The effect of mycorrhizal inoculation on the rhizosphere properties of trifoliate orange (Poncirus trifoliata L. Raf.)

Scientia Horticulturae 170 (2014) 137–142

Contents lists available at ScienceDirect

Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti

The effect of mycorrhizal inoculation on the rhizosphere properties of trifoliate orange (Poncirus trifoliata L. Raf.) Shuang Wang a , A.K. Srivastava b , Qiang-Sheng Wu a,∗ , R. Fokom c a b c

College of Horticulture and Gardening, Yangtze University, No. 88 Jingmi Road, Jingzhou 434025, Hubei, People’s Republic of China National Research Centre for Citrus, Amravati Road, Nagpur 440 010, Maharashtra, India Institute of Fisheries and Aquatic Sciences, University of Douala, PO Box 00812, Yaoundé, Cameroon

a r t i c l e

i n f o

Article history: Received 5 December 2013 Received in revised form 27 February 2014 Accepted 2 March 2014 Available online 25 March 2014 Keywords: Citrus Glomalin Mycorrhizal hyphae Soil aggregate stability Soil organic carbon

a b s t r a c t Key rhizosphere properties influenced by microorganism-mediated processes need to be identified for better understanding of their possible role in improving crop performance. This study monitored the changes in concentration of Bradford-reactive soil protein (BRSP), soil organic carbon (SOC) content, hyphal length, aggregate stability [fractal dimension (D), geometric mean diameter (GMD), and mean weight diameter (MWD)] and distribution of water-stable aggregate (WSA) in rhizosphere of trifoliate orange (Poncirus trifoliata L. Raf.) infected by five arbuscular mycorrhizal fungal species (Diversispora spurca, Glomus intraradices, Glomus mosseae, Glomus versiforme, and Paraglomus occultum). After four months of mycorrhizal inoculation, all the mycorrhizal plants showed higher shoot and root biomass but the increase was a function of the tested fungal species. The induced changes in rhizosphere properties were of much higher magnitude in mycorrhizal treatment than in non-mycorrhizal treatment. Mycorrhizal inoculation induced significant increases in the percentage of WSA at 1.00–2.00 mm size, fraction 1 of BRSP, SOC, and hyphal density, collectively aiding in improving the indices of soil aggregate stability, like GMD and MWD. Higher MWD and GMD conferred better soil structure in mycorrhizosphere of trifoliate orange. Correlation analysis further revealed that fraction 1 of BRSP as a new and more active glomalin may take part in stabilizing WSA but fraction 2 of BRSP as an older and more stable glomalin may contribute SOC pools. Our results suggest that mycorrhizal-mediated better soil aggregate stability might mainly be due to soil hyphal length, integrated with SOC and fraction 1 of BRSP. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Soil microorganisms are a major driving force in soil fertility transformations (Srivastava et al., 2002; Six et al., 2004; Wu et al., 2013b). And of them, arbuscular mycorrhizal fungi (AMF) are the ubiquitous root symbiotic fungi that belong to the phylum Glomeromycota (Schüßler et al., 2001; Smith and Read, 2008), possess a distinctive role in soil processes such as nutrient cycling and soil structural improvements (Miller and Jastrow, 2000) due to their widespread presence in soils. The rhizosphere properties governing the crop performance are characterized by changes in soil fertility (available nutrients), nature of proteins binding soil particles into different aggregate sizes (Nichols and Toro, 2011; Peng et al., 2013) commonly known as water-stable aggregate (WSA), and soil carbon pool (Rillig et al., 2001).

∗ Corresponding author. Tel.: +86 716 8066262; fax: +86 716 8066262. E-mail address: [email protected] (Q.-S. Wu). http://dx.doi.org/10.1016/j.scienta.2014.03.003 0304-4238/© 2014 Elsevier B.V. All rights reserved.

Different species or communities of AMF have been observed to promote soil aggregation to varying degrees (Schreiner et al., 1997; Bedini et al., 2009; Fokom et al., 2012). However, the mechanisms involved are still inconclusively understood. Extensive hyphae of AMF enmesh soil particles (Wilson et al., 2009; Peng et al., 2013) and increase soil water repellency (Rillig et al., 2010; Martin et al., 2012b), thus, facilitating the formation and stabilization of soil aggregates. These hyphae also release an insoluble N-linked glycoprotein, called as glomalin, which contains ∼60% carbohydrates and showed 3–10 times higher soil aggregating ability than hotwater-extractable carbohydrates (Wright and Upadhyaya, 1998). Glomalin in soils is defined as glomalin-related soil protein (GRSP) (Rillig, 2004). Studies have proven the highly positive correlation of GRSP with aggregate stability, irrespective of soil types (Spohn and Giani, 2010; Fokom et al., 2012; Wu et al., 2013a), even in soils rich in calcium carbonate (Hontoria et al., 2009). In citrus rhizosphere, we have found significantly positively correlation of GRSP with WSA at the size of 0.25–0.50 mm in field (Wu et al., 2012), and the contribution of GRSP to soil aggregate stability lied on WSA

