Enhancing the tomato plant defense against Fusarium wilt by using Trichoderma harzianum

Enhancing the tomato plant defense against Fusarium wilt by using Trichoderma harzianum | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enhancing the tomato plant defense against Fusarium wilt by using Trichoderma harzianum Khalaf Alhussaen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7317989/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Fusarium oxysporum is a soil-borne fungal pathogen that causes wilt diseases in a wide variety of plants. Fusarium oxysporum poses a significant threat to agriculture due to its ability to cause severe yield losses in a variety of crops. Management of Fusarium wilt is challenging because the fungus persists in the soil for many years. and there are limited effective chemical control options. This research is designed to study the effect of Trichoderma harzianum in reducing the effect of Fusarium oxysporum f. sp. lycopersici (Fol) on tomato plants. Fusarium oxysporum was isolated from diseased tomato plants and identified by using morphology and ITS sequencing of DNA. Three biological treatments were applied to examine the effect of Trichoderma harzianum on controlling disease wilt caused by Fusarium oxysporum . The results related to disease severity showed that the control plants appeared healthy according to disease severity scale of 1–2 with an average of (1.3) in contrast to plants treated with Fol which showed diseased plants according to disease severity scale of 3–4 with an average of (3.7). While the plants treated with Fol and to which Trichoderma harzianum was added showed healthy plants according to disease severity scale of 1–2 with an average of (1.8). Fusarium wilt Trichoderma harzianum Fusarium oxysporum f. sp. cubense Figures Figure 1 Figure 2 Figure 3 Introduction Fusarium oxysporum is a soil-borne fungal pathogen that causes wilt diseases in a wide variety of plants, including many economically important crops. Fusarium oxysporum is a filamentous fungus that primarily inhabits the soil. It can survive for long periods as chlamydospores, which are thick-walled, resting spores that allow the fungus to persist in the absence of a host. The fungus infects plants through the roots, colonizing the vascular system and leading to blockage of water transport, which results in wilting and often plant death (Jackson et. al., 2024). Fusarium oxysporum is known for its broad host range, affecting hundreds of plant species across numerous families. However, it is typically host-specific at the forma specialis (f. sp.) level, with different forma specialis adapted to specific hosts (Edel-Hermann and Lecomte, 2019). Some well-known forma specialis include Fusarium oxysporum f. sp. lycopersici causes wilt in tomatoes (Jamil et. al,. 2021; Komissarov et. al., 2025), Fusarium oxysporum f. sp. cubense causes Panama disease in bananas (Magdama et. al., 2020) and Fusarium oxysporum f. sp. vasinfectum infects cotton (Asif et. al., 2023). The primary symptom of Fusarium wilt is wilting of the plant, often starting with lower leaves and progressing upwards (Pandey et. al., 2023). Other symptoms include yellowing of the foliage, stunted growth, and vascular discoloration, which can be observed as brown streaks in the stem when it is cut open. Fusarium oxysporum spreads through infected soil, water, plant debris, and contaminated tools. It can also be transmitted via infected seedlings and plant material. The fungus thrives in warm, moist conditions, making it particularly problematic in tropical and subtropical regions. Fusarium oxysporum poses a significant threat to agriculture due to its ability to cause severe yield losses in a variety of crops. Management of Fusarium wilt is challenging because the fungus persists in the soil for many years. and there are limited effective chemical control options. Effective management of Fusarium wilt involves an integrated approach include planting resistant or tolerant crop varieties, implementing crop rotation, proper sanitation, and soil health management, utilizing antagonistic microorganisms that inhibit Fusarium oxysporum and limited use of fungicides, often as part of an integrated pest management (IPM) strategy (Lal et. al., 2024). Trichoderma is a genus of fungi that is widely recognized for its beneficial properties in agriculture and biotechnology. Trichoderma species are filamentous fungi commonly found in soil, decaying wood, and other organic matter. They are known for their rapid growth and prolific spore production. Trichoderma species are characterized by their green spores and white to greenish mycelium (Jorge et. al., 2024). Trichoderma fungi exhibit several mechanisms that make them beneficial for plant health and soil quality. Trichoderma species can parasitize other fungi, including many plant pathogens, by directly attacking and degrading their cell walls. They produce various secondary metabolites, including antibiotics, which inhibit the growth of competing microorganisms. Moreover, Trichoderma competes effectively for nutrients and space, outcompeting pathogenic fungi (Waleed et. al., 2024). Trichoderma can stimulate plant defense mechanisms, enhancing the plant's own ability to resist diseases. Trichoderma species are widely used as biological control agents and biofertilizers due to their ability to enhance plant growth and suppress soil-borne pathogens. Trichoderma is used to control various plant diseases caused by fungi, such as Fusarium, Rhizoctonia , and Pythium (Awal et. al., 2024). Trichoderma enhances nutrient uptake, promotes root growth, and improves plant vigor. Moreover, beyond agriculture, Trichoderma species have significant industrial applications, Trichoderma is known for its ability to produce a wide range of enzymes, including cellulases, hemicellulases, and chitinases, which are used in various industries such as biofuel production, paper, and pulp processing (Lima et. al., 2024). Ongoing research is focused on enhancing the efficacy of Trichoderma-based products and understanding the molecular mechanisms underlying their beneficial effects. Genetic and genomic studies are providing insights into the pathways involved in mycoparasitism, antibiosis, and