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Arbuscular mycorrhizal fungal species composition, propagule density, and soil alkaline phosphatase activity in response to continuous and alternate no-tillage in Northern China
Catena 133 (2015) 215–220
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Arbuscular mycorrhizal fungal species composition, propagule density, and soil alkaline phosphatase activity in response to continuous and alternate no-tillage in Northern China Junli Hu a,c,1, Anna Yang b,1, Junhua Wang a,c, Anning Zhu a, Jue Dai a,c, Ming Hung Wong c,d, Xiangui Lin a,c,⁎ a
State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, East Beijing Road 71, Nanjing 210008, PR China Provincial Key Laboratory of Biotic Environment and Ecological Security in Anhui, College of Life Sciences, Anhui Normal University, East Beijing Road 1, Wuhu 241000, PR China c Joint Open Laboratory of Soil and the Environment, Hong Kong Baptist University & Institute of Soil Science, Chinese Academy of Sciences, East Beijing Road 71, Nanjing 210008, PR China d Croucher Institute for Environmental Sciences, Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, PR China b
a r t i c l e
i n f o
Article history: Received 14 January 2015 Received in revised form 19 May 2015 Accepted 22 May 2015 Available online xxxx Keywords: Diversity External mycelium Organic C Relative abundance Species richness Spore density
a b s t r a c t Purpose: Arbuscular mycorrhizal (AM) fungi provide a direct link between soil and roots, and are renowned for their ability to increase nutrient phytoavailability, notably phosphorus (P). The objective of this study was to evaluate influences of continuous and alternate no-tillage on soil AM fungal species composition and soil Psupply parameters. Materials and methods: In June 2006, a long-term field experiment was established in a sandy loam soil under the rotation of summer maize (Zea mays L.) and winter wheat (Triticum aestivum L.) in Northern China, including conventional tillage (CT), no-tillage (NT) and alternate tillage (AT — tillage in the wheat season and no-tillage in the maize season). Top soil samples (0–15 cm) from four individual plots per treatment were collected before maize harvest on 18 September 2010. Soil AM fungal spores were isolated and identified, and the external mycelium length, soil alkaline phosphatase (ALP) activity and soil P content were also determined. Results and discussion: Thirty species of AM fungi within seven genera, Acaulospora, Claroideoglomus, Funneliformis, Glomus, Racocetra, Rhizophagus, and Scutellospora, were recovered. Some species sporulated differentially across the three treatments, and the lowest Jaccard index (J) of similarity in species composition was recorded between NT and CT (J = 0.767), but there were no significant differences in soil total P and available P contents, as well as in AM fungal spore density (SD), species richness (SR) and diversity indices, including Shannon–Wiener index (H′), Evenness (E) and Simpson's index (D). Compared with CT, NT rather than AT significantly increased (P b 0.05) the external mycelium length, soil ALP activity, and soil organic C content. Conclusions: Our results demonstrated the vital role of NT in maintaining external hyphae growth and soil ALP activity rather than in promoting AM fungal species diversity and spore density, and suggested that 4-year continuous NT will not cause degradation in either AM fungal community or soil P-supply efficiency. © 2015 Published by Elsevier B.V.
1. Introduction Soil microorganisms play a key role in biogeochemical cycles, and are responsible for a vast number of terrestrial functions (Garbeva et al., 2004). For instance, arbuscular mycorrhizal (AM) fungi, as ubiquitous mutualists which provide a direct link between soil and roots, and also as integral components of terrestrial ecosystems (Rillig, 2004), are renowned for their ability to increase plant growth and nutrient acquisition, notably phosphorus (P) (Hu et al., 2014; Smith and Read, 2008). Besides direct contribution of external hyphae to the transport reported for some elements (Rufyikiri et al., 2003), the enhancement ⁎ Corresponding author at: State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, PR China. E-mail address: [email protected] (X. Lin). 1 Joint first author with equal contribution.
http://dx.doi.org/10.1016/j.catena.2015.05.023 0341-8162/© 2015 Published by Elsevier B.V.
of soil enzymatic activities is another major mechanism involved in “mycorrhization effect” on plant nutrition, since AM propagules themselves can synthesize such enzymes, and mycorrhizal roots may release more root exudates containing enzymes because of the larger root system and/or improved nutrition (Wang et al., 2006). The extraradical hyphae of AM fungi also contribute to the formation and maintenance of soil aggregates (Bedini et al., 2009; Rillig et al., 2010) and in turn keep stored soil carbon (C) from escaping (Rillig et al., 2001; Wright and Upadhyaya, 1998). However, AM fungi are sensitive to changes in land-use patterns and management practices (Martinez and Johnson, 2010; Oehl et al., 2010). Therefore, there is an urgent concern about responsible maintenance of AM fungi in cultivated soils for sustainable management (Dai et al., 2013). The North China Plain (NCP) is a highly grain-producing area. The sustainable utilization of agricultural soil in this area may affect China's food security (Gong et al., 2009). In order to reduce diesel fuel
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usage and soil erosion, modern agriculture has greatly reduced the use of tillage relative to conventional agriculture. With the least disturbance of soil and consequent beneficial impacts on crop productivity, notillage (NT) seems to be superior for increasing C deposits into the soil (Alvear et al., 2005) and conserving soil and water (Blanco-Canqui and Lal, 2008). However, NT practice may also affect soil characteristics and microbial properties (Hu et al., 2013; van Groenigen et al., 2010). For instance, continuous NT may result in problems such as surface hardening of soil, leading to a more limited O2 supply for soil microorganisms (Álvaro-Fuentes et al., 2008). It seems to be unfavorable for AM fungal propagule distribution. For example, Curaqueo et al. (2011) observed decreases in AM fungal propagules after a long-term program of NT. Such variance could in turn influence the growth of cultivated crops, suggesting that moderate tillage may be more beneficial than continuous NT. In other words, it is needed to determine the suitable period of NT for common agroecosystems. More field experiments are thus necessary to improve our knowledge of the changes in AM fungal community with various tillage practices, and may help to ensure an opportunity for popularizing NT farm in arable soils. Because of concerns regarding soil quality degradation resulting from the replacement of conventional tillage (CT) with conservation tillage, a number of long-term field experiments were set up in agricultural regions to monitor changes in soil fertility and quality in China (He et al., 2011; Wang et al., 2008). However, uncertainties still remain about the influences of continuous and alternate NT on AM fungal parameters, including propagule composition and density. It was hypothesized that alternate NT would be more helpful than continuous NT in maintaining AM fungal species diversity, propagule growth, and related soil functions. In this study, a long-term field experiment, which was set up in a sandy loam soil in the NCP, was selected to determine soil AM fungi. The objectives of this study were to evaluate the influences of continuous and alternate NT practice on AM fungal propagules and soil P availabilities, and to find out the main factors that affect these parameters. This work investigated NT's superiority not only in AM fungal propagules themselves, but also in soil P-supply efficiency, and might contribute in developing soil microbial resource management schemes based on specific agronomic and economic demands.
