The Improvement of Morphological, Biochemical and Antioxidant Characterizes in Mexican lime Seedlings, Inoculated by Fungal Endophyte Extracted from Green Seaweed

Document Type : Research Article

Authors

• Leila Baghazadeh Daryaii ¹
• Davood Samsampoor ¹
• Abdolnabi Bagheri ²
• Jelveh Sohrabipour ³

¹ Agriculture and Natural Resources College, University of Hormozgan, Bandar Abbas, Iran

² Plant Protection Research Department, Hormozgan Agricultural and Natural Resources Research and Education Center, Bandar Abbas, Iran

³ Department of Natural Resources Researches, Agricultural and Natural Resources Research and Education Center, Bandar Abbas, Iran

https://doi.org/10.22067/jhs.2021.69603.1039

Abstract

   Introduction
 Fungal Endophytes have symbiosis life within the plant tissues without causing any obvious negative effects. Seaweeds are one of the large and diverse groups of marine plants that play an essential role in marine and oceans ecosystems. Seaweeds show rich diversity of associated microorganisms compare with the other multicellular organisms. Citrus species, are amongst the most important evergreen fruit trees, cultivated in many countries worldwide. There are several obstacles for citrus production in southern of Iran that limiting continuity of citrus production. Lack of suitable soil, is one of the main challenges threatening citrus industry in southern of Iran. Similar to other citrus species, the production of Mexican lime is threatened by certain biological stresses (such as pests, plant diseases and weeds) and non-biological stresses (such as salinity, drought, floods, cold and heat stress). Here, we have evaluated the potential of inoculating Mexican lime seedlings with seaweeds fungi endophyte, Aspergillus niger, to improve morphological, biochemical, antioxidant and photosynthesis pigments characterizes. Endophytes are advantageous group of microorganisms that protect plants from biotic and abiotic stresses. One of the alternative ways to restore normal plant growth may be to use plant growth to stimulate endophytes. Endophytes can play an important role in plant growth. Endophytes from marine environment are gaining special interest because of their existence in the harsh conditions of marines and ocean ecosystem such as temperature, light availability, high salinity and osmotic stress. Fungi have already been isolated from various marine habitat, including marine plants, marine invertebrates and vertebrates. Among these organisms, seaweeds are one of the most prevalent sources of marine-derived fungi for chemical studies. The purpose of this study was the isolation of associated fungi with seaweed species in Persian Gulf to investigate morphological and molecular characterization by using PCR amplifications ITS1-5.8S-ITS4 regions and primitive assessment of their potential as bio-fertilizer.
Materials and Methods
 The main aim of this study was investigation the role of endophytic fungi (Aspergillus niger), in improving the growth of Mexican lime seedlings. Cladophoropsis membranacea, green seaweed, was collected from coastal region of Bushehr province. Fungal endophytes were isolated and identified based on morphological and molecular methods. Molecular characteristic was investigated using PCR amplification of ITS1-5.8S-ITS4 regions. Mexican lime seeds were sterilized with 0.5% sodium hypochlorite for 20 minutes and then completely distilled three times with distilled water. Seedlings pots containing autoclaved soil were placed in the greenhouse of the Faculty of Agriculture, Hormozan University. Isolated fungi by MT420720 accession number was used as bio-fertilizing agents in eight months old Mexican lime seedlings. The suspension was adjusted to a concentration of 1×10⁶ cell per ml. For better contact of seedlings with fungi, inoculation was performed three times. After three months, morphological (trunk diameter, stem length, root length and width, leaf and branch number, leaf, stem and root dry and fresh weight), biochemical (Protein, MDA and SPAD), antioxidant (CAT, POD, SOD, APX and Gr enzymes activity) and photosynthesis pigments (Chlorophyll a, Chlorophyll b, Total Chlorophyll and Carotenoids) characterizes in treated Mexican lime seedlings were analyzed. The experiment was arranged in randomized complete design with three replications. Analysis of variance of traits was performed using SAS software version 9.4 and the means were compared using LSD method with a probability level of P≤0.05.
Results and Discussion
 The genera of Aspergillus was the most frequent isolates of the isolated fungi. The results show that most traits were significant compared with control. For example, leaf number (144.42%), root fresh weight (144.13%), stem fresh weight (94.85%) and root width (105.55%) were significantly higher compared with control (P>0.001). Fungal inoculation can significantly improve the photosynthesis pigments such as chlorophyll a (10.98%) and carotenoids (40.62%) (P>0.001) compared with control. In antioxidant capacity of seedling, CAT, POD, SOD, Gr and APX enzymes were analyzed. Fungal inoculation can increase the enzymes activity. For biochemical traits, fungal inoculation can significantly increase SPAD number and decrease MDA in inoculated seedlings compare with control (P>0.001). 
Conclusion
 The results showed that the use of entophytic fungi increased the growth of Mexican lime seedlings. Thereby it can be used as an effective tool for growing salinity-sensitive plants such as Mexican lime in saline conditions.

