Relationships Between Arbuscular Mycorrhizal Symbiosis and Soil ...

Pedosphere 25(1): 160–168, 2015 ISSN 1002-0160/CN 32-1315/P c 2015 Soil Science Society of China ° Published by Elsevier B.V. and Science Press

Relationships Between Arbuscular Mycorrhizal Symbiosis and Soil Fertility Factors in Citrus Orchards Along an Altitudinal Gradient WANG Peng1,2 , WANG Yin2 , SHU Bo1 , LIU Jin-Fa1 and XIA Ren-Xue1,∗ 1 Key

Laboratory of Horticultural Plant Biology, Ministry of Education, Huazhong Agricultural University, Wuhan 430070 (China) of Citrus Research, Zhejiang Academy of Agricultural Sciences, Taizhou 318026 (China)

2 Institute

(Received February 23, 2014; revised October 16, 2014)

ABSTRACT Arbuscular mycorrhizal (AM) symbionts are able to greatly affect soil fertility. However, the relationships between AM symbiosis development levels and citrus mycorrhizosphere soil fertility remain weakly known in field. In our study, AM colonization, spore density, hyphal length density, and glomalin-related soil protein (GRSP) content in citrus (Robertson naval orange grafted on Citrus reticulata Blanco) orchards along an altitudinal gradient were investigated seasonally in southern China. The results showed that AM colonization and abundances of spore and hyphae fluctuated significantly in different seasons and altitudes. The highest AM colonization (83.03%) was observed in orchards at 200 m above sea level in summer, spore density (16.8 spores g−1 soil) in orchards at 400 m in autumn, and hyphal length density (2.36 m g−1 soil) in orchards at 600 m orchards in summer; while the lowest values (43.60%, 2.7 spores g−1 soil and 0.52 m g−1 soil of AM colonization, spore density, and hyphal length density, respectively) were all observed in orchards at 800 m in winter. Correlation analyses demonstrated that the soil properties such as soil organic matter, alkali-hydrolyzable N, available P, and pH were significantly (P < 0.05) positively correlated with either citrus total AM colonization or the abundances of spore and hyphae. GRSP was significantly (P < 0.05) positively correlated with soil organic matter and pH. Redundancy analysis supported that soil environmental factors such as altitude, GRSP, soil organic matter, and alkali-hydrolyzable N severely (Monte Carlo permutation tests, P = 0.002) influenced AM colonization and abundances of spore and hyphae in citrus orchards. Our data demonstrated that soil environmental factors are vital in determining AM symbiosis development in citrus orchards. Key Words:

environmental factor, glomalin, hyphae, interaction, soil organic matter, spore

Citation: Wang, P., Wang, Y., Shu, B., Liu, J. F. and Xia, R. X. 2015. Relationships between arbuscular mycorrhizal symbiosis and soil fertility factors in citrus orchards along an altitudinal gradient. Pedosphere. 25(1): 160–168.

INTRODUCTION Arbuscular mycorrhizal (AM) fungi are ubiquitous component of most agroecosystems, colonizing ∼80% of the higher plants and forming symbiotic association with host roots (Smith and Read, 2008). AM fungi benefit from this association by obtaining photosynthetically fixed carbon. In return, AM fungi have beneficial impacts on plants and soils. AM symbiosis can promote host plant growth by increasing the uptake of mineral nutrition such as P, Zn, and Cu (Liu et al., 2000; Javot et al., 2007) and the resistance to biotic and abiotic stresses like drought, cold, saline, and pathogens (Aug´e, 2004; Whipps, 2004; Wu and Zou, 2010). They also play an important role in the formation and stability of soil aggregates and contribute to soil quality by producing a special immunoreactive glycoprotein, glomalin, quantified in soil as glomalinrelated soil protein (GRSP) (Wright et al., 2000; Wu et al., 2012). Their extra radical mycelia can translo∗ Corresponding

author. E-mail: peter [email protected].

