Effects of Soil and Climatic Factors on Arbuscular Mycorrhizal Fungi in Rhizosphere Soil Under Robinia Pseudoacacia in the Loess Plateau, China

European Journal of Soil Science, November 2016, 67, 847–856 doi: 10.1111/ejss.12381

Effects of soil and climatic factors on arbuscular mycorrhizal fungi in rhizosphere soil under Robinia pseudoacacia in the Loess Plateau, China

F. Hea,b,c,M.Tanga,b, S. L. Zhongd,R.Yangb,L.Huangd & H. Q. Zhangb aState Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, Northwest A&F University, Yangling, Shaanxi 712100, China, bCollege of Forestry, Northwest A&F University, Yangling, Shaanxi 712100, China, cSchool of Modern Agriculture and Biotechnology, Ankang University, Ankang, Shaanxi 725000, China, and dCollege of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, China

Summary We explored arbuscular mycorrhizal fungi (AMF) in the rhizosphere soil under Robinia pseudoacacia L., Leguminosae (black locust), and evaluated the relations between their diversity and soil and climatic factors in the semi-arid Loess Plateau of northwest China. A total of 23 AMF species were identified at six sites. Of these, six belonged to the genus Funneliformis (Glomeraceae), five to Rhizophagus (Glomeraceae), three to Glomus (Glomeraceae), two to Acaulospora (Acaulosporaceae), two to Claroideoglomus (Claroideoglomeraceae), two to Septoglomus (Glomeraceae), one to Ambispora (Ambisporaceae), one to Scutellospora (Gigasporaceae) and one to Sclerocystis (Glomeraceae). The species that occurred in the largest proportion of soil samples were Rhizophagus intraradices (75%) and Funneliformis dimorphicum (66.7%). Soil-available potassium was positively correlated with mycorrhizal colonization and species richness. Spore density was negatively correlated with soil pH, whereas the Shannon–Wiener diversity index was positively correlated with total potassium. Most of the soil and climatic factors measured affected the AMF indices directly rather than indirectly. The relative abundances of 13 AMF species were strongly affected by precipitation, temperature, total nitrogen, ammonium nitrogen and available potassium. This suggests that AMF species are diverse and Funneliformis was the most common and dominant AMF genus in the rhizosphere soil under black locust in the Loess Plateau. Precipitation directly affected AMF status and diversity in this distinctive semi-arid ecosystem.

Highlights • Analysis of the effects of soil and climatic factors on mycorrhizal colonization and AMF diversity of black locust. • Improved understanding of AMF communities and their function in soil under black locust. • Precipitation was the strongest direct factor that affected AMF in the semi-arid region. • Funneliformis was the most common AMF genus in rhizosphere soil under black locust.

Introduction et al., 2012). With the global rise in awareness of environmental protection, there has been increasing recognition of the need to The Loess Plateau of north-central China is a region with distinctive improve the ecological environment in the semi-arid regions of the topographical and geological features. This area has experienced Loess Plateau. severe soil erosion and degradation, partly because of industrial Under semi-arid conditions, plant nutrients become less effective pollution, extreme weather and the destruction of vegetation (Feng et al., 2011). Desiccation has occurred extensively across the Loess because of drought and plant survival depends on the plants’ Plateau, which adversely affects the restoration of vegetation (Yao strategies to overcome deficiencies in water and nutrients (Yao et al., 2012). Arbuscular mycorrhizal fungi (AMF) are ubiquitous Correspondence: M. Tang. E-mail: [email protected] and ecologically important soil microorganisms that belong to Received 22 October 2015; revised version accepted 26 July 2016 the phylum Glomeromycota (Schüßler et al., 2001) and form

