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Article

First Report of Tripartite Symbiosis Potential among Soybean, Bradyrhizobium japonicum, and Dark Septate Endophytes

by
Ni Luh Putu Citra Innosensia
1,2,
I Putu Wirya Suputra
3,
Gusti Ngurah Alit Susanta Wirya
3 and
Kazuhiko Narisawa
1,2,*
1
Department of Symbiotic Science of Environment and Natural Resources, United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Fuchu 183-0054, Japan
2
Department of Bioresource Science, College of Agriculture, Ibaraki University, Ami, Inashiki 300-0331, Japan
3
Faculty of Agriculture, Udayana University, Denpasar 80234, Indonesia
*
Author to whom correspondence should be addressed.
Agronomy 2023, 13(7), 1788; https://doi.org/10.3390/agronomy13071788
Submission received: 4 June 2023 / Revised: 20 June 2023 / Accepted: 26 June 2023 / Published: 3 July 2023
(This article belongs to the Section Soil and Plant Nutrition)
Browse Figures
Versions Notes

Abstract

:
Successful soybean and rhizobium interaction is characterized by the formation of root nodules. This symbiosis significantly affects the growth and yield of soybeans and has long been recognized as a key factor in sustainable agricultural systems. Naturally, soybeans could form a tripartite symbiotic relationship with rhizobium and arbuscular mycorrhizal fungi (AMF). However, there is no information regarding the symbiotic potential of soybean, nodulating bacteria, and dark septate endophytic (DSE) fungi. This study aims to delve into new tripartite symbiosis potential, shedding light on its efficacy in improving soybean growth while reducing environmental impacts. We examined the effect of incorporating two DSEs: Cladophialophora chaetospira SK51 (Cc) and Veronaeopsis simplex Y34 (Vs), into the classic soybean-rhizobium symbiosis. Under sterile conditions, the co-inoculation of DSEs with Bradyrhizobium japonicum IncB6 (Bj) significantly increased the nodule number and dry weight, leading to the acceleration of soybean vegetative growth. Soybean nodule numbers under co-inoculation treatments-CcBj and VsBj-were significantly increased by 77.6 and 43.6%, respectively, compared to the Bj treatment. Furthermore, under the CcBj and VsBj treatments, the soybean nodule dry mass was significantly increased by 455 and 363%, respectively, compared to Bj. This finding represents the first report of new beneficial tripartite symbiosis potential for soybean nodulation and vegetative growth.

1. Introduction

Soybean (Glycine max (L.) Merr.) is one of the most valuable, versatile, and nutritionally important legume crops in the world [1]. Soybean contains nine essential amino acids necessary for human nutrition [2] and is thus an important dietary source of protein. Its versatility to be converted into vegetable oil, livestock feed, vegan meal, soy milk, and biodiesel has led to soybeans becoming one of the most economically important crops [3,4]. Therefore, the demand for this legume will undoubtedly increase with the global population surge [5]. Soybean requires an appropriate dose of nitrogen (N) as a structural component of the chlorophyll molecule, as well as phosphorus (P), which is essential for soybean nodulation [6,7,8].
Farmers tend to add excessive chemical fertilizers to increase soybean growth and production [9]. A continuous application of chemical fertilizers can affect the soil’s microbiome by modifying the soil’s chemical composition and physical character [10,11]. Moreover, only 30–50% of applied N fertilizer and 10–45% of P fertilizer are utilized by crops [12,13]. This ineffective fertilizer assimilation by crops leads to nutrient runoff and leaching, having deleterious environmental consequences such as the deterioration of soil health and surface and groundwater [14]. Furthermore, N fertilizer was reported to reduce the number and size of root nodules [15].
Soybean nodules are specialized organs developed to provide sanctuary for N-fixing bacteria [16]. The soybean–bacterial association begins with the recognition of compatible bacteria by the host through a signal exchange [17]. Some research estimated this association provides 40 to 70% of the N demand for soybean, depending on the interactions among the host, their bacterial symbiont, and plant growth conditions [18,19,20]. This biological N fixation potentially alleviates the risks of environmental pollution caused by intensive synthetic N fertilizer use and production costs [21]. Consequently, maintaining and improving root nodule quality are essential for enhancing soybean growth and sustainable agriculture.
Several methods in genetic-based research to optimize nodule N fixation were reported to be less successful [22]. On the other hand, the symbiosis of soybean, rhizobium, and arbuscular mycorrhizal fungi (AMF) was reported to have an essential effect on soybean growth and nodulation under limited N and P conditions [23]. Such tripartite symbiosis might positively affect plants through biological N fixation and an improvement in P uptake by AMF [24,25]. In addition to the symbiotic relationship with both rhizobium and AMF, recent research has highlighted the involvement of dark septate endophytic (DSE) fungi in soybean microbiome [26]. Some studies suggested that DSEs could provide host plants with nutrients, accessing complex N and P compounds within the soil [27,28].
Dark septate endophyte colonization being frequently reported plays an important role in increasing plant growth under various environmental conditions [29,30,31]. This fungi has been reported to colonize a broad range of host plants, including around 600 species [32]. Despite being positively reported in many studies, information about the effect of DSE application on soybean growth and nodulation is currently unavailable. In this study, we investigated the effect of two DSE isolates: Cladophialophora chaetospira SK51 (Cc) and Veronaeopsis simplex Y34 (Vs), incorporated with nodulating bacteria on the growth of soybean in their vegetative stage. Therefore, nodule number (NN), nodule dry mass (NDM), shoot dry mass (SDM), root dry mass (RDM), fully expanded trifoliate leaf number (TFLN), and chlorophyll content (CC) were observed. Interestingly, the incorporation of DSEs with Bradyrhizobium japonicum IncB6 (Bj) significantly increased the nodule number and dry mass, leading to the acceleration of the soybean vegetative stage. Our study is the first to report the potential of successful tripartite symbiosis among soybean, B. japonicum, and DSEs. Further application of this information can lead to the development of sustainable agricultural practices, such as optimizing nodulation while reducing the need for synthetic fertilizers.

