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Article

The Endophytic Plant Growth Promoting Methylobacterium oryzae CBMB20 Integrates and Persists into the Seed-Borne Endophytic Bacterial Community of Rice

by
Denver I. Walitang
1,2,
Aritra Roy Choudhury
3,
Yi Lee
4,
Geon Choi
1,
Bowon Jeong
1,
Aysha Rizwana Jamal
1 and
Tongmin Sa
1,5,*
1
Department of Environmental and Biological Chemistry, Chungbuk National University, Cheongju 28644, Republic of Korea
2
College of Agriculture, Fisheries and Forestry, Romblon State University, Romblon 5505, Philippines
3
Microbiome Network and Department of Agricultural Biology, Colorado State University, Fort Collins, CO 80521, USA
4
Department of Industrial Plant Science and Technology, Chungbuk National University, Cheongju 28644, Republic of Korea
5
The Korean Academy of Science and Technology, Seongnam 13630, Republic of Korea
*
Author to whom correspondence should be addressed.
Agriculture 2023, 13(2), 355; https://doi.org/10.3390/agriculture13020355
Submission received: 16 January 2023 / Accepted: 29 January 2023 / Published: 31 January 2023
(This article belongs to the Special Issue Soil Microbial Communities in Agriculture and Environmental Resilience)
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Abstract

:
Endophytic persistence of inoculated plant growth promoting bacteria (PGPB) involves interaction with the host plant and the host’s indigenous endophytic bacterial communities. This study investigated the persistence of Methylobacterium oryzae CBMB20 into the rice endosphere together with the impact of inoculation on the diversity and community structure of the root and shoot bacterial endophytes in Oryza sativa L. spp. indica cv. IR29. Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis of the root and shoot showed that M. oryzae CBMB20 was able to integrate and persist in the rice endosphere without causing drastic shifts in bacterial endophytic diversity and community composition. The bacterial communities in the root and shoot are very similar to the seeds of IR29, suggesting that most of them are seed-borne. The root endosphere bacterial communities of inoculated and uninoculated IR29 plants are more diverse compared to the shoots in terms of richness and diversity indices. The dominant bacterial T-RFs of the root endosphere of IR29 belong to Microbacterium, Delftia, Pseudomonas, Xanthomonas and Stenotrophomonas, Herbaspirillum, Enterobacter, and Sphingomonas, as observed in the three restriction enzyme T-RFLP profiles. Bacterial clades identified as Curtobacterium, Enterobacter, Stenotrophomonas, and Xanthomonas were distinctly observed in both the root and shoot communities, and these bacterial groups are also the dominant endophytes of the shoot endosphere. This study showed that Methylobacterium oryzae CBMB20 could persist and incorporate into the endophytic bacterial community of the endosphere without causing long-term antagonistic interactions with its host plant and with the native microbiota.

1. Introduction

Rice harbors diverse genera of bacterial endophytes. The most dominant members comprise of Proteobacteria, mostly Enterobacter-related, as well as rhizobia, followed by Firmicutes and other lesser phylogenetic lineages [1]. The roots of rice are also colonized by bacterial groups such as Stenotrophomonas maltophilia [2], Dickeya zeae, Mycobacterium bolletii, Rhizobium radiobacter, and other groups which are found in many rice genotypes [3]. The seeds are also inhabited by potential core microbial groups including Curtobacterium, Enterobacter, Flavobacterium, Herbaspirillum, Microbacterium, Stenotrophomonas, and Xanthomonas [4,5]. These competent endophytes have inherent traits and adaptations, allowing them to successfully colonize and survive in the internal tissues of plants [6].
As putative bacterial endophytes migrate and persist inside the rice host, dynamic shifts in physiological processes in the plant because of biotic and abiotic factors could affect these colonizing microorganisms. They would also encounter populations of native microbial inhabitants, which may lead to competition or integration. For plant bioinoculants to confer beneficial effects, they generally have to integrate and persist into their host and maintain a viable population [7]. There have been some studies on the effects of inoculation on the bacterial structure and communities of some host plants. Conn and Franco [8] studied the impact of microbial bioinoculation on the indigenous endophytic community of wheat roots. They found that some microbes present in the mixed inoculum were able to outcompete the local actinobacterial inhabitants, leading to the reduced endophytic colonization of these native actinobacteria. Research by Andreote et al. [9] on the colonization of Pseudomonas putida P9 and its impact on microbial communities showed that the strain P9 actively migrates into potato plants, challenging pre-existing microbial populations. The strain P9R influenced bacterial groups, particularly P. azotoformans, P. veronii, and P. syringae. Another observation by Andreote et al. [10] revealed that changes in the structure of bacterial endophytic communities in inoculated plants may result in subsequent plant metabolic alterations.
The successful colonization, integration, and persistence of bacterial endophytes extends the potential for improved growth and health of plants, while maintaining microbial and plant interactions. The enhancement of growth, health, and yield is attributed to bacterial endophytes’ multifaceted plant growth promoting potentials under normal and stress conditions [11,12,13]. Inoculation of bacterial endophytes could also lead to effects such as changes in bacterial diversity and improvement in functional profiles, as observed in other systems [14].
Microbial diversity could be investigated through culture-dependent and culture-independent approaches. Isolation and identification of cultivable bacterial endophytes are essential in further investigations of the functions and properties of individual microbial communities [11]. However, culture-independent approaches such as T-RFLP analysis are helpful to gain insight and provide overviews of the overall community structure of the studied systems. T-RFLP in particular, although an older technique, remains relevant for observing trends in community structure and diversity comparable to the results of high-throughput sequencing technologies, such as MiSeq. This is also in consideration of its reliability, repeatability, and lower cost [15].
Bacterial competence in terms of successful colonization, integration, and persistence of any inoculum into the rhizosphere, phyllosphere, or endosphere system is potentially one of the most important factors before a PGPB could act as an effective bioinoculant. An inoculum that will not survive and will be outcompeted by autochthonous inhabitants will not be able to form plant associations and is less likely to confer plant growth promotion. Inoculation of a PGPB rice-associated endophytic bacteria could lead to shifts in the diversity and structure of microbial communities, while integrating into the endosphere of rice. Few investigations have been conducted on the impact of microbial inoculation on the endophytic bacterial communities of host plants, and the major contributing factors to the interactions of putatively beneficial bacteria and the indigenous endophytic community are not always well understood. Hence, this study investigates the effective integration and potential persistence of a PGPB into the indigenous endophytic bacterial communities of rice.

