Control of Tomato Early Blight (Alternaria solani) Disease by Aqueous Extract of Thyme (Thymus vulgaris)

پژوهش های حفاظت گیاهان ایران

Article 5, Volume 39, Issue 1 - Serial Number 67, March 2025, Pages 61-83   

Document Type: Research Article

DOI: 10.22067/jpp.2025.87475.1183

Authors

Ali Gholamzadeh-Bazarbash¹; Sahar Qoli Toloui¹; Tohid Alllahverdipour²; mehrdad salehzadeh^{* 3}

¹Department of Plant Protection, College of Agriculture, Afaq Institute of Higher Education, Urmia, Iran

²Department of Plant Protection, College Institute, Urof Agriculture Afagh Higher Education mia, Iran

³Plant Virology Research Center, School of Agriculture, Shiraz University, Shiraz, Iran.

Abstract

Introduction
Tomato (Solanum lycopersicum) is the second most valuable agricultural product after potatoes. Early blight, caused by Alternaria solani, is a major disease affecting tomatoes, damaging stems, leaves, and fruits. This disease significantly reduces photosynthesis and can lead to leaf drop in severe cases across tomato-growing regions in Iran. Common control methods often involve chemical pesticides, but their excessive use can result in environmental pollution, pathogen resistance, and the emergence of dangerous strains, exacerbating the damage. As a safer alternative, researchers have increasingly focused on using natural compounds, or phytometabolites, for pest and pathogen management. Plant biocides offer numerous advantages, including rapid decomposition, specific toxicity, reduced bioaccumulation, and minimal harm to beneficial insects. This study aims to evaluate the effectiveness of thyme extract in managing tomato spot disease as a substitute for chemical pesticides, alongside an assessment of morphological and physiological characteristics in infected versus healthy plants.
 
Materials and Methods
Five fungal isolates were collected from tomato fields in various locations of West Azarbaijan province. Extraction and isolation of A. solani followed the Rigotti method (2003), while DNA extraction and PCR were conducted using Ji Cho's method (2016), with the amplified fragments subsequently sequenced. A phylogenetic tree was created using Mega 8 software, positioning the Iranian isolate alongside Asian A. solani isolates. Thyme extract's effect on fungal colony growth was tested in the lab, and pathogenicity was assessed on tomato variety 4129. Morphological traits, including plant and root length and biomass, along with physiological traits such as chlorophyll content and the expression of defense genes (CAT, APX, POD), as well as phenol and flavonoid levels were measured for UrmiaB isolate, as the most virulent among the collected isolates.
 
Results and Discussion
All concentrations of thyme extract tested in this research exhibited significant inhibitory and antifungal effects at both 1% and 5% probability levels compared to the controls. Additionally, thyme extract showed moderate to high antifungal activity against A. solani, both in laboratory and greenhouse settings. In laboratory trials, thyme concentrations of 50, 100, and 150 mg mL^-1 markedly reduced fungal growth, particularly at higher concentrations (100 and 150 mg mL^-1), which also corresponded with a decrease in pathogenicity affecting tomato fruits of cultivar 4129. Morphological and physiological assessments indicated that thyme extract improved traits in tomato variety 4129, including root and shoot lengths, biomass, chlorophyll content, and the expression of defense-related enzymes (CAT, APX, POD), as well as phenolic and flavonoid levels under greenhouse conditions. These findings demonstrate that thyme extract effectively mitigates early blight disease in tomatoes by inhibiting fungal growth.
 
Conclusion
The results indicated that thyme extract effectively reduced the activity of the pathogen responsible for tomato early blight in both laboratory and greenhouse conditions. Its antifungal properties make thyme extract a promising candidate for commercialization in agricultural pest management, particularly when used in combination with other compounds.
 

