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Co-Inoculation with Rhizobia and AMF Inhibited Soybean Red Crown Rot: From Field Study to Plant Defense-Related Gene Expression Analysis

  • Xiang Gao,

    Affiliation State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Root Biology Center, South China Agricultural University, Guangzhou, China

  • Xing Lu,

    Affiliation State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Root Biology Center, South China Agricultural University, Guangzhou, China

  • Man Wu,

    Affiliation State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Root Biology Center, South China Agricultural University, Guangzhou, China

  • Haiyan Zhang,

    Affiliation State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Root Biology Center, South China Agricultural University, Guangzhou, China

  • Ruqian Pan,

    Affiliations State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Root Biology Center, South China Agricultural University, Guangzhou, China, Laboratory of Bacteria and Fungicides, South China Agricultural University, Guangzhou, China

  • Jiang Tian,

    Affiliation State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Root Biology Center, South China Agricultural University, Guangzhou, China

  • Shuxian Li,

    Affiliation Crop Genetics Research Unit, United States Department of Agriculture - Agricultural Research Service, Stoneville, Mississippi, United States of America

  • Hong Liao

    hliao@scau.edu.cn

    Affiliation State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Root Biology Center, South China Agricultural University, Guangzhou, China

Abstract

Background

Soybean red crown rot is a major soil-borne disease all over the world, which severely affects soybean production. Efficient and sustainable methods are strongly desired to control the soil-borne diseases.

Principal Findings

We firstly investigated the disease incidence and index of soybean red crown rot under different phosphorus (P) additions in field and found that the natural inoculation of rhizobia and arbuscular mycorrhizal fungi (AMF) could affect soybean red crown rot, particularly without P addition. Further studies in sand culture experiments showed that inoculation with rhizobia or AMF significantly decreased severity and incidence of soybean red crown rot, especially for co-inoculation with rhizobia and AMF at low P. The root colony forming unit (CFU) decreased over 50% when inoculated by rhizobia and/or AMF at low P. However, P addition only enhanced CFU when inoculated with AMF. Furthermore, root exudates of soybean inoculated with rhizobia and/or AMF significantly inhibited pathogen growth and reproduction. Quantitative RT-PCR results indicated that the transcripts of the most tested pathogen defense-related (PR) genes in roots were significantly increased by rhizobium and/or AMF inoculation. Among them, PR2, PR3, PR4 and PR10 reached the highest level with co-inoculation of rhizobium and AMF.

Conclusions

Our results indicated that inoculation with rhizobia and AMF could directly inhibit pathogen growth and reproduction, and activate the plant overall defense system through increasing PR gene expressions. Combined with optimal P fertilization, inoculation with rhizobia and AMF could be considered as an efficient method to control soybean red crown rot in acid soils.

Introduction

Soybean (Glycine max L. Merr.) is an important legume crop, supplying protein and oil for human and animals [1]. However, soybean growth has a lot of limitations which cause great yield losses, especially in tropical and subtropical areas where the warm, moist conditions and weathered acid soils not only favor the infection and reproduction of the pathogens [2], [3], but also easily lack of some essential nutrients, such as phosphorus (P) [4], [5]. Heavy application of chemical pesticides and fertilizers is commonly used to prevent such growth losses, but these amendments are highly cost and could cause severe environmental problems [6], [7]. Biocontrol approach should be a better alternative to solve these problems.

Red crown rot (also named black root rot) is caused by a soil-borne fungal pathogen Cylindrocladium parasiticum (teleomorph Calonectria ilicicola) [8], [9]. It was firstly found in peanut in 1965 and then in soybean in 1969 [2], [8]. Symptoms of soybean red crown rot were usually observed late in the season during or after pod set. The infected plants had a black root rot and its stalk tissue was grayish or reddish brown above soil surface for 5 to 10 cm, and leaves became cholorosis and interveinal necrosis followed by defoliation [3], [10]. Till now, more and more reports proved that red crown rot could be considered as one of the most serious soil-borne diseases for legumes around the world [3], [8], such as in China where it made soybean yield loss over 50% [2], [10].

Acid soils comprise up to 50% of the world's potentially arable land, of which syndrome causes severe yield losses in various crops, and thus significantly limit crop production worldwide [11]. In most acid soils, there are deficiencies of some essential elements, such as P, nitrogen (N), potassium (K) and some micronutrients [4], [5], [12]. Therefore, legumes like soybean grown in acid soils not only need to deal with diseases such as red crown rot but also need to face the nutrient deficiency problems.

Biocontrol approach by introducing microorganisms into soils has been proposed to suppress soil-borne diseases for improving plant health [13]. The disease suppression by biocontrol agents is regarded sustainable via harmonization of interactions among plants, pathogens, biocontrol agents and microbial communities within rhizosphere [13], [14]. Many reports found that inoculation with rhizobia and/or arbuscular mycorrhizal fungi (AMF) could promote plant growth and control fungal diseases, and thus be considered as one of the efficient biocontrol approaches [15], [16]. AMF inoculation not only helped host plant growth, but also enhanced defense system and subsequently reduced disease severity on many soil-borne pathogens [17][19]. Besides biological N fixation, many studies showed legumes inoculated with rhizobia could also increase host plant defense system against soil-borne diseases [20], [21]. Improvement of plant nutrition status, direct competition for invasion space, changes in root growth and morphology, as well as exudates of rhizobitoxine have been well documented as the main mechanisms underlying the inhibition of soil-borne pathogens by rhizobium and AMF inoculation [22][25]. Furthermore, increased transcripts of some plant pathogen defense-related (PR) gene expressions by inoculation with rhizobia and/or AMF might also be involved in plants against fungal pathogens [20], [26]. However, most of the studies described above had been conducted in greenhouse conditions and lacked of comprehensive investigations integrating field observations with physiological and molecular evidence.

Soybean could form tripartite symbiotic associations with rhizobia and AMF simultaneously for both P and N benefits [27], [28]. But until now, there is no report in whether rhizobium and/or AMF inoculation could inhibit soybean red crown rot in acid soils, and if so, what are the mechanisms underlying this inhibition? In this study, firstly, we investigated the disease incidence and index of soybean red crown rot with different P additions in acid soil field to elucidate whether the naturally inoculation of rhizobia and AMF could affect red crown rot. The field site has been growing soybean since 2000. Soybean red crown rot caused by C. parasiticum was observed in this field in 2006 and its disease incidence reached to 80% [10]. And then, we combined sand culture and fungal pathogen incubation experiments as well as some important PR gene expression analysis to evaluate the possible physiological and molecular mechanisms of rhizobium and AMF inoculation on soybean red crow rot inhibition.

