Evaluation of culture filtrate of Trichoderma isolates with potential to induce Fusarium wilt resistance and promote early flower and fruit development in tomato

Introduction

Tomato (Solanum lycopersicum L.) is one of the most widely cultivated and economically important crops globally, yet it remains vulnerable to various biotic stresses, including Fusarium wilt, a soil-borne disease caused by Fusarium oxysporum f. sp. lycopersici. Fusarium wilt severely affects plant health by obstructing water and nutrient flow, leading to significant yield losses. Managing this disease is challenging due to the pathogen's persistence in soil and its ability to rapidly adapt to chemical controls (Mudawi and Idris, 2015) [12] . Consequently, there is a growing interest in biological alternatives, particularly in the use of Trichoderma spp., which have emerged as a promising solution in sustainable agriculture. Climate change further exacerbates the vulnerability of crops like tomato, threatening global food security. In India, where agriculture is largely practiced by small and marginal farmers with limited resources, the impacts of climate change are particularly pronounced. The tropical climate, combined with these socioeconomic constraints, increases the risks to crop productivity and disease outbreaks (Bhardwaj et al., 2022) [2] . The use of biological control agents, such as Trichoderma spp., offers an eco-friendly and cost-effective solution, especially in regions like India where chemical inputs are not always accessible. Trichoderma spp. are well-known for their ability to suppress plant pathogens through multiple mechanisms, including mycoparasitism, competition, and the production of antimicrobial compounds (Tyśkiewicz et al., 2022) [18] . However, recent research has highlighted their additional role in inducing resistance in plants, which enhances the plant's defense response against pathogens like Fusarium. Culture filtrates of Trichoderma spp., containing bioactive secondary metabolites, have shown promise in triggering the plant's innate immune system, leading to enhanced resistance against Fusarium wilt (Shoresh et al., 2018) [4] . Beyond pathogen control, certain Trichoderma isolates have been found to positively influence plant growth by promoting early flowering and fruiting. This dual functionality, in which Trichoderma not only induces resistance but also stimulates early plant development, is particularly desirable for improving tomato yield (Mastouri et al., 2017) [11] . Studies have shown that the application of Trichoderma culture filtrates can result in enhanced photosynthesis, water-use efficiency, and nutrient uptake, all of which contribute to early flowering and fruit set (Vinale et al., 2019) [9] . In addition to improving crop yield, Trichoderma spp. have shown promise in enhancing the overall resilience of crops to environmental stresses such as drought and heat, which are becoming more prevalent due to climate change. Recent studies suggest that certain Trichoderma strains can improve water-use efficiency in crops, making them more tolerant to water deficits (Mastouri et al., 2017) [11] . Moreover, the application of Trichoderma culture filtrates can enhance the bioavailability of essential nutrients like nitrogen and phosphorus, further supporting plant growth and development (Woo et al., 2021) [21] . Inducing resistance through culture filtrates of Trichoderma spp. offers a sustainable and eco-friendly alternative to chemical fungicides, providing dual benefits of disease management and growth promotion. This approach aligns with the principles of sustainable agriculture, helping to reduce the reliance on synthetic chemicals while improving crop health and productivity. This study aims to explore new Trichoderma isolates with the ability to both induce resistance against Fusarium wilt and enhance early flowering and fruit development, ultimately contributing to the resilience and productivity of tomato cultivation.

Materials and Methods

The current experiment was conducted at Department of Plant Pathology and Student Instructional Farm, Chandra Shekhar Azad University of Agriculture and Technology, Kanpur during 2021-22 and 2022-23. The following work was conducted during the experiment described as below:- (Elad et al., 1981; Harman et al., 2004) [5,8] . The isolated Trichoderma spp. were identified under compound microscope on the basis of culture texture, colour, growth pattern of colony, conidiophores, phialides and conidia as per described by Harman (2000) [6] .

Isolation, purification and identification of

Preparation of culture filtrate of Trichoderma spp.

