Progress report for LNC23-490
Project Information
The purpose of this project is to increase the sustainability of specialty crop farming by reducing the incidence of an important food security issue such as Fusarium through identification and management with effective biocontrol agents. The hypothesis is that the molecular identification of local Fusarium species/strains, dual confrontation assays to assess biocontrol effectiveness of commercial Trichoderma species/strains, and its verification/demonstration in on-farm studies will facilitate adoption of effective management strategies that improve the sustainability of producing solanaceous crops.
Since there is no clear understanding of which Fusarium species/strains are currently infecting our solanaceous crops, identifying them is necessary to address this food security issue. This project will use molecular identification procedures to determine the diversity of pathogenic Fusarium in NCR fields. Sequence-based molecular identification of Fusarium is already well established but current sequence databases for local pathovars are missing, which may prevent accurate identification. To address this problem, we have partnered with INTA in Costa Rica, where they have decades of experience cataloging the diversity of Fusarium using similar approaches. INTA has agreed to assist in our development of molecular identification procedures and crop-specificity cataloging in the NCR.
Trichoderma spp. as biological control agents have become one promising option to mitigate the incidence of Fusarium. However, studies have shown that the effectiveness of Trichoderma depends on Fusarium species/strain and even commercial products are not always effective in the field. Thus, this project will validate, in the lab and on-farm, the effectiveness of different Trichoderma species/strains to inhibit or kill local pathogenic Fusarium strains. We will use already developed approaches. In-lab dual confrontation assays will be used to validate the biocontrol agent is effective. Then, we will take products to the original field for corroboration in situ.
The goal is to partner with NCR farmers to test the full potential of Trichoderma-based biocontrol to mitigate Fusarium outbreaks. Our extension specialists have already identified several farmers that are excited to participate, and more farmers will be identified during the course of this project. Once field applications are completed, we will take steps to fully evaluate the effectiveness of the field treatments by molecular detection of previously identified strains and by collecting farmers’ feedback regarding the health and productivity of subsequent crops. Steps will then be taken to disseminate the information to NCR farming communities through extension publications, website updates, webinars, workshops, presentations at farmers’ and professional meetings, and community field-day activities.
The Objectives / Outcomes for the proposed work are:
- Determine the diversity of Fusarium in NCR fields by identifying and differentiating local pathogenic Fusarium species, races, or strains affecting various solanaceous crops.
- Determine the biocontrol effectiveness and crop specificity of available Trichoderma species on locally identified Fusarium species/strains and characterize active compounds linked to the mode of action.
- Demonstrate/corroborate on-farm biocontrol effectiveness of Trichoderma species/strain to mitigate Fusarium outbreaks in the field.
- Disseminate new information on the biocontrol of Fusarium in solanaceous crops within the NCR to increase knowledge, adoption, and sustainability.
Overview: The project experienced significant delays in 2024 due to challenges associated with delayed funding. This resulted in the postponement of most proposed activities. Despite these setbacks, progress was achieved across several objectives, as outlined below.
Objective 1: Disease Diagnosis Dr. Ramón Arancibia and Dr. Dhruba Dhakal successfully collected 15 tomato samples from 8 Amish and Mennonite growers. These samples were submitted to the MU Plant Diagnostic Clinic for analysis. Both conventional and molecular diagnostic techniques were employed, confirming five tomato plants infected with Fusarium wilt, caused by Fusarium oxysporum. Seven Fusarium isolates were subsequently collected, purified, and transferred to Dr. Walter Gassmann’s laboratory for further analysis.
Objective 2: Antifungal Activity of Trichoderma Species Growth confrontation experiments were initiated to evaluate the antifungal activity of commercially available Trichoderma species. Investigations into the mode of action by which Trichoderma inhibits Fusarium pathogens are ongoing. This research aims to elucidate the biological mechanisms contributing to fungal suppression.
Objective 3: High Tunnel Biocontrol Trials Coordination efforts led by Dr. Ramón Arancibia resulted in agreements with two growers to establish high tunnel experiments. These trials will evaluate the efficacy of commercial biocontrol agents against Fusarium wilt, including Rootshield, Asparello, Rootshield Plus, Prestop, Lalstop, Theia, Mycostop, Actinovate, and Double Nickel. The trials are scheduled to commence in Spring 2025.
