Soybean (Glycine max L.) plays an essential role in human nutrition as a protein source, and in plant nutrition as a N source. The rate of N fixation varies depending on the cultivars and compatibility between the inoculated Rhizobium strain and the host cultivar. Characterizing the nodulation regulatory (Rj) genes is necessary to determine the compatibility of cultivars and Rhizobium strains. Rj genes were previously identified based on inoculation tests and PCR analyses. The six cultivars Yezin-3, Yezin-7, Yezin-11, Shan Seine (Local), Madaya (Local), and Hinthada (Local) were identified as harboring the Rj4 gene. Two cultivars, Yezin-6 and Yezin-8, were classified as non-Rj-gene harboring. Two other cultivars, Yezin-9 and Yezin-10, were identified as Rj3- and Rj2Rj3-gene harboring, respectively. Ours is the first report on Rj3- and Rj2Rj3-gene harboring cultivars in Myanmar. We evaluated Myanmar soybean cultivars for symbiotic effectiveness, relying on the standard strain Bradyrhizobium japonicum USDA110. In our first experiment, the soybean cultivar Yezin-11 (Rj4) showed the highest N fixing potential. Based on their potential for fixing N and nodulation, the top six soybean cultivars were Yezin-11 (Rj4), Yezin-9 (Rj3), Yezin-6 (non-Rj), Yezin-8 (non-Rj), Yezin-3 (Rj4) and Yezin-10 (Rj2Rj3). These cultivars were selected for a second experiment, which revealed that the N fixation, nodulation, and plant growth of Yezin-11 (Rj4) *Corresponding author. A. Z. Htwe et al. 2800 were superior to the other cultivars. We conclude that Yezin-11 (Rj4) is the most efficient cultivar for nodulation and N fixation when inoculated with B. japonicum USDA110.
Myanmar is an agricultural country, as agriculture is the backbone of its economy. Legumes are the second largest crops in Myanmar, following rice (Oryza sativa L.), in terms of cultivated hectares. Due to the relatively low cost of cultivation and increasing demand for domestic consumption and export, the total cultivated area of pulses has increased from 0.73 million hectares in 1988-89 to 4.4 million hectares in 2011-12 [
Nodule formation by a cultivar is often dependent on a specific Rhizobium strain [
Ishizuka et al. [
The Rj genes are identified by an inoculation test, which uses strains that restrict nodulation on specific Rj genotype soybean cultivars. As an accelerated method for molecularly identifying Rj genes, Yang et al. [
In Myanmar, many researchers have been focusing on selecting strains to increase soybean N fixation. Recently, the Department of Agricultural Research (DAR) has developed improved soybean varieties, such as Yezin-9, Yezin-10, and Yezin-11. However, Rj genes have not been identified in some of the released cultivars. To recommend the most efficient N-fixing cultivars, it is necessary to evaluate symbiotic effectiveness with inoculated strains and identify the nodulation Rj genes. In the past, Rj genes were identified based on inoculation tests. Therefore, our goal in this study was to identify Rj genes of Myanmar soybean cultivars based on inoculation tests and multiplex PCR analysis and to screen the cultivars for N-fixing efficiency by using the standard strain B. japonicum USDA 110.
Ten soybean varieties (Shan Seine [local], Hinthada [local], Madaya [local], Yezin-3, Yezin-6, Yezin-7, Yezin-8, Yezin-9, Yezin-10, Yezin-11) were collected from the Food Legume Section, Department of Agricultural Research, Yezin, Myanmar. These varieties were grown in the glasshouse of the Plant Nutrition Laboratory, Kyushu University, Japan from July to November 2013 to obtain genetically pure and viable seeds. The focus was to study the ability of cultivars to adapt to weather in Japan. Shan Seine [local], Hinthada [local], Madaya [local] were widely grown in Shan State, Ayeyawaddy Region, Mandalay and Sagaing Regions, respectively. Yezin cultivars used in this experiment were mainly grown in Yezin, Mandalya Region and Shan State, and recommended for farmers to improve soybean production. Flower color, days to maturity and origin of these varieties are shown in
The Rj genotypes of 10 soybean cultivars, including three reference cultivars D51 (Rj3), CNS (Rj2Rj3) and Hill (Rj4), were investigated to estimate their compatibility with native bradyrhizobia. These varieties were inoculated with the three bradyrhizobial strains B. japonicum Is-1, B. elkanii USDA33 and B. japonicum Is-34 [
The seeds were sterilized by soaking them in 2.5% sodium hypochlorite solution for 5 min, rinsing five times with 10 mL of 99.5% ethanol, and washing five times with sterilized half-strength modified Hoagland Nutrient (MHN) solution [
Variety | Flower color | Days to maturity | Origin |
---|---|---|---|
Yezin-3 | Violet | 115 | DAR, Yezin, MR |
Yezin-6 | Violet | 115 | DAR, Yezin, MR |
Yezin-7 | Violet | 100 | DAR, Yezin, MR |
Yezin-8 | Violet | 115 | DAR, Yezin, MR |
Yezin-9 | White | 120 | DAR, Yezin, MR |
Yezin-10 | White | 120 | DAR, Yezin, MR |
Yezin-11 | Violet | 110 | DAR, Yezin, MR |
Shan Seine | Violet | 120 | Shan State |
Hinthada | Violet | 120 | Hinthada, AR |
Madaya | Violet | 130 | Madaya, MR |
DAR: Department of Agricultural Research; AR: Ayeyawaddy Region; MR: Mandalay Region.