Potato dextrose broth (PDB) was poured into sterile conical flasks, filling them about 1/4 of their capacity. Sterilized the flasks containing the broth by autoclaving at 121.6 °C (15 psi) for 15 minutes. With the help of a cork borer, small bits were cut from the pure culture of Trichoderma in the Petri plate and then they were transferred to the potato Dextrose Broth (PDB) poured conical flask using an inoculation needle under Laminar air flow. Covered the conical flasks with cotton plug to allow air exchange while preventing contamination. Placed the inoculated flasks on a shaker set at 25±1 °C for 21 days but at every 5-6 days, flasks have to be manually shaken. After the incubation period, the flasks were removed from the shaker. The broth was filtered to separate the fungal biomass from the liquid culture filtrate using sterile Whatman filter paper no. 1. Alternatively, the broth was centrifuged at 2500-3000 RPM for 10-15 minutes to pellet the fungal biomass and then the supernatant was decanted. The supernatant was passed again through sterile Whatman filter paper no. 1 to remove any remaining spores or mycelial fragments to ensured that the culture filtrate was free of Trichoderma cells. The filtered culture filtrate was collected in sterile conical flask and labelled with the first three letter of name of location from where soil samples were collected for isolation of Trichoderma and date of preparation. The culture filtrate was stored at 4 °C until ready for use. All the collected Trichoderma isolates were used to prepare individual culture filtrates. These filtrates were applied as inducer in various treatments on tomato plants.

Isolation of Fusarium

The isolation of pathogen was performed in laboratory of Department of plant Pathology from infected plant sample collected from Student Instructional Farm, Chandra Shekhar Azad University of Agriculture and Technology, Kanpur. The identification of pathogen was performed under compound microscope as per description provided by Snyder and Hansen (1940) [17] . Seeds of tomato variety Aazad T-6 obtained from Department of Vegetable Science, C.S. Azad University of Agriculture and Technology, Kanpur used in this experiment and grown under wire house condition for raising nursery. Germination percentage, mortality rate, number of days taken for flowering, number of days taken for fruiting were tested by treating seeds with 10% concentration of culture filtrate of different isolates of Trichoderma spp. The pots seeded with tomato seed further inoculated with F. o. f. sp. lycopersici.

Bio-efficacy test

Bio-efficacy test of culture filtrate of different Trichoderma isolates has been performed at Laboratory of Department of Plant Pathology and by using Poison Food technique. The concentration of culture filtrate of Trichoderma @ 5 and 10% were tested, and radial growth of Fusarium measured in mm. The growth reduction through culture filtrate was calculated by given formulae:-

Where, C-Radial growth of Fuarium in control plate T-Radial growth of Fusarium in culture filtrate treated plate

Germination percent

Fourteen days after sowing (DAS), the number of treated seeds with culture filtrate @10%, that germinated in each treatment was counted and recorded. Germination percentage was calculated by given formula:-

Statistical analysis

The data were analyzed by following the procedure of Randomized Block Design (RBD) and Completely Block Design (CRD). Data recorded in percentage were first transformed at Arc sin value (Fisher and Yates, 1963) 1 sin before statistical analysis. Treatments were compared by means of critical difference (CD) at 5 percent level of significant. Mukherjee et al., 2020). A recent study reported significant inhibition of mycelial growth in Pythium undulatum and Phytophthora inundata through the use of culture filtrates from T. simmonsii and T. asperellum (Ben M'henni et al., 2022) [1] .

Result and Discussion

Effect of culture filtrate of Trichoderma isolates on Germination percent and Mortality rate