2. Materials and Methods

2.1. Dark Septate Endophytes

Two isolates of DSE were obtained from the culture collection of the Microbial Ecology Laboratory, Ibaraki University, Japan. These isolates were identified as Cladophialophora chaetospira SK51 and Veronaeopsis simplex Y34. The nucleotide sequences from the isolates used in this study can be found in the NCBI GenBank database under accession numbers MN811695 (C. chaetospira SK51) and LC632038 (V. simplex Y34). These two candidates were chosen because of their successful plant-growth-promoting effects, according to previous research [33,34]. The isolates were recovered from glycerol culture stock on ½ CMMY medium with a pH value of 5.6 (8.5 g of corn meal agar, 7.5 g of bacto agar, 10 g of malt extract, 1 g of yeast extract, and 1000 mL of distilled water) [35]. Each fungus was grown in 55 mm sterile Petri dishes at room temperature (approximately 23 °C) for four weeks.

2.2. Bradyrhizobium japonicum

2.2.1. Isolation

Bradyrhizobium japonicum was isolated using a baiting method by directly collecting nodules from soybean as a host plant. Soybean was cultivated in an organic field at the Microbial Ecology site at the Center for International Field Agriculture Research and Education, Ibaraki University, Ami, Japan. The root of soybean was excavated carefully and directly transferred to the laboratory. The root was washed under flowing water to remove soil debris. The nodules were then separated from the root area and sterilized. Nodule surface sterilization was conducted inside laminar airflow to minimize contamination. Nodules were submerged in 70% ethanol for one minute and then in 4% NaClO enriched with Tween 20 solution (10 µL/L) for three minutes. They were subsequently rinsed with sterile distilled water (SDW) six times. From the last rinse, nodules were aseptically crushed using blunt-nosed forceps on slightly modified yeast mannitol agar (YMA) medium enriched with Red Congo dye (25 mg/L) (0.5 g of K2HPO4, 0.2 g of MgSO4·7H2O, 0.1 g of NaCl, 0.4 g of yeast extract, 10 g of mannitol, 15 g of bacto agar, and 1000 mL of distilled water) [36]. They were macerated with SDW, and a drop was streaked on the medium. The inoculated plates were incubated inside an incubator at 28 °C for ten days. Translucent single colonies of suspected Bradyrhizobium were isolated and transferred onto a fresh plate for purification [36]. The purity of cultures was confirmed by repeatedly streaking the bacteria on YMA until a pure culture was successfully isolated.

