Regular Paper
Genetic Diversity of Endospore-forming Nitrogen-fixing Bacteria and Their Future Application as Biofertilizers in the Central Dry Zone of Myanmar
2025 Volume 40 Issue 4 Article ID: ME25033
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2025 Volume 40 Issue 4 Article ID: ME25033
In Myanmar, the application of both nitrogen-based chemical fertilizers and biofertilizers is limited and this low input has caused poor agricultural yields. The present study aimed to isolate indigenous endospore-forming nitrogen-fixing bacteria (EFNFB) and examine their potential for co-inoculation with agricultural waste. A total of 387 isolates were obtained from 42 different soil samples in the central dry zone of Myanmar using nitrogen-free Rennie medium. Nitrogen-fixing activity (NFA) assessed with the acetylene reduction assay was positive in 102 isolates. A phylogenetic analysis based on 16S rRNA sequences identified 25 different species, including the genera Paenibacillus, Priestia, Bacillus, Brevibacillus, Sporolactobacillus, Niallia, and Neobacillus. Among these genera, Paenibacillus spp. was the predominant genus, comprising 51 isolates (64%) across 16 different species (64%) that were prevalent in soils rotated with rice and pulses. Paenibacillus spp. showed different NFA levels in Rennie medium. Eleven species belonging to different genera had not been previously documented as nitrogen-fixing bacteria. NFA levels were evaluated in soil inoculated with EFNFB and rice straw or mung bean residue. The results obtained demonstrated that NFA levels were dependent on isolates and the type of agricultural waste. NFA in soil was significantly increased by inoculations with some isolates, suggesting their potential as biofertilizers. The inoculation of Priestia aryabhattai S10 with rice straw or mung bean resulted in significantly higher NFA levels in soil. These results indicate the potential of EFNFB as biofertilizer inoculants in Myanmar.
Nitrogen (N) is a major essential nutrient in crop production and is important for maintaining and improving crop growth and yield (Orchardson, 2020). Although the application of chemical N fertilizers enhances crop growth and yield, high rates may diminish the quality of agricultural products (Liu et al., 2019). In addition, the long-term excessive use of chemical fertilizers has resulted in serious negative impacts, such as soil hardening, acidification, and environmental pollution (Jiao et al., 2018). Therefore, a decrease in reliance on chemical fertilizers is expected for sustainable management and a reduction in their environmental impact (Alves et al., 2004). Biological nitrogen fixation (BNF) is attracting increasing attention as such an approach (Choudhury and Kennedy, 2004) and the use of efficient inoculants is regarded as an important strategy.
In Myanmar, legumes are important cash crops and approximately 70% of all legume crops are grown on 4.2–4.3 million ha during the winter season (October to January), with yields ranging between 1.0–1.3 t ha–1 (GAIN, 2020), which is markedly lower than those in Vietnam (14.3 t ha–1) and Thailand (12.2 t ha–1). Lower yields in Myanmar are attributed to inappropriate crop management practices and insufficient technology (Aye et al., 2013). In Myanmar, most farmers do not use chemical fertilizers due to their high price. According to the FAO (2021), Myanmar’s fertilizer consumption was only 38.7 kg ha–1, compared to 139 kg ha–1 in Thailand and 193 kg ha–1 in India. The average N fertilizer application rate in Myanmar for pulses is estimated to be only 10 kg of N ha–1, which is markedly lower than 90–170 kg ha–1 for Thailand and Vietnam (MAPSA, 2023). Therefore, N input other than chemical fertilizers may be needed to increase crop yields in Myanmar.
Crop residue return may increase yields and quality by improving soil organic matter, soil physical properties, water use efficiency, and soil structural stability and reducing soil bulk density (Soon and Lupwayi, 2012; Li et al., 2019). However, previous studies reported a negative impact on both the environment and crop yields (Lu, 2020). The decomposition of crop residues causes N starvation, which is not conducive to crop growth and yields (Wang et al., 2018). We interviewed farmers in Myanmar and found that many returned crop residues, such as rice straw and pulse straw, directly or after burning. Burning is more practical due to its low cost and feasibility, and enables fields to be cleared quickly for the next planting and also avoids N starvation. Some farmers consider burning to control pests and enhance soil fertility; however these benefits are limited and short-lived. While a small number of farmers are aware of the advantages of crop residue return, this practice alone is not sufficient to obtain better crop yields. Therefore, a technology that stimulates the soil N status is expected, and biofertilizers are attracting increasing attention (Zhao et al., 2024).
