Iranian Journal of Forest

Iranian Journal of Forest

Assessing Drought Stress Tolerance in Persian Oak Seedlings (Quercus brantii Lindl) Inoculated with Summer Truffle Ectomycorrhizal Fungus (Tuber aestivumVittad)

Document Type : Research Paper

Authors
ِD. Sepahvand 1
V. Etemad 2
A. Shirvany 2
M. Javan-Nikkhah 3
T. DeLuca 4
1 Ph.D. candidate., Dept. of Forestry, Faculty of Natural Resources, University of Tehran, Karaj, I. R. Iran
2 Associate Prof., Dept. of Forestry, Faculty of Natural Resources, University of Tehran, Karaj, I. R. Iran
3 Prof., Dept. Mycology and Plant Pathology, Faculty of Agriculture, University of Tehran, Karaj, I. R. Iran
4 Prof., Dept. of Forestry, , University of Oregon State, Corvallis, USA
10.22034/ijf.2025.466658.1996
Abstract
Introduction: Ectomycorrhizal truffle fungi (Tuber spp.) can establish a strong symbiotic relationship with trees in the beech family (Fagaceae), which enhances the trees growth and resilience against both biotic and abiotic stresses. Moreover, truffle fungi are economically valuable. This study aimed to assess the ectomycorrhizal  colonization between Persian oak (Quercus brantii)  and the summer truffle fungus (Tuber aestivum), as well as to evaluate the drought tolerance of Persian oak seedlings by measuring their morpho-physiological parameters.
Materials and Methods: Summer truffle samples were collected from a planted area of Quercus castaneifolia C.A.Mey., Crataegus monogyna Jacq., and Pinus nigra J.F.Arnold. in Mazandaran Province. Morphological, microscopic, and molecular assessments were conducted to confirm the species. Truffle inoculum was prepared and 10 mL of inoculum was injected into the roots of two-month-old Persian oak seedlings. After one year, during which mycorrhizae had formed, drought stress (25%, 50%, and 75% of field capacity) was applied for three months. Random sampling was performed by selecting five control seedlings and five inoculated with the summer truffle (T. aestivum). After washing the roots, they were examined under a stereomicroscope, and the percentage of colonization was recorded. DNA was extracted from the seedlings’ roots using the CTAB method, and the success of the inoculation  was confirmed by polymerase chain reaction (PCR). Finally, various growth and biochemical parameters of the seedlings were measured to assess plant health and development. These parameters included seedling height, collar diameter,  shoot and root biomass, number of leaves, relative water content (RWC), and chlorophyll content. Additionally, oxidative stress indices such as hydrogen peroxide (H₂O₂), malondialdehyde (MDA), and proline (as an index of stress tolerance) were determined. The activities of antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD), were also measured to evaluate the plant’s defense mechanisms against oxidative stress.
Results: Inoculating Persian oak seedlings with the summer truffle improved all growth traits compared to the control under all levels of drought stress, though the effect was less pronounced at 25% field capacity than at 50% field capacity. Inoculation with the summer truffle resulted in a 2.3-fold increase in shoot dry weight and a 1.8-fold increase in root dry weight under 50% field capacity drought stress, showing the most significant effect. Root inoculation with the summer truffle increased chlorophyll a, chlorophyll b, and total chlorophyll content by 17 mg, 11 mg, and 15 mg, respectively, compared to the control. Inoculation of Persian oak with the summer truffle increased CAT, POD, and SOD activities by 30%, 23%, and 13%, respectively, compared to the control. Additionally, inoculation with the summer truffle reduced MDA and H₂O₂ levels by 19% and 10%, respectively, under severe drought stress (25% field capacity).
 
Conclusion: The summer truffle (Tuber aestivum) appears to have alleviated the detrimental effects of water deficit on the physiological traits of Persian oak seedlings, even under moderate to severe drought stress. The positive response of Persian oak seedlings to ectomycorrhizal inoculation with the summer truffle and their enhanced drought tolerance, this fungus can be used for planting mycorrhizal seedlings. The positive effects of this experiment could benefit local communities economically and encourage public participation in the conservation and development of these seedlings.
Keywords
Antioxidant enzymes
Drought stress
Ectomycorrhizal symbiosis
Truffle
Zagros restoration

