بررسی کلنیزاسیون دوگانه Rhizophagus irregularis و Serendipita indica در جو در سطوح مختلف فسفر با به‌کارگیری آنتی‌بادی منوکلونال

نوع مقاله : مقالات پژوهشی

نویسندگان

• وحیده دین محمدی ¹
• ناصر علی اصغرزاد ²
• لیلی عاقبتی ملکی ³

¹ گروه علوم و مهندسی خاک، دانشکده کشاورزی، دانشگاه تبریز، تبریز، ایران

² گروه علوم و مهندسی خاک، دانشکده کشاورزی،استاد بیولوژی و بیوتکنولوژی خاک دانشگاه تبریز، تبریز، ایران

³ استادیار گروه ایمونولوژی، دانشکده پزشکی، مرکز تحقیقات ایمونولوژی، دانشگاه علوم پزشکی تبریز، تبریز، ایران

https://doi.org/10.22067/jsw.2023.79849.1230

چکیده

مطالعات اخیر نشان می‌دهد که اغلب گیاهان زراعی و باغی علاوه بر همزیستی با قارچ‌های میکوریز آربوسکولار می‌توانند همراه با قارچ‌ اندوفیت S. indica نیز ایجاد همزیستی کنند. مقدار بالای فسفر قابل‌جذب در خاک سبب محدودیت کلنیزاسیون قارچ‌های میکوریز آربوسکولار در ریشه می‌شود اما در خصوص تأثیر آن بر همزیستی قارچ اندوفیت اطلاعات کافی در دسترس نیست. در این آزمایش گیاه جو (Hordeum vulgare L.) با قارچ‌های R. irregularis (قارچ میکوریز آربوسکولار) و S. indica (قارچ اندوفیت) با سه سطح فسفر خاک (شامل صفر، 10، 20 میلی‌گرم فسفر بر کیلوگرم) از منبع سوپر فسفات تریپل تلقیح شدند. میزان گلومالین (گلیکوپروتئین) در ریشه‌ها و خاک با استفاده از آنتی‌بادی منوکلونال MAb32B11 اندازه‌گیری شد. نتایج نشان داد که در حالت تلقیح هر دو قارچ وزن تر و خشک بخش هوایی و ریشه نسبت به تلقیح انفرادی آن‌ها به‌طور معنی‌دار افزایش یافت. در سطح صفر فسفر، غلظت فسفر بخش هوایی و ریشه در تیمارهای دارای R. irregularis به‌طور معنی‌دار بیش‌تر از تیمار بدون قارچ بود. در تلقیح‌های انفرادی یا دوگانه، افزایش سطح فسفر تأثیر معنی‌داری بر غلظت فسفر بخش هوایی و ریشه نداشت. در تلقیح دوگانه قارچ‌ها، گرچه درصد کلنیزاسیون کل بیشتر بود اما سهم هریک از قارچ‌ها در مقایسه با تلقیح­های انفرادی کاهش یافت. با افزایش سطح فسفر درصد کلنیزاسیون در تلقیح­های منفرد و تلقیح دوگانه به‌طور معنی‌دار کاهش یافت. مقدار گلومالین خاک در تیمار R. irregularis زیاد بود اما تیمارهای شاهد بدون قارچ و تلقیح منفرد قارچ S. indica دارای مقدار اندکی گلومالین بودند. گلومالین ریشه‌ در حالت تلقیح دو قارچ (µg.g^-194/1006) کم‌تر از تیمار R. irregularis (µg.g^-149/1924) بود که نشان می‌دهد حضور قارچ S. indica در ریشه، کلنیزاسیون ریشه توسط قارچ R. irregularis را مهار می‌کند. هم‌چنین با افزایش سطح فسفر خاک، مقدار گلومالین ریشه به‌طور معنی‌دار کاهش یافت.

