Effect of Azotobacter and Azospirillum on Growth and Physiological Characteristics of Barley (Hordeum vulgare) under Salinity Stress

آب و خاک

Article 10, Volume 34, Issue 3 - Serial Number 71, July 2020, Pages 649-660   

Document Type: Research Article

DOI: 10.22067/jsw.v34i3.81093

Authors

R. Khodadadi¹; Reza Ghorbani nasrabadi^{* 2}; M. Olamaee¹; S.A. Movahedi Naini¹

¹Gorgan

²Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran.

Abstract

 
Introduction: Worldwide studies have shown that inappropriate land uses over the past 45 years have resulted in salinization of 6% of the world's land. Salinity has negative effects on soil physicochemical properties and microbial activities. The imbalance in nutrient uptake, ion toxicity and decreasing water consumption due to high osmotic pressure are resulted from high accumulation of solutes in soil solution. One of the strategies to mitigate soil salinity is the inoculation of crops with different types of beneficial soil bacteria and fungi. Plant growth promoting bacteria (PGPB) are a diverse group of bacteria capable of promoting growth and yield of many crops. The most important growth promoting mechanisms of bacteria are the ability to produce plant hormones, non-symbiotic nitrogen fixation, solubilization of insoluble phosphate and potassium, biocontrol of plants pathogens through producing hydrogen cyanide and siderophore production. Plant inoculation with growth promoting bacteria causes an increase in several indices such as shoot fresh and dry weight, root dry weight and volume as well as chlorophyll content. The synergetic effect of Azotobacter and Azospirillum on the plant has been documented by increasing the absorption of nutrients, production of hormones that stimulate plant growth such as auxin, and influencing the root morphology. Due to the wide area of saline soils, appropriate methods to reduce the negative effects of salinity are of great significance. Given the importance of using bacteria adapted with climatic conditions and soil ecosystems in each region, as well as the efficiency of the combined application of growth promoting bacteria, this study was conducted to investigate the effect of growth promoting bacteria as a single and combined application at two levels of salinity calculated based on the threshold of barley yield reduction (Karoon cultivar) and 50 % reduction in barley yield.
Materials and Methods: In order to record the Azotobacter isolates, 15 soil samples were collected from salt affected lands of Golestan province. Thirty two Azotobacter isolates were isolated by physiological and biochemical tests and cyst production in old culture. Then, their ability to grow in different concentrations of salinity, drought stress tolerance, polysaccharide production, auxin production, phosphorus and potassium solubilization, hydrogen cyanide synthesis and biological fixation of molecular nitrogen were investigated. Based on physiological and growth stimulation tests, Az13 isolate was selected as the superior isolate of Azotobacter for greenhouse test. Azospirillum superior isolate was then prepared from the microbial bank of Soil Science Department, Gorgan University of Agricultural Sciences and Natural Resources. A soil with 16 dS/m salinity was selected to determine the effects of experimental treatments at two threshold salinity levels of yield reduction and 50 % reduction of barley yield. Then, soil salinity was reduced to 8 dS/m (yield reduction threshold) by leaching. After reaching to the desired salinity, the soil was removed from the pots and air dried. The sample was sifted through a 2 - mm sieve and again transferred to the pots. The barley seeds, Karoon cultivar, were used. To prepare the inoculum, firstly the bacterial isolates were grown in the pre-culture nutrient broth medium, and then incubated at 120 rpm in a shaking incubator at 28°C for 48 hours. Afterwards, each seed was inoculated with one milliliter of the bacterial inoculant with a population of 109 CFU/ml. This experiment was conducted as factorial in a completely randomized design with three replications in the greenhouse at Gorgan University of Agricultural Sciences and Natural Resources. The treatments included four levels of bacteria (without inoculation, Azotobacter inoculation, Azospirillum inoculation, combined inoculation of Azotobacter and Azospirillum) and two levels of salinity (8 and 16 dS/m). After 70 days (late vegetative growth period), some growth and physiological indices and concentration of nutrients uptake were measured.
Results and Discussion: The results showed that salinity stress had a significant (p < 0.01) negative effect on growth and physiological traits and nutrient uptake of the plant. The combined application of Azotobacter and Azospirillum bacteria showed a positive significant influence (p < 0.01) on growth, dry weight, and root dry weight in the plant under salinity stress. The combined application of bacteria increased the chlorophyll a, b and a + b content at a salinity level of 16 dS/m by 136.49, 117.86 and 127.97 %, respectively. The combined application of bacteria resulted in a 65.39 and 55.94 % increase in proline amino acid content at salinity levels of 8 and 16 dS/m, respectively. The results revealed that nitrogen, phosphorus and potassium levels increased by 81.97, 80 and 66.67%, respectively, at 16 dS/m salinity level in combined application of both bacteria. Sodium ion accumulation in all bacterial treatments decreased in both salinity levels compared to control treatment and the highest reduction was observed in combined bacterial inoculation. These findings underline the positive effect of bacterial inoculation, particularly their combined application, on the growth and nutrients uptake of barley under salt stress.
Conclusion: Our results indicate that increasing salinity level significantly decreased shoot dry weight, root dry weight, plant height, chlorophyll content and nutrient concentrations of barley. Inoculation of salt-resistant bacteria, including Azotobacter and Azospirillum, reduced the adverse effects of salinity on growth and physiological traits, which was more pronounced in Azotobacter than Azospirillum. The combined application of Azotobacter and Azospirillum had a significant effect on root dry weight, plant height, chlorophyll content, increasing nutrient concentration efficiency (nitrogen, phosphorus, and potassium) and decreased sodium concentration at both salinity levels (8 and 16 dS/m) compared with the individually inoculated bacteria. Hence, the application of Azotobacter and Azospirillum isolates is an appropriate method for pot experiments with saline soils. To apply these results, field experiments in saline soils must be carried out to evaluate the effect of these bacterial isolates on the crop growth, yield and physiological characteristics.