2.2.2. DNA Extraction, Sequencing, and Phylogenetic Analysis

Genomic DNA extraction was performed using PrepMan™ Ultra Sample Preparation (Thermo Fisher Scientific, Warrington, UK). Polymerase chain reaction (PCR) was used to amplify the partial 16S rRNA gene with Primer 10F and Primer 907 R (Table S1). The reaction was carried out in a 50 µL reaction mixture containing 1 µL of 100 ng genomic DNA, 1.5 µL of 0.2 µM of each primer, 10 µL of 0.2 mM of deoxynucleoside triphosphate (dNTPs), 25 µL of 2× PCR buffer for KOD X Neo, 1 µL of KOD fx Neo, and 10 µL of SDW. Takara PCR Thermal Cycler Dice (Takara Bio INC., model TP 600, Kusatsu, Japan) was used according to the user’s manual with the following cycling conditions: initial denaturation at 94 °C for 4 min, followed by 35 cycles of 94 °C for 35 s, 52 °C for 55 s, and 72 °C for 2 min, then by a final 10 min extension at 72 °C.
PCR products were purified using 12 µL of 3 M sodium acetate (pH 4.8), 30 µL of 40% PEG, and 1.5 µL of 200 mM MgCl2. A sequencing reaction was carried out in a 10 µL reaction mixture containing 0.32 µL of each primer (0.5 µM), 1.5 µL of 5× sequencing buffer, 0.5 µL of BigDye, 6.68 µL of SDW, and 1.0 µL of purified DNA. The cycling conditions were 96 °C for 2 min as initial denaturation, then 25 cycles of 96 °C for 30 s, 50 °C for 15 s, and 60 °C for 3 min. Sequencing reaction products were purified using 3× volume of purification solution for PCR products, as described previously. Purified sequencing reaction products were resuspended in 20 µL of Hi-DiTM formamide solution (Applied Biosystems, Waltham, MA, USA), and sequence analysis was carried out using a BigDye Terminator v3.1 DNA sequencer instrument.
A basic local alignment search tool for nucleotides (BLASTN) was used to align the bacterial sequences with GenBank (NCBI) sequences. The nucleotide sequences from six bacterial isolates (IncB2, IncB3, IncB4, IncB5, IncB6, and IncB7) were deposited in the NCBI GenBank database under accession numbers OR078416-OR078421. Phylogenetic analyses of molecular datasets from 6 bacterial isolates and other selected sequences in this study (Table S1) were constructed and edited using MEGA11 [37]. Then, the neighbor-joining method [38] was used to construct a phylogenetic tree with 1000 bootstrap replications.

2.2.3. Preliminary Screening of Bacteria Nodulation Efficacy

The evaluation of the nodulation efficacy of suspected Bradyrhizobium was conducted. The isolates grown on YMA were transferred into yeast mannitol broth (YMB) medium and incubated inside an incubator shaker (Bio-Shaker BR-300LF, Taitec., Saitama, Japan) at 23 °C with 120 rpm shaking for three days to a cell density of approximately 1 × 109. The organic small-grain soybean seed cultivar Suzumaru was used in this research. This variety was chosen because of its small phenotypes suitable for cultivation inside the growth chamber. The seeds were surface-sterilized by soaking them in 70% ethanol for 40 s, soaking them in 1% NaClO solution for 15 min, and then rinsing them with SDW 3 times. Sterile seeds were then dried under laminar airflow overnight. Dried sterile seeds were placed on a 1% water agar (WA) medium and incubated for three days under 23 °C until the seeds germinated. Seeds were transplanted into a seedling tray containing 30 g of twice-sterilized organic seedling soil. In total, 2 mL of each bacterial suspension was added to the soil. Seedlings were incubated in a growth chamber under a 14/10 (light/dark) regime for 16 days. The temperature was set under 30 °C in light and 22 °C in dark, which is considered optimal for soybean seedling growth [39]. As a light source, LEDs were used at a photosynthetic photon flux density (PPFD) of 89.46 m−2 s−1.

2.3. Assessment of Soybean Vegetative Development under Various Treatments

2.3.1. Dark Septate Endophytes Materials’ Preparation

A fully grown fungal isolate was cut, diced, and transferred into a 250 mL flask containing 150 mL of 2% malt extract broth (MEB) (3 g of malt extract and 150 mL of distilled water) and incubated inside an incubator shaker (Bio-Shaker BR-300LF, Taitec.) at 23 °C with 120 rpm shaking for a month. The grown fungal mycelium was harvested and crushed using a sterile blender diluted with SDW to obtain a mycelial suspension. A total of 10 mL of each mycelial suspension (1.6 × 106 hyphal fragments/mL) was added to twice-sterilized (121 °C for 30 min) DSE carrier materials (50 g of wheat bran, 50 g of rice bran, 150 g of fermented leaves, and 170 mL of distilled water). Then, the DSE materials were incubated for a month inside a sealed plastic bag at room temperature until the fungus was fully grown (Figure S1).

2.3.2. Effect of Six Different Treatments on Soybean Vegetative Stage

Commercial organic seedling soil was sterilized twice by autoclaving it at 121 °C for 30 min prior to use. Each DSE material was mixed at 10% (w/w) into soil for DSE treatments. The organic soybean seed cultivar Suzumaru was used in this research. Seeds and a B. japonicum suspension were prepared as mentioned in the method described in Section 2.2.3. This experiment was conducted following a completely randomized design, with 15 biological replications for each treatment. Six treatments were given: soil only as a non-treatment control (NTC), C. chaetospira SK51 (Cc), V. simplex Y34 (Vs), B. japonicum IncB6 (Bj), C. chaetospira SK51+B. japonicum IncB6 (CcBj), and V. simplex Y34+B. japonicum IncB6 (VsBj). Plants were grown axenically inside the plant growth chamber. After 20 days of incubation, plants were harvested, and plant growth parameters were observed. Shoot dry mass (SDM), root dry mass (RDM), and nodule dry mass (NDM) were weighed after drying at 40 °C for a week. Nodule number (NN) and trifoliate leaf number (TFLN) were directly calculated. The first and second trifoliate leaves’ chlorophyll contents (CCs) were measured using a hand-held chlorophyll meter (SPAD-502; Minolta Camera Co., Osaka, Japan).