138

S. Wang et al. / Scientia Horticulturae 170 (2014) 137–142

fractions (Wu et al., 2013a). The role of GRSP on aggregate stability appeared on only hierarchically structured soils, where soil organic matter was the main binding agent, since GRSP correlated negatively with aggregate stability in the Lithic Calciorthid soil, where carbonates were the major binding agent (Rillig et al., 2003). Recently, GRSP was classified into three fractions by Koide and Peoples (2013): fraction 1 of Bradford-reactive soil protein (BRSP) (highly labile GRSP, corresponding to easily-extractable glomalinrelated soil protein), fraction 2 of BRSP (relatively more recalcitrant in soil, corresponding to difficultly-extractable glomaln-related soil protein), and total BRSP (fraction 1 + fraction 2 = T-BSRP). Plants can synthesize specific microbial structure according to their metabolic requirement and consequently the root exudates to varying composition, thereby, influencing rhizosphere properties on various accounts (Wu and Srivastava, 2012; Srivastava, 2013). On the other hand, besides GRSP, AMF also develop an extraradical hyphae network to enmesh soil aggregates (Bedini et al., 2009), and induced more SOC to cement aggregates (Liu et al., 2014). Trifoliate orange (Poncirus trifoliata L. Raf.) is a citrus relative species and widely used as citrus rootstock for raising citrus industry in southeast Asia (Srivastava et al., 2008). Our previous studies have shown the contribution of AMF-released GRSP to aggregate stability (Wu et al., 2008, 2012, 2013a), thereby, improving water relations of trifoliate orange (Wu et al., 2008). Nevertheless, different roles of all the three BRSP fractions on aggregate stability is lesser understood in plant rhizosphere including citrus plants. On the other hand, these reports less involved in the functions of different AMF species on aggregate stability under a controlled potted conditions. The controlled potted study will provide more reliable results on understanding AM functions. In addition, it is still not known whether the AMF-mediated contribution on aggregate stability of citrus rhizosphere depended on the type of AMF species, soil hyphal length, and SOC, besides GRSP. In this context, it also remains to be seen which BRSP fraction facilitates soil aggregation process. With these objectives, the present study was carried (i) to examine the effectiveness of different AMF species on rhizosphere properties of trifoliate orange through changes in plant biomass, soil hyphal length, concentration of BRSP fractions, distribution of WSA at different sizes, and aggregate stability, and (ii) to evaluate the role of BRSP fractions, SOC and soil hyphal length mediated by mycorrhization on aggregate stability.

Gi, Gm, Gv and Po (Source: the Bank of Glomeromycota in China, Beijing, China) were mixed with the soil and the non-AMF pots received the same amount of autoclaved mycorrhizal inoculum plus 2 ml filtrate (25 ␮m) of mycorrhizal inoculums for similar microflora except the AMF. Different test species of AMF were isolated from rhizosphere of diversified crops, e.g. Ds and Gi from the rhizosphere of Lycopersicon esculentum, Gm from Incarvillea younghusbandii, Gv from Astragalus adsurgens, and Po from Prunus persica. These isolated AMF species were propagated using the identified fungal spores through a pot culture utilizing Sorghum vulgare as the host plant for 16 weeks. The AMF and non-AMF seedlings were grown in a plastic greenhouse of Yangtze University campus in Jingzhou, China (Photosynthetic photon flux density: 338–982 ␮mol m−2 s−1 ; day/night temperature: 20–35 to 15–26 ◦ C; relative air humidity: 70–95%). After four months (April 6 to August 6, 2012) of AMF inoculation, the seedlings were harvested and subjected to various analyses. 2.3. Measurements of plant and soil variables Shoots were separated from the roots, and the soils adhered with the roots were gently shook out and collected as rhizosphere soil. Fine 1-cm long root segments were cleared in 10% (w/v) KOH at 90 ◦ C for 40 min, acidified in 20 mM HCl for 10 min, and finally stained with 0.05% (w/v) trypan blue in lactophenol for determination of root AM colonization (Phillips and Hayman, 1970). The AM colonization was computed using the following formula: AM colonization (%) = root length infected/root length observed × 100. The shoots and roots were dried at 75 ◦ C for 48 h followed by dry weight measurement. Analysis of soil hyphal length was performed according to the procedure described by Bethlenfalvay and Ames (1987). Soil organic carbon (SOC) was measured using the dichromate oxidation spectrophotometric method (Rowell, 1994). Water-stable aggregates (WSA at 2.00–4.00, 1.00–2.00, 0.50–1.00, 0.25–0.50, and >0.25 mm sizes) were analyzed according to the procedure suggested by Wu et al. (2008). The parameters of soil aggregate stability, like mean weight diameter (MWD), geometric mean diameter (GMD), and fractal dimension (D) were used to evaluate WSA stability. MWD and GMD were calculated by the following formula (Kemper and Rosenau, 1986): MWD =