cate nutrients from one host to another through a common mycelial net work (Simard et al., 1997; Robinson and Fitter, 1999). Due to their unique position at the root-soil interface, AM fungi have been described as “keystone mutualists (AM fungi species have been considered to be keystone because they are critical to mutualistic relationships with plants.)” in ecosystems (O’Neill et al., 1991). Citrus is one of the most economical fruit crops in China, indeed in the world. It has been shown that the citrus plants colonized by AM fungi had larger leaf area and higher leaf P concentration, and the tree growth and photosynthesis were more vigorous than those of non-colonized ones (Shrestha et al., 1995). Drought tolerance of trifoliate orange (Poncirus trifoliata L. Raf.) seedling inoculated with AM fungi was notably enhanced compared with non-inoculated ones (Wu et al., 2011). Wu et al. (2008) reported that inoculation of citrus seedlings with AM fungi improved the soil structure by increasing soil water-stable aggregates in a pot-

AM SYMBIOSIS AND SOIL FERTILITY FACTORS

culture experiment. Cruz et al. (2002) studied the rhizosphere network establishment of AM fungal hyphae between citrus trees and other mycorrhizal plants and reported that the citrus trees benefited when an adequate level of nutrient exchange was maintained in the network. Hence, citrus trees are fairly dependent on AM fungi to achieve a good plant growth. Numerous studies have showed that the AM symbiosis was of great importance in soil fertility directly from its influence on soil structure, organic carbon, and nutrient availability (Rillig et al., 2001; Cavagnaro et al., 2006; Wu et al., 2008) but also indirectly from its impact on soil microbial functioning (Duponnois et al., 2008). However, previous studies about AM effects on citrus fruit trees performance and mycorrhizosphere (the zone of soil influenced by both plant roots and AM fungi) environment mainly carried out in pot culture but not in field (Wang et al., 2008; Wu et al., 2012). Especially, the researches on examining the relationships between AM symbiosis development levels and citrus mycorrhizosphere soil fertility are limited. Additionally, Gai et al. (2012) reported that AM establishment was more difficult at higher altitudes. In the present study, survey was conducted to investigate AM colonization status, spore density, hyphal length density, GRSP content, and main soil fertility in order to clarify the relationships among these variables in mycorrhizosphere of citrus planted in field orchards along an altitudinal gradient. MATERIALS AND METHODS Study area The experimental sites were selected in the citrus orchards of Zigui County belonging to the Three Gorges Reservoir area, southern China. This area has a semi-tropical monsoon climate with annual sunlight of about 1 631.5 h, frost-free period of about 306 d, relative air humidity of about 72%, and mean precipitation of about 1 013.1 mm, but seasonal drought often occurs due to inconsistent rainfalls. The annual mean temperature was 16.7 ◦ C, with mean active accumulated temperature (≥ 10 ◦ C) of 5 723.6 ◦ C, mean temperature of 6.5 ◦ C in the coldest month, the highest reaching up to 42.9 ◦ C and the lowest dropping to −9.0 ◦ C in history. Citrus trees are planted according to the mountainous terrain. The calcareous purple soil (Regosol in FAO Taxonomy) accounts for 78.7% of the total plantation area, which has been eroded severely. A total of 15 orchards, each with 15 to 17-year-old citrus trees (Robertson naval orange grafted on Citrus reticulata Blanco) planted, were chosen along an