© 2016 British Society of Soil Science 847 848 F. H e et al. mutualistic symbioses with the roots of most terrestrial plants. Materials and methods The AMF can improve the abilities of plants to acquire water and Study sites nutrients and ensure their survival and growth; therefore, AMF play a crucial role in the restoration of vegetation (Yang et al., The Loess Plateau is a large region with deep loess, which makes 2014). Mycorrhizal plants have been shown to absorb water and it a unique region in the world. It suffers from widespread soil nutrients from the soil more efficiently than non-mycorrhizal plants. erosion that is the subject of large-scale soil protection projects. In addition, AMF protect host plants against biotic and abiotic The region has annual precipitation amounts of 350–600 mm, of stresses (e.g. drought and extreme temperatures) because of their which 60% occurs from July to September. The temperature ranges ∘ beneficial roles in improving growth and drought tolerance (Yang from 6 to 14 C(Fenget al., 2011). The study area was in the ∘ ′ ∘ ′ et al., 2014). north of Shaanxi province, northwest of China (36 35 –39 19 N, ∘ ′ ∘ ′ The spatial patterns and diversity of some AMF species have 109 15 –110 37 E), which is characterized by a semi-arid con- been shown to be associated with soil phosphorus concentrations, tinental climate. Precipitation and temperature range from 406.9 ∘ pH, soil disturbance, salinity, soil hydrological conditions and to 531.0 mm and 8.1 to 9.7 C, respectively. Six study sites were vegetation types (Escudero & Mendoza, 2005). In general, increases selected, Fugu (FG), Shenmu (SM), Yulin (YL), Mizhi (MZ), in plant nutrient status, salinity and pH of the soil lead to a decrease Yan’an (YA) and Ansai (AS), and the aridity indices were 3.6, in spore density or mycorrhizal colonization, and decreases in soil 3.0, 2.7, 2.5, 2.1 and 2.0, respectively (Table S1 in Supporting P lead to increases in AMF diversity (Yang et al., 2015). Although Information). there are diverse morphologies of AMF from different genera, The dominant vegetation at the six sites had previously com- most studies on AMF have used species from the genus Glomus prised Juniperus communis L., Ostryopsis davidiana Decaisne and Syringa oblata Lindl. However, these sites had been affected by only (Dodd et al., 2000). However, the mechanisms involved in the severe wind erosion, soil loss and removal of natural vegetation establishment of mycorrhizae might differ among genera (as well as because of mining during the 1980s. Therefore, they were reveg- species) of AMF and affect their subsequent function (Dodd et al., etated with the current vegetation of predominantly black locust 2000). Moreover, the symbiotic functions of AMF are numerous, and Amorpha fruticosa L. Other dominant species that were present for example in relation to the species level of the AMF, AMF from spontaneous colonization included Juniperus communis L. ecotype, host plant species and soil properties (Yang et al., 2015). and Pinus tabuliformis Carr. (Feng et al., 2011). The mean annual Despite the role of AMF in plant nutrition and physiology, and temperature and precipitation data for each study site, over 10 years in shaping plant communities (Escudero & Mendoza, 2005), the (2006–15), were obtained from the National Meteorological Center variables that affect AMF diversity, spore density and mycorrhizal of CMA (http://www.nmc.cn/publish/precipitation.html). colonization in soil are poorly understood. Robinia pseudoacacia L., Leguminosae (black locust) was intro- duced into China from North America in 1877 and was cultivated Collection of soil and root samples widely across the Loess Plateau in the 1980s (Tian et al., 2003). At each study site, six sampling plots (20 m × 20 m) were selected Since then, the woody tree has been considered a dominant species over 30-m apart, in October 2014. Then three healthy black locust for the restoration of vegetation in the soft rock areas of the Loess trees were selected randomly within each plot. The trees were Plateau. The tree species is well adapted to the environment: it mature: 18–20 years old and 8–10 m in height. Four samples were grows rapidly, is drought-tolerant and has deep and extensive root taken at random locations beneath each tree; each sample was systems, which reduce desertification and soil erosion (Tian et al., within 90 cm of the tree’s trunk to ensure that root fragments 2003). Black locust plantations on the Loess Plateau have ecologi- collected belonged to the correct tree species. At each sampling cal and economic value; they form shelterbelts that protect the soil location, the top 2 cm of soil was scraped off to remove litter and a from severe storms, serve as a source of honey and provide livestock large hole (diameter, 50 cm; depth, 50 cm) was excavated to collect forage (Yang et al., 2014). The AMF can establish symbioses with the roots. In addition, the roots were carefully excavated and traced black locust and promote its growth considerably, which is essen- from the tree to ensure their identity. Soil that was both tightly and tial for sustainable soil–plant ecosystems (Yang et al., 2014, 2015). loosely bound to the surface of the root fragments was scraped off Given that AMF are important for eco-restoration on the Loess with clean tweezers and defined as rhizosphere soil to determine Plateau, it is necessary to evaluate the effect of soil and climatic fac- spore density and for soil analyses. The rhizosphere soil samples tors on their status and diversity in the rhizosphere soil under black from beneath each tree were combined to form a composite sample locust. (approximately 1 kg). This resulted in six composite samples for In the present study, we analysed the diversity of AMF in each sampling site. The soil samples were packed into resealable the rhizosphere soil under black locust on the Loess Plateau plastic bags and transported to the laboratory, where they were with morphological identification. The soil and climatic factors stored at 4∘C until analysis. The samples were split into two that affected AMF most in soil were identified to provide evi- subsamples for analysing the AMF spores and measuring the dence for the use of AMF during eco-restoration of the Loess soil properties. The fine roots were washed gently with running Plateau. tap water and then immersed in formaldehyde-acetic acid alcohol

