Progress report for GNE24-310
Project Information
Mycotoxins (toxins produced by molds) contaminate approximately 60-80% of staple food and feed crops, with 25% exceeding regulatory limits. In the Northeast, economically essential crops like corn, crucial for animal feed and dairy production, are frequently impacted. Notably, aflatoxin, generated by Aspergillus spp., poses significant health risks, including liver and lung cancer, impacting global health and food security. Climate change projections indicate increased temperatures, prolonged droughts, and erratic precipitation patterns, promoting higher mycotoxin occurrence globally, including in the Northeast US. This project aims to assess beneficial fungi as a climate-smart solution to mitigate rising aflatoxin levels in corn amidst extreme climate conditions. Specifically, we will test and select the most resilient strains of Trichoderma (beneficial fungi) from current commercial biologicals and newly isolated strains found in PA soil environments. Their resilience will be gauged by their ability to effectively suppress Aspergillus flavus growth and aflatoxin production in corn under climate change-simulated temperature and water activity conditions. We will also develop need-specific informational outreach materials to enhance community awareness and engagement in the potential use of biologicals as a mycotoxin management practice. This study marks the initial step in identifying environmentally safe and climate resilient strategies to reduce mycotoxin levels, ensuring the sustainability of food and feed crops, and safeguarding the incomes of corn growers and dairy farms.
Objective 1: Investigate the synergistic effects of temperature and water activity combinations (T*Aw) on various biocontrol Trichoderma strains’ growth rate and development.
Hypothesis: Trichoderma strains with the fastest growth rates and highest spore production would be most resilient to T*Aw trials.
Expected outcomes: Trichoderma strains with highly resilient characteristics in T*Aw trials.
Deliverables: Spreadsheet with the genetic characterization of the various isolated Trichoderma strains; graphs showing the changes in growth rate and development (characterized by sporulation, germination/growth rate, and ATP of the different studied Trichoderma stains under the different temperature and water activity combinations.
Objective 2: Evaluate the synergistic effects of adverse T*Aw on the efficacy of select Trichoderma strains from obj 1 in reducing Aspergillus flavus growth and aflatoxin levels using three ecologically relevant models.
Hypotheses:
- In vitro, using the nonvolatile metabolite production and competitive exclusion models, changes in T*Aw won’t impact the inhibition of Aspergillus growth and aflatoxin levels by Trichoderma.
- In vitro, volatile organic compounds production by Trichoderma spp. is impacted by adverse T*Aw, affecting inhibition of Aspergillus growth and aflatoxin levels.
Expected outcomes: Discovery of Aspergillus growth and aflatoxin’s biocontrol agents whose bioactivity is highly resilient to adverse temperature and water activity combination trials and their potential best mode of action.
Deliverables: Spreadsheet with the Aspergillus growth percentage inhibition and aflatoxin levels by different Trichoderma strains using volatiles, non-volatiles, and competition models, with and without Trichoderma treatment under the various temperature and water activity combination trials.
Objective 3: Evaluate the synergistic effects of adverse T*Aw on the efficacy of Trichoderma strains in reducing aflatoxin levels in stored corn grains.
Hypothesis: Trichoderma strains will significantly reduce fungal growth and aflatoxin levels across all temperature and water activity combinations in storage grains.
Expected outcomes: Discovery of Aspergillus growth and aflatoxin’s biocontrol agents whose bioactivity is highly resilient to adverse temperature and water activity combination trials and can be used in storage or to coat seeds before planting.
Deliverables: Spreadsheet with aflatoxin level changes in corn grains with and without Trichoderma treatment under the various temperature and water activity combination trials.
The purpose of this project is to evaluate the use of beneficial fungi as a climate-smart and resilient mitigation strategy for the expected increase in aflatoxin levels in corn grains under extreme climate change conditions. This will ultimately bolster the economic sustainability of corn growers and dairy farms. The dairy industry leads the northeastern agricultural sector; in Pennsylvania (PA), it generates an estimated $8.3 Billion and creates 15400 direct jobs 1. Corn, a vital component in feed production, is pivotal in supporting the dairy industry. In PA alone, animal feed production for dairy cows, largely reliant on corn, generates $616 million 1.
Mycotoxins (“toxins secreted by fungi”) contaminate 60-80% of global food and feed crops, with 25% exceeding regulatory limits 2. This contamination not only threatens food security but also poses adverse health risks to humans and animals. In the northeastern region, crucial economic crops like corn, vital for animal feed, are often affected. Deoxynivalenol/vomitoxin (DON) from Fusarium graminearum and aflatoxin from Aspergillus flavus and Aspergillus parasiticus are among the economically significant mycotoxins found in PA’s corn grain and silage 3.