plant growth promotion (Alizadeh et. al., 2024). The use of Trichoderma in agriculture offers a sustainable and eco-friendly alternative to chemical pesticides and fertilizers. By promoting plant health and reducing dependency on synthetic chemicals, Trichoderma contributes to sustainable agricultural practices and environmental conservation. The current study aims to demonstrate the extent of the effect of using Trichoderma harzianum in reducing the effect of Fusarium oxysporum f. sp. lycopersici (Fol) on tomato plants. Materials and Methods Pathogen isolation: Fusarium oxysporum f. sp. lycopersici (Fol) was isolated from diseased tomato plants exhibiting typical wilt symptoms grown in Jordan Valley on November 2024. The isolation was carried out on potato dextrose agar (PDA) and incubated at 25–27°C for 5–7 days. Stock cultures of Fol were prepared and stored at 4°C. Pathogenicity tests for all Fol isolates were conducted according to Koch’s postulates, and a highly virulent isolate was selected for further experiments. The experiments were conducted in Biology department in Yarmouk University in Jordan on 2024. Isolation of Trichoderma spp. Trichoderma spp. were isolated from soil samples taken from the tomato rhizosphere across various locations in the agricultural field in Jordan Valley on 2024. The samples were stored at 4°C in the laboratory, and the isolation of different Trichoderma spp. was carried out using the serial dilution method (Zehra et al., 2017). Fungal DNA extraction and ITS sequencing Genomic DNA was extracted from the one-week-old fungal isolate using the method described by Doyle and Doyle (1987). The internal transcribed spacer (ITS) region of the rDNA was amplified using the universal primers ITS1/ITS4 through polymerase chain reaction (PCR) (Meena et al., 2017). The purified PCR products were then sequenced at both ends and analyzed. The resulting sequences were compared against those in GenBank using the National Center for Biotechnology Information's Basic Local Alignment Search Tool (NCBI-BLAST) ( https://www.ncbi.nlm.nih.gov/ ). Plant material: Tomato seeds ( Lycopersicon esculentum ) were obtained from local specialized store for selling seeds in the Jordan Valley on 2024. The seeds were surface-sterilized in a 4% sodium hypochlorite (NaOCl) solution containing 0.02% (v/v) Tween-20, thoroughly rinsed with sterilized water, and then allowed to germinate (Abdelshafy et al., 2020). Biological treatments and growth condition: The experiment was conducted in pots (20 cm in diameter), which were surface-sterilized with 0.1% mercuric chloride. The treatments were arranged as follows: ( 1 ) uninoculated as control, ( 2 ) pathogen (Fol) challenged, and ( 3 ) plants primed with Trichoderma harzianum spores and subsequently challenged with the pathogen (Fol). Seedlings were transferred to pots filled with a sterile soil and sand mixture (autoclaved at 121°C and 15 psi pressure for 15 minutes) in a 4:1 ratio, along with Trichoderma inoculum at a concentration of 10 6 conidia/g of soil. The experiment was carried out with three biological replicates (five seedlings per replicate) for each treatment. The same sand-soil mix, but without Trichoderma harzianum , was added to the uninoculated control plants. All plants were grown in a greenhouse maintained at a temperature of 25–29°C with 70% humidity, under a 14-hour light and 10-hour dark cycle. Three weeks after seedling transplantation, plants were inoculated with the Fol pathogen by adding a Fol spore suspension to the soil-sand mixture at a concentration of 10 6 conidia/g in all Fol-challenged and Trichoderma harzianum supplemented plants, while the uninoculated control plants were left untreated. All plants were kept for two weeks after transplantation. A scale of 1 to 4 was used to assess disease severity as follows: healthy plant; generally healthy but with start of wilting; unhealthy plant with sever wilt; and dead plant. Data analysis General Linear Model (GLM) ANOVA (SPSS Version 16.0) was used to find the differences ( P ≤ 0.05) between inoculated and non-inoculated (control) plants as regards the mean of wet weight and dry weight for shoots and roots. Data from shoots assessments (using ratings of 1 to 4) were also analyzed by GLM ANOVA to find the difference ( P ≤ 0.05) between inoculated and control plants as regards wilt disease levels. Results Pathogen identification: All the isolates obtained were identified as Fusarium oxysporum f. sp. lycopersici . Isolate tested morphological displayed pink-white fungal hyphae with a cottony appearance. Microscopic examination revealed asexual structures, including both microconidia and macroconidia when cultured on PDA plates. Koch’s postulates were applied to test the pathogenicity of all Fusarium oxysporum f. sp. lycopersici isolates. Based on the percentage of disease occurrence, highly virulent isolates were selected for further experiments. For more precise identification, molecular techniques of DNA sequences were conducted. The sequence obtained from the Fusarium oxysporum f. sp. lycopersici isolated was matched with Fusarium oxysporum f. sp. lycopersici (OQ244516) from GenBank. Identification of Trichoderma The isolated Trichoderma species was identified as Trichoderma harzianum based on its morphological and conidial characteristics, following the keys suggested by Rifai (1969) and Nagamani et al. (2002). The sequence obtained from the Trichoderma harzianum isolate matched Trichoderma harzianum (OR633234) from GenBank. Biological treatments: The disease severity scale of 1 to 4 could be used successfully to assess disease severity on tomato plants ratings of 1 and 2 were pooled to indicate generally healthy plants and ratings of 3 and 4 were pooled to indicate diseased plants. The results related to disease severity showed that the control plants appeared healthy according to disease severity scale of 1–2 with an average of (1.3) in contrast to plants treated with Fol which showed diseased plants according to disease severity scale of 3–4 with an average of (3.7) (Table 1 and Fig. 1 ). While the plants treated with Fol and to which Trichoderma harzianum was added showed healthy plants according to disease severity scale of 1–2 