. bicolor treatments, while the total biomass of Pin. massoniana was significantly lower in monoculture than when grown with Q. serrata in all the treatments (Fig. 1b).

In the Li. glaberQ. serrata combination, the total biomass of Li. glaber and Q. serrata were not significantly changed by any of the EM fungal inoculation treatments in monoculture and mixture, compared with the uninoculated control treatment (Fig. 1c). The Li. glaber responsiveness to EM fungi was −0.06g in monoculture and −0.07g in mixture with Q. serrata. The Q. serrata responsiveness to EM fungi was −0.06g in monoculture and −0.04g in mixture with Li. glaber. In addition, the total biomass of Li. glaber was not significantly different between monoculture and mixture in all treatments, while the total biomass of Q. serrata was significantly higher in monoculture than when grown with Li. glaber in EM fungus Ce. geophilum treatment (Fig. 1c).

Competitive balance index

All EM fungal inoculation significantly increased the competitive ability of the mid-successional tree Cy. glauca over the pioneer tree Pin. massoniana, as the Cb values were significantly higher in all EM fungal inoculation treatments than in the uninoculated control treatment (Fig. 2a). Furthermore, the highest Cb value was found in EM fungus Pis. tinctorius treatment, followed by Ce. geophilum, four fungal species mix, Pax. involutus and La. bicolor treatments (Fig. 2a). In the late-pioneer tree Q. serrata and Pin. massoniana combination, the Cb values in EM fungal inoculation treatments were not significantly different from the uninoculated control treatment (Fig. 2b). However, among the EM fungal treatments the Cb values were significantly higher in EM fungi Ce. geophilum and La. bicolor treatments than in Pis. tinctorius and three fungal mix treatments, but no significant difference between Ce. geophilum and La. bicolor treatments and between Pis. tinctorius and three fungal mix treatments (Fig. 2b). In addition, the Cb value was not significantly different among treatments in the late-successional tree Li. glaber and Q. serrata combination (Fig. 2c).

Figure 2:

competitive balance index for interspecific competition between Cyclobalanopsis glauca and Pinus massoniana (a), between Quercus serrata and Pin. massoniana (b) and between Lithocarpus glaber and Q. serrata (c) among different treatments. UI = uninoculated control, Pi = Paxillus involutus, Pt = Pisolithus tinctorius, Cg = Cenococcum geophilum, Lb = Laccaria bicolor and Mix = mixture of the three (Pis. tinctorius, Ce. geophilum and La. bicolor) or four fungal species. Means (n = 4 ± SE) shared the same letter are not significantly different according to a Tukey’s test at P < 0.05.

Figure 2:

competitive balance index for interspecific competition between Cyclobalanopsis glauca and Pinus massoniana (a), between Quercus serrata and Pin. massoniana (b) and between Lithocarpus glaber and Q. serrata (c) among different treatments. UI = uninoculated control, Pi = Paxillus involutus, Pt = Pisolithus tinctorius, Cg = Cenococcum geophilum, Lb = Laccaria bicolor and Mix = mixture of the three (Pis. tinctorius, Ce. geophilum and La. bicolor) or four fungal species. Means (n = 4 ± SE) shared the same letter are not significantly different according to a Tukey’s test at P < 0.05.

DISCUSSION

We found that the effect of EM fungi on the outcome of interspecific plant competition was dependent on plant pairs tested in this study. For example, all EM fungal inoculation treatments significantly promoted the competitive ability of seedlings of the mid-successional tree Cy. glauca against the pioneer tree Pin. massoniana in this study. Similarly, previous studies have found that EM fungi affected interspecific competition between trees (Pande et al. 2007; Perry et al. 1989) and between a tree and a grass (Pedersen et al. 1999). However, we found that EM fungal inoculation treatments had no significant effect on the outcomes of interspecific competition between the late-pioneer tree Q. serrata and Pin. massoniana and between the late-successional tree Li. glaber and Q. serrata. In this study, Cy. glauca was more responsive to EM fungi in mixture than in monoculture, yet Pin. massoniana was more responsive to EM fungi in monoculture than in mixture in the Cy. glaucaPin. massoniana combination. In contrast, plant species had a similar responsiveness to EM fungi in monoculture and mixture in the Q. serrataPin. massoniana and Li. glaberQ. serrata combinations, respectively. Therefore, the explanation of the different effects of EM fungi on the outcomes of interspecific competition in the three plant pairs may be because these plant species have different responses to EM fungi in monoculture and mixture in this study.

We found that plants had equal or even lower growth, while the plants had higher EM root colonization rates in some EM fungal inoculation treatments. For example, Cy. glauca grown in monoculture or mixture with Pin. massoniana had significantly higher EM root colonization rates, but had equal total biomass when inoculated with EM fungi Pax. involutus and Pis. tinctorius compared to La. bicolor. Furthermore, Pin. massoniana grown in mixture with Cy. glauca had higher EM root colonization rate, but had lower total biomass when inoculated with EM fungus Pis. tinctorius compared to Pax. involutus. Our results are in agreement with other studies that the degree of EM root colonization is not always coupled with the EM fungal effect on plant growth (Baum et al. 2006; Sell et al. 2005; van Scholl et al. 2005).

