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Coinoculation of Bacillus thuringeinsis-KR1 with Rhizobium leguminosarum enhances plant growth and nodulation of pea (Pisum sativum L.) and lentil (Lens culinaris L.)

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Nodulation and the subsequent nitrogen fixation are important factors that determine the productivity of legumes. The beneficial effects of nodulation can be enhanced when rhizobial inoculation is combined with plant-growth-promoting bacteria (PGPB). The PGPB strain Bacillus thuringiensis-KR1, originally isolated from the nodules of Kudzu vine (Pueraria thunbergiana), was found to promote plant growth of field pea (Pisum sativum L.) and lentil (Lens culinaris L.) under Jensen’s tube, growth pouch and non-sterile soil, respectively, when co-inoculated with Rhizobium leguminosarum-PR1. Coinoculation with B. thuringiensis-KR1 (at a cell density of 106 c.f.u. ml−1) provided the highest and most consistent increase in nodule number, shoot weight, root weight, and total biomass, over rhizobial inoculation alone. The enhancement in nodulation due to coinoculation was 84.6 and 73.3% in pea and lentil respectively compared to R. leguminosarum-PR1 treatment alone. The shoot dry-weight gains on coinoculation with variable cell populations of B. thuringiensis-KR1 varied from 1.04 to 1.15 times and 1.03 to 1.06 times in pea and lentil respectively, while root dry weight ratios of coinoculated treatments varied from 0.98 to 1.14 times and 1.08 to 1.33 times in pea and lentil respectively, those of R. leguminosarum-PR1 inoculated treatment at 42 days of plant growth. While cell densities higher than 106 c.f.u. ml−1 had an inhibitory effect on nodulation and plant growth, lower inoculum levels resulted in decreased cell recovery and plant growth performance. The results of this study indicate the potential of harnessing endophytic bacteria of wild legumes for improving the nodulation and growth of cultivated legumes.

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  1. Andrews JH, Harris RF (2003) The ecology and biogeography of microorganisms on plant surfaces. Annu Rev Phytopathol 38:145–180. doi:10.1146/annurev.phyto.38.1.145

  2. Araujo WL, Maccheroni W Jr, Aguilar-Vildosa CI et al (2001) Variability and interactions between endophytic bacteria and fungi isolated from leaf tissue of citrus rootstocks. Can J Microbiol 47:229–236. doi:10.1139/cjm-47-3-229

  3. Basu PS, Ghosh AC (1998) Indole acetic acid and its metabolism in root nodules of a monocotyledonous tree Roystonea regia. Curr Microbiol 37:137–140. doi:10.1007/s002849900352

  4. Bolton H Jr, Elliott LF, Turco RF et al (1990) Rhizoplane colonization of pea seedlings by Rhizobium leguminosarum and a deleterious root colonizing Pseudomonas sp. and effects on plant growth. Plant Soil 123:121–124

  5. Broughton WJ, Dilworth MJ (1971) Control of leghaemoglobin synthesis in snake beans. Biochem J 125:1075–1080

  6. Burns TA Jr, Bishop PE, Israel DW (1981) Enhanced nodulation of leguminous plant roots by mixed cultures of Azotobacter vinelandii and Rhizobium. Plant Soil 62:399–412. doi:10.1007/BF02374137

  7. Castejon-Munoz M, Oyarzun PJ (1995) Soil receptivity to Fusarium solani f. sp. pisi and biological control of root rot of pea. Eur J Plant Pathol 101:35–49. doi:10.1007/BF01876092

  8. Chattopadhyay A, Bhatnagar NB, Bhatnagar R (2004) Bacterial insecticidal toxins. Crit Rev Microbiol 30:33–54. doi:10.1080/10408410490270712

  9. Dashti N, Zhang F, Hynes R et al (1997) Application of plant growth-promoting rhizobacteria to soybean [Glycine max (L.) Merr.] increases protein and dry matter yield under short season conditions. Plant Soil 188:33–41. doi:10.1023/A:1004295827311

  10. Dong Y, Iniguez LA, Ahmer BMM et al (2003) Kinetics and strain specificity of rhizosphere and endophytic colonization by enteric bacteria on seedlings of Medicago sativa and Medicago truncatula. Appl Environ Microbiol 69:1783–1790. doi:10.1128/AEM.69.3.1783-1790.2003

  11. Dubeikovesky AN, Modokhova EA, Kocheskov VV et al (1993) Growth promotion of blackcurrant softwood cuttings by recombinant strain Pseudomonas fluorescens BSP53a synthesizing an increased amount of indole-3-acetic acid. Soil Biol Biochem 25:1211–1221. doi:10.1016/0038-0717(93)90217-Y