with an average of (1.8). There were significant differences in disease severity between control plants and plants treated with Fol after 14 days (p ≤ 0.05). Moreover, there were significant differences in in disease severity between plants treated with Fol and plants treated with Fol and Trichoderma harzianum after 14 days (p ≤ 0.05). However, the disease severity between control plats and plants treated with Fol and Trichoderma harzianum after 14 days showed healthy plant with average (1.3 and 1.8). Table 1 Disease severity on tomato plants ratings of 1 and 2 were pooled to indicate generally healthy plants and ratings of 3 and 4 were pooled to indicate diseased plants after 14 days. Within a column, means followed by the same letter are not significantly different from each other at P ≤ 0.05. Treatment Disease severity after 14 days Control 1.3 a With Fol 3.7 c With Fol and Trichoderma harzianum 1.8 b To confirm the effect of Trichoderma harzianum on controlling Fol, wet and dry weights of shoots and roots were measured after 14 days of adding the treatments. The treatments include a control group, a group treated with Fol, and a group treated with Fol along with Trichoderma harzianum . The key findings from the data are as follows: The control group, which received no additional treatments, showed the highest shoot and root weights, both wet and dry. For example, the wet shoot weight was 33.44 g and the dry shoot weight was 4.09 g. Similarly, the wet and dry root weights were 8.46 g and 1.56 g, respectively (Table 2 , Figs. 2 and 3 ). However, plants treatment with Fol significantly reduced both the shoot and root weights compared to the control. The wet shoot weight dropped to 13.11 g, and the dry weight to 1.74 g, indicating a notable reduction in plant growth. The root weights also decreased, with the wet root weight at 4.12 g and the dry root weight at 0.97 g (Table 2 , Figs. 2 and 3 ). Nevertheless, plants treated with the combination of Fol and Trichoderma harzianum improved plant growth compared to Fol alone but remained lower than the control. The wet shoot weight was 28.56 g and the dry weight was 3.45 g, showing a recovery in growth. The root weights also increased, with a wet root weight of 6.29 g and a dry weight of 1.13 g. Significant differences in all four growth parameters measured (both wet and dry weights of both shoots and roots) were found between plants inoculated with Fol and the control (uninoculated) plants (Table 2 ). Furthermore, there were significant differences in all four parameters between plants inoculated with combination of Fol and Trichoderma harzianum and the control plants. However, no significant differences were found between plants inoculated with Fol and Trichoderma harzianum and control plants as regards the dry weight of shoot parameter (Table 2 ). Table 2 Mean wet and dry weights (g) of roots and shoots after 14 days with three treatments (two inoculated and one control). Within a column, means followed by the same letter are not significantly different from each other at P ≤ 0.05. Treatment Shoot weight (g) Root weight (g) Wet Dry Wet Dry Control 33.44 a 4.09 a 8.46 a 1.56 a With Fol 13.11 c 1.74 b 4.12 c 0.97 c With Fol and Trichoderma harzianum 28.56 b 3.45 a 6.29 b 1.13 b Discussion Microbial biopriming is a versatile approach to enhancing plant defense systems, resulting in improved resistance and resilience to biotic and abiotic stresses (Zehra et al., 2017). In this study, tomato plants were primed with Trichoderma harzianum to improve their defense response against Fusarium wilt disease caused by Fusarium oxysporum f. sp. lycopersici . Tomato plants grew for 14 days after inoculation showed that the control plants appeared healthy according to disease severity scale of 1–2 with an average of (1.3) in contrast to plants treated with Fol which showed diseased plants with an average of (3.7) (Table 1 and Fig. 1 ). While the plants treated with Fol and to which Trichoderma harzianum was added showed healthy plants with an average of (1.8). Trichoderma species serve as potent biocontrol agents against Fusarium, a harmful soil-borne pathogen responsible for wilts, rots, and damping-off in crops. These beneficial fungi outcompete Fusarium for nutrients and space, produce antifungal compounds, and stimulate systemic resistance in plants. Notably, Trichoderma harzianum and Trichoderma viride are highly effective, releasing enzymes such as chitinases and β-1,3-glucanases that break down Fusarium cell walls (Abdelaziz et. al., 2023). Moreover, they enhance plant growth by facilitating better nutrient absorption. As a sustainable alternative to chemical fungicides, Trichoderma-based formulations are widely adopted in integrated pest management (IPM) strategies to suppress Fusarium infections and foster robust crop health (Zandyavari et. al., 2024). The Trichoderma harzianum priming significantly reduced disease incidence on tomato plants grown for 14 days after inculation. These findings align with the study by Saravanakumar et al. (2017), which demonstrated that a Trichoderma harzianum strain induces systemic resistance (ISR) in maize plants against Fusarium graminearum . This highlights its potential as a biocontrol agent. Numerous studies have reported the effective control of various plant pathogens, including Fusarium spp., by Trichoderma spp. through the elicitation of systemic or localized resistance. This resistance is triggered by the interaction of bioactive molecules, such as Avr-like proteins, extracellular enzymes, and cell wall fragments released during mycoparasitic activity (Can et al., 2022). Thangavelu and Mustaffa (2010) demonstrated that applying Trichoderma viride NRCB1 as a rice chaffy grain formulation, followed by challenge inoculation with Fusarium oxysporum f. sp. cubense (Foc) in the banana cultivar Rasthali, induced defense-related enzymes like peroxidase and phenylalanine ammonia-lyase (PAL). Additionally, total phenolic content increased significantly (> 50%) compared to control and Foc-inoculated plants, with peak induction observed between the 4th and 6th days post-treatment. They suggested that the enhanced activity of these lytic enzymes and the increased phenolic content in Trichoderma