Keywords

Plant growth promoting; Proline; salinity; Chlorophyll

References

1- Ali Ayyayi M. 1997. Descriptions of methods for soil chemical analysis. Volume II No. 1024, Soil and Water Research Institute, Tehran. (In Persian)

2- Ahmad P.A., ozturk M.U., and satyawati S.H. 2014. Effect of sodium carbonate-indaced salinity alkalinity on some osmoprotectahts, protein profile antioxidant enzymes and lipid peroxidantion in two mulberry. Plant Interactions 9:460-467.

3- Ashraf M., Hasnain S., and Hussain F. 2005. Exopolysaccharides(exopolysaccharide)producing biofilm bacteria in improving physicochemical characteristics of the saltaffected soils. Proceedings of the International Conference on EnvironmentallySustainable Development.

4- Arora N.K., Tewari S., Singh S., Lal N., and Maheshwari D.K. 2012. PGPR for protection of plant health under saline conditions. Bacteria in Agrobiology, Stress Management 239–258.

5- Arnon D.I. 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiology 24-1: 1.

6- Arzanesh M.H., Alikhani H.A., Khavazi K., Rahimian H.A., and Miransari M. 2011. Wheat (Triticum aestivum L.) growth enhancement by Azospirillum sp. under drought stress. World Journal Microbiology Biotechnology 27: 197-205.

7- Munns R., and Tester M. 2008. Mechanisms of salinity tolerance. Annu. Rev. Plant Biology 59: 651-681.

8- Asghari B., and Musarrt J. 2009. Salt tolerance in Zea mays (L). following inoculation with Rhizobium and Pseudomonas. Biology and Fertility of Soils 45: 405-413.

9- Bates L.S., Waldren R.P. and Teare, I.D. 1973. Rapid determination of free proline for water-stress studies. Plant and Soil 39(1): 205-207.

10- Bhardwaj D., Ansari MW., Sahoo RK., and Tuteja N. 2014. Biofertilizers function as key player in sustainable agriculture by improving soil fertility, plant tolerance and crop productivity. Microbial Cell Factories 13: 66.

11- Bashan Y., Holguin G., and de-Bashan L. E. 2004. Azospirillumplantrelationships: physiological, molecular, agricultural, and environmental advances (1997-2003). Canadian Journal of Microbiology 50-8: 521-577.

12- Bargaz A., Nassar R.M.A., Rady M.M., Gaballah M.S., and Thompsn S.M. 2016 . Improved Salinity Tolerance by Phosphorus Fertilizer in Two Phaseolus vulgaris Recombinant Inbred Lines Contrasting in Their P-Efficiency. Agronomy and Crop Science 202: 497-507.

13- Beinsan C., Camen D., Sumalan R., and Babou M. 2000. Study concerning salt stress effect on leaf area dynamics and chlorophyll content infour bean local landraces from Banat area. International symposium on Agriculture, Romania.