Keywords

Antifungal properties; Early blight; Morpho-physiological traits; Plant metabolite

References

1. Abd-Ellatif, S., Ibrahim, A.A., Safhi, F.A., Abdel Razik, E.S., Kabeil, S.S., Aloufi, S., & Elshafie, H.S. (2022). Green synthesized of Thymus vulgaris chitosan nanoparticles induce relative WRKY-genes expression in Solanum lycopersicum against Fusarium solani, the causal agent of root rot disease. Plants, 11(22), 3129. https://doi.org/10.3390/plants11223129
2. Abu-Bader, S.H. (2021). Using Statistical Methods in Social Science Research: With a complete SPSS Guide. Oxford University Press, USA.
3. Abdelgawad, Z.A., Mohamed, T.R., Afiah, S., & Al-Agwany, H. (2015). Effect of drought and salt stress on growth, osmolytes, protein, and isozymes in Vicia faba genotypes. Egyptian Journal of Agronomy, 37, 93-119. https://doi.org/10.21608/agro.2015.67
4. Aghazadeh Naeini, S.S., Maleki, M., Gholamnezhad, J., & Shirmardi, M. (2022). Evaluation of the effect of some plant extracts in controlling Rhizoctonia rot in the greenhouse cucumber. BioControl in Plant Protection, 9(2), 87-113. htp//doi.org/10.22092/BCPP.2022.128595.
5. Ahmed, M., Sajid, A. R., Javeed, A., Aslam, M., Ahsan, T., Hussain, D., & Ji, M. (2022). Antioxidant, antifungal, and aphicidal activity of the triterpenoids spinasterol and 22, 23-dihydrospinasterol from leaves of Citrullus colocynthisScientific Reports, 12(1), 4910. https://doi.org/10.1038/s41598-022-08999-z  .
6. Aksit, H., Bayar, Y., Simsek, S., & Ulutas, Y. (2022). Chemical composition and antifungal activities of the essential oils of thymus species (Thymus pectinatus, Thymus convolutus, Thymus vulgaris) against plant pathogens. Journal of Essential Oil Bearing Plants, 25(1), 200-207. https://doi.org/10.1080/0972060X.2022.2043189 .
7. Amini, J., Farhang, V., Javadi, T., & Nazemi, J. (2018). Antifungal effect of plant essential oils on controlling PhytophthoraThe Plant Pathology Journal, 32(1), 16. https://doi.org/10.5423/ppj.oa.05.2015.0091
8. Amini, M., Safaie, N., Salmani, M. J., & Shams-Bakhsh, M. (2012). Antifungal activity of three medicinal plant essential oils against some phytopathogenic fungi. Trakia Journal Sciences, 10(1), 1-8.
9. Al-Rahmah, A. N., Mostafa, A. A., Abdel-Megeed, A., Yakout, S. M., & Hussein, S. A. (2013). Fungicidal activities of certain methanolic plant extracts against tomato phytopathogenic fungi. African Journal of Microbiology Research, 7(6), 517-524. https://doi.org/5897/AJMR12.1902 .
10. Ashrafi, A., Salehzadeh, M., & Khezrinezhad, N. (2020). Detection and identification of tomato wilt disease in East Azerbaijan province and controlling it using antagonist bacteria. Genetic Engineering and Biosafety Journal, 9(1), 28-39. http://dorl.net/dor/20.1001.1.25885073.1399.9.1.2.5 .
11. Bahraminejad, S., Seifolahpour, B., & Amiri, R. (2016). Antifungal effects of some medicinal and aromatic plant essential oils against Alternaria solani. Journal of Crop Protection, 5(4), 603-616. http://dorl.net/dor/20.1001.1.22519041.2016.5.4.14.9.
12. Balkan, B., Balkan, S., Aydoğdu, H., Güler, N., Ersoy, H., & Aşkın, B. (2017). Evaluation of antioxidant activities and antifungal activity of different plants species against pink mold rot-causing Trichothecium roseum. Arabian Journal for Science and Engineering, 42(6), 2279-2289. https://doi.org/10.1007/s13369-017-2484-4.