viride treated plants likely strengthened physical barriers or rendered tissues chemically resistant to the hydrolytic enzymes produced by the pathogen. In this study, all isolates of Fusarium oxysporum were identified by morphological methods and DNA sequencing as Fusarium oxysporum f. sp. lycopersici . These methods are considered among the most successful methods for identifying fungi, and there are many studies that have used them. Renga (et. al., 2024) used traditional and DNA sequencing techniques to identify Fusarium oxysporum f. sp. lycopersici when isolated from tomato diseased plants. Other researchers performed RNA-seq analysis for Fusarium oxysporum identification (Sun et. al., 2022; Ye et. al., 2022; Pimentel et. al., 2025). In conclusion, this study demonstrated that wilt disease caused by Fusarium oxysporum f. sp. lycopersici on tomato plants can be controlled by Trichoderma harzianum . Tomato plants treated with Fol and Trichoderma harzianum showed healthy plants compare with plants treated with Fol. Moreover, the DNA sequencing techniques of ITS give accurate identification of Fusarium oxysporum f. sp. lycopersici and Trichoderma harzianum . Declarations Author Contribution Author Contributions: Khalaf Alhussaen conceptualized this study, conducted the research, analyzed the data, and wrote the whole manuscript. References Abdelaziz AM, Sharaf MH, Hashem AH, AlAskar AA, Marey SA, Mohamed FA, Abdelstar MN, Zaki MA, Abdelgawad H, Attia MS. 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Egypt J Biol Pest Control. 2024;34:1. https://doi.org/10.1186/s41938-023-00765-1 . Zehra A, Dubey M, Meena M, Upadhyay R. Effect of different environmental conditions on growth and sporulation of some Trichoderma species. J Environ Biol. 2017;38(2):197–203. 10.22438/jeb/38/2/MS-251 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7317989","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":498999584,"identity":"f40fd7bc-73f0-402e-9597-6fe4ccdafb28","order_by":0,"name":"Khalaf Alhussaen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIiWNgGAWjYBACgxsMjIehbMYHYOoAAS2GMxgYYFqYDRgSiNBiLIHQwiZBlBYz6eYHhwvb6uz523uPVfP+YJDju5HA/OEHHi02MscMDs9sO5w448y5tNs8CQzGkjcS2CR78GmRSDA4zNt2IIHhRo4ZSEviBqAWBh58DpNI/wDUUmcvf/+NWTFQSz1QC/PHP3i0GEvkgGxhZtxwg8eMGaglweBGAoM0PlsMZ+QUHOY5dzhx45kcY8k5aRKGM888bJOWwaPF4Eb6xsc8ZXX2csfPGH54Y2Mjz3c8+fDHN3i0oANgNDEwNpCgYRSMglEwCkYBNgAA/NBPvFpLtjYAAAAASUVORK5CYII=","orcid":"","institution":"University of Tabuk","correspondingAuthor":true,"prefix":"","firstName":"Khalaf","middleName":"","lastName":"Alhussaen","suffix":""}],"badges":[],"createdAt":"2025-08-07 10:53:30","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-7317989/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7317989/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88907284,"identity":"1155f779-3214-43a0-90a5-1dcabb15ad02","added_by":"auto","created_at":"2025-08-12 14:46:17","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":30600,"visible":true,"origin":"","legend":"\u003cp\u003eDisease severity on tomato plants ratings of 1 and 2 were pooled to indicate generally healthy plants and ratings of 3 and 4 were pooled to indicate diseased plants after 14 days. Error bars are the standard error of the mean.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7317989/v1/282586172caac6c2645110df.jpg"},{"id":88907283,"identity":"a30dc41a-e0f9-4609-b785-587ddfb1c4ef","added_by":"auto","created_at":"2025-08-12 14:46:17","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":34379,"visible":true,"origin":"","legend":"\u003cp\u003eMean wet and dry weights (g) of shoots after 14 days with three treatments (two inoculated and one control). Within a column, means followed by the same letter are not significantly different from each other at \u003cem\u003eP\u003c/em\u003e≤ 0.05.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7317989/v1/15cb25cafc71ad856c6fc3af.jpg"},{"id":88907285,"identity":"e6e423b3-4479-4fab-b36d-86e24047a2a6","added_by":"auto","created_at":"2025-08-12 14:46:17","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":31046,"visible":true,"origin":"","legend":"\u003cp\u003eMean wet and dry weights (g) of roots after 14 days with three treatments (two inoculated and one control). Within a column, means followed by the same letter are not significantly different from each other at P≤ 0.05.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7317989/v1/081fffbde1792df69f5a2cff.jpg"},{"id":88908570,"identity":"64a34812-5b48-46ed-bea7-2a6ce49579b9","added_by":"auto","created_at":"2025-08-12 14:54:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":538294,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7317989/v1/9399d0ae-35f9-4614-87be-e3aa9f3f9c82.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhancing the tomato plant defense against Fusarium wilt by using Trichoderma harzianum","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eFusarium oxysporum\u003c/em\u003e is a soil-borne fungal pathogen that causes wilt diseases in a wide variety of plants, including many economically important crops. \u003cem\u003eFusarium oxysporum\u003c/em\u003e is a filamentous fungus that primarily inhabits the soil. It can survive for long periods as chlamydospores, which are thick-walled, resting spores that allow the fungus to persist in the absence of a host. The fungus infects plants through the roots, colonizing the vascular system and leading to blockage of water transport, which results in wilting and often plant death (Jackson et. al., 2024).\u003c/p\u003e\u003cp\u003e\u003cem\u003eFusarium oxysporum\u003c/em\u003e is known for its broad host range, affecting hundreds of plant species across numerous families. However, it is typically host-specific at the forma specialis (f. sp.) level, with different forma specialis adapted to specific hosts (Edel-Hermann and Lecomte, 2019). Some well-known forma specialis include \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e causes wilt in tomatoes (Jamil et. al,. 2021; Komissarov et. al., 2025), \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003ecubense\u003c/em\u003e causes Panama disease in bananas (Magdama et. al., 2020) and \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003evasinfectum\u003c/em\u003e infects cotton (Asif et. al., 2023).