Humik Asit ve Liken Türevli Endofitik Bakterilerin Kombine Uygulaması, Yoncada (Medicago sativa L.) Tuz Stresi Kaynaklı Oksidatif Hasarı Yatıştırır

Abstract

Bu çalışma, leonardit kaynaklı humik asit (HA) ile liken kökenli bir endofitik bakteri (Bacillus wiedmannii) arasındaki sinerjik etkinin, yonca bitkisinde (Medicago sativa L., cv. Konya) tuz stresini hafifletmedeki rolünü araştırdı. Yonca tohumları, 100 mM NaCl, HA (500 veya 1000 mg L-1) ve B. wiedmannii’nin farklı kombinasyonlarını içeren turba-perlit karışımında ekildi. 35 gün sonra, bağıl su içeriği (RWC), reaktif oksijen türleri (H2O2, O.-), MDA (malondialdehit), antioksidan enzim aktiviteleri ve fotosentetik pigment düzeyleri ölçülmüştür. Tuz stresi, tek başına uygulandığında RWC, fotosentetik pigment miktarı ve antioksidan enzim aktivitelerinde azalmaya, H2O2, O.-, ve MDA birikiminde ise artışa neden olmuştur. HA veya B. wiedmannii tek başına bu olumsuz etkileri kısmen hafifletmiştir. Ancak, özellikle 1000 mg L-1 HA+B. wiedmannii+NaCl kombinasyonu, RWC’yi artırmış ve ROS ile MDA seviyelerini kontrol grubuna yakın düzeylere düşürmüştür. Ayrıca, bu kombine uygulama antioksidan enzim aktiviteleri ile fotosentetik pigment içeriklerini, sadece tuz uygulamasına kıyasla artırmıştır. Bu sonuçlar, HA ile B. wiedmannii arasındaki sinerjik etkinin, su dengesini iyileştirerek, oksidatif zararı azaltarak ve antioksidan savunma sistemlerini aktive ederek tuz stresini hafiflettiğini göstermektedir. Bu nedenle HA ve B. wiedmannii’nin birlikte uygulanması, yem bitkilerinde tuz toleransını artırmak için umut verici bir strateji olarak değerlendirilebilir.

Keywords

• Tuz stresi
• Yonca
• Humik asit
• Endofit
• Antioksidan
• Liken

Combined Humic Acid and Lichen-Derived Endophytic Bacteria Application Alleviates Salt Stress-Induced Oxidative Damage in Alfalfa (Medicago sativa L.)

Abstract

This study investigates the synergistic effects of leonardite-sourced humic acid (HA) and a lichen-derived endophytic bacterium (Bacillus wiedmannii) in mitigating salt stress in alfalfa (Medicago sativa L., cv. Konya). Alfalfa seeds were grown in a peat-perlite mix with different combinations of 100 mM NaCl, HA (500 or 1000 mg L-1), and B. wiedmannii. After 35 days, relative water content (RWC), ROS (H2O2, O₂.-), MDA, antioxidant enzyme activities, and photosynthetic pigments were measured. The salt stress alone reduced RWC, the photosynthetic pigment amount, and the antioxidant enzyme activities, while elevating H2O2 and O2.-, and MDA accumulation. HA or B. wiedmannii (Bw) individually partially ameliorated these effects, however, the combined HA+Bw treatment under saline conditions (especially 1000 mg L⁻¹ HA+Bw+NaCl) elevated RWC, and reduced ROS and MDA levels close to the control values. In addition, the antioxidant enzyme activities and the photosynthetic pigment contents were enhanced under the combined treatments compared to the salt alone. These results demonstrate a synergistic effect between HA and B. wiedmannii in alleviating salt stress in the alfalfa by enhancing water status, reducing oxidative damage, and activating antioxidant defenses, suggesting that the co‐application of HA and B. wiedmannii is a promising strategy for improving salt tolerance in forage crops.