13. Campolo, O., Giunti, G., Russo, A., Palmeri, V., & Zappalà, L. (2018). Essential oils in stored product insect pest control. Journal of Food Quality, 2018, 1-18. https://doi.org/10.1155/2018/6906105.
14. Chance, B., & Maehly, A. C. (1955). Assay of catalases and peroxidases. Methodes in Enzymology, 2 (1),  764-765. https://doi.org/10.1016/S0076-6879(55)02300-8.
15. Danaei, M., Baghizadeh, A., Pourseyedi, S., Amini, J., & Yaghoobi, M. M. (2014). Biological control of plant fungal diseases using volatile substances of Streptomyces griseus. Europian Journal of Experimental Bioogyl, 4(1), 334-339.
16. Doehlemann, G., Ökmen, B., Zhu, W., & Sharon, A. (2017). Plant pathogenic fungi. Microbiology Spectrum, 5(1), 5-1. https://doi.org/10.1128/microbiolspec.funk-0023-2016
17. Dev, U., Devakumar, C., Mohan, J., & Agarwal, P. C. (2004). Antifungal activity of aroma chemicals against seed-borne fungi. Journal of essential oil Research, 16(5), 496-499. https://doi.org/10.1080/10412905.2004.9698780.
18. Driscoll, W. C. (1996). Robustness of the ANOVA and Tukey-Kramer statistical tests. Computers and Industrial Engineering, 31(1-2), 265-268. https://doi.org/10.1016/0360-8352(96)00127-1
19. Farashah, S. D., & Salehzadeh, M. (2023). Effect of antagonistic bacterial agents isolated from the pistachio orchards on Aspergilus flavus. Journal of Microbial World, 16(2), 143-155. https://doi.org/10.30495/jmw.2023.1968442.2038.
20. Fatemi, M., Azadi, H., Rafiaani, P., Taheri, F., Dubois, T., Van Passel, S., & Witlox, F. (2018). Effects of supply chain management on tomato export in Iran: Application of structural equation modeling. Journal of Food Products Marketing, 24(2), 177-195. https://doi.org/10.1080/10454446.2017.1266552
21. Ferreira, R. B., Monteiro, S. A. R. A., Freitas, R., Santos, C. N., Chen, Z., Batista, L. M., Duarte, j., Borges, A., & Teixeira, A. R. (2007). The role of plant defence proteins in fungal pathogenesis. Molecular Plant Pathology, 8(5), 677-700. https://doi.org/10.1111/j.1364-3703.2007.00419.x.
22. Ferrigo, D., Mondin, M., Ladurner, E., Fiorentini, F., Causin, R., & Raiola, A. (2020). Effect of seed biopriming with Trichoderma harzianum strain INAT11 on Fusarium ear rot and Gibberella ear rot diseases. Biological Control, 147, 104286. https://doi.org/10.1016/j.biocontrol.2020.104286.
23. Fravel, D. R. (2005). Commercialization and implementation of biocontrol. Annual Review of Phytopathology Journal, 43(1), 337-359. https://doi.org/10.1146/annurev.phyto.43.032904.092924.
24. Gandomi, H., Misaghi, A., Basti, A. A., Bokaei, S., Khosravi, A., Abbasifar, A., & Javan, A. J. (2009). Effect of Zataria multiflora essential oil on growth and aflatoxin formation by Aspergillus flavus in culture media and cheese. Food and chemical toxicology, 47(10), 2397-2400. https://doi.org/10.1016/j.fct.2009.05.024.
25. Gholamnezhad, J., Arsalani, S., & Maleki, M. (2019). The investigation of the effect of garlic and thyme extracts on orange green mold (Penicillium digitatum), defense enzymes and genes expression. Plant Protection (Scientific Journal of Agriculture), 42(1), 91-118. https://doi.org/10.22055/ppr.2019.14495
26. Gupta, P., Gupta, H., Tripathi, S., & Poluri, K. M. (2023). Biochemical and metabolomic insights into antifungal mechanism of berberine against Candida glabrata. Applied Microbiology and Biotechnology, 107(19), 6085-6102. https://doi.org/10.1007/s00253-023-12714-x.