(50% ethanol, 5% glacial acetic acid and 3.7% formaldehyde) to Analysis of soil properties fix the tissues before determining the mycorrhizal colonization. Soil properties were determined on subsamples of soil that were air-dried at 15–25∘C to constant weight and then passed through Measurement of mycorrhizal colonization a 1-mm sieve to remove coarse roots, stones and other litter. Soil water content (SWC) was determined gravimetrically by drying the Mycorrhizal colonization was measured following Phillips & samples at 105∘C for 24 hours (Gardner & Klute, 1986). Soil pH Hayman (1970), but with some minor modifications. The fine was measured with a digital pH meter (PHS-3D, Leici, Shanghai, roots were washed gently again with tap water to remove the China) using a 1:5 (10 g soil and 50 ml water) soil : water suspension formaldehyde-acetic acid alcohol, and were cut into segments of (Peech, 1965). Soil organic carbon (SOC) was determined by oxi- about 1 cm. The segments were bleached with 10% KOH at 90∘C dization with potassium dichromate (K Cr O ) in the presence of for 1 hour, softened with alkalized H O for 20 minutes, acidified 2 2 7 2 2 concentrated sulphuric acid (H SO ) (Nelson & Sommers, 1982). with 1% HCl at room temperature for 24 hours, stained with trypan 2 4 Total nitrogen (TN) was estimated according to the semi-micro blue solution (500 ml glycerol, 50 ml 1% HCl, 0.05% trypan blue Kjeldahl method (Bremner & Mulvaney, 1982). Nitrate nitrogen and 450 ml water) at 90∘C for 1 hour, and finally destained with (NO −-N) and ammonium nitrogen (NH +-N) were extracted with lactic acid–glycerin (1:1, 10 ml lactic acid and 10 ml glycerin) at 3 4 2 m potassium chloride (KCl) and analysed with the flow injec- room temperature for at least 24 hours. Mycorrhizal colonization tion analyser FIAStar 5000 (Foss Tecator AB, Höganäs, Sweden) was measured by the grid-line intersection method under a light (Raave et al., 2014). Total phosphorus (TP) was determined col- microscope and defined as the percentage of root length colonized orimetrically after wet digestion of soil samples with hydrofluo- (Giovannetti & Mosse, 1980). ric and perchloric acids (HF–HClO4) (Jackson, 1958). Available phosphorus (AP) was extracted with 0.5 m sodium bicarbonate

Extraction and analysis of AMF spores (NaHCO3; pH 8.5) for 30 minutes and estimated colorimetrically with a spectrophotometer (UVmini-1240, Shimadzu, Kyoto, Japan) The AMF spores were isolated from subsamples (100 g) of the (Page, 1982). For total potassium (TK), the soil was digested with fresh soil by a wet-sieving and decanting method (Gerdemann & HF–HClO4, and for available potassium (AK), it was extracted with Nicolson, 1963). The AMF spores were observed with a stereo- 1 m ammonium acetate (CH3COONH4;pH7).TotalKandAKwere scopic microscope and quantified by the grid-line method at determined with a PerkinElmer flame photometer (PerkinElmer, 40 times magnification (Gerdemann & Nicolson, 1963). Before Oakbrook, IL, USA) (Oyedele et al., 2008). identification, the spores were mounted on slides using polyvinyl alcohol with Melzer’s reagent (Morton, 1988). The spores were Data analysis identified by their morphological characteristics, such as spore wall structure (i.e. number of layers, colour, size, flexibility, Data were tested for normality and homogeneity of variances. If refractivity, ornamentation, histological reactivity, and so on), the data were normally distributed and met the assumption of subtending hyphae and germination structures, according to taxo- homogeneity of variances, the comparison between multiple groups nomic criteria from Oehl et al. (2011a,2011b,2011c), the Manual was done with a one-way analysis of variance (anova) and Fisher’s for the Identification of VA Mycorrhizal Fungi (Schenck & Pérez, least significant difference (LSD) at a significance level of P < 0.05. 1990) and data from INVAM (International Culture Collection of If the variances were not homogeneous, the comparison between Arbuscular and Vesicular-Arbuscular Mycorrhizal Fungi, 2010) multiple groups was done with a nonparametric test. Probability (http://www.invam.caf.wvu.edu/taxonomy). The names used for values, P, less than 0.05 were considered statistically significant. the AMF species identified conform with the Index Fungorum To examine the multivariate relations between the soil, climatic (2008) (http://www.indexfungorum.org). variables and relative abundance of AMF species, redundancy The following indices were calculated for each soil sample: analysis (RDA) was carried out with soil and climate-related traits (i) spore density (the total number of spores in 100 g fresh soil), as the predictor variables and relative abundance of AMF species (ii) frequency of occurrence (the percentage of samples that con- as the response variables with Canoco 4.5 (Centre for Biometry, tained a particular AMF species), (iii) AMF species richness Wageningen, the Netherlands). (SR; the population of AMF species forming spores in each soil Pearson’s correlation coefficients were calculated to evaluate sample), (iv) relative spore abundance (the number of spores of a the relations among the soil and climatic factors, mycorrhizal species or genus/total number of spores in a sample) and (v) the colonization, spore density, AMF SR and the Shannon–Wiener Shannon–Wiener diversity index (H′) (Shannon & Weaver, 1949): diversity index at a significance of P < 0.05 with spss 17.0 and sas 8.0 (SAS Institute Inc., Cary, NC, USA). n ∑ ( ) ( ) Path analysis is a technique used to examine the strength of H′ =− n ∕N ln n ∕N ,(1) i i indirect and direct relations among variables. To examine the effects i=1 of soil and climatic factors on AMF, we carried out path analysis where ni is the number of individuals of AMF species i and N is the with soil and climate-related traits as the predictor variables and total number of individuals of all AMF species. AMF-related indices as the response variables. The correlation