Keywords

• Salt stress
• Alfalfa
• Humic acid
• Endophyte
• Antioxidant
• Lichen

References

1. Stavi İ, Thevs N, Priori S. Soil salinity and sodicity in drylands: A review of causes, effects, monitoring, and restoration measures. Front Environ Sci. 2021;9:712831.
2. Parida AK, Das AB. Salt tolerance and salinity effects on plants: a review. Ecotoxicol Environ Saf. 2005;60(3):324-349.
3. Hernández JA. Salinity tolerance in plants: trends and perspectives. Int J Mol Sci. 2019;20:2408.
4. Yılmaz E, Tuna AL, Bürün B. Bitkilerin tuz stresi etkilerine karşı geliştirdikleri tolerans stratejileri. Celal Bayar Univ J Sci. 2011;7(1):47-66.
5. Hasanuzzaman M, Raihan MRH, Masud AAC, Rahman K, Nowroz F, Rahman M, Fujita M. Regulation of reactive oxygen species and antioxidant defense in plants under salinity. Int J Mol Sci. 2021;22(17):9326.
6. Akıncı Ş. Hümik asitler, bitki büyümesi ve besleyici alımı. Marmara Univ Fen Bilimleri Dergisi. 2011;23(1):46-56.
7. Canellas LP, Teixeira Junior LRL, Dobbss LB, Silva CA, Medici LO, Zandonadi DB, Façanha AR. Humic acids cross-interactions with root and organic acids. Ann Appl Biol. 2008;153(2):157-166.
8. Abbas G, Rehman S, Siddiqui MH, Ali HM, Farooq MA, Chen Y. Potassium and humic acid synergistically increase salt tolerance and nutrient uptake in contrasting wheat genotypes through ionic homeostasis and activation of antioxidant enzymes. Plants. 2022;11(3):263.