27. Haghpanah, M., Najafi-Zarini, H., & Babaeian-Jelodar, N. (2023). Differential physiological and molecular responses of susceptible and resistant tomato genotypes to Alternaria solaniJournal of Crop Protection, 12(3), 227-240. http://dorl.net/dor/20.1001.1.22519041.2023.12.3.1.3.
28. Hemeda, H. M., & Klein, B. P. (1990). Effects of naturally occurring antioxidants on peroxidase activity of vegetable extracts. Journal of Food Science, 55(1), 184-185. https://doi.org/10.1111/j.1365-2621.1990.tb06048.x.
29. He, Y., Yan, H., Hua, W., Huang, Y., & Wang, Z. (2016). Selection and validation of reference genes for quantitative real-time PCR in Gentiana macrophylla. Frontiers in Plant Science, 7, 945. https://doi.org/10.3389/fpls.2016.00945
30. Hong, J. K., Jo, Y. S., Ryoo, D. H., Jung, J. H., Kwon, H. J., Lee, Y. H., & Park, C. J. (2018). Alternaria spots in tomato leaves differently delayed by four plant essential oil vapours. Research in Plant Disease, 24(24), 292-301. https://doi.org/10.5423/RPD.2018.24.4.292
31. Iraji, A., Yazdanpanah, S., Alizadeh, F., Mirzamohammadi, S., Ghasemi, Y., Pakshir, K., & Zomorodian, K. (2020). Screening the antifungal activities of monoterpenes and their isomers against CandidaJournal of Applied Microbiology, 129(6), 1541-1551. https://doi.org/10.1111/jam.14740.
32. ji Cho, H., Hong, S. W., Kim, H. J., & Kwak, Y. S. (2016). Development of a multiplex PCR method to detect fungal pathogens for quarantine on exported cacti. The Plant Pathology Journal, 32(1), 53. https://doi.org/10.5423%2FPPJ.NT.09.2015.0184.
33. Jia, J., Ford, E., Hobbs, S. M., Baird, S. M., & Lu, S. E. (2022). Occidiofungin is the key metabolite for antifungal activity of the endophytic bacterium Burkholderia MS455 against Aspergillus flavus. Phytopathology®, 112(3), 481-491. https://doi.org/10.1094/PHYTO-06-21-0225-R
34. Karimi, S., Gholamnezhad, J., & Maleki, M. (2020). Control of Aspergillus and related aflatoxin production by using different plant extracts. BioControl in Plant Protection, 8(1), 117-136. https://doi.org/10.22092/bcpp.2020.124008.
35. Kamangar, H., Hemmati, R., Yazdinejad, A., & Movahedi Fazel, M. (2014). Study on antifungal effects of five plant species extract against Fusarium solani and Rhizoctonia solani on bean. Iranian Journal of Plant Protection Science, 45(1), 49-58. https://doi.org/10.22059/ijpps.2014.52246.
36. Kasahara, K., Miyamoto, T., Fujimoto, T., Oguri, H., Tokiwano, T., Oikawa, H., & Fujii, I. (2010). Solanapyrone synthase, a possible Diels–Alderase and iterative type I polyketide synthase encoded in a biosynthetic gene cluster from Alternaria solani. ChemBioChem, 11(9), 1245-1252. https://doi.org/10.1002/cbic.201000173.
37. Kumar, V., Haldar, S., Pandey, K. K., Singh, R. P., Singh, A. K., & Singh, P. C. (2008). Cultural, morphological, pathogenic and molecular variability amongst tomato isolates of Alternaria solani in India. World Journal of Microbiology and Biotechnology, 24, 1003-1009. https://doi.org/10.1007/s11274-007-9568-3.
38. Lichtenthaler, H. K., & Wellburn, A. R. (1983). Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biological Society Transaction, 11 (5), 591-592. https://doi.org/10.1042/bst0110591.
39. Liu, W., Liu, K., Chen, D., Zhang, Z., Li, B., El-Mogy, M. M., & Chen, T. (2022). Solanum lycopersicum, a model plant for the studies in developmental biology, stress biology and food science. Foods, 11(16), 2402. https://doi.org/10.3390/foods11162402.