کلیدواژه‌ها

• تلقیح دوگانه
• قارچ اندوفیت
• قارچ میکوریز آربوسکولار
• منوکلونال آنتی‌بادی
• همزیستی همزمان

20.1001.1.20084757.1402.37.2.6.7

موضوعات

• علوم خاک

عنوان مقاله [English]

Study on Simultaneous Colonization of Rhizophagus irregularis and Serendipita indica in Barley under Different P Levels Using Monoclonal Antibody

نویسندگان [English]

• Vahideh Dinmohammadi ¹
• N. Aliasgharzad ²
• Leili Aghebati-Maleki ³

¹ Department of Soil Science and Engineering, Faculty of Agriculture, Tabriz University, Tabriz, Iran

² Department of Soil Science and Engineering, Faculty of Agriculture, Professor of Soil Biology & Biotechnology University of Tabriz, Tabriz, Iran

³ Assistant Professor of Immunology, Department of Immunology, Faculty of Medicine, Immunology research center, Tabriz University of Medical Sciences, Tabriz, Iran

چکیده [English]

Introduction
Recent studies show that most crops and horticultural plants can form symbiosis with the arbuscular mycorrhizal fungi (AMF) and the endophytic Serendepita indica, simultaneously. The endophytic fungus plays an important role in alleviating environmental stresses in plants. It has also been shown that excessive available phosphorus in soil limits the root colonization by arbuscular mycorrhizal fungi. No information is available on how soil phosphorus affects the establishment of endophytic fungus in root. Barley roots can be colonized by both mycorrhizal fungi and the endophytic fungus Serendipita indica. The objective of this study was to evaluate the effects of single or dual inoculation with Rhizophagus irregularis and Serendipita indica on barley roots under different phosphorus (P) levels. The researchers utilized a monoclonal antibody called MAb32B11 to assess the presence of glomalin, a signature molecule of arbuscular mycorrhizal (AM) fungi, in the roots. The glomalin content was quantified using the enzyme-linked immunosorbent assay (ELISA) method with the MAb32B11 antibody.
Materials and Methods
In this experiment, barley plants were inoculated with Rhizaphagus irregularis (AMF) and Serendepita indica (endophytic fungus) with three levels of phosphorus from triple super phosphate source. At the end of the vegetative growth period (about three months), the plants were harvested and phosphorus concentration in the plant were measured. A subsample from roots was stored in -20 ºC for determination of glomalin content. The glomalin content in the roots was analyzed using the monoclonal antibody MAb32B11. This antibody was employed to differentiate between the two fungi present in the roots and to quantify the abundance of arbuscular mycorrhizal fungi (AMF) specifically in plants treated with Rhizophagus irregularis. Additionally, the content of glomalin in the soil was determined at the end of the experiment using the same method as described above. The experiment was designed as a factorial completely randomized design (CRD) with three replications.
Results and Discussion
The results showed that the fresh and dry weights of shoot and root increased significantly in dual inoculation. At zero phosphorus level, shoot and root phosphorus concentrations were significantly higher in treatments with R. irregularis than in those without fungus (control). Under individual inoculation with R. irregularis, or S. indica as well as their dual inoculations, increasing level of phosphorus had no significant effect on shoot and root phosphorus concentration. In dual inoculation, the percentage of total colonization (88%) was significantly higher than that of individual inoculation treatment (68%) but the contribution of each fungus in root colonization under dual inoculation was significantly reduced as estimated by glomalin content of root and determination of total colonization. It was found that with increasing phosphorus level, total colonization percentage significantly decreased and the highest percentage of colonization (61%) was observed at zero level of phosphorus. By increasing phosphorus level, the percentage of root colonization was significantly decreased in individual inoculation by R. irregularis, or S. indica as well as dual inoculation. Results of glomalin assay in soil showed that the glomalin content was high in treatments of R. irregularis but control treatments without fungus and individual inoculation with S. indica had low glomalin. Antibody-reactive root glomalin was less in the dual inoculation treatment (1006.9 µg.g^-1) than in the R. irregularis treatment alone (1924.5 µg.g^-1) indicating that the presence of S. indica, in root inhibits, root colonization by R. irregularis. Moreover, the increasing of phosphorus level, significantly decreased root glomalin.
Conclusion
The increase of available phosphorus concentration in the soil caused to limit the expansion of the symbiosis of R. irregularis and S. indica, and this limitation was more for R. irregularis. In the case of dual inoculation with both Rhizophagus irregularis and Serendipita indica, the negative impact of phosphorus on colonization percentage was observed to be less compared to single inoculation. Although the percentage of colonization by each fungus decreased in the dual inoculation treatment compared to their individual inoculation, the overall colonization percentage increased significantly. It appears that in the dual inoculation scenario, while the total root colonization percentage increases, the presence of S. indica leads to a decrease in the colonization percentage specifically with R. irregularis. But in general, growth and nutrient absorption in the case of dual inoculation was better than the inoculation of each of them individually. It was also found that increasing the concentration of phosphorus in the soil caused a decrease in root colonization for both fungi, although the negative effect of phosphorus on the colonization of R. irregularis was more than that of S. indica. The measurement of glomalin in soil and root showed that the inhibitory effect of S. indica fungus on R. irregularis is less in soil than in root.
 

