- Email: [email protected]
Interactions between arbuscular mycorrhizal fungi and fungivorous nematodes on the growth and arsenic uptake of tobacco in arsenic-contaminated soils
Applied Soil Ecology 84 (2014) 176–184
Contents lists available at ScienceDirect
Applied Soil Ecology journal homepage: www.elsevier.com/locate/apsoil
Interactions between arbuscular mycorrhizal fungi and fungivorous nematodes on the growth and arsenic uptake of tobacco in arseniccontaminated soils Jianfeng Hua a,b , Qian Jiang b , Jianfeng Bai c , Feng Ding d, Xiangui Lin b , Yunlong Yin a, * a
Institute of Botany, Jiangsu Province and The Chinese Academy of Sciences, Nanjing 210014, China State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China c Waste Electrical and Electronic Equipment Recycling Centre, Shanghai Second Polytechnic University, Shanghai 201209, China d Qianxinan Tobacco Company, Guizhou 562400, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 February 2014 Received in revised form 7 July 2014 Accepted 9 July 2014 Available online xxx
The effects of inoculation with two AM fungi (M1, Glomus caledonium; M2, Glomus spp. and Acaulospora spp.) and a fungivorous nematode Aphelenchoides sp. on growth and arsenic (As) uptake of Nicotiana tabacum L. were investigated in soils contaminated with a range of As. The reproduction of Aphelenchoides sp. was triggered by the co-inoculation of AM fungi regardless of AM fungal isolates and As levels. Stimulative effects of Aphelenchoides sp. on the development of mycorrhiza, slightly different between two AM fungi, were found particularly at the lowest As level. Irrespective of mycorrhizal inoculi, increasing soil As level decreased plant growth, but increased plant As uptake. Co-inoculation of AM fungi and Aphelenchoides sp. led plants to achieving further growth and greater As accumulation at the lowest As level. Results showed that the interactions between AM fungi and fungivorous nematodes were important in plant As tolerance and phytoextraction at low level As-polluted soil. ã 2014 Published by Elsevier B.V.
Keywords: Mycorrhiza Fungivorous nematodes Fungus–nematode interactions Arsenic contamination Phytoextraction
1. Introduction Arbuscular mycorrhizal (AM) fungi are able to form a symbiotic association with approximately 80% of terrestrial plant species. In general, the growth of host plants will be improved through obtaining nutrients, especially phosphorus (P) via mycorrhizal hypha (Smith and Read, 2008). In addition, some AM fungi also play an important role in heavy metals uptake of host plants grown in contaminated soils (Gaur and Adholeya, 2004; Hildebrandt et al., 2007). For example, remarkable studies have found that mycorrhiza affected arsenic (As) uptake of plants since As transports across the plasma membrane via P transport systems (Li et al., 2013; Orlowska et al., 2012). As the main soil microorganism in rhizosphere, AM fungi will interact with a wide range of other organisms, including bacteria, fungi, protozoa, arthropods and nematodes (Fitter and Garbaye, 1994). Some of these interactions have shown to be able to influence mycorrhizal behavior and function (Durán et al., 2013; Siddiky et al., 2012). Larsen et al. (2009) found that plant growth promoting species of Paenibacillus may have suppressive effects on
* Corresponding author. Tel.: +86 25 84347059; fax: +86 25 84347066. E-mail addresses: [email protected] (J. Hua), [email protected] (Y. Yin). http://dx.doi.org/10.1016/j.apsoil.2014.07.004 0929-1393/ ã 2014 Published by Elsevier B.V.
AM fungi and growth of Cucumis sativus. Using 15N and 13CO2 labeling technology, Koller et al. (2013a,b) showed that both the root mycorrhizal colonization and the growth of Plantago lanceolata L. benefited from the presence of protozoa. As we know, soil nematodes are the most abundant multicellular animals on the planet. Some microbivorous nematodes could play important roles in the turnover of soil microbial biomass and availability of nutrients (Chen and Ferris, 2000; Mao et al., 2007). AM fungi producing substantial extraradical mycelium and spores are excellent feeding resource for fungivorous nematodes (Hussey and Roncadori, 1981). Today, many experiments have provided evidences that the presence of fungivorous nematodes could affect arbuscular mycorrhiza development and ‘mycorrhiza effects’ by grazing activity (Bakhtiar et al., 2001; Giannakis and Sanders, 1990; Ingham et al., 1985). Despite the ubiquity of the interplay between AM fungi and other organisms and their importance for plant nutrition and growth, only few studies investigated their interactions on the growth and metal accumulation of plant grown in contaminated soils. Using AM fungal and earthworm cultures, Yu et al. (2005) observed that earthworms modified the functioning of AM fungus– plant symbiosis in Cd polluted soils. Inoculation of earthworms increased mycorrhizal infection rates of roots, shoot yield and Cd uptake of mycorrhizal ryegrass at 5 and 10 mg Cd kg1 soils. Vivas
J. Hua et al. / Applied Soil Ecology 84 (2014) 176–184
et al. (2003) found that Trifolium repens dually inoculated with Brevibacillus sp. and AM fungi isolated from a Cd polluted soil attained further growth and nutrition and lower Cd concentration, particularly at 85.1 mg Cd kg1 soil. However, few observations have been reported on the interaction between AM fungi and fungivorous nematodes on plant growth and As uptake. In present study, we have investigated the effects of the inoculation with two isolates of AM fungi and a selected fungivorous nematode on the growth and As accumulation of tobacco plants (Nicotiana tabacum L.). AM fungi and nematodes were assayed in single or in co-inoculation in soils contaminated with a range of As. It is hypothesized that the interactions between AM fungi and fungivorous nematodes will stimulate the growth and As uptake of tobacco plants, and different fungal species and different soil As levels might induce differential outcomes. 2. Materials and methods 2.1. Soil and AM fungi and fungivorous nematodes inocula preparation Soils were collected from agricultural fields near an As sulphide mine (29 390 N, 111020 E), which has been exploited for more than 1500 years, in Shimen County, Hunan Province, China. This area has a North Asian subtropical maritime monsoon climate, with mean temperature 16.7 C and average rainfall of about 1500 mm and more than 260 frost-free days. Three sampling sites were selected: (1) an area at the foot of the mining area with high As content, later abbreviated as H; (2) a location about 2 km from the mining area, M; and (3) a site chosen about 8 km from the mining area, L. The land use in these areas was arable cropping. The dominant crop species was tobacco (N. tabacum L.). Soil samples were classified as Ali-Perudic Argosols, and the chemical properties are given in Table 1. All tested soils were air-dried and sieved with nylon mesh (4 mm) and then sterilized by 60Co-irradiation (25 kGy) before a pot experiment. Two AM fungal isolates were used in this study, Glomus caledonium (represented as M1) isolated from an unpolluted agricultural soil in Henan Province, and an indigenous combined inoculum of Glomus spp. and Acaulospora spp. (represented as M2), wet-sieved from an As-polluted soil in Shimen County, Hunan Province (Bai et al., 2008). These AM fungal species were identified morphologically according to current taxonomic criteria (Schenck and Perez, 1990) and internet information by INVAM (http://invam.caf.wvu.edu). Fungal inocula were propagated on clover (Trifolium subterraneum L.) grown in an autoclaved substrate with successive propagation cycle, for 4 months in a greenhouse of the Institute of Soil Science, Chinese Academy of Sciences. Inocula were air-dried and sieved (2 mm), and consisted of thoroughly mixed rhizosphere samples containing spores, hyphae and mycorrhizal root fragments. Fungivorous nematode Aphelenchoides sp. was used as a model species. It is eudominant in L, M and H soils with the presence ranging from 11.4% to 36.2% (Hua et al., 2009), and tolerant to As evidenced by a 10 mg L1 As solution test. Worms were collected by cotton wool (Oostenbrink, 1960; Townshend, 1963) from M soil and
177
cultured in petri dishes on Potato Dextrose Agar (PDA) with Botrytis cinerea Pers. at 25 0.5 C. They were collected by the cotton-wool filter method and washed twice with sterile distilled water before use in the experiments (Mao et al., 2007). 2.2. Experimental design The experiment was designed 3 2 3 factorial with the following factors: (1) mycorrhizal treatments including assay with two AMF isolates (M1 and M2) and one non-inoculated control treatment; (2) addition or not of fungivorous nematodes; and (3) three levels of As polluted soils, referred to as L, M and H. Tobacco (N. tabacum L.) was seeded into sterilized peat-based seeding substrate on August 4, 2011. At cross stage, the plantlets were carefully transplanted into 3 L plastic pots (one plant per pot) filled with 3.0 kg of dry soil. Each mycorrhizal treatment received 50 g AM fungi inocula at sowing stage. All non-mycorrhizal treatments were treated with the same amount of inocula that had been autoclaved twice at 121 C for 30 min together with about 20 mL filtrate (<20 mm) of 50 g AM fungi inocula to provide a general microbial population free of AM propagules (Vivas et al., 2003). One week after transplanting, each nematode-added treatment received 2000 Aphelenchoides sp. Pots were arranged in randomized complete block design with three replicates per treatment. Plants were grown in a sunlit greenhouse with natural light, a day/night temperature 33/22 C and relative humidity 40–60%. Plants were watered to maintain soil moisture at 60–70% of water holding capacity by adding deionised water during the experimental period. All plants did not receive any fertilizers during the study period. 2.3. Harvest and measurements Heights of tobacco plants were recorded, and then roots and shoots were harvested separately after 13 weeks. Sub-samples of fresh roots were taken to assess mycorrhizal colonization. Fresh weights of total roots and of sub-samples were measured. Shoots and remaining roots were first rinsed with tap water and then rinsed with deionised water. Tissues were then weighed after oven drying at 60 C for 72 h and then ground to <0.25 mm in a stainless steel mill. The water content (%) of remaining roots and total fresh weights of roots were used to estimate dry weights of roots. Root mycorrhizal colonization was evaluated after clearing and staining (Koske and Gemma, 1989), using the grid-line intersect method (Giovannetti and Mosse, 1980). The length of extraradical mycelium (ERM) in soil was estimated using a modified membrane filtration technique (Jakobsen et al., 1992). A sub-sample of 30 g moist soil was used to determine total numbers of spores at the end of the experiment, using the wet sieving and centrifugation technique (Bakhtiar et al., 2001). Aphelenchoides sp. was extracted from 100 g fresh soil of each sample with a cotton-wool filter method. At the same time, water content (%) of each soil sample was determined. After 48 h extraction at room temperature, total numbers of Aphelenchoides sp. were counted, and their populations were expressed as the number of individuals per 100 g dry weight soil.
Table 1 Chemical properties of the soils with different As concentrations. As level
Total As (mg kg1)
pH (H2O)
Organic C (g kg1)
Total N (g kg1)
Olsen P (mg kg1)
Available K (mg kg1)
L M H
12.7 0.31 36.8 0.52 148 4.01
5.98 0.05 6.57 0.07 5.18 0.02
31.0 1.03 28.2 0.75 19.6 0.63
1.55 0.11 1.46 0.08 0.88 0.09
49.5 5.30 39.8 2.62 22.1 1.62
373 25.2 353 26.1 140 19.4
Data presented are means standard deviation (n = 3). L, soil low in As level; M, soil medium in As level; H, soil high in As level.
178
J. Hua et al. / Applied Soil Ecology 84 (2014) 176–184
Table 2 F-value of an ANCOVA of tested parameters using soil Olsen P as covariate in this experiment. Factors
Height (cm)
Dry weight (g) Root
7.22** As Nematode 4.45* AMF isolate 1.32 As Nematode 10.7** As AMF isolate 1.57 Nematode AMF isolate 0.40 As Nematode AMF isolate 2.02 Covariate Soil Olsen P 1.70 * **
Shoot
As concentration (mg kg1)
As content (mg pot1)
Soil mineral N (mg kg1)
Root
Shoot
Root
Shoot
(NO3 + NO2)-N NH4+-N Colonization (%)
55.1** 1.88 20.4** 0.60 1.28 1.26 1.06
2.07 3.46 0.56 5.16* 2.26 0.63 2.77*
27.1** 9.06** 24.6** 10.8** 3.6* 3.12* 1.31
1.23 2.21 0.85 0.94 3.54* 2.65 1.08
4.26* 0.03 4.13* 0.16 3.39* 0.26 0.63
14.1** 0.75 45.1** 0.68 5.76** 0.01 0.91
1.36 8.06** 1.54 5.36* 7.29** 11.4** 1.40
13.0** 45.8** 2.35 18.2** 55.2** 11.7** 35.0**
2.94
0.47
0.76
0.32
3.08
0.01
0.86
0.81
5.51** 1.40 29.9** 2.34 14.9** 10.0** 1.14 1.41 4.23* 11.1** 16.7** 2.09 3.39* 2.91* 4.87** 0.56 0.39 1.53 2.09 3.46* 4.67** 1.28
0.01
0.59
Development of mycorrhiza ERM Spores (m g1) (100 g1)
P < 0.05. P < 0.01.