Subjects

Agerer, R. (2006). Fungal relationships and structural identity of their ectomycorrhizae. Mycological progress, 5, 67-107. https://doi.org/10.1007/s11557-006-0505-x
Álvarez-Lafuente, A., Benito-Matías, L.F., Peñuelas-Rubira, J.L., & Suz, L.M. (2018). Multi-cropping edible truffles and sweet chestnuts: Production of high-quality Castanea sativa seedlings inoculated with Tuber aestivum, its ecotype T. uncinatum, T. brumale, and T. macrosporum. Mycorrhiza, 28(1), 29-38. https://doi.org/10.1007/s00572-017-0805-9
Arnon, D.I. (1949). Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant physiology, 24(1), 1. https://doi.org/10.1104/pp.24.1.1
Bach, C., Beacco, P., Cammaletti, P., Babel-Chen, Z., Levesque, E., Todesco, F., Cotton, C., Robin, B., & Murat, C. (2021). First production of Italian white truffle (Tuber magnatum Pico) ascocarps in an orchard outside its natural range distribution in France. Mycorrhiza, 31, 383-388. https://doi.org/10.1007/s00572-020-01013-2
Bates, L.S., Waldren, R.P.A., & Teare, I.D. (1973). Rapid determination of free proline for water-stress studies. Plant and soil, 39, 205-207. https://doi.org/10.1007/BF00018060
Beauchamp, C., & Fridovich, I. (1971). Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Analytical biochemistry, 44(1), 276-287. https://doi.org/10.1016/0003-2697(71)90370-8
Bergmeyer, H.U. (Ed.). (2012). Methods of enzymatic analysis. Elsevier
Bradford, 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, 72(1-2), 248-254. https://doi.org/10.1016/0003-2697(76)90527-3
Cassel, D.K., & Nielsen, D.R. (1986). Field capacity and available water capacity. Methods of soil analysis: Part 1 Physical and mineralogical methods, 5, 901-926. https://doi.org/10.2136/sssabookser5.1.2ed.c36
Cotrozzi, L., Remorini, D., Pellegrini, E., Landi, M., Massai, R., Nali, C., Guidi, L., & Lorenzini, G. (2016). Variations in physiological and biochemical traits of oak seedlings grown under drought and ozone stress. Physiologia plantarum, 157(1), 69-84. https://doi.org/10.1111/ppl.12402
Dib, S., & Fortas, Z. (2019). Inoculation with desert truffles increases growth of the forest seedlings Quercus ilex L. and Pinus halepensis M. Asian Journal of Microbiology Biotechnology & Environmental Sciences, 21, 907-914.
Fu, Y., Li, X., Li, Q., Wu, H., Xiong, C., Geng, Q., Sun, H., & Sun, Q. (2016). Soil microbial communities of three major Chinese truffles in southwest China. Canadian Journal of Microbiology, 62(11), 970-979. https://doi.org/10.1139/cjm-2016-0139
Gardes, M., & Bruns, T.D. (1993). ITS primers with enhanced specificity for basidiomycetes‐application to the identification of mycorrhizae and rusts. Molecular ecology, 2(2), 113-118.  https://doi.org/10.1111/j.1365-294X.1993.tb00005.x
Ghanbary, E., Kouchaksaraei, M.T., Guidi, L., Mirabolfathy, M., Etemad, V., Sanavi, S.A.M.M., & Struve, D. (2018). Change in biochemical parameters of Persian oak (Quercus brantii Lindl.) seedlings inoculated by pathogens of charcoal disease under water deficit conditions. Trees, 32, 1595-1608. https://doi.org/10.1007/s00468-018-1736-6
Ghanbary, E., Tabari Kouchaksaraei, M., Zarafshar, M., Bader, K.F.M., Mirabolfathy, M., & Ziaei, M. (2020). Differential physiological and biochemical responses of Quercus infectoria and Q. libani to drought and charcoal disease. Physiologia plantarum, 168(4), 876-892. https://doi.org/10.1111/ppl.13027
Giannopolitis, C.N., & Ries, S.K. (1977). Superoxide dismutases: II. Purification and quantitative relationship with water-soluble protein in seedlings. Plant physiology, 59(2), 315-318. https://doi.org/10.1104/pp.59.2.315
Gryndler, M., Černá, L., Bukovská, P., Hršelová, H., & Jansa, J. (2014). Tuber aestivum association with non-host roots. Mycorrhiza, 24, 603-610. https://doi.org/10.1007/s00572-014-0580-9
Haase, D., & Hellwig, R. (2022). Effects of heat and drought stress on the health status of six urban street tree species in Leipzig, Germany. Trees, Forests and People, 8, 100252. https://doi.org/10.1016/j.tfp.2022.100252