The plants were cultivated in an environmentally-controlled room (25˚C and 75% Relative Humidity) under natural light for 4 weeks. Control pots were used to check for contamination by non-relevant strains and inoculated strains used in this experiment. Watering was done weekly with autoclaved deionized water. After 1 month, the formation of effective nodules was checked to identify nodulation types of all isolates being tested. This experiment was conducted three times, from January to June 2015.
Multiplex PCR analysis was used to identify Rj genes and confirm the Rj2 and Rj4 alleles, but it could not detect the Rj3 allele. For DNA extraction, the plants were cultivated in a growth chamber (28˚C for 16 hours for the light condition and 23˚C for 8 hours for the dark condition). Genomic DNA for PCR templates was extracted from the leaves of seedlings using Takara Bio, following the manufacturer’s instructions. Primers were designated from sequence information in reports identifying the Rj2 and Rj4 genes [
The seeds were surface-sterilized as described above. Six surface-sterilized seeds were sown in pots filled with 1 L of vermiculite and 0.6 L of N-free MHN solution. Bradyrhizobium japonicum USDA110 was cultured in A1E liquid media and incubated on a rotary shaker at 30˚C for 7 days. Inoculant was prepared as described above. Seeds were inoculated with the bacterial suspension at 5 mL per seed. The cultivation conditions were the same as described above. Three plants were chosen from each pot for data collection in the first experiment. Six plants were taken from three different pots for the second experiment.
For the acetylene reduction assay (ARA), the soybean plants with intact nodules were placed in 100-mL conical flasks, sealed with a serum stopper and injected with 12 mL of acetylene (C2H2) gas to replace air with acetylene.
Primer sets | Primer sequences | Annealing temperature (˚C) | Amplification site (bp) |
---|---|---|---|
Rj2 specific primers | |||
K452E-F | (5'-GCTTCAATAGATATGACTTGACAG-3') | 58.5 | 161 |
R490I-R | (5'-AATCAAGTCATGCATTGTAACTA-3') | 57.8 | |
rj2 specific primers | |||
K452-F | (5'-GCTTCAATAGATATGACTTGACAA-3') | 58.9 | 161 |
R490-R | (5'-ATCAAGTCATGCATTGTAACTC-3') | 58.2 | |
Rj4 specific primers | |||
T107A-F | (5'-TTGGAGGAAACGCCG-3') | 62.2 | 324 |
202-203AY-R | (5'-AATCATGAGAAGAACAAGTATAAGC-3') | 58.5 | |
rj4 specific primers | |||
T107-F | (5'-TTGGAGGAAACGCCA-3') | 59.9 | 318 |
202-203del-R | (5'-AATCATGAGAAGAACAAGTATGGA-3') | 60.4 |
The nitrogenase activity, in terms of ethylene (C2H4) concentration of the plants, was measured using a flame ionization gas chromatograph (GC-14A, Shimadzu, Kyoto, Japan) at 5 and 65 min after injecting with C2H2 gas as described by Soe et al. [