Results

Field experiments

The disease incidence and index of soybean red crown rot were significantly affected by P additions in field (Table 1). Compared to HP, the plants had lower disease incidence and index at NP. Under NP conditions, disease incidence and index was decreased by 28.56% and 18.80%, 16.68% and 19.08% in 2009 and 2010, respectively.

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Table 1. Disease incidence and index caused by Cylindrocladium parasiticum at different P levels in field.

https://doi.org/10.1371/journal.pone.0033977.t001

Plant growth in field was significantly affected by C. parasiticum infection as indicated by plant dry weight and grain yield (Table 2). Compared to the healthy plants, dry weight and grain yield of the infected plants had 39.82% and 40.09%, 39.06% and 47.80% reduction at NP and HP in 2009; 36.57% and 52.46%, 44.10% and 57.23% reduction in 2010, respectively. Interestingly, the growth of healthy plants was significantly affected by P addition, but not the infected plants. With P addition, the dry weight and grain yield of healthy plants increased 29.70% and 33.65%, 50.98% and 32.21% in 2009 and 2010, respectively. Same as plant growth, plant P and N content was significantly affected by C. parasiticum infection, but P and N content of the infected plants was not influenced by P level (Table S2). This suggested that the plant growth inhibition by C. parasiticum infection might be not directly affected by plant nutrient status.

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Table 2. Plant growth affected by Cylindrocladium parasiticum infection and P level in field.

https://doi.org/10.1371/journal.pone.0033977.t002

Mycorrhization and nodulation of roots were significantly inhibited by C. parasiticum infection as indicated by AMF colonization rate, nodule number and nodule dry weight in field, respectively (Figure 1). Compared to the healthy plants, the infected plants had 17.78%, 66.67% and 75.14%; 35.75%, 72.22% and 70.31% decrease of AMF colonization rate, nodule number and dry weight at NP and HP in 2009, 15.80%, 43.36% and 65.08%; 30.25%, 55.63% and 60.23% decrease in 2010, respectively. Compared to mycorrhization, nodulation showed more severe inhibition by C. parasiticum infection. But AMF colonization rate was more negatively affected by P addition, especially for the healthy plants. The AMF colonization rate of the healthy plants decreased 17.02% and 13.04% at HP in 2009 and 2010 compared to NP treatment, respectively. This indicated that there were complicate interactions among mycorrhization, nodulation, C. parasiticum infection and P status in the field of acid soils.

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Figure 1. AMF colonization and nodulation affected by C. parasiticum and P level in field.

A) and B) AMF colonization; C) and D) nodule number; E) and F) nodule dry weight. HP: 80 kg P2O5 ha−1 added as calcium superphosphate; NP: none P fertilizer added. Healthy plants were not infected by C. parasiticum, infected plants were infected by C. parasiticum with severe necrosis on the subterranean stem and roots, chlorosis of leaves. Each bar represented the mean of four replicates with standard error. * and ** stood the significant difference between two P levels analyzed by one-way ANOVA. *: significant at 0.05 (P<0.05); **: significant at 0.05 (P<0.01).

https://doi.org/10.1371/journal.pone.0033977.g001

Sand culture experiment

In order to understand the relationships among C. parasiticum pathogen infection, AMF colonization and nodulation as well as plant nutrient status, a sand culture experiment was further conducted. As expected, plant growth and nutrient status were negatively affected by pathogen infection but promoted by rhizobium and AMF inoculation under both LP and HP conditions as indicated by plant dry weight, N and P content (Figure S1). Furthermore, the suppression of plant growth and nutrient status derived from pathogen infection were better rescued by co-inoculation with rhizobia and AMF. Interestingly, co-inoculation with rhizobia and AMF further increased AMF colonization rate compared to AMF inoculation alone, and vice versa. The P availability imposed positive effects on nodulation but negative effects on mycorrhization as indicated by nodule number and nodule dry weight as well as AMF colonization rate, respectively (Figure S2).

We found that the disease incidence and index of soybean red crown rot were dramatically decreased after rhizobium and/or AMF inoculation in sand culture (Figure 2), indicating that symbiotic inoculation could not only lower the pathogen colonization but also reduce the root rot severity. Plants co-inoculated with rhizobia and AMF always had the lowest disease incidence and index, especially at low P level. Furthermore, the disease incidence and index decreased from 78.65% and 35.60 without inoculation to about 45% and 15, with rhizobial or AMF inoculation, and further decreased to 22.93% and 8.64 with co-inoculation at low P level, showing significant interactions between rhizobium and AMF inoculation on pathogen control. Interestingly, we found that both tap root and lateral roots where AMF and rhizobia colonized had less microsclerotia (Figure 2C and 2D), implying that space competition might be occurred among AMF, rhizobium and C. parasiticum infection.

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Figure 2. Disease incidence and index of soybean red crown rot affected by C. parasiticum infection, rhizobia and AMF inoculation as well as P level in the sand culture experiment.

A) disease incidence; B) disease index; C) pictures of C. parasiticum infection, rhizobia and AMF inoculation on tap root (left) and lateral roots (right); D) microsclerotia in the roots. LP, 15 µmol P added as KH2PHO4; HP, 500 µmol P added as KH2PHO4. Disease incidence and index was measured as described in Materials and Methods. All the roots were inoculated with C. parasiticum (see Materials and Methods for details). −R−A: roots without AMF and rhizobia inoculation; +R−A: roots inoculated by rhizobia; −R+A: roots inoculated by AMF; +R+A: roots inoculated by rhizobia and AMF. Each bar represented the mean of eight replicates with standard error. * and ** stood the significant difference between two P levels analyzed by one-way ANOVA. *: significant at 0.05 (P<0.05); **: significant at 0.05 (P<0.01).

https://doi.org/10.1371/journal.pone.0033977.g002

To further investigate the effects of symbiotic inoculation on C. parasiticum pathogen colony growth in roots, we measured CFU and found that the CFU caused by C. parasiticum was significantly inhibited by rhizobium and AMF inoculation (Figure 3). The CFU amounts were 57.78%, 52.89% and 73.33% lower under +R−A, −R+A and +R+A treatments compared to −R−A at LP level, respectively. P addition only significantly enhanced the C. parasiticum pathogen growth when inoculated with AMF as indicated by 58.02% and 40.42% increase of CFU under −R+A and +R+A treatments at HP, respectively.