2.4. Frequency of Soybean Root Colonization by DSEs

The re-isolation method was used to measure fungal colonization inside soybean roots. Three biological replications of soybean were harvested and washed under running tap water to remove adhering soil debris. The root was cut into one-centimeter-long fragments before being transferred into a sterile 50 mL conical tube (SPL Life Sciences Co., Ltd., Pocheon-si, Republc of Korea), submerged with 0.005% Tween 20 solution, and rinsed using SDW. Washing and rinsing were carried out using a high-speed vibrator (Eyela Cute Mixer CM-1000) (Tokyo Rikakikai Co., Ltd., Tokyo, Japan). The root fragments were then transferred into a 90 mm Petri dish with sterile filter paper (Advantec-Toyo Roshi Kaisha, Ltd., Tokyo, Japan) to dry the root fragments for 30 min. Fifty root fragments for each treatment were chosen randomly and then transferred onto ½ corn meal (CM) medium (8.5 g of corn meal agar, 7.5 g of bacto agar, and 1000 mL of distilled water) for two weeks at 23 °C. The frequency of re-isolation was calculated as the mean number of root segments colonized by the fungus. Each treatment’s remaining fresh soybean root fragments were prepared for light microscope observation (BX51; Olympus, Tokyo, Japan). The root was cleared in 10% (v/v) potassium hydroxide (KOH) in a water bath at 80 °C for 20 min. Subsequently, roots were acidified with 1 N hydrochloride (HCl) in a water bath at 80 °C for 20 min, followed by staining with 0.005% cotton blue at room temperature overnight.

2.5. Statistical Analysis

Raw data were compiled using Microsoft Office Excel. Statistical analysis was performed using one-way ANOVA of the population means, followed by mean comparisons using the Tukey HSD test at the 5% significance level. Pearson correlation was performed to analyze the correlation among variables. The statistical analysis used OriginPro, Version 2023 (OriginLab Corporation, Northampton, MA, USA).