14- Chandrasekar B.R., Ambrose G., and Jayabalan N. 2005. Influence of biofertilizers and nitrogen source level on the growth and yield of Echinochloa frumentacea. Journal of Agricultural Technology 1: 223–234.

15- Chaudhary D., Narula N.S.S., and Sindhu R.K., Behl. 2013. Plant growth stimulation of wheat (Triticum aestivum L.) by inoculation of salinity tolerant Azotobacter strains. Physiology and Molecular Biology of Plants 19(4): 515–519.

16- Dobbelaere S., Vanderleyden J., and Okon Y. 2003. Plant growth promoting effect of diazotrophs in the rhizosphere. Critical Review Inplant Science 22-2: 107-149.

17- Egamberdiyevaa D., and Hoflich, G. 2003. Influence of growth-promoting bacteria on the growth of wheat in different soils and temperatures. Soil Biology and Biochemistry 35: 973–978.

18- Egamberdiyeva D., and Lugtenverg B. 2015. Use of plant Growth- ptomoting Rhizobacteria to Alleviates alinity stress in plants. Use of Microbes for the Alleviation of Soil Stresses 1:73-96.

19- FAO. 2010. Extent and causes of salt-affected soils in participating countries. Available at http://www.fao.org/ag/AGL/agll/spuch/topic4.htm.

20- Glick B.R. 1995. The enhancement of plant growth by free-living bacteria. Canadian Journal of Microbiology 41: 109-117.

21- Harley J.L., and Smith S.E. 2000. Azotobacter Symbiosis. Academic Press, London.

22- Hamdi M.A., Shaddad M.A.K., and Doaa M.M. 2004. Mechanisms of salt tolerance and interactive effects of Azospirillum brasilense inoculation on maize cultivars grown under salt stress conditions. Plant Physiology 44: 165.

23- Han H.S., and Lee K.D. 2005. Physiological Responses of Soybean - Inoculation of Bradyrhizobium japonicum with PGPR in Saline Soil Conditions. Agricultural and Biological Sciences 1: 216-221.

24- Heydarian Z., Yu M., Gruber M., Glick BR., Zhou R., and Hegedus DD. 2016. Inoculation of Soil with Plant Growth Promoting Bacteria Producing 1-Aminocyclopropane-1-Carboxylate Deaminase or Expression of the Corresponding acdS Gene in Transgenic Plants Increases Salinity Tolerance in Camelina sativa. Frontiers in Microbiology 7: 1966.

25- Hoflich G., Wichc W., and Kuhn G. 1982. Plant growth stimulation by inoculation with symbiotic and associative rhizosphere microorganisms in salt stress. Experientia 50(10): 897-905.

26- Kafi M., and Khan M.A. 2008. Relatve salt tolerance of south Khorasan millets. Desert 14: 63-71.

27- Kao W.Y., Tasai, H.C. and Tasi T.T. 2001. Effect of Nacl and nitrogen availability on growth and photosynthesis of seedlings of a mangrove species Kandelia candel (L.) Druce. Journal of Plant Physiology 158: 841-846.

28- Krieg N. R. 2005. Bergey,s Manual of Systematic Bacteriology, Williams and Wilkins, 1136p.

29- Kumar T.S., Swaminathan V., and Kumar S. 2009. Influence of nitrogen phosphorus and biofertilizers on growth yield and essential oil constituents in ratoon crop of davana (Artemisia pallens Wall.). Electronic Journal of Environmental Agricultural and Food Chemistry 8:86-95.

30- Maas E.V., and Hoffman G.J. 1977. Crop salt tolerance–current assessment. Journal of the Irrigation and Drainage Division 103(2): 115-34.

31- Meena R.S., Meena V.S., Meena S.K., and Verma J.P .2015. Towards the plant stress mitigate the agricultural productivity: a Book Review 102: 552–553.

32- Marius S., Octavita A., Eugen U., and Vlad A. 2005. Study of a microbial inoculation on several biochemical indices in sunflower (Helianthus anuus L.) in salt stress. Genetica si Biologie Moleculara 11-14.

33- Mehta S., and Nautiyal C. S. 2001. An efficient method for qualitativa screening of phosphate-solubilizing bacteria. Current Microbiology 43: 51-56.

