Sustainable control of onion Fusarium basal rot using seed-applied Trichoderma spp

TL;DR

This study shows that using Trichoderma fungi on onion seeds can effectively and sustainably control a damaging disease caused by Fusarium.

Contribution

The study identifies specific Trichoderma strains that effectively suppress Fusarium pathogens in onions with minimal impact on plant growth.

Findings

Three Trichoderma strains (T. asperellum, T. longibrachiatum, T. beinertii) showed strong antagonism against Fusarium spp.

Trichoderma seed coatings increased plant growth and reduced disease symptoms under pathogen stress.

The treatment suppressed infection by up to 59% and improved shoot and bulb weights significantly.

Abstract

Fusarium Basal rot disease (FBR) poses a global threat to onion production, with damage occurring throughout the entire crop cycle. The present study evaluated Trichoderma-based protection against major Fusarium pathogens in Israel through a series of experiments, from in vitro antagonism and seed assays to a semi-field pot trial spanning a full growing season. In confrontation assays, 10 out of 15 Trichoderma strains exhibited statistically significant inhibitory activity against the Fusarium spp., with inhibition levels varying by strain and antagonistic mechanism. Among the most effective isolates, T. asperellum (P1), T. longibrachiatum (T7407), and T. beinertii (T14707) consistently demonstrated significant antagonism through both secreted metabolites and volatile compounds. Unlike other Trichoderma strains, these three caused moderate effects on seed germination and early growth. Seed assays further revealed cultivar-dependent sensitivity, with the Orlando yellow variety more susceptible than the Maadim red, and Neocosmospora falciformis emerging as the most aggressive pathogen. Seed coatings with selected Trichoderma strains, tested under semi-field conditions, significantly promoted plant growth, with effects evident as early as mid-season. Shoot fresh biomass increased by 54–131% compared with untreated controls, accompanied by parallel improvements in other growth parameters. At the season’s end, T. asperellum treatments showed consistent positive effects, significantly enhancing shoot weight (28–119%) and bulb weight (15–157%) under pathogen stress. These improvements were accompanied by up to 36% reduction in foliar symptoms and 59% suppression of infection (tracked by qPCR). Overall, the study demonstrates the promise of Trichoderma-based seed treatments as an effective and sustainable strategy for managing onion FBR.

The online version contains supplementary material available at 10.1007/s11274-026-04860-x.

Figures

Plate confrontation assay showing the percentage inhibition of *Fusarium* species (Table [1](#Tab1)) colony growth after 6 days of incubation with the *Trichoderma* biological control agents (Table [2](#Tab2)). Bars represent the mean of 4–5 biological replicates, and error bars indicate the standard error. Different lowercase letters (a–g) above the bars indicate statistically significant differences between treatments (*p* < 0.05), determined using the Kruskal–Wallis non-parametric test followed by uncorrected Dunn’s test

Secreted volatile compound inhibition assay showing the percentage reduction in *Fusarium* species (Table [1](#Tab1)) colony growth after 4 days of incubation in the presence of *Trichoderma* biological control agents (Table [2](#Tab2)). Bars represent the mean of 3–4 independent biological replicates, and error bars indicate the standard error. Different lowercase letters (a–c) above the bars denote statistically significant differences between treatments (*p* < 0.05), determined using the Kruskal–Wallis test followed by uncorrected Dunn’s multiple comparisons test for *F. proliferatum*, or o

Semi-field, open-enclosure, full-season pot trial. The experiment evaluated *Trichoderma*-based seed coating to protect the susceptible Orland variety against *Fusarium* basal rot. Experimental onion plants photographed at 60 (A) and 91 (B) days after sowing. Early disease symptoms are visible in 25-day-old sprouts (C), which may progress to damping-off (D). Panel E shows a symptomatic 76-day-old plantFig. 8Semi-field mid-season evaluation. Treatments included soil inoculation with *Fusarium* spp. (Table [1](#Tab1)) and *Trichoderma*-based seed coating (species details in Table [2](#Tab2)). Pa

Full text

Introduction

Onion (Allium cepa L.) is a major agricultural crop worldwide. In 2023, the global cultivation area of onions and shallots reached approximately 5,842,332 hectares, with an estimated production of about 111,273,600 tons of dry cultivars (FAO, 2023). Fusarium basal rot (FBR), primarily driven by Fusarium oxysporum f. sp. cepae (FOC), poses a serious universal threat to onion production, frequently leading to devastating yield and storage losses. In intensive onion-growing systems and high disease pressure, FBR has been reported to reduce yields by more than 50% (Lacy and Roberts 1982; Shin et al. 2023). In onion production regions, diverse *Fusarium *species have been detected, with nine species reported to date (Le et al. 2021b). While FBR can be incited by a single species, it is more often a disease complex involving multiple species (Haapalainen et al. 2023). The specific contribution of each species within this complex remains poorly understood, but those species interact synergistically or antagonistically, affecting the disease severity (Degani et al. 2024c; Dimant and Degani 2023). Moreover, the composition of the FBR complex varies depending on geographic region, plant host, and temporal shifts within and between growing seasons (Le et al. 2021a, b). For example, in Israel, the Fusarium pathogen population comprises FOC, F. proliferatum, F. acutatum, and Neocosmospora falciformis (Degani et al. 2024c). In that study, it was observed that although the overall distribution is relatively consistent across regions, it varies between onion cultivars. Interestingly, species from the Neocosmospora complex were detected in all onion samples, suggesting a generalist nature—widely distributed but typically less virulent than other species. However, within this complex, N. falciformis was identified as more aggressive compared to other species. In Finland, a pathogenic FOC isolate was reported, indicating that within the F. oxysporum forma specialis *cepae *population, both highly aggressive and less aggressive variants occur (Wang et al. 2019). Such pathogen diversity increases the disease pressure, hinders effective control measures, and amplifies crop losses (Degani et al. 2024c). FBR is expressed throughout the crop cycle, from seedling damping-off to bulb rotting at maturity. Typical symptoms include foliar yellowing, leaf tip necrosis, leaf curling, and basal plate rot, often progressing upward into the bulb scales (Shin et al. 2023). Beyond the field, FBR also reduces postharvest quality (Wesoly et al. 2024), with storage losses reaching 30–40% in some regions (Gupta and Gupta 2013; Mishra et al. 2014). The impact varies geographically with shifts in pathogen populations, and is further aggravated by the capacity of several Fusarium species to produce harmful mycotoxins (Le et al. 2021a; Stankovic et al. 2007). Together, these factors highlight the significant economic and food security implications of the disease.

Given the economic impact of FBR and considering increasing public concern over chemical pesticide use and the resulting regulatory constraints, recent research has prioritized the development of sustainable and resilient control strategies. Integrated disease management approaches combining genetic resistance, biocontrol agents, and precision diagnostics have demonstrated promising potential. For instance, advancements in resistant onion breeding have yielded genotypes with improved FBR tolerance, supported by marker-assisted selection using quantitative trait loci (QTLs) linked to resistance traits (Sharma and Cramer 2023). Simultaneously, rapid detection technologies—such as volatile organic compound (VOC) profiling—are being explored to enable early, non-destructive field or storage screening for FBR, offering pathways to limit spread and inform timely interventions (Labanska et al. 2022).

