Characterization of the Non-rhizobial Bacterial Community in the Nodule-Associated Soils of Akebono Soybeans and Isolation of Antibiotic Producing Amycolatopsis spp.

Abstract

The unique Akebono soybeans are cultivated in Minobu Town, Yamanashi Prefecture, Japan. The biogeography of Akebono soybeans and the microbial diversity associated with their root nodules remain unexplored. This study investigated the nodule-associated microbial community of Akebono soybeans using molecular techniques. The results showed that the family Bradyrhizobiaceae was dominant in soybeans obtained from Minobu town Imata (MI), Minobu town IItomi (MS), and Minobu town Hirase (MN). In contrast, members of the family Pseudonocardiaceae were isolated from the nodule samples from Minobu town Yasaiku (MY). The community structure of MY was different from that of the other sites and the genus Amycolatopsis was dominant. The bacterial community in the nodule associated soil obtained from the Minobu area was different from that of soybeans cultivated in Kofu City (approximately 35 km away from Minobu Town). The MY1 strain of Amycolatopsis spp. was isolated from the nodule associated soil in MY and was found to produce antibiotics. This study showed that the bacterial community in the nodules and the adjacent rhizosphere may be a regional characteristic of Akebono soybeans and the specific Amycolatopsis spp. dominant in the nodule associated soil of MY was implicated in determining the bacterial community structure.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12088-022-00999-x.

Introduction

The rhizosphere represents a hotspot for numerous organisms and is considered one of the most complex ecosystems on earth [1]. Rhizosphere bacteria have been extensively studied to determine their activities and functions due to their high diversity and direct involvement in plant growth and diseases [2]. The impact of plant growth, exudation, and nutrient availability in the rhizosphere physiochemical environment that influence rhizosphere microbial communities is of particular interest [3]. Root nodules for nitrogen fixation present in legume roots serve as an ecological niche characterized by a highly specific symbiotic relationship between leguminous plants and nodule-inducing bacteria [4]. Sharaf et al. [5] investigated the nodule-specific microbial community in soybean roots using high-throughput sequencing, and showed that the bacterial diversity at the family level varies based on the cultivar and water status. Therefore, the microbial community in soybean nodules is influenced by abiotic factors, soil geochemistry, and water availability. Studying the nodule microbial community in a specific environment may make it be possible to identify the relationship between the bacterial community in the nodule associated soil and plant genotype.

Compared to cereals such as corn (Zea mays), rice (Oryza sativa), and wheat (Triticum aestivum), soybean (Glycine max (L.) Merr.) produces seeds containing various proteins and lipids, which make soybean exceptionally nutritious [6]. A rare soybean called “Akebono soybean” is cultivated in Minobu Town, Yamanashi Prefecture, Japan (Fig. 1a). Akebono soybeans are white soybeans that are larger, sweeter, and taste better than other soybeans. Akebono soybean has been carefully maintained and preserved in very small farmlands for private use since the Meiji era (1868–1912). Therefore, it is a rare soybean with regional characteristics and limited production. This study investigated the geographical characteristics of bacterial communities in the nodule associated soil of Akebono soybean to protect the production area and the high quality. The regional characteristics of Akebono soybean cultivation with the soil environment are assessed. Finally, the genus Amicolatopsis dominant in the nodule associated soil of Minobu town Yasaiku (MY) was isolated, and we investigated its characteristics as an antibiotic producer.

Fig. 1.

a Sizes of different types of soybeans: (i) Akebono soybeans, (ii) normal white soybeans ‘Yuagari-musume,’ and (iii) green soybeans ‘Hayamidori.’ The upper photo shows dried soybeans, whereas the lower photo shows the soybeans soaked in water. b Map illustrating the locations of the sampling sites described in this study

Materials and Methods

Plant and Soil Samples

Plant samples were collected from four different contrasting localities. (1) a hilly and mountainous area (MY: 35°46′18′′ N, 138°40′49′′ E, altitude 630 m), (2) a non-mountainous field (Minobu town Imata (MI): 35°34′33′′ N, 138°42′46′′ E, altitude 300 m) where Akebono soybeans were cultivated in 2018, (3) an additional non-mountainous (Minobu town IItomi (MS): 35°43′15′′ N, 138°44′23′′ E) and, (4) one mountainous area (Minobu town Hirase (MN): 35°47′37′′ N, 138°40′54′′ E) was added to the investigation in 2019 (Fig. 1b). The soil and nodule associated soil samples were also obtained from Kofu area (KofuH: 35°65′01″ N, 138°60′70′′ E) in October, 2019. The primary chemical properties of the soils were determined and are shown in Table S1. Five plant samples were collected from each location together with the soil surrounding the roots. The nodules with nodule associated soil were harvested from the soybean roots immediately in the laboratory and stored at − 80 °C for DNA extraction and high-throughput sequencing.