One type of biofertilizer is a substance containing living microorganisms, such as Rhizobium and Trichoderma, which are applied to seeds, plant surfaces, or soil (Vessey, 2003). The use of biofertilizers has been shown to improve soil fertility by fixing atmospheric N, solubilizing insoluble phosphates, and/or producing plant growth-promoting substances in the soil (Mazid and Khan, 2015). In Myanmar, the use of biofertilizers is not widely practiced. Although some local products are available, e.g., Trichoderma and Rhizobium, the amount produced does not cover the whole agricultural area (Herridge et al., 2008). Although some commercial products, including Bacillus sp., are imported from Thailand and China, indicating prospects for yield increases by inoculation technology, most farmers cannot afford them due to their high costs. To address this issue, it is necessary to isolate indigenous microorganisms as an alternative biofertilizer for Myanmar’s agriculture.
Endospore-forming bacteria, such as Bacillus spp., Paenibacillus spp., and Brevibacillus spp., are major soil bacteria (Alexander, 1977). Several strains of Bacillus spp. exhibit antibacterial and antifungal activities against phytopathogens and, thus, may function as biocontrol agents (Fira et al., 2018). In addition, some endospore-forming bacteria have the ability to fix atmospheric N. For example, more than 30 members of Paenibacillus spp. have been isolated as N fixers from soils and rhizospheres and include Paenibacillus polymyxa, Paenibacillus macerans, Paenibacillus peoriae, Paenibacillus azotofixans, and Paenibacillus stellifer (Wang et al., 2013; Li et al., 2022). Among Bacillus spp., approximately 10 have the ability to fix atmospheric N, and Bacillus cereus, Bacillus pumilus, Bacillus circulans, Bacillus licheniformis, and Bacillus subtilis have been isolated from soils and rhizospheres (Ding et al., 2005). The genus Priestia used to belong to the genus Bacillus and some species fix N, such as Priestia endophytica, Priestia megaterium, and Priestia aryabhattai (Sharma et al., 2022; Liu et al., 2023; Pal et al., 2025). In the genus Niallia, Niallia circulans fixes N (Hashem et al., 2019). In the genus Brevibacillus, Brevibacillus agri fixes N (Suriani et al., 2022) and further studies are needed to assess this capability in the other species of this genus.
Bacteria survive for a long time after they form an endosphere due to resistance to extreme environmental conditions, drying, pH, and salinity in soil (Logan and De Vos, 2009). They then actively colonize the rhizosphere where they establish beneficial interactions with plants, indicating their potential contribution to BNF (Xie et al., 2016). Therefore, the present study focused on N-fixing bacteria that form endospores because they may enhance the soil N status and be readily applied to fields as an inoculum for biofertilizers.
In Myanmar, the screening of indigenous endospore-forming N-fixing bacteria (EFNFB) and their application as biofertilizers have not yet been examined. The study of effective indigenous EFNFB and feasible application methods with waste products represents a sound approach for future sustainable agriculture. Therefore, the aims of the present study were to screen indigenous EFNFB in Myanmar soil, examine the effects of bacterial inoculations on N-fixing ability in the soil, and propose how waste products may be managed by inoculations with effective indigenous EFNFB as biofertilizers.
Soil samples were collected from different cultivated fields (sesame, groundnut, green gram, cowpea, pigeon pea, chickpea, black gram, cranberry bean, cluster bean, rice bean, lima bean, hyacinth bean, winged bean, and yard long bean) in different regions (Magway, Mandalay, and Nay Pyi Taw), Myanmar (Fig. 1). Soil was sampled from five different locations in each field and a composite soil sample was made. Soil samples were sieved through a 5-mm mesh sieve and mixed well. Composite soil samples (100 g) were air dried and kept at room temperature (27°C).
The central dry zone (Magway, Mandalay, and Naypyitaw) of Myanmar (A) and soil collection sites in each region (B)
Ten grams of each soil sample was added to a 200-mL Erlenmeyer flask containing 90 mL of sterile tap water to make a 10–1 dilution. It was then heated in a water bath for 10 min at 80°C to obtain endospore-forming bacteria (Bendt, 1985). The flask was immediately transferred into a water bath. One-hundred microliters each of serially diluted suspensions (10–1, 10–2, 10–3, and 10–4) was inoculated into a vial containing semi-solid (0.3% agar) N-free Rennie medium (Elbeltagy et al., 2001).
A turbid medium a few days to one week after the inoculation indicated the presence of N-fixing bacteria. The turbid part was streaked onto several plates including N-free Rennie medium with 1.5% agar. Bacterial colonies of different shapes and colors on each plate were considered to be different strains and were separately purified on fresh solid N-free Rennie medium.
