Arbuscular mycorrhizal fungi combined with exogenous calcium improves the growth of peanut (Arachis hypogaea L.) seedlings under continuous cropping

CUI Li , GUO Feng , ZHANG Jia-lei , YANG Sha , MENG Jing-jing , GENG Yun , WANG Quan , ,LI Xin-guo , WAN Shu-bo,

1 Biotechnology Research Center, Shandong Academy of Agricultural Sciences/Key Laboratory of Crop Genetic Improvement and Ecological Physiology of Shandong Province, Jinan 250100, P.R.China

2 Scientific Observing and Experimental Station of Crop Cultivation in East China, Ministry of Agriculture, Jinan 250100, P.R.China

3 College of Life Sciences, Shandong Normal University, Jinan 250014, P.R.China

4 Shandong Academy of Agricultural Sciences, Jinan 250100, P.R.China

Abstract The growth and yield of peanut are negatively affected by continuous cropping. Arbuscular mycorrhizal fungi (AMF) and calcium ions (Ca2+) have been used to improve stress resistance in other plants, but little is known about their roles in peanut seedling growth under continuous cropping. This study investigated the possible roles of the AMF Glomus mosseae combined with exogenous Ca2+ in improving the physiological responses of peanut seedlings under continuous cropping.G. mosseae combined with exogenous Ca2+ can enhance plant biomass, Ca2+ level, and total chlorophyll content. Under exogenous Ca2+ application, the Fv/Fm in arbuscular mycorrhizal (AM) plant leaves was higher than that in the control plants when they were exposed to high irradiance levels. The peroxidase, superoxide dismutase, and catalase activities in AM plant leaves also reached their maximums, and accordingly, the malondialdehyde content was the lowest compared to other treatments. Additionally, root activity, and content of total phenolics and flavonoids were significantly increased in AM plant roots treated by Ca2+ compared to either G. mosseae inoculation or Ca2+ treatment alone. Transcription levels of AhCaM, AhCDPK, AhRAM1, and AhRAM2 were significantly improved in AM plant roots under exogenous Ca2+ treatment.This implied that exogenous Ca2+ might be involved in the regulation of G. mosseae colonization of peanut plants, and in turn, AM symbiosis might activate the Ca2+ signal transduction pathway. The combination of AMF and Ca2+ bene fitted plant growth and development under continuous cropping, suggesting that it is a promising method to cope with the stress caused by continuous cropping.

Keywords: Arachis hypogaea L., arbuscular mycorrhizal fungi, continuous cropping, exogenous calcium

1. Introduction

Peanut (Arachis hypogaeaL.) is an important oil and economic crop. The seeds contain essential proteins, vitamins, and minerals (Higgs 2003). Peanut plants can also be used for bio-energy (Liet al. 2011). Therefore, increasing peanut plant yield is a primary goal. However, peanut has increasingly been continuously cropped on the same land without any crop rotation because of the pressure on farmland from the increasing human population and agro-industrialization (Liet al. 2014), especially in China. Continuous cropping can potentially disrupt the structural and functional diversity of soil microbial communities and enzyme synthesis (Shipton 2003;Xionget al. 2015; Sheet al. 2017), which negatively affect the cycling of soil nutrients and soil functions. Furthermore,plant stress resistance is weakened, and disease pressure on peanut plants is increased by continuous cropping (Chenet al. 2012). In addition, autotoxic compounds from the plant accumulate in rhizosphere soil under continuous cropping,which destroys the rhizosphere environment (Huanget al.2013). Continuous cropping will thus cause a series of stresses in the soil environment that affect plant growth and development. These effects include decreases in plant biomass, photosynthesis, and yield, along with deterioration in plant quality. Fortunately, plants have some strategies to deal with adverse soil conditions, including the formation of plant-microbe symbiosis and improving signaling transduction pathways (Macleanet al. 2017).

Arbuscular mycorrhizal fungi (AMF), part of theGlomeromy cotinasubphylum, can form symbioses with about 80% of extant land plants (Smith and Read 2008). These symbioses are characterized by the formation of hyphal structures in the cortical cells of host plants, called arbuscules, which can provide numerous soil nutrients to plants in exchange for carbohydrates (K?hlet al. 2015). The formation of these symbiotic associations is a complex molecular process, and it has been reported that some specific marker genes are involved in the formation of AM symbiosis. For example,REDUCED ARBUSCULAR MYCORRHIZA 1(RAM1)encodes a GRAS-type transcription factor andRAM2encodes aglycerol-3-phosphate acyl transferase, both of which are necessary for the colonization of the root by AMF and arbuscule formation. Plants that are defective inRAM1andRAM2cannot be colonized by AMF (Wanget al. 2012;Richet al. 2015).