  12. Elvira-Recuenco M, van Vuurde JWL (2000) Natural incidence of endophytic bacteria in pea cultivars under weld conditions. Can J Microbiol 46:1036–1041. doi:10.1139/cjm-46-11-1036

  13. Garbeva P, Overbeek LS, Vuurde JW et al (2001) Analysis of endophytic bacterial communities of potato by plating and denaturing gradient gel electrophoresis (DGGE) of 16S rDNA based PCR fragments. Microb Ecol 41:369–383

  14. Goel AK, Sindhu SS, Dadarwal KR (2002) Stimulation of nodulation and plant growth of chickpea (Cicer arietinum L.) by Pseudomonas spp. antagonistic to fungal pathogens. Biol Fertil Soils 36:391–396. doi:10.1007/s00374-002-0554-5

  15. Hallmann J, Quadt-Hallmann A, Mahaffee WF et al (1997) Bacterial endophytes in agricultural crops. Can J Microbiol 43:895–914

  16. Halverson LJ, Handelsman J (1991) Enhancement of soybean nodulation by bacillus cereus UW85 in the field and in a growth chamber. Appl Environ Microbiol 57:2767–2770

  17. Handelsman J, Raffel S, Mester EH et al (1990) Biological control of damping-off of alfalfa seedlings with Bacillus cereus UW85. Appl Environ Microbiol 56:713–718

  18. Hans DY, Coplin DL, Bauer WD et al (2000) A rapid bioassay for screening rhizosphere microorganisms for their ability to induce systemic resistance. Phytopathol 90:327–332. doi:10.1094/PHYTO.2000.90.4.327

  19. Hirsch AM, Bhuvaneswari TV, Torrey JG et al (1989) Early nodulin genes are induced in alfalfa root outgrowths elicited by auxin transport inhibitors. Proc Natl Acad Sci USA 86:1244–1248. doi:10.1073/pnas.86.4.1244

  20. Inc SPSS (1999) SPSS Base 10.0 for Windows User’s Guide. SPSS Inc., Chicago

  21. Kloepper JW, Beauchamp CJ (1992) A review of issues related to measuring colonization of plant roots by bacteria. Can J Microbiol 38:1219–1232

  22. Kloepper JW, Leong J, Teintze M et al (1980) Enhanced plant growth by siderophores produced by plant growth-promoting rhizobacteria. Nature 286:883–884. doi:10.1038/286885a0

  23. Knight TJ, Langston-Unkefer PJ (1988) Enhancement of symbiotic dinitrogen fixation by a toxin-releasing plant pathogen. Science 241:951–954. doi:10.1126/science.241.4868.951

  24. Kobayashi DY, Palumbo JD (2000) Bacterial endophytes and their effects on plants and uses in agriculture. In: James CW, White JF (eds) Microbial endophytes, Marcel Dekker Inc. New York, pp 199–233

  25. Kondorosi A, Kondorosi E, Pankhurst CE et al (1982) Mobilization of a Rhizobium meliloti megaplasmid carrying nodulation and nitrogen fixation genes into other rhizobia and Agrobacterium. Mol Gen Genet 188:433–439. doi:10.1007/BF00330045

  26. Kuklinsky-Sobral J, Araujo WL, Mendes R et al (2004) Isolation and characterization of soybean-associated bacteria and their potential for plant growth promotion. Environ Microbiol 6:1244–1251. doi:10.1111/j.1462-2920.2004.00658.x

  27. Lifshitz R, Kloepper JW, Kozlowski M (1987) Growth promotion of canola (rapeseed) seedlings by a strain of Pseudomonas putida under gnotobiotic conditions. Can J Microbiol 33:390–395

  28. Liu ZL, Sinclair JB (1990) Enhanced soybean plant growth and nodulation by Bradyrhizobium japonicum in the presence of strains of Bacillus megaterium. (Abst). Plant Pathol 80:1024

  29. Liu ZL, Sinclair JB (1993) Colonization of soybean roots by bacillus megaterium B153–2-2. Soil Biol Biochem 25:849–855. doi:10.1016/0038-0717(93)90087-R

  30. Lodewyckx C, Vangronsveld J, Porteous F et al (2002) Endophytic bacteria and their potential applications. Crit Rev Plant Sci 21:583–606. doi:10.1080/0735-260291044377

  31. Martinez E, Palacios R, Sanchez F (1987) Nitrogen-fixing nodules induced by Agrobacterium tumefaciens harboring Rhizobium phaseoli plasmids. J Bacteriol 169:2828–2834

