Plant and Soil

, Volume 417, Issue 1–2, pp 349–362 | Cite as

Genetic diversity of indigenous soybean-nodulating Bradyrhizobium elkanii from southern Japan and Nueva Ecija, Philippines

  • Maria Luisa Tabing Mason
  • Shota Matsuura
  • Apolinario Laxamana Domingo
  • Akihiro Yamamoto
  • Sokichi Shiro
  • Reiko Sameshima-Saito
  • Yuichi SaekiEmail author
Regular Article



Understanding the factors that influence the diversity of soybean-nodulating rhizobia is important before doing inoculation. Since studies about this topic in tropical regions are limited, this could lay the groundwork for related research particularly on Bradyrhizobium elkanii.


To determine the genetic diversity of B. elkanii in different regions, we conducted Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and sequence analysis of 16S rRNA gene, internal transcribed spacer (ITS) region and rpoB gene. Also, sequence analysis of symbiotic nifD and nodD1 genes was conducted.


Analysis of the rpoB gene revealed a higher genetic diversity than the ITS region, and possible endemic B. elkanii strains were observed. Meanwhile, no variation was detected among the strains in both nifD and nodD1 phylogenies. Through rpoB gene analysis, variations in the ITS-rpoB type of B. elkanii strains were distinguished and differentiated with that of the closest reference strains. We identified potential soybean inoculants which possess symbiotic efficiency regardless of the Rj genotypes used, suggesting broad host-range of the strains.


We show how the genetic diversity of soybean-nodulating B. elkanii strains in subtropical and tropical regions might be influenced by temperature and soil pH and, provided some insights between the symbiotic genes and Rj genotypes.


Bradyrhizobium elkanii rpoB gene ITS region Philippines Symbiotic genes 



This study was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (B) no. 26310313) and the Japanese Government (MEXT) Scholarship program.

Supplementary material

11104_2017_3263_MOESM1_ESM.pdf (73 kb)
Table S1 List of primers used in this study for the PCR amplification and sequence analysis of 16S rRNA, ITS, rpoB, nifD and nodD1. (PDF 73 kb)
11104_2017_3263_MOESM2_ESM.pdf (89 kb)
Table S2 Shoot and nodule parameters of 19 representative B. elkanii isolates employing three Rj genotypes. Mean comparison was conducted in triplicates only between isolates within the same Rj genotype. (PDF 89 kb)
11104_2017_3263_MOESM3_ESM.pdf (80 kb)
Table S3 List of accession numbers for selected Bradyrhizobium USDA reference strains and isolates from the sequence analysis of 16S rRNA gene, 16S–23S rRNA gene ITS region, rpoB housekeeping gene and symbiotic genes nifD and nodD1. (PDF 79 kb)
11104_2017_3263_MOESM4_ESM.pdf (58 kb)
Figure S1 Indication of the usefulness of rpoB-RFLP analysis as shown by the similarity in the phylogeny and RFLP band patterns. (PDF 58 kb)
11104_2017_3263_MOESM5_ESM.pdf (139 kb)
Figure S2 Phylogenetic tree based on sequence analysis of 16S rRNA gene. The tree was constructed using the Neighbor- Joining method with the Kimura 2-parameter (K2P) distance correlation model and 1000 bootstrap replications in MEGA v.7 software. (PDF 138 kb)
11104_2017_3263_MOESM6_ESM.pdf (235 kb)
Figure S3 Phylogenetic tree based on sequence analysis of (A) nifD gene and (B) nodD1 gene. The tree was constructed using the Neighbor-Joining method with the Kimura 2-parameter (K2P) distance correlation model and 1000 bootstrap replications in MEGA v.7 software. The first letter of isolates’ name indicates the location as follows: H - Kumamoto; O - Okinawa; P - C. Luzon, Philippines. (PDF 235 kb)