Soil pH was determined with a glass electrode (soil/water = 1:2.5, m/V). Soil organic C was measured by the dichromate oxidation and total N by Kjeldahl titration. Olsen P was extracted with sodium bicarbonate, and determined by the molybdenum-blue method. Available K was extracted with 1.0 mol L1 ammonium acetate, and then measured by a flame photometry. Soil mineral N ((NO3 + NO2)-N and NH4+-N) was extracted with 2 mol L1 KCl (soil/ solution = 1:10, m/V) for 1 h and determined by an automated procedure (Skalar SANplus Segmented Flow Analyzer, Skalar Analytic B.V., De Breda, Netherlands). To determine total As concentration, soil was extracted with concentrated HNO3 and HCl at 1:3 (V/V), and analyzed by atomic fluorescence spectrometer (AFS-230E, Haiguang Instrumental Co., China). Dried sub-samples of roots and shoots were digested with concentrated HNO3 and HCl at 3:1 (V/V) to analyze As concentrations. The accuracy of the analyses was estimated by comparison with reference material GBW07406 (for soil) and GBW07603 (for plant) from Institute of Geophysical & Geochemical Exploration, Chinese Academy of Geosciences, and blanks were introduced regularly. The outcomes of the analyses on GBW07406 and GBW07603 were 217 10 mg As kg1 and 1.19 0.06 mg As kg1, respectively. The recovery rates of As were 98% and 95%, respectively.
2.4. Data analysis An analysis of covariance (ANCOVA) using soil Olsen P as covariate was applied to test the effects of As level, nematode addition and AM fungi inoculation as main factors and their interactions on growth and As uptake of tobacco. We focused our analysis on variances of root mycorrhizal colonization, extraradical mycelium length and spores numbers without the treatments noninoculated with AM fungi. An ANCOVA was also performed for the numbers of Aphelenchoides sp. using As level and AM fungi inoculation as factors, and soil Olsen P as covariate. Meanwhile, data of each As level were subjected to a one-way ANOVA. Statistical analyses were performed by SPSS 16.0 for Windows and a significant level of P < 0.05 was used. 3. Results 3.1. Growth of tobacco plants An ANCOVA of tested parameters using soil Olsen P as covariate demonstrated that there were no significant effects of soil Olsen P
Table 3 Effects of the inoculation of AM fungi (M1 or M2) and/or Aphelenchoides sp. on plant growth and soil mineral N under increasing soil As levels. Treatments
Height (cm)
Soil mineral N (mg kg1)
Dry weight (g) Root
Shoot
(NO3 + NO2)-N
NH4+-N
L Control M1 M2 Nem M1 + Nem M2 + Nem
45.6 4.00 c 48.7 3.21 c 48.2 3.97 c 53.7 2.52 bc 62.5 4.44 a 58.7 2.20 ab
1.61 0.13 bc 1.51 0.04 c 1.70 0.30 bc 2.04 0.32 ab 2.23 0.30 a 1.95 0.28 abc
8.69 0.61 c 9.31 0.43 c 11.3 0.41 b 12.7 0.37 ab 14.4 2.23 a 14.2 0.61 a
0.20 0.06 b 0.13 0.04 b 0.22 0.06 b 0.21 0.08 b 0.23 0.12 b 0.38 0.06 a
1.67 0.13 a 1.51 0.45 a 1.38 0.09 a 1.60 0.43 a 2.02 1.08 a 1.77 0.78 a
M Control M1 M2 Nem M1 + Nem M2 + Nem
48.0 2.00 ab 50.3 2.89 ab 53.0 4.58 a 49.0 1.73 ab 44.7 6.35 b 50.0 2.65 ab
1.76 0.15 ab 1.46 0.14 bc 1.88 0.33 a 1.64 0.12 ab 1.30 0.00 c 1.70 0.15 ab
10.2 0.83 a 8.97 1.15 a 10.4 0.81 a 10.2 0.48 a 8.70 1.39 a 9.38 0.70 a
0.19 0.02 a 0.07 0.05 a 0.16 0.08 a 0.21 0.14 a 0.14 0.07 a 0.18 0.06 a
1.95 0.47 ab 1.56 0.13 b 1.58 0.34 b 2.16 0.28 a 1.47 0.12 b 1.75 0.34 ab
H Control M1 M2 Nem M1 + Nem M2 + Nem
33.2 6.25 a 33.0 10.5 a 26.3 4.04 a 26.0 5.29 a 30.7 7.57 a 31.7 2.08 a
0.93 0.11 a 0.78 0.09 ab 0.61 0.10 b 0.75 0.11 ab 0.74 0.20 ab 0.74 0.05 ab
5.26 1.23 a 5.79 0.43 a 4.27 0.67 a 4.32 0.38 a 5.67 1.73 a 6.30 1.27 a
0.15 0.09 a 0.17 0.16 a 0.16 0.14 a 0.10 0.05 a 0.31 0.17 a 0.14 0.13 a
5.03 1.49 ab 6.75 4.41 ab 3.99 1.69 ab 5.70 3.01 ab 7.76 3.62 a 2.10 0.80 b
Data presented are means standard deviation (n = 3). Means followed by the different letters into each As level are significantly different according to Duncan’s multiple range test at 5% level, n = 3. L, soil low in As level; M, soil medium in As level; H, soil high in As level.
J. Hua et al. / Applied Soil Ecology 84 (2014) 176–184
10
2 1
bcd
bc
b
cd
d
a
a
8
0
6
1 2
a
a
3
a
a
a
a
4 5
Root
Sho ot
6
As contents (ug pot -1 )
As concentrations (mg kg -1 )
179
4
cd
2 0 2 4
a
a
Control
M1
M2
Nem
M1+Nem
M2+Nem
b
a
b
b
a
Control
2 1
a
6
1 2
c
c
bc
b
a
bc
4 5 6
Nem
M1+Nem
b
b
c
c
9
a b
b
a
0 2 c a
bc
ab
8
Control
M1
M2
ab
a
Nem
M1+Nem
b
b
M2+Nem
Control
2
M1
M2
Nem
M1+Nem
M2+Nem
10
b
ab 8
0
6 As contents (ug pot -1 )
1 2 3 4 5
M2+Nem
2
6
8
1
4
4
7
M
M2
8
0
3
M1
10
b
As contents (ug pot -1 )
As concentrations (mg kg -1 )
a
8
9
L
As concentrations (mg kg -1 )
a a
6
8
bc
d
7
c c
6
bc
abc
4
a
ab b
0 2 4 a
ab
a
M1
M2
ab
ab
ab
2
7 8
6
a
a
M1
M2
a
a
a
Nem
M1+Nem
M2+Nem
8
9
H
b
b
Control
Nem
M1+Nem
M2+Nem
Con trol
Fig. 1. Effects of the inoculation of AM fungi (M1 or M2) and/or Aphelenchoides sp. on As concentrations and contents of tobacco plants under increasing soil As levels. (L, soil in low As level; M, soil in medium As level; H, soil in high As level; control, non-inoculated with AM fungi or Aphelenchoides sp.; M1, inoculated with non-indigenous AM fungi Glomus caledonium; M2, inoculated with indigenous AM fungi Glomus spp. and Acaulospora spp.; Nem addition of Aphelenchoides sp. Bars represent standard errors. Different letters represent significant difference according to Duncan’s multiple range test at 5% level, n = 3.)
on the development of mycorrhiza, As uptake and growth of tobacco (Table 2). Soil As level (P = 0.002), nematode addition (P = 0.042) and their interaction (P < 0.0001) had significant effects on the height of tobacco plants (Table 2). Addition of Aphelenchoides sp. increased the height of mycorrhizal plants only in L soil. The most enhancing effect of these dual treatments was observed in M1 + Nem inoculated plants which increased 17 cm over control plants (Table 3). Root dry weight (P = 0.008) was influenced by soil As level. Decreasing plant height and dry weights were found with the increase of As concentration (Table 3). Obviously, high soil As levels inhibited tobacco growth. In addition, significant interaction effects between As and nematodes, and between As and AMF isolates were found on shoot (P < 0.0001, P = 0.035) and root (P < 0.0001, P = 0.019) dry weights (Table 2). Root dry weights of dually M1 + Nem inoculated plants were increased by 39% and 48% over control plants and M1 single inoculated mycorrhizal plants, respectively in L soil. Compared with control plants, shoot dry weights of all inoculated plants were increased in L soil. The greater positive effect was observed in the dual inoculated plants,
with shoot dry weights being 1.66 (M1 + Nem treatment) and 1.63 (M2 + Nem treatment) fold higher than that of control plants. In fact, tobacco plants better developed in L soil after co-inoculation of AM fungi and Aphelenchoides sp. (Table 3). 3.2. As concentrations and contents of tobacco plants Both soil As level and AMF isolate had large impacts on the As concentrations in tobacco plants (root, P < 0.0001, P = 0.023; shoot, P < 0.0001, P < 0.0001). In H soil, plants accumulated higher As concentrations irrespective of the inoculation of AM fungi and/or Aphelenchoides sp. nematodes (P = 0.003) and interaction effects between As and AMF isolate (P = 0.003) and between the three factors (P = 0.004) were observed for As concentrations in root. As contents in the roots were only affected by the interaction between As and nematodes (P = 0.011) and between As, AMF isolate and nematodes (P = 0.042), however, As contents in shoot were influenced by As (P < 0.0001), nematodes (P = 0.005), AMF isolate (P < 0.0001) as well as the interactions between As and nematodes (P < 0.0001), between As and AMF isolate (P = 0.014) and between nematodes and AMF isolate (P = 0.048) (Table 2).
J. Hua et al. / Applied Soil Ecology 84 (2014) 176–184
Soil As level (P = 0.022), AMF isolate (P = 0.024) and their interaction (P = 0.019) had significant effects on the concentrations of NH4+-N. However, concentrations of (NO3 + NO2)-N were only remarkably affected by the interaction effects between As and AMF isolate (P = 0.016) (Table 2). As shown in Table 3, nematode addition and AMF inoculation had no significant effects on the concentrations of (NO3 + NO2)-N in soils with higher As concentrations (M and H). At the lowest As level, the dual inoculation of M2 and Aphelenchoides sp. led to higher concentrations of (NO3 + NO2)-N (P = 0.025) than the other mycorrhizal treatments. Compared with the control, the inoculation of AM fungi (M1 or M2) and/or Aphelenchoides sp. did not effectively increase concentrations of NH4+-N irrespective of the As level in soils. 3.4. Development of nematodes and mycorrhiza Aphelenchoides sp. was absent or present at very low numbers in treatments without nematode addition, suggesting negligible background contamination, and thus, these data are not shown in Fig. 2. According to an ANCOVA using As level and AMF isolate as factors and soil Olsen P as covariate, the numbers of Aphelenchoides sp. were not influenced by covariate, but significantly affected by AMF isolate (P < 0.0001) and the interaction effects (P = 0.016). Following nematode addition to non-mycorrhizal treatments, the numbers of Aphelenchoides sp. increased to 3395, 3259 and 15,142 individuals per treatment in L, M and H soils, respectively. However, final number of Aphelenchoides sp. in dual inoculation treatments exceeded 38,000 individuals per treatment, and no
8
a
7
a
4
3.3. Soil mineral N
significant difference was found between M1 + Nem and M2 + Nem treatments in all tested soils. Obviously, growth of Aphelenchoides sp. was stimulated by AM fungi inoculation irrespective of the soil As level, with 12.1 and 13.9 times increase, respectively in the L soil, 20.0 and 12.5 times raise, respectively in the M soil, and 2.5 and 3.3 fold increase, respectively in the H soil (Fig. 2). Noticeable effects of As (P < 0.0001), AMF isolate (P < 0.0001) and their interaction (P = 0.009) were observed on the mycorrhizal colonization rate, while Aphelenchoides sp. had no significant effects according to an ANCOVA using Olsen P as covariate (Table 2). AM fungi M1 reached much greater AM colonization levels than M2 in L and H soils (L, P = 0.010; H, P = 0.010) both following single inoculation and co-inoculation with Aphelenchoides sp. (Fig. 3). The length of extraradical mycelium (ERM) in soil was markedly influenced by nematode addition (P = 0.009) and the interaction between each tested factor (As nematode, P = 0.012; As AMF isolate, P = 0.004; nematode AMF isolate, P = 0.003) (Table 2). With the increase of As exposure level, the ERM length showed an increasing trend. Compared with M2, M1 produced greater ERM length in M soil (P = 0.002) in the absence of Aphelenchoides sp., while an opposite trend was found in H soil (P = 0.032) in the presence of Aphelenchoides sp. Inoculation with Aphelenchoides sp. increased the ERM length of M2 by 49% in L soil and by 50% in M soil over the single inoculated mycorrhizal treatments (Fig. 3). As shown in Table 2, total numbers of AM fungal spores were significantly affected by all single and interaction effects except for the AMF isolate factor (As, P < 0.0001; nematode, P < 0.0001; As nematode, P < 0.0001; As AMF isolate, P < 0.0001; nematode AMF isolate, P = 0.002; As nematode AMF isolate, P < 0.0001). In the presence of Aphelenchoides sp., M1 produced more spores (P = 0.032) than M2 in L soil, while an opposite trend (P = 0.001) was observed in H soil. Regardless of the nematode addition, total numbers of spores were higher (P = 0.011) in M2 inoculated treatments than in M1 inoculated treatments in M soil. Total numbers of spores were differently affected by nematode addition according to As concentration in the soil. At the lowest As level, Aphelenchoides sp. increased spore numbers of M1 by 205% but did not affect that of M2. In contrast, at the highest As level,
-1
In general, As concentrations in shoot were increased by M2 but not affected by M1 both when inoculated single or dually with Aphelenchoides sp. Comparing the increasing effect of the two treatments involving M2 at different As levels, we can see that shoot As concentrations were 1.14 and 1.30 (L soil), 1.24 and 1.34 (M soil), 1.33 and 1.22 (H soil) fold higher than in control plants (Fig. 1). Inoculation with Aphelenchoides sp. had no effect on shoot As concentrations of mycorrhizal plants in M and H soils. In L soil, however, the co-inoculation of Aphelenchoides sp. into the two mycorrhizal treatments increased shoot As concentration by 16% over the M2 single inoculated treatment, but decreased it by 14% over the M1 single inoculated treatment. The effects of AM fungi and/or Aphelenchoides sp. on As contents in shoot varied depending on the As concentration in soil. At the highest As exposure level, AM fungi, in single inoculation or coinoculated with Aphelenchoides sp. was not effective in influencing the shoot As contents, but at the two lower As levels M2 increased such value by 15% (L soil) and 23% (M soil). Moreover, the positive effect was not affected by the dual addition of Aphelenchoides sp. in M soil, and was even more stimulated by 16% in L soil. Also, the coinoculation of M1 and Aphelenchoides sp. in L soil significantly enhanced shoot As contents when compared to the control and M1 single inoculated plants (Fig. 1). Both concentrations and contents of As in root were differently affected by the inoculation treatments in terms of the soil As levels. They showed a same trend at each As level (except at the high As concentration). In L soil, no differences in As concentrations and contents of As in roots were found among the tested treatments. In M soil, mycorrhizal plants had much lower concentrations and contents of As in roots compared with control plants, and Aphelenchoides sp. did not influence this effect. In H soil, As concentrations in roots were increased by the single inoculation wtih AM fungi, while this effect was neutralized by Aphelenchoides sp. In the case of As contents, no differences were observed among the tested treatments (Fig. 1).