Keywords

• Abiotic stress
• Fungal entophytes
• Photosynthesis pigments
• Seaweeds

20.1001.1.20084730.1401.36.2.6.5

Main Subjects

• Breeding and Biotechnology of Plant and Flower

References

1- Alscher R.G., Erturk N., Lenwood S., and Heath L.S. 2002. Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. Journal of Experimental Botany 53(372): 1331-1341.

2- Arnold A.E., and Lutsuzaki F. 2013. Diversity and Host Range of Fungal Endophytes: are rtopical leaves. Biodiversity hotspots Ecology 88: 541-549.

3- Arnon D.I. 1949. Copper enzymes in isolated chloroplast: polyphenoxidase in Beta vulgaris. Plant physiology 24: 1-15.

4- Asada K. 1994. Production and action of active oxygen species in photosynthetic tissues. In: Foyer C. & Mullineaux P.M. (eds), Causes of photooxidative stress and amelioration of defense systems in plants. CRC Press, Boca Raton, London 77- 100.

5- Auge R.M., Stodola A.J., Tims J.E., and Saxton A.M. 2001. Moisture retention properties of a mycorrhizal soil. Plant and Soil 230(1): 87-97.

6- Bacon C.W., and Hinton D.M. 2007. Bacterial endophytes: the endophytic niche, its occupants, and its utility. 155-194. In: Plant-associated bacteria. In: Gnanamanickam SS. Ed. Springer: USA.

7- Beauchamp C., and Fridovich I. 1971. Analytical Biochemistry 44(1): 276-287.

8- Bradfordm M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry.       

9- Bu N., Li, X., Li, Y., Ma, C., Ma L., and Zhang C. 2012. Effects of Na₂CO₃ stress on photosynthesis and antioxidative enzymes in endophyte infected and non-infected rice. Ecotoxicology Environmental Safety 78: 35-40. https://doi.org/10.1016/j.ecoenv.2011.11.007.

10- Bugni T.S., and Ireland C.M. 2004. Marine-drived fungi: a chemically and biologically drived group of microorganisms. Natural product Reports 21: 143-163. https://doi.org/10.1039/B301926H.

11- Chance B., and Maehly A.C. 1955. Assay of catalases and peroxidases 764–775.

In: Colowick S.P., and Kaplan N.O. (Eds.) Methods in Enzymology. Academic Press. New York. https://doi.org/10.1016/0003-9861(77)90165-5.

12- Dhindsa R.S., Plump-Dhinsa P., and Thorpe T.A. 1981. Leaf senescence: correlated with increased levels of membrane permeability and lipid peroxidation, and decreased levels of superoxide dismutase and catalase. Journal of Experiment Botany 32: 93–101. https://doi.org/10.1016/j.jarmap.2017.06.006.

13- Driscoll C.T., Whitall, D., Aber J., Boyer E., Castro, M., Cronan, C., Goodale C.L. Groffman P., Hopkinson C., and Lambert K. 2003. Nitrogen Pollution in the Northeastern United States: Sources, Effects, and Management Options. Bioscience 53: 357. https://doi.org/10.1641/0006-3568(2003)053[0357:NPITNU]2.0.CO;2.