For many crops, AMF can improve plant nutrient uptake,which increases plant biomass (Govindarajuluet al. 2005;Javotet al. 2007; Garciaet al. 2017). It has also been reported that AMF can positively improve plant growth by enhancing total chlorophyll and PSII ef ficiency (Yang Y Ret al. 2015), alleviating abiotic and biotic stresses by increasing scavenging capacity for reactive oxygen species(ROS) (Wu and He 2010; Chandrasekaranet al. 2014), and improving plant resistance against pathogens by enhancing defensive capacity (Kazan and Manners 2009; Cameronet al. 2013). It has been shown that AMF can improve peanut plant nutrient uptake and production (Carlinget al.1995). However, it is still unclear whether AMF can alleviate the stress caused by continuous cropping on peanut plants.

Calcium is an essential major element for plant growth and represents 0.1 to 5% of plant dry biomass (Jaffeet al.1975). It is the second messenger in signal transduction and participates in plant physiological and biochemical processes.Plants are subject to exchangeable Ca2+deficiency in many soils because Ca2+is not very mobile. Therefore,supplementation with exogenous Ca2+is often necessary.It has been shown that exogenous Ca2+application may protect plants against environmental stresses, e.g., drought stress (Bowler and Fluhr 2000), cold injury (Zhanget al.2014), and salt stress (Yinet al. 2015), by enhancing nutrient uptake, photosynthesis, membrane integrity, and antioxidant enzymes. Peanut is a calciphilous crop. In peanut, the application of exogenous Ca2+can enhance plant resistance to stress by improving PSII ef ficiency, and increase the activities of antioxidant enzymes (superoxide dismutase(SOD) and catalase (CAT)), and decrease malondialdehyde(MDA) content under stress (Yanget al. 2013; Liet al.2015). When exposed to a stressor, the transient elevation of free Ca2+in the cytoplast will trigger a full range of signal transduction pathwaysviaCa2+-binding proteins, such as calmodulins (CaM) and calcium-dependent protein kinases(CDPK). Previous studies have shown that exogenous Ca2+can increase the transcription ofAhCaMandAhCDPKunder environmental stress (Yanget al. 2013; Liet al. 2015), which suggests that these genes play an important role in protecting plants against stress. However, it is still not clear what role Ca2+plays in plant physiological and biochemical processes when peanut is being continuously cropped.

It has been reported that AM symbiosis can promote the Ca2+uptake of plants (Batiet al. 2015; Cabralet al.2016). Both AMF and exogenous Ca2+have important roles in alleviating plant stress. However, the role of AMF,exogenous Ca2+, and their combination during peanut growth in continuously cropped soils is not clear. In this study, we examined the impact of AMF, exogenous Ca2+,and their combination on plant biomass and physiological indicators. Our observations suggested that AMF combined with exogenous Ca2+could improve the growth of peanut seedlings under continuous cropping.