2. Materials and Methods

2.1. Plant and Bacterial Strain

The Rural Development Administration (RDA) of South Korea provided the rice seeds (Oryza sativa L. subsp. indica cv. IR29). Methylobacterium oryzae CBMB20, maintained in ammonium mineral salt medium with sodium succinate (0.05%), was utilized for this study. This strain was grown to OD600~0.8 and was harvested using 0.03 M MgSO4. For colony counting after inoculation, M. oryzae CBMB20-gfp [16] with kanamycin resistance as a selection marker to remove non-M. oryzae CBMB20 bacteria, was utilized for bioinoculation. It was prepared in a similar way to the wild-type M. oryzae CBMB20, but 50 µg mL−1 kanamycin was added to the AMS medium. M. oryzae CBMB20-gfp also appears as bright pink to red when grown in AMS medium. A total of 540 and 450 seeds were sown for the study of diversity and colony counting, respectively.

2.2. Seed Sterilization and Bacterial Inoculation

Rice seeds were dehusked under sterile conditions followed by surface-sterilization with 0.12% NaClO and salts (0.1% Na2CO3, 3% NaCl, and 0.15% NaOH) at 30 °C for 25 min at 200 rpm in an orbital shaker [17]. Washing the seeds twice for 10 min at 30 °C in a 200 rpm orbital shaker with sodium thiosulfate solution (2%) removed the sodium hypochlorite [18]. Sterile distilled water was used to rinse the seeds up to eight times before rehydrating the seeds for 1 h with sterile demineralized water at room temperature. The final rinse was used to confirm efficient seed surface sterilization by inoculating the final rinse onto R2A media and checking for contamination after 7 days.
Germinated seeds were transferred in a seedling tray with nursery soil, allowing seedling growth in the greenhouse, which was naturally illuminated. Bacterization of 14-day-old seedlings was carried out through foliar application [19] and the application of inoculum (5 mL) in the rhizosphere of rice. For non-inoculated plants, 0.03 M MgSO4 solution was used. This set-up was repeated for colony counting, but plants were applied with M. oryzae CBMB20-gfp.

2.3. Determination of Culturable Bacterial Endophytes in the Rice Microniches

To determine the putative endophytic bacterial groups found in the roots and shoots of IR29, surface-sterilized rice seeds were gnotobiotically grown in semi-solid MS media supplemented with 0.2% phytagel. The seeds were allowed to germinate for up to 7 days. The roots and shoots were separately harvested, and sterilized according to Hardoim et al. [2]. Culturable bacterial populations were noted after serial dilution and plating of 1 g crushed plant root or shoot in R2A media, incubated at 28 °C for 5 days. Thirty distinct colonies were randomly selected and purified. BOX PCR fingerprints were created and compared with previously isolated bacterial endophytes of IR29 seeds and related rice cultivars [20].

2.4. Root and Shoot Samples

Plants were carefully uprooted and soil particles attached to the soil were carefully washed with running water. A sterile scalpel was used to separate the root and the shoot tissues of the rice. For T-RFLP analysis, selected plant parts were divided into pieces (5 cm) followed by immediate freezing with liquid N2 and the samples were stored at −80 °C. Shoot samples were also collected as roots.

2.5. DNA Extraction

Selected plant parts for DNA extraction were surface sterilized. For instance, the snap-frozen roots were thawed and then sterilized with ethanol (70%) for 2 min, and then with NaClO solution (2%) for 2 min, followed by repeated rinsing with autoclaved demineralized water up to three times. To release the bacterial endophytes, the surface-sterilized roots (4 g) were homogenized by crushing and squeezing with a softheaded hammer in sterile plastic bags containing autoclaved MilliQ water (2 mL). A DNeasy plant extraction kit (Qiagen, Hilden, Germany) was employed for extracting DNA from 400 mL of homogenate following the manufacturer’s instructions, of which the disruption of cells was modified to 1 h to increase lysis of bacteria. The kit has been utilized in previous studies of plant microbial communities, including potatoes [9,10] and vine plants [21,22].

