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Analysis and effect of the use of biofertilizers on Trifolium rubens L., a preferential attention species in Castile and Leon, Spain, with the aim of increasing the plants conservation status

1 Department of Microbiology and Genetics, University of Salamanca, Salamanca, Castile & Leon, Spain
2 Spanish-Portuguese Institute for Agricultural Research (CIALE), Salamanca, Castile & Leon, Spain
3 Associated I + D Unit, USAL-CSIC (IRNASA), Salamanca, Castile & Leon, Spain

Special Issue: Plant probiotic bacteria: solutions to feed the World

Trifolium rubens L. is a leguminous plant “Preferential Attention”, according to the Catalog of Protected Flora of Castile and Leon (Spain). In this study we aimed to analyze the potential of three bacterial strains of the genus Rhizobium to improve the growth and development of this plant. All three strains produced 3-indoleacetic acid (IAA), but the strain ATCC 14480 produced the most. In addition, all strains produced biofilms and cellulases, although in different quantities. The synthesis of these products has been described as being related to the processes of the adherence of bacteria to the plant root surface and their entrance into the plant, respectively. In addition, in vitro assays and assays conducted under controlled and sterile conditions were performed, showing that the three strains were capable of nodulating T. rubens L. and effectively fixed nitrogen for the plant. These results were corroborated by morphological and histological analysis of nodules. Finally, greenhouse assays determined the effects of the strains under more competitive conditions, and it was concluded that inoculated plants presented greater lengths and weights, both aerial and radicular, and also chlorophyll and nitrogen content compared to the uninoculated controls.
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1. Valiente‐Banuet A, Aizen MA, Alcántara JM, et al. (2015) Beyond species loss: the extinction of ecological interactions in a changing world. Funct Ecol 29: 299–307.    

2. Catálogo de Flora Protegida de Castilla y León, Decreto 63/2007 de 14 de junio. B.O.C. y L.-N.º 119. Junta de Castilla y León, 2007. Available from: http://www.biodiversidade. eu/uploads/documentacion/ arquivo/e63/5249b68070-castilla-leon-decreto-flora-portegida.pdf.

3. Avellaneda Castro VA, Evaluación agronómica del Rhizobium con inoculación y fertilización nitrogenada en una pastura de trébol blanco (Trifolium repens) y ryegrass perenne (Lolium perenne). BS thesis, 2007. Available from: http://dspace.ups.edu.ec/handle/123456789/6714.

4. Williams JG, Kubelik AR, Livak KJ, et al. (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res 18: 6531–6535.    

5. Watson LE, Sayed-Ahmed H, Badr A (2000) Molecular phylogeny of Old World Trifolium (Fabaceae), based on plastid and nuclear markers. Plant Syst Evol 224: 153–171.    

6. Oleszek W, Stochmal A (2002) Triterpene saponins and flavonoids in the seeds of Trifolium species. Phytochemistry 61: 165–170.    

7. Vicioso C (1952) Tréboles españoles: Revisión del género Trifolium. Anal Inst Bol Cavanilles 10: 347–398.

8. Mayz-Figueroa J (2004) Fijación biológica de nitrógeno. Revista Científica UDO Agrícola 4: 1–20.

9. Izquierdo JA, Microbial communities involved in the nitrogen cycle at the soil aggregate scale. Doctoral Dissertations, 2007. Available from: http://scholarworks.umass.edu/dissertations/AAI32 89275.

10. Stanton-Geddes J, Anderson CG (2011) Does a facultative mutualism limit species range expansion? Oecologia 167: 149–155.    

11. Terpolilli J, Rui T, Yates R, et al. (2014) Genome sequence of Rhizobium leguminosarum bv trifolii strain WSM1689, the microsymbiont of the one flowered clover Trifolium uniflorum. Stand Genomic Sci 9: 527.

12. Reeve W, O'Hara G, Chain P, et al. (2010) Complete genome sequence of Rhizobium leguminosarum bv trifolii strain WSM2304, an effective microsymbiont of the South American clover Trifolium polymorphum. Stand Genomic Sci 2: 66–76.    