  1. Adhikari D, Kaneto M, Itoh K, Suyama K, Pokharel BB, Gaihre YK (2012) Genetic diversity of soybean-nodulating rhizobia in Nepal in relation to climate and soil properties. Plant Soil 357:131–145CrossRefGoogle Scholar
  2. Alam F, Bhuiyan MA, Alam SS, Waghmode TR, Kim PJ, Lee YB (2015) Effect of Rhizobium sp. BARIRGm901 inoculation on nodulation, nitrogen fixation and yield of soybean (Glycine max) genotypes in gray terrace soil. Biosci Biotechnol Biochem 79:1660–1668CrossRefPubMedGoogle Scholar
  3. Alves BJR, Boddey RM, Urquiaga S (2003) The success of BNF in soybean in Brazil. Plant Soil 252:1–9CrossRefGoogle Scholar
  4. Ansari PG, Rao DLN, Pal KK (2013) Diversity and phylogeny of soybean rhizobia in central India. Ann Microbiol 64:1553–1565CrossRefGoogle Scholar
  5. Barcellos FG, Menna P, Batista JS, Hungria M (2007) Evidence of horizontal transfer of symbiotic genes from a Bradyrhizobium japonicum inoculant strain to indigenous Diazotrophs Sinorhizobium (Ensifer) fredii and Bradyrhizobium elkanii in a Brazilian savannah soil. Appl Environ Micobiol 73:2635–2643CrossRefGoogle Scholar
  6. Bernstein L, MacKenzie AJ, Krantz BA (1955) Salt tolerance of field crops - soybeans. In: United States salinity laboratory report to collaborators. Riverside, CA, pp 35–36Google Scholar
  7. Carrascal OMP, Vanlnsberghe D, Juarez S, Polz MF, Vinuesa P, Gonzalez V (2016) Population genomics of the symbiotic plasmids of sympatric nitrogen-fixing Rhizobium species associated with Phaseolus vulgaris. Environ Microbiol 18:2660–2676CrossRefGoogle Scholar
  8. Cole MA, Elkan GH (1973) Transmissible resistance to penicillin G, neomycin, and chloramphenicol in Rhizobium japonicum. Antimicrob Ag Chemother 4:248–253CrossRefGoogle Scholar
  9. Degefu T, Wolde-Meskel E, Frostegård Å (2013) Phylogenetic diversity of Rhizobium strains nodulating diverse legume species growing in Ethiopia. Syst Appl Microbiol 36:272–280CrossRefPubMedGoogle Scholar
  10. Delamuta JRM, Ribeiro RA, Menna P, Bangel EV, Hungria M (2012) Multilocus sequence analysis (MLSA) of Bradyrhizobium strains: revealing high diversity of tropical Diazotrophic symbiotic bacteria. Braz J Microbiol 43:698–710CrossRefPubMedPubMedCentralGoogle Scholar
  11. Delamuta JR, Ribeiro RA, Ormeño-Orrillo E, Melo IS, Martinez-Romero E, Hungria M (2013) Polyphasic evidence supporting the reclassification of Bradyrhizobium japonicum group Ia strains as Bradyrhizobium diazoefficiens sp. nov. Int J Syst Evol Microbiol 63:3342–3351CrossRefPubMedGoogle Scholar
  12. Devine TE, Kuykendall LD (1996) Host genetic control of symbiosis in soybean (Glycine max L.) Plant Soil 186:173–187CrossRefGoogle Scholar
  13. Didelot X, Maiden MCJ (2010) Impact of recombination on bacterial evolution. Trends Microbiol 18:315–322CrossRefPubMedPubMedCentralGoogle Scholar
  14. Germano MG, Menna P, Mostasso FL, Hungria M (2006) RFLP analysis of the rRNA operon of a Brazilian collection of bradyrhizobial strains from 33 legume species. Int J Syst Evol Microbiol 56:217–229CrossRefPubMedGoogle Scholar
  15. Gordon BR, Klinger CR, Weese DJ, Lau JA, Burke PV, Dentinger BTM, Heath KD (2016) Decoupled genomic elements and the evolution of partner quality in nitrogen-fixing rhizobia. Ecol Evol 6:1317–1327CrossRefPubMedPubMedCentralGoogle Scholar
  16. Guimarães AA, Florentino LA, Almeida KA, Lebbe L, Silva KB, Willems A, Moreira FM (2015) High diversity of Bradyrhizobium strains isolated from several legume species and land uses in Brazilian tropical ecosystems. Syst Appl Microbiol 38:433–441CrossRefGoogle Scholar