2.6. T-RFLP PCR Amplification

The amplification of bacterial endophytic genomic DNA was carried out following the PCR conditions by Johnston-Monje and Raizada [23]. The components of the PCR master mix contained 25 mM of each of the dNTP mix (0.8 μL), Standard Taq Buffer (2.0 μL), 0.5 μL of 10 µM of each forward and reverse primers, Standard Taq (0.25 μL), total DNA (20 ng), and the final volume was adjusted to 20 μL with sterile MilliQ H2O. The 27 F-Degen primer and the 1492r primer sequences were AGRRTTYGATYMTGGYTYAG and GGTTACCTTGTTACGACTT [24], respectively, where R = A + G, Y = C + T, M = A + C. The PCR program included 96 °C DNA denaturation for 3 min, 25x (94 °C for 30 s, 48 °C for 30 s, 72 °C for 1 min 30 s), and a final extension at 72 °C for 7 min. DNA amplification was conducted in a DNA Thermal Cycler (PTC200, MJ Scientific, Foster City, CA, USA).
A second nested PCR with a 50 μL total volume was performed to increase the amount of the total amplified DNA. The master mix contained 25 mM each of the dNTP mix (4.0 μL), Standard Taq Buffer (5.0 μL), 2.0 μL of 10 µM of each forward and reverse primers, Standard Taq (0.3 μL), 10−1 PCR product from the first PCR reaction (2.0 μL), and the final volume was adjusted to 50.0 μL with sterile MilliQ H2O. The 799f primer was labeled with 6FAM with sequence AACMGGATTAGATACCCKG (where M = A + C, K = G + T) [25]. The 1492r primer had the same sequence as stated above. The 799f primer was originally designed to limit the amplification of the 16S rDNA from the chloroplast while amplifying 16S rDNA of bacterial samples [25]. The amplified mitochondrial 18S was identified and removed from the analysis. The PCR program included 95 °C DNA denaturation for 3 min, 25× (94 °C for 20 s, 53 °C for 40 s, 72 °C for 40 s), and a final extension at 72 °C for 7 min. DNA amplification was conducted in a DNA Thermal Cycler (PTC200, MJ Scientific, USA).

2.7. Restriction Enzyme Digestion

DdeI, HaeIII, and HhaI restriction enzymes (Promega, Madison, WI, USA) were used to separately digest the purified DNA PCR products. For the digestion mixture, the components were 4 U each (0.8 µL) restriction enzyme, 10× buffer (2.0 µL), 10× BSA (2.0 µL), and the mixture was adjusted to a total volume (20.0 uL) with MilliQ based on the volume of the PCR purification product containing 1.0 µg/µL. Digestion with the restriction enzymes was carried out for 16 h at 37 °C. To check for digestion, electrophoresis was carried out to separate and detect the digested products, of which 5.0 µL of the digestion mixture was loaded into 2% agarose gel.

2.8. Sizing

Terminal restriction fragments (T-RF) were analyzed through a DNA sequencer (ABI 3130, Applied Biosystems, Waltham, MA, USA). Final samples were prepared by mixing PCR digested products (1.5 µL), Hi-Di™ formamide (ABI) (9 µL), and size standard (500ROX, Bioventures) (0.6 µL) then denatured (95 °C, 3 min) and then put on ice (5 min). With the aid of the size mapper (500 ROX), the 5′ T-RFs fluorescently labeled with 6FAM were identified, detected, analyzed, and quantified with Genemapper, ver. 3.7 (Applied Biosystems).

2.9. Analysis

For T-RF community profiles, the individual T-RF peaks were identified and then abundances were compiled, arranged, and then finally normalized. The abundance or quantity of individual T-RF fragments was normalized by dividing the individual peak area by the total peak areas of each sample [26]. Shannon diversity index (H’), Simpson index (1/D), Richness (S), and Shannon evenness (J′) were accordingly calculated [4]. One-way ANOVA and Tukey’s test were carried out for the comparison of treatment means of CFU counts and diversity indices (SAS, Ver 9.4).

3. Results

3.1. The Seed-Borne Endophytic Community in Roots and Shoots of Gnotobiotically Grown IR29

Seed-borne endophytic communities actively and rapidly colonize the roots and shoots of their IR29 host plant. The seeds of IR29 contain 5.46 log CFU g−1 bacterial endophytes. When the seeds were grown in gnotobiotic conditions, seed-borne bacterial endophytes in the roots reached a population of 6.89 log CFU g−1 FW and 8.16 log CFU g−1 FW in the shoots (Table 1). BOX-PCR of bacterial isolates shows that the root endosphere is mainly dominated by Flavobacterium, followed by Pseudomonas, Microbacterium, and Pantoea. On the other hand, the shoot endosphere is dominated by Pantoea followed by Flavobacterium, Enterobacter and Microbacterium (Figure 1 and Figure S1).