2. Materials and methods 2.1. Description of the experimental site and soil sampling The tillage experiment was conducted in a well-drained field within the State Experimental Station for Agro-Ecology, Fengqiu County (35°00′N, 114°24′E), Henan Province, China. With a sandy loam texture, the soil is derived from alluvial sediments of the Yellow River and is classified as an Aquic Inceptisol. Maize (Zea mays L.) was sown in June and harvested in late September. Then wheat (Triticum aestivum L.) was sown in October and harvested in early June of the next year. In June 2006, three treatments, conventional tillage (CT, tillage in both wheat and maize seasons), no-tillage (NT, no-tillage in both wheat and maize seasons), and alternate tillage (AT, tillage in wheat season and no-tillage in maize season), were randomly arranged with four replicates (each plot 14 × 6.5 m). The CT plots were plowed with a moldboard to a depth of 20 cm. Later, the soil was disked twice, with a disk harrow before seed sowing. Both NT and AT plots were managed similarly to the CT plots, except for tillage which continued and alternated, respectively, to be NT with a no-tillage planter for seed sowing. Each plot received the same management every year since 2006. On 18 September 2010, before the harvesting of mature maize, soil samples were collected from 16 points per plot at a depth of 0–15 cm, and then mixed and homogenized by passing through a 2-mm mesh sieve to remove above-ground plant materials and stones. Soil samples were divided into two subsamples: one for soil enzyme activity determination and external mycelium extraction was stored at 4 °C, and
the other one for soil chemical analysis and AM fungal spore extraction was air-dried for about two weeks and then stored at 4 °C. 2.2. External mycelium length and soil chemical/enzymatic property analysis The external mycelium of AM fungi was extracted from soil samples using the method described by Malcová et al. (2002). Briefly, 5 g of soil was placed in a household blender containing 500 ml of water and blended for 30 s. Then, 1 ml of the resulting suspension was pipetted onto a membrane filter (24 mm diameter, 0.4 μm pore size) and vacuum filtered. The mycelium retained on the membrane filter was stained with a drop of 0.05% trypan blue in lactoglycerol, and total length of the mycelium was assessed using the grid-line intersect method (microscope equipped with focal plate grid 100× magnification). Soil pH was determined with a glass electrode using a soil-to-water ratio of 1:2.5. Soil organic C was determined by dichromate oxidation (Mebius, 1960). Soil total P was digested by HF-HClO4 (Jackson, 1958), while soil available P was extracted by sodium bicarbonate (Olsen et al., 1954), and then both of them were determined using the molybdenum blue method. Soil alkaline phosphatase (ALP) activity was determined by incubation at 37 °C with borate buffer (pH 9) according to Tabatabai (1982), and is given in units of mg pnitrophenol produced g−1 soil 24 h−1. All these results were expressed on an oven-dried soil weight basis by correcting for water content in the soil (105 °C, 24 h). 2.3. Recovery and counting of AM fungal spores The AM fungal spores were extracted from 20 g soil using the wetsieving method described by An et al. (1990), and were collected by 70, 100, 150 and 900 μm sieves, filtered onto a filter paper and placed in a Petri dish for examination under a binocular stereomicroscope. The spore density (SD) was calculated from direct counts of AM fungal spores (per 20 g air-dried soil). The intact healthy spores were sorted into groups and counted. The AM fungal spores were mounted in polyvinyl lactic acid (PVA) and PVA mixed 1:1 (v/v) with Meltzer's reagent (Morton, 1988; Morton and Benny, 1990) for species identification. The identification was based on morphological descriptions provided by the international collection of vesicular and arbuscular mycorrhizal fungi (http://invam.caf.wvu.edu), and recent advances in Glomeromycota taxonomy (Krüger et al., 2012; Oehl et al., 2011; Schüßler and Walker, 2010). The AM fungi species were identified using a light microscope (Olympus CX31). If the species is not yet described, it was recognized as an unknown species belonging to the most possible genus, and marked as Glomus-like sp. I, Scutellospora-like sp. II, etc. 2.4. AM fungal community structure and diversity analysis The structure of the AM fungal community was evaluated using the five selected parameters (Table 1) that have been widely employed in Table 1 Ecological diversity parameters for evaluating soil AM fungal community structure. Parameters
Formula and definition
Relative abundance (RA)
The percentage of the spore number of a species or a genus Species number per soil sample H′ = −∑Pi ln Pi E = H′ / H′max D = 1 − ∑[ni(ni − 1) / N(N − 1)] J = c / (a + b + c)
Species richness (SR) Shannon–Wiener index (H′) Evenness (E) Simpson's index (D) Jaccard index (J)
Pi = ni/N, where ni is the spore numbers of a species and N is the total number of identified spore samples. H′max = ln S, where S is the total number of identified species. J is a measure of the similarity of the spore community between treatments A and B, where a and b are the numbers of species occurring only at treatment A or treatment B, and c is the number of species occurring at both treatments.
J. Hu et al. / Catena 133 (2015) 215–220 Table 2 Soil properties in different tillage management treatments. Soil properties
Conventional tillage (CT)
No tillage (NT)
Alternating tillage (AT)
pH Organic C (g kg−1) Total P (g kg−1) Available P (mg kg−1)
8.57 ± 0.09 a 5.50 ± 0.59 b 0.97 ± 0.02 a 3.95 ± 0.77 a
8.55 ± 0.05 a 6.31 ± 0.17 a 1.02 ± 0.07 a 6.12 ± 1.57 a
8.54 ± 0.08 a 6.13 ± 0.21 ab 0.95 ± 0.05 a 5.68 ± 1.46 a
Data are mean values with standard deviation. Data within a row followed by different letters indicate a significant (P b 0.05) difference.
previous studies (Wang et al., 2011a; Yang et al., 2011). Spore specimens identified to species were used in the analysis of the relative abundance (RA) and the species richness (SR). Then, three AM fungal diversity indices, including Shannon–Wiener index (H′), Evenness (E), and Simpson's index (D), were evaluated as respectively sensitive indicators for the relative richness of species, the species evenness of the community, and the dominance of the most common species in the community. The Jaccard index (J) of similarity was calculated to compare AM fungal species composition under different treatments. 2.5. Statistical analyses The means and standard deviations of four replicates were computed. Analysis of variance (ANOVA) was carried out with SPSS software package (version 13.0). Significance of the parameters was tested using Least Significant Difference (LSD) multiple range test at P b 0.05
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after one-way ANOVA. To this end, the Pearson correlation coefficients were calculated among soil basic chemical properties, AM fungal community parameters (including SR, diversity indices, SD, and external mycelium length), and soil ALP activity. 3. Results 3.1. Soil pH, organic C, total P, and available P contents Soil pH and nutrient contents with different tillage management treatments are shown in Table 2. Compared with the CT treatment, the NT rather than the AT treatment significantly increased (P b 0.05) soil organic C content, but both NT and AT had no significant effects on soil pH, total P and available P contents. 3.2. AM fungal species composition and relative abundance (RA) A total of 30 species within seven genera, including three previously undescribed species, were recorded (Table 3). Acaulospora (35.1–38.2%), Scutellospora (15.2–23.7%, including Scutellospora-like sp. I and II), Funneliformis (16.9–20.9%), and Racocetra (10.3–18.5%) had relatively higher RAs than Claroideoglomus (6.7–6.9%), Rhizophagus (1.6–2.1%), and Glomus (0.6–1.2%, including Glomus-like sp. І). There were 22 species observed simultaneously in all the three treatments. The most abundant one was Acaulospora denticulata (21.9–25.1%), followed by Scutellospora reticulata (8.0–8.3%), Racocetra persica (6.3–8.7%), Funneliformis caledonium (4.6–6.5%), Claroideoglomus etunicatum (4.3–6.4%), Racocetra