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altitudinal gradient at 200, 400, 600, 700, and 800 m above sea level (a.s.l.) and located at 110◦ 440 29.000 E, 30◦ 520 27.600 N; 110◦ 440 34.700 E, 30◦ 550 38.900 N; 110◦ 450 35.800 E, 30◦ 550 5.600 N; 110◦ 420 35.000 E, 30◦ 560 7.600 N; 110◦ 460 25.000 E, 30◦ 540 37.600 N, respectively. The consistent no-tillage management (natural grass between citrus tree rows, cutting grass to control the grass height, mulching under citrus trees, and rejecting competitive weeds) was used in all the surveyed orchards, where fertilizers (N 7%, P2 O5 4%, K2 O 4%, and organic matter 20%) were applied after fruit picking (50%) and before sprout (30%) and fruit setting (20%). The water storage tanks (20 m3 per 0.0667 ha orchard) were constructed to irrigate the orchards during the drought period, and the gravels and stones of the orchard soils were removed at the beginning of orchards establishment. Sampling Three replicated orchards were sampled at each altitude using the five point sampling mode. Three healthy citrus trees with similar growing vigor were selected as one sampling point of an orchard. Fine roots and rhizosphere soils of a single citrus tree were collected at four directions from 5–30 cm depth of the soil after removing upper vegetation within the dripping line of the tree canopy in March (spring), July (summer), October (autumn) 2009, and January (winter) 2010. Soil and root samples from each point were separately mixed thoroughly. Meanwhile, the soil samples for analyses of AM fungal spores and hyphae were air-dried for 2 weeks, passed through a 2-mm mesh screen, and stored at 4 ◦ C until analysis. Roots (Φ ≤ 1 mm) were carefully washed with tap water to remove soil, chopped into 1 cm long pieces, and fixed in FAA (formalin-acetic-alcohol, 13:5:200, v/v/v) for 24 h, and then stored at 4 ◦ C until analysis. Soil assessment Selected soil properties were analyzed using the methods described by Tan (1996). Soil total organic matter (OM) was measured by the procedure of K2 CrO7 -H2 SO4 humid oxidation, and alkali-hydrolysable N (AN) was determined by the alkaline hydrolysis diffusion method (Cornfield, 1960). Available P (Olsen P) was extracted with NaHCO3 following the Olsen method (POlsen ) (Olsen et al., 1954) and determined with spectrophotometer (UV-2450, Shimadzu, Japan) (Rodriguez et al., 1994), by reacting with (NH4 )2 MoO4 using ascorbic acid as a reductant in the presence of antimony (Murphy and Riley, 1962). Soil pH was determined using a Mettler Toledo 320 pH me-

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ter (Mettler-Toledo Ltd., Switzerland) in a suspension at the soil-to-water ratio of 1:2.5 (w/v).

metrically with a Bradford protein assay using bovine serum albumin as a standard (Rosier et al., 2006).

Determination of mycorrhizal colonization

Statistical analysis

After FAA fixation, the roots were washed several times in tap water, and cleared in 10% (w/v) KOH by heating to approximately 90 ◦ C in a water bath for 1–2 h. The cooled root samples were washed, and then stained with 0.05% (w/v) trypan blue in lactophenol. Colonization of various AM fungal structures in roots was examined according to Koske and Gemma (1989) under a compound-light microscope (OlympusBH-2, Tokyo, Japan). Fungal colonization was estimated using the magnified intersection method (McGonigle et al., 1990). Total colonization rate (RLT), arbuscular colonization rate (RLA), and vesicular colonization rate (RLV) were quantified by examining 200 intersections per sample.

Data on percentage of AM colonization were transformed by arcsin(x)1/2 . The normality of the data and the homogeneity of variances were tested before analysis of variance (ANOVA). Repeated measures ANOVA was used to test the different significance probability of AM status and soil fertility among samples collected in various sites and times by comparing the means in the SAS 9.1. Pearson’s correlation coefficients between AM symbiosis and soil fertility were performed using the Proc Corr’s procedure in the SAS. The influences of selected environmental variables, including altitude (ALT), OM, AN, POlsen , pH, and GRSP on AM symbiosis were analyzed by redundancy analysis (RDA) using the CANOCO 4.5 software (ter Braak ˇ and Smilauer, 2002).

Quantification of spores and hyphae in the rhizosphere soils Spores were extracted from soils using the wet sieving and sucrose gradient centrifugation technique (Gerdemann and Nicolson, 1963). The number of AM fungal spores with intact surface and contents and without parasitism of each sample was counted with the stereoscopic microscope (Tech-XTS-30, Beijing, China). If spores were tightly grouped in a sporocarp, the sporocarp was considered as one unit, as it was difficult to count the number of the spores in such case. The spore density (SD) was expressed as the numbers of spores and sporocarps per g dry weight of soil. Soil hyphal length density (HLD) was determined as described by Bethlenfalvay and Ames (1987). Mycorrhizal and non-mycorrhizal hyphae were distinguished under a compound-light microscope according to Miller et al. (1995) using similar criteria as for internal hyphae. Hyphal lengths were determined with the aid of an ocular micrometer, and the lengths of hyphae per g dry weight of soil were calculated.