13. Alarcón A, Ferrera R, Ecología, fisiología y biotecnología de la micorriza arbuscular (No. 579.5 E2). Universidad Autónoma de San Luis Potosí, México, 2000. Available from: http://www.sidalc.net/cgibin/wxis.exe/?IsisScript=librosslp.xis&method=post&formato=2&cantidad=1&expresion=mfn=0022 09.

14. Cuervo J, Aislamiento y caracterización de Bacillus spp. como fijadores biológicos de nitrógeno y solubilizadores de fosfatos en dos muestras de biofertilizantes comerciales. Pontificia Universidad Javeriana, Facultad de Ciencias Básicas, Carreara de Microbiología Agrícola y Veterinaria, Bogotá-Colombia, 28, 2010. Available from: www.javeriana.edu.co/biblos/tesis/ ciencias/tesis404.pdf.

15. Selma MV, Martínez-Sánchez A, Allende A, et al. (2010) Impact of organic soil amendments on phytochemicals and microbial quality of rocket leaves (Eruca sativa). ‎J Agr Food Chem 58: 8331–8337.    

16. Schönian G, Meusel O, Tietz HJ, et al. (1993) Identification of clinical strains of Candida albicans by DNA fingerprinting with the polymerase chain reaction. Mycoses 36: 171–179.

17. O'hara GW, Goss TJ, Dilworth MJ, et al. (1989) Maintenance of intracellular pH and acid tolerance in Rhizobium meliloti. Appl Environ Microb 55: 1870–1876.

18. Fujishige NA, Kapadia NN, De HPL, et al. (2006) Investigations of Rhizobium biofilm formation. F EMS Microbiol Ecol 56: 195–206.    

19. Mateos PF, Jimenez-Zurdo JI, Chen J, et al. (1992) Cell-associated pectinolytic and cellulolytic enzymes in Rhizobium leguminosarum biovar trifolii. Appl Environ Microb 58: 1816–1822.

20. Fåhraeus G (1957) The infection of clover root hairs by nodule bacteria studied by a simple glass slide technique. Microbiology 16: 374–381.    

21. Mulcahy DL, Cresti M, Sansavini S, et al. (1993) The use of random amplified polymorphic DNAs to fingerprint apple genotypes. Sci Hortic-Amsterdam 54: 89–96.    

22. Stêpniak E, Zagalska MM, Switonski M (2002) Use of RAPD technique in evolution studies of four species in the family Canidae. J Appl Genet 43: 489–500.

23. Moschetti G, Peluso A, Protopapa A, et al. (2005) Use of nodulation pattern, stress tolerance, nodC gene amplification, RAPD-PCR and RFLP-16S rDNA analysis to discriminate genotypes of Rhizobium leguminosarum biovar viciae. Syst Appl Microbiol 28: 619–631.    

24. Valverde A, Igual JM, Peix A, et al. (2006) Rhizobium lusitanum sp. nov. a bacterium that nodulates Phaseolus vulgaris. Int J Syst Evol Microbiol 56: 2631–2637.

25. Alves-Santos FM, Ramos B, García-Sánchez MA, et al. (2002) A DNA-based procedure for in planta detection of Fusarium oxysporum f. sp. phaseoli. Phytopathology 92: 237–244.    

26. Castillo G, Altuna B, Michelena G, et al. (2005) Cuantificación del contenido de ácido indolacético (AIA) en un caldo de fermentación microbiana. An Biol 27: 137–142.

27. Duca D, Lorv J, Patten CL, et al. (2014) Indole-3-acetic acid in plant-microbe interactions. A Van Leeuw 106: 85–125.    

28. Machado RG, de Sá ELS, Bruxel M, et al. (2013) Indoleacetic acid producing rhizobia promote growth of Tanzania grass (Panicum maximum) and Pensacola grass (Paspalum saurae). Int J Agric Biol 15: 827–834.