Objective 4: Outreach and Application Objective 4 remains pending, as the majority of research activities are still in progress. Future efforts will focus on disseminating findings and providing recommendations to growers once sufficient data has been collected.
Conclusion: While financial constraints impacted the timeline, the research team has made significant strides in achieving key objectives. Diagnostic efforts, laboratory experiments, and field trial preparations are on track to continue into the next phase. The team remains committed to advancing sustainable biocontrol strategies to mitigate Fusarium wilt in tomatoes.
Research
- Different isolates of Fusarium that cause Fusarium wilt disease of solanaceous crops will be differentiated based on the morphological and genetical characteristics.
- The growth confrontation assay on the identified Fusarium isolates against commercially available Trichoderma will indicate the effectiveness of potential biological control candidates.
The purpose of this project is to increase the sustainability of specialty crop farming by reducing the incidence of soil-borne diseases such as Fusarium through identification and management with effective biocontrol agents. The hypothesis is that the generated and extended information on molecular identification of local Fusarium species/strains, dual confrontation assays to assess the specific Fusarium growth suppression and/or destruction by selected Trichoderma species/strains, and its verification/demonstration in on-farm studies will facilitate adoption of effective management strategies that will improve economic, environmental, and social sustainability of solanaceous crop production. This project connects up-to-date research on local Fusarium pathogens, their impacts on solanaceous crops, and the biocontrol promises of selected Trichoderma strains to arm produce and underserved farmers with the knowledge required to improve their own sustainable land management practices and to prevent the environmental damage that can be caused by current approaches to Fusarium eradication.
Objective / Outcome 1: Determine the diversity of Fusarium in NCR fields by identifying and differentiating local pathogenic Fusarium species, races, or strains affecting various solanaceous crops.
Farmers who have been producing tomato and other solanaceous crops in high tunnels for several years in Missouri have been identified (see farmers in Team Experience and role) and are willing to work on solutions to soilborne diseases. Infected plant samples will be collected from these sites to identify Fusarium species/strains and the infested area will be marked for later biocontrol trials. We will identify and work with additional produce farmers (including young and/or beginning farmers) in Missouri and surrounding states for sampling and to provide recommendations of biocontrol agent and later evaluation.
Field sample collection from problematic fields/high tunnels in the NCR will be performed by University of Missouri Extension and Lincoln University Extension field specialists, under the direction of Dr. Arancibia, Dr. Dhakal and Dr. Valliyodan, followed by isolation and identification in the MU Plant Diagnostic Clinic. At the MU Plant Diagnostic Clinic, diseased plant tissues will be selected for isolation of soilborne pathogens. Initial screening for Fusarium spp. will be performed based on morphological traits of fungal spores and fruiting structures from pure culture. Subsequently, fungal DNA will be extracted from each unknown Fusarium sample under the direction of Dr. Peng Tian. Next, specific DNA regions will be amplified using Polymerase Chain Reactions and sequenced at the Bond Life Sciences Center Genomics Technology Core to produce identity-specific information that is compared with known species, races and strains found in various databases. The Genomics Technology Core specializes in various types of sequencing (Sanger sequencing, Illumina Hi-seq and Oxford Nanopore Long Read sequencing), genotyping, SNP detection and analysis, and provides support for computer analysis of nucleic acid and data that can be used for this project. In addition, our collaborators at INTA in Costa Rica have agreed to assist with any problematic sequence identifications using their own developed Fusarium identification procedures and databases.
A database of Fusarium present in the NCR will be developed over the course of the project. The identified local Fusarium species/strains will be grouped by molecular traits. Based in this information, three fields with different Fusarium species/strain will be selected for on-farm studies (Objective / Outcome 3).
Objective / Outcome 2: Determine the biocontrol effectiveness and crop specificity of available Trichoderma species on locally identified Fusarium species/strains and characterize active compounds linked to the mode of action.