  32. Motsara MR, Bhattacharyya P, Srivastava B (1995) Biofertilizer Technology, marketing and usage—A sourcebook-cum-glossary. Fertilizer Development and Consultation Organization, 1st edn. New Delhi, India, pp184 + viii

  33. Raverker KP, Konde BK (1988) Effect of Rhizobium and Azospirillum lipoferum inoculation on nodulation, yield and nitrogen uptake of peanut cultivars. Plant Soil 106:249–252. doi:10.1007/BF02371220

  34. Reyes-Ramirez A, Escudero-Abarca BI, Aguilar-Uscanga G et al (2004) Antifungal activity of Bacillus thuringiensis chitinase and its potential for the biocontrol of phytopathgenic fungi in soybean seeds. J Food Sci 69:M131–M134

  35. Russell AD, Hugo WB, Ayliffo GAJ (1982) Principles and practices of disinfection, preservation & sterilization. Black Wall Scientific, London

  36. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, New York

  37. Schroth MN, Hancock JG (1981) Selected topics in biological control. Annu Rev Microbiol 35:453–476. doi:10.1146/annurev.mi.35.100181.002321

  38. Schwinghamer EA (1971) Antagonism between strains of R. trifolii in culture. Soil Biol Biochem 3:355–363. doi:10.1016/0038-0717(71)90046-0

  39. Selvakumar G, Kundu S, Gupta Anand D et al (2008) Isolation and characterization of nonrhizobial plant growth-promoting bacteria from nodules of kudzu (Pueraria thunbergiana) and their effect on wheat seedling growth. Curr Microbiol 56:134–139. doi:10.1007/s00284-007-9062-z

  40. Sindhu SS, Gupta SK, Dadarwal KR (1999) Antagonistic effect of Pseudomonas spp. on pathogenic fungi and enhancement of plant growth in green gram (Vigna radiata). Biol Fertil Soils 29:62–68. doi:10.1007/s003740050525

  41. Somasegaran P, Hoben HJ (1985) Methods in legume Rhizobium technology. Niftal University of Hawaii, Hawaii

  42. Srinivasan M, Peterson DJ, Holl FB (1996) Influence of IAA producing Bacillus isolates on the nodulation of Phaseolus vulgaris by Rhizobium etli. Can J Microbiol 42:1006–1014

  43. Strobel G, Daisy B, Castillo U et al (2004) Natural products from endophytic microorganisms. J Nat Prod 67:257–268. doi:10.1021/np030397v

  44. Sturz AV, Christie BR, Matheson BG et al (1997) Biodiversity of endophytic bacteria that colonize red clover nodules, roots, stems and foliage and their influence on host growth. Biol Fertil Soils 25:13–19. doi:10.1007/s003740050273

  45. Sturz AV, Christie BR, Nowak J (2000) Bacterial endophytes: potential role in developing sustainable systems of crop production. Crit Rev Plant Sci 19:1–30. doi:10.1016/S0735-2689(01)80001-0

  46. Valverde A, Velazquez E, Fernandez-Santos F et al (2005) Phyllobacterium trifolii sp nov. nodulating Trifolium and Lupinus in Spanish soils. Int J Syst Evol Microbiol 55:1985–1989. doi:10.1099/ijs.0.63551-0

  47. Zhang F, Dashti N, Hynes H et al (1997) Plant growth-promoting rhizobacteria and soybean [Glycine max (L.) Merr.] nodulation and nitrogen fixation at suboptimal root zone temperatures. Ann Bot (Lond) 77:453–459. doi:10.1006/anbo.1996.0055

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Thanks are due to Dr. Yogesh Shouche, National Centre for Cell Sciences, Pune, India, for the sequencing of the 16S rRNA gene of Bacillus thuringiensis-KR1. Dr. G. Singh, V·I.H.A., Almora, Uttarakhand, India is acknowledged for providing the seed material used in this study.

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Correspondence to Pankaj K. Mishra.

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Mishra, P.K., Mishra, S., Selvakumar, G. et al. Coinoculation of Bacillus thuringeinsis-KR1 with Rhizobium leguminosarum enhances plant growth and nodulation of pea (Pisum sativum L.) and lentil (Lens culinaris L.). World J Microbiol Biotechnol 25, 753–761 (2009). https://doi.org/10.1007/s11274-009-9963-z

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  • Bacillusthuringiensis-KR1
  • Endophytic bacteria
  • Lensculinaris L.
  • Pisumsativum L.
  • Plant growth promotion
  • Rhizobiumleguminosarum-PR1