The Effect of Nodulation Inducers on Alfalfa (Medicago sativa L.) Yield and Nodulation under Optimal and Salinity Conditions

Document Type : Research Article

Authors

University of Tehran

Abstract

Introduction
Luteolin is one of the most important flavonoids, which release from seeds during the first four hours of imbibition. On the other hand, at present, salinity is one of the most important factors in reducing crop production. The results of some studies show that the use of external flavonoids increases expression of nod genes, yield and nodulation in some legumes species under stress conditions. Therefore, this experiment was conducted with the aim of investigating the effect of luteolin and alfalfa seed exudates external inducers on the expression of Rhizobium nod gene and the yield and nodulation of alfalfa in normal and salinity condition.
Materials and Methods
We studied the effect of luteolin on the induction of nod genes in R. Meliloti carrying a plasmid with a translational fusion between R. Meliloti nodA and lacZ of Escherichia coli, and the expression activity was measured by β-galactosidase activity. Luteolin strongly induced the expression of nod genes inhibitory effects. We further studied the effect of luteolin and Seed exudate on alfalfa (Medicago sativa L.) yield and nodulation under optimal and salinity condition. One greenhouse was conducted to determine whether the pre-incubation of Rhizobium meliloti with luteolin and application of luteolin and seed exudate directly on to the seed surface can increase alfalfa nodulation and yield. The factorial experiment was arranged based on randomized complete block design, with three replications. Treatments were two cultivars, two bacterial strains (Sensitive and resistant strains), two levels of salt (0 and 15 dS.m-1 of NaCl) and two levels of application of inducers along with control.
Results and Discussion
The results from this experiment clearly indicated that inoculation of alfalfa seeds with luteolin and seed exudate increase alfalfa nodulation (47%) and yield (30%) significantly under salinity condition that these treats affect on traits more under normal condition. But significant effect was not observed on chlorophyll content. In this experiment a significant difference was not observed between the control and pre-incubation of R. meliloti. Also, salinity increased proline four times compared to normal condition. Luteolin and seed exudates increased proline 1.66 and 1.35 times respectively in salinity condition, but under normal condition they did not have significant effect. Salinity increased the content of sodium in the leaves (7 times) and roots (8.5 times) and decreased the content of potassium 29% and 24% in these organs of the plant, respectively, that, luteolin and seed exudate partially moderated these changes.
Conclusions
The results of this experiment indicated that luteolin and seed exudate can be used as exogenous inducers to improve the growth and nodulation of alfalfa under salinity and normal condition. Flavonoid inducers act in low concentration, and their negative effects relate to reduced germination and growth. In general, it seems that the application of flavonoid inducers is more suitable to improve nodulation, but the direct application of them in agriculture should be used only in specified cases and when flavonoids are as a limiting factor.