Using already documented and optimized laboratory procedures, Dual Confrontation Assay will assess the effect of Trichoderma species / strains on the growth of the collected Fusarium spp. from Objective / Outcome 1 (Tian et al., 2020; Matarese et al., 2012). The specific species and strains of Trichoderma will be based on commercial availability, documented reports as well as through evidence collected during multi-year investigations from our INTA partners. In the dual confrontation assays, both organisms are grown on Potato Dextrose Agar (PDA) plates (see example figure from MU’s past work with INTA). If the strain of Trichoderma is an effective biocontrol, a reduction in growth rate will be observed, and in some cases, death of the Fusarium may occur. Such results will be documented microscopically using facilities in the Molecular Cytology Core facility located in the Bond Life Science Center (LSC). Those Trichoderma strains that reduce or stop the growth of the identified Fusarium will be used to explore potential active compounds and to corroborate its effectiveness in on-farm field studies explained in Objective / Outcome 3. In addition, recommendations based on the dual confrontational trial will be given to the farmers with Fusarium infested field that were not selected for on-farm studies.
Confrontation assay plates that show biocontrol effects will have secreted metabolites extracted for analysis at the MU Metabolomics Center facilities also located in the LSC. These procedures have been previously developed and optimized during a Borlaug Fellowship project for the impacts of Trichoderma on banana Fusarium races in Costa Rica as well as other investigations (Shahriar et al., 2022). Here, four samples will be required to identify and produce counts of the unique metabolites produced during confrontation using both GC-MS and LC-MS platforms (1 – control Fusarium grown alone; 2 – control Trichoderma grown alone; 3 - confrontation of Fusarium with Trichoderma; and 4 - analysis of the media itself to allow subtraction of metabolites from the media alone). The aim is to make use of identified Trichoderma metabolites to interrogate existing metabolomic databases from similar projects to narrow the list of candidate compounds with biocontrol activity.
Objective / Outcome 3: Demonstrate/corroborate on-farm biocontrol effectiveness of Trichoderma species/strain to mitigate Fusarium outbreaks in the field.
Commercial biocontrol Trichoderma species will be tested in on-farm trials to determine if the biocontrol effectiveness observed in the lab translates to fields.
High tunnels and/or field areas with identified Fusarium problems will be treated with commercially available biocontrol agents (Trichoderma harzianum-strain-T22, Trichoderma-virens-strain-G-41, Trichoderma asperellum, strain T34, Trichoderma gamsii, and Trichoderma Viride). Two non-Trichoderma biocontrol agents (Streptomyces lydicus and Gliocladium catenulatum) will be included as a request of one grower. Bacillus amyloliquefaciens will not be included because it has not worked for this grower. Sections of the area will be separated for treatments with the biocontrol agents and an untreated control. Application will follow the label recommendations for each product. When combinations of different Trichoderma species are available in the same product, additional treatment areas will be considered. A randomized plot design with two to four replications depending on available space will be used. Tomato disease symptoms, survival, growth, and production will be recorded for evaluation. In addition, a recommendation based on the dual confrontational trials will be given to farmers that have Fusarium but were not selected for on-farm studies. We will follow up with these farmers for feedback on adoption and effectiveness of the biocontrol agents.
A challenge trial with soil collected from three Fusarium infested fields will be used to challenge the effectiveness of the Trichoderma spp. selected for the best activity against that site specific pathogenic Fusarium. Tomato plants of a susceptible variety (heirloom) grown in pots will be treated with the selected Trichoderma. Four treatments with four replicates will be used in a randomized block design for statistical purposes: soil, soil inoculated with locally isolated and identified Fusarium, soil treated with the selected Trichoderma preparation, and Fusarium inoculated soil treated with the selected Trichoderma preparation. Trichoderma preparations of chlamydospores and conidia from cultures diluted to appropriate concentrations will be used to drench the soil. Tomato disease symptoms, survival, growth, and production will be recorded for evaluation.