16S rRNA genes were amplified using extracted DNA as a template with the universal primers 27f (5′-AGAGTTTGATCMTGGCTCA-3′) (Frank et al., 2008) and 1378r (5′-CGGTGTGTACAAGGCCCGGGACG-3′) (Heuer et al., 1999). DNA extraction was conducted using the method reported by Miyashita (1992) and PCR amplification was performed using an Ex premier DNA polymerase (Takara Bio) with the conventional method (Jamily et al., 2018). PCR products were purified with a FavorPrepTM GEL/PCR Purification Mini Kit (Chiyoda Science) and were sent for sequencing at FASMAC. Eighty isolates were sequenced for ca. 800 bp using the 1378r primer. Regarding 8 isolates (F1, F2, F4, F12, F14, S3, S10, and S12) showing low similarity values to the closest species, an additional ca. 800 bp were sequenced using the primer 27f and the resulting sequences were aligned and combined using ClustalW v2.1. The resulting 16S rRNA sequences were analyzed using BLAST against the DDBJ/ENA/GenBank database (https://blast.ncbi.nlm.nih.gov/Blast.cgi), applying a 98.7% sequence similarity threshold as the species-level cut-off (Kim et al., 2014). Sequences were deposited in the DDBJ/ENA/GenBank database under accession numbers LC872246 to LC872325. A phylogenetic tree was constructed in MEGA v12 (Kumar et al., 2024) using the maximum likelihood (ML) method based on ca. 800 bp of 16S rRNA gene sequences aligned with ClustalW, applying the general time reversible (GTR) nucleotide substitution model and 1,000 bootstrap replicates.
Confirmation of nitrogen-fixing activity (NFA) with the acetylene reduction assay (ARA)To confirm the NFA of bacterial isolates, ARA was conducted (Knowles and Barraquio, 1994). Three milliliters of semi-solid N-free Rennie medium was added to a 13-mL vial. One loopful of a bacterial isolate suspended in sterilized saline water (0.85% NaCl) was inoculated into the vial. The vial was closed with a butyl rubber stopper and incubated at 30°C for 24 h. Before the incubation, 1 mL of the headspace air was removed and replaced with an equal volume of pure acetylene gas to create a 10% acetylene atmosphere. The ethylene concentration was measured after 1 and 7 days with a Shimadzu GC-14B gas chromatograph (Shimadzu) equipped with a hydrogen flame ionization detector (FID). The amount of ethylene in the headspace was quantified and activity was expressed as 3 different NFA levels: low (0–0.99 nmol C2H4 culture–1 h–1), middle (1–9.99 nmol C2H4 culture–1 h–1), and high (>10 nmol C2H4 culture–1 h–1).
Evaluation of NFA in soilMung bean residue and rice straw, which are typical rotation crops in the central dry zone in Myanmar, were used to examine the effects of the crop residue on N-fixing ability. Triplicate 30-mL vials were prepared, each containing 5 g of air-dried soil (1.4 g N kg–1, C/N ratio 9.5). Then, 15 mg of each crop residue powder (rice straw: 7.7 g N kg–1, 44.2 C/N ratio; mung bean: 12 g N kg–1, 34.7 C/N ratio) was placed on the soil surface. Tap water and the bacterial suspension were then added to the soil surface in order to adjust its moisture content to 60% of the maximum water holding capacity (MWHC) and its bacterial density to 1×107 CFU (g soil)–1. Bacterial suspensions were prepared by an overnight culture in 10–1 strength of DifcoTM nutrient broth. Vials were covered with aluminum foil and incubated at 30°C. After 6 days, vials were sealed with a rubber septum and 10% of the headspace was then replaced with pure acetylene gas. The vials were incubated again at 30°C for 24 h and ethylene was measured as described above. After the ethylene measurement, the rubber septum was replaced with aluminum foil and the incubation was continued at 30°C. Measurements were conducted periodically at 7-day intervals four times and the moisture content was adjusted to 60% MWHC every week. Rice straw was used in Exp. 1 and 2 and mung bean residue in Exp. 3 to examine the effects of different crop residues on the efficiency of EFNFB in the soil. Although different isolates were used in each experiment at different times, the inoculation method, incubation conditions, and ARA measurement procedures were consistent across all experiments. Results were expressed as the cumulative NFA for ethylene in 1 month.
Statistical analysisThe N-fixing ability of EFNFB in the soil with crop residue was compared to the control (-), and the significance of differences was analyzed using the Student’s t-test (IBM SPSS Statistics 20) and Microsoft Excel (ver. 2111).
A total of 387 isolates were isolated from 42 soil samples cultivated with different crops in different regions. Of these, 102 isolates exhibited NFA in ARA. Among the 102 NFA-positive isolates, 90 were identified at the genus level. These included 10 non-spore-forming bacteria, such as Azospirillum spp., Pandoraea spp., Burkholderia spp., and Microbacterium sp. The remaining 80 isolates were endospore-forming bacteria, i.e., the genera Paenibacillus (64%), Priestia (26%), Bacillus (3%), Sporolactobacillus (3%), Neobacillus (3%), Brevibacillus (1%), and Niallia (1%) (Table 1 and Fig. 2).