Individuals treatment (×10 )
180
6
a
a a
5
a
4 3 b 2 1
b
b
0 M Soils
L Nem
M1+Nem
H M2+Nem
Fig. 2. Numbers of Aphelenchoides sp. per treatment in nematode addition treatments. (L, soil in low As level; M, soil in medium As level; H, soil in high As level; M1, inoculated with non-indigenous AM fungi Glomus caledonium; M2, inoculated with indigenous AM fungi Glomus spp. and Acaulospora spp.; Nem addition of Aphelenchoides sp. Bars represent standard errors and different letters indicate significant differences at the same soil by Duncan’s multiple range test at 5% level, n = 3. Significance (F-value) of an ANCOVA using soil Olsen P as covariate, soil Olsen P: n.s. (1.79); As: n.s. (1.72); AMF isolate: 0.000 (63.51); As AMF isolate: 0.016 (4.13).)
J. Hua et al. / Applied Soil Ecology 84 (2014) 176–184
181
35 a
AM colonization (%)
30 25
a a
20
a
15 a b
b
10
b
a
b
a
5
a
0 L
M
H
0.9 a a
-1
ERM (m g )
0.7 0.6
a
a
0.8 a
ab b
a
a
ab
0.5
b b
0.4 0.3 0.2 0.1 0.0 L
M
H
800 a 700
-1
Spores (100 g )
600 500 400
a
300 200 b 100
a
a
b
b
b
b
b b
b
0 L
M
H
Soils M1
M2
M1+Nem
M2+Nem
Fig. 3. Effects of the inoculation of AM fungi (M1 or M2) and/or Aphelenchoides sp. on the development of mycorrhiza under increasing soil As levels. (L, soil in low As level; M, soil in medium As level; H, soil in high As level; control, non-inoculated with AM fungi or Aphelenchoides sp.; M1, inoculated with non-indigenous AM fungi Glomus caledonium; M2, inoculated with indigenous AM fungi Glomus spp. and Acaulospora spp.; Nem addition of Aphelenchoides sp. Bars represent standard errors. Different letters represent significant difference according to Duncan’s multiple range test at 5% level, n = 3.)
spores numbers of M2 were 5.60 fold increased by Aphelenchoides sp., while that of M1 were not affected (Fig. 3). 4. Discussion Artificially polluted soil creates an artificial environment where bound and free metals/metalloids are not in equilibrium (Heggo et al., 1990), and the effects of metals/metalloids on the tested organisms would be overestimated (Chaney et al., 1978). Instead of adding metals/metalloids as soluble salt, a more representative approach is using contaminated soil sampled near an arsenic sulphide mine as chosen in our study. Ineluctably, with different soil As levels also other chemical properties changed, especially the P concentrations. It is generally assumed that mycorrhizal symbiosis and plant growth may be affected by the availability of soil P (Lin et al., 2012), especially in soils with high As concentrations due to the interlink between As and P (Bai et al., 2008). Consequently, an ANCOVA of tested parameters using soil
Olsen P as covariate was conducted to work out the specific effect of As level, nematode, AM fungi and their interactions independent of soil Olsen P on As extraction and growth of tobacco. As shown in Table 2 and Fig. 2, the covariate soil Olsen P did not significantly affect the development of mycorrhiza and nematodes, growth and As uptake of tobacco plants in the present study. The results of this experiment provide insights into the interactions between Aphelenchoides sp. and AM fungi and the consequent effects on As uptake and growth of tobacco plants. 4.1. Interaction effects between AM fungi and nematodes on plants growth and As uptake AM fungi are one of the most abundant inhabitants of belowground ecosystems. They are able to promote the host plant growth, since in AM fungal symbiosis, the fungus receives C from the host and meanwhile, the host obtains nutrients (especially P) via the fungus. Nematodes are the most abundant metazoans in
182
J. Hua et al. / Applied Soil Ecology 84 (2014) 176–184
soil. Some of them feeding on fungi or bacteria occupy irreplaceable positions in the soil food web (Bernard, 1992). They play important roles in the decomposition of soil organic matter, mineralization of plant nutrients and nutrient cycling (Ingham et al., 1985; Mao et al., 2007). Many experiments have provided strong evidences that bacterivorous nematodes could improve the plant growth due to the increased N, mostly as ammonia, since the C:N ratio in the bacterial biomass (approximately 5:1) is lower compared with that of nematodes (approximately 10:1) (Anderson et al., 1980; Ferris et al., 1998). In this experiment, the height and dry weights of tobacco plants were unaffected or increased by M1 or M2 isolates in L soil. Nevertheless, the mycorrhizal effect improving plant growth was significantly enhanced by the presence of Aphelenchoides sp. This phenomenon could be explained by the feeding activities of Aphelenchoides sp. on AM fungi. In fact, the C:N ratio of fungivorous nematodes is similar to that of fungal mycelium (Ingham et al., 1985). As a result, there is no excess N associated with fungal tissues assimilated for nematode biomass production as happens when bacterivores feed on bacteria. However, a considerable proportion of the C consumed is used in respiration, about 40% of the food intake (Ingham et al., 1985). The N associated with respired C that is in excess of structural needs is also excreted. As reported by Coleman (1986), the fungivorous nematode Aphelenchus avenae could obtain 6.1 ng N through fungal mycelium per day, regardless of the amount of 1.45 ng N in the body of nematodes. Consequently, most of the N was released to soil. Likewise, Chen and Ferris (1997) found that the amount of NH4+ released from the microcosms in which barley straw was colonized by Rhizoctonia sp. was always greater when A. avenae was added. Further, nematodes could deposit feces enriched with organic material into the soil (Chen and Ferris, 2000). Apparently, the activities of nematodes improved available nutrition in soil solution which could be easily absorbed by both plants and microbes. For example, increased length of ERM of which could extend the absorption area of roots was observed in the present investigation. Similar results have been obtained in experiments involving fungivorous Collembola (Hanlon, 1981). This effect was also beneficial to plant growth. Surprisingly, except for the M2 + Nem treatment in L soil, no differences in mineral N contents were found between non-nematode and nematode addition treatments. The strong N absorption by plants and AM fungi may partly explain this result. Besides the mycorrhizal effect on plant growth, the role of AM fungi in As uptake by plants has been well documented since As is transported across the plasma membrane via P transport systems (Ullrich-Eberius et al., 1989). In most cases, As concentrations in shoots were increased by indigenous AMF isolate M2 but not affected by non-indigenous AMF isolate M1 regardless of the addition of Aphelenchoides sp. Similarly, Bai et al. (2008) found that mycorrhizal effects on As uptake in maize grown in the same polluted soil were different between M1 and M2 isolates. The probable explanation, as stated by Wang et al. (2005) and Weissenhorn et al. (1994), is that AMF isolates from contaminated soils have great efficacy, but those from unpolluted soils have high sensitivity and are not adapted to the stress of contaminated soils. Nevertheless, few experiments were focused on the interaction effects between AM fungi and mycophagous animals on metal uptake in plants. As reported by Yu et al. (2005), dual inoculation with earthworms (Pheretima sp.) and AM fungi (Glomus mosseae and Glomus intraradices) increased shoot Cd accumulation of ryegrass (Lolium multiflorum) at low soil Cd concentrations. Our study also found that the addition of Aphelenchoides sp. substantially increased shoot As concentrations of M2 mycorrhizal plants, but decreased it of M1 mycorrhizal plants in L soil. This is the first report to show that fungivorous nematodes had significant effects
on As uptake by mycorrhizal plants grown in an As polluted soil. Like the bacterial isolate Brevibacillus sp., whose dual inoculation with G. mosseae achieved further plant (T. repens) growth and lower Cd concentrations (Vivas et al., 2003), Aphelenchoides sp. can act by direct or indirect mechanisms on the AM fungus as myzorrhizae helping organisms, enhancing the “mycorrhizal effect” on plant As uptake. Furthermore, the co-inoculation of AM fungi and Aphelenchoides sp. significantly increased the shoot As contents when compared to the control and M1 and M2 single inoculated plants at L soil. This finding was consistent with the result of shoot dry weights. Obviously, the positive effect of Aphelenchoides sp. on the As accumulation in mycorrhizal plants could be mainly linked to the plants biomass resulting from the development of mycorrhiza and variation of soil nutrition that was induced in turn by the grazing activities of Aphelenchoides sp. on AM fungi. 4.2. Interaction effects between AM fungi and nematodes on their own development Numbers of Aphelenchoides sp. were considerably higher in mycorrhizal pots than in non-mycorrhizal pots. Also, numbers of Aphelenchoides sp. significantly exceeded the initial inoculum number in mycorrhizal pots. Our previous field experiment also found that the abundance of Aphelenchoides sp. was significantly increased in the M2 inoculated treatment than in the noninoculated treatment in an As contaminated soil (Hua et al., 2010). Obviously, this is an indication of the suitability of both M1 and M2 as good food sources for Aphelenchoides sp. Likewise, Bakhtiar et al. (2001) reported that the fungivorous nematodes A. avenae Bastian could multiply by feeding on the AM fungi Gigaspora margarita and Glomus coronatum grown with clover (T. subterraneum L.) as evidenced by an increased population in mycorrhizal treatments but a reduced number (below the initial density value) in nonmycorrhizal treatments. In fact, Hussey and Roncadori (1981) have noted that AM fungi should be excellent hosts for mycophagous nematodes due to the production of extraradical mycelium. Recently, Brussaard et al. (2001) using petri dishes observed that Aphelenchoides sp. was feeding and reproducing on all tested ectomycorrhizal fungi. Likewise, Ruess et al. (2000) studied the growth of Aphelenchoides sp. populations in vitro on 17 different fungal species, and found that nematode populations developed on saprophytic (Agrocybe and Chaetomium) and especially on mycorrhizal fungi (Cenococcum, Hymenoscyphus and Laccaria). As we known, pure culture of AM fungi is still a big challenge for microbiologists, and thus the direct evidence that fungivorous nematodes could feed on AM fungi is undetected now. Furthermore, an experiment using a transparent microcosm, like a petri dish filled with sepharose gel, with a plant or a Ri-T-DNA transformed root (Diop, 2003) infected by AM fungi, should be conducted to find the direct proof that Aphelenchoides sp. is feeding and reproducing on AM fungi by a microscope. On the other hand, the grazing activity of nematodes on AM fungi, including mycelial network and spores may be of significance in affecting the inoculum production and therefore the root colonization. To date, positive, neutral and negative effects were observed (Bakhtiar et al., 2001). In this experiment, the stimulative effects of Aphelenchoides sp. on the development of AM fungi were slightly different between two tested AM fungi. The ERM length of M2 was remarkably enhanced by Aphelenchoides sp. in L and M soils. The total number of spores of M1 in L soil and that of M2 in H soil was increased by the nematodes. The development of AM fungi may be partly explained by the grazing of Aphelenchoides on AM fungi which could recycle minerals locked up in senescent fungal tissue. Interestingly, Aphelenchoides sp. did not affect root colonization in the present experiment. However,