2. Materials and methods

2.1. Plant growth condition, AMF inoculation, and exogenous Ca2+ treatment

The AMFG.mosseae(BEG HEB02) was maintained as soil-sand-based inoculums. Soil that had been continuously cropped with peanut for 5 years (0–15 cm depth) was twice sterilized at 121°C for 30 min with an interval of 24 h between the two sterilizations. Huayu 22, a typical peanut cultivar with large seeds and an upright phenotype, was used as the experimental material. Seeds were first surface-sterilized with 70% alcohol for 3 min and rinsed six times with sterile water. They were then germinated in the dark at 25°C for 3 days. The germinated seeds were transferred to pots filled with sterilized continuously cropped soil and inoculated with about 400G.mosseaespores contained in 10 g mycorrhizal inoculums. The same sterilized mycorrhizal inoculums withoutG.mosseaespores were used as the control. The peanut seedlings were grown in a greenhouse at 24°C/18°C with a 16/8 h photoperiod, at 70% relative humidity, and at a photosynthetic photo flux density of 700 μmol m–2s–1.One seedling was planted in each pot (the pot diameter and height were 12 and 11 cm, respectively). One week after sowing, each seedling was watered regularly with 100 mL of modi fied (+Ca2+, 20 mmol L–1Ca2+) Hoagland’s solution(20 mmol L–1Ca(NO3)2·4H2O, 5 mmol L–1KNO3, 2 mmol L–1MgSO4·7H2O, 1 mmol L–1KH2PO4, 0.1 mmol L–1EDTA-Na2,0.1 mmol L–1FeSO4·7H2O, 46 μmol L–1H3BO4, 0.32 μmol L–1CuSO4·5H2O, 0.77 μmol L–1ZnSO4·7H2O, and 0.11 μmol L–1H2MoO4) or solution without Ca2+(20 mmol L–1NH4NO3,5 mmol L–1KNO3, 2 mmol L–1MgSO4·7H2O, 1 mmol L–1KH2PO4, 0.1 mmol L–1EDTA-Na2, 0.1 mmol L–1FeSO4·7H2O,46 μmol L–1H3BO4, 0.32 μmol L–1CuSO4·5H2O, 0.77 μmol L–1ZnSO4·7H2O, and 0.11 μmol L–1H2MoO4). In this study,20 mmol L–1NH4NO3was used to balance nitrogen content from Ca(NO3)2. In total, there were four experimental treatments, including Ca0+AM, Ca0–AM, Ca20+AM, and Ca20–AM; 0 and 20 represent the Ca2+concentrations(mmol L–1). A toatal of 20 mmol L–1of Ca(NO3)2was optimal according to our previous experiments (unpublished data).Each treatment contained 12 peanut seedlings and was replicated three times. Six weeks later, the plants were harvested and analyzed.

2.2. Mycorrhizal quantification, and determination of dry weight and Ca2+ content

Ninety root sections per sample were examined by light microscopy (CX41; OLYMPUS, Japan) to estimate the extent to which the root had been colonized by hyphae and arbuscules (McGonigleet al. 1990). The fresh shoots and roots of plants were placed at 105°C for 30 min and then dried at 80°C until the weight was constant to determine the plant dry weight. The Ca2+content in the leaves of AM and nonmycorrhizal (NM) plants from the different treatments were measured according to Yanget al. (2013).

2.3. Measurements of total chlorophyll and chlorophyll fluorescence

Total chlorophyll content was measured according to Arnon(1949). Chlorophyll fluorescence was measured under high irradiance stress with a portable fluorimeter (FMS2,Hansatech, UK) according to Van Kooten and Snel (1990).To induce high irradiance stress, the detached leaves were floated on the water with the adaxial sidefacing up. Then they were irradiated with 1 200 μmol m–2s–1PFD at room temperature (25°C). Initial fluorescence (Fo) was obtained using modulated light (about 10 μmol m–2s–1). The maximal fluorescence (Fm) was determined by 0.8 s of saturating light at 8 000 μmol m–2s–1on a leaf that had been dark adapted for 15 min. Variable fluorescence (Fv) was calculated fromFv=Fm–Fo. Maximal photochemical ef ficiency (Fv/Fm) of PSII was calculated usingFv/Fm=(Fm–Fo)/Fm

2.4. Determination of antioxidant enzyme activities and malondialdehyde content

The third fully expanded leaves from the apical tips were collected to assay antioxidant enzyme activities. Peroxidase(POD) activity was measured according to Sung and Jeng(1994). Fresh leaves were hand-homogenized separately at 4°C in a mortar and pestle with 5 mL of 5% (v/v) trichloracetic acid to precipitate proteins, and then centrifuged at 14 000×g for 20 min. The supernatant was used for POD determination. SOD activity in the fresh leaves was assayed according to Stewart and Bewley (1980). CAT activity and MDA content were determined according to Linet al. (2006)and Yang Set al. (2015), respectively.

2.5. Determination of root activity, and total phenolics and flavonoids contents

Root activity was estimated by tetrazolium chloride (TTC)reduction according to the method reported by Comaset al.(2000). Total phenolics content in the roots was detected using the method described by Chunet al. (2003). Flavonoid content in the roots was measured according to Jiaet al.(1999).