(a) (b) 100

1.5 80 1.7

3.6 2.7 1.8 60 1.4

20 Mycorrhizal colonization /%

0 FG SM YL MZ YA AS Study site

Figure 1 Mycorrhizal colonization (a) and spore density (b) in the rhizosphere soil under black locust at each study site in the Loess Plateau (northern Shaanxi province). Sampling sites: FG, Fugu; SM, Shenmu; YL, Yulin; MZ, Mizhi; YA, Yan’an; AS, Ansai. Values are means (n = 6). Values on the error bar represent the standard error of the colonization and spore density. coefficients were decomposed into indirect and direct effects by are no marked differences in mean annual temperature among path analysis; indirect coefficients are those parts of a variable’s the six study sites. The mean annual precipitation ranged from total effect that are affected by intervening variables and the direct 406.9 mm (site YL) to 531.0 mm (site YA), and increased in the coefficient of one variable on another is that part of its total effect order YL < SM < MZ < FG < AS < YA. that is not affected by intervening variables (Pattanaik et al., 2014). The AMF status can be affected by factors such as growth stages of black locust, soil and climate. Mycorrhizal colonization observed < Results at sites FG and MZ was larger than at the other sites (P 0.05); it was 83.1% at FG and 74.7% at MZ (Figure 1a). However, there Soil and climatic factors were no significant differences in mycorrhizal colonization among > The main characteristics of the soil determined from the samples the other four sites (SM, YL, YA and AS; P 0.05). The spore taken from the six study sites are given in Table S2 in Supporting density in the fresh soil ranged from 60 spores per 100 g in FG to Information. The soil samples were slightly alkaline, with pH 413 spores per 100 g in SM; the spore density decreased in the order > > > > > between 7.3 (site SM) and 7.8 (site AS). Site YA had the largest SM MZ YA AS YL FG (Figure 1b). SWC (16.58%), SOC (7.76 g kg−1), AP (14.69 mg kg−1)andTN A total of 23 morphologically distinctive AMF species were (1.11 g kg−1) concentrations, whereas the largest TP (1.86 g kg−1), recorded at the six sites; these included one species of the genus −1 + −1 Scutellospora, one of Sclerocystis, one of Ambispora,twoof AK (13.11 mg kg )andNH4 -N (14.40 mg kg ) concentrations − Septoglomus,twoofClaroideoglomus,twoofAcaulospora,three were from the soil at site MZ. The largest TK and NO3 -N concentrations were observed at sites SM (17.02 g kg−1)andAS of Glomus,fiveofRhizophagus and six species of Funneliformis (22.25 mg kg−1), respectively. (Table 1). The most frequently observed genus was Funneliformis There were small, but significant, differences in SWC, AP, (83.3%), followed by Rhizophagus (55.6%). Among the 23 species − + of AMF observed, the frequency of occurrence varied from 5.6 NO3 -N, NH4 -N and TN among the study sites. There were no significant differences in soil pH, SOC, TP and TK among to 75.0%; the species that occurred in the largest proportion three of the sites, MZ, YA and AS (P > 0.05), and there were of soil samples was Rhizophagus intraradices (75%), followed no significant differences in soil TP and AK among the other by Funneliformis dimorphicum (66.7%), Rhizophagus aggregatum three sites, FG, SM and YL (P > 0.05). There was consider- (63.9%) and Funneliformis geosporum (61.1%). Among the six able variation in SWC among the six sites, which followed the sites, the maximum AMF SR was recorded at site SM (13), followed pattern FG < SM < MZ < YL < AS < YA (Table S2 in Supporting by sites MZ (10) and YL (9). Information). The spores of four AMF species were isolated from the rhizo- The mean annual temperature and precipitation (2006–15) at sphere soil under black locust (Figure S2 in Supporting Informa- each site are given in Figure S1 in Supporting Information. There tion): Claroideoglomus etunicatum (W.N. Becker & Gerdemann)

Table 1 Presence and frequency of arbuscular mycorrhizal fungi (AMF) genera and species in the rhizosphere soils under black locust at each study site

Study site Frequency of Genera and species of AMF FG SM YL MZ YA AS occurrence / %