J. Hua et al. / Applied Soil Ecology 84 (2014) 176–184
Bakhtiar et al. (2001) observed that the mycorrhizal colonization of clover roots was decreased as the population of fungivorous nematode A. avenae Bastian increased in mycorrhizal pots. This result might be due to the declined percentage of spores, because the spores with high lipid contents could be penetrated by nematodes to provide high-energy food sources for them (Bakhtiar et al., 2001). The lack of adverse effects in this study agreed with the results of Hussey and Roncadori (1981) and Giannakis and Sanders (1990), who found no effects of Aphelenchus on mycorrhizal infection and spores production. Actually, the effects depended on the relationship between nematodes and AM fungi, such as the differences in accessibility or palatability of the spores or hyphae to different nematodes. For example, the stylet of Aphelenchoides is shorter (<8 mm) (http://plpnemweb.ucdavis. edu/Nemaplex/Taxadata/G011.htm) than that of Aphelenchus (15 mm) (Hooper, 1974), and not all of this length is available to penetrate the spores. Thus, at some sense, the access to spores would be limited. Besides, the starting numbers of nematodes applied would significantly influence the results, which has been thoroughly discussed by Bakhtiar et al. (2001). Moreover, unlike other studies, contaminated soils were used in our experiment. Considering the fact that soil pollution significantly influenced the nematode communities (Li et al., 2006) and AM fungi development (Bai et al., 2008; Wu et al., 2009), we have sound reasons to believe that soil contamination would influence the interaction between AM fungi and fungivorous nematodes. In conclusion, Aphelenchoides sp. could feed and reproduce on the tested AM fungi, and the mycorrhiza was developed in turn by the grazing activities. The interaction of AM fungi and fungivorous nematodes has potential to elevate the phytoextraction efficiency in low level As polluted soils. The isolation of tolerant beneficial organisms from contaminated soils may be an ideal way to achieve soil decontamination goals. Moreover, the mechanisms by which AM fungi and mycophagous animals enhance metal uptake by the plants would allow the management of soil organisms with suitable characteristics to be used for bioremediation purposes. The knowledge of basic interactions between host plant–AM fungi–mycophagous animals in other metal polluted soils needs further studies. Acknowledgments This work is jointly supported by the National Natural Science Foundation of China (No. 41101232), the Natural Science Foundation of Jiangsu Province of China (BK2011688) and the Research Fund of State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Science (Y412201436). We also thank two anonymous reviewers for their critical comments which greatly improved the quality of the manuscript. References Anderson, R.V., Cole, C.V., Coleman, D.C., 1980. Quantities of plant nutrients in the microbial biomass of selected soils. Soil Sci. 130, 211–216. Bai, J.F., Lin, X.G., Yin, R., Zhang, H.Y., Wang, J.H., Chen, X.M., Luo, Y.M., 2008. The influence of arbuscular mycorrhizal fungi on As and P uptake by maize (Zea mays L.) from As-contaminated soils. Appl. Soil Ecol. 38, 137–145. Bakhtiar, Y., Miller, D., Cavagnaro, T., Smith, S., 2001. Interactions between two arbuscular mycorrhizal fungi and fungivorous nematodes and control of the nematode with fenamifos. Appl. Soil Ecol. 17, 107–117. Bernard, E.C., 1992. Soil nematode biodiversity. Biol. Fertil. Soils 14, 99–103. Brussaard, L., Kuyper, T.W., de Goede, R.G.M., 2001. On the relationships between nematodes, mycorrhizal fungi and plants: functional composition of species and plant performance. Plant Soil 232, 155–165. Chaney, R.L., Hundemann, P.T., Palmer, W.T., Small, R.J., White, M.C., Decker, A.M., 1978. Plant accumulation of heavy metals and phytotoxicity resulting from utilization of sewage sludge and sludge composts on cropland. Proceedings of the National Conference of Composting of Municipal Residues and Sludges. Information Transfer Inc., Rockville, MD, pp. 86–97.
183
Chen, J.A., Ferris, H., 1997. Nitrogen mineralization by Aphelenchus avenae associated with Rhizoctonia sp. and barley straw. J. Nematol. 29 (4), 572. Chen, J., Ferris, H., 2000. Growth and nitrogen mineralization of selected fungi and fungal-feeding nematodes on sand amended with organic matter. Plant Soil 218, 91–101. Coleman, D.C., 1986. The role of microfloral and faunal interactions in affecting soil processes. In: Mitchell, M.J., Nakas, J.P. (Eds.), Micronoral and Fauna Interaction in Natural and Agro-ecosystems. Martinos N. Junk W. Publishers, Dordrecht, pp. 317–348. Diop, T.A., 2003. In vitro culture of arbuscular mycorrhizal fungi: advances and future prospects. Afr. J. Biotechnol. 2 (12), 692–697. Durán, P., Acuña, J.J., Jorquera, M.A., Azcón, R., Borie, F., Cornejo, P., Mora, M.L., 2013. Enhanced selenium content in wheat grain by co-inoculation of selenobacteria and arbuscular mycorrhizal fungi: a preliminary study as a potential Se biofortification strategy. J. Cereal Sci. 57 (3), 275–280. Ferris, H., Venette, R.C., van der Meulen, H.R., Lau, S.S., 1998. Nitrogen mineralization by bacterial-feeding nematodes: verification and measurement. Plant Soil 203, 159–171. Fitter, A.H., Garbaye, J., 1994. Interaction between mycorrhizal fungi and other soil organisms. Plant Soil 159, 123–132. Gaur, A., Adholeya, A., 2004. Prospects of arbuscular mycorrhizal fungi in phytoremediation of heavy metal contaminated soils. Curr. Sci. 86 (4), 527–534. Giannakis, N., Sanders, F.E., 1990. Interactions between mycophagous nematodes, mycorrhizal and other soil fungi. Agric. Ecosyst. Environ. 29, 163–167. Giovannetti, M., Mosse, B., 1980. An evaluation of techniques for measuring vesicular arbuscular mycorrhizal infection in roots. New Phytol. 84, 489–500. Hanlon, R.D.G., 1981. Influence of grazing by Collembola on the activity of senescent fungal colonies grown on medium of different nutrient concentrations. Oikos 36, 363–367. Heggo, A., Angle, J.S., Chaney, R.L., 1990. Effects of vesiculararbuscular mycorrhizal fungi on heavy metal uptake by soybeans. Soil Biol. Biochem. 22, 865–869. Hildebrandt, U., Regvar, M., Bothe, H., 2007. Arbuscular mycorrhiza and heavy metal tolerance. Phytochemistry 68 (1), 139–146. Hooper, D.J., 1974. Aphelenchus avenae. In: Willmott, S., Gooch, P.S., Siddiqi, M.R., Franklin, M.T. (Eds.), C.I.H. Descriptions of Plant–Parasitic Nematodes. Commonwealth Agricultral Bureaux, Farnham Royal, Slough, UK Set 4, No. 50. Hua, J.F., Lin, X.G., Yin, R., Jiang, Q., 2009. Effects of arsenic contamination on structure of soil nematode community in mining area. J. Ecol. Rural Environ. 25, 79–84 (in Chinese). Hua, J.F., Lin, X.G., Bai, J.F., Shao, Y.F., Yin, R., Jiang, Q., 2010. Effects of arbuscular mycorrhizal fungi and earthworm on nematode communities and arsenic uptake by plants in arsenic-contaminated soils. Pedosphere 20, 163–173. Hussey, R.S., Roncadori, R.W., 1981. Influence of Aphelenchus avenae on vesicular– arbuscular endomycorrhizal growth response in cotton. J. Nematol. 13, 48–52. Ingham, R.E., Trofymow, J.A., Ingham, E.R., Coleman, D.C., 1985. Interactions of bacteria, fungi, and their nematode grazers: effects on nutrient cycling and plant growth. Ecol. Monogr. 55, 119–140. Jakobsen, I., Abbott, L.K., Robson, A.D., 1992. External hyphae of vesicular– arbuscular mycorrhizal fungi associated with Trifolium subterraneum L. 1. Spread of hyphae and phosphorus inflow into roots. New Phytol. 120, 371–380. Koller, R., Rodriguez, A., Robin, C., Scheu, S., Bonkowski, M., 2013a. Protozoa enhance foraging efficiency of arbuscular mycorrhizal fungi for mineral nitrogen from organic matter in soil to the benefit of host plants. New Phytol. 199 (1), 203–211. Koller, R., Scheu, S., Bonkowski, M., Robin, C., 2013b. Protozoa stimulate N uptake and growth of arbuscular mycorrhizal plants. Soil Biol. Biochem. 65, 204–210. Koske, R.E., Gemma, J.N., 1989. A modified procedure for staining roots to detect VA mycorrhizas. Mycol. Res. 92, 486–505. Larsen, J., Cornejo, P., Barea, J.M., 2009. Interactions between the arbuscular mycorrhizal fungus Glomus intraradices and the plant growth promoting rhizobacteria Paenibacillus polymyxa and P. Macerans in the mycorrhizosphere of Cucumis sativus. Soil Biol. Biochem. 41 (2), 286–292. Li, Q., Jiang, Y., Liang, W.J., 2006. Effect of heavy metals on soil nematode communities in the vicinity of a aetallurgical factory. J. Environ. Sci. 18 (2), 323–328. Li, H., Man, Y.B., Ye, Z.H., Wu, C., Wu, S.C., Wong, M.H., 2013. Do arbuscular mycorrhizal fungi affect arsenic accumulation and speciation in rice with different radial oxygen loss? J. Hazard. Mater. 262, 1098–1104. Lin, X.G., Feng, Y.Z., Zhang, H.Y., Chen, R.R., Wang, J.H., Zhang, J.B., Chu, H.Y., 2012. Long-term balanced fertilization decreases arbuscular mycorrhizal fungal diversity in an arable soil in North China revealed by 454 pyrosequencing. Environ. Sci. Technol. 46, 5764–5771. Mao, X.F., Hu, F., Griffiths, B., Chen, X.Y., Liu, M.Q., Li, H.X., 2007. Do bacterial-feeding nematodes stimulate root proliferation through hormonal effects? Soil Biol. Biochem. 39, 1816–1819. Oostenbrink, M., 1960. Estimating nematode populations by some selected methods. Nematology 6, 85–102. Orlowska, E., Godzik, B., Turnau, K., 2012. Effect of different arbuscular mycorrhizal fungal isolates on growth and arsenic accumulation in Plantago lanceolata L. Environ. Pollut. 168, 121–130. Ruess, L., Garcia Zapata, E.J., Dighton, J., 2000. Food preferences of a fungal-feeding Aphelenchoides species. Nematology 2, 223–230. Schenck, N.C., Perez, Y., 1990. Manual for Identification of Vesicular Arbuscular Mycorrhizal Fungi, third ed. Synergistic Publication, University of Florida, Gainesville, Florida, USA, pp. 286. Siddiky, M.R.K., Schaller, J., Caruso, T., Rillig, M.C., 2012. Arbuscular mycorrhizal fungi and collembola non-additively increase soil aggregation. Soil Biol. Biochem. 47, 93–99.
184
J. Hua et al. / Applied Soil Ecology 84 (2014) 176–184
Smith, S.E., Read, D.J., 2008. Mycorrhizal Symbiosis, third ed. Academic Press and Elsevier, London. Townshend, J.L., 1963. A modification and evaluation of the apparatus for the Oostenbrink direct filter extraction method. Nematologica 9, 106–110. Ullrich-Eberius, C.I., Sanz, A., Novacky, A.J., 1989. Evaluation of arsenate and vanadate-associated changes of electrical membrane potential and phosphate transport in Lemna gibba G1. J. Exp. Bot. 40, 119–128. Vivas, A., Vörös, I., Biró, B., Campos, E., Barea, J.M., Azcón, R., 2003. Symbiotic efficiency of autochthonous arbuscular mycorrhizal fungus (G. mosseae) and Brevibacillus sp. isolated from cadmium polluted soil under increasing cadmium levels. Environ. Pollut. 126, 179–189.
Wang, F.Y., Lin, X.G., Yin, R., 2005. Heavy metal uptake by arbuscular mycorrhizas of Elsholtzia splendens and the potential for phytoremediation of contaminated soil. Plant Soil 269, 225–232. Weissenhorn, I., Glasho, A., Leyval, C., Berthelin, J., 1994. Differential tolerance to Cd and Zn of arbuscular mycorrhizal (AM) fungal spores isolated from heavy metalpolluted and unpolluted soils. Plant Soil 167, 189–196. Wu, F.Y., Ye, Z.H., Wong, M.H., 2009. Intraspecific differences of arbuscular mycorrhizal fungi in their impacts on arsenic accumulation by Pteris vittata L. Chemosphere 76, 1258–1264. Yu, X.Z., Cheng, J.M., Wong, M.H., 2005. Earthworm–mycorrhiza interaction on Cd uptake and growth of ryegrass. Soil Biol. Biochem. 37, 195–201.