3.2. Persistence of M. oryzae CBMB20 in Association with Rice

Methylobacterium oryzae CBMB20 was observed to be persistent in the rhizosphere, root and shoot endosphere of Oryza sativa spp. indica cv IR29. In general, there was a significant decrease in the M. oryzae CBMB20 population from the first day of inoculation to the 28th day. The rhizosphere region had the highest population, with 5.50 log CFU g−1 soil. On the 28th day post-inoculation, the population dropped to 4.64 log CFU g−1 FW. The populations of M. oryzae CBMB20 after one day of inoculation in the root and shoot endosphere were 4.58 and 4.13 log CFU g−1 FW, respectively. Although the colony counts of M. oryzae CBMB20 significantly dropped at the 28th day post-inoculation in the root and shoot endosphere, the population was still relatively high at 3.24 and 3.32 log CFU g−1 FW (Table 2).

3.3. Effects of M. oryzae CBMB20 Inoculation on the Root Endophytic Community of Rice

T-RFLP profiles of the roots of inoculated and uninoculated IR29 from day 1 to day 14 post-inoculation showed no significant change in terms of bacterial T-RFs present (Figure 2, Figures S2 and S3). There were some general trends when looking into the details of the bacterial community and diversity in the DdeI, HaeIII, and HhaI TRFLP profiles. Aside from the similar bacterial T-RFs present between inoculated and uninoculated IR29, the dominant bacteria in the uninoculated roots were also the dominant bacterial genera in the inoculated plants, there was a general increase in richness in the bacterial community and a decrease in evenness, and diversity indices showed a general increase in diversity or no significant difference between inoculated and uninoculated roots (Table 3). In general, the identity of bacterial endophytes found in the roots was very similar to the seed endophytic community when we compared the community profile in this study to the previous study of the endophytic community in seeds of IR29 [4].
The dominant bacterial T-RFs, as seen in the DdeI community profile, belong to Microbacterium, Delftia, Pseudomonas, Xanthomonas, and Stenotrophomonas. The HaeIII community profile shows Stenotrophomonas, Xanthomonas, Herbaspirillum, and Enterobacter to be the dominant groups, while the HhaI profile shows Sphingomonas and Pseudomonas to be the dominant bacterial groups in the root endosphere of IR29 (Figure 2, Figures S2 and S3). There seems to be a possible negative interaction between T-RF 240 (DdeI—Flavobacterium, Pantoea) and M. oryzae CBMB20, as this T-RF decreased in relative abundance when M. oryzae CBMB20 colonized the root endosphere. T-RF 105 (HaeIII) and T-RF 188, 304 (HhaI) seem to also have negative interactions with M. oryzae CBMB20, as populations decrease when the relative abundance of M. oryzae CBMB20 increases and vice versa. The relative abundance of M. oryzae CBMB20 in the roots is below 5% of the total relative abundance and constitutes a more minor bacterial community in the roots of inoculated IR29. The relative abundance of M. oryzae CMB20 was maintained from day 1 to day 14 post-inoculation, as seen in all restriction enzyme profiles.

3.4. Effects of M. oryzae CBMB20 Inoculation on the Shoot Endophytic Community of Rice

The endophytic bacterial community of the shoot showed reduced richness and evenness of bacterial communities dominated by very few groups of endophytes (Figure 3; Table S1). Bacterial community richness was 4, 3–5, and 5–7 in the DdeI, HaeIII, and HhaI profiles, respectively. Evenness also decreased when compared to the root community. Evenness between inoculated and uninoculated plants was only significant 14 days post inoculation. Shannon and Simpson’s diversity indices between the inoculated and uninoculated IR29 shoots showed no statistically significant differences from day 1 to day 14 post inoculation.
The dominant bacterial groups in the shoots were represented by a few endophytic groups and other unidentified groups (Figure 3). Curtobacterium was the dominant group in the DdeI profile, T-RF 143, Enterobacter, Stenotrophomonas, and Xanthomonas were the dominant groups in the HaeIII profile, and Enterobacter, T-RF 175, T-RF 353, and T-RF 189 were the dominant bacterial groups in the HhaI profile. M. oryzae CBMB20 was undetected in the DdeI community profile, one of the relatively abundant bacterial groups detected in HaeIII, and a less dominant group in HhaI.

4. Discussion

This study attempted to understand the effects of inoculating a plant growth promoting rice endophyte, Methylobacterium oryzae CBMB20, on its interaction with the indigenous seed-borne endophytes in the roots and shoots of the rice host. The root and the shoot rice endosphere is inhabited by considerably abundant populations of bacterial endophytes, which M. oryzae CBMB20 was able to incorporate and persist into. The indigenous bacterial endophytes of the roots and shoots of IR29 are mainly seed-borne. T-RFLP analysis showed that M. oryzae CBMB20 was able to integrate with the indigenous bacterial endophytic communities of the roots and shoots of IR29 with potential fluctuations in the richness, evenness, and diversity indices during colonization and persistence in the root and shoot endosphere.