14- Ennab H.A. 2016. Effect of humic acid on growth and productivity of Egyptian lime trees (Citrus aurantifolia swingle) under salt stress conditions. Journal of Agriculture Research Kafr El-Sheikh Universisty 42(4): 494-505.

15- Flewelling A.J., Currie, J., Gray C.A., and Johnson J.A. 2015. Endophytes from marine macroalgae:  promising sources of novel natural products. Current Science 109(1): 88-111.

16- Gerwick W.H., and Moore B.S. 2012. Lessons from the past and charting the future of marine natural products drug discovery and chemical biology. Chemistry and Biology 19: 85-98. https://doi.org/10.1016/j.chembiol.2011.12.014.

17- Gill S.S., Anjum, N.A., Hasanuzzaman M., Gill, R., Trivedi, D.K., Ahmad, I., Pereira E., and Tuteja N. 2013. Glutathione and glutathione reductase: a boon in disguise for plant abiotic stress defense operations. Plant Physiology and Biochemistry 1(70): 204–212. http://dx.doi.org/10.1016/j.plaphy.2013.05.032.

18- Golparyan F., Azizi A., and Soltani J. 2018. Endophytes of Lippia citriodora (Syn. Aloysia triphylla) enhance its growth and antioxidant activity. European Journal of Plant Pathology152: 759–768. https://doi.org/10.1007/s10658-018-1520-x.

19- Halo B.A., Khan A.L., Waqas M., Al-Harrasi A., Hussain J., Ali L., Adnan M., and Lee I.J. 2015. Endophytic bacteria (Sphingomonas sp. LK11) and gibberellin can improve Solanum lycopersicum growth and oxidative stress under salinity. Journal of Plant Interactions 10(1): 117–125. https://doi.org/10.1080/17429145.2015.1033659.

20- Jogawat A., Saha S., Bakshi M., Dayaman V., Kumar M., Dua M., Varma A., Oelmüller R., Tuteja N., and Johri A.K. 2013. Piriformospora indica rescues growth diminution of rice seedlings during high salt stress. Plant signaling and behavior 8(10): e26891. https://doi.org/10.4161/psb.26891.

21- Kaaria P., Wakibia J., and Matiru V. 2015. Antimicrobial Screening of Marin Endophytes and Epiphytes Isolated from Marin Algae of Kenyan Indian Ocean. Journal of Applied and Environmental Microbiology 3(3): 70-74. https://doi.org/10.12691/jaem-3-3-2.

22- Kjer J., Debbab A., Aly A.H., and Proksch P. 2010. Methods for isolation of marine derived endophyticfungy and their bioactive secondary products. Nature Protocols 5: 479-490. https://doi.org/10.1038/nprot.2009.233.

23- Krings M., Taylor T.N., Hass H., Kerp H., Dotzler N., and Hermsen E.J. 2007. Fungal endophytes in a 400-million-year-old land plant: infection pathways, spatial diseribution, and host responses. New Phytology 174(3): 648-57. https://doi.org/10.1111/j.1469-8137.2007.02008.x.

24- Lee K.E., and Pankhurst C.E. 1992. Soil organisms and sustainable productivity. Australian Journal of Soil Research 30: 85592. https://doi.org/10.1071/SR9920855.

25- Mayer A.M., Rodrigguez A.D., Berlinck R.G., and Fusetani N. 2011. Marine pharmacology in 2007-8: marine compounds with antibacterial, anticoagulant, antifungal, anti-inflammatory, antimalarial, antiprotozoal, antituberculosis and antiviral activities; affecting the immune and nervous system and other miscellaneous mechanisms of action, Comparative biochemistry and physiology. Toxicology and Pharmacology 153: 191-222. https://doi.org/10.1016/j.cbpc.2010.08.008.

26- Mohammadi K. 2010. Ecophysiological response of canola (Brassica napus L.) to different fertility systems in crop rotation. Ph.D thesis. Agronomy Department. Tarbiat Modares University, Tehran, Iran. 354.

27- Mohammadi K., and Sohrabi Y. 2012. Bacterial biofertilizers for sustainable crop production: a review, ARPN. Journal of Agriculture and Biolology Science 7(5): 307-316

28- Molinski T.F., Dalisay D.S., Lievens S.L., and Saludes J.P. 2009. Drug development from marine natural products. Nature Reviews Drug Discovery 8: 69-85. https://doi.org/10.1038/nrd2487.