Nitrogen-fixing activities of 80 endospore-forming bacterial isolates from cultivated soils in Myanmar and their similarity percentages to closest species
| NFA levels | Isolates | Closest species | Similarity % | C2H4 culture–1 h–1 | NFA levels | Isolates | Closest species | Similarity % | C2H4 culture–1 h–1 |
|---|---|---|---|---|---|---|---|---|---|
| High | S59 | Paenibacillus kribbensis | 99.8 | 42.5 | Middle | F6 | Paenibacillus polymyxa | 99.8 | 1.1 |
| T73 | Paenibacillus polymyxa | 99.9 | 33.2 | F17 | Paenibacillus typhae | 99.0 | 1.0 | ||
| S11 | Paenibacillus jamilae | 99.6 | 31.6 | S25 | Priestia aryabhattai | 99.8 | 1.0 | ||
| F4 | Paenibacillus polymyxa | 99.8 | 28.4 | S34 | Priestia megaterium | 99.6 | 1.0 | ||
| F3 | Paenibacillus peoriae | 99.8 | 19.1 | F9 | Paenibacillus sonchi | 99.5 | 1.0 | ||
| S22 | Paenibacillus xylanilyticus | 99.9 | 19.0 | Low | S4 | Paenibacillus typhae | 99.6 | 0.8 | |
| S23 | Paenibacillus sp. | 81.2 | 17.6 | F10 | Paenibacillus sonchi | 99.9 | 0.7 | ||
| F14 | Paenibacillus peoriae | 99.9 | 17.3 | F11 | Bacillus safensis | 99.8 | 0.6 | ||
| S38 | Paenibacillus sp. | 85.9 | 17.1 | S56 | Paenibacillus jilunlii | 99.0 | 0.6 | ||
| S17 | Paenibacillus sp. | 97.8 | 16.9 | S27 | Paenibacillus sp. | 92.6 | 0.5 | ||
| Middle | S15 | Paenibacillus sinensis | 99.3 | 8.5 | T81 | Paenibacillus stellifer | 99.4 | 0.5 | |
| F5 | Paenibacillus polymyxa | 99.8 | 6.2 | S57 | Paenibacillus jilunlii | 99.3 | 0.5 | ||
| S66 | Priestia megaterium | 99.7 | 6.1 | S12 | Paenibacillus sp. | 97.1 | 0.5 | ||
| F16 | Paenibacillus sp. | 94.3 | 5.2 | S41 | Paenibacillus sp. | 98.6 | 0.4 | ||
| S9 | Paenibacillus stellifer | 99.6 | 5 | F18 | Paenibacillus triticicola | 99.6 | 0.4 | ||
| S28 | Paenibacillus stellifer | 99.9 | 4.7 | S55 | Paenibacillus polymyxa | 100.0 | 0.3 | ||
| T75 | Paenibacillus sinensis | 99.9 | 4.4 | S39 | Priestia aryabhattai | 100.0 | 0.3 | ||
| S44 | Paenibacillus sabinae | 99.4 | 4.4 | S33 | Priestia megaterium | 99.9 | 0.2 | ||
| F13 | Paenibacillus peoriae | 99.4 | 4.1 | F1 | Niallia sp. | 96.4 | 0.2 | ||
| F12 | Paenibacillus sp. | 96.5 | 3.6 | S72 | Paenibacillus polymyxa | 99.8 | 0.2 | ||
| S36 | Paenibacillus sophorae | 99.4 | 3.3 | S42 | Priestia sp. | 96.9 | 0.1 | ||
| S24 | Paenibacillus stellifer | 99.9 | 3 | S69 | Paenibacillus beijingensis | 99.4 | 0.1 | ||
| S10 | Priestia aryabhattai | 100 | 2.9 | S1 | Brevibacillus nitrificans | 99.8 | 0.1 | ||
| T78 | Priestia aryabhattai | 99.9 | 2.9 | S54 | Priestia megaterium | 99.1 | 0.1 | ||
| S21 | Priestia megaterium | 100 | 2.7 | S65 | Neobacillus cucumis | 99.5 | 0.1 | ||
| S5 | Paenibacillus sp. | 93.1 | 2.6 | S18 | Paenibacillus xylanilyticus | 99.3 | 0.1 | ||
| S20 | Priestia megaterium | 100 | 2.2 | S63 | Priestia megaterium | 99.1 | 0.1 | ||
| S13 | Paenibacillus pabuli | 99.8 | 2.1 | S30 | Paenibacillus sp. | 93.9 | 0.05 | ||
| T83 | Paenibacillus stellifer | 99 | 2.1 | S62 | Priestia aryabhattai | 99.6 | 0.05 | ||
| S53 | Priestia sp. | 81.2 | 1.9 | S29 | Sporolactobacillus laevolacticus | 99.6 | 0.05 | ||
| S19 | Paenibacillus stellifer | 99.8 | 1.8 | S64 | Paenibacillus stellifer | 99.6 | 0.04 | ||
| F8 | Paenibacillus polymyxa | 99.5 | 1.7 | S37 | Paenibacillus sp. | 97.9 | 0.04 | ||
| T84 | Paenibacillus stellifer | 99.5 | 1.5 | S47 | Priestia megaterium | 100.0 | 0.03 | ||
| F15 | Paenibacillus peoriae | 99.9 | 1.4 | S52 | Priestia megaterium | 99.9 | 0.02 | ||
| F7 | Paenibacillus polymyxa | 99.9 | 1.3 | S71 | Sporolactobacillus laevolacticus | 98.8 | 0.02 | ||
| S35 | Priestia megaterium | 99.6 | 1.3 | S48 | Priestia sp. | 82.0 | 0.02 | ||
| F2 | Priestia sp. | 98 | 1.3 | S49 | Paenibacillus mucilaginosus | 99.5 | 0.01 | ||
| S3 | Paenibacillus sp. | 96.4 | 1.2 | S32 | Priestia aryabhattai | 99.9 | 0.01 | ||
| S16 | Paenibacillus stellifer | 98.8 | 1.2 | S31 | Neobacillus drentensis | 99.9 | 0.01 | ||
| S26 | Priestia megaterium | 99.5 | 1.2 | S50 | Bacillus salipaludis | 99.9 | 0.01 |
Three different nitrogen-fixing activity levels: low (0–0.99 nmol C2H4 culture–1 h–1), middle (1–9.99 nmol C2H4 culture–1 h–1), and high (>10 nmol C2H4 culture–1 h–1).