The highest germination rate (94.61%) was observed in treatment T 9 (Culture filtrate of Trichoderma Nag), with the lowest among treated groups in T 3 ( [7,4] . Moreover, the reduction in seed mortality observed in treatments T 9 (Culture filtrates of Trichoderma Nag) T 6 (Culture filtrates of Trichoderma Ban) and T 7 (Culture filtrates of Trichoderma Jab) indicates the efficacy of culture filtrate in controlling wilt incidence, which is often attributed to soilborne fungal pathogens such as F. o. f. sp. lycopersici. This result is consistent with the findings of Chagas et al. (2020) [3] , reported that Trichoderma spp. effectively reduce disease incidence by outcompeting pathogenic fungi, producing antifungal metabolites and enhancing the plant's own defense mechanisms (65%). In both 2021-22 and 2022-23, treatments with culture filtrates of Trichoderma spp. significantly reduced seed mortality compared to the control (T 13 ). In 2021-22, the lowest mortality (6%) was observed in T 9 (Culture filtrate of Trichoderma Nag), while the control had the highest (37%). Among treated groups, T 12 (Culture filtrate of Trichoderma Mot) and T 3 (Culture filtrate of Trichoderma Har) showed the highest mortality (19.67%). Treatment T 6 (Culture filtrate of Trichoderma Ban) showed low mortality (7%) after T 9 . In 2022-23, T 9 again showed the lowest mortality (7.33%), with T 6 , T 7 (Culture filtrate of Trichoderma Jab), and T 10 (Culture filtrate of Trichoderma Ana) following. The control exhibited 35% mortality. Overall, Trichoderma filtrates reduced seed mortality and enhanced germination in tomato plants. This result is consistent with the findings of Chagas et al. (2020) [3] , reported that Trichoderma spp. effectively reduce disease incidence by outcompeting pathogenic fungi, producing antifungal metabolites and enhancing the plant's own defense mechanisms. The variability in the performance of culture filtrate of different isolates of Trichoderma spp., as seen in the lower germination rates and higher mortality rates in treatments T 3 (Culture filtrate of Trichoderma Har) and T 12 (Culture filtrate of Trichoderma Mot), highlights the importance of selecting the right isolate for specific applications. This variability is well-documented in literature, where the effectiveness of Trichoderma isolates can vary depending on factors such as the strain, environmental conditions, and the crop being treated (Mastouri et al., 2017; Singh et al., 2021) [11,16] .

Effect of culture filtrate of Trichoderma isolates on number of days taken for flowering and fruiting