Identifying the Rj genes of soybean varieties is important to determine their host specificity and compatibility with specific bradyrhizobia. We evaluated the Rj genotypes of soybean cultivars from Myanmar to identify their nodulation Rj genes and estimate their compatibility with strains to be inoculated. Among the tested cultivars, six (Shan Seine [local], Hinthada [local], Madaya [local], Yezin-3, Yezin-7 and Yezin-11) were identified as harboring the Rj4-gene. Only two cultivars, Yezin-6, Yezin-8, were classified as non-Rj-gene harboring cultivars. Yezin-9 and Yezin-10 were identified as Rj3- and Rj2Rj3-gene harboring cultivars, respectively. The results of inoculation testing are shown in
Among the tested cultivars, six (Shan Seine [local], Hinthada [local], Madaya [local], Yezin-3, Yezin-7 and Yezin-11) harbored Rj4 gene alleles. Yezin-6, Yezin-8 and Yezin-9 did not harbor Rj4 or Rj2 genes, although Yezin-6 and Yezin-8 harbored the recessive alleles rj4 and rj2, and Yezin-9 and Yezion-10 harbored the recessive allele rj4. We found the Rj2 gene in Yezin-10. The results from the inoculation test and PCR analysis are shown in
The number of nodules produced was significantly different when inoculated with B. japonicum USDA 110 (
Cultivar | Nodule No. plant−1 on inoculated strains | Rj gene | ||
---|---|---|---|---|
USDA 33 | Is-1 | Is-34 | ||
Shan Seine | Medium | Medium | None | Rj4 |
Madaya | Medium | Medium | Low | Rj4 |
Hinthada | Medium | High | None | Rj4 |
Yezin-3 | Medium | High | Low | Rj4 |
Yezin-6 | Medium | Medium | Medium | non-Rj |
Yezin-7 | Medium | High | None | Rj4 |
Yezin-8 | Medium | Medium | Medium | non-Rj |
Yezin-9 | Low | Medium | Medium | Rj3 |
Yezin-10 | None | None | High | Rj2Rj3 |
Yezin-11 | Medium | Medium | None | Rj4 |
High = 10 - 15 nodules plant−1; Medium = 4 - 9 nodules plant−1; Low = 1 - 3 nodules plant−1; None = No nodulation. This division was based on Htwe et al. [
Cultivars | NN (No. plant−1) | NDW (mg∙plant−1) | SDW (g∙plant−1) | RDW (g∙plant−1) | ARA (μmol C2H4 h−1 plant−1) |
---|---|---|---|---|---|
Yezin-3 | 10.67 ab | 15.70 ab | 0.22 ab | 0.09 cde | 0.49 ab |
Yezin-6 | 9.67 ab | 15.40 ab | 0.23 ab | 0.13 ab | 0.52 ab |
Yezin-7 | 10.67 ab | 12.60 ab | 0.16 bc | 0.08 de | 0.35 ab |
Yezin-8 | 9.00 ab | 19.20 a | 0.23 ab | 0.11 bcd | 0.51 ab |
Yezin-9 | 5.33 ab | 11.40 ab | 0.21 ab | 0.12 abc | 0.58 ab |
Yezin-10 | 13.00 a | 10.00 ab | 0.20 ab | 0.11 bcd | 0.43 ab |
Yezin-11 | 12.00 ab | 23.80 a | 0.29 a | 0.15 a | 0.65 a |
Shane Seine | 8.00 ab | 11.80 ab | 0.14 bc | 0.08 de | 0.11 ab |
Hinthada | 10.67 ab | 16.30 ab | 0.27 a | 0.12 abc | 0.41 ab |
Madaya | 5.00 b | 2.80 b | 0.09 c | 0.05 e | 0.08 b |
Mean values in each column followed by the same letters are not significantly different at P > 0.05 (Tukey’s test). NN: nodule number; NDW: nodule dry weight; SDW: shoot dry weight; RDW: root dry weight; ARA: acetylene reduction activity. Yezin-6 was used as control. Nodule number, nodule dry weight and ARA value for control were zero. Shoot and root dry weight of control was 0.20 and 0.12 g∙plant−1, respectively.
produced the fewest nodules. Nodule dry weights also differed significantly among the cultivars. The nodule dry weights of Yezin-11 (Rj4) and Yezin-8 (non-Rj) were greater than weights of the other cultivars but, with the exception of Madaya Local (Rj4), these differences were not statistically significant.