4.1. Interaction of Methylobacterium oryzae CBMB20 with the Host Plant

The colonization of putative endophytes in plants primarily depends on the host plant and its associated microbial communities [10]. Methylobacterium oryzae CBMB20, as a putative endophyte of Oryza sativa spp. indica cv IR29, has to be competent in order to survive and colonize the rhizosphere and endosphere of its potential host [6]. We observed that M. oryzae CBMB20 persists in the rhizosphere, root and shoot endosphere, even after 28 days post inoculation with culturable populations of 4.64, 3.24 and 3.32 log CFU g−1 fresh weight. This indicates its survival and competence, and is probably attributed to its high association with rice, since M. oryzae CBMB20 was originally isolated as a stem endophyte in Oryza sativa L. subsp. japonica cv. Nam-Pyeong [27]. Studies have also proven that M. oryzae CBMB20 is a PGPB that could endophytically colonize [16,28,29] in other plant species, benefitting their host plant in the process. M. oryzae CBMB20 also has consistently enhanced plant growth when used as a bioinoculant under normal and stress conditions in multiple host plants, including rice japonica cultivars [30,31,32] and rice indica cultivars, IR29 and FL478 [33].
One of the major challenges in bioinoculant application is its effectiveness when especially applied in field conditions. Once introduced, in the soil for instance, bioinoculants face competition and usually challenging conditions resulting in potential elimination or decreased population, thus reducing its beneficial effects [34]. This study showed that M. oryzae CBMB20 was able to persist in the rhizosphere, root endosphere, and shoot endosphere, although there was a progressive decline in its population. Since M. oryzae CBMB20 is a rice-associated microbial community, it is possible that the M. oryzae CBMB20 population could attain optimal densities depending on plant tissue [35], or progressively decline in population over a longer period of time. Both of these scenarios are related to recent observations on the applications of Methylobacterium strains under field conditions on different rice landraces [36]. Methylobacterium strains reinoculated into their host during the seedling stage were able to be detected even after the seed harvesting stage, indicating the persistence of the closely associated Methylobacterium during the entirety of its host life cycle. However, the study also showed that Methylobacterium strains inoculated in non-original host plants were not detected, indicating natural selection as well as other factors involved in plant-microbe interactions.

4.2. The Seed-Borne Indigenous Community of the Root and Shoot of Oryza sativa spp. Indica cv IR29

In a previous study investigating seed endophytic communities of different genotypes of Oryza sativa spp. indica [4], the identified members of the bacterial communities of IR29 seeds seem to be the same endophytic bacterial community of the roots. This indicates that most of the bacterial endophytes in the roots of the uninoculated IR29 detected in the present study are seed-borne. Some of the identified bacterial endophytes, which are potentially seed-borne and found in the roots, belong to members of Herbaspirillum, Xanthomonas, Stenotrophomonas (HaeIII), Delftia, Microbacterium, Pseudomonas, Xanthomonas, Stenotrophomonas and Curtobacterium (DdeI), and Enterobacter, Sphingomonas, and Pseudomonas (HhaI), along with other seed-borne T-RFs that were unidentified (Figure S3). The shoots of IR29, though with reduced richness, also seem to be colonized by seed-borne endophytes belonging to Curtobacterium, Enterobacter, Stenotrophomonas, and Xanthomonas, along with other unidentified T-RFs previously observed in IR29 seeds. Seed-borne endophytes seem to be the majority of indigenous bacterial endophytes of the root and shoot endosphere, with which any new colonizers need to interact. It has already been found that rice seeds are major sources of bacterial endophytes in the roots and shoots of rice seedlings [2]. It was also observed that rice plants grown in the field had similar communities of bacterial endophytes in the leaves and seeds [37]. Haroim et al. [2] also found that diversity in the roots is higher compared to the shoots when seedlings were grown in natural soil, and the opposite was observed when seedlings were gnotobiotically grown. This was also observed in this study with nursery soil and gnotobiotically grown seedlings. Ferrando et al. [38] also observed that the leaves of different rice cultivars contained similar yet reduced groups of associated bacteria. It is highly possible that rice seedlings prefer seed-borne endophytes over soil-borne putative endophytes, since many of these seed-borne endophytes are plant growth promoters that enhance the germination and seedling growth of rice [20]. Many of these seed-borne endophytes are also vertically transmitted and are conserved in succeeding generations of their rice hosts [5].

4.3. Interaction of Methylobacterium oryzae CBMB20 with the Indigenous Bacterial Communities of the Root and Shoot Endosphere