Biological control has emerged as a promising approach for managing onion FBR. Different biological agents have shown efficacy against the disease in various Allium species (Sharma et al. 2024). For instance, yeast species (Saccharomyces cerevisiae, Candida tropicalis) have demonstrated strong inhibitory effects against FOC in vitro and under storage, greenhouse, and field conditions, achieving results comparable to fungicides (Ahmed et al. 2021). Likewise, soilborne bacteria, including Bacillus, Pseudomonas, Enterobacter, and Citrobacter species, have shown significant antagonism through mechanisms such as antibiosis, siderophore and protease production, and phytohormone (IAA) synthesis, in some cases reducing disease incidence by over 70% (Sharma et al. 2024). An increasing number of studies have demonstrated the efficacy of Trichoderma species, their bioactive metabolites, and their synergistic interactions with other beneficial microorganisms, underscoring their strong potential as biological control agents (Coşkuntuna and Özer 2008; Kredics et al. 2024). Coşkuntuna and Özer (2008) reported that T. harzianum inhibited the growth of FOC by 73% in vitro, showing efficacy comparable to prochloraz, a broad-spectrum chemical fungicide. In both growth chamber and field trials, this treatment exhibited antifungal activity against FOC, reducing the disease incidence significantly. Similarly, Bunbury-Blanchette and Walker (2019) evaluated several Trichoderma species—T. harzianum, T. hamatum, T. gamsii, and T. atroviride—isolated from local soils for their ability to suppress onion FBR caused by FOC. In culture bioassays, these isolates exhibited antagonistic mechanisms, including hyphal barrier formation and inhibition of FOC growth. To further enhance their effectiveness, other studies explored synergistic applications with botanical extracts. For instance, Astiko and Sudantha (2023) evaluated a liquid biofungicide derived from Legundi leaves (Vitex trifolia) fermented with T. harzianum for managing Fusarium wilt of shallots caused by FOC. The formulation was applied after planting by pipette into the rhizosphere at defined doses. Under these conditions, the biofungicide reduced disease incidence relative to the untreated control.

The antifungal activity of Trichoderma spp. can be further strengthened through synergistic interactions with other beneficial microorganisms such as arbuscular mycorrhizal fungi (AMF) and plant growth-promoting rhizobacteria (PGPR). Recent studies have demonstrated that combining Trichoderma with AMF, particularly Funneliformis mosseae, significantly reduced the severity of FBR in onions by ca. 20%, while also enhancing plant growth and phosphorus uptake (Yağmur et al. 2024). Similar benefits were reported in shallots, where AMF–Trichoderma treatments lowered wilt severity to below 2% and tended to improve bulb development (Afiefah et al. 2020). Beyond AMF, dual applications of Trichoderma with PGPR, such as Arthrobacter ureafaciens, have shown improved control of Fusarium crown rot in wheat and increased crop yields (Yang et al. 2025), while AMF–PGPR combinations enhanced plant defense responses in cucumber Fusarium wilt (Oulad Ziane et al. 2023). Together, these findings highlight the potential of integrated biocontrol strategies that combine Trichoderma with AMF and PGPR to achieve more stable and resilient suppression of FBR and other soilborne Fusarium diseases under field conditions. Although these studies emphasize the potential of diverse biocontrol agents, further optimization of application strategies and their integration with conventional methods are required to achieve consistent and durable management of FBR.

Fusarium basal rot management continues to face numerous challenges that are further intensified by global changes, including climatic fluctuations and shifting agricultural practices. Effective solutions must therefore be tailored to local pathogen populations, onion cultivars, and specific environmental conditions. In this context, the present study provides an integrated evaluation of Trichoderma-based seed-applied strategies for managing onion FBR under Israeli growing conditions. To address this, we designed a series of experiments ranging from in vitro antagonism assays—examining mycoparasitism, secretion of metabolites, and volatile compound activity—to seed bioassays and a full-season open-enclosure potted trial. The in vivo trial was further supported by detailed phenological monitoring and real-time PCR-based molecular tracking of pathogens’ dynamics within onion tissues, providing an integrated framework to assess the potential of Trichoderma as a sustainable biocontrol agent under local conditions.

Materials and methods

Rationale and research design

The study followed a stepwise experimental design aimed at identifying the most effective Trichoderma strain for advanced evaluation. Initially, 15 strains were screened using an in vitro plate confrontation assay, from which five were further assessed for inhibition via secreted metabolites and volatile compounds. After confirming that seed coatings did not impair germination or early growth, three strains were selected for full-season potted trials. Conducting the trial in pots under open-field, semi-field conditions provided several advantages over conventional open-field (farm-scale) experiments. The semi-field system consisted of a potted, open-enclosure setup conducted outdoors under natural environmental conditions (ambient temperature, solar radiation, and humidity), while maintaining experimental control over soil type, inoculum density, irrigation, and fertilization. Unlike greenhouse experiments, where environmental parameters are partially controlled but often still influenced by external conditions, this approach exposed plants to realistic field-like environmental fluctuations. At the same time, growing plants in pots rather than in-ground plots allowed tighter control of the soil conditions and manipulation of pathogen pressure. Moreover, this setup enabled the incorporation of mock controls—plants grown in natural local soil without additional inoculation—thereby providing a robust baseline for comparison.

Fusarium species origin used in this study

Four Fusarium species were included in this study: F. proliferatum (B1), F. acutatum (B5), F. oxysporum f. sp. cepae (FOC, B14), and N. falciformis (E3) (Table 1). All isolates originated from diseased onion plants collected in commercial fields in the Golan Heights, north-eastern Israel. Species identification was based on morphological and microscopic characterization combined with PCR amplification using fungi-specific and Fusarium-specific markers, including ITS, β-tubulin, TEF1α, SIX3, and CLPRO, with additional confirmation by ISSR markers and phylogenetic analyses (Degani et al. 2024c; Kalman et al. 2020). Sequence data are available online and in GenBank (accession numbers in Table 1). Pathogenicity was confirmed according to Koch’s postulates using seedling and bulb inoculation assays, and virulence and fungicide sensitivity were evaluated as described previously (Degani et al. 2022; Degani and Kalman 2021; Dimant and Degani 2023).Table 1. The Fusarium isolates used in this studyFusarium spp.IsolateGenBank AccessionNumbersCollection SiteSourceReferenceF. proliferatumB1OR206094.1Kibbutz OrtalRiverside (yellow var.)(Kalman et al. 2020)F. acutatumB5OR206092.1Kibbutz OrtalRiverside(Kalman et al. 2020)F. oxysporum f. sp. cepaeB14OR206089.1Moshav EliadHa3 (red var.)(Kalman et al. 2020)Neocosmospora falciformisE3OR206086.1, PP429275.1Givat YoavHa1 (yellow var.)(Degani et al. 2024c)