Table 3 Species composition and relative abundance (%) of soil AM fungi in different treatments. AM fungal species Acaulospora (10 species) A. bireticulata A. denticulata A. elegans A. foveata A. lacunosa A. laevis A. mellea A. cavernata A. spinosa A. tuberculata Claroideoglomus (2 species) C. claroideum C. etunicatum Funneliformis (5 species) F. caledonium F. constrictum F. geosporum F. mossea F. verruculosum Glomus (4 species) G. aggregatum G. globiferum G. monosporum Glomus-like sp. I Racocetra (2 species) Ra. persica Ra. verrucosa Rhizophagus (2 species) Rh. clarus Rh. intraradices Scutellospora (5 species) S. heterogama S. nigra S. reticulata Scutellospora-like sp. I Scutellospora-like sp. II
Conventional tillage (CT)
No tillage (NT)
Alternating tillage (AT)
0.65 ± 0.75 a 21.86 ± 5.32 a 0.30 ± 0.60 a 0.59 ± 0.68 a 1.20 ± 1.70 a 1.47 ± 2.23 a 3.43 ± 1.60 a 1.88 ± 2.30 a 1.17 ± 1.66 a 2.54 ± 1.87 a
2.72 ± 2.38 a 24.31 ± 5.89 a 1.52 ± 1.54 a 0.32 ± 0.63 a 1.79 ± 2.06 a 0.33 ± 0.66 a 2.24 ± 1.95 a 0.63 ± 1.27 a 1.22 ± 1.00 a 1.56 ± 1.88 a
2.71 ± 1.47 a 25.09 ± 7.98 a 2.11 ± 1.49 a 0.92 ± 1.11 a 2.68 ± 0.50 a 0.27 ± 0.54 a 1.68 ± 1.47 a – 0.65 ± 0.76 a 2.04 ± 2.62 a
2.37 ± 1.68 a 4.30 ± 2.77 a
1.88 ± 1.67 a 4.99 ± 3.37 a
0.28 ± 0.56 a 6.39 ± 2.25 a
4.73 ± 3.77 a 0.29 ± 0.59 c 1.29 ± 1.50 7.17 ± 3.59 a 7.45 ± 3.43 a
6.49 ± 1.76 a 5.24 ± 1.02 a – 3.50 ± 2.44 a 1.64 ± 3.29 b
4.55 ± 2.58 a 3.32 ± 1.22 b – 7.66 ± 4.83 a 3.42 ± 2.49 ab
– – – 1.18 ± 1.66 a
0.32 ± 0.63 a 0.32 ± 0.63 0.30 ± 0.61 –
0.27 ± 0.54 a – – 0.37 ± 0.74 a
6.31 ± 7.36 a 4.02 ± 5.34 a
6.92 ± 4.36 a 7.96 ± 2.89 a
8.70 ± 4.67 a 9.80 ± 4.83 a
1.47 ± 2.94 a 0.59 ± 0.68 a
– 1.55 ± 0.60 a
0.28 ± 0.56 a 1.65 ± 2.58 a
0.95 ± 1.18 a 4.54 ± 2.49 a 8.26 ± 5.68 a 3.31 ± 1.71 ab 6.67 ± 3.99 a
– 3.10 ± 0.73 a 8.29 ± 2.32 a 6.26 ± 1.96 a 4.59 ± 2.81 ab
0.27 ± 0.54 a 3.50 ± 1.36 a 8.01 ± 3.99 a 1.70 ± 2.19 b 1.67 ± 1.12 b
Data are mean values with standard deviation. Data within a row followed by different letters indicate a significant (P b 0.05) difference.
J. Hu et al. / Catena 133 (2015) 215–220
a
560
a a
-1
Table 4 Species richness and diversity indices of soil AM fungal community in different treatments.
Soil AM fungal spore density (20 g )
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Ecological parameters Species richness (SR) Shannon–Wiener index (H′) Evenness (E) Simpson's index (D)
Conventional tillage (CT)
No tillage (NT)
Alternating tillage (AT)
17.8 ± 1.9 a 2.53 ± 0.18 a
18.3 ± 2.2 a 2.55 ± 0.10 a
18.3 ± 1.5 a 2.49 ± 0.13 a
0.880 ± 0.031 a 0.905 ± 0.025 a
0.882 ± 0.037 a 0.903 ± 0.019 a
0.856 ± 0.033 a 0.890 ± 0.028 a
420
280
Data are mean values with standard deviation. Data within a row followed by different letters indicate a significant (P b 0.05) difference.
140
0 CT
-1
There were no significant differences in SR, Shannon–Wiener index (H′), Evenness (E), and Simpson's index (D) of soil AM fungal community among the three treatments (Table 4). The Jaccard index (J) of similarity in species composition indicated that the largest difference was between the NT and CT treatments (J = 0.767), followed by that between the NT and AT treatments (J = 0.793), while the lowest one was between the AT and CT treatments (J = 0.893).
4. Discussion The objective of this study was to compare the effects of continuous no-tillage (NT) and alternate no-tillage (AT) on species composition of soil arbuscular mycorrhizal (AM) fungi and soil P-supply related
b
70
0 CT
NT
AT
a
ab
NT
AT
0.4
c -1
Soil ALP activity (mg g 24h )
The Pearson correlation coefficients between soil properties and AM fungal parameters are shown in Table 5. Among soil chemical parameters, the available P content significantly correlated (P b 0.05) to the organic C content. Among AM fungal parameters, the SR of AM fungi significantly correlated (P b 0.05) to H′, while H′, E, and D significantly correlated (P b 0.01) to each other. Additionally, soil ALP activity significantly correlated to both SD (P b 0.01) and SR (P b 0.05) of AM fungi, and the available P content (P b 0.05). No other remarkable correlations were apparent for either AM fungal parameters or soil properties.
ab
140
3.4. AM fungal spore density (SD), external mycelium length, and soil alkaline phosphatase (ALP) activity
3.5. Pearson correlation coefficients
AT
a
210
3.3. AM fungal species richness (SR), diversity, and similarity
0.3
b
-1
There were no significant differences in SD of soil AM fungal community among the three treatments (Fig. 1a). External mycelium length was significantly higher (P b 0.05) under the NT practice than under the CT practice, but the AT treatment only resulted in a trend towards higher external mycelium length (Fig. 1b). In accordance with the increased external mycelium length, soil ALP activity was also significantly increased (P b 0.05) with the NT treatment, and tended to increase with the AT treatment (Fig. 1c).
NT
280
b
External mycelium length (mm g ) .
verrucosa (4.0–9.8%), Funneliformis mosseae (3.5–7.7%), Scutellospora nigra (3.1–4.5%), and so on. Glomus globiferum and Glomus monosporum were only recorded in the NT treatment, while Funneliformis geosporum was only recorded in the CT treatment. Rhizophagus clarum, Glomus-like sp. I and Scutellospora heterogama were absent with the NT treatment, while Acaulospora cavernata and Glomus aggregatum were absent with the AT and CT treatments, respectively. Some species sporulated differentially with the three treatments. Specifically, the RAs of Funneliformis constrictum were significantly different (P b 0.05) with the three treatments. The RAs of Funneliformis verruculosum, Scutellospora-like sp. I, and Scutellospora-like sp. II were significantly different (P b 0.05) between CT and NT, NT and AT, and CT and AT, respectively.
a
0.2
0.1
0.0 CT
Fig. 1. Arbuscular mycorrhizal fungal spore density (a), external mycelium length (b), and soil alkaline phosphatase (ALP) activity in different treatments. CT, conventional tillage; NT, no-tillage; AT, alternating tillage. Vertical T bars indicate standard deviations. Values not topped by a same letter differ significantly (P b 0.05).
J. Hu et al. / Catena 133 (2015) 215–220
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Table 5 The Pearson correlation coefficients between soil properties and AM fungal community parameters.
Organic C Total P Available P Species richness (SR) Shannon-Wiener index (H′) Evenness (E) Simpson's index (D) Spore density (SD) External mycelium length Soil alkaline phosphatase (ALP) activity
pH
Organic C
Total P
Available P
SR
H′
E
D
SD
External mycelium length
−0.097 0.374 −0.057 −0.216 −0.099 0.059 −0.608 0.218 −0.138 0.006
0.206 0.584⁎ −0.181 −0.406 −0.360 −0.426 0.110 0.470 0.436
0.330 0.122 0.457 0.519 0.515 0.209 0.132 0.523
−0.074 −0.159 −0.136 −0.167 0.495 0.473 0.606⁎
0.657⁎ 0.002 0.326 0.494 0.116 0.602⁎
0.754⁎⁎ 0.902⁎⁎ 0.331 −0.095 0.422
0.914⁎⁎ 0.004 −0.221 0.050
0.145 −0.246 0.243
0.006 0.746⁎⁎
0.393
⁎ P = 0.05 (significant). ⁎⁎ P = 0.01 (significant).