3. Results

Six B. japonicum isolates were successfully collected in this study (IncB2, IncB3, IncB4, IncB5, IncB6, and IncB7). Our screening data suggested that all bacterial isolates could form a symbiotic interaction with soybean, markedly by successful nodule formation. These B. japonicum isolates were sequenced and subjected to phylogenetic analysis (Figure 1).
Preliminary screening of nodulation efficacy was carried out for the six successfully isolated B. japonicum samples. All isolates successfully formed a symbiotic association with soybean var. Suzumaru as the host, indicated by visible nodules observed on the root. Bradyrhizobium japonicum IncB6 showed the highest nodule number, with significantly different values than the other isolates (Table 1) (Figure S2). Therefore, B. japonicum IncB6 will be used for the downstream experiment in this research.
Soybean vegetative growth under six different treatments was observed after 20 days of incubation inside a plant growth chamber. We found that soybean treated with CcBj showed the highest nodule number, which was significantly different compared with the others, followed by VsBj and Bj. Under the CcBj treatment, the soybean nodule numbers increased by 23.6 and 77.6% compared with VsBj and Bj, respectively (Figure 2A). Nodules’ dry mass data show that soybean plants treated with CcBj had the highest dry mass, followed by VsBj, and Bj (Figure 2B). Co-inoculation treatment CcBj led to no significantly different nodule biomass compared with VsBj, but both treatments showed significantly different values than Bj. The nodule dry mass of soybean treated with CcBj and VsBj was increased by 455 and 363% compared with soybean treated with Bj. Our observation results regarding nodule number and nodule dry weight indicated that incorporating DSEs can increase both the nodule number and dry mass of soybean compared with Bj treatment alone (Figure 3).
We observed the differences in soybean vegetative development among treatments, based on the number of fully expanded trifoliate leaves [40]. We found that under co-inoculation treatments, CcBj- and VsBj-treated soybean showed a significantly higher number of fully expanded trifoliate leaves compared with the other treatments (Figure 4A). These results indicate that co-inoculation treatments can accelerate soybean vegetative developmental stages compared with single inoculation treatments and the NTC. The number of fully expanded trifoliate leaves from the highest to lowest was observed under CcBj, VsBj, Vs, Cc, NTC, and Bj, respectively (Figure 4B).
After being harvested and dried for a week, soybean shoots and roots were weighed to obtain SDM and RDM data. SDM values from the highest to lowest were, respectively, observed in Vs, Cc, CcBj, VsBj, Bj, and NTC. Soybean treated with Vs, Cc, CcBj and VsBj showed a higher SDM, being significantly different to Bj and NTC. Soybean SDM increased by 53.5, 48.7, 45.5, and 39.1% after being treated with Vs, Cc, CcBj, and VsBj compared with NTC, respectively. Soybean roots, after being separated from nodules, were dried for RDM measurement. The RDM of soybean from the highest to lowest was, respectively, observed in Vs, Cc, CcBj, VsBj, Bj, and NTC. Soybean roots treated with Vs, Cc, CcBj, and VsBj showed higher RDM values, being significantly different when compared with NTC plants. However, NTC- and Bj-treated roots did not show significantly different RDM. Soybean RDM increased by 52.1, 42.9, 41.8, and 37.1% after being treated with Vs, Cc, CcBj, and VsBj compared with NTC, respectively (Figure 5).
The chlorophyll contents of the first and second fully expanded trifoliate leaves were measured in this study. In the first trifoliate leaf, the chlorophyll contents of NTC- and Bj-treated soybean showed significantly higher values than Cc-, Vs-, CcBj-, and VsBj-treated plants. A linear pattern was observed in the second trifoliate leaf chlorophyll content of soybean: NTC and Bj plants showed higher chlorophyll contents, being significantly different to the others (Figure 6).
To examine the correlation among variables, Pearson correlation was employed. Data show that NN was positively related to the increase in NDM and TFLN, with significantly different values. NDM was positively related to the increase in TFLN, SDM, and RDM, with significantly different values. Trifoliate leaf numbers showed a positive correlation with the increase in SDM. Moreover, SDM showed a positive correlation with RDM. On the other hand, we found that SDM was negatively correlated with the CC in both the first and second trifoliate leaves, being significantly different. Root dry mass was also negatively correlated with the first and second trifoliate leaf CC. Also, both chlorophyll contents were positively correlated with each other, being significantly different (Figure 7A). The frequency of soybean roots colonized by DSEs was examined. As shown in Figure 7B, the frequency of soybean root colonization after being treated with Cc, Vs, CcBj, and VsBj was 80.8, 82.7, 77, and 81.5%, respectively. We found no significantly different values among treatments (Figure 7B). Moreover, we also observed the roots of NTC- and Bj-treated plants. We found no DSE-like mycelium appeared, indicating no cross-contamination among treatments during the cultivation period.
To investigate DSE colonization inside the roots, microscope observations were conducted. The results indicated that DSE treatments led to the successful colonization of the treated soybean roots and corroborated the findings in Figure 7B. Roots treated with DSEs showed successful plant–fungus interaction. In general, the colonization pattern with DSE treatments showed the connection of soybean roots with fungal hyphae (Figure 8B,C,E,F). Meanwhile, in NTC- and Bj-treated roots, the fungus–plant interaction did not occur as expected (Figure 8A,D).