29- Morsy M., Cleckler B., and Armuelles-Millican H., 2020. Fungal Endophytes Promote Tomato Growth and Enhance Drought and Salt Tolerance. Plants 9(7): 877. https://doi.org/10.3390/plants9070877.

30- Morton J. 2013. Mexican Lime. 168-172. In: Julia F. Morton. Fruits of Warm Climates. Miami, FL.

31- Nakano Y., and Asada K. 1981. Hydrogen peroxide is scavenged by ascorbate specific peroxidase in spinach chloroplasts. Plant Cell Physiology 22: 867–880. https://doi.org/10.1093/oxfordjournals.pcp.a076232.

32- Nanda S., Mohanty B., and Joshi R.K. 2018. Endophyte-mediated host stress tolerance as a means for crop improvement. Endophyte Secondery Metabolits 2: 1-25.

33- Naveed M., Mitter B., Reichenauer T.G., Wieczorek K., and Sessitsch A. 2014. Increased drought stress resilience of maize through endophytic colonization by Burkholderia phytofirmans PsJN and Enterobacter sp. FD17, Environment. Experment Botany 97: 30–39. http://dx.doi.org/10.1016/j.envexpbot.2013.09.014.

34- Newman J.A., Abner M.L., Dado R.G., Gibson D.J., Brookings A., and Parsons A.J. 2003. Effects of elevated CO₂, nitrogen and fungal endophyte-infection on tall fescue: growth, photosynthesis, chemical composition and digestibility. Global Change Biology 9: 425–437. https://doi.org/10.1046/j.1365-2486.2003.00601.x.

35- Newman D.J., and Cragg G.M. 2014. Marine-sourced anti-cancer and cancer pain control agents in clinical and late preclinical development. Marine Drugs 12: 255-278. https://doi.org/10.3390/md12010255.

36- Noorjahan A., Aiyamperumal B., and Anantharaman P. 2019. Isolation and Charecterisation of Seaweed Endophytic Fungi as an Efficient Phosphate Solubiizers. Bioscience and Biotechnology Research. ASIA 16(1): 33-39. http://dx.doi.org/10.13005/bbra/2718.

37- Peng Z., Xin L., and Bin-Gui W. 2016. Secondary Metabolites from the Marine Algal-Derived Endophytic Fungi. Chemical Diversity and Biological Activity 82(09/10): 832–842. https://doi.org/10.1055/s-0042-103496.

38- Quan L.J., Zhang B., Shi W., and Li H.Y. 2008. Hydrogen peroxide in plants: a versatile molecule of the reactive oxygen species network. Journal of Integrative Plant Biology 50: 2-18. https://doi.org/10.1111/j.1744-7909.2007.00599.x.

39- Rateb M.E., and Ebel R. 2011. Secondary metabolites of fungi form marine habitats. Natural Product Reports 28: 290-344.

40- Robarge W.P., Walker J.T., McCulloch R.B., and Murray G. 2002. Atmospheric concentrations of ammonia and ammonium at an agricultural site in the southeast United States. Atmospheric Environment 36: 1661-1674.

41- Rodrigues Costapinto L.S., Azevedo J.L., Pereira J.O., Carneiro Viera M.L., and Labate C.A. 2000. Symptomless infection of banana and maize by endophytic fungi impairs photosynthetic efficiency. New Phytology 147: 609-615.

42- Rokhzadi A., Asgharzadeh A., Darvish F., Nourmohammadi G., and Majidi E. 2008. Influence of plant growth-promoting rhizobacteria on dry matter accumulation and yield of chickpea (Cicer arietinum L.) under field condition. American-Eurasian Journal of Agricultural and Environmental Sciences 3(2): 253-257.

43-Sadeghi F., Samsampour D., Seyahooei M.A., Bagheri A., and Soltani, J., 2020. Fungal endophytes alleviate drought-induced oxidative stress in mandarin (Citrus reticulata L.): Toward regulating the ascorbate–glutathione cycle. Scientia Horticulturae 261: 1089-1091. https://doi.org/10.1016/j.scienta.2019.108991.