2. Materials and methods 2.1. Experimental design The pot experiment under controlled conditions consisted of six AMF inoculation treatments comprising, (i). Diversispora spurca ¨ (Ds) (Preiff, Walker & Bloss) Walker & Schußler, (ii). Glomus intraradices (Gi) Schenck and Smith, (iii). Glomus mosseae (Gm) Nicol. and Gerd. Gerd. and Trappe, (iv). Glomus versiforme (Gv) Karsten Berch, (v). Paraglomus occultum (Po) Walker Morton and Redecker, and (vi). non-AMF as control. Each treatment had three replicates, for a total of eighteen pots. 2.2. Plant set-up The seeds of trifoliate orange (P. trifoliata L. Raf.) were germinated on moist filter paper at 28 ◦ C for 7 days and then transferred into the plastic pot (size dimension: 15.5 cm upper diameter × 10.5 cm bottom diameter × 13 cm height) filled with autoclaved Xanthi-udic ferralsol soil (0.11 MPa, 121 ◦ C, 2 h), whose characteristics are pH 6.4, soil organic matter 9.8 g kg−1 , and OlsenP 16.6 mg kg−1 . At the time of transplanting, ∼1000 spores of Ds,

n 

 n

WiXi

and

GMD = exp

i=1



WilgXi

i=1 n Wi i=1





where, Xi and Wi stand for mean diameter of the i sieve opening (mm) and proportion of i size fraction in the total sample mass, respectively. Fractal dimension (D) was assessed by the following formula (Yang et al., 1993):



lg

¯ W (ı < di) Wo







= (3 − D)lg





¯ di d¯ max



¯ d¯ max and lg W (ı < di)/Wo ¯ using lg di/ as abscissa and ordinate to polt, respectively, with 3 − D as the slope of the experiment ¯ as the mean diamstraight line. Other symbols correspond to: di ¯ as weight of the eter of the i sieve opening aggregate, W (ı < di) particle size less than the aggregate of di , Wo as total weight of the aggregate, and d¯ max as maximum particle size of aggregate. Fraction 1, fraction 2, and total BRSP were determined following the procedure as suggested by Koide and Peoples (2013). A 0.5 g of the sieved (4 mm) soil sample was mixed with 8 mL 20 mM citrate buffer (pH 7.0), autoclaved for 30 min at 121 ◦ C (0.11 MPa), and centrifuged at 10,000 × g for 5 min. The supernatants were taken to another centrifuge tube to determine fraction 1 of BRSP. Eight mL

S. Wang et al. / Scientia Horticulturae 170 (2014) 137–142

2.4. Statistical analysis

5

(a)

4 MWD (mm)

50 mM citrate solution (pH 8.0) was added in the same soil residues, autoclaved for 60 min at 121 ◦ C, and centrifuged at 10,000 × g for 5 min. The supernatants were then used to analyze fraction 2 of BRSP. The two supernatants were analyzed for Bradford (1976) assay using bovine serum albumin as the standard. The sum of fraction 1 and fraction 2 was assigned as T-BRSP.

139

The data were analyzed by one-way variance (ANOVA) with SAS software (SAS Institute Inc, 2001). Duncan’s multiple range tests were performed to compare the significant differences among six treatments at the 5% level. Pearson correlation was applied to analyze the correlation between the different variables on the basis of Proc Corr’s procedure (SAS Institute Inc, 2001).

a

3

b

b

b

c 2

d

1

0

(b)

1.0

3. Results and discussion GMD (mm)

The test plant species, trifoliate orange, was observed to be highly dependent on all the five AMF species, as evident from 32.1 to 59.1% root colonization (Table 1). The extent of root AMF colonization ranked as the trend of Gm > Gv ≥ Po > Ds > Gi. Soil hyphal length varied from 0.105 to 0.181 m g−1 soil and followed the response pattern similar to root AMF colonization. A highly positive correlation (r = 0.97, P < 0.01) was found between root AMF colonization and soil hyphal length. AMF colonization in rhizosphere is the result of selective enrichment of the organism best adapted to the ecological niche formed by the root environment where beneficial effects have been postulated to be partially due to production of phytohormones (Bashan and Holguin, 1995). Resultantly, shoot and root dry weight of AMF-seedlings was significantly higher than that of non-AMF seedlings (Table 1). The effect of the different AMF species could be ranked as: Gm > Po > Gv ≈ Ds ≈ Gi and Gm > Gi > Po ≈ Gv ≈ Ds on the basis of shoot and root dry weight, respectively. Inoculation with Gm showed higher plant biomass than the other AMF treatments, implying that Gm is more efficient AMF species in trifoliate orange. This result is in accordance with that of Wu et al. (2008), who found that Gm was relatively more efficient AMF species in stimulating plant biomass than Gv and Glomus diaphanum. The observation also demonstrated that like other plants such as Prunus cerasifera (Berta et al., 1995) and Stylosanthes leiocarpa (Saif, 1987), AMF-induced biomass increments in trifoliate orange were completely dependent on AMF species, suggesting that the combination of AMF and trifoliate orange had the compatibility (Smith and Read, 2008). Greater shoot and root biomass of AMF plants may attribute to improvement of soil structure and/or enhancement in nutrient and water uptake (Bouwmeester et al., 2007; Nichols, 2008). Inoculation with different AMF species induced significant changes in size fractions of WSA, with Gi as an exception, as compared with non-AMF treatment (Table 2). The percentage of WSA2.00–4.00 mm fraction was the highest in Ds inoculated soils. On the other hand, AMF inoculation with Gv, Po, Gm, Gi, and Ds increased the relative percentage of WSA1.00–2.00 mm fraction by 87, 139, 152, 19, and 221%, respectively, as compared with non-AMF treatment. With reducing size fraction of WSA from WSA0.50–1.0 mm to WSA>0.25 mm , the influence of different AMF species, especially between Ds, Gm, and Gi became statistically more distinctive (Table 2). Different AMF treatments significantly increased the percentage of WSA1.00–2.00 mm size since the amount of WSA1.00–2.00 mm size is most sensitive to short-term management of soils according to Kemper and Rosenau (1986).