parameters. A total of 30 AM fungal species was recorded in this study, slightly lower than the number of 35 reported from the same arable land (Wang et al., 2011a). The genus Acaulospora registered the largest species number, while Acaulospora denticulata was the most abundant species with a relative abundance (RA) of 21.8–25.1% (Table 3). The dominance of AM fungi was related to their sporogenous characteristics. Acaulospora species produced the smallest sized spores in large numbers in a short time, which distribute easily (Hepper, 1984). On the other hand, different tillage regimes also influence the RAs of AM fungi in the soil. For instance, compared with conventional tillage (CT), NT caused higher sporulation of Funneliformis constrictum, Glomus aggregatum, Glomus globiferum, and Glomus monosporum, and lower sporulation of Funneliformis geosporum, Funneliformis verruculosum, Glomus-like sp. I, Rhizophagus clarus, and Scutellospora heterogama, while AT also promoted the sporulation of Glomus aggregatum and Funneliformisconstrictum, but inhibited Acaulospora scrobiculata, Funneliformis geosporum, and Scutellospora-like sp. II. It can be deduced that some species were more sensitive to tillage regimes relative to other ones. Nevertheless, due to the contrary alterations of different species (Table 3), there were no significant differences in spore density (SD), species richness (SR), and diversity indices (H′, E, and D) of soil AM fungal community among the three treatments (Fig. 1a; Table 4). With regard to the mean values, both NT and AT seemed to be helpful in increasing the SD and the SR of AM fungi, which could be advantageous for ecosystems. For instance, soil alkaline phosphatase (ALP) activity, which closely and positively correlated to both the SD and the SR of AM fungi (Table 5), was significantly higher with NT than with CT (Fig. 1c). Unlike AM fungal spore parameters but similar to soil ALP activity, external mycelium length was also substantially higher with the continuous NT system (Fig. 1b). The causing mechanisms may be due in part to reduction in destruction by tillage on soil physico-chemical properties (Borie et al., 2006; Tabaglio et al., 2009). For example, soil organic C content was significantly higher under NT than under CT (Table 2), similar to the results reported from other studies (Alvear et al., 2005; Tabaglio et al., 2009). A long-term period of NT may increase soil bulk density by compacting processes derived from the use of planting machinery (Botta et al., 2009). It may reduce soil porosity and decrease O2 supply for heterotrophic microbial decomposition (Álvaro-Fuentes et al., 2008), explaining the accumulation of a high soil organic C (Curaqueo et al., 2011). Nevertheless, the reduction in physical disturbance to the system upon NT not only slows the decomposition rate of organic matter, but also allows AM fungi to grow undisturbed and/or contributes to the longevity of their extraradical hyphae in the soil. Therefore, the filament network left intact can readily serve as AM inoculum and colonize the roots of post-harvest germinating seedlings (Castillo et al., 2006), increasing nutrient (e.g., P) uptake by crop (Galvez et al., 2001) through enhanced root mycorrhizal colonization (Bilalis and Karamanos, 2010) and related soil enzyme activities (Wang et al., 2011b). In this study, the significant increase in soil ALP activity (Fig. 1c) suggested that NT
can play an important role in function enhancement of P-supply efficiency of the soil, due to the significant and positive correlation between soil available P content and soil ALP activity (Table 5). As a result, there were very similar altering trends of soil organic C and available P in response to NT, resulting in a significant correlation between them (Table 5), which was previously obtained from a long-term fertilization experiment in Northern China (Hu et al., 2010). Similar to the continuous NT system, the AT system also tended to increase external mycelium length (Fig. 1b), soil ALP activity (Fig. 1c) and organic C content (Table 2) at the end of the maize season relative to the CT system. All these three parameters fell in middle levels with the AT treatment. It was previously hypothesized that CT employed in the wheat season may increase the distribution of AM fungal propagules, while NT employed in the following maize season allowed the external mycelium to grow undisturbed. However, this integrated system of CT–NT showed no better beneficial effect on the density and activity of AM fungal extraradical hyphae compared to the NT system after 4 years with nine crop seasons. This observation demonstrated that 4-year continuous NT would not cause degradation in AM fungal community in this arable soil in Northern China. Furthermore, this research also provided valuable data on relationships between AM fungi and soil function, and may improve land-use sustainability by allowing farmers to better manage tillage with both economic and agronomic demands. However, further work is necessary to determine whether the sustainability and productivity in this system depend on the abundance of particular AM fungal species and to elucidate the mechanisms involved in the interactions between AM fungi and soil organic C sequestration. 5. Conclusions Compared with CT, 4-year continuous NT affected the species composition, but not the spore density, species richness and diversity indices (H′, E, and D), of AM fungal community at the maize harvest stage in the sandy loam soil in Northern China. However, NT significantly increased the external mycelium length, soil ALP activity and organic C content, while AT (no-tillage in the maize season and tillage in the wheat season) showed no better beneficial effects compared to NT. The results demonstrated that 4-year continuous NT would not cause degradation in either AM fungal community or soil P-supply efficiency in this region. Acknowledgments This work was supported by the National Basic Research Program (2011CB100505) and the National Natural Science Foundation (No.40801090) of China, and the Provincial Natural Science Foundation (1308085QD68) of Anhui, China. We wish to acknowledge Qi'ao Jiang, Linyun Zhou, Jinfang Wang, and Jian Liu, of the Fengqiu AgroEcological Experimental Station, Institute of Soil Science, Chinese
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Academy of Sciences, for their excellent field management and support on soil sampling. We are also grateful to the two anonymous reviewers, whose comments and suggestions greatly improved the quality of this manuscript. References Álvaro-Fuentes, J., Lopez, M.V., Cantero-Martinez, C., Arrue, J.L., 2008. Tillage effects on soil organic carbon fractions in Mediterranean dry land agroecosystems. Soil Sci. Soc. Am. J. 72, 541–547. Alvear, M., Rosas, A., Rouanet, J.L., Borie, F., 2005. Effects of three soil tillage systems on some biological activities in an Ultisol from southern Chile. Soil Tillage Res. 82, 195–202. An, Z.Q., Hendrix, J.W., Hershman, D.E., Henson, G.T., 1990. Evaluation of the “most probable number” (MPN) and wet-sieving methods for determining soil-borne populations of endogonaceous mycorrhizal fungi. Mycologia 82, 516–581. Bedini, S., Pellegrino, E., Avio, L., Pellegrini, S., Bazzoffi, P., Argese, E., Giovanetti, M., 2009. Changes in soil aggregation and glomalin-related soil protein content as affected by the arbuscular mycorrhizal fungal species Glomus mosseae and Glomus intraradices. Soil Biol. Biochem. 41, 1491–1496. Bilalis, D.J., Karamanos, A.J., 2010. Organic maize growth and mycorrhizal root colonization response to tillage and organic fertilization. J. Sustain. Agric. 