4. Discussion

Soybean root nodules are special organs needed for the sanctuary of symbiotic rhizobia to perform N fixation. Our study successfully revealed that incorporating DSEs and B. japonicum leads to better soybean nodulation. The nodule number and dry weight of soybean inoculated with CcBj and VsBj treatments showed significantly different values to single Bj inoculation. This finding supports the theory that DSE–plant symbioses are multifunctional and indefinite to nutrient acquisition and the resultant positive host growth response [41]. Dark septate endophytes might act as a bridge between soybean and B. japonicum interaction and promote bacterial colonization inside the root. It becomes more interesting when we note that B. japonicum and DSEs co-inhabit soybean roots without showing negative symptoms. Instead, they develop mutualism by enhancing nodulation.
This study is considered the first to reveal that DSEs play a vital role in supporting growth and the classic interaction between soybean and B. japonicum. Meanwhile, the available sources were limited to soybean, rhizobium, and AMF interaction [23,42]. Such tripartite interactions are crucial for soybeans, especially under organic or eco-friendly agricultural practices which rely on biological processes rather than agrochemical input. Tripartite symbiosis among plants, bacteria, and fungi occurs naturally in soil ecosystems. These interkingdom interactions are promoted by several factors, such as antibiotics, signaling molecules, cooperative metabolism, and physical interactions [43].
Recently, a mutualistic symbiosis between Aspergillus nidulans and Bacillus subtilis was discovered [44]. The authors demonstrated that A. nidulans mycelium is a highway for bacteria to migrate, disperse, and proliferate. The endophytic fungus Serendipita indica, an auxotrophy for thiamine, can be fulfilled by symbiosis with a soil-dwelling bacteria, B. subtilis [45]. Veronaeopsis simplex Y34 was previously reported to bind in mutualistic symbiosis with a rhizobium-related bacterium called Rhizobium sp. Y9 [46]. The co-inoculation of Rhizobium sp. Y9 and V. simplex Y34 tended to increase plant biomass and lead to higher fungal colonization in the roots of tomatoes, compared to V. simplex Y34 treatment alone (unpublished data). Moreover, based on the re-isolation method, no bacteria were isolated from plant roots when treated with only Rhizobium sp. Y9. This finding suggests that the bacteria cannot invade and colonize plant roots themselves. In our study, despite rhizobia naturally being able to invade soybean roots, we consider that the presence of DSEs might act as a conduit for bacteria to enter the roots expeditiously.
Fully expanded trifoliate leaf numbers are used to determine the vegetative development of soybean [40]. Under CcBj and VsBj treatments, fully expanded trifoliate leaf numbers were significantly higher than in soybean treated with other treatments. The increase in TFLN was positively correlated with NN and NDM. It is suggested that CcBj and VsBj improve soybean nodulation, leading to the acceleration of the soybean vegetative stage. Soybean biomass, shoots, and roots were significantly increased upon a single inoculation with DSEs (Cc and Vs) and co-inoculation treatment (CcBj and VsBj). These results suggest that in this stage, DSE fungi potentially increase soybean biomass regardless of the presence of Bj. Although DSE and co-inoculation treatments contribute positively to soybean growth, it is important to note that the early establishment of these associations might require higher symbiotic costs. The cost might be manifested as a temporary reduction in the soybean chlorophyll content, as we observed in our study.
Establishing effective Rhizobium symbiosis is crucial for soybean production and long-term soil fertility [47]. Soybean possesses the ability to harbor Rhizobium species to undertake biological nitrogen fixation, ranging between 40 and 70% of the total soybean N uptake [7,19]. Due to the importance of nodules for soybean yield, studies have been carried out to support or increase the success of the nodulation process. Bacillus cereus UW85 and seed treatment with non-thermal plasmas have been used to enhance soybean nodulation [48,49]. Genetically based research in N2 fixation has also been performed, although incorporating some variation was reported as less successful [22]. However, our study exploits the tripartite symbiosis potential among soybean, B. japonicum, and DSEs, leading to an increase in soybean nodulation in a relatively more practical and environmentally friendly way.
The positive response we observed to dual inoculation with B. japonicum and DSEs was possibly facilitated by increased nutrient supply to soybean. Heteroconium chaetospira (syn. C. chaetospira) was reported to provide N to the Chinese cabbage rather than the plant mineralizing available organic N [50]. Meanwhile, the interaction between B. japonicum-DSE might share similar traits to the well-known B. japonicum-AMF symbiosis associated with increased P supply to the nodules [51]. A previous study has proven that DSE fungi contributed to the decomposition of organic P in soil [28]. Phosphorus was reported as essential to regulating nodule growth, nitrogenase activity, and metabolic pathways and enhancing the capacity of N-fixing root nodules [52,53,54,55,56]. Those previous studies suggest that increased N and P supply to soybean possibly facilitated the upsurge of observed growth parameters under co-inoculation treatments.
Information about the tripartite symbiosis potential among soybeans, B. japonicum, and DSEs provides new insight regarding the use of microbes to support sustainable agricultural practices. Employing this interkingdom interaction offers opportunities for optimizing organic agricultural practices and developing sustainable strategies for soybean production. Moreover, relying on microbial associations is always important in organic soybean production, whose demand keeps increasing for direct human consumption. However, continued investigations into the mechanisms and broader ecological implications of this tripartite symbiosis will contribute to a better understanding of their potential for agricultural systems in the future.

5. Conclusions

In conclusion, our study is the first to reveal that incorporating dark septate endophytic (DSE) fungi along with Bradyrhizobium japonicum (Bj) enhances soybean nodulation, leading to an increase in nodule number and nodule dry weight. Furthermore, the co-inoculation treatments of both Cladophialophora chaetospira SK51 and Veronaeopsis simplex Y34 with Bradyrhizobium japonicum IncB6 (CcBj and VsBj) significantly increased the number of fully expanded trifoliate leaves, which is an indicator of soybean vegetative development. Soybean biomass, both shoots and roots, increased with single DSE inoculation and co-inoculation treatments. Our study revealed the vital role of DSEs in supporting the growth and interaction of soybean with B. japonicum, which are crucial in organic agricultural practices. This study highlights the new potential of utilizing microbial associations to support sustainable agricultural practices.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy13071788/s1, Table S1: Primer sequences used for bacteria identification, Table S2: GenBank accession numbers of sequences used for phylogenetic analyses, Figure S1: Ready-to-use DSE materials. (A) Cladophialophora chaetospira SK51 material and (B) Veronaeopsis simplex Y34 material. Figure S2: Bacteria screening, root of soybean treated with (A) Bradyrhizobium japonicum IncB2, (B) Bradyrhizobium japonicum IncB3, (C) Bradyrhizobium japonicum IncB4, (D) Bradyrhizobium japonicum IncB5, (E) Bradyrhizobium japonicum IncB6, (F) Bradyrhizobium japonicum IncB7.