a d

c

b

b

a

b

bc

c

d

0.8 0.7 0.6 0.5 0.4 2.9

(c)

2.8 2.7

a d

2.6 D

3.1. AMF effects on plant biomass, distribution of WSA fractions, and aggregate stability

0.9

2.5 2.4 2.3 2.2 2.1 2.0

a es seae MF urc rme ltum d ic s n-A D. sp ntrara . mo ersifo . occu G v No i P . G G.

Fig. 1. Mean weight diameter (MWD) (a), geometric mean diameter (GMD) (b), and fractal dimension (D) (c) of water-stable aggregate in rhizosphere of trifoliate orange (Poncirus trifoliata) seedlings inoculated with or without AMF. Date (means ± SE, n = 3) followed by different letters above the bars are significantly different at P < 0.05.

AMF can potentially influence soil aggregation at different levels, namely plant communities, plant roots (individual host), and the effects are mediated by the mycelial network itself (Rillig, 2004; Rillig and Mummey, 2006). In the present study, soil aggregate stability was measured through the indicators like MDW, GMD, and D. MWD and GMD values in our studies were significantly higher, but D values lower in AMF than in non-AMF soils (Fig. 1). On the basis of MWD and GMD values, different AMF species were rated as: Ds > Gm ≈ Gv ≈ Po > Gi and Ds ≈ Po > Gm ≈ Gv > Gi (Fig. 1a and b), respectively. These observations were entirely different with respect to D values (Ds ≈ Po < Gv ≤ Gm ≤ Gi) (Fig. 1c). Following four months of AMF inoculation, increase in MWD and GMD values and decrease in D values within the rhizosphere of trifoliate orange plant (Fig. 1), was regulated through improved aggregate stability or soil structural improvement of response. Previous studies also observed such pattern from 16 weeks (Wu et al., 2008; Cho et al.,

140

S. Wang et al. / Scientia Horticulturae 170 (2014) 137–142

Table 1 Root AM colonization, soil hyphal length, and shoot and root dry weight in AMF and non-AMF trifoliate orange (Poncirus trifoliata) seedlings. Inoculation

Root AM colonization (%)

Non-AMF D. spurca G. intraradices G. mosseae G. versiforme P. occultum

0 43.0 32.1 59.1 52.2 50.8

± ± ± ± ± ±

0e 1.4c 3.5d 4.8a 1.0b 4.6b

Soil hyphal length (m g−1 ) 0 0.132 0.105 0.181 0.143 0.159

± ± ± ± ± ±

Shoot dry weight (g plant−1 )

0e 0.008c 0.015d 0.013a 0.014bc 0.005b

0.86 1.08 0.99 1.65 1.10 1.23

± ± ± ± ± ±

Root dry weight (g plant−1 )

0.05d 0.05c 0.06c 0.07a 0.04c 0.11b

0.30 0.35 0.43 0.62 0.36 0.38

± ± ± ± ± ±

0.01d 0.02c 0.02b 0.03a 0.02c 0.02c

Note: Date (means ± SE, n = 3) followed by different letters show significant differences at P < 0.05 among mycorrhizal treatments using Duncan’s multiple range test. Table 2 Effect of different AMF treatments on the percentage of soil water-stable aggregates at different sizes in rhizosphere of trifoliate orange (Poncirus trifoliata) seedlings. Inoculation

Water-stable aggregate (%) 2.00–4.00 mm

Non-AMF D. spurca G. intraradices G. mosseae G. versiforme P. occultum

0.16 0.29 0.19 0.19 0.23 0.20

± ± ± ± ± ±

0.01d 0.01a 0.01c 0.01c 0.01b 0.01c

1.00–2.00 mm 0.23 0.74 0.29 0.58 0.42 0.55

± ± ± ± ± ±

0.02d 0.08a 0.02d 0.01b 0.04c 0.01b

0.50–1.00 mm 0.60 1.16 0.77 0.78 0.83 0.74

± ± ± ± ± ±

0.25–0.50 mm

0.04c 0.06a 0.02b 0.08b 0.05b 0.08b

1.06 1.76 1.19 1.49 1.29 1.25

± ± ± ± ± ±

>0.25 mm

0.04d 0.01a 0.02c 0.08b 0.05c 0.09c

2.06 3.95 2.43 3.04 2.77 2.74

± ± ± ± ± ±

0.11e 0.08a 0.01d 0.12b 0.10c 0.19c

Note: Date (means ± SE, n = 3) followed by different letters show significantly differences at P < 0.05 among mycorrhizal treatments using Duncan’s multiple range test.