2.6. Quantitative real-time PCR

Total RNA was isolated from the roots, and cDNA was synthesized for qRT-PCR analyses using SYBR PremixEx TaqPolymerase (TaKaRa, Japan) according to the manufacturer’s protocol. The selected genes were analyzed using a Bio-Rad iQ1 Real-Time PCR machine (Bio-Rad,USA). The primers are shown in Table 1. The control reactions were conducted using primers Tua5-F and Tua5-R,which were reported by Chiet al. (2012). At least three replicates were tested per sample. Relative mRNA (fold)differences were assessed with the 2–ΔΔCtformula (Livak and Schmittgen 2001).

2.7. Statistical analysis

Analysis of variance was performed using SSPS Software version 16.0 for Windows. One-way analysis of variance(ANOVA) was used, followed by Duncan’s test. The values obtained are the mean±SE for the three replicates in each treatment. AP-value≤0.05 was considered to be significant.

3. Results

3.1. Effects of AM symbiosis combined with exogenous Ca2+ on root colonization

Six weeks afterG.mosseaeinoculation, 34.34 and 34.56%of the plant roots were colonized at 0 and 20 mmol L–1of Ca2+, respectively (Fig. 1-A). The application of Ca2+seemed to have little effect on the degree ofG.mosseaecolonization.

3.2. AM association combined with exogenous Ca2+improved plant biomass yield and Ca2+ content

Root dry weight per plant significantly increased in the Ca0+AM treatment compared to the Ca0–AM treatment,and it increased even more when Ca2+was applied. The AM plants treated with 20 mmol L–1Ca2+had the highest root dry weight among all treatments (Fig. 1-B). The shoot dry weight per plant had the same trend as the roots(Fig. 1-C). Additionally, the Ca2+content was significantly higher in AM plants than in NM plants under 0 and 20 mmol L–1Ca2+treatments (Fig. 1-D), which showed that AM symbiosis increases Ca2+uptake. Furthermore, the application of exogenous Ca2+significantly enhanced Ca2+content of plants compared with Ca0–AM and Ca0+AM plants (Fig. 1-D). This indicated that supplemental exogenous Ca2+is the main Ca2+source for plants growing in continuously cropped soil, and AM association could further increase Ca2+content.

3.3. AM association combined with exogenous Ca2+increased total chlorophyll and chlorophyll fluorescence

Total chlorophyll content significantly increased in Ca0+AMplants compared with Ca0–AM plants, and increased even more in Ca20+AM treatment (Fig. 2-A). As an indicator of photoinhibition, the maximum photochemical ef ficiency of PSII (Fv/Fm) has been widely studied. When exposed to the high irradiance stress,Fv/Fmdecreased in both AM and NM plant leaves. At the end of the stress period,Fv/Fmin the Ca0–AM, Ca0+AM, Ca20–AM, and Ca20+AM treatments decreased by 21.45, 14.82, 8.20, and 4.75% of their initial values, respectively (Fig. 2-B).

Table 1 Primer sequences for quantitative real-time PCR amplification

Fig. 1 Effects of arbuscular mycorrhizal (AM) symbiosis on plant growth following exogenous Ca2+ treatment. The fungal colonization rates were estimated in the roots of AM plants under different concentrations of Ca(NO3)2 (A). Root (B) and shoot (C) dry weight per plant for AM and non-mycorrhizal (NM) plants under 0 and 20 mmol L–1 Ca2+ treatments were measured. The Ca2+ content(D) was determined in AM and NM plant leaves treated with Ca2+ for 6 wk. Different letters show that the columns are significantly different (P≤0.05). n=6. Experiments were independently replicated three times. Bars mean SD.

3.4. AM association combined with exogenous Ca2+enhanced antioxidant enzyme activities

POD, SOD, and CAT are important antioxidant defense system enzymes. Their activities significantly increased in Ca0+AM plant leaves, and exogenous Ca2+application further significantly increased their activities (Fig. 3-A–C).The activities of these enzymes were the highest in the Ca20+AM treatment. The MDA content, which re flects the degree of membrane lipid peroxidation, significantly decreased in Ca0+AM plants (Fig. 3-D), and decreased even further in AM plants after Ca2+treatment compared to those with no application of exogenous Ca2+.