In current study the earliest flowering was observed in treatment T 9 (Seedling treatment + One foliar spray of culture filtrate of Trichoderma Nag), where flowering occurred at 31.29 days after transplanting (Table -3). This is significantly earlier than the control (T 13 ), where flowering took 55.78 days (in 2021-22 season). The efficacy of culture filtrate of Trichoderma Nag (T 9 ) in promoting early flowering could be attributed to its ability to enhance nutrient uptake and hormonal balance, which may promote the transition from vegetative to reproductive stages. Similar trends were observed in the 2022-23 cropping period, where treatment T 9 again showed the earliest flowering at 33.59 days, followed by culture filtrate of Trichoderma Ban (T 6 ) at 36.06 days and culture filtrate of Trichoderma Jha (T 2 ) at 38.73 days. In terms of number of says taken for first fruiting among all the treatments T 9 treatment (Seedling treatment + One foliar spray of culture filtrate of Trichoderma Nag) showed the earliest fruit appearance at 38.96 days, while the control (T 13 ) has taken 63.78 days in 2021-22 (Table -3). This suggests that culture filtrate of Trichoderma spp. not only accelerates flowering but also promotes early fruit set, likely due to improved plant vigor and increased resistance to stress conditions (Woo et al., 2006) [22] . In the 2022-23 season, T 9 once again led to the earliest fruit appearance at 41.26 days, followed by T 6 (Seedling treatment + One foliar spray of culture filtrate of Trichoderma Ban) at 43.06 days, and T 7 (Seedling treatment + One foliar spray of culture filtrate of Trichoderma Jab) at 49.39 days. The control treatment took 66.08 days, confirming the superiority of these culture filtrate treatments in reducing the time to fruiting. These findings supporting the role of Trichoderma spp. as plant growth promoters, which could be attributed their abilities to enhance nutrient availability, modulate plant hormone levels, and induce systemic resistance (Vinale et al., 2008) [20] . Current findings also aligns with findings of Siddiquee et al. (2012) [15] they concluded that Trichoderma culture filtrates can be effectively used to accelerate the reproductive phase of plants, leading to earlier fruiting and flowering. @ 10%, Treatment 2 = Seedling treatment + One foliar spray of culture filtrate of Trichoderma (Jha) @ 10%, Treatment 3 = Seedling treatment + One foliar spray of culture filtrate of Trichoderma (Har) @ 10%, Treatment 4 = Seedling treatment + One foliar spray of culture filtrate of Trichoderma (Lak) @ 10%, Treatment 5 = Seedling treatment + One foliar spray of culture filtrate of Trichoderma (Var) @ 10%, Treatment 6 = Seedling treatment + One foliar spray of culture filtrate @ 10% of Trichoderma (Ban), Treatment 7 = Seedling treatment + One foliar spray of culture filtrate of Trichoderma (Jab) @ 10%, Treatment 8 = Seedling treatment + One foliar spray of culture filtrate of Trichoderma (Usn) @ 10%, Treatment 9 = Seedling treatment + One foliar spray of culture filtrate of Trichoderma (Nag) @ 10%, Treatment 10 = Seedling treatment + One foliar spray of culture filtrate of Trichoderma (Ana) @ 10%, Treatment 11 = Seedling treatment + One foliar spray of culture filtrate of Trichoderma (Lud) @ 10%, Treatment 12 = Seedling treatment + One foliar spray of culture filtrate of Trichoderma (Mot) @ 10%, Treatment T13 = Control (Untreated) = Seedling treatment + One foliar spray of culture filtrate of Trichoderma (Ayo) @ 10%, Treatment 2 = Seedling treatment + One foliar spray of culture filtrate of Trichoderma (Jha) @ 10%, Treatment 3 = Seedling treatment + One foliar spray of culture filtrate of Trichoderma (Har) @ 10%, Treatment 4 = Seedling treatment + One foliar spray of culture filtrate of Trichoderma (Lak) @ 10%, Treatment 5 = Seedling treatment + One foliar spray of culture filtrate of Trichoderma (Var) @ 10%, Treatment 6 = Seedling treatment + One foliar spray of culture filtrate @ 10% of Trichoderma (Ban), Treatment 7 = Seedling treatment + One foliar spray of culture filtrate of Trichoderma (Jab) @ 10%, Treatment 8 = Seedling treatment + One foliar spray of culture filtrate of Trichoderma (Usn) @ 10%, Treatment 9 = Seedling treatment + One foliar spray of culture filtrate of Trichoderma (Nag) @ 10%, Treatment 10 = Seedling treatment + One foliar spray of culture filtrate of Trichoderma (Ana) @ 10%, Treatment 11 = Seedling treatment + One foliar spray of culture filtrate of Trichoderma (Lud) @ 10%, Treatment 12 = Seedling treatment + One foliar spray of culture filtrate of Trichoderma (Mot) @ 10%, Treatment T13 = Control (Untreated)

Conclusion

The study highlights the effectiveness of culture filtrates from Trichoderma isolates in controlling Fusarium oxysporum f. sp. lycopersici and enhancing tomato plant growth. Treatments with culture filtrate of Trichoderma Nag (T 9 ) consistently resulted in the highest germination rates, lowest seed mortality, and greatest inhibition of fungal mycelial growth. Additionally, T 9 accelerated flowering and fruiting, outperforming other isolates and the control in both the 2021-22 and 2022-23 seasons. Culture filtrate of other isolates, such as Trichoderma Ban (T 6 ) and Trichoderma Jab (T 7 ), also showed promising results. The study underscores the biocontrol potential of Trichoderma spp., attributed to their production of antifungal metabolites, improvement of nutrient uptake and enhancement of plant defense mechanisms, making them valuable candidates for sustainable agriculture and pathogen management.

Acknowledgement

AcknowledgementThe authors are highly grateful to Department of Plant Pathology, C.S.A. University of Agriculture and Technology, Kanpur for all kinds of research facilities throughout the work.

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