Origin of the trichoderma species isolates used in this study

Fifteen Trichoderma strains representing multiple species and ecological origins were evaluated (Table 2). Ten isolates originating from marine sources were kindly provided by Prof. Oded Yarden (Hebrew University of Jerusalem, Israel) and have been characterized previously (Gal-Hemed et al. 2011; Panizel et al. 2013). Two additional isolates of T. virens (Pachauri et al. 2023) were obtained by courtesy of Prof. Benjamin A. Horwitz (Technion – Israel Institute of Technology, Haifa, Israel). The species collection also included well-studied biocontrol strains (e.g., T203, P1), previously shown to suppress major soilborne pathogens such as Magnaporthiopsis maydis and Macrophomina phaseolina (Degani and Dor 2021; Degani et al. 2021c; Gal-Hemed et al. 2011; Gortikov et al. 2022; Samuels et al. 2010). Their prior biocontrol efficacy provided the rationale for testing them against onion FBR in this study. Finally, a new strain of T. harzianum (isolate ED10B) was included in this work.Table 2. List of Trichoderma spp. isolates used in this studyTrichodermaSpeciesDesignationOriginReferencesAssay post plate confrontationT. asperellumP1Maize, Prelude cv.(Degani et al. 2021a)Soluble & volatile Metabolites, semi-fieldT. asperelloidesT203ATCC 36,042, CBS 396.92(Gortikov et al. 2022; Samuels et al. 2010)-T. asperelloidesT1607Psammocinia sp. ^1^(Gal-Hemed et al. 2011)-T. atrovirideT3807Psammocinia sp. ^1^(Gal-Hemed et al. 2011)-T. atrovirideNF16 (IL isolate)Axinella sp. ^1^(Panizel et al. 2013)-**T. longibrachiatumT5007Psammocinia sp. ^1^(Gal-Hemed et al. 2011)-T. longibrachiatumT6607Psammocinia sp. ^1^(Gal-Hemed et al. 2011)Soluble & volatile MetabolitesT. longibrachiatumT7407Psammocinia sp. ^1^(Degani and Dor 2021; Gal-Hemed et al. 2011)Soluble & volatile Metabolites, semi-fieldT. longibrachiatumT7507Psammocinia sp. ^1^(Gal-Hemed et al. 2011)-T. virensGvW (P islate/IMI304061)Soil, Uttarakhand, India(Pachauri et al. 2023)Soluble & volatile MetabolitesT. virensGv29.8 (Q isolate)Soil, Texas, USA(Pachauri et al. 2023)-T. harzianumED10BCotton ^2^, Goliath V-6 cv.Current work-T. beinertiiT7107Psammocinia sp. ^1^(Gal-Hemed et al. 2011)-T. beinertiiT14707Psammocinia sp. ^1^(Gal-Hemed et al. 2011)Soluble & volatile Metabolites, semi-fieldT. sp. nov. 1(Strictipilosa clade)T1007Psammocinia sp. ^1^(Gal-Hemed et al. 2011)-^1^ Mediterranean sponge. ^2^ Gossypium barbadense

Trichoderma isolate ED10B was obtained from the roots of apparently healthy cotton plants (Gossypium barbadense, cv. Goliath V-6) grown in agricultural fields in Israel, as previously described (Degani et al. 2024a, b). The isolate was purified, morphologically characterized, and molecularly identified based on ITS and TEF1α sequencing (Rodríguez-Martínez et al. 2025), confirming its identity as T. harzianum. Detailed isolation procedures, morphological observations, and molecular identification steps are provided in the Supplementary Material (Supplementary Methods S1).

Growth of the fungi

Colonies were grown on a potato dextrose agar (PDA) medium in 90 mm plates. The plates were kept in an incubator at a temperature of 28 ± 1˚C in the dark for 4–5 days. To transfer the fungal colony to a fresh growth plate, a 6 mm diameter disk, obtained from the perimeter of a 5–7-day-old colony, was removed and placed in the center of a new PDA medium and incubated at the above conditions. For submerged cultures, seven fungal disks were sown in a 500 mL Erlenmeyer flask containing 210 mL of potato dextrose broth (PDB). The flasks were plugged with a breathable cover and incubated for 6 days, shaken in the dark at 130 rpm at 28 ± 1 °C.

Plate confrontation assay

The Trichoderma species (Table 2) were evaluated for their biocontrol potential against Fusarium spp. (Table 1) using direct dual-culture confrontation assays, following the method described in Degani et al. (2024a). Antagonistic activity was assessed based on antifungal compound secretion, inhibition of hyphal growth upon contact, or overgrowth of the Fusarium colony. For the assay, 6-mm agar disks from the margins of growing Fusarium cultures were first placed 0.5 cm from the edge of 90-mm PDA plates. Six-millimeter Trichoderma disks (also from growing colony margins) were introduced on the opposite side 2 days later. Plates were incubated at 28 ± 1 °C in darkness and monitored for 6 days following Trichoderma inoculation. Control plates were seeded with Fusarium alone on both sides of the Petri dishes and incubated under identical conditions. Interactions between the biocontrol agents and pathogens were recorded and photographed. Trichoderma strains that successfully suppressed Fusarium growth were classified as having mycoparasitic potential. Each strain was tested in five replicates. The inhibition percentage of the Fusarium spp. was determined as:

\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\%I=\frac{R-T}R\times100$$\end{document}

where R is the radial growth of the control, and T is the radial growth under treatment (de Oliveira et al. 2022). To assess the overall biocontrol capacity, the mean score of each Trichoderma strain against all four Fusarium species was calculated.

Secreted volatile compounds inhibition assay

The volatile-mediated antagonistic effect of Trichoderma spp. (Table 2) on the target pathogen (Fusarium spp., Table 1) was assessed using a sealed face-to-face “sandwich” plate assay (Li et al. 2018). In brief, 6-mm mycelial plugs from actively growing margins of 1- to 2-day-old Trichoderma colonies were placed centrally on PDA (90 mm diameter), and analogous plugs of the pathogen were inoculated onto separate PDA plates. After initial one-day growth, the lids were removed, agar surfaces of the “source” (Trichoderma, lower plate) and “receiver” (pathogen, upper plate) plates were opposed and sealed together with two layers of Parafilm to create a shared, enclosed headspace without direct contact. Pathogen-only plates (incubated with the surface down) and pathogen–pathogen paired plates were used as negative controls. Assemblies were incubated at 28 ± 1 °C in darkness for 72 h, until the pathogen in control plates reached ~ 2/3 of the plate radius. At termination, pathogen colony diameters were measured across two perpendicular axes, averaged, and used to calculate percent growth inhibition (%I) relative to controls: %I = [(R₍control₎ – R₍VOCs₎)/R₍control₎] × 100. Each treatment included 5 independent biological replicates.

Secreted soluble metabolites assay

This method enables the assessment of antifungal activity mediated by secreted metabolites independent of direct mycoparasitic interaction. The effect of metabolites secreted by Trichoderma isolates was evaluated using a cellophane overlay technique as previously described (Li et al. 2019). Briefly, sterile sheets of cellophane were placed on the surface of PDA plates, and each Trichoderma isolate was grown on the overlaid medium for 3 days at 28 ± 1 °C in darkness until it covered three-quarters of the membrane. After incubation, the cellophane sheets bearing Trichoderma colonies were carefully removed, leaving the culture medium potentially enriched with secreted soluble metabolites but free of fungal biomass. Subsequently, the plates were inoculated at the center with agar plugs (6 mm diameter) of the test pathogen species. Control plates, consisting of PDA overlaid with sterile cellophane without prior Trichoderma growth, were treated identically. Pathogen radial growth was measured after an additional 4 days of incubation at 28 ± 1 °C, and the inhibition percentage was calculated relative to the control treatment (as in Sect. 2.5). Each treatment was performed in five independent biological replicates.