  17. Gyaneshwar P, Hirsch AM, Moulin L, Chen W, Elliott GN, Bontemps C, de los Santos PE, Gross E, dos Resi FB Jr, Sprent JI, Young JPW, James EK (2011) Legume-nodulating Betaproteobacteria: diversity, host range, and future prospects. MPMI 24:1276–1288CrossRefPubMedGoogle Scholar
  18. Hiraishi A, Kamagata Y, Nakamura K (1995) Polymerase chain reaction amplification and restriction fragment length polymorphism analysis of 16S rRNA genes from methanogens. J Ferment Bioeng 79:523–529CrossRefGoogle Scholar
  19. Htwe AZ, Yamakawa T, Sarr PS, Sakata T (2015) Diversity and distribution of soybean-nodulating bradyrhizobia isolated from major soybean-growing regions in Myanmar. Afr J Microbiol Res 9:2183–2196CrossRefGoogle Scholar
  20. Ikeda S, Rallos LEE, Okubo T, Eda S, Inaba S, Mitsui H, Minamisawa K (2008) Microbial community analysis of field-grown soybeans with different nodulation phenotypes. Appl Environ Microbiol 74:5704–5709CrossRefPubMedPubMedCentralGoogle Scholar
  21. Ikeda S, Okubo T, Kaneko T, Inaba S, Maekawa T, Eda S et al (2010) Community shifts of soybean stem-associated bacteria responding to different nodulation phenotypes and N levels. ISME J 4:315–326CrossRefPubMedGoogle Scholar
  22. Jordan DC (1982) Transfer of Rhizobium japonicum Buchanan 1980 to Bradyrhizobium gen. Nov., a genus of slow-growing, root nodule bacteria from leguminous plants. Int J Syst Bacteriol 32:136–139CrossRefGoogle Scholar
  23. Keyser HH, Bohlool BB, Hu TS, Weber DF (1982) Fast-growing rhizobia isolated from root nodules of soybean. Science 215:1631–1632CrossRefPubMedGoogle Scholar
  24. Kimura M (1980) A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120CrossRefPubMedGoogle Scholar
  25. Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874CrossRefPubMedGoogle Scholar
  26. Kuykendall LD, Saxena B, Devine TE, Udell SE (1992) Genetic diversity in Bradyrhizobium Jordan 1982 and a proposal for Bradyrhizobium elkanii sp. nov. Can J Microbiol 38:501–505CrossRefGoogle Scholar
  27. Laguerre G, Nour SM, Macheret V, Sanjuan J, Drouin P, Amarger N (2001) Classification of rhizobia based on nodC and nifH gene analysis reveals a close phylogenetic relationship among Phaseolus vulgaris symbionts. Microbiol 147:981–993CrossRefGoogle Scholar
  28. Ling J, Wang H, Wu P, Li T, Tang Y, Naseer N, Zheng H, Masson-Boivin C, Zong Z, Zhu J (2016) Plant nodulation inducers enhance horizontal gene transfer of Azorhizobium caulinodans symbiosis island. Proc Natl Acad Sci U S A 113:13875–13880CrossRefPubMedPubMedCentralGoogle Scholar
  29. Loureiro M, Kaschuk G, Alberton O, Hungria M (2007) Soybean [Glycine max (L.) Merrill] rhizobial diversity in Brazilian oxisols under various soil, cropping, and inoculation managements. Biol Fertil Soils 43:665–674CrossRefGoogle Scholar
  30. Maj D, Wielbo J, Marek-Kozaczuk M, Skorupska A (2010) Response to flavonoids as a factor influencing competitiveness and symbiotic activity of Rhizobium leguminosarum. Microbiol Res 165:50–60CrossRefPubMedGoogle Scholar
  31. Man CX, Wang H, Chen WF, Sui XH, Wang ET, Chen WX (2008) Diverse rhizobia associated with soybean grown in the subtropical and tropical regions of China. Plant Soil 310:77–87CrossRefGoogle Scholar