34, 836–849. Blanco-Canqui, H., Lal, R., 2008. No-tillage and soil-profile carbon sequestration: an onfarm assessment. Soil Sci. Soc. Am. J. 72, 693–701. Borie, F., Rubio, R., Rouanet, J.L., Morale, A., Borie, G., Rojas, C., 2006. Effects of tillage systems on soil characteristics, glomalin and mycorrhizal propagules in a Chilean Ultisol. Soil Tillage Res. 88, 253–261. Botta, G.F., Becerra, A.T., Tourn, F.B., 2009. Effect of the number of tractor passes on soil rut depth and compaction in two tillage regimes. Soil Tillage Res. 103, 381–386. Castillo, C.G., Rubio, R., Rouanet, J.L., Borie, F., 2006. Early effects of tillage and crop rotation on arbuscular mycorrhizal fungal propagules in an Ultisol. Biol. Fertil. Soils 42, 83–92. Curaqueo, G., Barea, J.M., Acevedo, E., Rubio, R., Cornejo, P., Borie, F., 2011. Effects of different tillage system on arbuscular mycorrhizal fungal propagules and physical properties in a Mediterranean agroecosystem in central Chile. Soil Tillage Res. 113, 11–18. Dai, J., Hu, J., Lin, X., Yang, A., Wang, R., Zhang, J., Wong, M.H., 2013. Arbuscular mycorrhizal fungal diversity, external mycelium length, and glomalin-related soil protein content in response to long-term fertilizer management. J. Soils Sediments 13, 1–11. Galvez, L., Douds Jr., D.D., Wagoner, P., 2001. Tillage and farming system affect AM fungus populations, mycorrhizal formation, and nutrient uptake by winter wheat in a high-P soil. Am. J. Altern. Agric. 16, 152–160. Garbeva, P., Van Veen, J.A., Van Elsas, J.D., 2004. Microbial diversity in soil: selection of microbial populations by plant and soil type and implications for disease suppressiveness. Annu. Rev. Phytopathol. 42, 243–270. Gong, W., Yan, X., Wang, J., Hu, T., Gong, Y., 2009. Long-term manure and fertilizer effects on soil organic matter fractions and microbes under a wheat–maize cropping system in northern China. Geoderma 149, 318–324. He, J., Li, H., Rasaily, R.G., Wang, Q., Cai, G., Su, Y., Qiao, X., Liu, L., 2011. Soil properties and crop yields after 11 years of no tillage farming in wheat–maize cropping system in North China Plain. Soil Tillage Res. 113, 48–54. Hepper, C.M., 1984. Isolation and culture of VA mycorrhizal (VAM) fungi. In: Powell, C.L., Bagyaraj, D.J. (Eds.), VA Mycorrhizae. CRC Press, Florida, USA, pp. 95–112. Hu, J., Lin, X., Wang, J., Cui, X., Dai, J., Chu, H., Zhang, J., 2010. Arbuscular mycorrhizal fungus enhances P-acquisition of wheat (Triticum aestivum L.) in a sandy loam soil with long-term inorganic fertilization regime. Appl. Microbiol. Biotechnol. 88, 781–787. Hu, J.L., Zhu, A.N., Wang, J.H., Dai, J., Wang, J.T., Chen, R.R., Lin, X.G., 2013. Soil microbial metabolism and invertase activity under crop rotation and no-tillage in North China. Plant Soil Environ. 59, 511–516. Hu, J., Wu, F., Wu, S., Lam, C.L., Lin, X., Wong, M.H., 2014. Biochar and Glomus caledonium influence Cd accumulation of upland kangkong (Ipomoea aquatica Forsk.) intercropped with Alfred stonecrop (Sedum alfredii Hance). Sci. Rep. 4, 4671. Jackson, M.L., 1958. Soil Chemical Analysis. Prentice-Hall Inc., Englewood Cliffs, NJ, pp. 111–133. Krüger, M., Krüger, C., Walker, C., Stockinger, H., Schüßler, A., 2012. Phylogenetic reference data for systematics and phylotaxonomy of arbuscular mycorrhizal fungi from phylum to species level. New Phytol. 193, 970–984.
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Arbuscular mycorrhizal fungal species composition, propagule density, and soil alkaline phosphatase activity in response to continuous and alternate no-tillage in Northern China Junli Hu a,c,1, Anna Yang b,1, Junhua Wang a,c, Anning Zhu a, Jue Dai a,c, Ming Hung Wong c,d, Xiangui Lin a,c,⁎ a
State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, East Beijing Road 71, Nanjing 210008, PR China Provincial Key Laboratory of Biotic Environment and Ecological Security in Anhui, College of Life Sciences, Anhui Normal University, East Beijing Road 1, Wuhu 241000, PR China c Joint Open Laboratory of Soil and the Environment, Hong Kong Baptist University & Institute of Soil Science, Chinese Academy of Sciences, East Beijing Road 71, Nanjing 210008, PR China d Croucher Institute for Environmental Sciences, Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, PR China b
a r t i c l e
i n f o
Article history: Received 14 January 2015 Received in revised form 19 May 2015 Accepted 22 May 2015 Available online xxxx Keywords: Diversity External mycelium Organic C Relative abundance Species richness Spore density
a b s t r a c t Purpose: Arbuscular mycorrhizal (AM) fungi provide a direct link between soil and roots, and are renowned for their ability to increase nutrient phytoavailability, notably phosphorus (P). The objective of this study was to evaluate influences of continuous and alternate no-tillage on soil AM fungal species composition and soil Psupply parameters. Materials and methods: In June 2006, a long-term field experiment was established in a sandy loam soil under the rotation of summer maize (Zea mays L.) and winter wheat (Triticum aestivum L.) in Northern China, including conventional tillage (CT), no-tillage (NT) and alternate tillage (AT — tillage in the wheat season and no-tillage in the maize season). Top soil samples (0–15 cm) from four individual plots per treatment were collected before maize harvest on 18 September 2010. Soil AM fungal spores were isolated and identified, and the external mycelium length, soil alkaline phosphatase (ALP) activity and soil P content were also determined. Results and discussion: Thirty species of AM fungi within seven genera, Acaulospora, Claroideoglomus, Funneliformis, Glomus, Racocetra, Rhizophagus, and Scutellospora, were recovered. Some species sporulated differentially across the three treatments, and the lowest Jaccard index (J) of similarity in species composition was recorded between NT and CT (J = 0.767), but there were no significant differences in soil total P and available P contents, as well as in AM fungal spore density (SD), species richness (SR) and diversity indices, including Shannon–Wiener index (H′), Evenness (E) and Simpson's index (D). Compared with CT, NT rather than AT significantly increased (P b 0.05) the external mycelium length, soil ALP activity, and soil organic C content. Conclusions: Our results demonstrated the vital role of NT in maintaining external hyphae growth and soil ALP activity rather than in promoting AM fungal species diversity and spore density, and suggested that 4-year continuous NT will not cause degradation in either AM fungal community or soil P-supply efficiency. © 2015 Published by Elsevier B.V.
1. Introduction Soil microorganisms play a key role in biogeochemical cycles, and are responsible for a vast number of terrestrial functions (Garbeva et al., 2004). For instance, arbuscular mycorrhizal (AM) fungi, as ubiquitous mutualists which provide a direct link between soil and roots, and also as integral components of terrestrial ecosystems (Rillig, 2004), are renowned for their ability to increase plant growth and nutrient acquisition, notably phosphorus (P) (Hu et al., 2014; Smith and Read, 2008). Besides direct contribution of external hyphae to the transport reported for some elements (Rufyikiri et al., 2003), the enhancement ⁎ Corresponding author at: State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, PR China. E-mail address: [email protected] (X. Lin). 1 Joint first author with equal contribution.
http://dx.doi.org/10.1016/j.catena.2015.05.023 0341-8162/© 2015 Published by Elsevier B.V.