Contents lists available at ScienceDirect
Applied Soil Ecology journal homepage: www.elsevier.com/locate/apsoil
Interactions between arbuscular mycorrhizal fungi and fungivorous nematodes on the growth and arsenic uptake of tobacco in arseniccontaminated soils Jianfeng Hua a,b , Qian Jiang b , Jianfeng Bai c , Feng Ding d, Xiangui Lin b , Yunlong Yin a, * a
Institute of Botany, Jiangsu Province and The Chinese Academy of Sciences, Nanjing 210014, China State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China c Waste Electrical and Electronic Equipment Recycling Centre, Shanghai Second Polytechnic University, Shanghai 201209, China d Qianxinan Tobacco Company, Guizhou 562400, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 February 2014 Received in revised form 7 July 2014 Accepted 9 July 2014 Available online xxx
The effects of inoculation with two AM fungi (M1, Glomus caledonium; M2, Glomus spp. and Acaulospora spp.) and a fungivorous nematode Aphelenchoides sp. on growth and arsenic (As) uptake of Nicotiana tabacum L. were investigated in soils contaminated with a range of As. The reproduction of Aphelenchoides sp. was triggered by the co-inoculation of AM fungi regardless of AM fungal isolates and As levels. Stimulative effects of Aphelenchoides sp. on the development of mycorrhiza, slightly different between two AM fungi, were found particularly at the lowest As level. Irrespective of mycorrhizal inoculi, increasing soil As level decreased plant growth, but increased plant As uptake. Co-inoculation of AM fungi and Aphelenchoides sp. led plants to achieving further growth and greater As accumulation at the lowest As level. Results showed that the interactions between AM fungi and fungivorous nematodes were important in plant As tolerance and phytoextraction at low level As-polluted soil. ã 2014 Published by Elsevier B.V.
Keywords: Mycorrhiza Fungivorous nematodes Fungus–nematode interactions Arsenic contamination Phytoextraction
1. Introduction Arbuscular mycorrhizal (AM) fungi are able to form a symbiotic association with approximately 80% of terrestrial plant species. In general, the growth of host plants will be improved through obtaining nutrients, especially phosphorus (P) via mycorrhizal hypha (Smith and Read, 2008). In addition, some AM fungi also play an important role in heavy metals uptake of host plants grown in contaminated soils (Gaur and Adholeya, 2004; Hildebrandt et al., 2007). For example, remarkable studies have found that mycorrhiza affected arsenic (As) uptake of plants since As transports across the plasma membrane via P transport systems (Li et al., 2013; Orlowska et al., 2012). As the main soil microorganism in rhizosphere, AM fungi will interact with a wide range of other organisms, including bacteria, fungi, protozoa, arthropods and nematodes (Fitter and Garbaye, 1994). Some of these interactions have shown to be able to influence mycorrhizal behavior and function (Durán et al., 2013; Siddiky et al., 2012). Larsen et al. (2009) found that plant growth promoting species of Paenibacillus may have suppressive effects on
* Corresponding author. Tel.: +86 25 84347059; fax: +86 25 84347066. E-mail addresses: [email protected] (J. Hua), [email protected] (Y. Yin). http://dx.doi.org/10.1016/j.apsoil.2014.07.004 0929-1393/ ã 2014 Published by Elsevier B.V.
AM fungi and growth of Cucumis sativus. Using 15N and 13CO2 labeling technology, Koller et al. (2013a,b) showed that both the root mycorrhizal colonization and the growth of Plantago lanceolata L. benefited from the presence of protozoa. As we know, soil nematodes are the most abundant multicellular animals on the planet. Some microbivorous nematodes could play important roles in the turnover of soil microbial biomass and availability of nutrients (Chen and Ferris, 2000; Mao et al., 2007). AM fungi producing substantial extraradical mycelium and spores are excellent feeding resource for fungivorous nematodes (Hussey and Roncadori, 1981). Today, many experiments have provided evidences that the presence of fungivorous nematodes could affect arbuscular mycorrhiza development and ‘mycorrhiza effects’ by grazing activity (Bakhtiar et al., 2001; Giannakis and Sanders, 1990; Ingham et al., 1985). Despite the ubiquity of the interplay between AM fungi and other organisms and their importance for plant nutrition and growth, only few studies investigated their interactions on the growth and metal accumulation of plant grown in contaminated soils. Using AM fungal and earthworm cultures, Yu et al. (2005) observed that earthworms modified the functioning of AM fungus– plant symbiosis in Cd polluted soils. Inoculation of earthworms increased mycorrhizal infection rates of roots, shoot yield and Cd uptake of mycorrhizal ryegrass at 5 and 10 mg Cd kg1 soils. Vivas
J. Hua et al. / Applied Soil Ecology 84 (2014) 176–184
et al. (2003) found that Trifolium repens dually inoculated with Brevibacillus sp. and AM fungi isolated from a Cd polluted soil attained further growth and nutrition and lower Cd concentration, particularly at 85.1 mg Cd kg1 soil. However, few observations have been reported on the interaction between AM fungi and fungivorous nematodes on plant growth and As uptake. In present study, we have investigated the effects of the inoculation with two isolates of AM fungi and a selected fungivorous nematode on the growth and As accumulation of tobacco plants (Nicotiana tabacum L.). AM fungi and nematodes were assayed in single or in co-inoculation in soils contaminated with a range of As. It is hypothesized that the interactions between AM fungi and fungivorous nematodes will stimulate the growth and As uptake of tobacco plants, and different fungal species and different soil As levels might induce differential outcomes. 2. Materials and methods 2.1. Soil and AM fungi and fungivorous nematodes inocula preparation Soils were collected from agricultural fields near an As sulphide mine (29 390 N, 111020 E), which has been exploited for more than 1500 years, in Shimen County, Hunan Province, China. This area has a North Asian subtropical maritime monsoon climate, with mean temperature 16.7 C and average rainfall of about 1500 mm and more than 260 frost-free days. Three sampling sites were selected: (1) an area at the foot of the mining area with high As content, later abbreviated as H; (2) a location about 2 km from the mining area, M; and (3) a site chosen about 8 km from the mining area, L. The land use in these areas was arable cropping. The dominant crop species was tobacco (N. tabacum L.). Soil samples were classified as Ali-Perudic Argosols, and the chemical properties are given in Table 1. All tested soils were air-dried and sieved with nylon mesh (4 mm) and then sterilized by 60Co-irradiation (25 kGy) before a pot experiment. Two AM fungal isolates were used in this study, Glomus caledonium (represented as M1) isolated from an unpolluted agricultural soil in Henan Province, and an indigenous combined inoculum of Glomus spp. and Acaulospora spp. (represented as M2), wet-sieved from an As-polluted soil in Shimen County, Hunan Province (Bai et al., 2008). These AM fungal species were identified morphologically according to current taxonomic criteria (Schenck and Perez, 1990) and internet information by INVAM (http://invam.caf.wvu.edu). Fungal inocula were propagated on clover (Trifolium subterraneum L.) grown in an autoclaved substrate with successive propagation cycle, for 4 months in a greenhouse of the Institute of Soil Science, Chinese Academy of Sciences. Inocula were air-dried and sieved (2 mm), and consisted of thoroughly mixed rhizosphere samples containing spores, hyphae and mycorrhizal root fragments. Fungivorous nematode Aphelenchoides sp. was used as a model species. It is eudominant in L, M and H soils with the presence ranging from 11.4% to 36.2% (Hua et al., 2009), and tolerant to As evidenced by a 10 mg L1 As solution test. Worms were collected by cotton wool (Oostenbrink, 1960; Townshend, 1963) from M soil and
177
cultured in petri dishes on Potato Dextrose Agar (PDA) with Botrytis cinerea Pers. at 25 0.5 C. They were collected by the cotton-wool filter method and washed twice with sterile distilled water before use in the experiments (Mao et al., 2007). 2.2. Experimental design The experiment was designed 3 2 3 factorial with the following factors: (1) mycorrhizal treatments including assay with two AMF isolates (M1 and M2) and one non-inoculated control treatment; (2) addition or not of fungivorous nematodes; and (3) three levels of As polluted soils, referred to as L, M and H. Tobacco (N. tabacum L.) was seeded into sterilized peat-based seeding substrate on August 4, 2011. At cross stage, the plantlets were carefully transplanted into 3 L plastic pots (one plant per pot) filled with 3.0 kg of dry soil. Each mycorrhizal treatment received 50 g AM fungi inocula at sowing stage. All non-mycorrhizal treatments were treated with the same amount of inocula that had been autoclaved twice at 121 C for 30 min together with about 20 mL filtrate (<20 mm) of 50 g AM fungi inocula to provide a general microbial population free of AM propagules (Vivas et al., 2003). One week after transplanting, each nematode-added treatment received 2000 Aphelenchoides sp. Pots were arranged in randomized complete block design with three replicates per treatment. Plants were grown in a sunlit greenhouse with natural light, a day/night temperature 33/22 C and relative humidity 40–60%. Plants were watered to maintain soil moisture at 60–70% of water holding capacity by adding deionised water during the experimental period. All plants did not receive any fertilizers during the study period. 2.3. Harvest and measurements Heights of tobacco plants were recorded, and then roots and shoots were harvested separately after 13 weeks. Sub-samples of fresh roots were taken to assess mycorrhizal colonization. Fresh weights of total roots and of sub-samples were measured. Shoots and remaining roots were first rinsed with tap water and then rinsed with deionised water. Tissues were then weighed after oven drying at 60 C for 72 h and then ground to <0.25 mm in a stainless steel mill. The water content (%) of remaining roots and total fresh weights of roots were used to estimate dry weights of roots. Root mycorrhizal colonization was evaluated after clearing and staining (Koske and Gemma, 1989), using the grid-line intersect method (Giovannetti and Mosse, 1980). The length of extraradical mycelium (ERM) in soil was estimated using a modified membrane filtration technique (Jakobsen et al., 1992). A sub-sample of 30 g moist soil was used to determine total numbers of spores at the end of the experiment, using the wet sieving and centrifugation technique (Bakhtiar et al., 2001). Aphelenchoides sp. was extracted from 100 g fresh soil of each sample with a cotton-wool filter method. At the same time, water content (%) of each soil sample was determined. After 48 h extraction at room temperature, total numbers of Aphelenchoides sp. were counted, and their populations were expressed as the number of individuals per 100 g dry weight soil.
Table 1 Chemical properties of the soils with different As concentrations. As level
Total As (mg kg1)
pH (H2O)
Organic C (g kg1)
Total N (g kg1)
Olsen P (mg kg1)
Available K (mg kg1)
L M H
12.7 0.31 36.8 0.52 148 4.01
5.98 0.05 6.57 0.07 5.18 0.02
31.0 1.03 28.2 0.75 19.6 0.63
1.55 0.11 1.46 0.08 0.88 0.09
49.5 5.30 39.8 2.62 22.1 1.62
373 25.2 353 26.1 140 19.4
Data presented are means standard deviation (n = 3). L, soil low in As level; M, soil medium in As level; H, soil high in As level.
178
J. Hua et al. / Applied Soil Ecology 84 (2014) 176–184
Table 2 F-value of an ANCOVA of tested parameters using soil Olsen P as covariate in this experiment. Factors
Height (cm)
Dry weight (g) Root
7.22** As Nematode 4.45* AMF isolate 1.32 As Nematode 10.7** As AMF isolate 1.57 Nematode AMF isolate 0.40 As Nematode AMF isolate 2.02 Covariate Soil Olsen P 1.70 * **
Shoot
As concentration (mg kg1)
As content (mg pot1)
Soil mineral N (mg kg1)
Root
Shoot
Root
Shoot
(NO3 + NO2)-N NH4+-N Colonization (%)
55.1** 1.88 20.4** 0.60 1.28 1.26 1.06
2.07 3.46 0.56 5.16* 2.26 0.63 2.77*
27.1** 9.06** 24.6** 10.8** 3.6* 3.12* 1.31
1.23 2.21 0.85 0.94 3.54* 2.65 1.08
4.26* 0.03 4.13* 0.16 3.39* 0.26 0.63
14.1** 0.75 45.1** 0.68 5.76** 0.01 0.91
1.36 8.06** 1.54 5.36* 7.29** 11.4** 1.40
13.0** 45.8** 2.35 18.2** 55.2** 11.7** 35.0**
2.94
0.47
0.76
0.32
3.08
0.01
0.86
0.81
5.51** 1.40 29.9** 2.34 14.9** 10.0** 1.14 1.41 4.23* 11.1** 16.7** 2.09 3.39* 2.91* 4.87** 0.56 0.39 1.53 2.09 3.46* 4.67** 1.28
0.01
0.59
Development of mycorrhiza ERM Spores (m g1) (100 g1)
P < 0.05. P < 0.01.