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Figure 3. C. parasiticum pathogen growth affected by rhizobia and AMF inoculation as well as P level in the sand culture experiment.

LP, 15 µmol P added as KH2PHO4; HP, 500 µmol P added as KH2PHO4. All the roots were inoculated with C. parasiticum (see Materials and Methods for details). CFU: colony forming units per gram root sample. −R−A: roots without AMF and rhizobia inoculation; +R−A: roots inoculated by rhizobia; −R+A: roots inoculated by AMF; +R+A: roots inoculated by rhizobia and AMF. Each bar represented the mean of four replicates with standard error. * and ** stood the significant difference between two P levels analyzed by one-way ANOVA. *: significant at 0.05 (P<0.05); **: significant at 0.05 (P<0.01).

https://doi.org/10.1371/journal.pone.0033977.g003

The growth of C. parasiticum pathogen was significantly inhibited by the exudates from the roots inoculated with rhizobia and/or AMF under different P conditions as indicated by pathogen colony diameter and sporulation, especially at LP (Figure 4). In comparison with control, the addition of 2 mL root exudates from plants with rhizobium and/or AMF inoculation dramatically inhibited pathogen growth (Figure 4A). Under LP conditions, the pathogen colony diameter and sporulation of C. parasiticum decreased 27.36% and 55.31% after co-inoculation with rhizobia and AMF compared to control, showing the strong synergistic interactions between rhizobium and AMF inoculation on inhibiting C. parasiticum growth. This suggested that the symbiotic inoculation induced/enhanced the ability of host plants to secrete some or more defense-compounds to control pathogen infection. Furthermore, there were four phenolic acids detected in the root exudates (Table 3). Among them, roots from the −R−A treatment only released gallic acid, and the concentration was enhanced by rhizobia and/or AMF inoculation. Interestingly, the other three detected phenolic acids, including ferulic acid, cinnamic acid and salicylic acid were only secreted from the roots inoculated with rhizobia and/or AMF at both P levels, indicating that the changes of phenolic acids in root exudates might be the main compounds controlling the pathogen infection in current conditions.

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Figure 4. Mycelial radial growth and sporulation of C. parasiticum affected by the exudates from the roots with rhizobia and/or AMF inoculation as well as P level.

LP, 15 µmol P added as KH2PHO4; HP, 500 µmol P added as KH2PHO4. a) pictures of pathogen colony at LP; b) colony diameter of pathogen; c) number of spores. −R−A: roots without AMF and rhizobia inoculation; +R−A: roots inoculated by rhizobia; −R+A: roots inoculated by AMF; +R+A: roots inoculated by rhizobia and AMF. Each bar represented the mean of four replicates with standard error. * and ** stood the significant difference between two P levels analyzed by one-way ANOVA. *: significant at 0.05 (P<0.05); **: significant at 0.05 (P<0.01).

https://doi.org/10.1371/journal.pone.0033977.g004

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Table 3. The concentration of phenolic acids (µg·g−1 root DW) in root exudates from different treatments at flowering stage detected by HPLC.

https://doi.org/10.1371/journal.pone.0033977.t003

Plant defense-related gene expression analysis

Quantitative RT-PCR was used to assay expression patterns of 8 pathogen defense-related (PR) genes in both roots and leaves. Results showed that the transcripts of the most tested genes in roots were significantly increased by rhizobium and/or AMF inoculation, which was partly dependent on P availability (Figure 5). Except that significant responses of PR12 to rhizobium and/or AMF inoculation were only observed at 3 DAI (Day After Inoculation), transcripts of the other 7 genes were significantly increased at both 1 and 3 DAI. Furthermore, the genes responded differentially to rhizobium and/or AMF inoculation. Among them, transcription levels of PR2, PR3, PR4 and PR10 reached the highest level with co-inoculation of rhizobium and AMF. However, the most transcripts of PR12, PPO and PAL were observed at AMF inoculation. Interestingly, transcripts of some genes with rhizobium and/or AMF inoculation were up-regulated by P application, especially at 3 DAI. For example, PR3, PR4, PR10 and PAL with rhizobium inoculation, PR10, PPO and PAL at AMF inoculation, PR3 and PR4 at rhizobium and AMF co-inoculation exhibited higher expression levels with P application at 3 DAI.

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Figure 5. Expression changes of eight defense-related genes in roots of soybean in response to C. parasiticum infection and rhizobia and/or AMF inoculation as well as P level.

LP, 15 µmol P added as KH2PHO4; HP, 500 µmol P added as KH2PHO4. Except CK, all the roots were inoculated with C. parasiticum (see Materials and Methods for details). CK: roots without C. parasiticum, AMF and rhizobia inoculation; −R−A: roots without AMF and rhizobia inoculation; +R−A: roots inoculated by rhizobia; −R+A: roots inoculated by AMF; +R+A: roots inoculated by rhizobia and AMF. Each bar represented the mean of three biological replicates with standard error.

https://doi.org/10.1371/journal.pone.0033977.g005

Similar responses of the genes to rhizobium and/or AMF inoculation were also observed in leaves (Figure S3). However, the highest responses of 6 genes to rhizobium inoculation were observed, including PR2, PR3, PR4, PR10, PR12 and PPO. The other 2 genes responded mostly to AMF inoculation, including PR1 and PAL. Furthermore, P availability was also involved in regulating expression patterns of the genes. Except PAL, transcripts of all the other genes were increased by P application at rhizobium inoculation at 1DAI. At 3 DAI, P application also resulted in increasing transcripts of the genes, including PR1, PR2 and PR3 at rhizobium inoculation, PR1, PR2, PR4 and PAL at AMF inoculation.

Discussion

The severe yield losses caused by red crown rot have been estimated to be as high as 50% [2], [10]. In earlier reports, the research of the relationship between symbionts and pathogens was mostly conducted in greenhouse or laboratory under better controlled conditions [29], [30], but no report has been carried out in both field and greenhouse. Therefore, our study should be the first comprehensive research combined field, sand culture as well as pathogen incubation experiments, from phenotype to physiological and molecular analysis to elucidate the underlying relationships and mechanisms among symbionts and soybean red crown rot as well as P application in acidic red soils.