کلیدواژه‌ها [English]

• Endophytic fungus
• Monoclonal antibody
• Co-inoculation
• Simultaneous symbiosis
• Arbuscular mycorrhizal fungus

مراجع

1. Achatz, B., Von Ruden, S., Andrade, D., Neumann, E., Pons-Kuhnemann, J., Franken, P., Kogel, K.H., & Waller, F. (2010). Root colonization by Piriformospora indica enhances grain yield in barley under diverse nutrient regimes by accelerating early plant development. Plant and Soil, 333, 59-70. http://doi.org/10.1007/s11104-010-0319-0
2. Aliasgharzad, N. (1997). Soil Microbiology and Biochemistry (translation), First edition, Tabriz University Publications.
3. Aliasgharzad, N., Rastin, SN., Towfighi, H., & Alizadeh, A. (2001). Occurrence of arbuscular mycorrhizal fungi in saline soils of Tabriz Plain of Iran in relation to some physical and chemical properties of soil. Mycorrhiza, 11, 119-122. http://doi.org/10.1007/s005720100113
4. Cottenie, A. (1980). Soil and Plant Testing. FAO soils Bullention, 38/2: 94-100.
5. Deshmukh, S., & Kogel, K. (2007). Piriformospora indica protects barley from root disease caused by Fusarium. Journal of Plant Disease and Protection, 114(6), 236-268. http://doi.org/10.1007/BF03356227
6. Dolatabadi, H.K., Mohammadi Goltape, A., Moeini, A., & Verma, A. (2012). Evaluation of different investigations of auxin and fungus Piriformospora indica and Sebacina vermifera on peppermint (Mentha piperita) and thyme (Thymus vulgaris) in vitro. Quarterly Journal of Medicinal Plants, 2(9), 13-22.) In Persian (
7. Feddermann, N., Finaly, R., Boller, T., & Elfstrand, M. (2010). Functional diversity in arbuscular mycorrhiza-the role of gene expression, phosphorus nutrition and symbiotic efficiency. Fungal Ecology, 3, 1-8. http://doi.org/10.1016/j.funeco.2009.07.003
8. Ghabuli, M., Shahriari, F., Sepehari, M., Marashi, H., & Hosseini Salekdeh, A. (2011). The effect of the endophytic fungus Piriformospora indica on some characteristics of barley Hordeum vulgare under drought stress conditions, Journal of Agroecology, 3(3), 328-336.) In Persian(
9. Gholami, A., & Kochaki, A. (2010). Mycorrhiza in Sustainable Agriculture (translation), Shahrood University Publications.
10. Hallasgo, A.M., Spangl, B., Steinkellner, S., & Hage-Ahmad, K. (2020). The fungal endophyte Serendipita williamsii does not affect Phosphorus status but carbon and nitrogen dynamics in Arbuscular Mycorrhizal tomato plants. Journal of Fungi, 6(4), 233. http://doi.org/10.3390/jof6040233
11. Heidarianpour, MB., Aliasgharzad, N., & Olsson, PA. (2020). Positive effects of co-inoculation with Rhizophagus irregularis and Serendipita indica on tomato growth under saline conditions, and their individual colonization estimated by signature lipids. Mycorrhiza, 30(4), 455-466. http://doi.org/10.1007/s00572-020-00962-y
12. Jansa, J., Finlay, R., Wallander, H., Smith, F.A., & Smith, S.E. (2011). Role of mycorrhizal symbioses in phosphorus cycling. p.137-168. In :Phosphorus in action, vol 6. Springer, Berlin, Heidelberg.
13. Junker, A., Muraya, M.M., Weigelt-Fischer, K., Arana-Ceballos, F., Klukas, Ch., Melchinger, A.E., Meyer, Rh.C., Riewe, D., & Altmann, Th. (2015). Optimizing experimental procedures for quantitative evaluation of crop plant performance in high throughput phenotyping systems. Plant Science, 5, http://doi.org/ 10.3389/fpls.2014.00770