44- Selosse M.A., and Strullu-Derrien C. 2015. Origins of the terrestrial flora: A symbiosis with fungi? EDP sciences 4, 00009. https://doi.org/1051/bioconf/ 20150400009.

45- Schulz B., and Boyle C. 2006. What are endophytes? 1–14. In Microbial Root Endophytes; Sieber, T., Eds.; Springer: Berlin/Heidelberg, Germany.

46- Shukla N., Awasthi R.P., Rawat L., and Kumar J. 2012. Biochemical and physiological responses of rice (Oryza sativa L.) as influenced by Trichoderma harzianum under drought stress. Plant Physiology and Biochemistry 54: 78–88.

47- Smith I.K., Vierheller T.L., and Thorne C.A. 1988. Assay of glutathione-reductase in crude tissue-homogenates using 5, 5′-dithiobis (2-nitrobenzoic acid). Analytical Biochemistry 175: 408–413.

48- Sohrabipour J., and Rabiei R. 2007. The checklist of green algae of the Iranian coast lines of the Persian Gulf and Gulf of Oman. Iranian Journal of Botany 23(1): 146-149.

49- Sohrabipour J., and Rabiei R. 2008. Rhodophyta of Oman Gulf (South east of Iran). The Iranian journal of botany 14(1): 70-74.

50- Soltani J., ZaheriShoja M., Hamzei J., Hosseyni-Moghaddam M.S., and Pakvaz S. 2016. Diversity and bioactivity of endophytic bacterial community of Cupressaceae. Forest Pathology 46: 353–361. https://doi.org/10.1111/efp.12270.

51- Strobel G., and Daisy B. 2003. Bioprospecting for microbial endophytes and their natural products, Microbiology and Molecular Biology Reviews 67(4): 491-502. https://doi.org/10.1128/MMBR.67.4.491–502.2003.

52- Strobel G., Daisy B., Castillo U., and Harper J. 2004. Natural products from endophytic microorganism. Journal of Natural Product 67: 257-268.

53- Tan R.X., and Zou W.X. 2001. Endophytes: a rich source of functional metabolites. Natural Products Reports 18: 448–459.

54- Venkatachalam A., Guvinda R.M.B., Thirunavukkarasu N., and Suryanarayanan T.S. 2015. Endophytic fungi of marine algae and seagrasses: a novel source of chitin modifying enzymes. Mycosphere 6(3): 345-355. https://doi.org/10.5943/mycosphere/6/3/10.

55- Wani S.P., Rego T.G., Rajeshwari S., and Lee K.K. 1995. Effect of legume–based cropping systems on nitrogen mineralization potential of Vertisol. Plant Soil 175(2): 265-274.

56- White T.J., Bruns T., Lee S., and Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. 315–322. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic Press, San Diego.

57- White J.F.J., Morrow A.C., Morgan-Jones G., and Chambless D.A. 1991. Endophyte-host associations in forage grasses. IV. Primary stromata formation and seed transmission in Epichloë typhina: developmental and regulatory aspects. Mycologia 83: 72-81. https://doi.org/10.1080/00275514.1991.12025979.

58- Zhang Y.P., and Nan Z.B. 2007. Growth and anti-oxidative systems change in elymus dahuricus is affected by neotyphodium endophyte under contrasting water availability. Journal of Agronomy and Crop Science 193: 377–386. https://doi.org/10.1111/j.1439-037X.2007.00279.x.

59- Zhang X., Li C., and Nan Z. 2010. Effects of cadmium stress on growth and antioxidative systems in achnatherum inebrians symbiotic with neotyphodium gansuense. Journal of Hazardous Materials 175: 703–709. https://doi.org/10.1016/j.jhazmat.2009.10.066.

60- Zhanyuan D., and William J. 1992. Modified Thiobarbituric acid assey for measuring lipid oxidative in sugar rich plant tissue extracts. Journal of Agricultural and Food Chemistry 40(9): 1566-1570. https://doi.org/10.1021/jf00021a018.