Participating farmers with the assistance of Dr. Arancibia and Dr. Dhakal will contribute to the evaluation of the on-farm studies by comparing crop performance and disease incidence, survival, growth, and production with untreated controls. Subsequent testing of crop growth, survival, and production over the next two years, and recovery of Trichoderma from treated soils will provide evidence on survival and the potential of extended biocontrol activity. We will also test for recovery of the pathogenic Fusarium species/strain.
Objective / Outcome 4: Disseminate new information on the biocontrol of Fusarium in solanaceous crops within the NCR to increase knowledge, adoption, and sustainability.
Several venues for dissemination of the generated information on Fusarium biocontrol in solanaceous crops within NCR will be considered.
Workshops and/or on-farm field days will be organized to showcase the effectiveness of biocontrol agents against Fusarium diseases. University of Missouri Extension and Lincoln University Extension will collaborate on the outreach efforts to extend the information to growers in Missouri. In addition, efforts to collaborate with extension personnel from other states in the NCR will be made to extend the information beyond Missouri. The information will be delivered through presentations at grower meetings such as the annual Great Plains Growers Conference (organized by MO IA, KS and NE), the Missouri Organic Association meeting, and several grower meetings organized by the Mennonite produce communities. This photo shows Dr. Dhakal provided on-site plant diagnostic service during a Mennonite Auction event in 2020. In addition, journal and extension publications, website updates, and MUIPM video posts will be developed to disseminate newly generated information. While some underserved farmers, such as minority and women farmers, have internet access, print publications such as the Missouri Produce Growers Bulletin published by MU Extension IPM is critical to reach most of the Mennonite/Amish farmers. These activities will involve information derived from the research and directly from farmer involvement to create effective resources on disease management and crop growing practices that will reduce losses and improve the economic and social sustainability of these specialty crop farmers. In addition, building upon existing MU and LU outreach programs will allow our program to have an outreach legacy that will be functional and expandable beyond the scope of this research. This program takes science on the road, traveling to the underrepresented communities in Missouri to educate both the public and the educators who will in turn touch many in the future.
In addition, Mennonites and Amish farmers have well established communication among them, and they share their problems and solutions. Therefore, participating farmers will also contribute to the dissemination of the results to the rest of their community members and to other communities that produce solanaceous crops in their fields and high tunnels.
PROJECT TIMELINE
| YEAR 1 | YEAR 2 | YEAR 3 |
| Upon funding award, meeting with participating growers to coordinate on-farm studies | On-farm challenge studies with potted tomato plants | Continue with on-farm challenge studies with potted tomato plants |
| Collection of Fusarium infected samples and field marking |
Extension activities: Field days, workshops, and presentations at grower meetings. Video post in YouTube-MUIPM channel Presentations at professional meetings. |
Continue with Extension activities: Field days, workshops, and presentations at grower meetings. Video post in YouTube-MUIPM channel Presentations at professional meetings. |
| Molecular identification of local pathogenic Fusarium spp. and database | Continue molecular identification of local pathogenic Fusarium spp. and database | Publications of results in the Missouri Produce Growers bulletin, HortTechnology, and/or Plant Disease. |
| Dual confrontation tests in the lab | Continue dual confrontation test in the lab | |
| Metabolomic analysis of selected Trichoderma strain during Fusarium confrontation | Bioinformatic analysis of Trichoderma metabolite data to determine active compounds | |
| On-farm field evaluation of commercially available biocontrol agents (Trichoderma) and strategies. | Continue with on-farm field evaluation of commercially available biocontrol agents (Trichoderma) and strategies. | Follow up with evaluation of treated on-farm fields. Isolation of Fusarium spp. and Trichoderma spp. from the soil |
References:
Matarese, F., Sarrocco, S., Gruber, S., Seidl-Seiboth, V., & Vannacci, G. (2012). Biocontrol of Fusarium head blight: interactions between Trichoderma and mycotoxigenic Fusarium. Microbiology, 158(1), 98-106.
Shahriar, S. A., Islam, M. N., Chun, C. N. W., Kaur, P., Rahim, M. A., Islam, M. M., ... & Siddiquee, S. (2022). Microbial metabolomics interaction and ecological challenges of Trichoderma species as biocontrol inoculant in crop rhizosphere. Agronomy, 12(4), 900.