3.2. BRSP fractions BRSP fractions have been known to play a significant role in soil aggregate stability (Rillig and Mummey, 2006). Wu et al. (2008) reported a significant increase of total BRSP concentration induced by Gm, Gv, and G. diaphanum in trifoliate orange rhizosphere. In the present study, all the AMF treatments compared to non-AMF treatment, increased the concentration of different BRSP fractions viz., fraction 1, fraction 2, and total BRSP (Fig. 2a–c). The differences among five AMF species were non-significant with respect to fraction 1 of BRSP, but inoculation with Gm showed significantly higher concentration of fraction 2 of BRSP and total BRSP in comparison to other AMF species (Fig. 2). Such varying response of AMF species is ascribed to their hyphal morphological characteristics in terms of hyphal diameter, wall thickness, and branching patterns, collectively influencing the soil aggregate stability (Rillig and Mummey, 2006). Peng et al. (2011) observed direct involvement of GRSP on MWD in Purpli-Udic Cambosols, based on the path coefficient analysis. AMF species have the ability to induce branching in roots of host plants (Wu et al., 2012), that facilitates the infection points offer soil aggregate stability by AMF, thereby, greater extraradical hyphae bind soil particles to different soil aggregates (Piotrowski et al., 2004). AMF species differ in a variety of life history traits (Hart and Reader, 2002), and therefore AMF species may differ in

their ability to produce BRSPs. In the present work, however, no significant differences of fraction 1 production, occurred among AMF species. This result is in agreement with the findings of Bedini et al. (2009), who observed no significant differences among AMF strains to influence variation in fraction 1 of BRSP. The no significant differences of fraction 1 among AMF species may be due to that four months duration of the experiment was probably not enough to induce significant differences of fraction 1 among AMF species for release of new glomalin (namely fraction 1). 3.3. Changes in SOC Soil organic carbon (SOC) is observed pivotal to decline in soil health and an important index of rhizosphere quality assessment (Srivastava et al., 2002). All the AMF inoculation profoundly increased the SOC concentration in the plant rhizosphere compared to non-AMF treatment due to improvements in soil hyphal length and BRSP concentration (Fig. 3). An improvement in SOC to the extent of 42, 44, 56, 58, and 74% was observed with Gi, Ds, Po, Gv, and Gm, respectively, as compared with non-AMF treatment.

2.0 BRSP concentration (mg g -1 dry soil)

2009), but not from only 49 days (Martin et al., 2012a) after AMF inoculation. Leifhei et al. (2014) using a meta-analysis showed that duration of inoculation had significant influence on stability of WSA and the influence started at 2.2 months. Our study had performed four months of AMF inoculation, and the AMF-mediated improvement of aggregate stability in rhizosphere of trifoliate orange was significant. AMF species reporting improved WSA stability through increase in WSA2.00–4.00 mm size fraction with G. etunicatum, Gm, and Gigaspora rosea (Schreiner et al., 1997). Bedini et al. (2009) in this context, observed that not only interspecific (Gi versus Gm) but also intraspecific species of AMF (different isolates of Gi) displayed a differential ability towards soil aggregate stability. Rillig and Mummey (2006), in their review on mycorrhizas and soil structure, suggested a suite of mechanisms by which AMF can influence soil aggregation. By extension of these mechanisms to the question of fungal diversity, it was recognized that different species or communities of fungi can promote soil aggregation to different degrees.

Non-AMF D. spurca G. intraradices G. mosseae G. versiforme P. occultum

1.5

c

a bb b b

1.0 b

aaaaa cbb

0.5

abb

0.0 Fraction 1

Fraction 2

Total

Fig. 2. Fraction 1, fraction 2 and total (fraction 1 + fraction 2) of Bradford-reactive soil protein (BRSP) concentrations in rhizosphere of trifoliate orange (Poncirus trifoliata) seedlings inoculated with or without AMF. Date (means ± SE, n = 3) followed by different letters above the bars are significantly different at P < 0.05.

S. Wang et al. / Scientia Horticulturae 170 (2014) 137–142 Table 3 Correlation coefficients among part variables involved in aggregate stability (n = 18).

Fraction 1 of BRSP Fraction 2 of BRSP Total of BRSP SOC Hyphal length

MWD

GMD

D

SOC

Hyphal length

0.53* 0.42 0.45 0.51* 0.71**

0.52* 0.41 0.44 0.58* 0.60**

–0.48* –0.34 –0.48* –0.56* –0.70**

0.60** 0.66** 0.77** 1.00 0.93***

0.70** 0.70** 0.85*** 0.93*** 1.00

Abbreviation: Bradford-reactive soil protein, BRSP; fractal dimension, D; geometric mean diameter, GMD; mean weight diameter, MWD; soil organic carbon, SOC. * Indicate significant differences at P < 0.05. ** Indicate significant differences at P < 0.01. *** Indicate significant differences at P < 0.001.