Seed health validation

The FBR-susceptible onion cultivars Orlando (Riverside) and Maadim (marketed by Hazera Seeds Ltd.) were selected for this study, and their germination percentage under Trichoderma and Fusarium treatments was evaluated as described by (Degani et al. 2024b). Seeds were thoroughly washed with tap water and surface-sterilized by immersion in 0.91% sodium hypochlorite (NaOCl), followed by 1 min in 70% ethanol and rinsing with sterile double-distilled water (DDW). After sterilization, seeds were air-dried and placed in Petri dishes (10 seeds per plate) lined with sterile Whatman paper moistened with sterile tap water. Plates were inoculated with either Fusarium spp. or Trichoderma spp. by placing a single agar disk, excised from the margin of an actively growing colony, at the center of each seeds’ plate. Control plates were left untreated. All plates were incubated in complete darkness at 28 ± 1 °C for 8 days. Radicle and cotyledon emergence were recorded at days 4 and 8 post-inoculation. The seeds’ wet biomass was assessed on day 8. Each treatment consisted of four independent biological replicates (plates containing 10 seeds each).

Virulence trials in semi-field, open-enclosure, full-season potted conditions

The trial architecture

The semi-field potted experiments were conducted in a completely randomized design. The trial comprised 16 treatments, each inoculated with one of the Fusarium species under study—F. proliferatum, F. acutatum, F. oxysporum f. sp. cepae, and N. falciformis (strains B1, B5, B14, and E3, respectively)—either alone or in combination with Trichoderma strains. The Trichoderma treatments applied via seed coating included T. asperellum (P1), T. longibrachiatum (T7407), and T. beinertii (T14707). A local field soil control group was included for comparison. This soil was selected to closely simulate local field conditions and had no documented history of FBR. Baseline Fusarium inoculum levels were not quantified prior to the experiment; therefore, the natural presence of Fusarium spp., if present, was assumed to reflect minimal to moderate background populations typical of the agricultural area (Degani et al. 2024c). Each treatment and control consisted of 12 biological replicates (pots), distributed in a fully randomized design. Due to mortality caused by FBR, the final number of surviving replicates is indicated in each figure.

Key dates and meteorological conditions

The semi-field experiment was carried out during the spring-summer of 2025, whose key dates are provided in Table 3. The temperature and humidity conditions throughout the onion growing season were typical and favorable for disease development, as described previously (Degani et al. 2022). Detailed meteorological data are presented in Table 4.Table 3. The dates of the semi-field open enclosure pot experimentDateSoil inoculation, sowing, and monitoringDays from sowing19 March 2025 1 st inoculation (sterilized millet grains)−827 March 2025Sowing, 2nd inoculation (colony disks)03 April 20253rd inoculation (colony disks)710 April 2025Sprouting assessment, I1417 April 2025Sprouting assessment, II2124 April 2025Sprouting assessment, III2826 May 2025Mid-sampling (leaving one plant/pot)6001 July 2025Final sampling96Table 4Meteorological data for semi-field experiments ^1^ParametersValueDates19/03/2025–01/07/2025Temperature (°C)22.4 ± 7.4 (min 2.5, max 41.2)Above soil temp (°C)28.6 ± 10.6 (min 12.7, max 50.3)Humidity (%)55.5 ± 20.8 (min 10, max 96)Precipitations (mm)0.0 (min 0.0, max – 1.1)^1^ Average data (± standard deviation) according to the Israel Meteorological Service, Ministry of Transport and Road Safety, Kfar Blum Meteorological Station

Growth conditions

Each pot was sown with ten onion seeds (Orlando variety), derived from the same seed batch used in the seed health validation assay (Sect. 2.8). Plants were cultivated in 10-L plastic pots filled with heavy soil collected from the Northern R&D experimental farm in the Hula Valley, Upper Galilee, Israel (33°09′08.2″ N, 35°37′21.6″ E). Soil aeration was improved by incorporating 25% (v/v) Perlite No. 4. Fertilizers and pest control measures were applied throughout the growing season to prevent diseases other than those induced by Fusarium spp. Sowing was carried out on 27 March 2025. Above-ground emergence (“peeking”) was recorded at 14, 21, and 28 days after sowing (DAS). Thinning was performed on 21 and 28 DAS, reducing the number of plants per pot to seven and five, respectively. At mid-season sampling (60 DAS), plants were further thinned to one per pot. Harvest was conducted 96 DAS. Irrigation was applied through a computerized drip-line system equipped with emitters delivering water at a rate of 2 L h⁻¹ per dripper. Daily water volumes gradually increased over the course of the season to accommodate plant growth. From sowing until 48 DAS, each plant receives approximately 400 mL of water per day. Between 49 and 60 DAS, the irrigation level increased to ~ 800 mL per day, and from 61 to 96 DAS, plants were irrigated with ~ 1 L per day.

Preparation of infected, sterilized millet grains

Sterilized millet grains (Panicum miliaceum) were applied as the inoculum carrier for Fusarium spp., following a modified version of a previously described protocol (Gordani et al. 2023). In brief, 3 kg of millet kernels were boiled in ca. 6 L of tap water for 100 min. After cooling, 39 g of gypsum (CaSO₄·2 H₂O) was incorporated into the mixture. The kernels were then divided between four 3.5 L glass jars and sterilized by autoclaving at 121 °C for 20 min, followed by 134 °C for 40 min. After sterilization, each jar was supplemented with 120 mL of sterile DDW and inoculated with 7-day-old Fusarium cultures at a ratio of one colony per kilogram of wet millet grains. The colonies, grown on PDA and comprising the entire agar surface of 9-cm Petri plates, were cut into small fragments before being added. The contents were then homogenized using a sterile spatula. Jars were loosely capped to allow gas exchange, covered with aluminum foil, and incubated in darkness at 28 ± 1 °C for 13–15 days, until the grains were fully colonized by the fungus, as evidenced by the development of fungal hyphae.

Soil inoculation and Trichoderma protective treatment

Soil infestation with Fusarium spp. was initiated 8 days prior to sowing by incorporating 64 g of sterilized millet grains colonized by the selected Fusarium species into the upper 5 cm soil layer of each pot, following the protocol of Gordani et al. (2023). After inoculation, the soil was irrigated to maintain mild humidity. Additional soil inoculations were applied at sowing and again one week later (seedling emergence). For each supplementary inoculation event, three fungal culture discs (6 mm in diameter), obtained from Fusarium spp. grown as described in Sect. 2.4, were placed into the soil around each sprout (3 discs per seed; 30 discs per pot). Repeated Fusarium inoculations were applied to ensure sustained and sufficiently high disease pressure under pot conditions using field soil, where pathogen establishment is often lower than under open-field conditions.