Acaulospora 13.9 Acaulospora denticulate Sieverding & S. Toro −− +− −− 5.6 Acaulospora rehmii Sieverding & S. Toro −+ ++ +−27.8 Ambispora 8.3 Ambispora leptoticha (N.C. Schenck & G.S. Smith) C. Walker, Vestberg & A. Schüßler −+ −− −− 8.3 Claroideoglomus 13.9 Claroideoglomus claroideum (N.C. Schenck & G.S. Smith) C. Walker & A. Schüßler −− −+ −− 8.3 Claroideoglomus etunicatum (W.N. Becker & Gerdemann) C. Walker & A. Schüßler −+ ++ −+55.6 Funneliformis 83.3 Funneliformis coronatum (M. Giovannetti) C. Walker & A. Schüßler −+ +− −− 8.3 Funneliformis dimorphicum (Boyetchko & J.P. Tewari) Oehl, G.A. Silva & Sieverding −+ −+ −−66.7 Funneliformis geosporum (T.H. Nicolson & J.W. Gerdemann) C. Walker & A. Schüßler −+ ++ ++61.1 Funneliformis monosporum (J.W. Gerdemann & Trappe) Oehl, G.A. Silva & Sieverding −− −− ++55.6 Funneliformis mosseae (T.H. Nicolson & J.W. Gerdemann) C. Walker & A. Schüßler +− −+ −+ 8.3 Funneliformis multiforum (Tadych & Błaszkowski) Oehl, G.A. Silva & Sieverding −+ +− −−50.0 Glomus 22.2 Glomus microcarpum Tulasne & Tulasne −− −− +−27.8 Glomus multicaule J.W. Gerdemann & B.K. Bakshi −− +− −− 8.3 Glomus reticulatum Bhattacharjee & Mukerji ++ −− −−33.3 Rhizophagus 55.6 Rhizophagus aggregatum N.C. Schenck & G.S. Smith ++ −− −+63.9 Rhizophagus diaphanus (C. Cano & Y. Dalpé) C. Walker & A. Schüßler −− −+ −− 5.6 Rhizophagus fasciculatum (Thaxter) C. Walker & A. Schüßler ++ +− −+36.1 Rhizophagus intraradices (N.C. Schenck & G.S. Smith) C. Walker & A. Schüßler ++ ++ ++75.0 Rhizophagus manihotis (R.H. Howeler, Sieverding & N.C. Schenck) C. Walker & A. Schüßler −− −+ −− 5.6 Sclerocystis 2.8 Sclerocystis rubiforme (J.W. Gerdemann & Trappe) R.T. Almeida & N.C. Schenck −+ −− −− 5.6 Scutellospora 8.3 Scutellospora rubra Sturmer & J.B. Morton −− ++ +−27.8 Septoglomus 11.1 Septoglomus constrictum (Trappe) Sieverding, G.A. Silva & Oehl −− −+ ++27.8 Septoglomus deserticola Trappe, Bloss & J.A. Menge −+ −− −+ 8.3 Species richness 51391068

+,present;−, absent. Sampling sites: FG, Fugu; SM, Shenmu; YL, Yulin; MZ, Mizhi; YA, Yan’an; AS, Ansai.

C. Walker & A. Schüßler (Figure S2a in Supporting Information), (P < 0.05). The AMF SR was positively correlated with soil AK Rhizophagus manihotis (R.H. Howeler, Sieverding & N.C. (P < 0.01) and negatively correlated with H′, soil pH, AP, annual Schenck) C. Walker & A. Schüßler (Figure S2b in Support- temperature and precipitation (P < 0.05). The Shannon–Wiener ing Information), Funneliformis coronatum (M. Giovannetti) C. diversity index (H′) was positively correlated with soil TK, annual Walker & A. Schüßler (Figure S2c in Supporting Information) and temperature and precipitation, and negatively correlated with SOC, + < Rhizophagus aggregatum (N.C.Schenck&SmithG.S.)C.Walker AK and NH4 -N (P 0.05). & A. Schüßler (Figure S2d in Supporting Information).

Path analysis Correlation analysis Although the correlation coefficients give the relations between Mycorrhizal colonization was positively correlated with soil AK AMF indices and soil and climatic factors, they do not reveal − and negatively correlated with spore density, SR, pH, NO3 -N, TN the direct and indirect effects of different soil and climatic and annual precipitation (P < 0.05; Table 2). Spore density was factors on AMF indices, whereas path analysis partitions the − + positively correlated with soil SR,TP,NO3 -N, NH4 -N, TN and correlations into their direct and indirect effects (Table S3 in annual precipitation, and negatively correlated with H′ and soil pH Supporting Information). Table S3 in Supporting Information,

Table 2 Correlation coefficients between soil and climatic factors and myc- –1.5 orrhizal colonization, spore density, arbuscular mycorrhizal fungi (AMF) species richness and Shannon–Wiener diversity index among the six study sites S13 Group 3 P S12 T Variable MC SD SR H′ NO – 3 AP MC 1.000 TN TP S1 − + SD 0.618 1.000 NH4 pH SR −0.388 0.733 1.000 SOC S3 Group 2 H′ −0.250 −0.377 −0.556 1.000 S11 S10 − pH −0.265 −0.300 0.668 −0.018 S2 S5 S7 SOC −0.314 0.262 −0.170 −0.375 TKS4

− RDA axis 2 (24.1%) AP 0.180 −0.013 0.630 0.198 Group1 S8 TP 0.020 0.400 0.049 −0.221 S6 S9 AK 0.370 0.058 0.537 −0.518 AK TK 0.180 −0.280 0.165 0.363 − − NO3 -N 0.501 0.500 0.171 −0.070 + NH4 -N −0.288 0.396 −0.085 −0.421 TN −0.574 0.808 0.228 −0.191 –1.5 T 0.038 −0.020 −0.467 0.806 –1.5 1.5 RDA axis 1 (54.5%) P −0.268 0.323 −0.464 0.602