Soil pH was determined with a glass electrode (soil/water = 1:2.5, m/V). Soil organic C was measured by the dichromate oxidation and total N by Kjeldahl titration. Olsen P was extracted with sodium bicarbonate, and determined by the molybdenum-blue method. Available K was extracted with 1.0 mol L1 ammonium acetate, and then measured by a flame photometry. Soil mineral N ((NO3 + NO2)-N and NH4+-N) was extracted with 2 mol L1 KCl (soil/ solution = 1:10, m/V) for 1 h and determined by an automated procedure (Skalar SANplus Segmented Flow Analyzer, Skalar Analytic B.V., De Breda, Netherlands). To determine total As concentration, soil was extracted with concentrated HNO3 and HCl at 1:3 (V/V), and analyzed by atomic fluorescence spectrometer (AFS-230E, Haiguang Instrumental Co., China). Dried sub-samples of roots and shoots were digested with concentrated HNO3 and HCl at 3:1 (V/V) to analyze As concentrations. The accuracy of the analyses was estimated by comparison with reference material GBW07406 (for soil) and GBW07603 (for plant) from Institute of Geophysical & Geochemical Exploration, Chinese Academy of Geosciences, and blanks were introduced regularly. The outcomes of the analyses on GBW07406 and GBW07603 were 217 10 mg As kg1 and 1.19 0.06 mg As kg1, respectively. The recovery rates of As were 98% and 95%, respectively.
2.4. Data analysis An analysis of covariance (ANCOVA) using soil Olsen P as covariate was applied to test the effects of As level, nematode addition and AM fungi inoculation as main factors and their interactions on growth and As uptake of tobacco. We focused our analysis on variances of root mycorrhizal colonization, extraradical mycelium length and spores numbers without the treatments noninoculated with AM fungi. An ANCOVA was also performed for the numbers of Aphelenchoides sp. using As level and AM fungi inoculation as factors, and soil Olsen P as covariate. Meanwhile, data of each As level were subjected to a one-way ANOVA. Statistical analyses were performed by SPSS 16.0 for Windows and a significant level of P < 0.05 was used. 3. Results 3.1. Growth of tobacco plants An ANCOVA of tested parameters using soil Olsen P as covariate demonstrated that there were no significant effects of soil Olsen P
Table 3 Effects of the inoculation of AM fungi (M1 or M2) and/or Aphelenchoides sp. on plant growth and soil mineral N under increasing soil As levels. Treatments
Height (cm)
Soil mineral N (mg kg1)
Dry weight (g) Root
Shoot
(NO3 + NO2)-N
NH4+-N
L Control M1 M2 Nem M1 + Nem M2 + Nem
45.6 4.00 c 48.7 3.21 c 48.2 3.97 c 53.7 2.52 bc 62.5 4.44 a 58.7 2.20 ab
1.61 0.13 bc 1.51 0.04 c 1.70 0.30 bc 2.04 0.32 ab 2.23 0.30 a 1.95 0.28 abc
8.69 0.61 c 9.31 0.43 c 11.3 0.41 b 12.7 0.37 ab 14.4 2.23 a 14.2 0.61 a
0.20 0.06 b 0.13 0.04 b 0.22 0.06 b 0.21 0.08 b 0.23 0.12 b 0.38 0.06 a
1.67 0.13 a 1.51 0.45 a 1.38 0.09 a 1.60 0.43 a 2.02 1.08 a 1.77 0.78 a
M Control M1 M2 Nem M1 + Nem M2 + Nem
48.0 2.00 ab 50.3 2.89 ab 53.0 4.58 a 49.0 1.73 ab 44.7 6.35 b 50.0 2.65 ab
1.76 0.15 ab 1.46 0.14 bc 1.88 0.33 a 1.64 0.12 ab 1.30 0.00 c 1.70 0.15 ab
10.2 0.83 a 8.97 1.15 a 10.4 0.81 a 10.2 0.48 a 8.70 1.39 a 9.38 0.70 a
0.19 0.02 a 0.07 0.05 a 0.16 0.08 a 0.21 0.14 a 0.14 0.07 a 0.18 0.06 a
1.95 0.47 ab 1.56 0.13 b 1.58 0.34 b 2.16 0.28 a 1.47 0.12 b 1.75 0.34 ab
H Control M1 M2 Nem M1 + Nem M2 + Nem
33.2 6.25 a 33.0 10.5 a 26.3 4.04 a 26.0 5.29 a 30.7 7.57 a 31.7 2.08 a
0.93 0.11 a 0.78 0.09 ab 0.61 0.10 b 0.75 0.11 ab 0.74 0.20 ab 0.74 0.05 ab
5.26 1.23 a 5.79 0.43 a 4.27 0.67 a 4.32 0.38 a 5.67 1.73 a 6.30 1.27 a
0.15 0.09 a 0.17 0.16 a 0.16 0.14 a 0.10 0.05 a 0.31 0.17 a 0.14 0.13 a
5.03 1.49 ab 6.75 4.41 ab 3.99 1.69 ab 5.70 3.01 ab 7.76 3.62 a 2.10 0.80 b
Data presented are means standard deviation (n = 3). Means followed by the different letters into each As level are significantly different according to Duncan’s multiple range test at 5% level, n = 3. L, soil low in As level; M, soil medium in As level; H, soil high in As level.
J. Hua et al. / Applied Soil Ecology 84 (2014) 176–184
10
2 1
bcd
bc
b
cd
d
a
a
8
0
6
1 2
a
a
3
a
a
a
a
4 5
Root
Sho ot
6
As contents (ug pot -1 )
As concentrations (mg kg -1 )
179
4
cd
2 0 2 4
a
a
Control
M1
M2
Nem
M1+Nem
M2+Nem
b
a
b
b
a
Control
2 1
a
6
1 2
c
c
bc
b
a
bc
4 5 6
Nem
M1+Nem
b
b
c
c
9
a b
b
a
0 2 c a
bc
ab
8
Control
M1
M2
ab
a
Nem
M1+Nem
b
b
M2+Nem
Control
2
M1
M2
Nem
M1+Nem
M2+Nem
10
b
ab 8
0
6 As contents (ug pot -1 )
1 2 3 4 5
M2+Nem
2
6
8
1
4
4
7
M
M2
8
0
3
M1
10
b
As contents (ug pot -1 )
As concentrations (mg kg -1 )
a
8
9
L
As concentrations (mg kg -1 )
a a
6
8
bc
d
7
c c
6
bc
abc
4
a
ab b
0 2 4 a
ab
a
M1
M2
ab
ab
ab
2
7 8
6
a
a
M1
M2
a
a
a
Nem
M1+Nem
M2+Nem
8
9
H
b
b
Control
Nem
M1+Nem
M2+Nem
Con trol
Fig. 1. Effects of the inoculation of AM fungi (M1 or M2) and/or Aphelenchoides sp. on As concentrations and contents of tobacco plants under increasing soil As levels. (L, soil in low As level; M, soil in medium As level; H, soil in high As level; control, non-inoculated with AM fungi or Aphelenchoides sp.; M1, inoculated with non-indigenous AM fungi Glomus caledonium; M2, inoculated with indigenous AM fungi Glomus spp. and Acaulospora spp.; Nem addition of Aphelenchoides sp. Bars represent standard errors. Different letters represent significant difference according to Duncan’s multiple range test at 5% level, n = 3.)
on the development of mycorrhiza, As uptake and growth of tobacco (Table 2). Soil As level (P = 0.002), nematode addition (P = 0.042) and their interaction (P < 0.0001) had significant effects on the height of tobacco plants (Table 2). Addition of Aphelenchoides sp. increased the height of mycorrhizal plants only in L soil. The most enhancing effect of these dual treatments was observed in M1 + Nem inoculated plants which increased 17 cm over control plants (Table 3). Root dry weight (P = 0.008) was influenced by soil As level. Decreasing plant height and dry weights were found with the increase of As concentration (Table 3). Obviously, high soil As levels inhibited tobacco growth. In addition, significant interaction effects between As and nematodes, and between As and AMF isolates were found on shoot (P < 0.0001, P = 0.035) and root (P < 0.0001, P = 0.019) dry weights (Table 2). Root dry weights of dually M1 + Nem inoculated plants were increased by 39% and 48% over control plants and M1 single inoculated mycorrhizal plants, respectively in L soil. Compared with control plants, shoot dry weights of all inoculated plants were increased in L soil. The greater positive effect was observed in the dual inoculated plants,
with shoot dry weights being 1.66 (M1 + Nem treatment) and 1.63 (M2 + Nem treatment) fold higher than that of control plants. In fact, tobacco plants better developed in L soil after co-inoculation of AM fungi and Aphelenchoides sp. (Table 3). 3.2. As concentrations and contents of tobacco plants Both soil As level and AMF isolate had large impacts on the As concentrations in tobacco plants (root, P < 0.0001, P = 0.023; shoot, P < 0.0001, P < 0.0001). In H soil, plants accumulated higher As concentrations irrespective of the inoculation of AM fungi and/or Aphelenchoides sp. nematodes (P = 0.003) and interaction effects between As and AMF isolate (P = 0.003) and between the three factors (P = 0.004) were observed for As concentrations in root. As contents in the roots were only affected by the interaction between As and nematodes (P = 0.011) and between As, AMF isolate and nematodes (P = 0.042), however, As contents in shoot were influenced by As (P < 0.0001), nematodes (P = 0.005), AMF isolate (P < 0.0001) as well as the interactions between As and nematodes (P < 0.0001), between As and AMF isolate (P = 0.014) and between nematodes and AMF isolate (P = 0.048) (Table 2).
J. Hua et al. / Applied Soil Ecology 84 (2014) 176–184
Soil As level (P = 0.022), AMF isolate (P = 0.024) and their interaction (P = 0.019) had significant effects on the concentrations of NH4+-N. However, concentrations of (NO3 + NO2)-N were only remarkably affected by the interaction effects between As and AMF isolate (P = 0.016) (Table 2). As shown in Table 3, nematode addition and AMF inoculation had no significant effects on the concentrations of (NO3 + NO2)-N in soils with higher As concentrations (M and H). At the lowest As level, the dual inoculation of M2 and Aphelenchoides sp. led to higher concentrations of (NO3 + NO2)-N (P = 0.025) than the other mycorrhizal treatments. Compared with the control, the inoculation of AM fungi (M1 or M2) and/or Aphelenchoides sp. did not effectively increase concentrations of NH4+-N irrespective of the As level in soils. 3.4. Development of nematodes and mycorrhiza Aphelenchoides sp. was absent or present at very low numbers in treatments without nematode addition, suggesting negligible background contamination, and thus, these data are not shown in Fig. 2. According to an ANCOVA using As level and AMF isolate as factors and soil Olsen P as covariate, the numbers of Aphelenchoides sp. were not influenced by covariate, but significantly affected by AMF isolate (P < 0.0001) and the interaction effects (P = 0.016). Following nematode addition to non-mycorrhizal treatments, the numbers of Aphelenchoides sp. increased to 3395, 3259 and 15,142 individuals per treatment in L, M and H soils, respectively. However, final number of Aphelenchoides sp. in dual inoculation treatments exceeded 38,000 individuals per treatment, and no
8
a
7
a
4
3.3. Soil mineral N
significant difference was found between M1 + Nem and M2 + Nem treatments in all tested soils. Obviously, growth of Aphelenchoides sp. was stimulated by AM fungi inoculation irrespective of the soil As level, with 12.1 and 13.9 times increase, respectively in the L soil, 20.0 and 12.5 times raise, respectively in the M soil, and 2.5 and 3.3 fold increase, respectively in the H soil (Fig. 2). Noticeable effects of As (P < 0.0001), AMF isolate (P < 0.0001) and their interaction (P = 0.009) were observed on the mycorrhizal colonization rate, while Aphelenchoides sp. had no significant effects according to an ANCOVA using Olsen P as covariate (Table 2). AM fungi M1 reached much greater AM colonization levels than M2 in L and H soils (L, P = 0.010; H, P = 0.010) both following single inoculation and co-inoculation with Aphelenchoides sp. (Fig. 3). The length of extraradical mycelium (ERM) in soil was markedly influenced by nematode addition (P = 0.009) and the interaction between each tested factor (As nematode, P = 0.012; As AMF isolate, P = 0.004; nematode AMF isolate, P = 0.003) (Table 2). With the increase of As exposure level, the ERM length showed an increasing trend. Compared with M2, M1 produced greater ERM length in M soil (P = 0.002) in the absence of Aphelenchoides sp., while an opposite trend was found in H soil (P = 0.032) in the presence of Aphelenchoides sp. Inoculation with Aphelenchoides sp. increased the ERM length of M2 by 49% in L soil and by 50% in M soil over the single inoculated mycorrhizal treatments (Fig. 3). As shown in Table 2, total numbers of AM fungal spores were significantly affected by all single and interaction effects except for the AMF isolate factor (As, P < 0.0001; nematode, P < 0.0001; As nematode, P < 0.0001; As AMF isolate, P < 0.0001; nematode AMF isolate, P = 0.002; As nematode AMF isolate, P < 0.0001). In the presence of Aphelenchoides sp., M1 produced more spores (P = 0.032) than M2 in L soil, while an opposite trend (P = 0.001) was observed in H soil. Regardless of the nematode addition, total numbers of spores were higher (P = 0.011) in M2 inoculated treatments than in M1 inoculated treatments in M soil. Total numbers of spores were differently affected by nematode addition according to As concentration in the soil. At the lowest As level, Aphelenchoides sp. increased spore numbers of M1 by 205% but did not affect that of M2. In contrast, at the highest As level,
-1
In general, As concentrations in shoot were increased by M2 but not affected by M1 both when inoculated single or dually with Aphelenchoides sp. Comparing the increasing effect of the two treatments involving M2 at different As levels, we can see that shoot As concentrations were 1.14 and 1.30 (L soil), 1.24 and 1.34 (M soil), 1.33 and 1.22 (H soil) fold higher than in control plants (Fig. 1). Inoculation with Aphelenchoides sp. had no effect on shoot As concentrations of mycorrhizal plants in M and H soils. In L soil, however, the co-inoculation of Aphelenchoides sp. into the two mycorrhizal treatments increased shoot As concentration by 16% over the M2 single inoculated treatment, but decreased it by 14% over the M1 single inoculated treatment. The effects of AM fungi and/or Aphelenchoides sp. on As contents in shoot varied depending on the As concentration in soil. At the highest As exposure level, AM fungi, in single inoculation or coinoculated with Aphelenchoides sp. was not effective in influencing the shoot As contents, but at the two lower As levels M2 increased such value by 15% (L soil) and 23% (M soil). Moreover, the positive effect was not affected by the dual addition of Aphelenchoides sp. in M soil, and was even more stimulated by 16% in L soil. Also, the coinoculation of M1 and Aphelenchoides sp. in L soil significantly enhanced shoot As contents when compared to the control and M1 single inoculated plants (Fig. 1). Both concentrations and contents of As in root were differently affected by the inoculation treatments in terms of the soil As levels. They showed a same trend at each As level (except at the high As concentration). In L soil, no differences in As concentrations and contents of As in roots were found among the tested treatments. In M soil, mycorrhizal plants had much lower concentrations and contents of As in roots compared with control plants, and Aphelenchoides sp. did not influence this effect. In H soil, As concentrations in roots were increased by the single inoculation wtih AM fungi, while this effect was neutralized by Aphelenchoides sp. In the case of As contents, no differences were observed among the tested treatments (Fig. 1).