The Trichoderma-based protective treatment was applied as described previously (Degani et al. 2024b), with minor modifications. Briefly, Trichoderma liquid cultures grown in PDB (Sect. 2.4) were homogenized for 1 min using a blender to obtain a uniform suspension. In addition, colony-forming units (CFU), comprising mycelial fragments and spores, were prepared from 10-day-old Trichoderma cultures grown on PDA by scraping the colony surface with a Drigalski spatula in 5 mL of sterile DDW. CFUs obtained from two PDA plates were then added to each of three homogenized PDB-grown cultures. Tween 80 (Sigma, Rehovot, Israel) was included in the final coating suspension at a concentration of 0.05% (v/v). The bio seed-coating suspension contained approximately 3 × 10⁶ CFU mL⁻¹ of Trichoderma and was applied at a volume of 35 mL per gram of onion seeds. A sterile DDW solution containing only Tween 80 served as the control treatment. Prior to coating, onion seeds (26 g) were rinsed thoroughly with tap water, surface sterilized in 2% bleach for 1 min, and rinsed again with water. The seeds were divided into four groups: three were incubated for 7 min in the Trichoderma coating suspension, while the fourth was incubated in the control solution. The coating process was performed at room temperature. Seeds were then spread on paper towels and air-dried overnight at room temperature.

Experimental measurements

The effects of the treatments on plant development and health were evaluated at the pot level, with the surviving number of replicates for each treatment and time point reported in the figure captions. Seedling emergence (“aboveground peeking”) was monitored at 14, 21, and 28 DAS. Aboveground peeking (%) was calculated as the number of seedlings with visible shoots above the soil surface divided by the total number of seeds sown, multiplied by 100. Plant growth parameters were recorded at mid-season thinning (60 DAS) and at the final harvest (96 DAS). These included survival rate, plant height, fresh weight of the aboveground tissues, bulb fresh biomass, phenological stage (number of leaves), and, at harvest, the number of dry leaves. Root samples were collected at both time points, immediately frozen at − 20 °C, and later processed for DNA extraction and purification to enable quantification of fungal DNA in root tissues by quantitative real-time PCR (qPCR).

Fusarium infection molecular diagnostic

DNA extraction

Basal plates were thoroughly rinsed with tap water, cut into ~ 2 cm segments, and the sample weight was standardized to 0.7 g per plant. Each sample was placed in a BioMed universal extraction bag (Bioreba, Reinach, Switzerland) containing 4 mL of cetyltrimethylammonium bromide (CTAB) buffer. Tissue homogenization was performed for 5 min using an electric tissue homogenizer (Bioreba, Reinach, Switzerland). DNA was then extracted following a previously published protocol (Degani et al. 2019). The final DNA extracts were resuspended in 100 µL of HPLC-grade water and stored at − 20 °C until further qPCR analysis.

Real-time PCR molecular evaluation

Molecular tracking of Fusarium spp. infection within the basal plate tissue was performed in the semi-field trial at two sampling points, 60 and 96 DAS. qPCR analyses were performed on genomic DNA, rather than cDNA, as the aim was to quantify fungal biomass for infection assessment rather than gene expression. The assays were performed using an ABI-7900HT system (384-well format; Applied Biosystems, Foster City, CA, USA) following a standard SYBR Green-based protocol optimized for fungal DNA detection (Dimant and Degani 2023). Each 5 µl reaction (run in quadruplicate) contained 0.25 µl of each primer (10 µM), 2.5 µl of iTaq™ Universal SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA), and 2 µl of DNA template. Cycling conditions included an initial denaturation at 95 °C for 60 s, followed by 40 cycles of 95 °C for 15 s and 59 °C for 30 s, and a final melting curve analysis. The Fus-for/rev primers, designed in this study, amplified a 115 bp fragment of the Fusarium translation elongation factor 1-alpha (TEF1) gene. The cytochrome oxidase (COX) gene served as the internal control using specific primers targeting a mitochondrial housekeeping gene. Primer sequences are provided in Supplementary Table 2. Relative fungal DNA levels were calculated using the ΔCt method, assuming equal amplification efficiencies across samples.

Statistical analysis

Statistical analyses were performed using GraphPad Prism software, version 10.6.0 (890) (GraphPad Software Inc., San Diego, CA, USA). Data normality was assessed with the Shapiro–Wilk test. When datasets met the assumption of normality (p > 0.05), a one-way analysis of variance (ANOVA) was conducted, followed by Fisher’s least significant difference (LSD) test, with significance set at p < 0.05. For datasets that did not meet the normality assumption (p < 0.05), the nonparametric Kruskal–Wallis test was applied, followed by Dunn’s multiple comparisons test (uncorrected). Multiple-comparison corrections were not applied in order to avoid overly conservative outcomes and an increased risk of Type II errors. Outliers were identified using the ROUT method in GraphPad Prism with conservative settings applied uniformly across datasets, and their removal did not affect the overall conclusions.

Results

This study investigated eco-friendly, Trichoderma-based management strategies against the major causal agents of onion FBR in Israel. As an initial step, to identify the most promising antagonistic isolates, all strains were evaluated in an in vitro plate confrontation assay (Fig. 1; Supplementary Figures S1-S5). Ten out of the fifteen Trichoderma strains tested demonstrated significant pathogen suppression (approximately 30–40%, p < 0.05), manifested through various antagonistic mechanisms such as the formation of hyphal barriers and direct overgrowth of the Fusarium colony. Notably, the inhibitory capacity of individual Trichoderma species varied with the pathogen tested. For example, T. asperellum (P1) was the most effective antagonist against F. proliferatum (B1), yet it showed a weak inhibitory effect against N. falciformis (E3).Fig. 1. Plate confrontation assay showing the percentage inhibition of Fusarium species (Table 1) colony growth after 6 days of incubation with the Trichoderma biological control agents (Table 2). Bars represent the mean of 4–5 biological replicates, and error bars indicate the standard error. Different lowercase letters (a–g) above the bars indicate statistically significant differences between treatments (p < 0.05), determined using the Kruskal–Wallis non-parametric test followed by uncorrected Dunn’s test