40. Lotfi, A., Kottb, M., Elsayed, A., & Shafik, H. (2021). Antifungal activity of some Mediterranean seaweed against Macrophomina phaseolina and Fusarium oxysporum in vitro. Alfarama Journal of Basic and Applied Sciences, 2(1), 81-96. https://doi.org/10.21608/ajbas.2020.41969.1031.
41. Mahmoudi, E., Ahmadi, A., & Naderi, D. (2012). Effect of Zataria multiflora essential oil on Alternaria alternata in vitro and in an assay on tomato fruits. Journal of Plant Diseases and Protection, 119, 53-58. https://doi.org/10.1007/BF03356420.
42. Meena, B. R., Meena, S., Chittora, D., & Sharma, K. (2021). Antifungal efficacy of Thevetia peruviana leaf extract against Alternaria solani and characterization of novel inhibitory compounds by Gas Chromatography-Mass Spectrometry analysis. Biochemistry and Biophysics Reports, 25, 100914. https://doi.org/10.1016/j.bbrep.2021.100914.
43. Moghaddam, M., & Mehdizadeh, L. (2020). Chemical composition and antifungal activity of essential oil of Thymus vulgaris grown in Iran against some plant pathogenic fungi. Journal of Essential Oil Bearing Plants, 23(5), 1072-1083. https://doi.org/10.1080/0972060X.2020.1843547.
44. Nejhad, A. A., Behbahani, B. A., Hojjati, M., Vasiee, A., & Mehrnia, M. A. (2024). Investigation of the inhibitory, fungicidal and interactive effects of the aqueous extract of Calotropis procera on Alternaria alternata, Alternaria solani, Saccharomyces cerevisiae, and Fusarium solani “in vitro”. Journal of Food Science and Technology (2008-8787), 20(143).
45. Narware, J., Singh, S. P., Manzar, N., & Kashyap, A. S. (2023). Biogenic synthesis, characterization, and evaluation of synthesized nanoparticles against the pathogenic fungus Alternaria solani. Frontiers in Microbiology, 14, 1159251. https://doi.org/10.3389/fmicb.2023.1159251.
46. Nateqi, M., & Mirghazanfari, S. M. (2018). Determination of total phenolic content, antioxidant activity and antifungal effects of Thymus vulgaris, Trachyspermum ammi and Trigonella foenum-graecum extracts on growth of Fusarium solani. Cellular and Molecular Biology, 64(14), 39-46. https://doi.org/10.14715/cmb/2018.64.14.7.
47. Pusztahelyi, T., Holb, I. J., & Pócsi, I. (2015). Secondary metabolites in fungus-plant interactions. Frontiers in plant Science, 6, 573. https://doi.org/10.3389/fpls.2015.00573.
48. Rana, K. M., Maowa, J., Alam, A., Dey, S., Hosen, A., Hasan, I., & Kawsar, S. M. (2021). In silico DFT study, molecular docking, and ADMET predictions of cytidine analogs with antimicrobial and anticancer properties. In Silico Pharmacology, 9, 1-24. https://doi.org/10.1007/s40203-021-00102-0.
49. Rehmany, A. P., Grenville, L. J., Gunn, N. D., Allen, R. L., Paniwnyk, Z., Byrne, J., & Beynon, J. L. (2003). A genetic interval and physical contig spanning the Peronospora parasitica (At) avirulence gene locus ATR1Nd. Fungal Genetics and Biology, 38(1), 33-42. https://doi.org/10.1016/S1087-1845(02)00515-7.
50. Ribera, A. E., & Zuñiga, G. (2012). Induced plant secondary metabolites for phytopatogenic fungi control: A review. Journal of Soil Science and Plant Nutrition, 12(4), 893-911. http://dx.doi.org/10.4067/S0718-95162012005000040.
51. Rigotti, S., Viret, O., & Gindrat, D. (2003). Fungi from symptomless strawberry plants in Switzerland. Phytopathologia Mediterranea, 42(1), 85-88.