In this study, we found that inoculation with rhizobia or AMF could inhibit soybean red crown rot happening and development, and co-inoculation get more effective results than using a single microsymbiont in sand culture (Figure 2). From the field experiment, we also found that mycorrhization and nodulation of roots were significantly inhibited by C. parasiticum infection (Figure 1), implying that under the condition of simultaneously naturally inoculated by indigenous rhizobia, AMF and pathogen in field, there might be vital competition and antagonism effects occurred. Firstly, rhizosphere colonization was important not only as the first step in pathogenesis of soil borne microorganisms but also was crucial in the application of microorganisms for beneficial purposes [31]. Rhizobia and AMF should be able to compete with pathogens through efficiently colonizing the rhizosphere of the plants [22], [32]. Secondly, rhizobia and AMF might compete for the colonizing sites of plant roots with pathogens, and thus protect plants out of pathogen infection as the roles of most biocontrol agents [32], [33]. This competition effect could be supported by our results from sand culture that tap root and lateral roots colonized by AMF and rhizobia had less pathogen microsclerotia (Figure 2C). Furthermore, preventing growth and proliferation of phytopathogens through the toxic metabolites produced by rhizobium and AMF is another well accepted mechanism of biocontrol agents [34][36]. We found that the root exudates from nodulated and/or AMF colonized roots directly interfered with the growth and proliferation of pathogens in vitro (Figure 4), which suggested that more antibiotics might be produced by plants when inoculated with rhizobia and/or AMF, and thus reduce pathogen growth and proliferation. In our study, we found that the phenolic acids in the root exudates were coincidently enhanced by rhizobia and/or AMF inoculation (Table 3). Therefore, we speculated that the phenolic acids in the root exudates might be the critical biological control substances for C. parasiticum infection in soybean. The detailed mechanisms are needed to be further elucidated.

Moreover, it has been well documented that inoculation with rhizobia and/or AMF had potential impacts to the disease occurrence and development [37], [38], which probably due to the increase of plant growth and nutrient status after roots colonized by rhizobia and AMF [39], [40]. However, in our study, we found that even P fertilizer addition in both field and sand culture experiments enhanced plant growth, N and P nutrition (Table 2 and Table S2), but also increased the disease incidence and index of red crown rot no matter inoculation with rhizobia and AMF or not (Table 1 and Figure 2), indicating that direct promotion of plant growth and nutrient status is not the mechanism of the inhibition effect of rhizobium and AMF inoculation on C. parasiticum infection. The similar results are also found in many garden plants that P application increase the severity of disease caused by Sclerotinia sclerotiorum [41], [42]. Furthermore, high P availability has been demonstrated to play opposite roles in nodulation and mycorrhization as indicated by enhanced nodulation but suppressed mycorrhization with increasing P availability [28]. We also found that P addition significantly decreased AMF colonization rate in both field and sand culture (Figure 1 and Figure S2), implying that the increased disease incidence and index of C. parasiticum could be partly due to the decrease of AMF colonization rate. This is also supported by the results of root pathogen colony growth from sand culture. The CFU only enhanced by high P when inoculated with AMF (Figure 3). Therefore, for using rhizobia and AMF inoculation to control soybean red crown rot in acid soils, proper P application should be further considered.

In addition, activation of specific plant defense mechanisms in response to symbiont colonization is also an obvious basis for the protective behavior of rhizobia and AMF [20], [43], [44]. Compounds associated with plant defense included, but not limited to, phytoalexins, chitinases, β-1, 3-glucanases, pathogenesis related proteins, callose and phenolics [17], [45], [46]. In legumes, a number of genes encoding pathogenesis related proteins have been showed to be locally and systemically up-regulated after inoculation with pathogens [47][49]. Furthermore, the increase of some specific plant defense-related (PR) gene expressions to rhizobium and/or AMF colonization has been reported to against fungal pathogens [24], [26]. For example, inoculation with rhizobium could induce the PR genes involved in phytoalexin synthesis and made plants primed for against the fungal pathogen, and then reduce the severity of disease [26]. Phenolics were observed in rhizobium treated seedlings against the pathogen infection, which might be controlled by PAL [50]. Mycorrhization also amplified the accumulation of the PR genes and phytoalexins synthesis in fungal pathogen infected [51][53]. Our results through q-PCR analysis found that inoculation with rhizobia and/or AMF could significantly increase the expression of most selected PR genes in both roots and leaves at 1 and 3 DAI (Figure 5), especially under co-inoculation conditions. This indicated that inoculation with rhizobia and/or AMF in our study activated both local and systemic defense systems in a short- and long-term way in soybean plants. The similar elicitation via AMF and rhizobia symbiosis to activate specific defense reactions and predispose an early response when attacked by a root pathogen has been found in legumes [17], [53]. Interestingly, we also found that inoculation with rhizobia up-regulated more PR genes in roots, but AMF up-regulated more in leaves (Figure 5 and Figure S3), suggesting that the plant responds to rhizobium and AMF inoculation through different defense systems. Plants prefer to activate local defense systems to rhizobium inoculation, but systemic defense systems to AMF inoculation. Co-inoculation could activate both defense systems and result in better pathogen inhibition.

Take together, our study firstly showed that inoculation with rhizobia and AMF could decrease soybean red crown rot occurrence and development, through direct evidence in inhibition of pathogen growth and reproduction, and enhancement of some PR gene expressions. The study indicated that the biocontrol method and nutrient management, like inoculation with rhizobia and AMF and optimal P fertilization, could be considered as an efficient method to control soybean red crown rot in acid soils, and therefore apply practical measure for sustainable soybean production.

Materials and Methods

Field experiments

Soybean genotype HN112 was grown on acidic red soils at Boluo (E114.28°, N23.18°) experimental site of South China Agricultural University in Guangdong Province of China in 2009 and 2010. Basic soil chemical characteristics were as follows: pH, 5.37; organic matter, 17.63 g kg−1; available P (Bray I method), 15.68 mg P kg−1; available nitrogen, 86.64 mg N kg−1; available potassium, 75.28 mg K kg−1.