The secretion of volatile metabolites plays a critical role in Trichoderma–pathogen interactions. To evaluate this mode of antagonism, a double-plate inhibition assay was performed (Fig. 2; Supplementary Figure S6A). Five Trichoderma isolates were selected for testing based on their superior performance in the plate confrontation assay (Fig. 1). The effectiveness of volatile metabolite-mediated inhibition varied depending on the Fusarium pathogen species. Among the isolates, T. asperellum (P1) and T. longibrachiatum (T7407) exhibited the strongest antagonistic activity, achieving approximately 25–45% inhibition compared with the untreated control (p < 0.05). In contrast, T. virens (GvW) showed little (non-significant) inhibitory effect against F. acutatum, and T. longibrachiatum (T6607) was a weak antagonist against both F. oxysporum f. sp. cepae (FOC) and F. proliferatum.Fig. 2. Secreted volatile compound inhibition assay showing the percentage reduction in Fusarium species (Table 1) colony growth after 4 days of incubation in the presence of Trichoderma biological control agents (Table 2). Bars represent the mean of 3–4 independent biological replicates, and error bars indicate the standard error. Different lowercase letters (a–c) above the bars denote statistically significant differences between treatments (p < 0.05), determined using the Kruskal–Wallis test followed by uncorrected Dunn’s multiple comparisons test for F. proliferatum, or one-way ANOVA followed by Fisher’s least significant difference (LSD) post hoc test for all other species Similar to the volatile compound assessment, the secretion of soluble metabolites represents another important antagonistic mode of action that distinguishes between the different Trichoderma species (Fig. 3; Supplementary Figures S6B). In this assay, the inhibitory effect was nearly twice as strong as that observed in both the confrontation and volatile metabolite assays, reaching up to ~ 80% growth suppression in the most effective antagonists. Notably, T. asperellum (P1) and T. longibrachiatum (T7407) consistently exhibited strong inhibitory activity against three of the Fusarium pathogens tested, while T. virens (GvW) demonstrated pronounced inhibition of F. acutatum, in clear contrast to its negligible effect in the volatile metabolite assay with this pathogen (Fig. 2).Fig. 3. Inhibition assay of secreted soluble metabolites showing the percentage reduction in Fusarium species (Table 1) colony growth after 4 days of incubation in the presence of Trichoderma biological control agents (Table 2). Bars represent the mean of five independent biological replicates, and error bars indicate the standard error. Different letters (a–d) above the bars denote statistically significant differences between treatments (p < 0.05), determined using one-way ANOVA followed by Fisher’s least significant difference (LSD) post hoc test for F. oxysporum f. sp. cepae, or the Kruskal–Wallis test followed by uncorrected Dunn’s multiple comparisons test for all other species As a pre-sowing evaluation to assess both the safety of the Trichoderma-based seed coating for germinating seeds and the pathogenicity of the applied Fusarium species, an in vitro seed assay was performed (Figs. 4, 5 and 6; Supplementary Figures S7). In this assay, the Orlando yellow onion cultivar exhibited markedly higher sensitivity than the Maadim red var., manifested by severely reduced sprout development under pathogen stress. Among the tested pathogens, N. falciformis (E3) was the most aggressive toward seedlings, followed by FOC (B14) and F. acutatum (B5). In contrast, F. proliferatum (B1) was the least aggressive, showing no statistically significant effect compared with the control in the Maadim var., but causing significant growth suppression (p < 0.05) in the Orlando cultivar.Fig. 4. Seed health assay: photos. The upper panel shows the red Maadim onion variety, and the lower panel shows the yellow Orlando variety. Each Petri dish contained 10 seeds placed on moist filter paper and was inoculated with a 6-mm agar disk excised from the actively growing margin of 5–7-day-old Fusarium (Table 1) or Trichoderma (Table 2) cultures, positioned at the center of the plate. Control plates (highlighted in green) received no fungal inoculation. All plates were incubated at 28 ± 1 °C in complete darkness. Seed germination and early seedling development are indicated by radicle and shoot emergence, whereas reduced or absent sprouting reflects inhibitory or pathogenic effects of the treatments. Representative Petri plates of each treatment were photographed 4 days after inoculationFig. 5Seed health assay: germination rates. Radicle and epicotyl emergence (% relative to the untreated control) were evaluated in onion seeds of the Maadim var. after 4 (A,** B**) and 8 (C,** D**) days of incubation at 28 ± 1 °C in darkness. Corresponding results for the Orlando var. are shown in panels E–H. Each plate contained 10 seeds and was inoculated with a 6-mm colony agar disk taken from the actively growing margin of 5–7-day-old Fusarium (Table 1) or Trichoderma (Table 2) cultures. Values represent the mean of four biological replicates. Error bars indicate the standard error. Different letters (a–f) above the bars indicate statistically significant differences between treatments (p < 0.05), as determined using the Kruskal–Wallis test followed by Dunn’s uncorrected multiple comparisons testFig. 6Seed health assay: fresh biomass. Wet weight (% relative to the untreated control) was measured in onion seedlings of the Maadim variety (A) and the Orlando variety (B) after 8 days of incubation at 28 ± 1 °C in darkness. Each plate contained 10 seeds and was inoculated with a 6-mm colony agar disk excised from the actively growing margin of 5–7-day-old Fusarium (Table 1) or Trichoderma (Table 2) cultures. Values represent the mean of four biological replicates. Error bars indicate the standard error. Different letters (a–e) above the bars denote statistically significant differences between treatments (p < 0.05), as determined using the Kruskal–Wallis test followed by Dunn’s uncorrected multiple comparisons test

Most of the Trichoderma species tested were less harmful to seeds than the most harmful Fusarium species, N. falciformis (E3), particularly in the Maadim onion cultivar. In contrast, few isolates negatively affected seed germination and early seedling development in both cultivars. Notably, T. virens (GvW) and T. longibrachiatum (T7507), and to a lesser extent T. asperellum (P1), significantly suppressed sprout growth. Consistently, on the fourth day of incubation, T. beinertii (T14707) did not adversely affect seedling development (Fig. 5, Supplementary Figure S7). By the eighth day, both T. beinartii strains (T14707 and T7107) and T. longibrachiatum (T7407) remained safe for sprout growth in the Maadim cultivar, whereas only T7107 and T7407 were also safe for the Orlando cultivar. These biofriendly species showed the greatest statistical separation from the other tested Trichoderma spp. in terms of sprout biomass (Fig. 6). The assessment of sprouts’ fresh biomass at day 8 revealed a pattern consistent with the radicle and epicotyl germination percentages.