High-throughput DNA Sequencing and Data Analysis

DNA was extracted from each nodule sample three times using the FastDNA™ Spin Kit for Soil (MP Biomedicals Japan, Tokyo, Japan). The DNA samples isolated in 2018 were pooled to constitute one sample. Similarly, DNA was extracted three times from each sample in 2019 and pooled. The 2018 DNA samples were sequenced with the Ion Torrent S5 XL system, whereas the 2019 samples were sequenced with the Illumina MiSeq system. Similar number of reads were obtained for 2018 and 2019 samples (total read: 90,173 ± 136 in 2018 and 90,688 ± 5,920 in 2019). However, as the read length obtained by Illumina MiSeq (approximately 450 nt) analysis is longer than that of Ion (approximately 250 nt), the former was used in 2019. DNA preparation and the experimental procedures were performed as described by Mahmood et al., [7].

The reads were uploaded to the DNA Data Bank of Japan (DDBJ) Sequence Read Archive (https://www.ddbj.nig.ac.jp/dra/index-e.html) in a Bioproject and are available under accession number DRA012380. Beta-diversity analysis was performed to evaluate differences in species complexity, and the beta-diversity of both weighted and unweighted variants of UniFrac was calculated using the QIIME software (Version 1.7.0, http://qiime.org/scripts/beta_diversity.html). Non-metric multidimensional scaling (NMDS) was used to visualize the pairwise UniFrac distances among samples using the R package, vegan (https://cran.r-project.org/web/packages/vegan/index.html).

Isolation of Amycolatopsis Bacteria

Bacteria from the nodules and the surrounding soil was isolated using the dilution plating technique. Briefly, five nodules and 4.0 mL of sterile distilled water were placed in a test tube and mixed thoroughly using a vortex. Subsequently, 50-µL dilutions from the first tube were spread onto humic acid-vitamin agar media [8] using a disposable spreader; the plates were then incubated at 25 °C for 1 month. Colonies appearing after 1 month were re-streaked until a single pure-colony type per plate was obtained.

Polymerase Chain Reaction (PCR) Amplification of Amycolatopsis genes

Colony PCR was used to identify Amycolatopsis spp. Specific primers AMY2 (positions 832–854): 5′-GGTGTGGGCGACATCCACGTTGT-3′ and ATOP (positions 1267–1289): 5′-GTATCGCAGCCCTCTGTACCAGC-3′ reported by [9] were used for amplification using a TaKaRa PCR Thermal Cycler Dice® system (TakaRa Bio Inc., Shiga, Japan). The cycling conditions included: 95 °C for 5 min; 30 cycles at 95 °C for 1 min, 65 °C for 1 min, and 72 °C for 1 min; and a final extension at 72 °C for 10 min. The 16S rRNA DNA of the Amycolatopsis isolates was amplified using the following PCR cycles: 95 °C for 5 min; 30 cycles at 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min; and a final extension at 72 °C for 7 min. Thus, the 25 µL of PCR mixture was comprised of 1 µL of sample DNA, 10 mM primers 341F (5′- CCTACGGGAGGCAGCAG-3′) and 1378R (5′- TGTGCAAGGAGCAGGGAC-3′), 9.5 µL of nuclease-free water, and 12.5 µL of GoTaq® Green Master Mix (Promega, Madison, WI). The amplification products (5 µL) were separated via electrophoresis on a 1% (w/v) agarose gel in Tris–acetate-ethylenediaminetetraacetic acid buffer at 100 V for 25 min and visualized via GelRed™ staining (1:20,000 dilution; Biotium, Fremont, CA). The DNA sequences obtained were compared to those uploaded previously to the DNA DDBJ (http://blast.ddbj.nig.ac.jp/). The sequence of the strain was submitted to the DDBJ.

Antibiotic production by Amycolatopsis spp. isolates

Amycolatopsis spp. were cultured in 200 mL of potato dextrose broth (PDB) at 25 °C for 1 week with shaking at 120 rpm. PDB without Amycolatopsis spp. was prepared as a control. After culturing, 100 mL of the culture was transferred to a separating funnel and extracted thrice using 50 mL of ethyl acetate. The extracts (150 mL) were concentrated to dryness using a rotary evaporator (Rotavapor R-3 system equipped with vacuum pump V-700, Nihon BUCHI K.K., Tokyo, Japan) and then resuspended in 50% aqueous methanol solution. The crude extract was sterilized by filtering through a 0.2 µm filter and 80 µL of which was used to soak a paper disc (8 mm diameter). The paper discs were placed in the center of potato dextrose agar plates (pH 7) spread with a bacterial suspension (Bacillus subtilis NBRC 3134, Escherichia coli NBRC 3301), Saccharomyces cerevisiae NBRC 100,929, Aspegillus niger NBRC 6341, Fusarium spp., Sclerotium rolfsii, and cultured at 25 °C. The antimicrobial activity was evaluated based on the development of clear zones. The experiment was carried out in triplicate.