14. Koide, R.T., & Kabir, Z. (2000). Extraradical hyphae of the mycorrhizal fungus Glomus intraradices can hydrolyse organic phosphate. New Phytologists, 148(3), 511-517. http://doi.org/10.1046/j.1469-8137.2000.00776.x
15. Kormanic, P.P., & Graw, M.C. (1982). Methods and Principles of Mycorrhizal Research. Quantification of VA mycorrhizae in plant roots. p. 37-45. In: Schenck NC (Ed). Saint Paul Minnesota American, Phytopathological Society.
16. Kumari, R., Kishan, H., Bhoon, YK., & Varma, A. (2003). Colonization of cruciferous plants by Piriformospora indica. Current Science, 85, 1672-1674.
17. Malekzadeh, E., Majidi, J., Aliasgharzad, N., & Abdolalizadeh, J. (2016). The effect of lead on the glomalin content of hypha and root reactive with monoclonal antibody and Bradford in both in vitro and pot culture conditions. Journal of Water and Soil, 30(2), 605-618. (In Persian with English abstract). http://doi.org/10.22067/JSW.V30I2.47802
18. Najafi, N., Mostafaei, M., Dabagh Mohammadi Nasab, A., & Ostan, S. (2013). Effect of intercropping and farmyard manure on the growth, yield and protein concentration of corn, bean and bitter Vetch, Science of Agriculture and Sustainable Production, 23(1), 115-99. (In Persian with English abstract)
19. Neumann, E., & George, E. (2004). Colonization with the arbuscular mycorrhizal fungus Glomus mosseae (Nicol & Gred) enhanced phosphorus uptake from dry soil in Sorghum bicolor (L.). Plant and Soil, 261, 245-255. http://doi.org/10.1023/B:PLSO.0000035573.94425.60
20. Nichols, K.A. (2003). Characterization of glomalin, a glycoprotein produced by arbuscular mycorrhizal fungi.D Dissertation. p.285.University of Maryland, College Park, Mryland.
21. Nichols, K.A., & Wright, S.F. (2004). Contribution of fungi to soil organic matter in agroecosystems. p. 179-198. In: F Magdoff and RP Weil (eds). Soil Organic Matter in Sustainable Agriculture. CRC Press, Florida.
22. Norrif, IR., Read, D.J., & Varma, A.K. (1992). Methods in Microbiology Techniques for Study of Mycorrhiza. Academic press, London.
23. Olsson, PA., Rahm, J., & Aliasgharzad, N. (2010). Carbon dynamics in mycorrhizal symbioses is linked to carbon costs and phosphorus benefits. FEMS Microbiology Ecology, 72(1), 125–131. http://doi.org/10.1111/j.1574-6941.2009.00833.x
24. Rai, M., Acharya, D., Singh, A., & Varma, A. (2001). Positive growth responses of the medicinal plants Spilanthes calva and Withania somnifera to inoculation by Piriformospora indica in a field trial. Mycorrhiza, 11(3), 123–128. http://doi.org/10.1007/s005720100115
25. Rai, M., & Varma, A. (2005). Arbuscular mycorrhiza-like biotechnological potential of Piriformosporaindica, which promotes the growth of Adhatodavasica. Journal of Biotechnology, 8, 107-111.
26. Ratnayake, M., Leonard, R.T., & Menge, J.A. (1978). Root exudation in relation to supply of phosphorus and its possible relevance to mycorrhizal formation. New Phytologist, 81, 543-552.
27. Richardson, A.E., Barea, J.M., McNeil, A.M., & Prigent-Combaret, C. (2009). Acquisition of phosphorus and nitrotrogen in rhizosphere and plant growth promotion by microorganisms. Plant and Soil, 321, 305-339. http://doi.org/10.1007/s11104-009-9895-2