\u003c/p\u003e\u003cp\u003eThe primary symptom of Fusarium wilt is wilting of the plant, often starting with lower leaves and progressing upwards (Pandey et. al., 2023). Other symptoms include yellowing of the foliage, stunted growth, and vascular discoloration, which can be observed as brown streaks in the stem when it is cut open.\u003c/p\u003e\u003cp\u003e\u003cem\u003eFusarium oxysporum\u003c/em\u003e spreads through infected soil, water, plant debris, and contaminated tools. It can also be transmitted via infected seedlings and plant material. The fungus thrives in warm, moist conditions, making it particularly problematic in tropical and subtropical regions.\u003c/p\u003e\u003cp\u003e\u003cem\u003eFusarium oxysporum\u003c/em\u003e poses a significant threat to agriculture due to its ability to cause severe yield losses in a variety of crops. Management of Fusarium wilt is challenging because the fungus persists in the soil for many years. and there are limited effective chemical control options. Effective management of Fusarium wilt involves an integrated approach include planting resistant or tolerant crop varieties, implementing crop rotation, proper sanitation, and soil health management, utilizing antagonistic microorganisms that inhibit \u003cem\u003eFusarium oxysporum\u003c/em\u003e and limited use of fungicides, often as part of an integrated pest management (IPM) strategy (Lal et. al., 2024).\u003c/p\u003e\u003cp\u003eTrichoderma is a genus of fungi that is widely recognized for its beneficial properties in agriculture and biotechnology. Trichoderma species are filamentous fungi commonly found in soil, decaying wood, and other organic matter. They are known for their rapid growth and prolific spore production. Trichoderma species are characterized by their green spores and white to greenish mycelium (Jorge et. al., 2024).\u003c/p\u003e\u003cp\u003eTrichoderma fungi exhibit several mechanisms that make them beneficial for plant health and soil quality. Trichoderma species can parasitize other fungi, including many plant pathogens, by directly attacking and degrading their cell walls. They produce various secondary metabolites, including antibiotics, which inhibit the growth of competing microorganisms. Moreover, Trichoderma competes effectively for nutrients and space, outcompeting pathogenic fungi (Waleed et. al., 2024). Trichoderma can stimulate plant defense mechanisms, enhancing the plant's own ability to resist diseases.\u003c/p\u003e\u003cp\u003eTrichoderma species are widely used as biological control agents and biofertilizers due to their ability to enhance plant growth and suppress soil-borne pathogens. Trichoderma is used to control various plant diseases caused by fungi, such as \u003cem\u003eFusarium, Rhizoctonia\u003c/em\u003e, and \u003cem\u003ePythium\u003c/em\u003e (Awal et. al., 2024). Trichoderma enhances nutrient uptake, promotes root growth, and improves plant vigor. Moreover, beyond agriculture, Trichoderma species have significant industrial applications, Trichoderma is known for its ability to produce a wide range of enzymes, including cellulases, hemicellulases, and chitinases, which are used in various industries such as biofuel production, paper, and pulp processing (Lima et. al., 2024).\u003c/p\u003e\u003cp\u003eOngoing research is focused on enhancing the efficacy of Trichoderma-based products and understanding the molecular mechanisms underlying their beneficial effects. Genetic and genomic studies are providing insights into the pathways involved in mycoparasitism, antibiosis, and plant growth promotion (Alizadeh et. al., 2024).\u003c/p\u003e\u003cp\u003eThe use of Trichoderma in agriculture offers a sustainable and eco-friendly alternative to chemical pesticides and fertilizers. By promoting plant health and reducing dependency on synthetic chemicals, Trichoderma contributes to sustainable agricultural practices and environmental conservation.\u003c/p\u003e\u003cp\u003eThe current study aims to demonstrate the extent of the effect of using \u003cem\u003eTrichoderma harzianum\u003c/em\u003e in reducing the effect of \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e (Fol) on tomato plants.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003ePathogen isolation:\u003c/p\u003e\u003cp\u003e\u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e (Fol) was isolated from diseased tomato plants exhibiting typical wilt symptoms grown in Jordan Valley on November 2024. The isolation was carried out on potato dextrose agar (PDA) and incubated at 25\u0026ndash;27\u0026deg;C for 5\u0026ndash;7 days. Stock cultures of Fol were prepared and stored at 4\u0026deg;C. Pathogenicity tests for all Fol isolates were conducted according to Koch\u0026rsquo;s postulates, and a highly virulent isolate was selected for further experiments. The experiments were conducted in Biology department in Yarmouk University in Jordan on 2024.\u003c/p\u003e\u003cp\u003eIsolation of Trichoderma spp.\u003c/p\u003e\u003cp\u003e\u003cem\u003eTrichoderma\u003c/em\u003e spp. were isolated from soil samples taken from the tomato rhizosphere across various locations in the agricultural field in Jordan Valley on 2024. The samples were stored at 4\u0026deg;C in the laboratory, and the isolation of different \u003cem\u003eTrichoderma\u003c/em\u003e spp. was carried out using the serial dilution method (Zehra et al., 2017).\u003c/p\u003e\u003cp\u003eFungal DNA extraction and ITS sequencing\u003c/p\u003e\u003cp\u003eGenomic DNA was extracted from the one-week-old fungal isolate using the method described by Doyle and Doyle (1987). The internal transcribed spacer (ITS) region of the rDNA was amplified using the universal primers ITS1/ITS4 through polymerase chain reaction (PCR) (Meena et al., 2017). The purified PCR products were then sequenced at both ends and analyzed. The resulting sequences were compared against those in GenBank using the National Center for Biotechnology Information's Basic Local Alignment Search Tool (NCBI-BLAST) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePlant material:\u003c/p\u003e\u003cp\u003eTomato seeds (\u003cem\u003eLycopersicon esculentum\u003c/em\u003e) were obtained from local specialized store for selling seeds in the Jordan Valley on 2024. The seeds were surface-sterilized in a 4% sodium hypochlorite (NaOCl) solution containing 0.02% (v/v) Tween-20, thoroughly rinsed with sterilized water, and then allowed to germinate (Abdelshafy et al., 2020).