Shaded letters indicate isolates belonging to Paenibacillus spp. Bold letters indicate isolates exhibiting nitrogen-fixing activity for the first time in this study.
Phylogenetic tree based on 16S rRNA gene sequences, drawn using 800 bp from the reverse primer. Stains shown in red were sequenced for 1,400 bp. Reanalyzed results with BLAST are shown in Table 1. The scale bar indicates 10% sequence divergence. Reference strains were obtained from the DDBJ database. Staphylococcus aureus was used as the outgroup. Bootstrap value ≥50%.
The genus Paenibacillus was prevalent across different NFA levels (Table 1). Specifically, the strain with the highest NFA level belonged to the genus Paenibacillus. In the middle range of NFA isolates, Paenibacillus accounted for 68%, followed by Priestia (32%). Among the low NFA strains, Paenibacillus was present at 49%, followed by other genera, such as Priestia (29%), Bacillus (6%), Sporolactobacillus (6%), Neobacillus (6%), Niallia (3%), and Brevibacillus (3%).
Fifty-one isolates were the closest to the genus Paenibacillus and were distributed across 16 distinct species. Pa. stellifer (9 isolates), Pa. polymyxa (8 isolates), and Pa. peoriae (4 isolates) were frequently found in soil samples and were prominent among high NFA isolates. The remaining 13 Paenibacillus species were less frequent across different NFA levels. Among them, Paenibacillus kribbensis exhibited the highest NFA level, followed by Pa. polymyxa. Only two isolates (3%) were the closest to Bacillus (Bacillus safensis and Bacillus salipaludis), both of which showed low NFA levels. Priestia was also frequently detected, with 21 isolates (26%) across Pr. megaterium (11 isolates) and Pr. aryabhattai (6 isolates), both of which exhibited middle and low NFA levels.
A single species was detected in the genera Brevibacillus (Brevibacillus nitrificans) and Niallia sp. Additionally, two isolates were the closest to the genera Neobacillus and Sporolactobacillus. These 4 genera showed low NFA levels.
The majority of isolates belonged to the genus Paenibacillus spp., and Pa. stellifer (9 strains), the dominant species in this genus, had a similarity range of 98.8 to 99.9% (Table 1). Isolates closest to Priestia spp. were related to Pr. aryabhattai (99.5–100%) and Pr. megaterium (99.1–100%). Bacillus spp. isolates showed the highest similarity to Ba. safensis (99.8%) and Bacillus salipaludis (99.9%). Neobacillus spp. isolates were very similar to Neobacillus drentensis (99.9%) and Neobacillus cucumis (99.5%). The Sporolactobacillus spp. isolate was the closest to Sporolactobacillus laevolacticus (>98.8%) and that of Brevibacillus sp. was the closest to Br. nitrificans (99.8%).
Effects of cropping patterns on the diversity of endospore-forming nitrogen-fixing bacteriaAmong the 80 isolates, 36 were found in soils under rice-pulses rotations and 18 were predominantly detected in a pulses-sesame rotations (Table 2). Additionally, 11 isolates were identified in pulses-chili rotations, 9 in pulses-onion rotations, 5 in rice-sesame rotations, and 1 in pulses-only soil. Within the rice-pulses rotation, Paenibacillus (53%) and Priestia (39%) were the most frequently isolated genera, followed by Neobacillus (6%) and Sporolactobacillus (3%). In the pulses-sesame crop rotation, Paenibacillus was the dominant genus (83%), while Bacillus, Priestia, and Niallia were each detected at 6%. Additionally, Paenibacillus spp. occupied 89% of isolates from pulses-onion rotations, 64% from pulses-chili rotations, and 40% from rice-sesame rotations.