There were two P levels, including high P (80 kg P2O5 ha−1 added as calcium superphosphate, HP) and low P (none P fertilizer added, NP). The P fertilizer was applied to the topsoil by spread application. Each treatment had four replicates in a randomized complete block design with 8 plots in total. Each plot had an area of 18 m2 and the planting density was 21 plants m−2 (40 cm between rows, 15 cm between plants). The experimental field was managed following local farmer's practices. Seventy days after planting, 100 plants were examined in each plot for the damage caused by red crown rot. Two representative healthy and infected plants were harvested from each replicate for further analysis. Plant dry weight, grain yield, plant N and P content, nodule number, nodule dry weight and AMF colonization rate were measured after harvest. Plants and nodules were dried at 105°C for 30 min, and then kept at 75°C till completely dry to determine dry weight. Total N and P content were measured using the semi-micro Kjedahl procedure with a N analyzer (Kjedahl 2300; FOSS, Hoganas, Sweden) and phosphorus–molybdate blue color reaction, respectively [54].

Disease incidence was defined as the percentage of diseased subterranean stems. Disease severity was recorded for each plant on a 0–5 scale according to Nishi [55]: i.e., 0 = no visible symptoms; 1 = small necrotic lesions on the subterranean stem; 2 = necrotic lesions extending around the subterranean stem, 3 = necrotic lesion on the subterranean stem extending to the groundline; 4 = severe necrosis on the subterranean stem and roots, leaf chlorosis; 5 = plant dead. Disease index was summarized within each plot as {[(n1×1)+(n2×2)+(n3×3)+…+(nN×N)]/[N×(n1+n2+n3…+nN)]}×100, where n1…nN was the number of subterranean stems in each of the respective disease categories, N was the highest scoring of the disease [56]. Fifty roots of each plot with 1 cm length were sampled for AMF assay. The roots were cleared by 10% KOH for 7 days, and stained with 5% ink-vinegar solution according to the procedure of Vierheilig [57]. Total AMF colonization rate of roots was determined as the percentage of root length colonized by AMF using the intersection method.

Sand culture experiment

The sand culture experiment was conducted in the greenhouse of Root Biology Center at South China Agricultural University using soybean genotype HN112 as plant materials. Each pot consisted of 2 kg of silica sand and 6 soybean plants. The silica sand was sterilized by autoclave on two consecutive days for 40 min at 121°C. There were 3 factors including P level, pathogen infection and symbiotic inoculation, and thus 16 treatments in total. Two P levels were HP (500 µM P added as KH2PO4) and LP (15 µM P added as KH2PO4). Pathogen infection was treated as added C. parasiticum spore suspensions or sterilized spore suspensions as control. For symbiotic inoculation, there were 4 inoculation levels: rhizobium inoculation alone (+R−A, 20 mL rhizobia liquid per pot), AMF inoculation alone (−R+A, 100 g AMF inoculants per pot), co-inoculation with rhizobium and AMF (+R+A, 20 mL rhizobia liquid and 100 g AMF inoculants per pot) and none inoculation as control (−R−A, 100 g sterilized AMF inoculants and 20 mL sterilized rhizobia liquid per pot). Inoculants with rhizobium and/or AMF was mixed with silica sand before planting. There were 8 replicates for each treatment in this study.

The pathogen used in this study was C. parasiticum (GenBank Accession No. GU073284) isolated from the infected soybean roots in Boluo field site [10]. Spore suspensions of plant pathogens were obtained from 14-day-old V8-juice media which were collected by adding 10 mL of sterile water to each Petri dish and rubbing the surface with a sterile L-shaped spreader. The suspension was subsequently filtered through 3-layers of cheesecloth. The spore concentration was determined using a hemacytometer and adjusted to 1×105 spores per mL.

The rhizobium inoculant was made by Bradyrhizobium sp. BXYD3 with MPN value of 1×109 rhizobia per mL of liquid [58]. The original AMF inoculum was Glomus mosseae from maize (Zea mays L.) with MPN value as 300 progagules per gram of soils. The AMF inoculants were a mixture of the infected maize roots, spores and mycelium [58]. AMF and rhizobium inoculants were mixed into sand before planting. Thirty days after planting, plant stem base was infected with 20 mL C. parasiticum spore suspensions each pot. Plants were irrigated once every day with modified 1/2 strength Hoagland nutrient solution with either of the two P additions as mentioned above [12]. Sixty days after planting, plants were investigated the damage caused by red crown rot as described above, and then harvested. Plant dry weight, N and P content, nodule number, nodule dry weight, AMF colonization were measured as mentioned above. The microsclerotia in the roots were observed under a Stereo microscope (Leica, M165C, Germany).

Determination of root infection by Cylindrocladium parasiticum

In order to determine the root infection by C. parasiticum, root colony forming unit (CFU) was measured 30 days after pathogen inoculation in sand, randomly selected soybean roots were cut into 1 cm segments and washed thoroughly and surface-sterilized with 0.5% (v/v) NaClO for 3 min, rinsed three times in sterilized water, and then blotted on sterilized filter paper. About 1 g root segments were ground in 50 mL of sterile deionized water using a blender at high speed for 1 min [59]. The homogenized root suspension was diluted 10 folds with sterile deionized water. 100 µL dilution was placed and evenly spread on Rose Bengal Medium with a sterile glass rod for 4 plates. There were 4 replicates and thus 16 plates for each treatment. All plates were incubated at 28°C in dark for 4 days. Colonies of C. parasiticum were identified and counted on each plate to determine the CFUs per gram of roots for each treatment.

Root exudates of soybean seedlings infected by C. parasiticum from the sand culture experiments were collected at flowering stage, and then added to V8-juice medium during C. parasiticum incubation for studying the effects of root exudates on mycelial radial growth and sporulation of C. parasiticum. Roots were gently taken out of the sand and washed with deionized water. The cleaned roots were submerged in a plastic cup containing 500 mL of 0.5 µM CaCl2 to collect exudates for 6 hours. Root exudates were filtered by 0.45 µm Millipore membrane and stored at −20°C. During collection, each cup containing 3 seedlings were covered by a black plastic lid to avoid contamination and light. The grown diameter and sporulation of pathogen was conducted by adding 2 mL root exudates to V8-juice medium before it was solidified to give a total volume of 20 mL per Petri dish. Plates were incubated at 28°C in dark, the colony diameter and sporulation of C. parasiticum was determined on 5 and 14 days after incubation. For phenolic acid detection, the root exudates were firstly evaporated in a rotary evaporator (Free Zone Freeze Dry Systems, LABCONCO, USA), and then analyzed by HPLC (Agilent 1200, USA) according to Banwart [60].