Miller and Kling (2000) reported that 15% SOC was derived from AMF out of total SOC pool in the grassland. Therefore, Cheng et al. (2012) proposed that AMF obtain up to 20% of net plant photosynthates, contribute SOC pools through the turnover of mycorrhizal hyphae, GRSP, and chitin. However, the stimulation of AMF to soil C sequestration could be reversed under elevated atmospheric CO2 , resulting in considerable soil C losses. 3.4. Correlation analysis Soil aggregation has important influences on soil C storage. In the light of their abundance in rhizosphere, AMF directly access to plant C and hyphal growth for long-term aggregate stabilization (Miller and Jastrow, 2000). Correlation analyses showed that MWD and GMD values as an indicator of soil aggregate stability were significantly positively correlated with hyphal length, agreed with the results of Bedini et al. (2009). Soil mycorrhizal hyphae can enmesh soil particles and thus bind microaggregates into macroaggregates, primarily resulting in stabilizing macroaggregates (Degens et al., 1996; Bearden and Petersen, 2000; Peng et al., 2013). The present study also showed that SOC was highly positively correlated with the GMD and MWD values and negatively with the D values (Table 3), demonstrating the important role of SOC on soil aggregate stability (Tisdall and Oades, 1982). Studies showed that GRSP presented a direct effect on soil aggregate stability (Peng et al., 2011). In field, fraction 1 and total BRSP were significantly positively correlated with WSA only at size of 0.25–0.50 mm in citrus rhizosphere (Wu et al., 2012). Our study further indicated that, in BRSP fractions, only fraction 1 was significantly positively correlated with MWD and GMD values and

SOC concentration (mg g

-1

dry soil)

16 14 12 cd

d

b

bc

8 e 6 4

4. Conclusion The response of trifoliate orange was observed highly dependent on AMF species, which largely depended on changes in WSA sizes with consequent improvements in MWD, GMD, fraction 1 of BRSP and SOC. AMF-mediated aggregate stability in trifoliate orange rhizosphere was related with fraction 1 but not fraction 2, soil hyphal length, and SOC. Fraction 1 as a new and more active glomalin may take part in stabilizing WSA but fraction 2 as an older and more stable glomalin may contribute SOC pools. Such AMF-induced changes in rhizosphere activated hyphal growth through acquisition of nutrients mobilized towards plant roots. Additional data on the thresholds of these rhizosphere properties would provide better insight in understanding the rhizosphere-host plant-AMF relationship, thereby, offering better opportunities for the engineering rhizosphere environment according to plant metabolic demand.

This study was supported by the National Natural Science Foundation of China (31372017), the Key Project of Chinese Ministry of Education (211107), and the Key Project of Natural Science Foundation of Hubei Province (2012FFA001). References

2 0

negatively with D (Table 3). In general, fraction 1 of BRSP is newly produced and relatively more labile, and fraction 2 of BRSP is older and more stable in soil (Koide and Peoples, 2013). We guess that the fraction 1 as the more active glomalin might take part in stabilizing WSA but the fraction 2, as an older and more stable glomalin, might contribute SOC pools. A higher correlation coefficient between fraction 2 and SOC (r = 0.66) than between fraction 1 and SOC (r = 0.60) showed a greater potential role of fraction 2 on SOC contribution than fraction 1. In addition, different fractions of BRSP viz., fraction 1, fraction 2 and total were significantly positively correlated with hyphal length and SOC (Table 3), since BRSPs originated from a component of hyphae and/or spore walls (Driver et al., 2005) and contributed 15% of total SOC pool (Miller and Kling, 2000). These observations supported the fact that AMF have a definite role in increase of SOC via various secretions and extraradical hyphae loaded into the rhizosphere. According to Rillig and Mummey (2006), rhizodeposition of organic C by the roots of host plant can fuel microbial activity that in turn, contributes largely to the soil aggregate formation and stabilization. In addition, the correlation (r = 0.71) between MWD and soil hyphal length was stronger than between MWD and fraction 1 (r = 0.53) or SOC (r = 0.51), in the present study. Peng et al. (2011) also found higher correlation between MWD and soil hyphal length than between MWD and fraction 1 in Gi- and Gm-infected Triticum aestivuml plants. Moreover, the effects of soil hyphal length on MWD was higher than that of root volume (Bedini et al., 2009). Therefore, we concluded that AMF-mediated aggregate stability mainly ascribed to soil hyphal length, combined with SOC and fraction 1, but not fraction 2.

Acknowledgements

a

10

141

a es sseae rme tum MF urc dic l o n-A D. sp ntrara G. mo versif . occu o N . i P . G G

Fig. 3. Soil organic carbon (SOC) concentration in rhizosphere of trifoliate orange (Poncirus trifoliata) seedlings inoculated with or without AMF. Date (means ± SE, n = 3) followed by different letters above the bars are significantly different at P < 0.05.

Bashan, Y., Holguin, G., 1995. Inter-root movement of Azospirillum brasilense and subsequent root colonization of crop and weed seedlings growing in soil. Microbiol. Ecol. 29, 269–281. Bearden, B.N., Petersen, L., 2000. Influence of arbuscular mycorrhizal fungi on soil structure and aggregate stability of a verticol. Plant Soil 218, 173–183. Bedini, S., Pellegrino, E., Avio, L., Pellegrini, S., Bazzoffi, P., Argese, E., Giovannetti, 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. Berta, G., Trotta, A., Fusconi, A., Hooker, J.E., Munro, M., Atkinson, D., Giovannetti, M., Morini, S., Fortuna, P., Tisserant, B., Gianinazzi-Pearson, V., Gianinazzi, S.,