9. Abu-Ria M, Shukry W, Abo-Hamed S, Albaqami M, Almuqadam L, Ibraheem F. Humic acid modulates ionic homeostasis, osmolytes content, and antioxidant defense to improve salt tolerance in rice. Plants. 2023;12(9):1834.
10. Boydak E, Enes B, Demirkıran AR. Effect of leonardite material doses on the elimination of salt stress in groundnut. Türk Doğa ve Fen Dergisi. 2025;14(1):1-8.
11. Tiwari J, Ramanathan AL, Bauddh K, Korstad J. Humic substances: Structure, function and benefits for agroecosystems-A review. Pedosphere. 2023;33(2):237-249.
12. Gülmez O, Tiryaki D, Atıcı Ö, Baris O. Boron-resistant Alternaria alternata (OG14) mitigates boron stress by improving physiological and antioxidative response in wheat (Triticum aestivum L.). Plant Physiol Biochem. 2023;202:107911.
13. Li B, Mamuti R, Xiao L, Qian B, Wang Y, Wei X. The adaptation of lichen symbiosis to desert saline-alkali stress depends more on their symbiotic algae. Physiol Plantarum. 2024;176(5):e14510.
14. Patil PS, Gore NS. Botanical symbiosis unveiled: a review of mycorrhizas, lichen, and plant consorts. Bot Rev. 2025;1-24.
15. Bianchi E, Benesperi R, Colzi I, Coppi A, Lazzaro L, Paoli L, Gonnelli C. The multi-purpose role of hairiness in the lichens of coastal environments: Insights from Seirophora villosa (Ach.) Frödén. Plant Physiol Biochem. 2019;141:398-406.
16. Ma Y, Freitas H, Dias MC. Strategies and prospects for biostimulants to alleviate abiotic stress in plants. Front Plant Sci. 2022;13:1024243.
17. Emilia DA, Luisa DA, Stefania DP, Petronia C. Use of Biostimulants to Improve Salinity Tolerance in Agronomic Crops. In: Hasanuzzaman, M. (eds) Agronomic Crops. Springer, Singapore. 2020;423-441.
18. Wu S, Lu YC, McMurtrey JE, Weesies G, Devine TE, Foster GR. Soil conservation benefits of large biomass soybean (LBS) for increasing crop residue cover. J Sustain Agric. 2004;24(1):107–128.
19. Brahmbhatt NH, Kalasariya HS. Effect of algae on seedling growth of “Queen of Forages.” Int J Eng Res Gen Sci. 2015;3:827-833.
20. Özkurt M, Saygılı İ, Dirik KÖ. Bazı yonca çeşitlerinin erken gelişme dönemindeki kuraklık toleransının belirlenmesi. KSÜ Tar Doga Derg. 2019;22:557-562.
21. Yamamoto Y, Kinoshita Y, Thor GR, Hasumi M, Kinoshita K, Koyama K, et al. Isofuranonaphthoquinone derivatives from cultures of the lichen Arthonia cinnabarina (DC.) Wallr. Phytochemistry. 2002;60(7):741-745.
22. Witham FH, Blaydes BF, Devlin RM. Experiments in plant physiology. Van Nostrand Reinhold Company; 1971. p. 167-200.
23. Hodges DM, DeLong JM, Forney CF, Prange RK. Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta. 1999;207:604-611.
24. Atıcı Ö, Aydın İ, Karakuş S, Tiryaki D. Inoculating maize (Zea mays L.) seeds with halotolerant rhizobacteria from wild halophytes improves physiological and biochemical responses of seedlings to salt stress. Biologia Futura. 2025;1-16.
25. Elstner EF, Heupel A. Inhibition of nitrite formation from hydroxylammonium chloride: a simple assay for superoxide dismutase. Anal Biochem. 1976;70:616-620.
26. Esim N, Atıcı Ö. Nitric oxide alleviates boron toxicity by reducing oxidative damage and growth inhibition in maize seedlings (Zea mays L.). Aust J Crop Sci. 2013;7(8):1085-1092.
27. Agarwal S, Pandey V. Antioxidant enzyme responses to NaCl stress in Cassia angustifolia. Biologia Plantarum. 2004;48(4):555-560.
28. Nakano Y, Asada Y. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 1981;22:867-880.
29. Foyer CH, Halliwell B. The presence of glutathione and glutathione reductase in chloroplasts: a proposed role in ascorbic acid metabolism. Planta. 1976;133:21-25.
30. İlay R, Aktaş M, Aslantekin NB, Özcan H. Farklı kaynaklardan elde edilen organik materyalin kumlu tın bünyeli toprağın bazı özellikleri üzerine etkileri. Toprak Bilimi ve Bitki Besleme Dergisi. 2021;9(1):30-38.
31. Pekcan T, Esetlili BÇ, Turan HS, Aydoğdu E. Leonardit kökenli organik materyallerin bazı fiziksel ve kimyasal özelliklerinin belirlenmesi. Uludağ Üniversitesi Ziraat Fakültesi Dergisi. 2018;32:31-41.
32. Tahir MM, Khurshid M, Khan MZ, Abbasi MK, Kazmi M. H. Lignite-derived humic acid effect on growth of wheat plants in different soils. Pedosphere 2011;21;124-131.