RESULTS Soil characteristics The contents of OM, AN, and POlsen increased in orchards from 200 m a.s.l. to 600 m a.s.l., and then declined (Fig. 1a, b, c). The pH values decreased with increasing altitude in each season (Fig. 1d). The higher OM and AN were found in the warm and wet (summer and autumn) seasons (Fig. 1a, b), but the higher POlsen and pH were observed in the cold and dry (spring and winter) seasons at each altitude (Fig. 1c, d). The values of OM ranged from 17.30 to 23.91 g kg−1 , AN from 97.42 to 140.38 mg kg−1 , POlsen from 5.23 to 25.14 mg kg−1 , and pH from 5.51 to 6.70 in all investigated orchards during the study period (Fig. 1). All the selected soil properties varied significantly in citrus orchards in different seasons and altitudes, except the OM content, which showed no obvious changes in different seasons, but significant alterations among orchards at different altitudes (Table I).

Test of GRSP in the rhizosphere soils

Arbuscular mycorrhizal colonization

Total GRSP was extracted based on the protocols of Wright and Jawson (2001). Briefly, 1.0 g of wellmixed soil was suspended in 8 ml of 50 mmol L−1 trisodium salt of citric acid (pH 8.0, adjusted with HCl). Samples were vortexed, autoclaved for 60 min at 121 ◦ C and 0.11 MPa, and centrifuged at 10 000 × g for 3 min to remove soil particles. After three cycles of extraction and centrifugation, the supernatant was clear and had a light yellow colour. The GRSP concentration in the extracts was determined colori-

In the present study, all citrus tree roots surveyed were colonized by AM fungi. The formed typical AM structures characterized AM fungi colonization levels. Intra- and inter-cellular hyphae, arbuscules, vesicles, and occasional intraradical spores were observed alone or together in citrus root tissues. The AM fungi hyphae in citrus roots were usually observed in all root samples, and the arbuscules were abundant, and sometimes occurred in clusters. However, the vesicles were rarely observed in citrus roots.


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Fig. 1 Soil organic matter (a), available N (b), available P (c), and pH (d) in citrus orchards along an altitudinal gradient. Error bars represent the standard errors of the means (n = 15). Bars with the same letter(s) are not significantly different (P < 0.05) according to least significant difference (LSD) test. SP = spring; SU = summer; AU = autumn; WI = winter. TABLE I F -values from repeated measures analysis of variance (ANOVA) for selected soil propertiesa) in citrus orchards (n = 60) Variable

Season Altitude Season × Altitude

Degree of freedom 3 4 12

F -value OM 1.35NSb) 184.65*** 0.45NS

AN

POlsen

pH

GRSP

46.39*** 42.17*** 0.36NS

94.79*** 2 040.39*** 6.49***

197.73*** 1 814.98*** 21.20***

64.73*** 86.04*** 20.05***

*, **, *** Significant at 0.05, 0.01, and 0.001 levels of probability, respectively. a) OM = soil organic matter; AN = alkali-hydrolyzable N; P Olsen = Olsen P; GRSP = glomalin-related soil protein. b) Not significant.

The RLT and RLA decreased with increasing altitude in each season, and showed the higher values in warm and wet season in orchards at different altitudes. Especially in summer, the RLT and RLA reached to the peak values in orchards at each altitude (Fig. 2a, b). The RLT ranged from 83.03% to 43.60%, and the RLA ranged from 51.59% to 4.67% in surveyed orchards during the study period. Compared with the RLT, RLV was continuously lower during the study period, varied from 18.77% to 1.19% in all surveyed orchards, and increased with the increasing altitude (Fig. 2c). The time with maximum and minimum of RLV for each altitude appeared contrary to the RLT and RLA (Fig. 2). Repeated measures ANOVA showed that RLT, RLA, and RLV fluctuated significantly with the changing seasons, and they notably differed in or-