Individuals treatment (×10 )
180
6
a
a a
5
a
4 3 b 2 1
b
b
0 M Soils
L Nem
M1+Nem
H M2+Nem
Fig. 2. Numbers of Aphelenchoides sp. per treatment in nematode addition treatments. (L, soil in low As level; M, soil in medium As level; H, soil in high As level; M1, inoculated with non-indigenous AM fungi Glomus caledonium; M2, inoculated with indigenous AM fungi Glomus spp. and Acaulospora spp.; Nem addition of Aphelenchoides sp. Bars represent standard errors and different letters indicate significant differences at the same soil by Duncan’s multiple range test at 5% level, n = 3. Significance (F-value) of an ANCOVA using soil Olsen P as covariate, soil Olsen P: n.s. (1.79); As: n.s. (1.72); AMF isolate: 0.000 (63.51); As AMF isolate: 0.016 (4.13).)
J. Hua et al. / Applied Soil Ecology 84 (2014) 176–184
181
35 a
AM colonization (%)
30 25
a a
20
a
15 a b
b
10
b
a
b
a
5
a
0 L
M
H
0.9 a a
-1
ERM (m g )
0.7 0.6
a
a
0.8 a
ab b
a
a
ab
0.5
b b
0.4 0.3 0.2 0.1 0.0 L
M
H
800 a 700
-1
Spores (100 g )
600 500 400
a
300 200 b 100
a
a
b
b
b
b
b b
b
0 L
M
H
Soils M1
M2
M1+Nem
M2+Nem
Fig. 3. Effects of the inoculation of AM fungi (M1 or M2) and/or Aphelenchoides sp. on the development of mycorrhiza under increasing soil As levels. (L, soil in low As level; M, soil in medium As level; H, soil in high As level; control, non-inoculated with AM fungi or Aphelenchoides sp.; M1, inoculated with non-indigenous AM fungi Glomus caledonium; M2, inoculated with indigenous AM fungi Glomus spp. and Acaulospora spp.; Nem addition of Aphelenchoides sp. Bars represent standard errors. Different letters represent significant difference according to Duncan’s multiple range test at 5% level, n = 3.)
spores numbers of M2 were 5.60 fold increased by Aphelenchoides sp., while that of M1 were not affected (Fig. 3). 4. Discussion Artificially polluted soil creates an artificial environment where bound and free metals/metalloids are not in equilibrium (Heggo et al., 1990), and the effects of metals/metalloids on the tested organisms would be overestimated (Chaney et al., 1978). Instead of adding metals/metalloids as soluble salt, a more representative approach is using contaminated soil sampled near an arsenic sulphide mine as chosen in our study. Ineluctably, with different soil As levels also other chemical properties changed, especially the P concentrations. It is generally assumed that mycorrhizal symbiosis and plant growth may be affected by the availability of soil P (Lin et al., 2012), especially in soils with high As concentrations due to the interlink between As and P (Bai et al., 2008). Consequently, an ANCOVA of tested parameters using soil
Olsen P as covariate was conducted to work out the specific effect of As level, nematode, AM fungi and their interactions independent of soil Olsen P on As extraction and growth of tobacco. As shown in Table 2 and Fig. 2, the covariate soil Olsen P did not significantly affect the development of mycorrhiza and nematodes, growth and As uptake of tobacco plants in the present study. The results of this experiment provide insights into the interactions between Aphelenchoides sp. and AM fungi and the consequent effects on As uptake and growth of tobacco plants. 4.1. Interaction effects between AM fungi and nematodes on plants growth and As uptake AM fungi are one of the most abundant inhabitants of belowground ecosystems. They are able to promote the host plant growth, since in AM fungal symbiosis, the fungus receives C from the host and meanwhile, the host obtains nutrients (especially P) via the fungus. Nematodes are the most abundant metazoans in
182
J. Hua et al. / Applied Soil Ecology 84 (2014) 176–184
soil. Some of them feeding on fungi or bacteria occupy irreplaceable positions in the soil food web (Bernard, 1992). They play important roles in the decomposition of soil organic matter, mineralization of plant nutrients and nutrient cycling (Ingham et al., 1985; Mao et al., 2007). Many experiments have provided strong evidences that bacterivorous nematodes could improve the plant growth due to the increased N, mostly as ammonia, since the C:N ratio in the bacterial biomass (approximately 5:1) is lower compared with that of nematodes (approximately 10:1) (Anderson et al., 1980; Ferris et al., 1998). In this experiment, the height and dry weights of tobacco plants were unaffected or increased by M1 or M2 isolates in L soil. Nevertheless, the mycorrhizal effect improving plant growth was significantly enhanced by the presence of Aphelenchoides sp. This phenomenon could be explained by the feeding activities of Aphelenchoides sp. on AM fungi. In fact, the C:N ratio of fungivorous nematodes is similar to that of fungal mycelium (Ingham et al., 1985). As a result, there is no excess N associated with fungal tissues assimilated for nematode biomass production as happens when bacterivores feed on bacteria. However, a considerable proportion of the C consumed is used in respiration, about 40% of the food intake (Ingham et al., 1985). The N associated with respired C that is in excess of structural needs is also excreted. As reported by Coleman (1986), the fungivorous nematode Aphelenchus avenae could obtain 6.1 ng N through fungal mycelium per day, regardless of the amount of 1.45 ng N in the body of nematodes. Consequently, most of the N was released to soil. Likewise, Chen and Ferris (1997) found that the amount of NH4+ released from the microcosms in which barley straw was colonized by Rhizoctonia sp. was always greater when A. avenae was added. Further, nematodes could deposit feces enriched with organic material into the soil (Chen and Ferris, 2000). Apparently, the activities of nematodes improved available nutrition in soil solution which could be easily absorbed by both plants and microbes. For example, increased length of ERM of which could extend the absorption area of roots was observed in the present investigation. Similar results have been obtained in experiments involving fungivorous Collembola (Hanlon, 1981). This effect was also beneficial to plant growth. Surprisingly, except for the M2 + Nem treatment in L soil, no differences in mineral N contents were found between non-nematode and nematode addition treatments. The strong N absorption by plants and AM fungi may partly explain this result. Besides the mycorrhizal effect on plant growth, the role of AM fungi in As uptake by plants has been well documented since As is transported across the plasma membrane via P transport systems (Ullrich-Eberius et al., 1989). In most cases, As concentrations in shoots were increased by indigenous AMF isolate M2 but not affected by non-indigenous AMF isolate M1 regardless of the addition of Aphelenchoides sp. Similarly, Bai et al. (2008) found that mycorrhizal effects on As uptake in maize grown in the same polluted soil were different between M1 and M2 isolates. The probable explanation, as stated by Wang et al. (2005) and Weissenhorn et al. (1994), is that AMF isolates from contaminated soils have great efficacy, but those from unpolluted soils have high sensitivity and are not adapted to the stress of contaminated soils. Nevertheless, few experiments were focused on the interaction effects between AM fungi and mycophagous animals on metal uptake in plants. As reported by Yu et al. (2005), dual inoculation with earthworms (Pheretima sp.) and AM fungi (Glomus mosseae and Glomus intraradices) increased shoot Cd accumulation of ryegrass (Lolium multiflorum) at low soil Cd concentrations. Our study also found that the addition of Aphelenchoides sp. substantially increased shoot As concentrations of M2 mycorrhizal plants, but decreased it of M1 mycorrhizal plants in L soil. This is the first report to show that fungivorous nematodes had significant effects
on As uptake by mycorrhizal plants grown in an As polluted soil. Like the bacterial isolate Brevibacillus sp., whose dual inoculation with G. mosseae achieved further plant (T. repens) growth and lower Cd concentrations (Vivas et al., 2003), Aphelenchoides sp. can act by direct or indirect mechanisms on the AM fungus as myzorrhizae helping organisms, enhancing the “mycorrhizal effect” on plant As uptake. Furthermore, the co-inoculation of AM fungi and Aphelenchoides sp. significantly increased the shoot As contents when compared to the control and M1 and M2 single inoculated plants at L soil. This finding was consistent with the result of shoot dry weights. Obviously, the positive effect of Aphelenchoides sp. on the As accumulation in mycorrhizal plants could be mainly linked to the plants biomass resulting from the development of mycorrhiza and variation of soil nutrition that was induced in turn by the grazing activities of Aphelenchoides sp. on AM fungi. 4.2. Interaction effects between AM fungi and nematodes on their own development Numbers of Aphelenchoides sp. were considerably higher in mycorrhizal pots than in non-mycorrhizal pots. Also, numbers of Aphelenchoides sp. significantly exceeded the initial inoculum number in mycorrhizal pots. Our previous field experiment also found that the abundance of Aphelenchoides sp. was significantly increased in the M2 inoculated treatment than in the noninoculated treatment in an As contaminated soil (Hua et al., 2010). Obviously, this is an indication of the suitability of both M1 and M2 as good food sources for Aphelenchoides sp. Likewise, Bakhtiar et al. (2001) reported that the fungivorous nematodes A. avenae Bastian could multiply by feeding on the AM fungi Gigaspora margarita and Glomus coronatum grown with clover (T. subterraneum L.) as evidenced by an increased population in mycorrhizal treatments but a reduced number (below the initial density value) in nonmycorrhizal treatments. In fact, Hussey and Roncadori (1981) have noted that AM fungi should be excellent hosts for mycophagous nematodes due to the production of extraradical mycelium. Recently, Brussaard et al. (2001) using petri dishes observed that Aphelenchoides sp. was feeding and reproducing on all tested ectomycorrhizal fungi. Likewise, Ruess et al. (2000) studied the growth of Aphelenchoides sp. populations in vitro on 17 different fungal species, and found that nematode populations developed on saprophytic (Agrocybe and Chaetomium) and especially on mycorrhizal fungi (Cenococcum, Hymenoscyphus and Laccaria). As we known, pure culture of AM fungi is still a big challenge for microbiologists, and thus the direct evidence that fungivorous nematodes could feed on AM fungi is undetected now. Furthermore, an experiment using a transparent microcosm, like a petri dish filled with sepharose gel, with a plant or a Ri-T-DNA transformed root (Diop, 2003) infected by AM fungi, should be conducted to find the direct proof that Aphelenchoides sp. is feeding and reproducing on AM fungi by a microscope. On the other hand, the grazing activity of nematodes on AM fungi, including mycelial network and spores may be of significance in affecting the inoculum production and therefore the root colonization. To date, positive, neutral and negative effects were observed (Bakhtiar et al., 2001). In this experiment, the stimulative effects of Aphelenchoides sp. on the development of AM fungi were slightly different between two tested AM fungi. The ERM length of M2 was remarkably enhanced by Aphelenchoides sp. in L and M soils. The total number of spores of M1 in L soil and that of M2 in H soil was increased by the nematodes. The development of AM fungi may be partly explained by the grazing of Aphelenchoides on AM fungi which could recycle minerals locked up in senescent fungal tissue. Interestingly, Aphelenchoides sp. did not affect root colonization in the present experiment. However,