29. García-Fraile P, Carro L, Robledo M, et al. (2012) Rhizobium promotes non-legumes growth and quality in several production steps: towards a biofertilization of edible raw vegetables healthy for humans. PLoS One 7: e38122.    

30. Flores‐Félix JD, Menéndez E, Rivera LP, et al. (2013) Use of Rhizobium leguminosarum as a potential biofertilizer for Lactuca sativa and Daucus carota crops. J Plant Nutr 176: 876–882.    

31. Rubio-Canalejas A, Celador-Lera L, Cruz-González X, et al. (2016) Rhizobium as potential biofertilizer of Eruca sativa, In: Biological Nitrogen Fixation and Beneficial Plant-Microbe Interaction, Springer, 213–220.

32. Garg V, Kukreja K, Gera R, et al. (2015) Production of indole-3-acetic acid by berseem (Trifolium alexandrinum L.) rhizobia isolated from Haryana, India. Agric Sci Digest 35: 229–232.

33. Bhattacharjee RB, Jourand P, Chaintreuil C, et al. (2012) Indole acetic acid and ACC deaminase-producing Rhizobium leguminosarum bv. trifolii SN10 promote rice growth, and in the process undergo colonization and chemotaxis. Biol Fert Soils 48: 173–182.

34. Velázquez E, Carro L, Flores-Félix JD, et al. (2017) The legume nodule microbiome: A source of plant growth-promoting bacteria, In: Probiotics and Plant Health, Springer Singapore, 41–70.

35. Serra DO, Richter AM, Hengge R (2013) Cellulose as an architectural element in spatially structured Escherichia coli biofilms. ‎J Bacteriol 195: 5540–5554.    

36. Donlan RM (2002) Biofilms: microbial life on surfaces. Emerg Infect Dis 8: 881–890.    

37. Costerton JW (1995) Overview of microbial biofilms. J Ind Microbiol Biot 15: 137–140.    

38. Pasmore M, Costerton JW (2003) Biofilms, bacterial signaling, and their ties to marine biology. J Ind Microbiol Biotechnol 30: 407–413.    

39. Martínez Y, Evaluación de un bioensayo para medir la inhibición de biopelículas bacterianas como indicativo de la actividad antifouling de compuestos de origen natural. (Tesis inédita). Facultad de Ciencia, Universidad Nacional de Colombia, 2010. Available from: http://www.bdigital.unal.edu.co/5100/.

40. Sainz-Rozas H, Echeverría H (1998) Uso del medidor de clorofila para el monitoreo de la nutrición nitrogenada del cultivo de maíz. Rev Fac Agron La Plata 103: 37–44.

41. Novoa SA, Villagran A (2002) Evaluación de un instrumento medidor de clorofila en la determinación de niveles de nitrógeno foliar en maíz. Agricultura Técnica 62: 166–171.

42. Mendoza M, González GA, Santelises AA, et al. (1998) Estimación de la concentración de nitrógeno y clorofila en tomate mediante un medidor portátil de clorofila. Terra 16: 135–141.

43. Barbazán M, de Suelos ADF, Análisis de plantas y síntomas visuales de deficiencia de nutrientes, Facultad de agronomía de la Universidad de la República de Montevideo-Uruguay. Montevideo, Uruguay, 1998. Available from: www.fagro.edu.uy/fertilidad/publica/AnPlantas.pdf.

44. Schwieger F, Tebbe CC (2000) Effect of field inoculation with Sinorhizobium meliloti L33 on the composition of bacterial communities in rhizospheres of a target plant (Medicago sativa) and a non-target plant (Chenopodium album)-linking of 16S rRNA gene-based single-strand conformation polymorphism community profiles to the diversity of cultivated bacteria. Appl Environ Microb 66: 3556–3565.    

Copyright Info: © 2017, Xavier Cruz-González, et al., licensee AIMS Press. This is an open access article distributed under the terms of the Creative Commons Attribution Licese (http://creativecommons.org/licenses/by/4.0)

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