Putative endophytes successfully colonizing the internal structures of plants have to establish and maintain association with native bacterial endophytes and their host plant [39]. There have been a few studies on the effect of PGPB inoculation on endophytic communities. Conn and Franco [8] showed that some microbes in a mixed microbial inoculant competed with indigenous actinobacterial endophytes. The PGP Pseudomonas putida P9 also competed with bacterial groups, including Pseudomonas azotoformans, Pseudomonas veronii, and Pseudomonas syringae [9]. Another study by Andreote et al. [10] observed that plants inoculated with Paenibacillus sp. Strain E119 and Methylobacterium mesophilicum strain SR1.6/6 resulted in alterations in communities, especially Alphaproteobacteria and Paenibacillus species, leading to shifts in the metabolism of the plant.
The present study showed that M. oryzae CBMB20, a stem endophyte isolated in rice, was able to integrate into the endophytic community of the roots and shoots of IR29 without causing dramatic changes in the diversity and community structure of the native bacterial endophytes. In summary, the bacterial endophytic community profile of inoculated and non-inoculated IR29 plants mainly contain the same bacterial communities, the dominant groups in non-inoculated plants are also the dominant groups in the inoculated plants, and the richness of the endophytic microbial community increased due to the inclusion of M. oryzae CBMB20; however, the fluctuations in diversity indices were not significant between inoculated and non-inoculated IR29 plants. M. oryzae CBMB20 was also able to persist and be potentially maintained in the host endosphere for a longer period of time. These observations were similarly seen in the phyllosphere microbiome of rice landraces inoculated with Methylobacterium strains where the application of closely associated Methylobacterium did not alter the phyllosphere microbiome [36]. In a similar study [2], reinoculation of an ‘artificial’ community that resembled microbial communities in the endosphere of rice roots did not cause shifts in the community composition of indigenous root and shoot endophytes of the studied rice host. This is potentially the case for microbial communities that are highly adapted or associated with the host plant. It is possible that M. oryzae CBMB20 can coexist well with both the host plant and its native endophytes, since it is a PGP, rice-associated bacterium [27,40].
The dominant endophytic bacterial communities in the uninoculated and inoculated roots of IR29 include Microbacterium, Delftia, Pseudomonas, Xanthomonas, Stenotrophomonas, Herbaspirillum, and Sphingomonas. Those in the shoot endosphere include Curtobacterium, Enterobacter, Stenotrophomonas, and Xanthomonas. Pseudomonas strains from IR29 and related rice genotypes are known for high siderophore production and phosphate solubilization potential. Xanthomonas and Microbacterium strains have high pectinase and cellulase activities with lower production of IAA and a lower phosphate solubilization index with potential biocontrol activities. Herbaspirillum is a well-known nitrogen-fixing rice-associated bacterial group [20]. Stenotrophomonas and Sphingomonas are potential plant growth promoters in other rice genotypes [41,42]. Since the endophytic microbial communities of inoculated IR29 plants were not drastically altered, we could extend that the inclusion of M. oryzae CBMB20 into the rice microbiome, although not becoming a dominant member in terms of relative abundance, was the main factor responsible for the physical and physiological changes in the host plants leading to plant growth promotion. A recent study by Roy Choudhury et al. [32] showed that M. oryzae CBMB20 increased photosynthetic pigments and altered proteome composition related to metabolic pathways, riboflavin metabolism, protein synthesis, and chaperonin-related proteins under normal conditions, and altered carbohydrate metabolic process and response to stimulus under salt stress conditions. Bioinoculation with M. oryzae CBMB20 potentially enriched the gene ontology terms of inoculated host plants, leading to observed plant growth promotion and stress alleviation in previous studies with M. oryzae CBMB20.

4.4. Methylobacterium oryzae CBMB20 as a PGP Bioinoculant

The present study supports the high association of Methylobacterium oryzae CBMB20 with the rice host, such as Oryza sativa spp. indica cv. IR29, since it was able to persist in the rhizosphere and endosphere, and the presence of M. oryzae CBMB20 did not cause dramatic changes in the community profiles of the pre-existing indigenous bacterial endophytes of the rice host. We could also extrapolate that the observations seen in this study could be some of the reasons why M. oryzae CBMB20 is a proven plant growth promoting bacteria capable of showing beneficial interaction with multiple plant hosts. Its viability and maintenance in the rhizosphere and endosphere show that it is a competent bioinoculum, and competence is a major factor required for exhibiting beneficial effects [6,7]. M. oryzae CBMB20 inoculation in tomatoes showed that it was able to persist in the rhizosphere and exhibited plant growth [43]. M. oryzae CBMB20 also improved defense responses when tomato plants were later challenged with plant pathogens [16,29], and this was mainly attributed to its ACC deaminase activity as well as the potential involvement of priming. This is the first study showing the integration and persistence of M. oryzae CBMB20 with the native endophytic bacterial community of rice without apparent shifts and changes in the overall diversity and community composition of bacterial endophytes in the plant host. All of these could contribute to the plant growth promotion and stress alleviation of M. oryzae CBMB20 observed in different studies [28,43,44,45,46].
The increasing use of bioinoculants as enhancers or biocontrol agents has provided avenues for improved sustainable agricultural practices. However, we are still compounded by the complexities of plant-microbe interactions and the mechanisms governing these interactions. High throughput sequencing involving omics studies has given possibilities to investigate these mechanisms and could help us design more effective and reliable applications of bioinoculants. This study showed that M. oryzae CBMB20 was able to integrate into the endophytic bacterial community of rice; however, its interaction in other plant systems may show different trends, and mechanisms of interactions have not been fully elucidated. Aside from these, other concerns such as biosafety issues [47] and shelf life remain vital aspects of bioinoculant technologies.