MC, mycorrhizal colonization; SD, spore density; SR, AMF species rich- Figure 2 Plot of the relative abundance of arbuscular mycorrhizal fungi ness; H′, the Shannon–Wiener diversity index; SOC, soil organic carbon; (AMF) species and soil and climatic factors in the plane of the first two AP, available phosphorus; TP, total phosphorus; AK, available potassium; axes from redundancy analysis (RDA). The lengths of the arrows indicate − + TK, total potassium; NO3 -N, nitrate nitrogen; NH4 -N, ammonium nitro- the relative importance of each soil and climatic factor in explaining the gen; TN, total nitrogen; T, mean annual temperature (2006–15); P, mean relative abundances of the AMF species, whereas the angles between the annual precipitation (2006–15). Bold is P < 0.01 and italic P < 0.05 values. arrows indicate the degree to which they are correlated. The red arrows Correlations above 0.75 are strong, 0.35–0.75 moderate and less than 0.35 ( ) show the soil and climatic factors (pH; SOC, soil organic carbon; weak. AP, available phosphorus; P, mean annual precipitation (2006–15); T, mean annual temperature (2006–15); TP, total phosphorus; AK, available potas- − + for mycorrhizal colonization, shows that the contribution of the sium; TK, total potassium; NO3 -N, nitrate nitrogen; NH4 -N, ammo- soil and climatic factors follows the pattern AP > TP > AK > nium nitrogen; and TN, total nitrogen. The blue arrows ( ) show the + > > > > > − > AMF species analysed (S1, Acaulospora rehmii;S2,Glomus microcarpum; NH4 -N TK SOC temperature precipitation NO3 -N pH > TN, and that six of these were involved directly with S3, Scutellospora rubra;S4,Glomus reticulatum;S5,Rhizophagus fasci- mycorrhizal colonization (AP, TP, AK, TN, temperature and pre- culatum;S6,Claroideoglomus etunicatum;S7,Funneliformis geosporum; cipitation). The contributions of each soil and climatic factor to S8, Funneliformis dimorphicum;S9,Funneliformis multiforum; S10, Rhi- zophagus aggregatum; S11, Rhizophagus intraradices; S12, Septoglomus spore density were very different from those of mycorrhizal colo- constrictum; S13, Funneliformis monosporum). Groups are based on soil nization. For example, the direct path coefficient of AP was 0.846 and climatic factors. for mycorrhizal colonization, but 0.212 for spore density (Table S3 in Supporting Information). In addition to pH, AP, TK, temperature and precipitation, the other three soil factors also had considerable spore density, AMF species richness and the Shannon–Wiener − diversity index (Table S3 in Supporting Information). direct effects on spore density, including AK, NO3 -N and TN (Table S3 in Supporting Information). SR For AMF , the contribution of the soil and climatic fac- Redundancy analysis − > > > > > tors followed the pattern NO3 -N AK TN SOC TP > > + > > > temperature AP NH4 -N TK precipitation pH, which is To resolve further the effects of soil and climatic factors on the most different from that observed for the Shannon–Wiener diversity frequent AMF species observed, we carried out an RDA analysis; index (TK > precipitation > pH > TN > AP > SOC > temperature > the 13 most frequent (≥ 10%) AMF species were included in the > − > > + TP NO3 -N AK NH4 -N). Six soil and climatic factors had analysis. In Figure 2, the lengths of the arrows indicate the relative − direct effects on AMF species richness (pH, TP, NO3 -N, TN, importance of each soil and climatic factor in explaining the relative temperature and precipitation), whereas the Shannon–Wiener abundances of the AMF species, whereas the angles between the − + diversity index was directly affected by AK, TK, NO3 -N, NH4 -N arrows indicate the degree to which they are correlated. On the and precipitation. RDA plot, the relative abundance of AMF species and soil and In general, most of the soil and climatic factors contributed climatic factors were included in the plane of the first two axes; directly rather than indirectly to the AMF indices. Mean annual the two canonical axes explain 78.6% of the variance observed in precipitation had the most direct effect on mycorrhizal colonization, the relative abundances of the AMF species; the first canonical axis