Plant defense-related gene expression analysis

In order to study the responses of plant defense-related genes to C. parasiticum infection, AMF and rhizobia inoculation, total RNA from leaves and roots of soybean plants were separately extracted according to Guo [61]. Thirty-day soybean seedlings grown in sand at both low P and high P levels were used in this study. Leaves and roots for each treatment were randomly harvested at 0, 1, 2, 3 days after pathogen inoculation, respectively. Leaves and roots were washed with tap water and immediately frozen in liquid nitrogen. Total RNA was extracted and isolated according to the method of Plant RNA Mini Kit as described by the manufacturer (Omega, GA, USA). First strand cDNA was synthesized from the 1 µg of total RNA using ImProm-II™ Reverse transcription system according to the manufacturer's instructions (Promega, Madison, USA). All the reactions were done on a Rotor-Gene 3000 (Corbett Research, Australia). The soybean housekeeping gene EF1-α (Accession number: X56856) was used as endogenous control to normalize the samples. Eight plant defense-related genes were selected to verify their expression patterns by quantitative real time-PCR (qRT-PCR). The specific primer sequences and putative functions of the tested plant defense-related genes were listed in Table S1.

Data Analysis

All the data in the experiments were statistically analyzed by ANOVA using Excel 2003 software (Microsoft Corporation, 1985–2003) and SAS 8.1 (SAS Inc., Cary, NC, USA).

Supporting Information

Figure S1.

Plant dry weight, P and N content affected by C. parasiticum infection, rhizobia and AMF inoculation as well as P level in sand culture experiment. A) plant dry weight; B) plant P content; C) plant N content. LP, 15 µmol P added as KH2PHO4; HP, 500 µmol P added as KH2PHO4. Besides CK, all the roots were inoculated with C. parasiticum (see Materials and Methods for details). CK: roots without C. parasiticum, AMF and rhizobia inoculation; −R−A: roots without AMF and rhizobia inoculation, +R−A: roots inoculated by rhizobia, −R+A: roots inoculated by AMF, +R+A: roots inoculated by rhizobia and AMF. Each bar represents the mean of four replicates with standard error.

https://doi.org/10.1371/journal.pone.0033977.s001

(TIF)

Figure S2.

AMF colonization, nodule number and dry weight affected by C. parasiticum, rhizobia and AMF inoculation as well as P level in sand culture experiment. A) nodule number; B) nodule dry weight; C) AMF colonization. LP: 15 µmol P added as KH2PHO4; HP: 500 µmol P added as KH2PHO4. Besides CK, all the roots were inoculated with C. parasiticum (see Materials and Methods for details). CK: roots without C. parasiticum, AMF and rhizobia inoculation; −R−A: roots without AMF and rhizobia inoculation, +R−A: roots inoculated by rhizobia, −R+A: roots inoculated by AMF, +R+A: roots inoculated by rhizobia and AMF. Each bar represents the mean of four replicates with standard error.

https://doi.org/10.1371/journal.pone.0033977.s002

(TIF)

Figure S3.

Expression changes of eight defense-related genes in leaves of soybean in response to C. parasiticum infection and rhizobia and/or AMF inoculation as well as P level. LP, 15 µmol P added as KH2PHO4; HP, 500 µmol P added as KH2PHO4. Except CK, all the roots were inoculated with C. parasiticum (see Materials and Methods for details). CK: roots without C. parasiticum, AMF and rhizobia inoculation; −R−A: roots without AMF and rhizobia inoculation; +R−A: roots inoculated by rhizobia; −R+A: roots inoculated by AMF; +R+A: roots inoculated by rhizobia and AMF. Each bar represented the mean of three biological replicates with standard error.

https://doi.org/10.1371/journal.pone.0033977.s003

(TIF)

Table S1.

Real Time PCR primers designed for this study.

https://doi.org/10.1371/journal.pone.0033977.s004

(DOC)

Table S2.

Plant N and P content affected by C. parasiticum infection and P level in field.

https://doi.org/10.1371/journal.pone.0033977.s005

(DOC)

Acknowledgments

We thank Mingfang Guan, Hui Jiang, Xinping Chen, Ruineng Xu and Zuotong He for helps in field and lab experiments, and Dr. Zide Jiang for his guidance on pathogen analysis.

Author Contributions

Conceived and designed the experiments: XG HL. Performed the experiments: XG MW HZ. Analyzed the data: XG XL MW JT HL. Contributed reagents/materials/analysis tools: XG HL. Wrote the paper: XG XL JT HL. Guided the plant pathology experiments: RP SL.