Results and Discussion

Geographic Characteristics of the Bacterial Community in/on the Soil Associated with Nodules of Akebono Soybean Plants

Legumes, such as soybeans, participate in plant-rhizobia-microbe interactions via the nodule associated soil. Rhizobia and other bacteria form a community within the root nodule and enhance plant health and survival, particularly under environmental stress [10]. The bacterial community in the soybean rhizosphere has been studied by Sugiyama and Hamid et al. [6, 11]. Microorganisms isolated from nodules, previously referred to as root nodule bacteria [12], are now known as non-rhizobia endophytes [13], nodule endophytes [14], or nodule-associated bacteria [15]. High-throughput sequencing analysis of the DNA from the soybean nodules from the MI, MS, and MN sites showed that the family Bradyrhizobiaceae was the most dominant bacteria in the nodules and nodule associated soil, followed by the family Comamonadaceae. Members of the family Comamonadaceae are known to dominate the soybean rhizosphere [16]. Okutani et al. [17] also reported that the addition of daidzein, an exudate from soybean roots, modulates the bacterial community in vitro and increases the abundance of Comamonadaceae. Therefore, members of the family Comamonadaceae may show a high preference for soybean rhizosphere and nodule associated soil. In contrast, Pseudonocardiaceae—a family of bacteria belonging to the class Actinobacterium—were isolated from the nodule samples obtained from MY. It showed a different community structure than those of the other soils (Fig. 2b). The genus Amycolatopsis was dominant in the community. A similar result was obtained from the investigation on this microbial population in 2018 (Fig. S1). Similar trends were seen for other families in 2018 and 2019, especially in the Top 20 families, where Xanthomonadaceae was more common in 2018 but decreased in 2019. In addition, Ruminococcaceae and Flavobacteriaceae also showed an increase or decrease in relative abundance depending on the year of collection.

Fig. 2.

a Unweighted Pair-group Method with Arithmetic Mean (UPGMA) cluster tree based on (i) unweighted and (ii) weighted unifrac distance. b Relative abundance of taxa of Actinobacterium class, which arepresent on and in soybean nodules

The composition of microbial communities varies remarkably depending on the environmental conditions and the resulting interspecific interactions [18]. Moreover, the composition of microbial communities has a remarkably impact on the selection and maintenance of specific communities according to the survival needs of the population [18]. The composition of a bacterial community is remarkably correlated with differences in soil pH [19, 20]. However, the bacterial diversity and structure in Akebono soybean nodules have not been characterized extensively. Liu et al. [21] found that soil environmental parameters including pH, total carbon, total nitrogen, and moisture may account for 52.27% of the variation in bacterial communities, whereas 47.73% of the variation remains unexplained. Geographic distance may account for 14.75% of the variation in bacterial communities [21]. While diversity indices are useful in determining community characteristics using a single vector, they cannot evaluate important compositional features of biodiversity related to the abundance of shared taxa [22]. Therefore, NMDS was performed in this study to assess the β-diversity among the locations. The result of the NMDS analysis was based on the weighted UniFrac algorithm. The results showed that the bacterial community in the nodule associated soil in Kofu City differed from that of the Minobu area (Fig. 3). Kofu City is approximately 35 km away from Minobu Town (Fig. 1b). The chemical properties of the soil, including pH, are similar in the two areas. However, soil pH was lower in MY than other sites; moreover, higher amount of humus and of available manganese were detected in MY (Table S1). Higher humus and lower pH may be one of the reasons why there were many Actinomycetes in MY. Further research is needed on this point. The different bacterial communities in the nodule- associated soil of Akebono soybeans in the two areas may be due to geographic factors and cultural practices [6].

Fig. 3.