chards at different altitudes (Table II). In synthesis, the AM symbionts associated with citrus roots in orchards located at lower altitude (< 600 m a.s.l.) were markedly better established than in orchards located at higher altitude, especially in warm and wet seasons in our investigation. Hyphal length density, spore density and GRSP content Our study showed that hyphal length density in warm and wet seasons was always higher than that in cold and dry seasons in all orchards (Fig. 3a). The peak value in summer season appeared in orchards at 600 m a.s.l., with autumn and spring at 400 m a.s.l., and winter at 200 m a.s.l. The lowest HLDs were always observed in orchards at 800 m a.s.l. in each season. Similar to HLD, spore density in warm and wet

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TABLE II F -values from repeated measures analysis of variance (ANOVA) for arbuscular mycorrhizal colonizationa) in citrus roots, spore density (SD) and hyphal length density (HLD) in citrus orchards (n = 60) Variable

Season Altitude Season × Altitude

Degree of freedom 3 4 12

F -value RLT

RLA

RLV

SD

HLD

24.71*** 196.25*** 1.00NSb)

764.09*** 60.37*** 4.78**

548.17*** 63.21*** 20.23***

124.02*** 454.18*** 8.83***

262.05*** 105.17*** 12.89***

*, **, *** Significant at 0.05, 0.01, and 0.001 levels of probability, respectively. a) RLT = total colonization rate; RLA = arbuscular colonization rate; RLV = vesicular colonization rate. b) Not significant.

Fig. 2 Arbuscular mycorrhizal colonization, i.e., total colonization rate (a), arbuscular colonization rate (b), and vesicular colonization rate (c), of different structures in citrus roots from different altitudinal orchards. Error bars represent the standard errors of the means (n = 15). Bars with the same letter(s) are not significantly different (P < 0.05) according to least significant difference (LSD) test. SP = spring; SU = summer; AU = autumn; WI = winter.

seasons was also generally higher than that in cold and

Fig. 3 Hyphal length density (a), spore density (b) and glomalin-related soil protein (GRSP) (c) content in citrus orchards along an altitudinal gradient. Error bars represent the standard errors of the means (n = 15). Bars with the same letter(s) are not significantly different (P < 0.05) according to least significant difference (LSD) test. SP = spring; SU = summer; AU = autumn; WI = winter.

dry seasons in all orchards (Fig. 3b). The highest values in different seasons were all observed in orchards


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at 400 m a.s.l., while the lowest values were present in orchards at 800 m a.s.l. in each season (Fig. 3b). The GRSP content decreased with the increasing altitude in all seasons except summer (Fig. 3c). There was no similar trend observed for GRSP seasonality in orchards located at different altitudes (Fig. 3c). The ranges of HLD, SD, and GRSP content were from 2.36 to 0.52 m g−1 soil, 16.8 to 2.7 spores g−1 soil, and 1.39 to 0.85 mg g−1 soil in all orchards during the study period. Repeated measures ANOVA showed that HLD, SD, and GRSP content highly varied throughout the seasons and remarkably differed among orchards located at different altitudes (Tables I and II). Correlation analysis Correlation analysis indicated that RLT had positive correlations with OM, pH, and GRSP at P < 0.001 level, positive correlations with AN and POlsen at P < 0.05 level. RLA positively correlated with AN and GRSP at P < 0.01 level. RLV exhibited negative correlation with AN at P < 0.01 level. Significantly positive correlations were also found between SD or HLD and OM (P < 0.01 or 0.05) or AN (P < 0.001) or GRSP (P < 0.01). GRSP positively correlated with OM at P < 0.05 level and pH at P < 0.01 level in the present study (Table III). Redundancy analysis Redundancy analysis showed that the coordinate from the first two ordination axes explained 61.1% (56.2% and 4.8% of the first and second axes, respectively) of the total variance, and the significance (Monte Carlo permutation tests) of all canonical axes was found at P = 0.002 (Fig. 4). The results indicated that soil fertility factors significantly impacted the AM symbiosis development in citrus orchards. In Fig. 4, the angle between the vectors shows degree of correlation of the environmental factor and AM symbiosis deve-