Sources of 80 bacterial isolates from cultivated soils under different crop rotations in this study
| Closest species | No. of isolates | Rice-pulses | Rice-sesame | Pulses-sesame | Pulses-onion | Pulses-chili | Pulses |
|---|---|---|---|---|---|---|---|
| Paenibacillus | |||||||
| Pa. stellifer | 9 | 3 | 1 | 3 | 2 | ||
| Pa. polymyxa | 8 | 2 | 1 | 5 | |||
| Pa. peoriae | 4 | 4 | |||||
| Pa. sinensis | 2 | 2 | |||||
| Pa. xylanilyticus | 2 | 2 | |||||
| Pa. typhae | 2 | 1 | 1 | ||||
| Pa. sonchi | 2 | 2 | |||||
| Pa. sabinae | 1 | 1 | |||||
| Pa. jilunlii | 2 | 2 | |||||
| Pa. beijingensis | 1 | 1 | |||||
| Pa. triticicola | 1 | 1 | |||||
| Pa. sophorae | 1 | 1 | |||||
| Pa. pabuli | 1 | 1 | |||||
| Pa. mucilaginosus | 1 | 1 | |||||
| Pa. jamilae | 1 | 1 | |||||
| Pa. kribbensis | 1 | 1 | |||||
| Paenibacillus sp. | 15 | 5 | 2 | 3 | 2 | ||
| Priestia | |||||||
| Pr. megaterium | 11 | 9 | 1 | 1 | |||
| Pr. aryabhattai | 6 | 4 | 1 | 1 | |||
| Priestia sp. | 4 | 1 | 1 | 1 | 1 | ||
| Bacillus | |||||||
| Ba. safensis | 1 | 1 | |||||
| Ba. salipaludis | 1 | 1 | |||||
| Neobacillus | |||||||
| N. cucumis | 1 | 1 | |||||
| N. drentensis | 1 | 1 | |||||
| Sporolactobacillus | |||||||
| S. laevolacticus | 2 | 1 | 1 | ||||
| Brevibacillus | |||||||
| Br. nitrificans | 1 | 1 | |||||
| Niallia | |||||||
| Niallia sp. | 1 | 1 | |||||
| Total | 80 | 36 | 5 | 18 | 9 | 11 | 1 |
Furthermore, most isolates from groundnut and black gram cultivated soils were Paenibacillus spp. Priestia spp. were mainly isolated from groundnut fields and less frequently from sesame, black gram, pea, and cluster bean soils. Ba. safensis and Ba. salipaludis were isolated from sesame and pea cultivated fields, respectively. Neobacillus spp. were isolated from groundnut soils under the rice-pulses rotation in the Naypyitaw region. Two Sporolactobacillus laevolacticus strains were isolated from groundnut and chickpea cultivated soils. Niallia sp. was isolated from sesame soil and Br. nitrificans from pigeon pea cultivated soil. N-fixing isolates from groundnut and sesame soils exhibited the highest NFA levels across various genera.
Evaluation of nitrogen-fixing ability in the soilN-fixing ability was higher in soil inoculated with any bacterial isolate than in the control without an inoculation (Table 3). Additionally, the rice straw amendment increased NFA in some isolates. In Exp. 1, Pa. peoriae F13 exhibited a significant (P<0.05) increase in NFA upon the addition of rice straw. Conversely, Pa. polymyxa F4 and Paenibacillus sp. F16 did not exhibit marked changes in NFA, regardless of the presence or absence of the residue. In Exp. 3, Paenibacillus typhae S4 and Pr. aryabhattai S10 showed a significant (P<0.05 and P<0.01) increase in NFA upon the addition of rice straw. In the absence of crop residues, Pr. megaterium F2, Paenibacillus sonchi F10, Paenibacillus sp. S27, and Pa. typhae S4 all exhibited significantly higher NFA levels (P<0.01 and P<0.05). When combined with mung bean residue, Pa. polymyxa F6 and Pr. aryabhattai S10 exhibited significant (P<0.05 and P<0.01) increases in NFA. Pr. aryabhattai S10 also showed a significantly (P<0.01) higher NFA level in the soil upon the addition of rice straw or mung bean residues than that with organic amendment. There was no correlation between the NFA of isolates in N-free media in vials (Table 1) and in soil (Table 3).