NMDS plot with distances determined using the weighted Unifrac algorithm, MY (white square), MS (black square), MI (black triangle), MN (black circle) and KofuH (white circle). MY had different bacterial community compared to other areas among Minobu area. Moreover, the bacterial community of entire Minobu area was significantly different from that found in the nodule associated soil of Akebono soybeans cultivated in the Kofu area. The distance between data points reflects the extent of variation. Error bar represents the mean ± standard deviation of triplicates. NMDS, Non-metric multidimensional scaling

Isolation of Amycolatopsis spp. and its Antibiotic Production

This study isolated the MY1 strain of the target Amycolatopsis spp. from the nodule associated soil. Scanning electron microscopy showed that the cells of MY1 strain were filamentous-shaped and approximately < 1 µm in diameter (Fig. 4c). The aerial hyphae of the strain were whitish and dense and produced pink or wine-red pigments when cultured on potato dextrose agar (pH 7) plates (Fig. 4a, B). The 16S rRNA sequence of strain MY1 (979 bp; GenBank accession No. LC627096) exhibited high sequence similarity to Amycolatopsis spp. MY1 shared the highest sequence similarity (100%) with A. kentuckyensis strain S1 (MK192019). Amycolatopsis spp. have been reported to act as a plant growth regulator [23], and a biocontrol agent [24]. In this study, Amycolatopsis spp., a rare actinomycete of the family Pseudonocardiaceae, was the predominant genus in the nodule associated soil collected from MY (Fig. 4). Moreover, the strain MY1 was found to produce antibiotics (Fig. 5). The antibiotic produced was effective only against B. subtilis and did not affect fungi such as Fusarium spp. and Sclerotium spp. (Fig. S2). Amycolatopsis spp. are widely used in the commercial production of several antibiotics, including the important broad-spectrum antibiotics rifamycin and vancomycin [25, 26]. This strain can also produce various other secondary metabolites and bioactive molecules [27], and can be exploited for a range of biotechnological applications [28]. The role of the Amycolatopsis spp. and its effect on the bacterial community in the nodule associated soil of Akebono soybeans is not clear. Hamid et al. [11], showed that soybeans alter the structural and functional composition of the rhizosphere microbiota to suppress pathogen infection in long-term monoculture cropping soils. Sugiyama [6] also argues soybean’s secretion of specific metabolites is developmentally and nutritionally regulated and may alter the microbial community in the rhizosphere. The predominance of Amycolatopsis spp. in MY is, perhaps, due to a similar reason. However, further experiments on the rhizosphere of the soybean growing environment of the MY fields, including the secretion of metabolites and the reasons for the dominance of Amycolatopsis spp. in the area are required.

Fig. 4.

Morphological characteristics of Amyclotopsis sp. strain MY1 cultured for 7 d at 25˚C on potato dextrose agar (pH 7.0). a A mature colony with entire to undulate margin and white aerial hyphae. b The reverse of the colony showing a pale orange to pink color. c The cells of strain MY1 observed via electron microscopy (scale bars: C = 5 µm). Straight, elongated, smooth surfaced aerial hyphae differentiating into ellipsoidal spore-like structures

Fig. 5.

Antibacterial activity of the crude extract of Amycolatopsis sp. MY1 spent broth culture. a Potato dextrose broth (pH 7) without bacteria was used as the control. b Zone of clearance—an expression of antibiotic activity—obtained using extracts by ethyl acetate from spent culture broth of Amycolatopsis sp. MY1 culture

In conclusion, Akebono soybeans passed down from generation to generation in Minobu Town show varietal characteristics including regional characteristics. Moreover, this study suggested that the bacterial community in the nodule associated soil may be a regional characteristic. And Amycolatopsis spp. in MY may contribute to establishing the bacterial community and growth and health of the soybean plants. Further clarification on the regional characteristics of Akebono soybeans, the relationship between metabolites secreted from roots and microbial communities, and the identification of antibiotics produced by Amycolatopsis spp., is necessary.

Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Table S1: Soil chemical properties. Figure S1: Relative abundance of (A) the top 30 bacterial families and (B) only Actinobacterium present on and in soybean nodules in 2018. Figure S2: Inhibition effect of Amycolatopsis spp. strain MY1 on Fusarium spp. and Sclerotium rolfsii. Confrontation assay using culture (a) and extracts (b) against Fusarium spp. (Right plates) and Sclerotium rolfsii (Left plates). Down paper disk dipped to extracts and upper one dipped to 50% MeOH.

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contributions

Conceptualization, R.K. and H.Y.; methodology, R.K.; investigation, R.K. and M.H.; writing—original draft preparation, R.K.; writing—review and editing, R.K., H.Y. and M.H.; visualization, R.K.; supervision, R.K.; project administration, R.K.; funding acquisition, R.K. All authors have read and agreed to the published version of the manuscript.

Funding

Please add: This research was funded by a regional promotion research project at the University of Yamanashi and the Rekura Minobu Agricultural Production Corporation.

Data Availability Statement

The data presented in this study are available on request from the corresponding author. The data presented in this study are deposited in DNA Data Bank Japan (DDBJ). Accession numbers are written in this text.

Declarations

Conflict of interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.