5. Conclusions

The persistence and integration of native bacterial communities support the competence, endophytic nature, and high association of Methylobacterium oryzae CBMB20 with the rice plant. These result in intimate plant-microbe interactions, leading to the observed plant growth promoting effects and stress alleviation conferred by Methylobacterium oryzae CBMB20 across multiple plant species previously demonstrated in past studies.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture13020355/s1, Figure S1: BOX PCR of seed borne endophytes in root and shoot of rice identified using BOX PCR profiles of a previous study [20]; Figure S2: TRFLP profiles of root endophytes in HaeIII; Figure S3: TRFLP profiles of root endophytes in HhAI; Table S1. Diversity indices of bacterial endophytes inhabiting the shoots in 1-, 7- and 14-days post-inoculation of M. oryzae CBMB20 inoculated IR29 and uninoculated control.

Author Contributions

Conceptualization, D.I.W. and T.S.; methodology, D.I.W.; validation, D.I.W. and A.R.C.; formal analysis, D.I.W.; investigation, D.I.W.; resources, T.S. and Y.L.; data curation, D.I.W., G.C., B.J. and A.R.J.; writing—original draft preparation, D.I.W.; writing—review and editing, D.I.W. and A.R.C.; visualization, D.I.W.; supervision, T.S. and Y.L.; project administration, T.S.; project support, G.C., B.J. and A.R.J.; funding acquisition, T.S. and A.R.C. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Basic Science Research Program, National Research Foundation of Korea (NRF), Ministry of Education, Science and Technology [2021R1A2C1006608], Republic of Korea.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

D.I.W. would like to thank Meriam P. Catajay-Mani, the Romblon State University President, for facilitating his invited Post-Doctoral Fellowship.

Conflicts of Interest

The authors declare no conflict of interest.