\u003c/p\u003e\u003cp\u003eBiological treatments and growth condition:\u003c/p\u003e\u003cp\u003eThe experiment was conducted in pots (20 cm in diameter), which were surface-sterilized with 0.1% mercuric chloride. The treatments were arranged as follows: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) uninoculated as control, (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) pathogen (Fol) challenged, and (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) plants primed with \u003cem\u003eTrichoderma harzianum\u003c/em\u003e spores and subsequently challenged with the pathogen (Fol).\u003c/p\u003e\u003cp\u003eSeedlings were transferred to pots filled with a sterile soil and sand mixture (autoclaved at 121\u0026deg;C and 15 psi pressure for 15 minutes) in a 4:1 ratio, along with Trichoderma inoculum at a concentration of 10\u003csup\u003e6\u003c/sup\u003e conidia/g of soil. The experiment was carried out with three biological replicates (five seedlings per replicate) for each treatment. The same sand-soil mix, but without \u003cem\u003eTrichoderma harzianum\u003c/em\u003e, was added to the uninoculated control plants. All plants were grown in a greenhouse maintained at a temperature of 25\u0026ndash;29\u0026deg;C with 70% humidity, under a 14-hour light and 10-hour dark cycle.\u003c/p\u003e\u003cp\u003eThree weeks after seedling transplantation, plants were inoculated with the Fol pathogen by adding a Fol spore suspension to the soil-sand mixture at a concentration of 10\u003csup\u003e6\u003c/sup\u003e conidia/g in all Fol-challenged and \u003cem\u003eTrichoderma harzianum\u003c/em\u003e supplemented plants, while the uninoculated control plants were left untreated. All plants were kept for two weeks after transplantation.\u003c/p\u003e\u003cp\u003eA scale of 1 to 4 was used to assess disease severity as follows:\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003ehealthy plant;\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003egenerally healthy but with start of wilting;\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eunhealthy plant with sever wilt; and\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003edead plant.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eData analysis\u003c/h2\u003e\u003cp\u003eGeneral Linear Model (GLM) ANOVA (SPSS Version 16.0) was used to find the differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05) between inoculated and non-inoculated (control) plants as regards the mean of wet weight and dry weight for shoots and roots. Data from shoots assessments (using ratings of 1 to 4) were also analyzed by GLM ANOVA to find the difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05) between inoculated and control plants as regards wilt disease levels.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003ePathogen identification:\u003c/h2\u003e\u003cp\u003eAll the isolates obtained were identified as \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e. Isolate tested morphological displayed pink-white fungal hyphae with a cottony appearance. Microscopic examination revealed asexual structures, including both microconidia and macroconidia when cultured on PDA plates.\u003c/p\u003e\u003cp\u003eKoch\u0026rsquo;s postulates were applied to test the pathogenicity of all \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e isolates. Based on the percentage of disease occurrence, highly virulent isolates were selected for further experiments.\u003c/p\u003e\u003cp\u003eFor more precise identification, molecular techniques of DNA sequences were conducted. The sequence obtained from the \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e isolated was matched with \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e (OQ244516) from GenBank.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eIdentification of Trichoderma\u003c/h3\u003e\n\u003cp\u003eThe isolated \u003cem\u003eTrichoderma\u003c/em\u003e species was identified as \u003cem\u003eTrichoderma harzianum\u003c/em\u003e based on its morphological and conidial characteristics, following the keys suggested by Rifai (1969) and Nagamani et al. (2002). The sequence obtained from the \u003cem\u003eTrichoderma harzianum\u003c/em\u003e isolate matched \u003cem\u003eTrichoderma harzianum\u003c/em\u003e (OR633234) from GenBank.\u003c/p\u003e\n\u003ch3\u003eBiological treatments:\u003c/h3\u003e\n\u003cp\u003eThe disease severity scale of 1 to 4 could be used successfully to assess disease severity on tomato plants ratings of 1 and 2 were pooled to indicate generally healthy plants and ratings of 3 and 4 were pooled to indicate diseased plants.\u003c/p\u003e\u003cp\u003eThe results related to disease severity showed that the control plants appeared healthy according to disease severity scale of 1\u0026ndash;2 with an average of (1.3) in contrast to plants treated with Fol which showed diseased plants according to disease severity scale of 3\u0026ndash;4 with an average of (3.7) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). While the plants treated with Fol and to which \u003cem\u003eTrichoderma harzianum\u003c/em\u003e was added showed healthy plants according to disease severity scale of 1\u0026ndash;2 with an average of (1.8).\u003c/p\u003e\u003cp\u003eThere were significant differences in disease severity between control plants and plants treated with Fol after 14 days (p\u0026thinsp;\u0026le;\u0026thinsp;0.05). Moreover, there were significant differences in in disease severity between plants treated with Fol and plants treated with Fol and \u003cem\u003eTrichoderma harzianum\u003c/em\u003e after 14 days (p\u0026thinsp;\u0026le;\u0026thinsp;0.05). However, the disease severity between control plats and plants treated with Fol and \u003cem\u003eTrichoderma harzianum\u003c/em\u003e after 14 days showed healthy plant with average (1.3 and 1.8).