  32. Manuel PC, Huelgas R, Espanto LH (1986) Adoption of soybean in Lupao, Nueva Ecija, Philippines. UN/ESCAP CGPRT Centre, regional coordination Centre for Research and Development of coarse grains, roots and tuber crops in the humid tropics of Asia and the Pacific. CGPRT no. 7: 62 ppGoogle Scholar
  33. Martens M, Dawyndt P, Coopman R, Gillis M, De Vos P, Willems A (2008) Advantages of multilocus sequence analysis for taxonomic studies: a case study using 10 housekeeping genes in the genus Ensifer (including former Sinorhizobium). Int J Syst Evol Microbiol 58:200–214CrossRefPubMedGoogle Scholar
  34. Martinez-Romero E, Caballero-Mellado J (1996) Rhizobium phylogenies and bacterial genetic diversity. Crit Rev Plant Sci 15:113–140CrossRefGoogle Scholar
  35. Minami M, Yamakawa T, Yamamoto A, Akao S, Saeki Y (2009) Estimation of nodulation tendency among Rj-genotype soybeans using the bradyrhizobial community isolated from an andosol. Soil Sci Plant Nutr 55:65–72CrossRefGoogle Scholar
  36. Minamisawa K, Itakura M, Suzuki M, Ichige K, Isawa T, Yuhashi K, Mitsui H (2002) Horizontal transfer of nodulation genes in soils and microcosms form Bradyrhizobium japonicum to B. elkanii. Microbes Environ 2:82–90CrossRefGoogle Scholar
  37. Njira KOW, Nalivata PC, Kanyama-Phiri GY, Lowole MW (2013) An assessment for the need of soybean inoculation with Bradyrhizobium japonicum in some sites of Kasungu district, Central Malawi. Int J Curr Microbiol App Sci 2:60–72Google Scholar
  38. Nguyen MT, Akiyoshi K, Nakatsukasa M, Saeki Y, Yokoyama K (2010) Multiple occupancy of nodules by nodulating rhizobia on field-grown soybeans with attendance of Sinorhizobium spp. Soil Sci Plant Nutr 56:382–389CrossRefGoogle Scholar
  39. Peix A, Ramirez-Bahena MH, Velazquez E, Bedmar EJ (2015) Bacterial associations with legumes. Crit Rev Plant Sci 34:17–42CrossRefGoogle Scholar
  40. Ramirez-Bahena MH, Peix A, Rivas R, Camacho M, Rodriguez-Navarro DN, Mateos PF, Martinez-Molina E, Willems A, Velazquez E (2009) Bradyrhizobium pachyrhizi sp. nov. and Bradyrhizobium jicamae sp. nov., isolated from effective nodules of Pachyrhizus erosus. Int J Syst Evol Microbiol 59:1929–1934CrossRefPubMedGoogle Scholar
  41. Redmond M, Batley M, Djordjevic MA, Innes RW, Kuempel PL, Rolfe BG (1986) Flavones induce expression of nodulation genes in Rhizobium. Nature 323:632–634CrossRefGoogle Scholar
  42. Risal CP, Yokoyama T, Ohkama-Ohtsu N, Djedidi S, Sekimoto H (2010) Genetic diversity of native soybean bradyrhizobia from different topographical regions along the southern slopes of the Himalayan Mountains in Nepal. Syst Appl Microbiol 33:416–425CrossRefPubMedGoogle Scholar
  43. Rivas R, Martens M, de Lajudie P, Willems A (2009) Multilocus sequence analysis of the genus Bradyrhizobium. Syst Appl Microbiol 32:101–110CrossRefPubMedGoogle Scholar
  44. Sameshima R, Isawa T, Sadowsky MJ, Hamada T, Kasai H, Shutsrirung A et al (2003) Phylogeny and distribution of extra-slow-growing Bradyrhizobium japonicum harboring high copy numbers of RSα, RSβ and IS1631. FEMS Microbiol Ecol 44:191–202CrossRefPubMedGoogle Scholar
  45. Sanz-Sáez A, Heath K, Burke P, Ainsworth E (2015) Inoculation with an enhanced N2O-fixing Bradyrhizobium japonicum strain (USDA110) does not alter soybean (Glycine max Merr.) response to elevated [CO2]. Plant Cell Environ 38:2589–2602CrossRefPubMedGoogle Scholar
  46. Saeki Y, Akagi I, Takaki H, Nagatomo Y (2000) Diversity of indigenous Bradyrhizobium strains isolated from three different Rj-soybean cultivars in terms of randomly amplified polymorphic DNA and intrinsic antibiotic resistance. Soil Sci Plant Nutr 46:917–926CrossRefGoogle Scholar