Fig. 2 Effects of arbuscular mycorrhizal (AM) symbiosis combined with exogenous Ca2+ on total chlorophyll (A) and on the maximum photochemical ef ficiency of PSII (Fv/Fm) (B) in peanut plants. Ca0–AM, peanut plants without AM association and Ca2+ application;Ca0+AM, peanut plants with AM symbiosis, but without Ca2+ application; Ca20–AM, peanut plants were only treated by 20 mmol L–1 Ca(NO3)2; Ca20+AM, peanut plants with AM symbiosis and 20 mmol L–1 Ca(NO3)2 was applied. Different letters show significant difference at P≤0.05. n=6. Experiments were independently replicated three times. Bars mean SD.

Fig. 3 Effects of arbuscular mycorrhizal (AM) symbiosis combined with exogenous Ca2+ on the peroxidase (POD, A), superoxide dismutase (SOD, B), and catalase (CAT, C) activities, and malondialdehyde (MDA) content (D) in the leaves of plants growing in continuously cropped soil. Different letters show that the columns are significantly different at P≤0.05. n=6. Experiments were independently replicated three times. The data presented are the mean value±SD of three individual experiments.

3.5. AM association combined with exogenous Ca2+improved root activity and increased the content of total phenolics and flavonoids

AM symbiosis has no obvious effect on root activity under no exogenous Ca2+application. However, Ca2+significantly increased root activity, which was the highest in the Ca20+AM treatment (Fig. 4-A). Meanwhile, the total phenolics content in the roots of AM plants was not significantly different from the NM plants under no supply of exogenous Ca2+, but it was significantly accumulated in the Ca20–AM treatment, and its content was the highest in the Ca20+AM treatment (Fig. 4-B).A similar trend was observed for the total flavonoids content(Fig. 4-C).

3.6. AM symbiosis combined with exogenous Ca2+changed the transcript level of genes involved in AM symbiosis and the Ca2+ signal transduction pathway

The transcript levels of two AM-specific marker genes and two Ca2+-related genes were assayed to estimate the impact of AMF combined with Ca2+on AM formation and the Ca2+signal transduction pathway.AhRAM1andAhRAM2expression levels were significantly up-regulated in roots of AM plants under both 0 and 20 mmol L–1Ca2+treatments, but their expression levels were the highest in the Ca20+AM treatment (Fig. 5-A and B). In addition, theAhCaMexpression level was significantly up-regulated in AM plants after applying Ca2+, and it was the highest in plant roots with Ca20+AM treatment (Fig. 5-C). TheAhCDPKtranscript level was only up-regulated in AM plant roots and was not affected by exogenous Ca2+(Fig. 5-D).These results indicated that AM symbiosis was not only regulated by exogenous Ca2+, but also participated in the Ca2+signal transduction pathway.

4. Discussion

When subject to continuous cropping, peanut seedlings are seriously compromised by decreases in the activities of antioxidant enzyme and in leaf photosynthesis, leading to decreased plant biomass and yield (Liuet al. 2015). AM fungi not only enhance the ability of plants to absorb mineral nutrition, but also provide non-nutritional bene fits to the host, including tolerance to abiotic stress and resistance against pathogens (Nadeemet al. 2014). Moreover, Ca2+can also protect plants against environmental stresses (Yinet al. 2015).

Fig. 4 Effects of arbuscular mycorrhizal (AM) symbiosis on plant roots supplied with exogenous Ca2+. The root activity (A),total phenolics content (B), and flavonoids content (C) were determined in the roots of AM and NM plants under 0 and 20 mmol L–1 Ca2+ conditions. TTC, tetrazolium chloride; A, activity;GAE, gallic acid equivalent. Different letters show that the columns are significantly different at P≤0.05. n=6. Experiments were independently replicated three times. The data presented are the mean value±SD of three individual experiments.

4.1. AM symbiosis combined with exogenous Ca2+improves plant growth and development

The combination of AM symbiosis and Ca2+increases mineral nutrition uptake, since both Ca2+content (Fig. 1-D)and potassium content in plants were increased with the supply of exogenous Ca2+(Leiet al. 2014), thus increasing the root and shoot dry weights. Besides nutrient uptake,significant increases in the root and shoot dry weight of peanut might also be related to increases in photosynthesis(Fig. 2) and root activity (Fig. 4-A) due to AM symbiosis combined with exogenous Ca2+. The results indicated thatG.mosseaecombined with exogenous Ca2+improves plant dry biomass when peanut is continuously cropped.