We also found that some EM fungal species had significantly different effects on the outcomes of competition between Cy. glauca and Pin. massoniana and between Q. serrata and Pin. massoniana. For example, the competitive ability of Cy. glauca over Pin. massoniana was significantly more promoted by EM fungus Pis. tinctorius than that by Ce. geophilum, La. bicolor and Pax. involutus. In addition, the competitive ability of Q. serrata over Pin. massoniana was significantly more promoted by EM fungi Ce. geophilum and La. bicolor than that by Pis. tinctorius. Similarly, Pande et al. (2007) showed that EM fungus R. vesca significantly increased the competitive ability of Q. leucotrichophora against Pin. roxburghii, while A. hemibapha reversed the outcome of interspecific competition in the Himalaya region of India. These results indicate that some EM fungi are more beneficial to a host plant than others due to genetic and physiological compatibilities between an EM fungus and its host (Timonen et al. 1997; Baum et al. 2009).

In addition, a similar effect of some EM fungi on the outcome of interspecific plant competition was found in this study. For example, in the Cy. glaucaPin. massoniana combination, there was a similar effect between EM fungal species Pax. involutus and Ce. geophilum and between Pax. involutus and La. bicolor on enhancing the competitive ability of Cy. glauca against Pin. massoniana. In the Q. serrataPin. massoniana combination, a similar effect on enhancing the competitive ability of Q. serrata over Pin. massoniana was found in EM fungi Ce. geophilum and La. bicolor. Furthermore, all EM fungal species had similar effects on interspecific competition between Li. glaber and Q. serrata. These results indicate that some EM fungal species may have a similar physiological effect on the outcome of interspecific plant competition.

Although Wagg et al. (2011) have demonstrated the complementary effect of AM fungal diversity on the outcome of interspecific plant competition, we did not find the complementary effect of EM fungal diversity on the outcome of interspecific plant competition, as the effect of EM fungal mix treatment never exceeded the range of respective single-species inoculation in all the three plant pairs. Similarly, no complementary effect of EM fungal diversity (Laccaria laccata, Hebeloma crustuliniforme, Rhizopogon vinicolor and Rhizopogon ochraceorubens) was observed in the outcome of interspecific competition between Pse. menziesii and Pin. ponderosa in a pot study in southwestern Oregon of USA (Perry et al. 1989). The possible explanation may be because the relatively homogenous conditions occur in pot experiments, or different EM fungi are in constant competition for accessing to host resources and soil nutrients (Koide et al. 2005; Pickles et al. 2012).

We also realized that the replacement design did not distinguish the effect of EM fungi on interspecific plant competition from intraspecific plant competition (Scheublin et al. 2007; Vandermeer 1981; Wagg et al. 2011). Furthermore, in this study the uninoculated plant species were not free from mycorrhizal colonization contaminated by air-dispersed inocula, as reported in some previous studies (Perry et al. 1989; Pedersen et al. 1999). Indeed, contamination by contaminant such as Thelephora terrestris in greenhouses is particularly common due to its worldwide distribution and broad host range (Smith and Read 2008). However, in our study, most plant roots had higher EM colonization rates in EM fungal inoculation treatments than in the uninoculated control treatment, thus our results of the effect of EM fungal inoculation on interspecific plant competition are still believable, as previous studies (e.g. Perry et al. 1989; Pedersen et al. 1999). In addition, we conducted the pot experiment in 6 months, which may do not reflect the situation in natural forest ecosystems. Therefore, future studies of the effect of EM fungi on interspecific plant competition should be carried out in situ over large temporal and spatial scales.

CONCLUSIONS

The effect of EM fungi on the outcome of interspecific plant competition was dependent on the plant pairs tested. All EM fungal inoculation treatments significantly promoted a competitive advantage of the mid-successional tree Cy. glauca over the pioneer tree Pin. massoniana, and the extent to which EM fungi affected the outcome of interspecific plant competition was dependent on EM fungal identity. Although all EM fungal inoculation treatments had no significant effect on the outcomes of interspecific competition between the late-pioneer tree Q. serrata and Pin. massoniana and between the late-successional tree Li. glaber and Q. serrata compared with the uninoculated control treatment, EM fungi Ce. geophilum and La. bicolor were significantly more effective than Pis. tinctorius in promoting the competitive ability of Q. serrata over Pin. massoniana in the EM fungal inoculation treatments. In addition, we did not observe the complementary effect of EM fungal diversity on the outcome of the interspecific competition in all three plant pairs. As tree seedling competition has an important function in plant regeneration (Paine et al. 2008), our findings indicate that EM fungi may play an important role in driving plant community assembly during subtropical forest succession.

FUNDING

National Natural Science Foundation of China (31210103910, 30930005, 31470545, 31570499).

ACKNOWLEDGMENTS

We thank Mr. Zhi-Yong Jiang for help in collecting plant seeds in the GNNR. We sincerely thank Professor François Buscot from UFZ-Helmholtz Centre for Environmental Research, Germany for providing EM fungus Laccaria bicolor and Dr Yong-Jun Fan from Inner Mongolia Normal University, China for providing EM fungi Paxillus involutus, Pisolithus tinctorius and Cenococcum geophilum.

Conflict of interest statement. None declared.

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Author notes

*Correspondence address. State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District, Beijing 100101, China. Tel/Fax: +86-10-64807510; E-mail: guold@sun.im.ac.cn