The open-enclosure pot trial constituted the final stage of the evaluation and was designed to assess the protective treatment under field-simulated conditions while maintaining partial control over experimental variables. Given the natural environmental fluctuations and the inherent difficulty of achieving fully uniform pathogen inoculation across pots, considerable variability in the data was anticipated. This variability, reflected by elevated standard error values, may have reduced the ability to detect statistically significant differences among treatments. FBR disease symptoms were reflected in sprout dehydration and mortality, yellowing of the leaves starting from the upper tip and spreading downwards (Fig. 7), roots decay, and basal rot. During the sprouting phase (14–28 DAS), the addition of inoculum to the naturally mild infested local field soil had no apparent effect (Supplementary Figure S8). By mid-season (60 DAS), however, soil infection induced an observable, though non-significant, suppression of plant growth, most pronounced in the F. proliferatum and FOC treatments (Fig. 8). Toward the end of the season, these effects became less evident (Fig. 9).Fig. 7. Semi-field, open-enclosure, full-season pot trial. The experiment evaluated Trichoderma-based seed coating to protect the susceptible Orland variety against Fusarium basal rot. Experimental onion plants photographed at 60 (A) and 91 (B) days after sowing. Early disease symptoms are visible in 25-day-old sprouts (C), which may progress to damping-off (D). Panel E shows a symptomatic 76-day-old plantFig. 8Semi-field mid-season evaluation. Treatments included soil inoculation with Fusarium spp. (Table 1) and Trichoderma-based seed coating (species details in Table 2). Parameters assessed were: (A) shoot fresh weight, (B) phenological development (leaf number), (C) bulb fresh biomass, (D) shoot height, and (E) relative quantity of Fusarium spp. (Fus) DNA in basal tissues, normalized to the plant cytochrome c oxidase (Cox) gene. Red percentages indicate the percent change relative to untreated plants grown in naturally infested local field soil (green bars), which were set at 100%; pathogen-only controls without protective treatment are shown in black. Data represent the mean ± standard error of 9–12 biological replicates. In the qPCR analysis (E), outliers were removed using the ROUT method (Q = 1%) in GraphPad Prism. Different letters (a–f) above the bars denote statistically significant differences between treatments (p < 0.05), determined using one-way ANOVA followed by Fisher’s LSD test (A–D) or the Kruskal–Wallis test followed by Dunn’s uncorrected multiple comparisons test (E)Fig. 9. Semi-field end-season evaluation. Treatments included soil inoculation with Fusarium spp. (Table 1) and Trichoderma-based seed coating (species details in Table 2). Parameters assessed were: (A) shoot fresh weight, (B) phenological development (leaf number), (C) bulb fresh biomass, (D) shoot height, (E) dry leaf count, and (F) relative quantity of Fusarium spp. (Fus) DNA in root tissues, normalized to the plant cytochrome c oxidase (Cox) gene. Red percentages indicate the percent change relative to untreated plants grown in naturally infested local field soil (green bars), which were set at 100%; pathogen-only controls without protective treatment are shown in black. Data represent the mean ± standard error of 9–12 biological replicates. In the dry leaves analysis (E), outliers were removed using the ROUT method (Q = 1%) in GraphPad Prism. Different letters (a–f) above the bars denote statistically significant differences between treatments (p < 0.05), determined using one-way ANOVA followed by Fisher’s LSD test (A–D) or the Kruskal–Wallis test followed by Dunn’s uncorrected multiple comparisons test (E, F)

The sprouting (above-ground emergence) assessment at 14 DAS revealed only minor, non-significant differences among treatments (p > 0.05). Continued monitoring at 21 and 28 DAS gradually highlighted the benefits of the protective treatments. Sprouting rates were up to 40% and 27% higher than those recorded in the natural field soil control under T. longibrachiatum (T7407) and T. beinertii (T14707) shielding, respectively.

By mid-season (60 DAS), seed coatings with selected Trichoderma strains markedly mitigated FBR stress and promoted plant growth. Shoot and bulb fresh biomass increased by 54–131% relative to untreated controls (Fig. 8A, C), accompanied by parallel improvements in additional growth parameters. Among the treatments, T. asperellum (P1) was the most effective, enhancing biomass by 92% and 114% against F. acutatum, and by 131% and 83% against N. falciformis. In terms of shoot phenological development (leaf number) and height (Fig. 8B, D), T. beinertii (T14707) and T. longibrachiatum (T7407) performed best, achieving 7–44% improvement compared with untreated controls. Molecular real-time (qPCR) monitoring at 60 DAS (Fig. 8E) enabled evaluation of the disease at this asymptomatic stage (Dimant and Degani 2023). Although some changes were observed, such as a 34% increase in pathogen infection levels in the F. proliferatum control treatment and a 29% suppression of FOC under T. longibrachiatum (T7407) protection, these differences did not reach statistical significance, which may reflect the very high sensitivity of the method.

By the end of the season (Fig. 9), some of the biological seed coatings kept rescuing the plant growth indexes, despite the continuous FBR pressure. Particularly, T. asperellum (P1) treatments performed best, significantly enhancing shoot weight by 28–119% and bulb weight by 15–157% under all four Fusarium spp. stress. These growth improvements were further associated with up to 36% reduction in foliar symptoms and 59% suppression of pathogen infection, as quantified by qPCR. Interestingly, bio-shielding with T. beinertii (T14707) against F. acutatum and T. longibrachiatum (T7407) against FOC was associated with an unexpected increase in the number of dry leaves (57% and 32% above the control, respectively). Nevertheless, these treatments reduced pathogen infection by 9% and 39%, respectively. Moreover, both interventions showed moderate benefits against other pathogen species—for example, T. beinertii (T14707) promoted growth under F. proliferatum stress, while T. longibrachiatum (T7407) was effective against N. falciformis.

Discussion

This study provides the first evidence in Israel that selected Trichoderma strains can suppress FBR in onion. In vitro and semi-field trials showed strong antagonistic activity, particularly via soluble metabolites, with T. asperellum (P1) consistently promoting growth and reducing disease-related outcomes. These results highlight Trichoderma as an eco-friendly tool for integrated FBR management, while also emphasizing the need for optimized formulations and field validation. FBR, caused by F. oxysporum f. sp. cepae (FOC) and related species (Le et al. 2021b), is difficult to control because the pathogen persists in soil and symptoms can remain latent. Integrated management approaches that combine resistant cultivars, biological agents, and low-dose fungicides remain the most realistic path forward. Bio-agents such as Trichoderma play a central role through mechanisms including antibiosis, mycoparasitism, competition, and induction of host resistance (Ghanbarzadeh et al. 2016; Singh et al. 2024). In this context, dual-culture assays, though limited, provided an efficient first screen for antagonistic mechanisms such as mycoparasitism, metabolite secretion, and volatile inhibition (Williams et al. 2025). Moreover, the distinct antagonistic modes of action that differentiate Trichoderma species can provide critical insights for designing sustainable management strategies, particularly those that integrate multiple bio-agents in complementary combinations (Pastor et al. 2023). The strong in vitro activity of several isolates paralleled their performance in the semi-field trial, underscoring the potential of strain–pathogen matching for durable control. It also emerges from multiple studies that a successful, strong biocontrol agent can effectively act against different pathogen species that cause diseases in different plant species (D’Ambrosio et al. 2022).

Indeed, the specific Trichoderma strains evaluated in this study proved effective against the fungal agents of FBR. These strains were previously tested and shown to be highly efficient against the maize late wilt pathogen Magnaporthiopsis maydis (Degani and Dor 2021; Degani et al. 2021c) and the cotton charcoal rot pathogen Macrophomina phaseolina (Degani et al. 2023a, b). Notably, some Trichoderma species can function as endophytes—living in symbiotic association within the host plant—and thereby provide continuous protection throughout the growing season. (Carro-Huerga et al. 2020; Giordano et al. 2023) A prime example is the T. asperellum (P1) isolate, which demonstrated strong growth-promotion and infection-suppression capabilities under FBR stress in this study. Originally isolated in our laboratory from maize seeds (Degani et al. 2021a), this strain secretes the potent antifungal metabolite 6-pentyl-α-pyrone (Degani and Gordani 2022; Degani et al. 2021b). The identification of Trichoderma strains with protective potential against soilborne pathogens paves the way for their combined application with low-dosage chemical fungicides, enhancing both efficacy and stability under fluctuating environmental conditions and high pathogen pressure. Such an integrated disease-management approach has already been demonstrated in various cultivars (Ons et al. 2020; Ruano-Rosa et al. 2018), including the combined use of the specific Trichoderma strains tested here with azoxystrobin in maize and cotton (Degani et al. 2024b; Gordani et al. 2023).