of soil enzymatic activities is another major mechanism involved in “mycorrhization effect” on plant nutrition, since AM propagules themselves can synthesize such enzymes, and mycorrhizal roots may release more root exudates containing enzymes because of the larger root system and/or improved nutrition (Wang et al., 2006). The extraradical hyphae of AM fungi also contribute to the formation and maintenance of soil aggregates (Bedini et al., 2009; Rillig et al., 2010) and in turn keep stored soil carbon (C) from escaping (Rillig et al., 2001; Wright and Upadhyaya, 1998). However, AM fungi are sensitive to changes in land-use patterns and management practices (Martinez and Johnson, 2010; Oehl et al., 2010). Therefore, there is an urgent concern about responsible maintenance of AM fungi in cultivated soils for sustainable management (Dai et al., 2013). The North China Plain (NCP) is a highly grain-producing area. The sustainable utilization of agricultural soil in this area may affect China's food security (Gong et al., 2009). In order to reduce diesel fuel
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usage and soil erosion, modern agriculture has greatly reduced the use of tillage relative to conventional agriculture. With the least disturbance of soil and consequent beneficial impacts on crop productivity, notillage (NT) seems to be superior for increasing C deposits into the soil (Alvear et al., 2005) and conserving soil and water (Blanco-Canqui and Lal, 2008). However, NT practice may also affect soil characteristics and microbial properties (Hu et al., 2013; van Groenigen et al., 2010). For instance, continuous NT may result in problems such as surface hardening of soil, leading to a more limited O2 supply for soil microorganisms (Álvaro-Fuentes et al., 2008). It seems to be unfavorable for AM fungal propagule distribution. For example, Curaqueo et al. (2011) observed decreases in AM fungal propagules after a long-term program of NT. Such variance could in turn influence the growth of cultivated crops, suggesting that moderate tillage may be more beneficial than continuous NT. In other words, it is needed to determine the suitable period of NT for common agroecosystems. More field experiments are thus necessary to improve our knowledge of the changes in AM fungal community with various tillage practices, and may help to ensure an opportunity for popularizing NT farm in arable soils. Because of concerns regarding soil quality degradation resulting from the replacement of conventional tillage (CT) with conservation tillage, a number of long-term field experiments were set up in agricultural regions to monitor changes in soil fertility and quality in China (He et al., 2011; Wang et al., 2008). However, uncertainties still remain about the influences of continuous and alternate NT on AM fungal parameters, including propagule composition and density. It was hypothesized that alternate NT would be more helpful than continuous NT in maintaining AM fungal species diversity, propagule growth, and related soil functions. In this study, a long-term field experiment, which was set up in a sandy loam soil in the NCP, was selected to determine soil AM fungi. The objectives of this study were to evaluate the influences of continuous and alternate NT practice on AM fungal propagules and soil P availabilities, and to find out the main factors that affect these parameters. This work investigated NT's superiority not only in AM fungal propagules themselves, but also in soil P-supply efficiency, and might contribute in developing soil microbial resource management schemes based on specific agronomic and economic demands.
2. Materials and methods 2.1. Description of the experimental site and soil sampling The tillage experiment was conducted in a well-drained field within the State Experimental Station for Agro-Ecology, Fengqiu County (35°00′N, 114°24′E), Henan Province, China. With a sandy loam texture, the soil is derived from alluvial sediments of the Yellow River and is classified as an Aquic Inceptisol. Maize (Zea mays L.) was sown in June and harvested in late September. Then wheat (Triticum aestivum L.) was sown in October and harvested in early June of the next year. In June 2006, three treatments, conventional tillage (CT, tillage in both wheat and maize seasons), no-tillage (NT, no-tillage in both wheat and maize seasons), and alternate tillage (AT, tillage in wheat season and no-tillage in maize season), were randomly arranged with four replicates (each plot 14 × 6.5 m). The CT plots were plowed with a moldboard to a depth of 20 cm. Later, the soil was disked twice, with a disk harrow before seed sowing. Both NT and AT plots were managed similarly to the CT plots, except for tillage which continued and alternated, respectively, to be NT with a no-tillage planter for seed sowing. Each plot received the same management every year since 2006. On 18 September 2010, before the harvesting of mature maize, soil samples were collected from 16 points per plot at a depth of 0–15 cm, and then mixed and homogenized by passing through a 2-mm mesh sieve to remove above-ground plant materials and stones. Soil samples were divided into two subsamples: one for soil enzyme activity determination and external mycelium extraction was stored at 4 °C, and
the other one for soil chemical analysis and AM fungal spore extraction was air-dried for about two weeks and then stored at 4 °C. 2.2. External mycelium length and soil chemical/enzymatic property analysis The external mycelium of AM fungi was extracted from soil samples using the method described by Malcová et al. (2002). Briefly, 5 g of soil was placed in a household blender containing 500 ml of water and blended for 30 s. Then, 1 ml of the resulting suspension was pipetted onto a membrane filter (24 mm diameter, 0.4 μm pore size) and vacuum filtered. The mycelium retained on the membrane filter was stained with a drop of 0.05% trypan blue in lactoglycerol, and total length of the mycelium was assessed using the grid-line intersect method (microscope equipped with focal plate grid 100× magnification). Soil pH was determined with a glass electrode using a soil-to-water ratio of 1:2.5. Soil organic C was determined by dichromate oxidation (Mebius, 1960). Soil total P was digested by HF-HClO4 (Jackson, 1958), while soil available P was extracted by sodium bicarbonate (Olsen et al., 1954), and then both of them were determined using the molybdenum blue method. Soil alkaline phosphatase (ALP) activity was determined by incubation at 37 °C with borate buffer (pH 9) according to Tabatabai (1982), and is given in units of mg pnitrophenol produced g−1 soil 24 h−1. All these results were expressed on an oven-dried soil weight basis by correcting for water content in the soil (105 °C, 24 h). 2.3. Recovery and counting of AM fungal spores The AM fungal spores were extracted from 20 g soil using the wetsieving method described by An et al. (1990), and were collected by 70, 100, 150 and 900 μm sieves, filtered onto a filter paper and placed in a Petri dish for examination under a binocular stereomicroscope. The spore density (SD) was calculated from direct counts of AM fungal spores (per 20 g air-dried soil). The intact healthy spores were sorted into groups and counted. The AM fungal spores were mounted in polyvinyl lactic acid (PVA) and PVA mixed 1:1 (v/v) with Meltzer's reagent (Morton, 1988; Morton and Benny, 1990) for species identification. The identification was based on morphological descriptions provided by the international collection of vesicular and arbuscular mycorrhizal fungi (http://invam.caf.wvu.edu), and recent advances in Glomeromycota taxonomy (Krüger et al., 2012; Oehl et al., 2011; Schüßler and Walker, 2010). The AM fungi species were identified using a light microscope (Olympus CX31). If the species is not yet described, it was recognized as an unknown species belonging to the most possible genus, and marked as Glomus-like sp. I, Scutellospora-like sp. II, etc. 2.4. AM fungal community structure and diversity analysis The structure of the AM fungal community was evaluated using the five selected parameters (Table 1) that have been widely employed in Table 1 Ecological diversity parameters for evaluating soil AM fungal community structure. Parameters
Formula and definition
Relative abundance (RA)
The percentage of the spore number of a species or a genus Species number per soil sample H′ = −∑Pi ln Pi E = H′ / H′max D = 1 − ∑[ni(ni − 1) / N(N − 1)] J = c / (a + b + c)
Species richness (SR) Shannon–Wiener index (H′) Evenness (E) Simpson's index (D) Jaccard index (J)
Pi = ni/N, where ni is the spore numbers of a species and N is the total number of identified spore samples. H′max = ln S, where S is the total number of identified species. J is a measure of the similarity of the spore community between treatments A and B, where a and b are the numbers of species occurring only at treatment A or treatment B, and c is the number of species occurring at both treatments.
J. Hu et al. / Catena 133 (2015) 215–220 Table 2 Soil properties in different tillage management treatments. Soil properties
Conventional tillage (CT)
No tillage (NT)
Alternating tillage (AT)
pH Organic C (g kg−1) Total P (g kg−1) Available P (mg kg−1)
8.57 ± 0.09 a 5.50 ± 0.59 b 0.97 ± 0.02 a 3.95 ± 0.77 a
8.55 ± 0.05 a 6.31 ± 0.17 a 1.02 ± 0.07 a 6.12 ± 1.57 a
8.54 ± 0.08 a 6.13 ± 0.21 ab 0.95 ± 0.05 a 5.68 ± 1.46 a
Data are mean values with standard deviation. Data within a row followed by different letters indicate a significant (P b 0.05) difference.