One-month cumulative nitrogen-fixing activities of endospore-forming bacteria inoculated to Tatkon soil in Myanmar with rice straw or mung bean crop residue (0.3%)
| C2H4 produced (n mol–1 day–1 [g soil]–1) | |||||
|---|---|---|---|---|---|
| Isolates | NFA levels in N-free media |
No Residue | +Rice straw | +Mung bean | |
| Exp. 1 | No inoculation | 0 | 0.29±0.45 | NT | |
| F4 (Paenibacillus polymyxa) | high | 0.71±0.61 | 0.71±0.58 | NT | |
| F13 (Paenibacillus peoriae) | middle | 1.22±1.08 | 1.75±0.58* | NT | |
| F14 (Paenibacillus peoriae) | high | 0.2±0.25 | 0.53±0.19 | NT | |
| F16 (Paenibacillus sp.) | middle | 0.58±0.41 | 0.53±0.38 | NT | |
| Exp. 2 | No inoculation | 0 | 0.38±0.66 | NT | |
| S3 (Paenibacillus sp.) | middle | 3.89±0.92 | 2.69±0.06 | NT | |
| S9 (Paenibacillus stellifer) | middle | 1.66±1.27 | 0.56±0.96 | NT | |
| S11 (Paenibacillus jamilae) | high | 2.44±1.08 | 0 | NT | |
| S59 (Paenibacillus kribbensis) | high | 1.50±1.41 | 2.92±3.49 | NT | |
| S64 (Paenibacillus stellifer) | low | 2.09±1.06 | 1.69±0.42 | NT | |
| S66 (Priestia megaterium) | middle | 0.29±0.51 | 1.22±1.59 | NT | |
| S69 (Paenibacillus beijingensis) | low | 1.13±1.30 | 1.84±1.39 | NT | |
| S72 (Paenibacillus polymyxa) | low | 0.92±1.60 | 0.46±0.80 | NT | |
| T78 (Priestia aryabhattai) | middle | 1.92±1.69 | 2.93±1.68 | NT | |
| Exp. 3 | No inoculation | 0 | 0.10±0.18 | 0.19±0.33 | |
| F1 (Niallia circulans) | low | 1.31±0.60 | 1.20±0.63 | 0.27±0.23 | |
| F2 (Priestia megaterium) | middle | 0.76±0.29* | 1.24±0.86 | 0.41±0.36 | |
| F6 (Paenibacillus polymyxa) | middle | 0.43±0.75 | 0.20±0.35 | 0.55±0.21* | |
| F10 (Paenibacillus sonchi) | low | 2.16±0.19** | 1.38±1.28 | 0.44±0.39 | |
| S4 (Paenibacillus typhae) | low | 1.08±0.06** | 1.53±0.80* | 1.28±1.16 | |
| S10 (Priestia aryabhattai) | middle | 0.26±0.44 | 1.86±0.15** | 1.44±0.23** | |
| S12 (Paenibacillus sp.) | low | 1.19±1.11 | 0.26±0.44 | 0.29±0.26 | |
| S27 (Paenibacillus sp.) | low | 1.64±0.19** | 0.62±1.07 | 0.23±0.21 | |
This experiment was repeated three times using different isolates (Exp. 1, 2, and 3) and soil samples were incubated at 60% of the maximum water holding capacity.
Nitrogen-fixing activity in soil represents mean values and the standard deviation of triplicate measurements.
Significant differences between the inoculated treatments (bacteria, residues, and bacteria + residues) and control (no bacteria or residues) were examined with a given comparison (independent samples t-test: * P<0.05, ** significant at P<0.01).
NT: Not tested.
This is the first study to isolate EFNFB from soils cultivated with different crops in Myanmar and examine their application as biofertilizers. To isolate EFNFB, soil samples were heat-treated at 80°C for 10 min (Bendt, 1985). Molecular identification showed that 89% of the isolates were endospore-forming bacteria (Fig. 2), while 11% did not form spores. These results suggest that the heat treatment method was not perfect, but effectively selected endospore-forming bacteria, similar to previous studies. Paenibacillus spp. were the predominant genus (Table 1), which is consistent with previous findings showing that Paenibacillus spp. are ubiquitous in nature and capable of forming resistant endospores (Bloemberg and Lugtenberg, 2001). Many studies have already reported that some endospore-forming bacteria, e.g., Bacillus spp. and Paenibacillus spp., exhibit NFA (Ding et al., 2005; Li et al., 2022). The present results confirmed the role of endospore-forming bacteria in N fixation and their potential as biofertilizers.
The high abundance of EFNFB in the rice-pulses rotation and pulses-sesame rotation may be due to the favorable conditions created by legume cultivation. Legumes fix atmospheric N through symbiosis and enhance soil microbial diversity and activity and the establishment of free-living N-fixing bacteria (Qiao et al., 2024). In addition, rice cultivation often involves temporary flooding, which creates an anaerobic environment in the soil (Wang et al., 2021), whereas pulses restore aerobic conditions during their growth cycle. This rotation creates alternating soil redox conditions and may promote the survival of facultative anaerobes, such as Paenibacillus (Kiran et al., 2017) and Neobacillus (Patel and Gupta, 2020; Schober et al., 2025). In the present study, Paenibacillus was the predominant genus and was highly prevalent in soils associated with rice and pulses-based crop rotations (Table 2). In addition, two strains of Neobacillus, N. cucumis and N. drentensis, were isolated from soils under the rice-pulses crop rotation (Table 2). Strains belonging to these two facultative anaerobic genera may survive well under anaerobic conditions.