Keywords


1. Abd Alla, M. H., Bagy, M. K., El-enany, A. S., and Bashandy, S. R. 2014. Activation of Rhizobium tibeticum With Flavonoids Enhances Nodulation, Nitrogen Fixation, and Growth of Fenugreek (Trigonella foenum-graecum L.) Grown in Cobalt-Polluted Soil. Archives of Environmental Contamination and Toxicology 66: 303-315.
2. Arnon, D. I. 1967. Copper anzymes in isolated chloroplasts polyphenoloxidase in Beta Vulgaris. Pant Physiology 24: 1-10.
3. Asch, F., Dingkuhn, M., and Droffling, K. 2000. Salinity increases CO2 assimilation but reduces growth in field growth irrigated rice. Plant and Soil 218: 1-10.
4. Bates, L. S., Waldren, R. P., and Teare, I. D. 1973. Rapid determination of free proline for water stress studies. Plant and Soil 39: 205-207.
5. Begum, A. A., Leibovitch, S., Migner, P., and Zhang, F. 2001a. Specific flavonoids induced nod gene expression and pre-activated nod genes of Rhizobium leguminosarum increased pea (Pisum sativum L.) and lentil (Lens culinaris L.) nodulation in controlled growth chamber environments. Journal of Experimental Botany 52: 1537-1543.
6. Begum, A. A., Leibovitch, S., Migner, P., and Zhang, F. 2001b. Inoculation of pea (Pisum sativum L.) by Rhizobium leguminosarum bv. viceae preincubated with naringenin and hesperetin or application of naringenin and hesperetin directly into soil increased pea nodulation under short season conditions. Plant and Soil 237: 71-80.
7. Bouhmouch, I., Souad-Mouhsine, B., Brhada, F., and Aurag, J. 2005. Influence of host cultivars and rhizobium species on the growth and symbiotic performance of Phaseolus vulgaris under salt stress. Journal of Plant Physiology 162: 1103-1113.
8. Bruning, B., and Rozema, J. 2012. Symbiotic nitrogen fixation in legumes: Perspectives for saline agriculture. Environmental and Experimental Botany 92: 134-143.
9. Campanelli, A., Ruta, C., Morone-Fortunato, I. and Mastro, G. D. 2013. Alfalfa (Medicago sativa L.) clones tolerant to salt stress: in vitro selection. Central European Journal of Biology 8 (8): 765-776.
10. Cooper, J. E. 2007. Early interactions between legumes and rhizobia: disclosing complexity in a molecular dialogue. Journal of Applied Microbiology 103: 1355-1365.
11. Dadkhah, A. 2011. Effect of Salinity on Growth and Leaf Photosynthesis of Two Sugar Beet (Beta vulgaris L.) Cultivars. Journal of Agricultural Science and Technology 13: 1001-1012.
12. Deaker, R., Roughley, R. J., and Kennedy, I. R. 2004. Legume seed inoculation technology. Soil Biology and Biochemistry 36: 1275-1288.
13. Farhangian, S. 2009. The effect of salinity on chlorophyll content of Onobrychis sativa and Medicago sativa. Plant and Ecosystem 18: 77- 89.
14. Farissi, M., Faghire, M., Bargaz, A., Bouizgaren, A., Makoudi, B., Sentenac, H., and Ghoulam, C. 2014. Growth, nutrients concentrations, and enzymes involved in plants nutrition of alfalfa populations under saline conditions. Journal of Agricultural Science and Technology 16: 301-314.
15. Garg, B., Dogra, R. C., and Shama, P. K. 1999. High-efficiency transformation of Rhizobium leguminosarum by electroporation. Applied and Environmental Microbiology 65: 2802-4.
16. Ghasem, F., Poustini, K., Besharati, H., Mohammadi, V. A., Abooei Mehrizi, F., and Goettfert, M. 2012. Pre-incubation of Sinorhizobium meliloti with Luteolin, Methyl jasmonate and Genistein Affecting Alfalfa (Medicago sativa L.) Growth, Nodulation and Nitrogen Fixation under Salt Stress Conditions. Journal of Agricultural Science and Technology 14: 1255-1264.
17. Ghoulam, C., Foursy, A., and Fares, K. 2002. Effects of Salt Stress on Growth, Inorganic Ions and Proline Accumulation in Relation to Osmotic Adjustment in Five Sugar Beet Cultivars. Environmental and Experimental Botany 47: 39-50.
18. Hartwig, U. A., Maxwell, C. A., Joseph, C. M., and Phillips, D. A. 1990. Chrysoeriol and Luteolin Released from Alfalfa Seeds Induce nod Gene sin Rhizobium meliloti. Plant Physiology 92: 116-122.
19. Hirsch, A. M. 1992. Developmental biology of legume nodulation. New Phytologist 122: 211-237.
20. Hungria, M., and Phillips, D. A. 1993. Effects of a seed color mutation on rhizobial nod- gene- inducing flavonoids and nodulation in common bean. Molecular Plant-Microbe Interactions 6: 418-22.
21. Juan, M., Rivero, R. M., Romero, L., and Ruiz, J. M. 2005. Evaluation of Some Nutritional and Biochemical Indicators in Selected Salt Resistance Tomato Cultivars. Environmental and Experimental Botany 54: 193-201.