Hackbarth, C., Soffiatti, P., Zanette, F., Floh, E.I.S., Macedo, A.F., & Laureano, H.A. (2018). Free amino acid content in trunk, branches and branchlets of Araucaria angustifolia (Araucariaceae). Journal of Forestry Research, 29, 1489-1496. https://doi.org/10.1007/s11676-017-0581-6
Hall, I.R., & Zambonelli, A. (2012). Laying the Foundations, (eds) Edible Ectomycorrhizal Mushrooms. Soil Biology, 34. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-33823-61
He, F., Sheng, M., & Tang, M. (2017). Effects of Rhizophagus irregularis on photosynthesis and antioxidative enzymatic system in Robinia pseudoacacia L. under drought stress. Frontiers in Plant Science, 8, 183. https://doi.org/10.3389/fpls.2017.00183
Hodges, D.M., DeLong, J.M., Forney, C.F., & Prange, R.K. (1999). Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta, 207, 604-611. https://doi.org/10.1007/s004250050524
Huang, Y., Zou, J., Kang, Z., Zhang, X., Penttinen, P., & Li, X. (2021). Effects of truffle inoculation on a nursery culture substrate environment and seedling of Carya illinoinensis. Fungal Biology, 125(7), 576-584.
Iotti, M., Piattoni, F., & Zambonelli, A. (2013). Techniques for host plant inoculation with truffles and other edible ectomycorrhizal mushrooms. In Edible Ectomycorrhizal Mushrooms: Current Knowledge and Future Prospects (145-161). Berlin, Heidelberg: Springer Berlin Heidelberg. https://doi.org/10.1007/978-3-642-33823-6_9
Jafarnia, S., Akbarinia, M., Hosseinpour, B., Modarres Sanavi, S.A.M., & Salami, S.A. (2018). Effect of drought stress on some growth, morphological, physiological, and biochemical parameters of two different populations of Quercus brantii. Forest-Biogeosciences and Forestry, 11(2), 212. https://doi.org/10.3832/ifor2496-010
Jamali, S. (2017). First report of identification and molecular characterization of Tuber aestivum in Iran. Agroforestry Systems, 91, 335-343. https://doi.org/10.1007/s10457-016-9932-0
JM, P. (1970). Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. British Mycological Society, 55, 158-160.
Karimi, A., Tabari, M., Javanmard, Z., & Bader, M.K.F. (2022). Drought effects on morpho-physiological and biochemical traits in persian oak and black poplar seedlings. Forests, 13(3), 399. https://doi.org/10.3390/f13030399
Kennedy, P. (2010). Ectomycorrhizal fungi and interspecific competition: species interactions, community structure, coexistence mechanisms, and future research directions. New Phytologist, 187(4), 895-910. https://doi.org/10.1111/j.1469-8137.2010.03399.x
Krishna, H., Singh, S.K., Sharma, R.R., Khawale, R.N., Grover, M., & Patel, V.B. (2005). Biochemical changes in micropropagated grape (Vitis vinifera L.) plantlets due to arbuscular-mycorrhizal fungi (AMF) inoculation during ex vitro acclimatization. Scientia horticulturae, 106(4), 554-567. https://doi.org/10.1016/j.scienta.2005.05.009
Lilleskov, E.A., Hobbie, E.A., & Horton, T.R. (2011). Conservation of ectomycorrhizal fungi: exploring the linkages between functional and taxonomic responses to anthropogenic N deposition. Fungal ecology, 4(2), 174-183. https://doi.org/10.1016/j.funeco.2010.09.008
Navarro-Ródenas, A., Bárzana, G., Nicolás, E., Carra, A., Schubert, A., & Morte, A. (2013). Expression analysis of aquaporins from desert truffle mycorrhizal symbiosis reveals a fine-tuned regulation under drought. Molecular Plant-Microbe Interactions, 26(9), 1068-1078. https://doi.org/10.1094/MPMI-07-12-0178-R
Pegler, D.N., Spooner, B.M., & Young, T.W. (1993). British truffles: a revision of british hypogeous fungi, Kew Royal Botanic Gardens.
Percudani, R., Trevisi, A., Zambonelli, A., & Ottonello, S. (1999). Molecular phylogeny of truffles (Pezizales: Terfeziaceae, Tuberaceae) derived from nuclear rDNA sequence analysis. Molecular phylogenetics and evolution, 13(1), 169-180. https://doi.org/10.1006/mpev.1999.0638
Reuveni, R. (2017). Biochemical markers for disease resistance. In Molecular methods in plant pathology. 99-114, CRC Press. https://doi.org/10.1201/9780203746523
Reyna, S., & Garcia-Barreda, S. (2014). Black truffle cultivation: a global reality. Forest systems, 23(2), 317-328. https://doi.org/10.5424/fs/2014232-04771