  47. Saeki Y, Aimi N, Hashimoto M, Tsukamoto S, Kaneko A, Yoshida N et al (2004) Grouping of Bradyrhizobium USDA strains by sequence analysis of 16S rDNA and 16S-23S rDNA internal transcribed spacer region. Soil Sci Plant Nutr 50:517–525CrossRefGoogle Scholar
  48. Saeki Y, Aimi N, Tsukamoto S, Yamakawa T, Nagatomo Y, Akao S (2006) Diversity and geographical distribution of indigenous soybean-nodulating bradyrhizobia in Japan. Soil Sci Plant Nutr 52:418–426CrossRefGoogle Scholar
  49. Saeki Y, Minami M, Yamamoto A, Akao S (2008) Estimation of the bacterial community diversity of soybean-nodulating bradyrhizobia isolated from Rj-genotype soybeans. Soil Sci Plant Nutr 54:718–724CrossRefGoogle Scholar
  50. Saeki Y, Ozumi S, Yamamoto A, Umehara Y, Hayashi M, Sigua GC (2010) Changes in population occupancy of bradyrhizobia under different temperature regimes. Microbes Environ 25:309–312CrossRefPubMedGoogle Scholar
  51. Saeki Y, Shiro S, Tajima T, Yamamoto A, Sameshima-Saito R, Sato T, Yamakawa T (2013) Mathematical ecology analysis of geographical distribution of soybean-Nodulating Bradyrhizobia in Japan. Microbes Environ 28:470–478CrossRefPubMedPubMedCentralGoogle Scholar
  52. Saeki Y, Shiro S (2014) Comparison of soybean-Nodulating Bradyrhizobia community structures along north latitude between Japan and USA, advances in biology and ecology of nitrogen fixation, prof. Takuji Ohyama (Ed.) InTech, pp. 195–224Google Scholar
  53. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425PubMedGoogle Scholar
  54. Scholla MH, Elkan HG (1984) Rhizobium fredii sp. nov. a fast-growing species that effectively nodulates soybeans. Int J Syst Bacteriol 34:484–486CrossRefGoogle Scholar
  55. Sharma M, Srivastava K, Sharma S (2010) Biochemical characterization and metabolic diversity of soybean rhizobia isolated from Malwa region of Central India. Plant Soil Environ 56:375–383Google Scholar
  56. Shiro S, Yamamoto A, Umehara Y, Hayashi M, Yoshida N, Nishiwaki A et al (2012) Effect of Rj genotype and cultivation temperature on the community structure of soybean-nodulating bradyrhizobia. Appl Environ Microbiol 78:1243–1250CrossRefPubMedPubMedCentralGoogle Scholar
  57. Shiro S, Matsuura S, Saiki R, Sigua GC, Yamamoto A, Umehara Y et al (2013) Genetic diversity and geographical distribution of indigenous soybean-nodulating bradyrhizobia in the United States. Appl Environ Microbiol 79:3610–3618CrossRefPubMedPubMedCentralGoogle Scholar
  58. Suzuki K, Oguro H, Yamakawa T, Yamamoto A, Akao S, Saeki Y (2008) Diversity and distribution of indigenous soybean - nodulating rhizobia in the Okinawa islands, Japan. Soil Sci Plant Nutr 54:237–246CrossRefGoogle Scholar
  59. Tian CF, Young JPW, Wang ET, Tamimi SM, Chen WX (2010) Population mixing of Rhizobium leguminosarum bv. viciae nodulating Vicia faba: the role of recombination and lateral gene transfer. FEMS Microbiol Ecol 73:563–576PubMedGoogle Scholar
  60. Tsurumaru H, Yamakawa T, Tanaka M, Sakai M (2008) Tn5 mutants of Bradyrhizobium japonicum is-1 with altered compatibility with Rj2-soybean cultivars. Soil Sci. Plant Nutr 54:197–203CrossRefGoogle Scholar
  61. van Berkum P, Fuhrmann JJ (2000) Evolutionary relationships among the soybean bradyrhizobia reconstructed from 16S rRNA gene and internally transcribed spacer region sequence divergence. Int J Syst Evol Microbiol 50:2165–2172CrossRefPubMedGoogle Scholar