22. Kapulnik, Y., Joseph, C. M., and Phillips, D. A. 1987. Flavone limitations to root nodulation and symbiotic nitrogen fixation in alfalfa. Plant Physiology 84: 1193-1196.
23. Lokhande, V. H., Nikam, T. D., Patade, V. Y., Ahire, M. L., and Suprasanna, P. 2011. Effects of optimal and supra-optimal salinity stress on antioxidative defence, osmolytes and in vitro growth responses in Sesuvium portulacastrum L. Plant Cell, Tissue Organ Culture 104: 41-49.
24. Mabood, F., and Smith, D. L. 2005. Pre-incubation of Bradyrhizobium japonicum with jasmonates accelerates nodulation and nitrogen fixation in soybean (Glycine max) at optimal and suboptimal root zone temperatures. Physiologia Plantarum 125: 311-323.
25. Maj, D., Wielbo, J., Marek-Kozaczuk, M., and Skorupska A. 2010. Response to flavonoids as a factor influencing competitiveness and symbiotic activity of Rhizobium leguminosarum. Microbiological Research 165: 50-60.
26. Meloni D. A., Gulotta, M. R., and Martinez, C. A. 2008. Salinity Tolerance in Schinopsis quebracho Colorado: Seed Germination, Growth, Ion Relations and Metabolic Responses. Journal of Arid Environments 72: 1785-1792.
27. Miller, J. 1972. Experiments in molecular genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
28. Miransari, M., and Smith, D. L. 2009. Alleviating salt stress on soybean (Glycine max (L.) Merr.) Bradyrhizobium japonicum symbiosis, using signal molecule genistein. European Journal of Soil Biology 45: 146-152.
29. Mulligan, J. T., and Long, S. R. 1989. A family of activator genes regulates expression of Rhizobium meliloti nodulation genes. Genetics 122: 7-18.
30. Novak, K., Chovance, P., Skrdleta, V., Kropacova, M., Lisa, L., and Nemcova, M. 2002. Effect of exogenous flavonoids on nodulation of pea (Pisum sativum L.). Journal of Experimental Botany 53 (375): 1735-1745.
31. Oldroyd, G. E. D., and Downie, J. A. 2004. Calcium, kinases and nodulation signalling in legumes. Nature Reviews Molecular Cell Biology 5: 566-576.
32. Perez-Montaño, F., Guasch-Vidal, B., Gonzalez-Barroso, S., Lopez-Baena, F. J., and Cubo, T. 2011. Nodulation-gene-inducing flavonoids increase overall production of autoinducers and expression of N-acyl homoserine lactone synthesis genes in rhizobia. Research in Microbiology 162: 715-723.
33. Perret, X., Staehelin, C., and Broughton, W. J. 2000. Molecular basis of symbiotic promiscuity. Microbiology and Molecular Biology Reviews 64: 180-201.
34. Soussi, M., Lluch, C., and Ocana, A. 1999. Comparative study of nitrogen fixation and carbon metabolism in two chickpea (Cicer arietinum L.) cultivars under salt stress. Journal of Experimental Botany 50: 1701-1708.
35. Tambalo, D. D., Vanderlinde, E. M., Robinson, S., Halmillawewa, A., Hynes, M. F., and Yost, C. K. 2013. Legume seed exudates and Physcomitrella patens extracts influences warming behavior in Rhizobium leguminosarum. Canandian Journal of Microbiology 60: 15-24.
36. Tu, J. C. 1981. Effect of salinity on rhizobium-root-hair interaction nodulation and growth of soybean. Canadian Journal of Plant Science 61: 231-239.
37. Valia, R. Z., Patel, V. K. and Kaadia, P. K. 1993. Physiological response of drumstick (Moringoolifera Lamk) to varying Levels of ESP. Indian Journal of Plant Physiology 36 (4): 261-262.
38. Wang, X., Chen, W., Zhou, Y., Han, J., Zhao, J., Decheng Shi, D., and Yang, C. 2012. Comparison of adaptive strategies of alfalfa (Medicago sativa L.) to salt and alkali stresses. Australian Journal of Crop Science 6 (2): 39-315.
39. Zaat, S. A., Wijffelman, C. A., Mulders, I. H. M., van Brussel, A. A. N., and Lugtenberg, B. J. J. 1988. Root exudates of various host plants of Rhizobium leguminosarum contain different sets of inducers of Rhizobium nodulation genes. Plant Physiology 86: 1298-303.
40. Zahran, H. H., and Sprent, J. I. 1986.Effects of sodium-chloride and polyethyleneglycol on root hair infection and nodulation of Vicia faba L. plants by Rhizobium leguminosarum. Planta 167: 303-309.
41. Zeng, Y., Li, L., Yang, R., Yi, X., and Zhang, B. 2015. Contribution and distribution of inorganic ions and organic compounds to the osmotic adjustment in Halostachys caspica response to salt stress. Scientific Reports 1-11.
42. Zhang, F., and Smith, D. L. 1996. Inoculation of soybean [Glycine max (L) Merrill] with genistein-preincubated Bradyrhizobium japonicum or genistein directly applied into soil increases soybean protein and dry matter yield under short season conditions. Plant and Soil 179: 33-241.
CAPTCHA Image