previous studies (Wang et al., 2011a; Yang et al., 2011). Spore specimens identified to species were used in the analysis of the relative abundance (RA) and the species richness (SR). Then, three AM fungal diversity indices, including Shannon–Wiener index (H′), Evenness (E), and Simpson's index (D), were evaluated as respectively sensitive indicators for the relative richness of species, the species evenness of the community, and the dominance of the most common species in the community. The Jaccard index (J) of similarity was calculated to compare AM fungal species composition under different treatments. 2.5. Statistical analyses The means and standard deviations of four replicates were computed. Analysis of variance (ANOVA) was carried out with SPSS software package (version 13.0). Significance of the parameters was tested using Least Significant Difference (LSD) multiple range test at P b 0.05
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after one-way ANOVA. To this end, the Pearson correlation coefficients were calculated among soil basic chemical properties, AM fungal community parameters (including SR, diversity indices, SD, and external mycelium length), and soil ALP activity. 3. Results 3.1. Soil pH, organic C, total P, and available P contents Soil pH and nutrient contents with different tillage management treatments are shown in Table 2. Compared with the CT treatment, the NT rather than the AT treatment significantly increased (P b 0.05) soil organic C content, but both NT and AT had no significant effects on soil pH, total P and available P contents. 3.2. AM fungal species composition and relative abundance (RA) A total of 30 species within seven genera, including three previously undescribed species, were recorded (Table 3). Acaulospora (35.1–38.2%), Scutellospora (15.2–23.7%, including Scutellospora-like sp. I and II), Funneliformis (16.9–20.9%), and Racocetra (10.3–18.5%) had relatively higher RAs than Claroideoglomus (6.7–6.9%), Rhizophagus (1.6–2.1%), and Glomus (0.6–1.2%, including Glomus-like sp. І). There were 22 species observed simultaneously in all the three treatments. The most abundant one was Acaulospora denticulata (21.9–25.1%), followed by Scutellospora reticulata (8.0–8.3%), Racocetra persica (6.3–8.7%), Funneliformis caledonium (4.6–6.5%), Claroideoglomus etunicatum (4.3–6.4%), Racocetra
Table 3 Species composition and relative abundance (%) of soil AM fungi in different treatments. AM fungal species Acaulospora (10 species) A. bireticulata A. denticulata A. elegans A. foveata A. lacunosa A. laevis A. mellea A. cavernata A. spinosa A. tuberculata Claroideoglomus (2 species) C. claroideum C. etunicatum Funneliformis (5 species) F. caledonium F. constrictum F. geosporum F. mossea F. verruculosum Glomus (4 species) G. aggregatum G. globiferum G. monosporum Glomus-like sp. I Racocetra (2 species) Ra. persica Ra. verrucosa Rhizophagus (2 species) Rh. clarus Rh. intraradices Scutellospora (5 species) S. heterogama S. nigra S. reticulata Scutellospora-like sp. I Scutellospora-like sp. II
Conventional tillage (CT)
No tillage (NT)
Alternating tillage (AT)
0.65 ± 0.75 a 21.86 ± 5.32 a 0.30 ± 0.60 a 0.59 ± 0.68 a 1.20 ± 1.70 a 1.47 ± 2.23 a 3.43 ± 1.60 a 1.88 ± 2.30 a 1.17 ± 1.66 a 2.54 ± 1.87 a
2.72 ± 2.38 a 24.31 ± 5.89 a 1.52 ± 1.54 a 0.32 ± 0.63 a 1.79 ± 2.06 a 0.33 ± 0.66 a 2.24 ± 1.95 a 0.63 ± 1.27 a 1.22 ± 1.00 a 1.56 ± 1.88 a
2.71 ± 1.47 a 25.09 ± 7.98 a 2.11 ± 1.49 a 0.92 ± 1.11 a 2.68 ± 0.50 a 0.27 ± 0.54 a 1.68 ± 1.47 a – 0.65 ± 0.76 a 2.04 ± 2.62 a
2.37 ± 1.68 a 4.30 ± 2.77 a
1.88 ± 1.67 a 4.99 ± 3.37 a
0.28 ± 0.56 a 6.39 ± 2.25 a
4.73 ± 3.77 a 0.29 ± 0.59 c 1.29 ± 1.50 7.17 ± 3.59 a 7.45 ± 3.43 a
6.49 ± 1.76 a 5.24 ± 1.02 a – 3.50 ± 2.44 a 1.64 ± 3.29 b
4.55 ± 2.58 a 3.32 ± 1.22 b – 7.66 ± 4.83 a 3.42 ± 2.49 ab
– – – 1.18 ± 1.66 a
0.32 ± 0.63 a 0.32 ± 0.63 0.30 ± 0.61 –
0.27 ± 0.54 a – – 0.37 ± 0.74 a
6.31 ± 7.36 a 4.02 ± 5.34 a
6.92 ± 4.36 a 7.96 ± 2.89 a
8.70 ± 4.67 a 9.80 ± 4.83 a
1.47 ± 2.94 a 0.59 ± 0.68 a
– 1.55 ± 0.60 a
0.28 ± 0.56 a 1.65 ± 2.58 a
0.95 ± 1.18 a 4.54 ± 2.49 a 8.26 ± 5.68 a 3.31 ± 1.71 ab 6.67 ± 3.99 a
– 3.10 ± 0.73 a 8.29 ± 2.32 a 6.26 ± 1.96 a 4.59 ± 2.81 ab
0.27 ± 0.54 a 3.50 ± 1.36 a 8.01 ± 3.99 a 1.70 ± 2.19 b 1.67 ± 1.12 b
Data are mean values with standard deviation. Data within a row followed by different letters indicate a significant (P b 0.05) difference.
J. Hu et al. / Catena 133 (2015) 215–220
a
560
a a
-1
Table 4 Species richness and diversity indices of soil AM fungal community in different treatments.
Soil AM fungal spore density (20 g )
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Ecological parameters Species richness (SR) Shannon–Wiener index (H′) Evenness (E) Simpson's index (D)
Conventional tillage (CT)
No tillage (NT)
Alternating tillage (AT)
17.8 ± 1.9 a 2.53 ± 0.18 a
18.3 ± 2.2 a 2.55 ± 0.10 a
18.3 ± 1.5 a 2.49 ± 0.13 a
0.880 ± 0.031 a 0.905 ± 0.025 a
0.882 ± 0.037 a 0.903 ± 0.019 a
0.856 ± 0.033 a 0.890 ± 0.028 a
420
280
Data are mean values with standard deviation. Data within a row followed by different letters indicate a significant (P b 0.05) difference.
140
0 CT
-1
There were no significant differences in SR, Shannon–Wiener index (H′), Evenness (E), and Simpson's index (D) of soil AM fungal community among the three treatments (Table 4). The Jaccard index (J) of similarity in species composition indicated that the largest difference was between the NT and CT treatments (J = 0.767), followed by that between the NT and AT treatments (J = 0.793), while the lowest one was between the AT and CT treatments (J = 0.893).
4. Discussion The objective of this study was to compare the effects of continuous no-tillage (NT) and alternate no-tillage (AT) on species composition of soil arbuscular mycorrhizal (AM) fungi and soil P-supply related
b
70
0 CT
NT
AT
a
ab
NT
AT
0.4
c -1
Soil ALP activity (mg g 24h )
The Pearson correlation coefficients between soil properties and AM fungal parameters are shown in Table 5. Among soil chemical parameters, the available P content significantly correlated (P b 0.05) to the organic C content. Among AM fungal parameters, the SR of AM fungi significantly correlated (P b 0.05) to H′, while H′, E, and D significantly correlated (P b 0.01) to each other. Additionally, soil ALP activity significantly correlated to both SD (P b 0.01) and SR (P b 0.05) of AM fungi, and the available P content (P b 0.05). No other remarkable correlations were apparent for either AM fungal parameters or soil properties.
ab
140
3.4. AM fungal spore density (SD), external mycelium length, and soil alkaline phosphatase (ALP) activity
3.5. Pearson correlation coefficients
AT
a
210
3.3. AM fungal species richness (SR), diversity, and similarity
0.3
b
-1
There were no significant differences in SD of soil AM fungal community among the three treatments (Fig. 1a). External mycelium length was significantly higher (P b 0.05) under the NT practice than under the CT practice, but the AT treatment only resulted in a trend towards higher external mycelium length (Fig. 1b). In accordance with the increased external mycelium length, soil ALP activity was also significantly increased (P b 0.05) with the NT treatment, and tended to increase with the AT treatment (Fig. 1c).