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Agriculture 13 00355 g001 550
Figure 1. Bacterial genera and their relative proportions found in the different microniches of 7-day rice seedlings gnotobiotically grown. Data from the seeds were from Walitang et al. [20].
Figure 1. Bacterial genera and their relative proportions found in the different microniches of 7-day rice seedlings gnotobiotically grown. Data from the seeds were from Walitang et al. [20].
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Figure 2. T-RFLP profiles of root endophytic community in uninoculated (blue) and M. oryzae CBMB20-inoculated (red) rice cultivar after digestion with DdeI restriction enzyme seen in 1-, 7-, and 14-days post inoculation (DPI); T-RF identity: ①—Pseudomonas, Xanthomonas, Stenotrophomonas; 119—M. oryzae CBMB20; DPI—days post inoculation.
Figure 2. T-RFLP profiles of root endophytic community in uninoculated (blue) and M. oryzae CBMB20-inoculated (red) rice cultivar after digestion with DdeI restriction enzyme seen in 1-, 7-, and 14-days post inoculation (DPI); T-RF identity: ①—Pseudomonas, Xanthomonas, Stenotrophomonas; 119—M. oryzae CBMB20; DPI—days post inoculation.
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Figure 3. T-RFLP profiles of shoot endophytic community in uninoculated (blue) and M. oryzae CBMB20-inoculated (red) rice cultivar after digestion with DdeI restriction enzyme seen in 1-, 7-, and 14-days post inoculation (DPI); T-RF identity: ①—Stenotrophomonas, Xanthomonas.
Figure 3. T-RFLP profiles of shoot endophytic community in uninoculated (blue) and M. oryzae CBMB20-inoculated (red) rice cultivar after digestion with DdeI restriction enzyme seen in 1-, 7-, and 14-days post inoculation (DPI); T-RF identity: ①—Stenotrophomonas, Xanthomonas.
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Table
Table 1. Population of culturable bacterial endophytes (log CFU g−1 FW) in the seed and then in the root and shoot of IR29 after 7 days of germination grown under gnotobiotic conditions.
Table 1. Population of culturable bacterial endophytes (log CFU g−1 FW) in the seed and then in the root and shoot of IR29 after 7 days of germination grown under gnotobiotic conditions.
CultivarSeedRootShoot
IR295.46 ± 0.09 C6.89 ± 0.006 B8.16 ± 0.02 A
Values given are the means of three replicates ± standard error. Values of the same letter are not statistically significant at p < 0.05 (Tukey’s test, SAS Version 9.4).
Table
Table 2. Persistence of Methylobacterium oryzae CBMB20 in the rhizosphere soil, root and shoot of IR29 days after inoculation.
Table 2. Persistence of Methylobacterium oryzae CBMB20 in the rhizosphere soil, root and shoot of IR29 days after inoculation.
NicheDays Post-Inoculation (DPI)
17142128
Log CFU g−1
Rhizosphere5.50 ± 0.025 A5.33 ± 0.015 B4.86 ± 0.007 C4.78 ± 0.02 D4.64 ± 0.0 E
Root4.58 ± 0.05 A4.65 ± 0.06 A4.39 ± 0.03 A3.79 ± 0.11 B3.24 ± 0.22 C
Shoot4.13 ± 0.01 A3.44 ± 0.03 B3.37 ± 0.03 BC3.35 ± 0.01 BC3.32 ± 0.03 C
Values given are the means of three replicates ± standard error. Values of the same letter are not statistically significant at p < 0.05 (Tukey’s test, SAS Version 9.4).
Table
Table 3. Diversity parameters of root bacterial endophytes in 1-, 7-, and 14-days post inoculation of M. oryzae CBMB20-inoculated IR29 and uninoculated control.
Table 3. Diversity parameters of root bacterial endophytes in 1-, 7-, and 14-days post inoculation of M. oryzae CBMB20-inoculated IR29 and uninoculated control.
Diversity
Parameter		Rice TreatmentT-RFLP Profile
DdeIHaeIIIHhaI
RichnessUninoculated (1 DPI)8.33 ± 1.20 C10.33 ± 0.33 B7.33 ± 0.33 BC
Inoculated (1 DPI)11.33 ± 0.67 AB13.33 ± 0.33 A8.00 ± 0.58 B
Uninoculated (7 DPI)10.67 ± 0.33 ABC13.00 ± 0.00 A4.67 ± 0.33 D
Inoculated (7 DPI)10.67 ± 0.33 ABC13.00 ± 0.00 A8.00 ± 0.00 B
Uninoculated (14 DPI)8.67 ± 0.33 BC8.67 ± 0.33 B6.00 ± 0.00 CD
Inoculated (14 DPI)11.67 ± 0.33 A14.33 ± 0.67 A11.00 ± 0.00 A
EvennessUninoculated (1 DPI)0.71 ± 0.03 BC0.86 ± 0.01 AB0.90 ± 0.02 A
Inoculated (1 DPI)0.64 ± 0.01 C0.83 ± 0.01 AB0.77 ± 0.02 B
Uninoculated (7 DPI)0.80 ± 0.01 AB0.76 ± 0.01 DC0.64 ± 0.02 C
Inoculated (7 DPI)0.73 ± 0.01 BC0.81 ± 0.00 BC0.73 ± 0.00 B
Uninoculated (14 DPI)0.72 ± 0.03 BC0.89 ± 0.01 A0.87 ± 0.02 A
Inoculated (14 DPI)0.84 ± 0.01 A0.75 ± 0.02 D0.77 ± 0.01 B
Shannon
Index		Uninoculated (1 DPI)1.49 ± 0.15 C2.01 ± 0.04 ABC1.79 ± 0.07 AB
Inoculated (1 DPI)1.56 ± 0.03 BC2.16 ± 0.02 A1.60 ± 0.05 BC
Uninoculated (7 DPI)1.88 ± 0.05 AB1.96 ± 0.02 BC0.97 ± 0.03 D
Inoculated (7 DPI)1.73 ± 0.02 AB2.08 ± 0.01 AB1.52 ± 0.01 C
Uninoculated (14 DPI)1.56 ± 0.03 BC1.91 ± 0.07 C1.55 ± 0.03 C
Inoculated (14 DPI)2.06 ± 0.03 A1.99 ± 0.01 BC1.84 ± 0.03 A
Simpson’s
Index		Uninoculated (1 DPI)0.66 ± 0.05 C0.84 ± 0.01 ABC0.80 ± 0.02 A
Inoculated (1 DPI)0.68 ± 0.01 C0.86 ± 0.00 A0.76 ± 0.01 AB
Uninoculated (7 DPI)0.81 ± 0.01 AB0.81 ± 0.00 BC0.50 ± 0.01 C
Inoculated (7 DPI)0.76 ± 0.01 AB0.84 ± 0.00 AB0.72 ± 0.00 B
Uninoculated (14 DPI)0.71 ± 0.01 BC0.84 ± 0.01 ABC0.75 ± 0.01 AB
Inoculated (14 DPI)0.84 ± 0.01 A0.81 ± 0.01 C0.77 ± 0.01 AB
Values given are the means of three replicates ± standard error. Values of the same letter are not statistically significant at p < 0.05 (Tukey’s test, SAS Version 9.4). DPI—days post inoculation.
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Walitang, D.I.; Roy Choudhury, A.; Lee, Y.; Choi, G.; Jeong, B.; Jamal, A.R.; Sa, T. The Endophytic Plant Growth Promoting Methylobacterium oryzae CBMB20 Integrates and Persists into the Seed-Borne Endophytic Bacterial Community of Rice. Agriculture 2023, 13, 355. https://doi.org/10.3390/agriculture13020355

AMA Style

Walitang DI, Roy Choudhury A, Lee Y, Choi G, Jeong B, Jamal AR, Sa T. The Endophytic Plant Growth Promoting Methylobacterium oryzae CBMB20 Integrates and Persists into the Seed-Borne Endophytic Bacterial Community of Rice. Agriculture. 2023; 13(2):355. https://doi.org/10.3390/agriculture13020355

Chicago/Turabian Style

Walitang, Denver I., Aritra Roy Choudhury, Yi Lee, Geon Choi, Bowon Jeong, Aysha Rizwana Jamal, and Tongmin Sa. 2023. "The Endophytic Plant Growth Promoting Methylobacterium oryzae CBMB20 Integrates and Persists into the Seed-Borne Endophytic Bacterial Community of Rice" Agriculture 13, no. 2: 355. https://doi.org/10.3390/agriculture13020355

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