J. Hua et al. / Applied Soil Ecology 84 (2014) 176–184
Bakhtiar et al. (2001) observed that the mycorrhizal colonization of clover roots was decreased as the population of fungivorous nematode A. avenae Bastian increased in mycorrhizal pots. This result might be due to the declined percentage of spores, because the spores with high lipid contents could be penetrated by nematodes to provide high-energy food sources for them (Bakhtiar et al., 2001). The lack of adverse effects in this study agreed with the results of Hussey and Roncadori (1981) and Giannakis and Sanders (1990), who found no effects of Aphelenchus on mycorrhizal infection and spores production. Actually, the effects depended on the relationship between nematodes and AM fungi, such as the differences in accessibility or palatability of the spores or hyphae to different nematodes. For example, the stylet of Aphelenchoides is shorter (<8 mm) (http://plpnemweb.ucdavis. edu/Nemaplex/Taxadata/G011.htm) than that of Aphelenchus (15 mm) (Hooper, 1974), and not all of this length is available to penetrate the spores. Thus, at some sense, the access to spores would be limited. Besides, the starting numbers of nematodes applied would significantly influence the results, which has been thoroughly discussed by Bakhtiar et al. (2001). Moreover, unlike other studies, contaminated soils were used in our experiment. Considering the fact that soil pollution significantly influenced the nematode communities (Li et al., 2006) and AM fungi development (Bai et al., 2008; Wu et al., 2009), we have sound reasons to believe that soil contamination would influence the interaction between AM fungi and fungivorous nematodes. In conclusion, Aphelenchoides sp. could feed and reproduce on the tested AM fungi, and the mycorrhiza was developed in turn by the grazing activities. The interaction of AM fungi and fungivorous nematodes has potential to elevate the phytoextraction efficiency in low level As polluted soils. The isolation of tolerant beneficial organisms from contaminated soils may be an ideal way to achieve soil decontamination goals. Moreover, the mechanisms by which AM fungi and mycophagous animals enhance metal uptake by the plants would allow the management of soil organisms with suitable characteristics to be used for bioremediation purposes. The knowledge of basic interactions between host plant–AM fungi–mycophagous animals in other metal polluted soils needs further studies. Acknowledgments This work is jointly supported by the National Natural Science Foundation of China (No. 41101232), the Natural Science Foundation of Jiangsu Province of China (BK2011688) and the Research Fund of State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Science (Y412201436). We also thank two anonymous reviewers for their critical comments which greatly improved the quality of the manuscript. References Anderson, R.V., Cole, C.V., Coleman, D.C., 1980. Quantities of plant nutrients in the microbial biomass of selected soils. Soil Sci. 130, 211–216. Bai, J.F., Lin, X.G., Yin, R., Zhang, H.Y., Wang, J.H., Chen, X.M., Luo, Y.M., 2008. The influence of arbuscular mycorrhizal fungi on As and P uptake by maize (Zea mays L.) from As-contaminated soils. Appl. Soil Ecol. 38, 137–145. Bakhtiar, Y., Miller, D., Cavagnaro, T., Smith, S., 2001. Interactions between two arbuscular mycorrhizal fungi and fungivorous nematodes and control of the nematode with fenamifos. Appl. Soil Ecol. 17, 107–117. Bernard, E.C., 1992. Soil nematode biodiversity. Biol. Fertil. Soils 14, 99–103. Brussaard, L., Kuyper, T.W., de Goede, R.G.M., 2001. On the relationships between nematodes, mycorrhizal fungi and plants: functional composition of species and plant performance. Plant Soil 232, 155–165. Chaney, R.L., Hundemann, P.T., Palmer, W.T., Small, R.J., White, M.C., Decker, A.M., 1978. Plant accumulation of heavy metals and phytotoxicity resulting from utilization of sewage sludge and sludge composts on cropland. Proceedings of the National Conference of Composting of Municipal Residues and Sludges. Information Transfer Inc., Rockville, MD, pp. 86–97.
183
Chen, J.A., Ferris, H., 1997. Nitrogen mineralization by Aphelenchus avenae associated with Rhizoctonia sp. and barley straw. J. Nematol. 29 (4), 572. Chen, J., Ferris, H., 2000. Growth and nitrogen mineralization of selected fungi and fungal-feeding nematodes on sand amended with organic matter. Plant Soil 218, 91–101. Coleman, D.C., 1986. The role of microfloral and faunal interactions in affecting soil processes. In: Mitchell, M.J., Nakas, J.P. (Eds.), Micronoral and Fauna Interaction in Natural and Agro-ecosystems. Martinos N. Junk W. Publishers, Dordrecht, pp. 317–348. Diop, T.A., 2003. In vitro culture of arbuscular mycorrhizal fungi: advances and future prospects. Afr. J. Biotechnol. 2 (12), 692–697. Durán, P., Acuña, J.J., Jorquera, M.A., Azcón, R., Borie, F., Cornejo, P., Mora, M.L., 2013. Enhanced selenium content in wheat grain by co-inoculation of selenobacteria and arbuscular mycorrhizal fungi: a preliminary study as a potential Se biofortification strategy. J. Cereal Sci. 57 (3), 275–280. Ferris, H., Venette, R.C., van der Meulen, H.R., Lau, S.S., 1998. Nitrogen mineralization by bacterial-feeding nematodes: verification and measurement. Plant Soil 203, 159–171. Fitter, A.H., Garbaye, J., 1994. Interaction between mycorrhizal fungi and other soil organisms. Plant Soil 159, 123–132. Gaur, A., Adholeya, A., 2004. Prospects of arbuscular mycorrhizal fungi in phytoremediation of heavy metal contaminated soils. Curr. Sci. 86 (4), 527–534. Giannakis, N., Sanders, F.E., 1990. Interactions between mycophagous nematodes, mycorrhizal and other soil fungi. Agric. Ecosyst. Environ. 29, 163–167. Giovannetti, M., Mosse, B., 1980. An evaluation of techniques for measuring vesicular arbuscular mycorrhizal infection in roots. New Phytol. 84, 489–500. Hanlon, R.D.G., 1981. Influence of grazing by Collembola on the activity of senescent fungal colonies grown on medium of different nutrient concentrations. Oikos 36, 363–367. Heggo, A., Angle, J.S., Chaney, R.L., 1990. Effects of vesiculararbuscular mycorrhizal fungi on heavy metal uptake by soybeans. Soil Biol. Biochem. 22, 865–869. Hildebrandt, U., Regvar, M., Bothe, H., 2007. Arbuscular mycorrhiza and heavy metal tolerance. Phytochemistry 68 (1), 139–146. Hooper, D.J., 1974. Aphelenchus avenae. In: Willmott, S., Gooch, P.S., Siddiqi, M.R., Franklin, M.T. (Eds.), C.I.H. Descriptions of Plant–Parasitic Nematodes. Commonwealth Agricultral Bureaux, Farnham Royal, Slough, UK Set 4, No. 50. Hua, J.F., Lin, X.G., Yin, R., Jiang, Q., 2009. Effects of arsenic contamination on structure of soil nematode community in mining area. J. Ecol. Rural Environ. 25, 79–84 (in Chinese). Hua, J.F., Lin, X.G., Bai, J.F., Shao, Y.F., Yin, R., Jiang, Q., 2010. Effects of arbuscular mycorrhizal fungi and earthworm on nematode communities and arsenic uptake by plants in arsenic-contaminated soils. Pedosphere 20, 163–173. Hussey, R.S., Roncadori, R.W., 1981. Influence of Aphelenchus avenae on vesicular– arbuscular endomycorrhizal growth response in cotton. J. Nematol. 13, 48–52. Ingham, R.E., Trofymow, J.A., Ingham, E.R., Coleman, D.C., 1985. Interactions of bacteria, fungi, and their nematode grazers: effects on nutrient cycling and plant growth. Ecol. Monogr. 55, 119–140. Jakobsen, I., Abbott, L.K., Robson, A.D., 1992. External hyphae of vesicular– arbuscular mycorrhizal fungi associated with Trifolium subterraneum L. 1. Spread of hyphae and phosphorus inflow into roots. New Phytol. 120, 371–380. Koller, R., Rodriguez, A., Robin, C., Scheu, S., Bonkowski, M., 2013a. Protozoa enhance foraging efficiency of arbuscular mycorrhizal fungi for mineral nitrogen from organic matter in soil to the benefit of host plants. New Phytol. 199 (1), 203–211. Koller, R., Scheu, S., Bonkowski, M., Robin, C., 2013b. Protozoa stimulate N uptake and growth of arbuscular mycorrhizal plants. Soil Biol. Biochem. 65, 204–210. Koske, R.E., Gemma, J.N., 1989. A modified procedure for staining roots to detect VA mycorrhizas. Mycol. Res. 92, 486–505. Larsen, J., Cornejo, P., Barea, J.M., 2009. Interactions between the arbuscular mycorrhizal fungus Glomus intraradices and the plant growth promoting rhizobacteria Paenibacillus polymyxa and P. Macerans in the mycorrhizosphere of Cucumis sativus. Soil Biol. Biochem. 41 (2), 286–292. Li, Q., Jiang, Y., Liang, W.J., 2006. Effect of heavy metals on soil nematode communities in the vicinity of a aetallurgical factory. J. Environ. Sci. 18 (2), 323–328. Li, H., Man, Y.B., Ye, Z.H., Wu, C., Wu, S.C., Wong, M.H., 2013. Do arbuscular mycorrhizal fungi affect arsenic accumulation and speciation in rice with different radial oxygen loss? J. Hazard. Mater. 262, 1098–1104. Lin, X.G., Feng, Y.Z., Zhang, H.Y., Chen, R.R., Wang, J.H., Zhang, J.B., Chu, H.Y., 2012. Long-term balanced fertilization decreases arbuscular mycorrhizal fungal diversity in an arable soil in North China revealed by 454 pyrosequencing. Environ. Sci. Technol. 46, 5764–5771. Mao, X.F., Hu, F., Griffiths, B., Chen, X.Y., Liu, M.Q., Li, H.X., 2007. Do bacterial-feeding nematodes stimulate root proliferation through hormonal effects? Soil Biol. Biochem. 39, 1816–1819. Oostenbrink, M., 1960. Estimating nematode populations by some selected methods. Nematology 6, 85–102. Orlowska, E., Godzik, B., Turnau, K., 2012. Effect of different arbuscular mycorrhizal fungal isolates on growth and arsenic accumulation in Plantago lanceolata L. Environ. Pollut. 168, 121–130. Ruess, L., Garcia Zapata, E.J., Dighton, J., 2000. Food preferences of a fungal-feeding Aphelenchoides species. Nematology 2, 223–230. Schenck, N.C., Perez, Y., 1990. Manual for Identification of Vesicular Arbuscular Mycorrhizal Fungi, third ed. Synergistic Publication, University of Florida, Gainesville, Florida, USA, pp. 286. Siddiky, M.R.K., Schaller, J., Caruso, T., Rillig, M.C., 2012. Arbuscular mycorrhizal fungi and collembola non-additively increase soil aggregation. Soil Biol. Biochem. 47, 93–99.
184
J. Hua et al. / Applied Soil Ecology 84 (2014) 176–184
Smith, S.E., Read, D.J., 2008. Mycorrhizal Symbiosis, third ed. Academic Press and Elsevier, London. Townshend, J.L., 1963. A modification and evaluation of the apparatus for the Oostenbrink direct filter extraction method. Nematologica 9, 106–110. Ullrich-Eberius, C.I., Sanz, A., Novacky, A.J., 1989. Evaluation of arsenate and vanadate-associated changes of electrical membrane potential and phosphate transport in Lemna gibba G1. J. Exp. Bot. 40, 119–128. Vivas, A., Vörös, I., Biró, B., Campos, E., Barea, J.M., Azcón, R., 2003. Symbiotic efficiency of autochthonous arbuscular mycorrhizal fungus (G. mosseae) and Brevibacillus sp. isolated from cadmium polluted soil under increasing cadmium levels. Environ. Pollut. 126, 179–189.
Wang, F.Y., Lin, X.G., Yin, R., 2005. Heavy metal uptake by arbuscular mycorrhizas of Elsholtzia splendens and the potential for phytoremediation of contaminated soil. Plant Soil 269, 225–232. Weissenhorn, I., Glasho, A., Leyval, C., Berthelin, J., 1994. Differential tolerance to Cd and Zn of arbuscular mycorrhizal (AM) fungal spores isolated from heavy metalpolluted and unpolluted soils. Plant Soil 167, 189–196. Wu, F.Y., Ye, Z.H., Wong, M.H., 2009. Intraspecific differences of arbuscular mycorrhizal fungi in their impacts on arsenic accumulation by Pteris vittata L. Chemosphere 76, 1258–1264. Yu, X.Z., Cheng, J.M., Wong, M.H., 2005. Earthworm–mycorrhiza interaction on Cd uptake and growth of ryegrass. Soil Biol. Biochem. 37, 195–201.