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDisease severity on tomato plants ratings of 1 and 2 were pooled to indicate generally healthy plants and ratings of 3 and 4 were pooled to indicate diseased plants after 14 days. Within a column, means followed by the same letter are not significantly different from each other at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDisease severity after 14 days\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.3 a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWith Fol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.7 c\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWith Fol and \u003cem\u003eTrichoderma harzianum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.8 b\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo confirm the effect of \u003cem\u003eTrichoderma harzianum\u003c/em\u003e on controlling Fol, wet and dry weights of shoots and roots were measured after 14 days of adding the treatments. The treatments include a control group, a group treated with Fol, and a group treated with Fol along with \u003cem\u003eTrichoderma harzianum\u003c/em\u003e. The key findings from the data are as follows:\u003c/p\u003e\u003cp\u003eThe control group, which received no additional treatments, showed the highest shoot and root weights, both wet and dry. For example, the wet shoot weight was 33.44 g and the dry shoot weight was 4.09 g. Similarly, the wet and dry root weights were 8.46 g and 1.56 g, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHowever, plants treatment with Fol significantly reduced both the shoot and root weights compared to the control. The wet shoot weight dropped to 13.11 g, and the dry weight to 1.74 g, indicating a notable reduction in plant growth. The root weights also decreased, with the wet root weight at 4.12 g and the dry root weight at 0.97 g (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eNevertheless, plants treated with the combination of Fol and \u003cem\u003eTrichoderma harzianum\u003c/em\u003e improved plant growth compared to Fol alone but remained lower than the control. The wet shoot weight was 28.56 g and the dry weight was 3.45 g, showing a recovery in growth. The root weights also increased, with a wet root weight of 6.29 g and a dry weight of 1.13 g.\u003c/p\u003e\u003cp\u003eSignificant differences in all four growth parameters measured (both wet and dry weights of both shoots and roots) were found between plants inoculated with Fol and the control (uninoculated) plants (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Furthermore, there were significant differences in all four parameters between plants inoculated with combination of Fol and \u003cem\u003eTrichoderma harzianum\u003c/em\u003e and the control plants. However, no significant differences were found between plants inoculated with Fol and \u003cem\u003eTrichoderma harzianum\u003c/em\u003e and control plants as regards the dry weight of shoot parameter (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMean wet and dry weights (g) of roots and shoots after 14 days with three treatments (two inoculated and one control). Within a column, means followed by the same letter are not significantly different from each other at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eShoot weight (g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eRoot weight (g)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWet\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDry\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWet\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eDry\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e33.44 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.09 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8.46 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.56 a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWith Fol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13.11 c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.74 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.12 c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.97 c\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWith Fol and \u003cem\u003eTrichoderma harzianum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e28.56 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.45 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.29 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.13 b\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMicrobial biopriming is a versatile approach to enhancing plant defense systems, resulting in improved resistance and resilience to biotic and abiotic stresses (Zehra et al., 2017). In this study, tomato plants were primed with \u003cem\u003eTrichoderma harzianum\u003c/em\u003e to improve their defense response against Fusarium wilt disease caused by \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e. Tomato plants grew for 14 days after inoculation showed that the control plants appeared healthy according to disease severity scale of 1\u0026ndash;2 with an average of (1.3) in contrast to plants treated with Fol which showed diseased plants with an average of (3.7) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). While the plants treated with Fol and to which \u003cem\u003eTrichoderma harzianum\u003c/em\u003e was added showed healthy plants with an average of (1.8).\u003c/p\u003e\u003cp\u003eTrichoderma species serve as potent biocontrol agents against Fusarium, a harmful soil-borne pathogen responsible for wilts, rots, and damping-off in crops. These beneficial fungi outcompete Fusarium for nutrients and space, produce antifungal compounds, and stimulate systemic resistance in plants. Notably, \u003cem\u003eTrichoderma harzianum\u003c/em\u003e and Trichoderma viride are highly effective, releasing enzymes such as chitinases and β-1,3-glucanases that break down Fusarium cell walls (Abdelaziz et. al., 2023). Moreover, they enhance plant growth by facilitating better nutrient absorption. As a sustainable alternative to chemical fungicides, Trichoderma-based formulations are widely adopted in integrated pest management (IPM) strategies to suppress Fusarium infections and foster robust crop health (Zandyavari et. al., 2024).