Application of the Toxin Produced by Esca-Associated Fungi as a Bioassay Method for Screening Grapevine Cultivars

پژوهش های حفاظت گیاهان ایران

Article 3, Volume 38, Issue 3 - Serial Number 65, September 2024, Pages 227-240   

Document Type: Research Article

DOI: 10.22067/jpp.2024.85310.1165

Author

Mahmoud Reza Karimishahri^*

n Department of Plant Protectio, Khorasan Razavi Agricultural and Natural Resources Research and Education Center, AREEO, Mashhad, Iran

Abstract

Introduction                                                                                                                 
Esca of grapevine (Vitis vinifera) is an important complex disease in almost all areas, where grapevines are grown. The symptoms of this disease may affect the trunk, branches, shoots (brown wood-streaking and white rot of trunk), leaves (light green or chlorotic, irregular areas between the veins or along the leaf margin, which gradually spread from the basal to the distal parts of the shoot) and fruit (tiny brown spots and sometimes wilt of berries). Toxins are secondary metabolites, which are important virulence factors of phytopathogenic fungi. Phaeoacremonium aleophilum (Pal), Phaeomoniella chlamydospora (Pch), and Fomitiporia mediterranea (Fme) produced two pentaketides (scytalone and isosclerone), and the α-glucan named pullulan. Several evidences indicated that at least some of these metabolites may induce the characteristic symptoms of diseases. The main objective of this research was to establish a clear efficient and cheap method using callus and extracted toxic metabolites in order to select susceptible, tolerant, or resistant commercial grapevine cultivars to the esca disease.
 
Materials and Methods
Esca-associated fungi were obtained from the Agricultural Research, Education and Extension Organization (AREEO), Khorasan Razavi province, Iran. Five mL of a suspension of three mentioned fungi (10-day-old) in 50 ml sterile water were added to 1 L flask containing 150 mL Czapek Dox medium amended with 0.1% yeast and 0.1% malt extract. They were incubated at 25^°C for 28 days in the dark. The mycelia were removed by filtration using Filter membranes, nitrocellulose 0.22 μm. The toxic secondary metabolites were extracted from this suspension. Briefly, the culture filtrates from each fungus (2 L per strain) were treated with equal volumes of cold ethanol-acetone and incubated in an ice batch for 4 to 6 hours. The resulting precipitates were extracted by centrifugation at 3000 rpm for 10 seconds. Recrystallization was done by dissolving the polymer in hot water and adding the same volume of ethanol to it, and the formed precipitate was filtered through a Whatman filter, dried at 40°C and weighed. The toxic metabolite obtained from liquid cultures of each fungal species was assayed on detached leaves of 5 grapevine commercial cultivars of Khorasan Razavi province, including Torkaman8(TU8), Kolahdari(KOL), Torkaman6(TU6), Fakhri Shahrood(FSH), and Keshmeshi-Quchan(KQU). The leaves with their petioles were immersed in a 3 mL solution until complete absorption, which usually took a few hours, and then were transferred to distilled water. Callus of the five grapevine cultivars, micro propagated shoot cultures were cultivated on modified MS media containing 15, 30, and 45% toxic metabolites. The grown callus was inoculated with different concentrations of toxic metabolites,  then dry weight of callus and vital cells were measured visually, as well as spectrophotometrically, by using Triphenyl Tetrazolium Chloride (TTC).
 
Results and Discussion                                                                
The Pal, Pch, and Fme fungi were all able to produce toxic secondary metabolites, but an isolate of the Pal fungus obtained from Memlejeh (a village in the Central District of Bojnord County, North Khorasan Province, Iran.) produced a large amount of pullulan. The symptoms produced on the detached leaves that absorbed a toxic solution were quite similar to those observed on the leaves of the same cultivars naturally infected by the same fungal species. Differences between cultivars in symptom severity were also observed under experimental conditions. The results showed that reduction of the callus dry weight in TU8 grapevine cultivar was the lowest (26%) compared to the other cultivars tested, followed by KOL (31%) and TU6 (40%) cultivars. The KQU and FSH cultivars showed the highest reduction in callus dry weight (48% and 44%, respectively). The effect of pullulan produced by three important esca-associated fungi indicated that the toxin produced by the Fme fungus had the least effect and the Pal fungus showed the greatest effect in reducing dry weight of the callus. Dry weight of the callus in 15% concentration of the toxic metabolite showed the least decrease and maximum decrease of the callus weight was obtained in 45% concentration. Among the cultivars tested, the callus cells of Torkaman 8 had the highest optical density, which means the number of living cells was more than other cultivars. The KQU cultivar with minimal live callus cells showed more sensitivity to the metabolite. Toxin of the Fme fungus had the least and that of the Pal fungus had the most effect on the callus cells. The percentage of survival rate in the inoculated callus cells of Torkaman 8 compared to the KQU cultivar at 45% concentration of the extract was about 50.36% and the survival rate in the KQU cultivar was about 15.51%. Also, the obtained results showed that the grapevine cultivar TU8 was comparatively more tolerant than other cultivars tested. On the other hand, the FSH and KQU cultivars were susceptible to secondary metabolite of the mentioned fungi.
 