  62. Vincent JM (1970) A manual for the practical study of the root-nodule bacteria. Blackwell Scientific, OxfordGoogle Scholar
  63. Vinuesa P, Rojas-Jiménez K, Contreras-Moreira B, Mahna SK, Prasad BN, Moe H et al (2008) Multilocus sequence analysis for assessment of the biogeography and evolutionary genetics of four Bradyrhizobium species that Nodulate soybeans on the Asiatic continent. Appl Environ Microbiol 74:6987–6996CrossRefPubMedPubMedCentralGoogle Scholar
  64. Vos M, Quince C, Pijl AS, de Hollander M, Kowalchuk GA (2012) A comparison of rpoB and 16S rRNA as markers in pyrosequencing studies of bacterial diversity. PLoS One 7:1–8CrossRefGoogle Scholar
  65. Wang J (2016) Analysis of the factors influencing Japan’s soybean import trade: based on gravity model. Am J Ind Bus Manag 6:109–116Google Scholar
  66. Yamakawa T, Hussain AKMA, Ishizuka J (2003) Soybean preference for Bradyrhizobium japonicum for nodulation. Soil Sci Plant Nutr 49:835–841CrossRefGoogle Scholar
  67. Yan J, Han XZ, Ji ZJ, Li Y, Wang ET, Xie ZH, Chen WF (2014) Abundance and diversity of soybean-Nodulating rhizobia in black soil are impacted by land use and crop management. Appl Environ Microbiol 80:5394–5402CrossRefPubMedPubMedCentralGoogle Scholar
  68. Yasuda M, Miwa H, Masuda S, Takebayashi Y, Sakakibara H, Okazaki S (2016) Effecter-triggered immunity determines host genotype-specific incompatibility in legume-rhizobium symbiosis. Plant Cell Physiol 57:1791–1800CrossRefPubMedGoogle Scholar
  69. Young JM (2003) The genus name Ensifer Casida 1982 takes priority over Sinorhizobium Chen et al. 1988, and Sinorhizobium morelense Wang et al. 2002 is a later synonym of Ensifer adhaerens Casida 1982. Is the combination ‘Sinorhizobium adhaerens’ (Casida 1982) Willems et al. 2003 legitimate? Request for an opinion. Int J Syst Evol Microbiol 53:2107–2110CrossRefPubMedGoogle Scholar
  70. Zaat SA, Schripsema J, Wijffelman CA, van Brussel AAN, Lugtenberg BJJ (1989) Analysis of the major inducers of the Rhizobium nodA promoter from Vicia sativa root exudate and their activity with different nodD genes. Plant Mol Biol 13:175–188CrossRefPubMedGoogle Scholar
  71. Zhang YM, Li Y Jr, Chen WF, Wang ET, Tian CF, Li QQ, Zhang YZ, Sui XH, Chen WX (2011) Biodiversity and biogeography of rhizobia associated with soybean plants grown in the North China plain. Appl Environ Microbiol 77:6331–6342CrossRefPubMedPubMedCentralGoogle Scholar
  72. Zhao L, Fan M, Zhang D, Yang R, Zhang F, Xu L, Wei X, Shen Y, Wei G (2014) Distribution and diversity of rhizobia associated with wild soybean (Glycine soja Sieb. & Zucc.) in Northwest China. Syst Appl Microbiol 37:449–456CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2017

Authors and Affiliations

  • Maria Luisa Tabing Mason
    • 1
    • 2
  • Shota Matsuura
    • 3
  • Apolinario Laxamana Domingo
    • 2
  • Akihiro Yamamoto
    • 3
  • Sokichi Shiro
    • 4
  • Reiko Sameshima-Saito
    • 5
  • Yuichi Saeki
    • 3
    Email author
  1. 1.Interdisciplinary Graduate School of Agriculture and EngineeringUniversity of MiyazakiMiyazakiJapan
  2. 2.College of Agriculture, Science City of MuñozCentral Luzon State UniversityNueva EcijaPhilippines
  3. 3.Faculty of AgricultureUniversity of MiyazakiMiyazakiJapan
  4. 4.Faculty of Life and Environmental ScienceShimane UniversityMatsueJapan
  5. 5.Faculty of AgricultureShizuoka UniversityShizuokaJapan

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