142

S. Wang et al. / Scientia Horticulturae 170 (2014) 137–142

1995. Arbuscular mycorrhizal induced changes to plant growth and root system morphology in Prunus cerasifera. Tree Physiol. 15, 281–293. Bethlenfalvay, G.J., 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. Bouwmeester, H.J., Roux, C., Lopez-Raez, J.A., Becard, G., 2007. Rhizosphere communication of plants, parasitic plants and AM fungi. Trends Plant Sci. 12, 224–230. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Cheng, L., Booker, F.L., Tu, C., Burkey, K.O., Zhou, L.S., David Shew, H., 2012. Arbuscular mycorrhizal fungi increase organic carbon decomposition under elevated CO2 . Science 337, 1084–1087. Cho, E.J., Lee, D.J., Wee, C.D., Kim, H.L., Cheong, Y.H., Cho, J.S., Sohn, B.K., 2009. Effects of AMF inoculation on growth of Panax ginseng C.A. Meyer seedlings and on soil structures in mycorrhizosphere. Sci. Hortic. 122, 633–637. Degens, B.P., Sparling, G.P., Abbott, L.K., 1996. Increasing the length of hyphae in a sandy soil increases the amount of water-stable aggregates. Appl. Soil Ecol. 3, 149–159. Driver, J.D., Holben, W.E., Rillig, M.C., 2005. Characterization of glomalin as a hyphal wall component of arbuscular mycorrhizal fungi. Soil Biol. Biochem. 37, 101–105. Fokom, R., Adamou, S., Teugwa, M.C., Begoude Boyogueno, A.D., Nana, W.L., Ngonkeu, M.E.L., Tchameni, N.S., Nwaga, D., Tsala Ndzomo, G., Amvam Zollo, P.H., 2012. Glomalin related soil protein, carbon, nitrogen and soil aggregate stability as affected by land use variation in the humid forest zone of south Cameroon. Soil Tillage Res. 120, 69–75. Hart, M.M., Reader, R.J., 2002. Taxonomic basis for variation in the colonization strategy of arbuscular mycorrhizal fungi. New Phytol. 153, 335–344. Hontoria, C., Velásquez, R., Benito, M., Almorox, J., Moliner, A., 2009. Bradfordreactive soil proteins and aggregate stability under abandoned versus tilled olive groves in a semi-arid calcisol. Soil Biol. Biochem. 41, 1583–1585. Kemper, W.D., Rosenau, R., 1986. Aggregate stability and size distribution. In: Klute, A. (Ed.), Methods of Soil Analysis, Part 1. Physical and Mineralogical Methods. Agronomy Monograph. American Society of Agronomy and Soil Science Society of America, US, pp. 425–442. Koide, R.T., Peoples, M.S., 2013. Behavior of Bradford-reactive substances is consistent with predictions for glomalin. Appl. Soil Ecol. 63, 8–14. Leifhei, E.F., Veresoglou, S.D., Lehmann, A., Kathryn Morris, E., Rillig, M.C., 2014. Multiple factors influence the role of arbuscular mycorrhizal fungi in soil aggregation-a meta-analysis. Plant Soil 374, 523–537. Liu, M.Y., Chang, Q.R., Qi, Y.B., Liu, J., Chen, T., 2014. Aggregation and soil organic carbon fractions under different land uses on the tableland of the Loess Plateau of China. Catena 115, 19–28. Martin, S.L., Mooney, S.J., Dickinson, M.J., West, H.M., 2012a. The effects of simultaneous root colonisation by three Glomus species on soil pore characteristics. Soil Biol. Biochem. 49, 167–173. Martin, S.L., Mooney, S.J., Dickinson, M.J., West, H.M., 2012b. Soil structural responses to alterations in soil microbiota induced by the dilution method and mycorrhizal fungal inoculation. Pedobiologia 55, 271–281. Miller, R.M., Jastrow, J.D., 2000. Mycorrhizal fungi influence soil structure. In: Kapulnik, Y., Douds, D.D. (Eds.), Arbuscular mycorrhizas: molecular biology and physiology. Kluwer Academic, Dordrecht, The Netherlands, pp. 3–8. Miller, R.M., Kling, M., 2000. The importance of integration and scale in the arbuscular mycorrhizal symbiosis. Plant Soil 226, 161–196. Nichols, K.A., 2008. Indirect contributions of AM fungi and soil aggregation to plant growth and protection. In: Mycorrhizae: Sustainable Agriculture and Forestry. Springer, The Netherlands, pp. 177–194. Nichols, K.A., Toro, M., 2011. A whole soil stability index (WSSI) for evaluating soil aggregation. Soil Tillage Res. 111, 99–104. Peng, S.L., Guo, T., Liu, G.C., 2013. The effects of arbuscular mycorrhizal hyphal networks on soil aggregations of purple soil in southwest China. Soil Biol. Biochem. 57, 411–417. Peng, S.L., Shen, H., Yuan, J.J., Wei, C.F., Guo, T., 2011. Impacts of arbuscular mycorrhizal fungi on soil aggregation dynamics of neutral purple soil. Acta Ecol. Sin. 31, 498–505 (in Chinese with English abstract). Phillips, J.M., Hayman, D.S., 1970. Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. Br. Mycol. Soc. 55, 158–161.