Conclusions
Occurrence of the esca diseases is increasing in grapevine all over the world, whereas efficient therapeutic strategies are lacking. The use of toxic metabolites of esca-associated fungi and tissue culture of the host plants under in vitro conditions could be cheap,easy, and helpful assays for controlling the disease via selection, improvement, and reproduction of the tolerant genotypes. According to the reaction of grapevine cultivars to the secondary metaboilte of esca-associated fungi, it can be concluded that the TU8 grapevine cultivar is less sensitive than the KQU grapevine cultivar against the esca disease.

Keywords

Callus; Grapevine decline; Secondary metabolite

References

1. Bahrabadi, M., Karimishahri, M.R., &Hashemi, M. (2012). Study on Genetic Variability in Fungi Associated with Esca Disease in North Khorasan Province Vineyards with RAPD-PCR. Journal of Plant Protection Research. 26:92-100.https://doi.org/ 22067/jpp.v26i1.12721.
2. Beris, E., Selim, M., Kechagia, D., & Evangelou, A. (2022). Overview of the esca complex as an increasing threat in vineyards worldwide: Climate change, control approaches and impact on grape and wine quality. In Recent Advances in Grapes and Wine Production-New Perspectives for Quality Improvement. IntechOpen.
3. Bolandi, A.R., Hamidi, H., & Rezagholy, A.A. (2016). Effects of culture media and growth regulators on propagation of rootstock GF677 in tissue culture conditions. Journal of Plant Research (Iranian Journal of Biology), 29(1), 1-14. (In Persian with English abstract)
4. Bruno, G., & Sparapano, L. (2006a). Effects of three esca-associated fungi on Vitis vinifera: II. Characterization of biomolecules in xylem sap and leaves of healthy and diseased vines. Physiological and Molecular Plant Pathology, 69(4-6), 195-208. https://doi.org/10.1016/j.pmpp.2007.04.007
5. Bruno, G., & Sparapano, L. (2006b). Effects of three esca-associated fungi on Vitis vinifera: I. Characterization of secondary metabolites in culture media and host responses to the pathogens in calli. Physiological and Molecular Plant Pathology, 69(4-6), 209-223. https://doi.org/10.1016/j.pmpp.2007.04.008
6. De Rafael, M.A., Valle, T., Babiano, M.J., & Corchete, P. (2001). Correlation of resistance and H₂O₂ production in Ulmus pumila and Ulmus campestris cell suspension cultures inoculated with Ophiostoma novo‐ulmi. Physiologia Plantarum, 111(4), 512-518. https://doi.org/10.1034/j.1399-3054.2001.1110411.x
7. Evidente, A., Sparapano, L., Andolfi, A., & Bruno, G. (2000). Two naphthalenone pentakides from liquid cultures of «Phaeoacremonium aleophilum», a fungus associated with esca of grapevine. Phytopathologia Mediterranea, 39(1), 162-168. https://doi.org/10.14601/Phytopathol_Mediterr-1559
8. Fischer, M., & Payghami Ashnaei, S. (2019). Grapevine, esca complex, and environment: The disease triangle. Phytopathologia Mediterranea, 58(1), 17-37. https://doi.org/10.14601/Phytopathol_Mediterr-25086
9. Fox, H. (2022). A review of blackfoot, petri, and esca; grapevine fungal diseases, their treatments and the impacts of copper based fungicides. Ecorestoration: RNS Technical Series, (1).
10. Gramaje, D., Mostert, L., & Armengol, J. (2011). Characterization of Cadophora luteo-olivacea and melinii isolates obtained from grapevines and environmental samples from grapevine nurseries in Spain. Phytopathologia Mediterranea, 50(4), 112-126. https://doi.org/10.14601/Phytopathol_Mediterr-8723
11. Graniti, A., Durbin, R.D., & Ballio, A. (Eds.). (2013). Phytotoxins and plant pathogenesis(Vol. 27). Springer Science & Business Media. https://doi.org/10.1007/978-3-642-73178-5
12. Ibrahim, A.M., & Quick, J.S. (2001). Heritability of heat tolerance in winter and spring wheat. Crop Science, 41(5), 1401-1405. https://doi.org/10.2135/cropsci2001.4151401x
13. Mostert, L., Groenewald, J.Z., Summerbell, R.C., Gams, W., & Crous, P.W. (2006). Taxonomy and pathology of Togninia (Diaporthales) and its PhaeoacremoniumStudies in Mycology, 54, 1-113. https://doi.org/10.1016/S0166-0616(14)60211-6