Author Contributions

K.N. supervised the research and provided research funding. N.L.P.C.I., I.P.W.S. and G.N.A.S.W. planned and designed the research, analyzed and interpreted the data. N.L.P.C.I. performed the experiments. N.L.P.C.I. assisted by I.P.W.S. and G.N.A.S.W., wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the research program on development of innovative technology grants (JPJ007097) from the Project of the Bio-oriented Technology Research Advancement Institution (BRAIN).

Data Availability Statement

Not applicable.

Acknowledgments

We thank Made Arimbawa, Dwi Sugiarta, and Shah Mahapati Dinarkaya for their help in phylogenetic tree construction, and Nursanti and Felix for their field and laboratory work assistance.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Phylogenetic tree of six bacterial isolates and reference sequences derived from the NCBI GenBank database. Brevibacillus invocatus strain B32 (MH587029) was used as an outgroup. The bar represents 0.1 substitutions per nucleotide position. The bacteria isolated from this study were presented in bold.
Figure 1. Phylogenetic tree of six bacterial isolates and reference sequences derived from the NCBI GenBank database. Brevibacillus invocatus strain B32 (MH587029) was used as an outgroup. The bar represents 0.1 substitutions per nucleotide position. The bacteria isolated from this study were presented in bold.
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Figure 2. (A) Nodule number of soybean and (B) nodule dry mass of soybean after 20 days of cultivation under sterile conditions. Median values are lines across the box, with lower and upper boxes indicating the 25th and 75th percentiles, respectively. Whiskers represent the maximum and minimum values. Diamonds refer to the data distribution of 15 biological replications for each treatment. Means followed by the same letter are not significantly different at p < 0.05 according to Tukey’s honestly significant difference test.
Figure 2. (A) Nodule number of soybean and (B) nodule dry mass of soybean after 20 days of cultivation under sterile conditions. Median values are lines across the box, with lower and upper boxes indicating the 25th and 75th percentiles, respectively. Whiskers represent the maximum and minimum values. Diamonds refer to the data distribution of 15 biological replications for each treatment. Means followed by the same letter are not significantly different at p < 0.05 according to Tukey’s honestly significant difference test.
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Figure 3. Soybean root performance under different treatments. (A) Root of soybean treated with Bradyrhizobium japonicum IncB6 (Bj), (B) root of soybean treated with C. chaetospira SK51+B. japonicum IncB6 (CcBj), (C) root of soybean treated with V. simplex Y34+B. japonicum IncB6 (VsBj), (D) nodules of soybean treated with Bj, (E) nodules of soybean treated with CcBj, and (F) nodules of soybean treated with VsBj.
Figure 3. Soybean root performance under different treatments. (A) Root of soybean treated with Bradyrhizobium japonicum IncB6 (Bj), (B) root of soybean treated with C. chaetospira SK51+B. japonicum IncB6 (CcBj), (C) root of soybean treated with V. simplex Y34+B. japonicum IncB6 (VsBj), (D) nodules of soybean treated with Bj, (E) nodules of soybean treated with CcBj, and (F) nodules of soybean treated with VsBj.
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Figure 4. (A) Soybean shoots’ morphological differences. (B) Fully expanded trifoliate leaf number after 20 days of cultivation. Means followed by the same letter are not significantly different at p < 0.05 according to Tukey’s honestly significant difference test (n = 15). Whiskers represent standard error (SE) value.
Figure 4. (A) Soybean shoots’ morphological differences. (B) Fully expanded trifoliate leaf number after 20 days of cultivation. Means followed by the same letter are not significantly different at p < 0.05 according to Tukey’s honestly significant difference test (n = 15). Whiskers represent standard error (SE) value.
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Figure 5. Shoot and root dry mass of soybean after 20 days of incubation. Median values are lines across the bar; whiskers represent the maximum and minimum values. Diamonds refer to the data distribution of 15 biological replications for each treatment. Means followed by the same letter are not significantly different at p < 0.05 according to Tukey’s honestly significant difference test.
Figure 5. Shoot and root dry mass of soybean after 20 days of incubation. Median values are lines across the bar; whiskers represent the maximum and minimum values. Diamonds refer to the data distribution of 15 biological replications for each treatment. Means followed by the same letter are not significantly different at p < 0.05 according to Tukey’s honestly significant difference test.