References

  1. 1. Messina MJ (1999) Legumes and soybeans: overview of their nutritional profiles and health effects. Am J Clin Nutr 70: 439–450.
  2. 2. Berggren GT, Snow JP (1989) Red crown rot. In: Sinclair JB, Backman PA, editors. Compendium of soybean disease. 3rd ed. American Phytopathological Society. St. Paul, MN. pp. 44–45.
  3. 3. Kuruppu P, Schneider R, Russin J (2004) Factors affecting soybean root colonization by Calonectria ilicicola and development of red crown rot following delayed planting. Plant Dis 88: 613–619.
  4. 4. Kochian LV, Hoekenga OA, Piñeros MA (2004) How do crop plants tolerate acid soils? Mechanisms of aluminum tolerance and phosphorous efficiency. Annu Rev Plant Biol 55: 459–493.
  5. 5. Yan XL, Wu P, Ling HQ, Xu GH, Xu FS, et al. (2006) Plant nutriomics in China: an overview. Ann Bot 98: 473–482.
  6. 6. Gan YT, Siddique KH, Macleod WJ, Jayakumar P (2006) Management options for minimizing the damage by ascochyta blight (Ascochyta rabiei) in chickpea (Cicer arietinum L.). Field Crops Res 97: 121–134.
  7. 7. Gil S, Haro R, Oddino C, Kearney M, Zuza M, et al. (2008) Crop management practices in the control of peanut diseases caused by soil-borne fungi. Crop Prot 27: 1–9.
  8. 8. Bell DK, Sobers EK (1966) A peg, pod and root necrosis of peanuts caused by a species of Calonectria. Phytopathology 56: 1361–1364.
  9. 9. Crous PW, Wingfield MJ, Alfenas AC (1993) Cylindrocladium parasiticum sp. nov, a new name for C.crotalariae. Mycol Res 97: 889–896.
  10. 10. Guan M, Pan R, Gao X, Xu D, Deng Q, et al. (2010) First report of red crown rot caused by Cylindrocladium parasiticum on soybean in Guangdong, Southern China. Plant Dis 94: 485.
  11. 11. von Uexküll HR, Mutert E (1995) Global extent, development and economic impact of acid soils. In: date RA, Grundom NJ, raymet GE, Probert ME, editors. Plant-Soil Interactions at Low pH: Principles and Management. Kluwer Academic Publisher, Dordrecht, The Netherlands. pp. 5–19.
  12. 12. Liao H, Wan HY, Shaff J, Wang XR, Yan XL, et al. (2006) Phosphorus and Aluminum interactions in soybean in relation to aluminum tolerance. Exudation of specific organic acids form different regions of the intact root system. Plant Physiol 141: 674–684.
  13. 13. Handelsman J, Stabb EV (1996) Biocontrol of soilborne plant pathogens. Plant Cell 8: 1855–1869.
  14. 14. Whipps JM (2001) Microbial interactions and biocontrol in the rhizosphere. J Exp Bot 52: 487–511.
  15. 15. Chandanie WA, Kubota M, Hyakumachi M (2006) Interactions between plant growth promoting fungi and arbuscular mycorrhizal fungus Glomus mosseae and induction of systemic resistance to anthracnose disease in cucumber. Plant Soil 286: 209–217.
  16. 16. Pozo MJ, Azcon–Aguilar C (2007) Unraveling mycorrhiza-induced resistance. Curr Opin Plant Biol 10: 393–398.
  17. 17. Gianinazzi-Pearson V, Gollotte A, Dumas-Gaudot E, Franken P, Gianinazzi S (1994) Gene expression and molecular modifications associated with plant responses to infection by arbuscular mycorrhizal fungi. In: Daniels M, Downic JA, Osbourn AE, editors. Advances in molecular genetics of plant microbes interactions. Kluwer, Dordrecht. pp. 179–186.
  18. 18. Kosuta S, Chabaud M, Lougnon G, Gough C, Denarie J, et al. (2003) A diffusible factor from arbuscular mycorrhizal fungi induces symbiosis-specific MtENOD11 expression in roots of Medicago truncatula. Plant Physiol 131: 952–962.
  19. 19. Chandanie WA, Kubota M, Hyakumachi M (2009) Interactions between the arbuscular mycorrhizal fungus Glomus mosseae and plant growth-promoting fungi and their significance for enhancing plant growth and suppressing damping-off of cucumber (Cucumis sativus L.). Appl Soil Ecol 41: 336–341.
  20. 20. Dar GH, Zargar MY, Beigh GM (1997) Biocontrol of Fusarium root-rot in the common bean (Phaseolus vulgaris L.) by using symbiotic Glomus mosseae and Rhizobium leguminosarum. Microb Ecol 34: 74–80.
  21. 21. Arfaoui A, Sifi B, El Hassni M, EI Hadrami I, Boudabous A, et al. (2005) Biochemical analysis of chickpea protection against Fusarium wilt afforded by two Rhizobium isolates. Plant Pathol J 4: 35–42.
  22. 22. Smith SE, Giananizzi-Pearson V (1988) Physiological interactions between symbionts in vesicular-arbuscular mycorrhizal plants. Annu Rev Plant Physiol Plant Mol Biol 39: 221–244.
  23. 23. Breil BT, Borneman J, Triplett EW (1996) A newly discovered gene, tfuA, involved in the production of ribosomally synthesized peptide antibiotic trifolitoxin. J Bacterial 178: 4150–4156.
  24. 24. Cordier C, Pozo MJ, Barea JM, Giannizazzi S, Gianinazzi-Pearson V (1998) Cell defense responses associated with localized and systemic resistance to Phytophthora parasitica induced in tomato by an arbuscular mycorrhizal fungus. Mol Plant Microbe Interact 11: 1017–1028.
  25. 25. Huang HC, Erickson RS (2007) Effect of seed treatment with Rhizobium leguminosarum on Pythium damping-off, seedling height, root nodulation, root biomass, shoot biomass, and seed yield of pea and lentil. J Phytopathol 155: 31–37.
  26. 26. Arfaoui A, EI Hadrami A, Mabrouk Y, Sifi B, Boudabous A, et al. (2007) Treatment of chickpea with Rhizobium isolates enhances the expression of phenylpropanoid defense-related genes in response to infection by Fusarium oxysporum f. sp. ciceris. Plant Physiol Bioch 45: 470–479.
  27. 27. Lisette J, Xavier C, Germida J (2003) Selective interactions between arbuscular mycorrhizal fungi and Rhizobium leguminosarum bv. viceae enhance pea yield and nutrition. Biol Fertil Soils 37: 261–267.
  28. 28. Wang XR, Pan Q, Chen FX, Yan XL, Liao H (2011) Effects of co-inoculation with arbuscular mycorrhizal fungi and rhizobia on soybean growth as related to root architecture and availability of N and P. Mycorrhiza 21: 173–181.
  29. 29. Estevez JC, Percich JA, Graham PH (2002) Integrated management strategies of bean root rot with Bacillus subtilis and Rhizobium in Minnesota. Field Crops Res 74: 107–115.