\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eTrichoderma harzianum\u003c/em\u003e priming significantly reduced disease incidence on tomato plants grown for 14 days after inculation. These findings align with the study by Saravanakumar et al. (2017), which demonstrated that a \u003cem\u003eTrichoderma harzianum\u003c/em\u003e strain induces systemic resistance (ISR) in maize plants against \u003cem\u003eFusarium graminearum\u003c/em\u003e. This highlights its potential as a biocontrol agent. Numerous studies have reported the effective control of various plant pathogens, including \u003cem\u003eFusarium\u003c/em\u003e spp., by \u003cem\u003eTrichoderma\u003c/em\u003e spp. through the elicitation of systemic or localized resistance. This resistance is triggered by the interaction of bioactive molecules, such as Avr-like proteins, extracellular enzymes, and cell wall fragments released during mycoparasitic activity (Can et al., 2022). Thangavelu and Mustaffa (2010) demonstrated that applying \u003cem\u003eTrichoderma viride\u003c/em\u003e NRCB1 as a rice chaffy grain formulation, followed by challenge inoculation with \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003ecubense\u003c/em\u003e (Foc) in the banana cultivar Rasthali, induced defense-related enzymes like peroxidase and phenylalanine ammonia-lyase (PAL). Additionally, total phenolic content increased significantly (\u0026gt;\u0026thinsp;50%) compared to control and Foc-inoculated plants, with peak induction observed between the 4th and 6th days post-treatment. They suggested that the enhanced activity of these lytic enzymes and the increased phenolic content in \u003cem\u003eTrichoderma viride\u003c/em\u003e treated plants likely strengthened physical barriers or rendered tissues chemically resistant to the hydrolytic enzymes produced by the pathogen.\u003c/p\u003e\u003cp\u003eIn this study, all isolates of \u003cem\u003eFusarium oxysporum\u003c/em\u003e were identified by morphological methods and DNA sequencing as \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e. These methods are considered among the most successful methods for identifying fungi, and there are many studies that have used them. Renga (et. al., 2024) used traditional and DNA sequencing techniques to identify \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e when isolated from tomato diseased plants. Other researchers performed RNA-seq analysis for \u003cem\u003eFusarium oxysporum\u003c/em\u003e identification (Sun et. al., 2022; Ye et. al., 2022; Pimentel et. al., 2025).\u003c/p\u003e\u003cp\u003eIn conclusion, this study demonstrated that wilt disease caused by \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e on tomato plants can be controlled by \u003cem\u003eTrichoderma harzianum\u003c/em\u003e. Tomato plants treated with Fol and \u003cem\u003eTrichoderma harzianum\u003c/em\u003e showed healthy plants compare with plants treated with Fol. Moreover, the DNA sequencing techniques of ITS give accurate identification of \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e and \u003cem\u003eTrichoderma harzianum\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthor Contributions: Khalaf Alhussaen conceptualized this study, conducted the research, analyzed the data, and wrote the whole manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdelaziz AM, Sharaf MH, Hashem AH, AlAskar AA, Marey SA, Mohamed FA, Abdelstar MN, Zaki MA, Abdelgawad H, Attia MS. Biocontrol of \u003cem\u003eFusarium\u003c/em\u003e wilt disease in pepper plant by plant growth promoting \u003cem\u003ePenicillium expansum\u003c/em\u003e and \u003cem\u003eTrichoderma harzianum\u003c/em\u003e. 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J Environ Biol. 2017;38(2):197\u0026ndash;203. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.22438/jeb/38/2/MS-251\u003c/span\u003e\u003cspan address=\"10.22438/jeb/38/2/MS-251\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Fusarium wilt, Trichoderma harzianum, Fusarium oxysporum f. sp. cubense","lastPublishedDoi":"10.21203/rs.3.rs-7317989/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7317989/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFusarium oxysporum is a soil-borne fungal pathogen that causes wilt diseases in a wide variety of plants. Fusarium oxysporum poses a significant threat to agriculture due to its ability to cause severe yield losses in a variety of crops. Management of Fusarium wilt is challenging because the fungus persists in the soil for many years. and there are limited effective chemical control options. This research is designed to study the effect of Trichoderma harzianum in reducing the effect of \u003cem\u003eFusarium oxysporum f. sp. lycopersici\u003c/em\u003e (Fol) on tomato plants. \u003cem\u003eFusarium oxysporum\u003c/em\u003e was isolated from diseased tomato plants and identified by using morphology and ITS sequencing of DNA. Three biological treatments were applied to examine the effect of \u003cem\u003eTrichoderma harzianum\u003c/em\u003e on controlling disease wilt caused by \u003cem\u003eFusarium oxysporum\u003c/em\u003e. The results related to disease severity showed that the control plants appeared healthy according to disease severity scale of 1\u0026ndash;2 with an average of (1.3) in contrast to plants treated with Fol which showed diseased plants according to disease severity scale of 3\u0026ndash;4 with an average of (3.7). While the plants treated with Fol and to which \u003cem\u003eTrichoderma harzianum\u003c/em\u003e was added showed healthy plants according to disease severity scale of 1\u0026ndash;2 with an average of (1.8).\u003c/p\u003e","manuscriptTitle":"Enhancing the tomato plant defense against Fusarium wilt by using Trichoderma harzianum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-12 14:46:12","doi":"10.21203/rs.3.rs-7317989/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"28857cb8-d066-44c3-a9d2-e1555ae33c45","owner":[],"postedDate":"August 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-12T14:46:12+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-12 14:46:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7317989","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7317989","identity":"rs-7317989","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}