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Figure 6. Soybean chlorophyll content of the first and second trifoliate leaves after 20 days of incubation. Median values are lines across the bar, and whiskers represent the maximum and minimum values. Diamonds refer to the data distribution of 15 biological replications for each treatment. Means followed by the same letter are not significantly different at p < 0.05 according to Tukey’s honestly significant difference test.
Figure 6. Soybean chlorophyll content of the first and second trifoliate leaves after 20 days of incubation. Median values are lines across the bar, and whiskers represent the maximum and minimum values. Diamonds refer to the data distribution of 15 biological replications for each treatment. Means followed by the same letter are not significantly different at p < 0.05 according to Tukey’s honestly significant difference test.
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Figure 7. (A) Pearson correlation heat map indicating the relationships among variables. NN: nodule number, NDM: nodule dry mass, TFLN: fully expanded trifoliate leaf number, SDM: shoot dry mass, RDM: root dry mass, CC1: chlorophyll content trifoliate leaf 1, CC2: chlorophyll content trifoliate leaf 2. Red indicates a positive correlation between two variables; blue indicates a negative correlation between two variables. The number of each cell indicates the Pearson correlation coefficient. The box with * means significantly different at p < 0.05, ** means significantly different at p < 0.01, and *** means significantly different at p < 0.001. (B) The frequency of roots colonized by DSE fungi; data were obtained via re-isolation method. Means followed by the same letter are not significantly different at p < 0.05 according to Tukey’s honestly significant difference test.
Figure 7. (A) Pearson correlation heat map indicating the relationships among variables. NN: nodule number, NDM: nodule dry mass, TFLN: fully expanded trifoliate leaf number, SDM: shoot dry mass, RDM: root dry mass, CC1: chlorophyll content trifoliate leaf 1, CC2: chlorophyll content trifoliate leaf 2. Red indicates a positive correlation between two variables; blue indicates a negative correlation between two variables. The number of each cell indicates the Pearson correlation coefficient. The box with * means significantly different at p < 0.05, ** means significantly different at p < 0.01, and *** means significantly different at p < 0.001. (B) The frequency of roots colonized by DSE fungi; data were obtained via re-isolation method. Means followed by the same letter are not significantly different at p < 0.05 according to Tukey’s honestly significant difference test.
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Figure 8. Light microscopic observation of soybean root (scale bar: 200 µm). (A) NTC plant root, (B) Cc-treated root, (C) Vs-treated root, (D) Bj-treated root, (E) CcBj-treated root, (F) VsBj-treated root. Arrows indicate fungal hyphae of DSE fungus connection with soybean root.
Figure 8. Light microscopic observation of soybean root (scale bar: 200 µm). (A) NTC plant root, (B) Cc-treated root, (C) Vs-treated root, (D) Bj-treated root, (E) CcBj-treated root, (F) VsBj-treated root. Arrows indicate fungal hyphae of DSE fungus connection with soybean root.
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Table
Table 1. Preliminary screening of six B. japonicum isolates used to inoculate soybean. Means followed by the same letter in the column are not significantly different at p < 0.05 according to Tukey’s honestly significant difference test (n = 6).
Table 1. Preliminary screening of six B. japonicum isolates used to inoculate soybean. Means followed by the same letter in the column are not significantly different at p < 0.05 according to Tukey’s honestly significant difference test (n = 6).
Bacterial IsolatesNodule Number Mean ± SE
Bradyrhizobium japonicum IncB25.2 ± 0.94 b
Bradyrhizobium japonicum IncB34.8 ± 1.07 b
Bradyrhizobium japonicum IncB46 ± 1.26 b
Bradyrhizobium japonicum IncB57.2 ± 0.94 b
Bradyrhizobium japonicum IncB615.2 ± 0.47 a
Bradyrhizobium japonicum IncB78b ± 1.39 b
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Innosensia, N.L.P.C.; Suputra, I.P.W.; Wirya, G.N.A.S.; Narisawa, K. First Report of Tripartite Symbiosis Potential among Soybean, Bradyrhizobium japonicum, and Dark Septate Endophytes. Agronomy 2023, 13, 1788. https://doi.org/10.3390/agronomy13071788

AMA Style

Innosensia NLPC, Suputra IPW, Wirya GNAS, Narisawa K. First Report of Tripartite Symbiosis Potential among Soybean, Bradyrhizobium japonicum, and Dark Septate Endophytes. Agronomy. 2023; 13(7):1788. https://doi.org/10.3390/agronomy13071788

Chicago/Turabian Style

Innosensia, Ni Luh Putu Citra, I Putu Wirya Suputra, Gusti Ngurah Alit Susanta Wirya, and Kazuhiko Narisawa. 2023. "First Report of Tripartite Symbiosis Potential among Soybean, Bradyrhizobium japonicum, and Dark Septate Endophytes" Agronomy 13, no. 7: 1788. https://doi.org/10.3390/agronomy13071788

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