Across dual-culture assays, several Trichoderma spp. (e.g., T. harzianum, T. asperellum, T. viride) consistently inhibit FBR pathogens, including FOC, via secreted and volatile metabolites (Bunbury-Blanchette and Walker 2019). Similarly, our results indicate that secreted soluble metabolites play a pivotal role in FBR suppression. Their inhibitory effect in vitro was nearly twice as strong as that observed in the confrontation and volatile metabolite assays, reaching up to ~ 80% growth suppression in the most effective antagonists.

The production of diverse secondary metabolites by Trichoderma species is strain-dependent and central to their antagonistic activity against pathogens (Vinale et al. 2008). Recent reviews provide comprehensive overviews of Trichoderma secondary metabolite diversity and ecological functions (Cortez-Lázaro et al. 2025; Jin and Alberti 2025), as well as modern methodologies for metabolite discovery—such as metabolomic dereplication, genome mining, and omics-based validation of biosynthetic pathways. These metabolites are central to the antagonistic activity of Trichoderma against plant-pathogenic fungi, where they inhibit spore germination, disrupt membrane integrity, interfere with enzymatic pathways, and modulate host defense mechanisms (Harman et al. 2004; Vinale et al. 2008). The present findings constitute an important step toward advancing our understanding of these bioactive molecules and lay the groundwork for developing novel management strategies that harness such metabolites as potential biocontrol agents against FBR pathogens.

Greenhouse trials with Trichoderma spp. to prevent FBR generally confirm pathogen suppression and growth promotion (Bunbury-Blanchette and Walker 2019). For instance, combining arbuscular mycorrhizal fungi (AMF) with T. harzianum reduced disease severity and improved biomass in growth-chamber/greenhouse tests, while highlighting cultivar-dependent responses (Yağmur et al. 2024). A further step in the establishment process is the semi-field setup, as gaps persist when translating controlled assay results to real soils and weather conditions. Yet, semi-field evaluations, while important for bridging lab and field, remain comparatively scarce in the onion/FBR literature. As demonstrated here, semi-field evidence is valuable to test strain (both the pathogens and the antagonistic bio-agents) performance under fluctuating abiotic conditions and complex inoculum.

Finally, agricultural field validation is the goal. Full field-scale trials are essential for optimizing application dose, formulation, and delivery methods, as well as for rigorously evaluating multi-strain or synergistic Trichoderma combinations and their interactions with native soil microbiota under commercial production conditions. Although several Trichoderma treatments were associated with improved plant growth parameters, no direct mechanistic analyses (e.g., phytohormone profiling or signaling pathway assays) were conducted, and growth promotion is therefore interpreted at the phenotypic level only. While field studies provide the most decision-relevant evidence yet require vast investment in time, manpower resources, and, thus, there are fewer such studies than lab/greenhouse reports. Previous study showed that T. harzianum seed treatment suppressed FBR and enhanced growth under naturally infested soils (field and pot), supporting the viability of seed-applied formulations (Coşkuntuna and Özer 2008). More recently, field testing of multiple Trichoderma strains on onion demonstrated significant yield benefits under commercial conditions, even when disease endpoints were not the primary readout (Dutta et al. 2024). Natural-field biocontrol trials in onion further support the Trichoderma application and its benefits (El-Mougy and Abdel-Kader 2019).

High-quality formulations and delivery (e.g., seed coatings vs. soil amendments) strongly affect persistence, colonization, and efficacy—points repeatedly emphasized in field-oriented biocontrol work. Mixed inocula (e.g., Trichoderma + AMF or Trichoderma + freshwater microalgae) can enhance outcomes but require compatibility testing (Elshahawy and El-Sayed 2018; Yağmur et al. 2024). Future works that explore the potential of bio-agents such as Trichoderma spp. should focus on matching strains to pathogens and cultivars across sites, optimizing formulation and dose for seed and soil applications as suggested by Le et al. (2021b). The use of sensitive molecular assays for early-season disease assessment—when infections may remain asymptomatic for extended periods—is an essential tool for evaluating protective applications (Dimant and Degani 2023). Nevertheless, qPCR measurements provide relative estimates of pathogen DNA abundance and do not distinguish between viable and non-viable fungal biomass; therefore, these data were used to compare treatment-associated differences in colonization dynamics.

The seed health assay employed in this study was designed to assess potential risks of seed bio-coating to plants and to evaluate the aggressiveness of pathogen species prior to soil inoculation. Interestingly, N. falciformis (E3) emerged as a major influencing species. This outcome was unexpected, as recent observations have shown that N. falciformis is only moderately pathogenic compared with F. acutatum, although more abundant and therefore considered a generalist species (Degani et al. 2024c). Examination of the control plants (unprotected and inoculated with E3) relative to those inoculated with other Fusarium species supports this interpretation: plants exposed to N. falciformis (E3) without protection displayed comparatively more vigorous and healthy growth. Thus, as anticipated, the in vitro seed assay had limited predictive power regarding the impact of treatments on plant performance under semi-field, full-season conditions. This limitation aligns with previous reports highlighting that indirect seed health tests—such as molecular or serological assays—do not necessarily reflect pathogen viability or actual disease outcomes in situ, underscoring the necessity for field-level validation (Cardwell et al. 2018; Hiddink et al. 2023).

The present study contributes novel insights by addressing onion FBR management under Israeli conditions, where the pathogen complex is dominated by FOC, F. proliferatum, F. acutatum, and N. falciformis. While FBR is commonly described as a disease complex, individual Fusarium species were evaluated separately in this study to enable controlled and reproducible assessment of antagonistic efficacy; potential synergistic or competitive interactions among co-infecting pathogens were not examined and warrant targeted investigation in future studies. Although Trichoderma-mediated suppression of FBR has been demonstrated in other regions (Bunbury-Blanchette and Walker 2019; Coşkuntuna and Özer 2008; Yağmur et al. 2024), the current work extends this knowledge by showing that strain–pathogen interactions are highly context dependent. Furthermore, the integration of qPCR-based molecular diagnostics with semi-field trials enabled the detection of pathogen dynamics at asymptomatic stages, providing a more sensitive measure of treatment efficacy than visible symptoms alone. These combined features—geographic specificity, pathogen strain–specific outcomes, and molecular validation— form the basis for future large-scale field applications in Israel and comparable environments.

Conclusions

Fusarium basal rot (FBR), primarily caused by Fusarium oxysporum f. sp. cepae, remains one of the most destructive onion diseases worldwide. This study provides the first evidence in Israel that selected Trichoderma strains, particularly T. asperellum (P1), can suppress FBR and promote plant growth under semi-field conditions. Integrating qPCR monitoring with phenotypic assessments revealed strain-specific effects and confirmed the value of combining molecular and agronomic endpoints. While these findings support Trichoderma as a promising, eco-friendly tool for onion FBR management, challenges remain. Strain performance varies with pathogen species, cultivar, soil, and climate, highlighting the need for optimized formulations, delivery methods, and large-scale field trials. A deeper understanding of strain–host–environment interactions and metabolite roles will be essential for translating experimental success into robust field solutions.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (DOCX 14.6 MB)