Tian, Y., Yu, D., Liu, N., Tang, Y., Yan, Z., & Wu, A. (2020). Confrontation assays and mycotoxin treatment reveal antagonistic activities of Trichoderma and the fate of Fusarium mycotoxins in microbial interaction. Environmental Pollution, 267, 115559.
Efforts concerning Objective 1 (Determine the diversity of Fusarium in NCR fields) have resulted in seven pathogenic Fusarium strains being conclusively identified from diseased tomato plants in Missouri crop fields based on the PCR analyses using three sets of primers targeting ITS, EF and RPB genes. The conventional and molecular identification and diagnostic work were performed at MU Plant Diagnostic Clinic.
As part of Objective 2 (Determine the biocontrol effectiveness available Trichoderma species) the Gassmann lab has been performing growth confrontation experiments on these pathovars against several commercially available Trichoderma species, T. asperellum strain T34, T. harzianum stain t22b, and T. viride. To date, analysis of three of the Fusarium strains with T. asperellum T34 have been completed. Results show that T. asperellum T34 is effective at inhibiting and stopping the growth of all Fusarium strains under laboratory conditions. This can be observed when the growth curve of Fusarium stops increasing in response to introduced Trichoderma (see figure1). 
Results from the T. harzianum stain t22b and T. viride and the remaining 4 Fusarium strains are also in progress.
Also, as part of Objective 2, the Gassmann lab has been pursuing the mode of action behind how Trichoderma species inhibit the growth of Fusarium pathogens. Stimulated by new findings by our INTA collaborators in Costa Rica, we are exploring the possibility that the inhibitory compounds involved are not just water-soluble metabolites but also involve volatile compounds released into the environment. To assess this, we have teamed up with a new lab at MU, run by Dr. Joseph Lynch, specializing in the collection and identification of volatile metabolites. Here, new approaches using PDMS Twister technology (Gerstel, Germany) were used to isolate volatile compounds during fungal confrontation. Twisters are essentially small stir bars coated with a sorptive material that can be held in place inside
the plates using small magnets (see example in figure 2). Volatiles collected on this material were isolated with a Centri 90 thermal desorption unit (Markes, Germany) and analyzed using an Agilent GC-MS apparatus (Agilent, CA).
Initial results identified 100s of compounds using this approach. 
Careful examination of the compounds coming from the Trichoderma / Fusarium confrontation experiments compared with controls containing either Fusarium or Trichoderma alone as, well as media alone, are allowing compounds of interest to be identified. One example GC-MS result is shown in the figure3. We are in the process of analyzing the complete set of PDMS Twisters to find unique and/or up-regulated compounds produced during confrontation.
Project Activities
Educational & Outreach Activities
Participation Summary:
Field specialists Dr. Dhruba Dhakal and Dr. Ramón Arancibia conducted site visits to approximately eight Amish and Mennonite growers, where they collected diseased tomato and pepper samples and provided consultations on plant disease management strategies. Additionally, Dr. Arancibia collaborated with two growers to establish high tunnel experiments designed to evaluate the effectiveness of various commercial biocontrol agents against Fusarium wilt. The selected biocontrol agents include Rootshield, Asparello, Rootshield Plus, Prestop, Lalstop, Theia, Mycostop, Actinovate, and Double Nickel. These experiments are scheduled to commence in Spring 2025. It is important to note that photographs documenting the interactions between specialists and growers were not obtained, respecting the specific requests of the participating growers.
Dr. Peng Tian, Principal Investigator (PI) of this project, presented the research and its progress at several prominent meetings, including the North Central Plant Diagnostic Network annual meeting, the National Plant Diagnostic Network national annual meeting, and the 2024 annual meeting of the Georgia Association of Plant Pathologists (GAPP). Furthermore, Dr. Tian was invited to present the project during his visits to the Department of Plant Pathology at the University of Georgia (UGA) and the Plant Diagnostic Center at the University of Florida (UF), where he delivered a lecture highlighting key aspects and findings of the study.