52. Sajjadi, S. A., & Assemi, H. (2014). Study of antifungal activity of plant extracts of catmint, tobacco and thyme on tobacco pathogens fungal. Biological Control of Pests and Plant Diseases, 3(1), 41-52.
53. Sánchez-Gómez, T., Santamaría, Ó., Martín-García, J., & Poveda, J. (2024). Seed extracts as an effective strategy in the control of plant pathogens: Scalable industry bioactive compounds for sustainable agriculture. Biocatalysis and Agricultural Biotechnology, 103332. https://doi.org/10.1016/j.bcab.2024.103332.
54. Sareena, S., Poovannan, K., Kumar, K. K., Raja, J. A. J., Samiyappan, R., Sudhakar, D., & Balasubramanian, P. (2006). Biochemical responses in transgenic rice plants expressing a defence gene deployed against the sheath blight pathogen, Rhizoctonia solani. Current Science, 91 (11), 1529-1532.
55. Sepehrvand, A., Ezatpour, B., Tarkhan, F., Bahmani, M., Khonsari, A., & Rafieian-Kopaei, M. (2017). Phytotherapy in fungi and fungal disease: A review of effective medicinal plants on important fungal strains and diseases. International Journal of Pharmaceutical Sciences and Research, 8(11), 4473-4495. https://doi.org/13040/IJPSR.0975-8232.8(11).4473-95.
56. Serag, A., Salem, M. A., Gong, S., Wu, J. L., & Farag, M. A. (2023). Decoding metabolic reprogramming in plants under pathogen attacks, a comprehensive review of emerging metabolomics technologies to maximize their applications. Metabolites, 13(3), 424. https://doi.org/10.3390/metabo13030424.
57. Shahriari, D., Alibeyk Tehrani, N., & Maleki, M. (2017). The inhibitory effect of Thymus vulgaris and Carum copticum essential oil on the growth of Rhizoctonia solani, the causal agent of potato stem canker in vitro and greenhouse conditions. Applied Plant Protection, 6(2), 97-107.
58. Shalaby, S., & Horwitz, B. A. (2015). Plant phenolic compounds and oxidative stress: Integrated signals in fungal–plant interactions. Current genetics, 61, 347-357. https://doi.org/10.1007/s00294-014-0458-6.
59. Soltani, J., & Moghaddam, M. S. H. (2014). Diverse and bioactive endophytic Aspergilli inhabit Cupressaceae plant family. Archives of Microbiology, 196, 635-644. https://doi.org/10.1007/s00203-014-0997-8.
60. Stepanova, M., & Korzhikova-Vlakh, E. (2022). Modification of cellulose micro-and nanomaterials to improve properties of aliphatic polyesters/cellulose composites: A review. Polymers, 14(7), 1477. https://doi.org/10.3390/polym14071477.
61. Sudhakar, N., Nagendra-Prasad, D., Mohan, N., & Murugesan, K. (2007). Induction of systemic resistance in Lycopersicon esculentum PKM1 (tomato) against cucumber mosaic virus by using ozone. Journal of Virological Methods, 139(1), 71-77. https://doi.org/10.1016/j.jviromet.2006.09.013.
62. Tamura, K., Dudley, J., Nei, M., & Kumar, S. (2007). MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution, 24(8), 1596-1599. https://doi.org/10.1093/molbev/msm092
63. Verma, P. K., Verma, S., Pandey, N., & Chakrabarty, D. (2021). Antimicrobial products from plant biodiversity. Bioprospecting of Plant Biodiversity for Industrial Molecules, 8 (1). 153-173. https://doi.org/10.1002/9781119718017.ch8.
64. Younesi, S., Salehzadeh, M., & Soleymani Pari, M. J. (2023). Control of strawberry gray mold fungus with combined application of different species of Trichoderma and salicylic acid. Journal of Microbial World, 16(1), 88-72.