lopment. Therefore, the development of AM symbiosis except for RLV showed a negative relationship with the ALT and positive relationships with the GRSP, OM, AN, and POlsen . Among all selected environmental variables, the RDA plot revealed that the most important soil fertility factors governing AM symbiosis development were GRSP, OM, and AN in the present study. DISCUSSION Arbuscular mycorrhizas are the most important microbial symbioses for the majority of plants and influence host plants development, nutrient uptake, water relations, and above-ground productivity, especially under conditions of phosphorus-limitation (Jeffries et al., 2003). In the present study, all citrus rootstocks were well colonized by native AM fungi in orchards where the POlsen content was the lower or medium (5.23–25.14 mg kg−1 ), and the AM colonization is heavy, indicating high dependency of citrus on AMs. In the present study, the higher level of AM colonization except the RLV was observed in summer and autumn, which is in agreement with previous studies that AM colonization peaks in wet and warm season in the terrestrial ecosystems (Sig¨ uenza et al., 1996; Li et al., 2005). Kennedy et al. (2002) also proposed that the AM colonization might fluctuate with the growth of plants. Well AM colonization of roots could enhance transportation of organic nutrient from hosts to fungal partner, thereby improving hyphal extension and sporulation (Sieverding, 1991). As a result, the higher SDs and HLDs were found in wet and warm season in our investigation. It is well known that common hyphal network of AM fungi is a greatly important source of the soil organic carbon and nitrogen (Rillig et al., 2001). In the present study, the HLD and SD were positively correlated with the OM and AN, implying that higher HLD

TABLE III Pearson’s correlation coefficients between the development of arbuscular mycorrhizal symbiosisa) and different soil parametersb) in the citrus orchards (n = 60) Variable

RLT

RLA

RLV

SD

HLD

GRSP

OM AN POlsen pH GRSP

0.762*** 0.493* 0.560* 0.789*** 0.844***

0.406NSc) 0.625** 0.101NS 0.238NSs 0.679**

−0.363NS −0.595** −0.074NS −0.104NS −0.402NS

0.619** 0.693*** 0.307NS 0.405NS 0.660**

0.513* 0.768*** 0.177NS 0.203NS 0.586**

0.521* 0.294NS 0.328NS 0.628** 1.000

*, **, ***Significant at 0.05, 0.01, and 0.001 levels of probability, respectively. = total colonization rate; RLA = arbuscular colonization rate; RLV = vesicular colonization rate; SD = spore density; HLD = hyphal length density. b) OM = soil organic matter; AN = alkali-hydrolyzable N; P Olsen = Olsen P; GRSP = glomalin-related soil protein. c) Not significant. a) RLT

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Fig. 4 Ordination diagram from the redundancy analysis of the relationships between the development of arbuscular mycorrhizal symbiosis and soil environmental factors in citrus orchards. The soil environmental factors and development of arbuscular mycorrhizal are represented by dashed and solid vectors, respectively. Soil sampling sites are indicated by rhombuses. The first number (× 10) of the sampling site represents the altitude of samplings, which are numbered in sequence. RLT = total colonization rate; RLA = arbuscular colonization rate; RLV = vesicular colonization rate; SD = spore density; HLD = hyphal length density; ALT = altitude; OM = soil organic matter; AN = alkali-hydrolyzable N; POlsen = Olsen P; GRSP = glomalin-related soil protein.

and SD might contribute to OM increasment and stabilization in citrus orchards. Olsson et al. (1999) have reported that AM fungi accounted for 5%–36% of the total biomass in soil and 9%–55% of the biomass of soil microorganisms. Therefore, AM fungi are now known to be a large biomass pool (Wallander et al., 2001), and the turnover of mycorrhizal external mycelium was the dominant pathway (62%) through which carbon entered the OM pool, exceeding the input via leaf litter and fine root turnover (Godbold et al., 2006). In the present study, the POlsen was positively correlated with the RLT. In addition, the POlsen content was observed relatively lower in warm and wet seasons, which might be associated with that high affinity of AM hyphae for specific ions such as phosphate resulted in depletion of the available pool of these ions (Li et al., 1991). When extraradical hyphae stopped transporting nutrients, their protective glomalin sloughed off into the soil. In our study, positive correlations between GRSP and HLD or SD were significant, which indicated that the higher AM level was favor in maintaining of higher content of GRSP in soil (Bedini et al., 2007; Wu et al., 2012). Glomalin can attach to particles of minerals (sand, silt, and clay) and organic matter, forming clumps of soil granules, and improving aggre-