© 2016 British Society of Soil Science, European Journal of Soil Science, 67, 847–856 Soil and climatic factors on AMF in black locust soil 853 explains 54.5% of the variance and the second explains 24.1% of inhibit AMF hyphal elongation and spore germination (Muthuku- + the variance. Temperature, precipitation, TN, NH4 -N and AK had mar et al., 1997). Excess SWC has been reported to be unfavourable strong effects on the relative abundances of various AMF species. for the development of mycorrhizae, whereas mild soil drought Soil AK (soil and climatic factors group 1) was positively cor- has little effect on AMF infection; this might partly explain the related with (and strongly affected) Glomus microcarpum (AMF observed negative correlation between mycorrhizal colonization species, S2) (Figure 2). Soil TK (soil and climatic factors group and precipitation (Hattori et al., 2014). 2) was positively correlated with (and strongly affected) a group of Spore density is a function of the relative rates of spore germi- AMF species that includes Rhizophagus fasciculatum (S5), G. retic- nation, mycelial sporulation and spore death (Slade et al., 1987). ulatum (S4), R. aggregatum (S10) and R. intraradices (S11). Soil Precipitation appears to affect spore density, but there have been + − SOC, NH4 -N,TP,TN,pH,AP,NO3 -N, temperature and precipi- conflicting observations in the literature (Aziz et al., 1995; Escud- tation (soil and climatic factors group 3) were all correlated with ero & Mendoza, 2005). Several reports suggest that because AMF a group of AMF species that included Acaulospora rehmii (S1), are obligate aerobes, excess SWC would decrease AMF sporula- Scutellospora rubra (S3), Septoglomus constrictum (S12) and Fun- tion and spore density would be negatively associated with pre- neliformis monosporum (S13). The AMF species Claroideoglomus cipitation or redox potential (Aziz et al., 1995). Others, however, etunicatum (S6), F. geosporum (S7), F. dimorphicum (S8) and F. have discovered larger values of spore density in wet than in dry multiforum (S9) occupied an intermediate position between groups soil and suggested that large sporulation is a stress response of 1 and 3 of the soil and climatic factors, although they were positively AMF to adverse soil and climatic conditions (Escudero & Mendoza, correlated with SOC (Figure 2). 2005). We observed a positive association between spore density Among the soil and climatic factors, soil TK was positively and precipitation (P = 0.021), which is consistent with the latter associated with the relative abundances of AMF species S4, S5, point of view. S10 and S11, but S2 was negatively correlated with precipitation, Several studies have proposed that large amounts of mycorrhizal − + AP, pH, NO3 -N,TP,TN,NH4 -N and SOC. Species S3 was colonization often coincide with a large spore density (Sigüenza + − positively correlated with SOC, NH4 -N, TN, TP, NO3 -N, pH, AP et al., 1996), whereas others have observed that when the spore den- and precipitation, but was negatively correlated with temperature, sity in soil is small the mycorrhizal density is large, and vice versa AK and TK. The AMF species S1, S12 and S13 were positively (Fontenla et al., 1998). We observed a negative correlation between − correlated with temperature, precipitation, AP, pH, NO3 -N, TP, spore density and mycorrhizal colonization in the semi-arid ecosys- + TN, NH4 -N and SOC, but negatively correlated with soil AK and tem (P = 0.003). The AMF spore density does not necessarily TK (Figure 2). always correlate with mycorrhizal colonization, partly because the number of spores produced does not reflect the abundance of AMF in roots. Discussion The large amount of mycorrhizal colonization and large spore Status of AMF and soil and climatic factors densities we observed demonstrate that well-established mutualistic associations exist between black locust and AMF in the In natural ecosystems, soil acidity is considered to have a major Loess Plateau, China. Yang et al. (2015) had similar results for effect on the distribution and composition of AMF communities black locust in Feng County, a semi-arid area in northwest (Dumbrell et al., 2011). In our study, mycorrhizal colonization and China. Glasshouse and field experiments have proved that myc- spore density in the rhizosphere soil under black locust had a orrhizal symbiosis protects the host plant against the adverse weak negative correlation with soil pH (r =−0.265 and −0.300, effects of infertile soil and water deficit (Yang et al., 2014). There- respectively) (Table 2). Our results support the general hypothesis fore, the formation and occurrence of AMF is important for the that AMF spore density decreases with increasing soil pH. This establishment of black locust in the semi-arid conditions of the is important, because neutral or slightly acidic soil rather than Loess Plateau. alkaline soil favours the formation and development of mycorrhizal arbuscules (Escudero & Mendoza, 2005). Although the correlation was weak between soil acidity and the Shannon–Wiener diversity Mycorrhizal colonization, spore density and precipitation index, pH had an effect on the relative abundance of certain AMF Mycorrhizal colonization represents the percentage of root seg- species. This could partly arise from the effect of soil pH on the ments colonized by arbuscules, vesicles or hyphae (Giovannetti & germination of mycorrhizal spores, germ tube growth, root infection Mosse, 1980). In this study, precipitation was negatively correlated by hyphae and the sporulation of AMF. with mycorrhizal colonization (P = 0.0312). Poor colonization The number of AMF spores is often limited by available soil P of mycorrhizae in wetter soil could be partly because AMF are (Guo et al., 2012). In our study, the AP concentrations of the soil at assumed to require well-aerated soil; their formation and develop- the six sites were less than 15 mg kg−1, a key limiting factor for the ment might be suppressed at small oxygen concentrations (Hattori growth and development of black locust; the positive association et al., 2014). Under anaerobic conditions, a complex series of toxic between spore density and soil TP might be partly because of the substances (e.g. organic acids, H2S) are liberated, which might small concentrations of soil AP.