NT
280
b
External mycelium length (mm g ) .
verrucosa (4.0–9.8%), Funneliformis mosseae (3.5–7.7%), Scutellospora nigra (3.1–4.5%), and so on. Glomus globiferum and Glomus monosporum were only recorded in the NT treatment, while Funneliformis geosporum was only recorded in the CT treatment. Rhizophagus clarum, Glomus-like sp. I and Scutellospora heterogama were absent with the NT treatment, while Acaulospora cavernata and Glomus aggregatum were absent with the AT and CT treatments, respectively. Some species sporulated differentially with the three treatments. Specifically, the RAs of Funneliformis constrictum were significantly different (P b 0.05) with the three treatments. The RAs of Funneliformis verruculosum, Scutellospora-like sp. I, and Scutellospora-like sp. II were significantly different (P b 0.05) between CT and NT, NT and AT, and CT and AT, respectively.
a
0.2
0.1
0.0 CT
Fig. 1. Arbuscular mycorrhizal fungal spore density (a), external mycelium length (b), and soil alkaline phosphatase (ALP) activity in different treatments. CT, conventional tillage; NT, no-tillage; AT, alternating tillage. Vertical T bars indicate standard deviations. Values not topped by a same letter differ significantly (P b 0.05).
J. Hu et al. / Catena 133 (2015) 215–220
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Table 5 The Pearson correlation coefficients between soil properties and AM fungal community parameters.
Organic C Total P Available P Species richness (SR) Shannon-Wiener index (H′) Evenness (E) Simpson's index (D) Spore density (SD) External mycelium length Soil alkaline phosphatase (ALP) activity
pH
Organic C
Total P
Available P
SR
H′
E
D
SD
External mycelium length
−0.097 0.374 −0.057 −0.216 −0.099 0.059 −0.608 0.218 −0.138 0.006
0.206 0.584⁎ −0.181 −0.406 −0.360 −0.426 0.110 0.470 0.436
0.330 0.122 0.457 0.519 0.515 0.209 0.132 0.523
−0.074 −0.159 −0.136 −0.167 0.495 0.473 0.606⁎
0.657⁎ 0.002 0.326 0.494 0.116 0.602⁎
0.754⁎⁎ 0.902⁎⁎ 0.331 −0.095 0.422
0.914⁎⁎ 0.004 −0.221 0.050
0.145 −0.246 0.243
0.006 0.746⁎⁎
0.393
⁎ P = 0.05 (significant). ⁎⁎ P = 0.01 (significant).
parameters. A total of 30 AM fungal species was recorded in this study, slightly lower than the number of 35 reported from the same arable land (Wang et al., 2011a). The genus Acaulospora registered the largest species number, while Acaulospora denticulata was the most abundant species with a relative abundance (RA) of 21.8–25.1% (Table 3). The dominance of AM fungi was related to their sporogenous characteristics. Acaulospora species produced the smallest sized spores in large numbers in a short time, which distribute easily (Hepper, 1984). On the other hand, different tillage regimes also influence the RAs of AM fungi in the soil. For instance, compared with conventional tillage (CT), NT caused higher sporulation of Funneliformis constrictum, Glomus aggregatum, Glomus globiferum, and Glomus monosporum, and lower sporulation of Funneliformis geosporum, Funneliformis verruculosum, Glomus-like sp. I, Rhizophagus clarus, and Scutellospora heterogama, while AT also promoted the sporulation of Glomus aggregatum and Funneliformisconstrictum, but inhibited Acaulospora scrobiculata, Funneliformis geosporum, and Scutellospora-like sp. II. It can be deduced that some species were more sensitive to tillage regimes relative to other ones. Nevertheless, due to the contrary alterations of different species (Table 3), there were no significant differences in spore density (SD), species richness (SR), and diversity indices (H′, E, and D) of soil AM fungal community among the three treatments (Fig. 1a; Table 4). With regard to the mean values, both NT and AT seemed to be helpful in increasing the SD and the SR of AM fungi, which could be advantageous for ecosystems. For instance, soil alkaline phosphatase (ALP) activity, which closely and positively correlated to both the SD and the SR of AM fungi (Table 5), was significantly higher with NT than with CT (Fig. 1c). Unlike AM fungal spore parameters but similar to soil ALP activity, external mycelium length was also substantially higher with the continuous NT system (Fig. 1b). The causing mechanisms may be due in part to reduction in destruction by tillage on soil physico-chemical properties (Borie et al., 2006; Tabaglio et al., 2009). For example, soil organic C content was significantly higher under NT than under CT (Table 2), similar to the results reported from other studies (Alvear et al., 2005; Tabaglio et al., 2009). A long-term period of NT may increase soil bulk density by compacting processes derived from the use of planting machinery (Botta et al., 2009). It may reduce soil porosity and decrease O2 supply for heterotrophic microbial decomposition (Álvaro-Fuentes et al., 2008), explaining the accumulation of a high soil organic C (Curaqueo et al., 2011). Nevertheless, the reduction in physical disturbance to the system upon NT not only slows the decomposition rate of organic matter, but also allows AM fungi to grow undisturbed and/or contributes to the longevity of their extraradical hyphae in the soil. Therefore, the filament network left intact can readily serve as AM inoculum and colonize the roots of post-harvest germinating seedlings (Castillo et al., 2006), increasing nutrient (e.g., P) uptake by crop (Galvez et al., 2001) through enhanced root mycorrhizal colonization (Bilalis and Karamanos, 2010) and related soil enzyme activities (Wang et al., 2011b). In this study, the significant increase in soil ALP activity (Fig. 1c) suggested that NT
can play an important role in function enhancement of P-supply efficiency of the soil, due to the significant and positive correlation between soil available P content and soil ALP activity (Table 5). As a result, there were very similar altering trends of soil organic C and available P in response to NT, resulting in a significant correlation between them (Table 5), which was previously obtained from a long-term fertilization experiment in Northern China (Hu et al., 2010). Similar to the continuous NT system, the AT system also tended to increase external mycelium length (Fig. 1b), soil ALP activity (Fig. 1c) and organic C content (Table 2) at the end of the maize season relative to the CT system. All these three parameters fell in middle levels with the AT treatment. It was previously hypothesized that CT employed in the wheat season may increase the distribution of AM fungal propagules, while NT employed in the following maize season allowed the external mycelium to grow undisturbed. However, this integrated system of CT–NT showed no better beneficial effect on the density and activity of AM fungal extraradical hyphae compared to the NT system after 4 years with nine crop seasons. This observation demonstrated that 4-year continuous NT would not cause degradation in AM fungal community in this arable soil in Northern China. Furthermore, this research also provided valuable data on relationships between AM fungi and soil function, and may improve land-use sustainability by allowing farmers to better manage tillage with both economic and agronomic demands. However, further work is necessary to determine whether the sustainability and productivity in this system depend on the abundance of particular AM fungal species and to elucidate the mechanisms involved in the interactions between AM fungi and soil organic C sequestration. 5. Conclusions Compared with CT, 4-year continuous NT affected the species composition, but not the spore density, species richness and diversity indices (H′, E, and D), of AM fungal community at the maize harvest stage in the sandy loam soil in Northern China. However, NT significantly increased the external mycelium length, soil ALP activity and organic C content, while AT (no-tillage in the maize season and tillage in the wheat season) showed no better beneficial effects compared to NT. The results demonstrated that 4-year continuous NT would not cause degradation in either AM fungal community or soil P-supply efficiency in this region. Acknowledgments This work was supported by the National Basic Research Program (2011CB100505) and the National Natural Science Foundation (No.40801090) of China, and the Provincial Natural Science Foundation (1308085QD68) of Anhui, China. We wish to acknowledge Qi'ao Jiang, Linyun Zhou, Jinfang Wang, and Jian Liu, of the Fengqiu AgroEcological Experimental Station, Institute of Soil Science, Chinese
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