14. Mamaghani, M., Rahnama, K., Mashayekhi, K., & Haghighi, H. (2012). Application of culture filtrates of Ophiostoma novo-ulmi and ulmi under in vitro condition as a bioassay to screen for disease tolerant Ulmus parvifolia and Ulmus campestris. Journal of Plant Production Research, 19(4). (In Persian with English abstract)
15. Mugnai, L., Graniti, A., & Surico, G. (1999). Esca (black measles) and brown wood-streaking: two old and elusive diseases of grapevines. Plant Disease, 83(5), 404-418. https://doi.org/10.1094/PDIS.1999.83.5.404
16. Murashige, T., & Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum, 15(3), 473-497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
17. Pelah, D., Kaushik, R.A., Nerd, A., & Mizrahi, Y. (2003). Validity of in vitro viability tests for predicting response of different vine cacti in the field to high and low temperatures. Journal of the Association for Cactus Development, Chapingo, 5, 65-71.
18. Pijut, P.M., Domir, S.C., Lineberger, R.D., & Schreiber, L.R. (1990). Use of culture filtrates of Ceratocystis ulmi as a bioassay to screen for disease tolerant Ulmus americana. Plant Science, 70(2), 191-196. https://doi.org/10.1016/0168-9452(90)90133-9
19. Shahpiri, A., Omidi, M., Tehrani, P.A., & Davoodi, D. (2004). A study of tissue culture and somaclonal variation in potato. Iranian Journal Agriculture Science, 35(2), 323-335. (In Persian with English abstract)
20. Shimomura, N., & Hasebe, K. (2004). Estimation of viability of inner bark tissue of Quercus serrata, a substrate for log cultivation of Lentinula edodes, using the TTC assay method. Mycoscience, 45(5), 362-365. https://doi.org/10.1007/S10267-004-0188-6
21. Sparapano, L., & Evidente, A.A. (1995). Studies on structure‐activity relationship of seiridins, phytotoxins produced by three species of Seiridium. Natural Toxins, 3(3), 166-173. https://doi.org/10.1002/nt.2620030308
22. Sparapano, L., & Bruno, G. (1997). Production of pullulans by virulent and hypovirulent strains of Cryphonecteria parasitica. Proccedings 10^th Congress of Mediterranean Phytopathogical Union, June 1-5, 1997. Montepellier, Societe Francasie de pathologie-ORSTOM, Montpellier, France, 488-493.
23. Sparapano, L., Bruno, G., & Graniti, A. (1998). Esopolisaccaridi fitotossici sono prodotii in coltura da due specie di Phaeoacremonium associate al complesso del mal dell esca della vite. Petria, 8, 210-212.
24. Sparapano, L., Bruno, G., Ciccarone, C., & Graniti, A. (2000a). Infection of grapevines with some esca-disease associated fungi. II. Interaction among Fomitiporia punctata, Phaeoacremonium chlamydosporum and aleophilum. Phytopathologia Mediterranea, 39, 53-58. https://doi.org/10.14601/Phytopathol_Mediterr-1543
25. Sparapano, L., Bruno, G., & Graniti, A. (2000b). Effects on plants of metabolites produced in culture by Phaeoacremonium chlamydosporum, P. aleophilum and Fomitiporia punctata. Phytopathologia Mediterranea, 39(1), 169-177. https://doi.org/14601/Phytopathol_Mediterr-1535
26. Sparapano, L., Bruno, G., & Campanella, A. (2001). Interactions between three fungi associated with esca of grapevine and their secondary metabolites. Phytopathologia Mediterranea (Italy). 40(4), 417-422. https://doi.org/10.14601/Phytopathol_Mediterr-1626
27. Surico, G., Mugnai, L., & Marchi, G. (2008). The esca disease complex. In Integrated management of diseases caused by fungi, phytoplasma and bacteria(pp. 119-136). Dordrecht: Springer Netherlands. https://doi.org/10.1007/978-1-4020-8571-0_6
28. Surico, G. (2009). Towards a redefinition of the diseases within the esca complex of grapevine. Phytopathologia Mediterranea, 48(1), 5-10. https://doi.org/10.14601/Phytopathol_Mediterr-2870
29. Towill, L.E., & Mazur, P. (1975). Studies on the reduction of 2, 3, 5-triphenyltetrazolium chloride as a viability assay for plant tissue cultures. Canadian Journal of Botany, 53(11), 1097-1102. https://doi.org/10.1139/b75-129