Shoot dry weights were significantly different between some of the cultivars. The highest shoot biomass (0.29 g∙plant−1) was obtained from Yezin-11 (Rj4), but this did not differ significantly from that of Yezin-3, Yezin-6, Yezin-8, Yezin-9, Yezin-10, or Hinthada local cultivars. Significantly greater root dry weight was recorded for Yezin-11 (Rj4), but it was not significantly different from Yezin-6, Yezin-9, or Hinthada local cultivars. The nitrogenase activities varied significant among soybean cultivars when inoculated with B. japonicum USDA110 (
The number of nodules, ranging from 10 to 17.67 per plant, differed significantly among cultivars when inoculated with B. japonicum USDA 110 (
Symbiotic N fixation of soybean could provide 65 to more than 160 kg fixed N∙ha−1 [
Compatibility between Rj soybean genotypes and soybean-nodulating bradyrhizobia must be considered in selecting the best varieties and strains for soybean cultivation. Therefore, nodulation Rj genes collected from soybean cultivars in Myanmar were evaluated to determine compatibility and preference between strains and cultivars. The Rj genes could be identified by inoculating with specific strains of Bradyrhizobium, such as B. japonicum Is-1, B. elkanii USDA33 and B. japonicum Is-34 [
In this study, we performed PCR analysis to confirm the Rj genotypes. Although we could not identify Rj3, the analysis was very useful in identifying the Rj4 and Rj2 alleles. The cultivars Shan Seine (local), Hinthada (local), Madaya (local), Yezin-3, Yezin-7 and Yezin-11 harbored Rj4 alleles. Yezin-6, Yezin-8, and Yezin-9 cultivars did not harbor other dominant Rj alleles, although they harbored the recessive alleles rj2 and rj4. Only Yezin-9 was identified as an Rj2-gene harboring cultivar. Based on PCR analysis, we confirmed that Yezin-3 and Madaya (local) were Rj4-genotype soybean cultivars, although they could also form a few nodules with their nodulation restricting strain Is-34. Contrary to Soe et al. [
According to the inoculation test and PCR results, the cultivars Shan Seine (local), Hinthada (local), Madaya (local), Yezin-3, Yezin-7 and Yezin-11 were identified as Rj4-genotype cultivars. Yezin-6 and Yezin-8 were identified as non-Rj genotype cultivars. Yezin-9 and Yezin-10 were identified as harboring the Rj3 and Rj2Rj3 genes, respectively. In Myanmar, the Rj4 genotype cultivars were the most widely grown cultivars, accounting for 60% of the tested cultivars. Devine and Kuykendall [
In Myanmar, DAR has developed improved soybean cultivars to replace local cultivars. However, some Myanmar farmers have continued to grow local soybean cultivars, such as Shan Seine (local), Madaya (local) and Hinthada (local). Proper matching of soybean cultivars and Rhizobia strains optimizes performance through enhanced N fixation. In this study, we screened improved and local cultivars using the strain B. japonicum USDA110, as several studies have reported significant increases in growth, yield, nodulation, and N fixation of Myanmar soybean cultivars due to inoculation with this strain [
We found that local cultivars such as Madaya (local) (Rj4), Shan Seine (local) (Rj4) and Yezin-7 (Rj4) showed lower nitrogenase activity, nodulation and plant growth. Hinthada (local) produced an increased number of nodules and nodule dry weight, but its N fixation was lower than other improved cultivars such as Yezin-11 and Yezin-3, despite having the Rj4 genes in common. Soe et al. [
In results from the second screening experiment, Yezin-11 (Rj4) was the most efficient cultivar, with the most nodules, and highest nodule, shoot and root dry weights, and the greatest nitrogenase activity. When we compared the N fixing rates, in terms of ARA per plant inoculated with B. japonicum USDA 110, Yezin-9 (Rj3) and Yezin-10 (Rj2Rj3) were most efficient in N fixation, though they did not differ significantly from the N fixation of Yezin-6 (non-Rj), Yezin-8 (non-Rj) and Yezin-3 (Rj4). This might be related to the Rj genes, which can affect both preference and compatibility for nodulation between the host cultivar and soybean Rhizobia [
Most Myanmar soybean cultivars were identified as harboring the Rj4 gene. A few cultivars were classified as non-Rj, Rj2Rj3 and Rj3 gene harboring. This was the first report of Rj2Rj3 and Rj3 genotype soybean cultivars in Myanmar. We evaluated the N fixation and nodulation of Myanmar soybean cultivars by using the standard strain B. japonicum USDA 110. In both experiments, Yezin-11 (Rj4) was the most efficient for N fixation and nodulation. It appeared that Yezin-11 (Rj4) was more compatible with B. japonicum USDA110 based on the results from both experiments. Yezin-11 had about two to three times higher symbiotic N fixation capacity than the other soybean cultivars. Our study provides useful information for breeders seeking to produce cultivars efficiently at N fixation. Further study is needed on the effectiveness of different Rj genotypes with indigenous bradyrhizobia to increase soybean productivity via symbiotic N fixation.
This work was supported by Ministry of Education, Culture, Sports, Sciences and Technology of Japan. We are grateful to Dr. Htun Shwe (Researcher, Food Legume Section, Department of Agricultural Research) for providing soybean seeds.
Sadiq, R., Maqbool, N. and Haseeb, M. (2017) Ameliorative Effect of Chelating Agents on Photosynthetic Attributes of Cd Stressed Sunflower. Agricultural Sciences, 8, 149-160. https://doi.org/10.4236/as.2017.82010