33. Sharif M, Khattak RA, Sarir MS. 2002). Effect of different levels of lignitic coal derived humic acid on growth of maize plants. Commun Soil Sci Plant Anal. 2002;33:3567-3580.
34. Gupta B, Huang B. Mechanism of salinity tolerance in plants: physiological, biochemical, and molecular characterization. Int J Genom. 2014:701596.
35. Egamberdieva D, Wirth SJ, Alqarawi AA, Abd_Allah EF, Hashem A. Phytohormones and beneficial microbes: essential components for plants to balance stress and fitness. Front Microbiol. 2017;8:2104.
36. Gill SS, Tuteja N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem. 2010;48(12):909-930.
37. Younesi O, Moradi A. Effect of priming of seeds of Medicago sativa “Bami” with gibberellic acid on germination, seedling growth and antioxidant enzymes activity under salinity stress. J Hortic Res. 2014:22(2).
38. Ali AYA, Ibrahim MEH, Zhou G, Nimir NEA, Jiao X, Zhu G, Yue W. Exogenous jasmonic acid and humic acid increased salinity tolerance of sorghum. Agronomy Journal. 2020;112(2): 871-884.
39. Ali Q, Ayaz M, Mu G, Hussain A, Yuanyuan Q, Yu C, Gao X. Revealing plant growth-promoting mechanisms of Bacillus strains in elevating rice growth and its interaction with salt stress. Frontiers in Plant Science. 2022;13: 994902.
40. Patani A, Prajapati D, Ali D, Kalasariya H, Yadav VK, Tank J. Evaluation of the growth-inducing efficacy of various Bacillus species on the salt-stressed tomato (Lycopersicon esculentum Mill.). Front Plant Sci. 2023;14:1168155.
41. Siddika A, Rashid AA, Khan SN, Khatun A, Karim MM, Prasad PV, Hasanuzzaman M. Harnessing plant growth-promoting rhizobacteria, Bacillus subtilis and B. aryabhattai to combat salt stress in rice: a study on the regulation of antioxidant defense, ion homeostasis, and photosynthetic parameters. Front Plant Sci. 2024;15:1419764.
42. Saeed SWZ, Naseer I, Zahir ZA, Hilger T, Shahid S, Iqbal Z, Ahmad M. Bacillus strains with catalase enzyme improve the physiology and growth of rice (Oryza sativa L.). Stresses. 2023;3(4):736-748.
43. da Silva MSRDA, dos Santos BDMS, da Silva CSRDA, da Silva CSRDA, Antunes LFDS, dos Santos RM, Rigobelo EC. Humic substances in combination with plant growth-promoting bacteria as an alternative for sustainable agriculture. Front Microbiol. 2021;12:719653.
44. Yıldız M, Terzi H, Akçalı N. Tuz stresi altındaki bitkilerin metabolik yollarındaki proteom değişimleri. Bitlis Eren Univ Fen Bilimleri Dergisi. 2014;3(1):81-93.
45. Hu LongXing HL, Li HuiYing LH, Pang HuangCheng PH, Fu JinMin FJ. Responses of antioxidant gene, protein and enzymes to salinity stress in two genotypes of perennial ryegrass (Lolium perenne) differing in salt tolerance. J Plant Physiol Biochem. 2012; 146-156.
46. Jabborova D, Zhang Y, Alhewairini SS, Jabbarov Z, Barasarathi J, Abdrakhmanov T, Saharan BS. Enhancing growth and physiological traits in alfalfa by alleviating salt stress through biochar, hydrogel, and biofertilizer application. Front Microbiol. 2025;16:1560762.
47. Sofi A, Ebrahimi M, Shirmohammadi E. Effect of humic acid on germination, growth, and photosynthetic pigments of Medicago sativa L. under salt stress. Ecopersia, 2018; 6(1):21-30
48. Han L, Zhang M, Du L, Zhang L, Li B. Effects of Bacillus amyloliquefaciens QST713 on photosynthesis and antioxidant characteristics of alfalfa (Medicago sativa L.) under drought stress. Agronomy. 2022;12(9):2177.
49. Ansari M, Shekari F, Mohammadi MH, Juhos K, Végvári G, Biró B. Salt-tolerant plant growth-promoting bacteria enhanced salinity tolerance of salt-tolerant alfalfa (Medicago sativa L.) cultivars at high salinity. Acta Physiol Plant. 2019;41:1-13.
50. Khan MA, Hamayun M, Asaf S, Khan M, Yun BW, Kang SM, Lee IJ. Rhizospheric Bacillus spp. rescues plant growth under salinity stress via regulating gene expression, endogenous hormones, and antioxidant system of Oryza sativa L. Front Plant Sci. 2021;12:665590.
51. Seven S, Akinci Ş. Effects of humic acid and salt stress on growth, physiological parameters and mineral substance uptake in Artemisia dracunculus L. (Tarragon). Turk J Bot. 2025;49(2), 64-79.
52. Bhagat N, Raghav M, Dubey S, Bedi N. Bacterial exopolysaccharides: Insight into their role in plant abiotic stress tolerance. J Microbiol Biotech. 2021;31:1045.