  30. 30. Akhtar MS, Shakeel U, Sidduqiu ZA (2010) Biocontrol of Fusarium wilt by Bacillus pumilus, Pesudomonas alcaligenes, and Rhizobium sp. on lentil. Turk J Biol 32: 1–7.
  31. 31. Lugtenberg BJ, Dekkers LC, Bloemberg GV (2001) Molecular determinants of rhizosphere colonization by Pseudomonas. Annu Rev Phytopathol 39: 461–490.
  32. 32. Weller DM (1983) Colonization of wheat roots by a Fluorescent Pseudomonad suppressive to take-all. Phytopathology 73: 1548–1553.
  33. 33. Vigo C, Norman JR, Hooker JE (2000) Biocontrol of pathogen Phytophthora parasitica by arbuscular mycorrhizal fungi is a consequence of effects on infection loci. Plant Pathol 49: 509–514.
  34. 34. Kraus J, Loper JE (1995) Characterization of a genomic region required for production of the antibiotic pyoluteorin by the biological control agent Pseudomonas fluorscens Pf-d. Appl Environ Microbiol 61: 849–854.
  35. 35. Raaijmakers JM, Weller DM, Thomashow LS (1997) Frequency of antibiotic-producing Pseudomonas spp. In natural environments. Appl Environ Microbiol 63: 881–887.
  36. 36. Compant S, Duffy B, Nowak J, Clément C, Barka EA (2005) Use of plant growth-promoting bacteria for biocontrol of plant diseases: principles, mechanisms of action, and future prospects. Appl Environ Microbiol 71: 4951–4959.
  37. 37. Parniske M (2000) Intracellular accommodation of microbes by plants: a common developmental program for symbiosis and disease? Curr Opin Plant Biol 3: 320–328.
  38. 38. Aysan E, Demir S (2009) Using arbuscular mycorrhizal fungi and Rhizobium legumiosarum biovar phaseoli against Sclerotinia sclerotiorum (Lib.) de bary in the common bean (Phaseolus vulgaris L.). Plant Pathol J 8: 74–78.
  39. 39. Clark RB, Zeto SK (2000) Mineral acquisition by arbuscular mycorrhizal plants. J Plant Nutr 23: 867–902.
  40. 40. Siddiqui ZA, Singh LP (2004) Effects of soil inoculants on the growth, transpiration and wilt disease of chickpea. J Plant Dis Protect 111: 151–157.
  41. 41. Huber DM (1980) The role of mineral nutrition in defense. In: Horsfall JG, Cowling EB, editors. Plant Disease. An advanced treatise, Volume 5, How plants defend themselves. Academic. New York. pp. 381–406.
  42. 42. Dordas C (2008) Role of nutrients in controlling plant disease in sustainable agriculture: A review. Agron Sustain Dev 28: 33–46.
  43. 43. Parnike M (2004) Molecular genetics of the arbuscular mycorrhizal symbiosis. Curr Opin Plant Biol 7: 414–421.
  44. 44. Arfaoui A, Sifi B, Boudabous A, EI Hadrami I, Cherif M, et al. (2006) Identification of Rhizobium isolates possessing antagonistic activity against Fusarium oxysporum f. sp. ciceris, the causal agent of Fusarium wilt of chickpea. J Plant Pathol 88: 67–75.
  45. 45. Ismail Y, McCormick S, Hijri M (2011) A fungal symbiont of plant-roots modulates mycotoxin gene expression in the pathogen Fusarium sambucinum. PLoS ONE 6(3): e17990.
  46. 46. Song YY, Zeng RS, Xu JF, Li J, Shen X, et al. (2010) Interplant communication of tomato plants through underground common mycorrhizal networks. PLoS ONE 5(10): e13324.
  47. 47. Robert GU, Martha ER (2010) Defense-related gene expression in soybean leaves and seeds inoculated with Cercospora kikuchii and Diaporthe phaseolorum var. Meridionalis. Physiol Mol Plant P 75: 64–70.
  48. 48. Samac DA, Graham MA (2007) Recent advances in legume-microbe interactions: recognition, defense response, and symbiosis from a genomic perspective. Plant Physiol 144: 582–587.
  49. 49. Liu J, Maldonado-Mendoze I, Lopez-Meyer M, Cheung F, Town CD, et al. (2007) Arbuscular mycorrhizal symbiosis is accompanied by local and systemic alterations in gene expression and an increase in disease resistance in the shoots. Plant J 50: 529–544.
  50. 50. Mishra RPN, Singh RK, Jaiswal HK, Kumar V, Maurya S (2006) Rhizobium mediated induction of phenolics and plant growth promotion in rice (Oryza sativa L.). Curr Microbiol 8: 383–389.
  51. 51. Pozo MJ, Cordier C, Dumas-Gaudot E, Gianinazzi S, Barea JM, et al. (2002) Localized vs systemic effect of arbuscular mycorrhizal fungi on defense response to Phytophthora infection in tomato plants. J Exp Bot 53: 525–534.
  52. 52. Yao MK, Desilets H, Charles MT, Boulanger R, Tweddell RJ (2003) Effect of mycrorrhization on the accumulation of rishitin and solavetivone in potato plantles challenged with Rhizoctonia solani. Mycorrhiza 13: 333–336.
  53. 53. Zhao S, Qi X (2008) Signaling in plant disease resistance and symbiosis. J Integr Plant Biol 50: 799–807.
  54. 54. Murphy J, Riley JP (1962) A modified single solution method for the determination of phosphate in natural waters. Anal Chim Acta 27: 31–36.
  55. 55. Nishi K, Sato F, Karasawa T, Sato T, Fukuta T, et al. (1999) Ecology and control of root necrosis of soybean caused by Calonectria crotalariae. Bull Nat Agric Res 30: 100–109.
  56. 56. Li C, He X, Zhu S, Zhou H, Wang Y, et al. (2009) Crop diversity for yield increase. PLoS ONE 4(11): e8049.
  57. 57. Vierheilig H, Coughlan AP, Wyss U, Piché Y (1998) Ink and vinegar, a simple staining technique for arbuscular-mycorrhizal fungi. Appl Environ Microb 64: 5004–5007.
  58. 58. Qin L, Jiang H, Tian J, Zhao J, Liao H (2011) Rhizobia enhance acquisition of phosphorus from different sources by soybean plants. Plant Soil.
  59. 59. Li S, Hartman GL, Domir LL, Boykin D (2008) Quantification of Fusarium solani f. sp. glycines isolates in soybean roots by colony-forming unit assays and real-time quantitative PCR. Theor Appl Genet 117: 343–352.
  60. 60. Banwart WL, Porter PM, Granato TC, Hassett JJ (1985) HPLC separation and wavelength area rations of more than 50 phenolic acids and flavonoids. J Chem Ecol 11: 383–395.
  61. 61. Guo WB, Zhao J, Li XX, Qin L, Yan XL, et al. (2011) A soybean β-expansin gene GmEXPB2 intrinsically involved in root system architecture responses to abiotic stresses. Plant J 66: 541–552.