In the present study, most of the EFNFB identified have already been reported as N-fixing bacteria (Wang et al., 2013; Li et al., 2022; Liu et al., 2023; Pal et al., 2025). To the best of our knowledge, the NFA of 11 isolates, i.e., Pa. kribbensis, Pa. typhae, Paenibacillus triticicola, Paenibacillus xylanilyticus, Paenibacillus jamilae, Ba. salipaludis, Ba. safensis, Br. nitrificans, N. cucumis, N. drentensis, and S. laevolacticus, have not been reported, although previous studies documented their beneficial activities for plants, such as enhancing nutrient uptake, providing biocontrol against pathogens, and promoting plant growth. Among them, Pa. kribbensis, with the highest NFA, has been associated with enhanced plant disease resistance against Rhizoctonia solani and tobacco mosaic virus (Canwei et al., 2020) and phosphate-solubilizing ability (Marra et al., 2012). Pa. xylanilyticus with a high NFA level was proposed as a chitin-degrading bacterium by Liao et al. (2019), which showed its potential in biotechnology, pharmaceuticals and phosphate solubilization (Pandya et al., 2015). Pa. jamilae is known for its highly antagonistic activity against soil-borne pathogens and for its ability to increase beneficial bacteria in the wheat rhizosphere (Wang et al., 2019). The results obtained herein newly demonstrated its high NFA. This study also identified a strain belonging to Br. nitrificans, which was previously proposed as a novel nitrifying bacterium species by Takebe et al. (2012), as a N fixer.
In the evaluation of NFA using soil, some strains exhibited increased NFA upon the addition of rice straw or mung bean (Table 3). Among them, Pr. aryabhattai S10 exhibited enhanced NFA following the addition of both rice straw and mung bean residue. This result indicates that Pr. aryabhattai S10 possessed the ability to effectively utilize different types of plant materials, which resulted in enhanced NFA. Pr. aryabhattai BPR-9 has been shown to produce extracellular enzymes, such as cellulases, amylases, pectinases, proteases, and lipases, facilitating the breakdown of complex polysaccharides in plant residues (Shahid et al., 2022). Additionally, the whole-genome sequencing of Pr. aryabhattai strain S2 revealed the presence of genes involved in plant growth promotion, such as indole-3-acetic acid synthesis, and in salinity stress resistance, further supporting its adaptability in various environmental conditions (Sliti et al., 2023). These findings indicate the potential of Pr. aryabhattai S10 as a biofertilizer for enhancing nitrogen input in rice-pulses crop rotation systems, particularly under residue management practices.
The addition of crop residues has been shown to enhance soil fertility and crop growth and change soil microbial diversity (He et al., 2024). Therefore, inoculating straw with efficient microorganisms may enhance N fixation more effectively than an inoculation only (Roper and Ladha, 1995). The NFA of Pr. aryabhattai S10 and Pa. peoriae F13 increased following the addition of rice straw or mung bean residue. This result indicates that the isolates competed with indigenous microbes and utilized the crop residues, resulting in increased NFA. NFA increased over the incubation period, possibly because the residues gradually decomposed, providing more nutrients for bacteria. In contrast, the results of other isolates did not change. Some strains, such as Pa. sonchi F10 and Paenibacillus sp. S27, had higher NFA in the bacterial inoculation without the crop residue. In soil, inoculants must compete with the native population and the addition of a crop residue may increase competition. Isolates that showed lower NFA in the presence of the crop residue were considered to be less competitive with the crop residue. These results support previous findings showing that straw-associated N fixation was affected by many environmental and management factors (Roper and Ladha, 1995). It currently remains unclear whether the response of NFA to the addition of rice straw or mung bean differed depending on strains. Therefore, further research is needed to evaluate NFA by EFNFB in different soil types, various organic residues, and inoculation strategies in order to obtain a more detailed understanding of their effects on N fixation in the soil. We plan to use these EFNFB in rice-legume rotation fields to improve crop yields in Myanmar.
Phoo, Y. M., Toyota, K., and Min, Y. Y. (2025) Genetic Diversity of Endospore-forming Nitrogen-fixing Bacteria and Their Future Application as Biofertilizers in the Central Dry Zone of Myanmar. Microbes Environ 40: ME25033.
https://doi.org/10.1264/jsme2.ME25033
The first author would like to express her gratitude to the Ministry of Education, Science, Sports and Culture of Japan for their financial support and the opportunity to study in Tokyo University of Agriculture and Technology, Japan. We also thank the staff members of the Department of Agricultural Microbiology, Yezin Agricultural University, Myanmar, for their valuable assistance with soil collection. The first author gratefully acknowledges the Ministry of Agriculture, Livestock and Irrigation of Myanmar for their support in providing official documents for the international transport of soil samples.