Fig. 5 Effects of arbuscular mycorrhizal (AM) symbiosis combined with exogenous Ca2+ on the expression levels of genes involved in AM function and the Ca2+ signal pathway. The transcript levels of the AM-specific marker genes AhRAM1 (A) and AhRAM2(B), and Ca2+ signal pathway genes AhCaM (C) and AhCDPK (D) were determined in AM and non-mycorrhizal (NM) peanut roots grown under the 0 and 20 mmol L–1 Ca2+ treatment conditions for 6 wk. Different letters show that the columns are significantly different at P≤0.05. n=6. Experiments were independently replicated three times. Bars mean SD.

4.2. AM symbiosis combined with exogenous Ca2+enhances plant stress-resistance

AM symbiosis can enhance photosynthetic ability (Andradeet al. 2015). For peanut seedlings in this study, AM symbiosis increased photosynthetic ability by increasing both chlorophyll content and the maximum photochemical quantum ef ficiency of PSII, which was further increased by Ca2+application (Fig. 2). These results suggest thatG.mosseaecombined with exogenous Ca2+is involved in the mechanism that protects peanut plants under continuous cropping from more severe PSII photoinhibition induced by environmental stress (Yanget al. 2013; Yang S 2015).It has been proven that the increase in photosynthetic ef ficiency stems from improved ROS scavenging ability(Yang Y Ret al. 2015; Garciaet al. 2017). In the present study, AM symbiosis combined with Ca2+application further increased the activities of antioxidant enzymes in peanut leaves, including POD, SOD, and CAT (Fig. 3),which are responsible for scavenging the ROS induced by continuous cropping. These results indicated thatG.mosseaecombined with exogenous Ca2+increases ROS scavenging and alleviates oxidative damage caused by continuous cropping.

Simultaneously, improved ROS scavenging ability was also an important factor for maintaining higher root activity (Fig. 4-A) induced by AM symbiosis combined with exogenous Ca2+compared with Ca2+application alone.Moreover, the increased root activity was closely related to the accumulation of phenolics and flavonoids, which improve root tolerance to abiotic stress, and to alterations in the soil environment (Normanet al. 1996; Leifheitet al. 2014).As secondary metabolites, phenolics and flavonoids could improve plant tolerance to stress by removing toxic radicals and maintaining membrane stability (Pietta 2000; Michalak 2006; Wahid and Ghazanfar 2006). It has been reported that AM symbiosis regulated the biosynthesis of phenylpropanoid derivatives, including increasing the flavonoids content(Adolfssonet al. 2017). In the present study, exogenous Ca2+further increased the content of total phenolics and flavonoids in AM plants (Fig. 4-B and C), suggesting thatG.mosseaecombined with exogenous Ca2+increased the synthesis of phenolics and flavonoids, which could enhance root development in continuously cropped soil.

4.3. Exogenous Ca2+ improves the formation of AM symbiosis

As AM-specific marker genes,AhRAM1andAhRAM2play an important role in formation of the AM symbiosis (Wanget al. 2012; Richet al. 2015). Exogenous Ca2+application significantly up-regulated the expression levels of these two genes, indicating that exogenous Ca2+positively affected AM association.CaMandCDPKwere important components in the Ca2+transduction pathway (Liese and Romeis 2013;Rugeet al. 2016), and the expression levels ofAhCaMandAhCDPKwere up-regulated by the application of exogenous Ca2+in peanut plants under stress (Yanget al. 2013; Liet al.2015). However, theAhCDPKtranscript was not affected by exogenous Ca2+(Fig. 5-D), it was consistent with the NtCDPK expression that was induced by fungal elicitors(Yoonet al. 1999). Interestingly,AhCaMandAhCDPKwere significantly up-regulated in AM plant roots, suggesting that AM symbiosis might be involved in the Ca2+signal transduction pathway.

5. Conclusion

Arbuscular mycorrhiza symbiosis enhanced the Ca2+content in peanut plants, and Ca2+participated in AM symbiosis signalingviathe Ca2+signal transduction pathway, which plays an important role in protecting plants against stresses.Therefore, the interaction between AM symbiosis and exogenous Ca2+can increase resistance to stress caused by continuous cropping and improved growth of peanut seedlings. Further studies will be needed to validate the molecular mechanism that operates whenG.mosseaecombined with exogenous Ca2+improves plant growth under continuous cropping.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31601261, 31601252, 31571581 and 31571605) and the China Postdoctoral Science Foundation(2016M592236).