Sánchez, F.J., Manzanares, M., de Andres, E.F., Tenorio, J.L., & Ayerbe, L. (1998). Turgor maintenance, osmotic adjustment and soluble sugar and proline accumulation in 49 pea cultivars in response to water stress. Field crops research, 59(3), 225-235. https://doi.org/10.1016/S0378-4290(98)00125-7
Savi, T., Cotrozzi, L., Beruzzi, S., Nali, C., & Bove, F. (2020). Ecophysiological and biochemical traits of Quercus ilex trees growing under urban stress conditions. Agrochimica: International Journal of Plant Chemistry, Soil Science and Plant Nutrition of the University of Pisa, 64(1), 41-57.
Schneider‐Maunoury, L., Deveau, A., Moreno, M., Todesco, F., Belmondo, S., Murat, C., & Selosse, M.A. (2020). Two ectomycorrhizal truffles, Tuber melanosporum and T. aestivum, endophytically colonise roots of non‐ectomycorrhizal plants in natural environments. New Phytologist, 225(6), 2542-2556.  https://doi.org/10.1111/nph.16321
Schütz, J.P. (1999). Close-to-nature silviculture: is this concept compatible with species diversity?. Forestry, 72(4), 359-366.  https://doi.org/10.1093/forestry/72.4.359
Sepahvand, D., Shirvany, A., Etemad, V., Javan-Nikkhah, M., DeLuca, T.H., & Khalilabad, A.A. (2021). The first report of Choiromyces venosus and a new report of Tuber aestivum from north Hyrcanian forest of Iran. Central Asian Journal of Plant Science Innovation, 1(3), 151-159. 10.22034/CAJPSI.2021.03.03
Slankis, V. (1974). Soil factors influencing formation of mycorrhizae. Annual Review of Phytopathology, 12(1), 437-457. 10.1146/annurev.py.12.090174.002253
Smirnoff, N. (1993). Tansley Review No. 52. The Role of Active Oxygen in the Response of Plants to Water Deficit and Desiccation. The New Phytologist, 125(1), 27–58. http://www.jstor.org/stable/2557905
Smith, S.E., & Read, D.J. (2010). Mycorrhizal symbiosis. Academic press.
Szabados, L., & Savouré, A. (2010). Proline: a multifunctional amino acid. Trends in plant science, 15(2), 89-97. 10.1016/j.tplants.2009.11.009
Trappe, J.M., & Castellano, M.A. (1991). Keys to the genera of truffles (Ascomycetes). McIlvanea, 10, 47-65.
Turgeman, T., Ben Asher, J., Roth-Bejerano, N., Kagan-Zur, V., Kapulnik, Y., & Sitrit, Y. (2011). Mycorrhizal association between the desert truffle Terfezia boudieri and Helianthemum sessiliflorum alters plant physiology and fitness to arid conditions. Mycorrhiza, 21, 623-630. https://doi.org/10.1007/s00572-011-0369-z
Velikova, V., Yordanov, I., & Edreva, A.J.P.S. (2000). Oxidative stress and some antioxidant systems in acid rain-treated bean plants: protective role of exogenous polyamines. Plant science, 151(1), 59-66. https://doi.org/10.1016/S0168-9452(99)00197-1
Xiong, S., Wang, Y., Chen, Y., Gao, M., Zhao, Y., & Wu, L. (2022). Effects of drought stress and rehydration on physiological and biochemical properties of four oak species in China. Plants, 11(5), 679. https://doi.org/10.3390/plants11050679
Yin, C., Wang, X., Duan, B., Luo, J., & Li, C. (2005). Early growth, dry matter allocation and water use efficiency of two sympatric Populus species as affected by water stress. Environmental and Experimental Botany, 53(3), 315-322. https://doi.org/10.1016/j.envexpbot.2004.04.007
Zamani, S.M., Kazerani, F., Rabbani Nasab, H., & Ghanaei, S. (2023). Diversity and population structure of ectomycorrhizal fungi in forest habitats of summer truffle (Tuber aestivum Vittad. Iranian Journal of Forest, 14(4), 407-424.in Persian 10.22034/ijf.2022.333059.1860
Zambonelli, A., Iotti, M., & Hall, I. (2015). Current status of truffle cultivation: recent results and future perspectives. Italian journal of mycology, 44, 31-40. ttps://doi.org/10.6092/issn.2465-311X/5593
Zhang, X., Ye, L., Kang, Z., Zou, J., & Li, X. (2019). Mycorrhization of Quercus acutissima with Chinese black truffle significantly altered the host physiology and root-associated microbiomes. PeerJ, 7, e6421. https://doi.org/10.7717/peerj.6421

  • Receive Date 20 July 2024
  • Revise Date 09 September 2024
  • Accept Date 05 October 2024