Piotrowski, J.S., Denich, T., Klironomos, J.N., Graham, J.M., Rillig, M.C., 2004. The effects of arbuscular mycorrhizas on soil aggregation depend on the interaction between plant and fungal species. New Phytol. 164, 365–373. Rillig, M.C., 2004. Arbuscular mycorrhizae, glomalin and soil quality. Can. J. Soil Sci. 84, 355–363. Rillig, M.C., Maestre, F.T., Lamit, L.J., 2003. Microsite differences in fungal hyphal length, glomalin, and soil aggregate stability in semiarid Mediterranean steppes. Soil Biol. Biochem. 35, 1257–1260. Rillig, M.C., Mardatin, N.F., Leifheit, E.F., Antunes, P.M., 2010. Mycelium of arbuscular mycorrhizal fungi increases soil water repellency and is sufficient to maintain water-stable soil aggregates. Soil Biol. Biochem. 42, 1189–1191. Rillig, M.C., Mummey, D.L., 2006. Mycorrhizas and soil structure. New Phytol. 171, 41–53. Rillig, M.C., Wright, S.F., Nichols, K.A., Schmidt, W.F., Torn, M.S., 2001. Large contribution of arbuscular mycorrhizal fungi to soil carbon pools in tropical forest soils. Plant Soil 233, 167–177. Rowell, D.L., 1994. Soil Science: Methods and Applications. Longman Group Limited, Longman Scientific and Technical, Harlow. SAS Institute, Inc., 2001. SAS User’s Guide: Statistics. Version 8.1. SAS Institute, Inc., Cary, NC. Saif, S.R., 1987. Growth responses of tropical forage plant species to vesiculararbuscular mycorrhizae. Plant Soil 97, 25–35. Schreiner, R.P., Mihara, K.L., McDaniel, H., Bethlenfalvay, G.J., 1997. Mycorrhizal fungi influence plant and soil functions and interactions. Plant Soil 188, 199–209. Schüßler, A., Schwarzott, D., Walker, C., 2001. A new fungal phylum, the Glomeromycota: phylogeny and evolution. Mycol. Res. 105, 1413–1421. Smith, S.E., Read, D.J., 2008. Mycorrhizal Symbiosis, third ed. Academic Press, London. Six, J., Bossuyt, B., Degryze, H., Denef, K., 2004. A history of research on the link between (micro) aggregates, soil biota and soil organic matter dynamics. Soil Tillage Res. 79, 7–31. Spohn, M., Giani, L., 2010. Water-stable aggregates, glomalin-related soil protein, and carbohydrates in a chronosequence of sandy hydromorphic soils. Soil Biol. Biochem. 42, 1505–1511. Srivastava, A.K., 2013. Nutrient management in Nagpur mandarin: frontier developments. Sci. J. Agric. 2, 1–14. Srivastava, A.K., Singh, S., Albrigo, L.G., 2008. Diagnosis and remediation of nutrient constraints in Citrus. Hortic. Rev. 34, 277–364. Srivastava, A.K., Singh, S., Marathe, R.A., 2002. Organic citrus: soil fertility and plant nutrition. J. Sustainable Agric. 19, 5–29. Tisdall, J.M., Oades, J.M., 1982. Organic matter and water stable aggregates in soil. J. Soil Sci. 33, 141–163. Wright, S.F., Upadhyaya, A., 1998. A survey of soils for aggregate stability and glomalin, a glycoprotein produced by hyphae of arbuscular mycorrhizal fungi. Plant Soil 198, 97–107. Wilson, G.W., Rice, C.W., Rillig, M.C., Springer, A., Hartnett, D.C., 2009. Soil aggregation and carbon sequestration are tightly correlated with the abundance of arbuscular mycorrhizal fungi: results from long-term field experiments. Ecol. Lett. 12, 452–461. Wu, Q.S., He, X.H., Cao, M.Q., Zou, Y.N., Wang, S., Li, Y., 2013a. Relationships between glomalin-related soil protein in water-stable aggregate fractions and aggregate stability in citrus rhizosphere. Int. J. Agric. Biol. 15, 603–606. Wu, Q.S., Srivastava, A.K., 2012. Rhizosphere microbial communities: Isolation, characterization and value addition for substrate development. In: Srivastava, A.K. (Ed.), Advances in Citrus Nutrition. Springer Verlag, The Netherlands, pp. 169–194. Wu, Q.S., Srivastava, A.K., Zou, Y.N., 2013b. AMF-induced tolerance to drought stress in citrus: a review. Sci. Hortic. 164, 77–87. Wu, Q.S., Xi, R.X., Zou, Y.N., 2008. Improved soil structure and citrus growth after inoculation with three arbuscular mycorrhizal fungi under drought stress. Eur. J. Soil Biol. 44, 122–128. Wu, Q.S., Zou, Y.N., Liu, C.Y., Lu, T., 2012. Interacted effect of arbuscular mycorrhizal fungi and polyamines on root system architecture of citrus seedlings. J. Integr. Agric. 11, 1675–1681. Yang, P.L., Luo, Y.P., Shi, Y.C., 1993. Fractal feature of soil on expression by weight distribution of particle-size. Chin. Sci. Bull. 38, 1896–1899 (in Chinese with English abstract).