28. Rillig, M.C. (2004). Arbuscular mycorrhizal and terrestrial ecosystem processes. Ecology Letters, 7(8), 740-754. https://doi.org/10.1111/j.1461-0248.2004.00620.x
29. Rillig, MC., & Mummey, DL. (2006). Mycorrhizas and soil structure. New Phytologist, 171(1), 41-53. https://doi.org/10.1111/j.1469-8137.2006.01750.x
30. Rosier, C.L., Hoye, A.T., & Rillig, M.C. (2006). Glomalin –related soil protein: assessment of current detection and quantification tools. Soil Biology and Biochemistry, 38(8), 2205-2211. http://doi.org/10.1016/j.soilbio.2006.01.021
31. Sanders, F.E., & Tinker, P.B.H. (1973). Phosphate flow into mycorrhizal roots. Pesticide Science, 4, 385-395.
32. Schenck, N.C., & Perez, Y. (1988). Mannual for the Identification of VA Mycorrhizal Fungi. p. 241. IN:Vesicule Arbuscular Mycorrhizal, 1453 Fifield Hall, University of Florida, Gainesville, Florida, USA.
33. Turner, B.L., Lambers, H., Condron, L.M., Carmer, M.D., Leake, J.R., Richardson, A.E., & Smith, S.E. (2013). Soil microbial biomass anh the fate of phosphorus during long-term ecosystem development. Plant and Soil, 367, 225-234. http://doi.org/ 10.1007/s11104-012-1493-z
34. Verma, S., Varma, A., Rexer, KH., Hassel, A., Kost, G., Sarabhoy, A., Bisen, P., Butehorn, B., & Franken, P. (1998). Piriformospora indica, gen. et sp. Nov., a new root-colonizing fungus. Mycologia, 90(5), 896-903. http://doi.org/10.1080/00275514.1998.12026983
35. Vlot, AC., Dempsey, DA., & Klessig, DF. (2009). Salicylic acid a multifaceted hormone to combat disease. Annual Review of Phytopathology, 47, 177-206.  http://doi.org/10.1146/annurev.phyto.050908.135202
36. Waller, F., Achatz, B., Blatruschat, H., Fodor, J., Becker, K., Fischer, M., Heier, T., Huckelhoven, R., Neumann, C., Wettstein, D., Franken, P., & Kogel, KH.( 2005). The endophytic fungus Piriformospora indica reprograms barley to salt-stress tolerance, disease resistance, and higher yield. Proceeding of the National Academy of Sciences, 102(38), 13386-13391. http://doi.org/10.1073/pnas.0504423102
37. Westerm, L.Z. (1990). Soil Testing and Plant Analysis. Soil Science Society of America Journal, Inc. Madison, Wisconsin USA.
38. Williams, S.C.K., Vestberg, M., & Uosukainen, M. (1992). Effects of fertilizers and arbuscular mycorrhizal fungi on the post-vitro growth of micropropageted strawberry. Agronomie, 12(10), 851-857. http://doi.org/10.1051/agro:19921020
39. Wright, S.F., Franke-Synder, M., Morton, J.B., & Upadhyaya, A. (1996). Time-course study and partial characterization of a protein on hyphae of arbuscular mycorrhizal fungi during active colonization of roots. Plant and Soil, 181(2), 193-203. https://doi.org/10.1007/BF00012053
40. Wright, S.F., & Upadhyaya, A. (1999). Quantification of arbuscular mycorrhizal fungi activity by the glomalin concentration on hyphal traps. Mycorrhiza, 8, 283–285 . https://doi.org/10.1007/s005720050247
41. Zhu, Y.G., & Miller, R.M. (2003). Carbon cycling by arbuscular mycorrhizal fungi in soil-plant system. Trends in Plant Science, 8, 407-409. https://doi.org/10.1016/s1360-1385(03)00184-5