gate stability; as well as extraradical hyphae can mechanically bind soil particles together (van der Heijden et al., 2006). It has been reported that the extraradical mycelium and glomalin play a crucial role in the contribution of AM fungi to the soil structure (Miller and Jastrow, 2000; Rillig et al., 2003). In our study, the GRSP also showed significantly positive correlation with the OM. Glomalin is thought to greatly reduce soil organic matter degradation by protecting labile compounds within soil aggregates thus enhancing carbon sequestration in soil ecosystems (Wright et al., 2000; Rillig, 2004). Besides, glomalin was considered as an insoluble N-linked glycoprotein with about 37% carbon and 3%–5% nitrogen, which also contributed to C pools (Lovelock et al., 2004). Additionally, the soil pH was notably positively correlated with GRSP and RLT in the present study. The result suggested that the higher AM symbiosis level could also inhibit a decrease in soil pH resulted from acid rain and chemical fertilizer in citrus orchards. These supported that GRSP might represent a useful biochemical parameter for the assessment of biological soil fertility in citrus orchards (Bedini et al., 2007). In our study, the RDA also indicated that soil environmental factors significantly impacted the AM


symbiosis development in citrus orchards. Wang et al. (2009) also reported that long-term fixed fertilization (e.g., manure, N, P, and K) significantly influenced mycorrhizal colonization percentage and SD. In consideration of the effective functions of AM symbiosis to improve soil quality (Li et al., 2012), it is proposed that organic farming management practices like improving sod culture, applying organic fertilizer instead of chemical fertilizer, excluding pesticides and herbicides, and using diverse rotations are employed in agricultural fields, which can enhance the AM symbiosis development level, increase the benefits that AM fungi bring to this type of farming system, and then promote a low chemical input agriculture (Gosling et al., 2006; Wang et al., 2012). CONCLUSIONS The citrus tree roots were heavily colonized by indigenous AM fungi in orchards. The AM colonization associated with citrus, abundance of hyphae and spore, and GRSP content maintained in higher level in wet and warm season and lower altitude (≤ 600 m a.s.l.) orchards. Correlation analysis demonstrated that the soil fertility factors like OM, POlsen , and AN were significantly positively correlated with citrus AM development status. Meanwhile, the RDA showed that ALT, GRSP, OM, and AN significantly influenced the AM symbiosis development in citrus orchards. However, the role and details of GRSP in citrus orchards are rarely completely known. In the future, more efforts are needed to explore the AM fungi community structure, important roles of GRSP, and mechanism in citrus orchard systems. ACKNOWLEDGEMENTS This research is supported by the China Spark Program of the Ministry of Science and Technology, China (No. 2007EA760023). We thank Mr. Song Wen-Hua, Dr. Li Zhi, and Mr. Cao Yu-Jiang, Huazhong Agricultural University, for their help in sample collection. REFERENCES Aug´ e, R. M. 2004. Arbuscular mycorrhizae and soil/plant water relations. Can. J. Soil Sci. 84: 373–381. Bedini, S., Avio, L., Argese, E. and Giovannetti, M. 2007. Effects of long-term land use on arbuscular mycorrhizal fungi and glomalin-related soil protein. Agr. Ecosyst. Environ. 120: 463–466. Bethlenfalvay, G. J., and Ames, R. N. 1987. Comparison of two methods for quantifying extraradical mycelium of vesicular arbuscular mycorrhizal fungi. Soil Sci. Soc. Am. J. 51: 834– 837. Cavagnaro, T. R., Jackson, L. E., Six, J., Ferris, H., Goyal, S., Asami, D. and Scow, K. M. 2006. Arbuscular mycorrhizas,

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