− + Correlations between spore density and soil NO3 -N, NH4 -N This indicates that the conditions at arid and semi-arid sites could and TN concentrations in the rhizosphere soil studied were strong stimulate the sporulation of R. intraradices and F. geosporum and positive (Table 2). Guo et al. (2012) presented similar results because of stress. for soil from a sandy ecosystem where AMF spore density was positively correlated with soil-available N. A possible explanation Expectation could be that AMF have a large nitrogen requirement for the syn- thesis of chitin and protein, the main components of their cell The identification of AMF species at the sites studied and quantifi- walls (Guo et al., 2012). Furthermore, AMF spores are considered cation of their diversity and dominance was limited to certain sporu- to be indicators of N-enriched soil, such as Paraglomus occultum lating species; therefore, late-sporulating, minor or non-sporulating (Egerton-Warburton & Allen, 2000), which largely sporulates in species might have been neglected (Tonin et al., 2001). Neverthe-

N-enriched C4 plants. By contrast, correlations between mycor- less, our morphological analysis revealed a unique AMF commu- − rhizal colonization and soil NO3 -N and TN contents were negative nity in the semi-arid study area, and path analysis demonstrated that in our study (Table 2). Under conditions where N supply is lim- precipitation had one of the strongest direct effects on AMF. There- iting, nitrogen-fixing bacteria that form symbioses with legumes fore, appropriate irrigation could contribute to the growth of AMF and non-legumes can act as ‘mycorrhization helper bacteria’, which and the host plant during eco-restoration. assists in the germination of many spores and leads to greater mycorrhizal infection of roots (Wu et al., 2005). This might partly Conclusions explain the negative correlations between mycorrhizal colonization − and NO3 -N and TN contents in the soil of this study. Our results indicated that a total of 23 AMF species were identified No significant correlations were observed between SOC and that belong to nine genera. Of these, Funneliformis was the domi- spore density, mycorrhizal colonization or AMF species richness nant AMF genus, and seven species were dominant in the coloniza- (r =−0.314, 0.262 and −0.170, respectively) (Table 2). How- tion of black locust roots at the six study sites; the species included ever, SOC was closely related to AMF diversity, with a nega- R. intraradices, R. aggregatum, F. dimorphicum, F. geosporum, tive correlation between SOC and the Shannon–Wiener diversity F. monosporum, F. multiforum and C. etunicatum. Soil and climatic index (r =−0.375) (Table 2). Soil SOC was also negatively corre- factors had a marked effect on AMF indices, and it is worth noting lated with the relative abundances of Glomus microcarpum (AMF that precipitation had a more direct effect on AMF status and diver- species, S2), G. reticulatum (S4), R. fasciculatum (S5), R. aggre- sity in the rhizosphere soil under black locust in the Loess Plateau gatum (S10) and R. intraradices (S11), but positively correlated than the other soil and climatic factors. Further studies are needed with the relative abundances of F. multiforum (S9), C. etunicatum to reveal how soil and climatic factors affect the AMF community. (S6), F. geosporum (S7), Scutellospora rubra (S3), Acaulospora rehmii (S1), Septoglomus constrictum (S12) and F. monosporum Supporting Information (S13). These results contrast with those of Guo et al. (2012), who reported that SOC had a positive relation with the spore density of The following supporting information is available in the online AMF in semi-arid regions. These conflicting results indicate that the version of this article: effects of SOC on the AMF might be related to the indirect effects of Figure S1. Mean annual temperature and mean annual precipitation other factors such as host plants, stage of plant development, AMF from 2006 to 2015 for the six study sites in the Loess Plateau species and environmental conditions (Guo et al., 2012). (northern Shaanxi province). Figure S2. Spores of arbuscular mycorrhizal fungi isolated from the rhizosphere soil under black locust in the Loess Plateau, China. Dominant AMF Table S1. Descriptions of study sites. The morphological analysis revealed that 23 AMF species belonged Table S2. Characteristics of the rhizosphere soil samples taken from to nine genera, and that Funneliformis was the most extensive and soil under black locust in the Loess Plateau, China. dominant genus in the AMF community of black locust at all of Table S3. Path coefficient analysis showing the direct and indirect the study sites. Our results are consistent with a separate study that effects of soil and climatic factors on the AMF indices. showed Funneliformis was extensive in semi-arid soil (Belay et al., Table S4. Summary results from one-way analyses of variance. 2015). The Funneliformis might have been the most dominant and Table S5. Summary results of mean value and standard error. common genus in our survey because they can produce more spores in a shorter period of time than other genera such as Scutellospora Acknowledgements (Belay et al., 2015). In the arid Indian Thar Desert, Panwar & Tarafdar (2006) This research was supported by the Special Fund for Forest Sci- observed a large occurrence of R. intraradices, F. geosporum, entific Research in the Public Welfare (201404217), the National Septoglomus constrictum, R. fasciculatum, F. mosseae and Scl. Natural Science Foundation of China (31270639, 41671268) rubiforme. In our study, R. intraradices and F. geosporum were and Shaanxi Science and Technology Innovation Project Plan also the most dominant species at most of our semi-arid sites. (2016KTCL02-07).

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