34- Mohapatra B., Verma D.K., Sen A., Panda B.B., and Asthie B. 2013. Biofertilizers- a gateway of sustainable agriculture. Popular Kheti 1: 97–106.

35- Mitra D., Sharma K., Uniyal N., Chauhan A., Sarkar P. 2016. Study on plant hormone (indole-3- acetic acid) producing level and other plant growth promotion ability (pgpa) by Asparagus racemosus rhizobacteria. Journal Chem Pharm Research 8: 995–1002.

36- Narula N., Kumar V., Behl R. K., Deubel A., Gransee A., and Merbach W. 2000. Effect of P-solubilizing Azotobacter chroococcum on N, P, K uptake in P-responsive wheat genotypes grown under greenhouse conditions. Plant Nutrient Soil Science 163: 393–398.

37- Nehra V., and Choudhary M. 2015. A review on plant growth promoting rhizobacteria acting as bioinoculants and their biological approach towards the production of sustainable agriculture. Journal of Applied and Natural Science 7(1): 540-556.

38- Nosrati R., Owlia P., Saderi H., Rasooli I., and Malboobi M.A. 2014. Phosphate solubilization characteristics of efficient nitrogen fixing soil Azotobacter strains. Iranian Journal of Microbiology 6(4): 285.

39- Panwar M., Tewari R., Gulati A., and Nayyar, H. 2016. Indigenous salt-tolerant rhizobacterium Pantoea dispersa (PSB3) reduces sodium uptake and mitigates the effects of salt stress on growth and yield of chickpea. Acta Physiologiae Plantarum 38(12): 278.

40- Parihar P., Singh S., Singh R., and Prased S. 2015. Effect of salinity stress on plants and its tolerance strategies. Enviromental science and Pollution Research 22:4056-4075.

41- Pessarakli M.ed., 2016. Handbook of plant and crop stress. CRc press.

42- Peng Y.L., Gao Z.W., Gao Y., Liu G.F., Sheng L.X. and Wang D.L. 2008. Ecophysiological characteristics of alfalfa seedlings in response to various mixed salt-alkaline stresses. Journal of Integrative Plant Biology 50 (1): 29-39.

43- Rais L., Masood A., Inam A., and Khan N. 2013. Sulfur and nitrogen co-ordinately improve photosynthetic efficiency, growth and proline accumulation in two cultivarsof mustard under salt stress. Plant Biochemistry and Physiologye.

44- Rai S.N., and Gaur A.C. 2001. Characterization of Azotobacter SPP and effect of Azotobacter and Azospirillum as inoculant on the yield and N Uptake of wheat crop. Plant Soil 109: 131-134.

45- Santi C., Bogusz D., and Franche C. 2013. Biological nitrogen fixation in non-legume plants. Annals of Botany 10: 1–25.

46- Sayed A.V., and Hossein A.F. 2011. Investigation of biofertilizers influence on quantity and quality characteristics in Nigella sativa L. Journal of Horticulture and Forestry 3- 3: 88–92.

47- Saxena B., Shukla K., and Giri B., 2017. Arbuscular Mycorrhizal Fungi and Tolerance of Salt Stress in Plants. Arbuscular Mycorrhizas and Stress Tolerance of Plants 24: 67-97.

48- Spaepen S., and Vanderleyden J. 2011. Auxin and plant-microbe interactions. Cold Spring Harbor Perspectives in Biology 3(4): a001438.

49- Yildirim E., Turan M., and Donmez M. 2008. Mitigation of salt stress in radish (Raphanus sativus L.) by plantgrowth promoting rhizobacteria. Rumanian Biotechnological Letters 13-5: 3933-3943.

50- Zaki H.E., and Yokoi S. 2016. A comparative in vitro study of salt tolerance in cultivated tomato and related wild species. Plant Biotechnology 33(5): 361-372.

51- Zarea M.J., Hajinia S., Karimi N., Mohammadi Goltapeh E., Rejali F., and Varma A. 2012. Effect of Piriformospora indica and Azospirillum strains from salineor non-saline soil on mitigation of the effects of NaCl. Soil Biology and Biochemistry 45: 139–146.

52- Zahir Z.A., Ghoni U., Naveed M., Nadeem S.M., and Asghar H.N. 2009. Comporative effectivness of pseudomonas and serratia sp. Containing ACC-diaminase for improving growth and yield of wheat (Triticum aestivum L.) under salt-stressed conditions. Archives of Microbiology 191(5): 415-424.