Fungal contamination and toxin production severity hinge on abiotic climatic factors. Current precipitation and low temperatures in the northeast favor high levels of DON and lower aflatoxin production 4. However, predicted climate change scenarios forecast a shift in mycotoxin contamination patterns. For example, severe weather fluctuations, such as temperature increases and droughts, have generated new aflatoxin hotspots in places where aflatoxin was never a problem 5. By 2050, significant increases in aflatoxin levels are anticipated in US corn belts 6. These climate change patterns are also reflected in the northeast predicted changes, with predicted high heat waves, 4.5-10°F increase in temperature, and more days above 90°F than experienced in the last century 7. Similarly, PA indicates heightened temperatures, prolonged droughts, and extreme rainfall, amplifying fungal contamination and aflatoxin risks 8–10. These climate-induced weather variations will impact corn yield due to growth time changes and foster ideal conditions for pests, Aspergillus growth, and aflatoxin production in corn ears.
Aflatoxin contaminations have been tied to the increase in genotoxicity and immunotoxicity, causing hepatocellular carcinomas in humans with increased risk to the population with Hepatitis B Virus and HIV 11,12. Dairy cows can also metabolize aflatoxin and carry it over in milk as aflatoxin M1, causing health impacts in people after milk consumption. Consumption of contaminated aflatoxin B1 by dairy cows reduces their milk quality and productivity by about 0.36 l/cow/day 13. Ultimately, the rise of aflatoxin levels in corn grains would lead to downgrading and rejection, creating an economic burden for corn growers and dairy farms. To address this, climate change-resilient strategies are needed to reduce Aspergillus contamination and enhance plant drought resistance.
The growing awareness of sustainable agriculture has led to a decline in consumers’ and farmers’ acceptance of synthetic fungicides due to their adverse environmental risk and fungal resistance issues 14,15. As a result, there’s a rising interest in biological alternatives to improve plant health and combat pathogens. These can be biological control agents (beneficial microorganisms) and their biologicals (microorganisms or plants and their naturally derived products). Trichoderma spp. have been extensively studied for their agricultural and industrial applications. These fungi are rapid-growing, widespread, versatile in nutrient utilization, resilient, suppress multiple plant diseases, and enhance plant resilience to drought, improving yields 16–18. Despite extensive studies on various plant pathogen reduction and individual abiotic condition resistance, research on Trichoderma spp. against Aspergillus and aflatoxin production remains limited. There is also no literature on how the combinations of the different climate change-predicted conditions will affect the development and bioactivity of Trichoderma spp. as aflatoxin biocontrols.
This project aims to evaluate and select climate-resilient Trichoderma spp. that can reduce and control aflatoxigenic fungal growth and toxin production. To accomplish this goal, I propose three research objectives and an outreach program (see Plan of Work). I will test Trichoderma spp. from current biologicals and newly isolated strains found in PA soil environments using three ecological relevant laboratory models. Within these models, we will simulate anticipated drought conditions using temperature (T) and water activity (Aw). If the results show merit, these novel biologicals can be tested in field trials for aflatoxin control with the potential to enhance plant resistance to drought. This study marks the initial step in identifying environmentally safe and climate resilient strategies to reduce mycotoxin levels, ensuring the sustainability of food and feed crops, and safeguarding the incomes of corn growers and dairy farms.
Cooperators
- (Educator)
Research
Objective 1: Compare the synergistic effects of T*Aw on various biocontrol Trichoderma strains' growth rate, sporulation, and ATP activity.
1.1 Selection of Trichoderma strains for use in the temperature and water activity trials
Diverse Trichoderma strains based on preliminary experiments, genetic diversity, and isolation sources will be used for this project and categorized as follows: Group 1: Trichoderma strains isolated from commercial biocontrol (TR 13, 34, 36, and 38). Group 2: NRRL ARS reference strains (NRRL 5243 and NRRL 2314). Group 3: Trichoderma spp. isolated from the soil of PA farmers in Dr. Wallace’s network (see letter attached).
When tested against Aspergillus parasiticus and A. flavus growth, our preliminary data showed that Trichoderma strains selected for group 1 reduced Aspergillus growth by 20-95% depending on the mechanism of action, with volatiles being the least effective and non-volatiles the most effective (data not published yet). Antifungal volatiles such as terpenes were shown to be exclusive to Trichoderma strains with the most inhibitory efficacy. Group 2 strains were selected due to the following reasons. Trichoderma viride (NRRL 6418) was isolated from a corn field, making them competitive for the substrate and potentially meaning they have cohabitated with other corn-inhabiting microorganisms such as Aspergillus. Trichoderma virens (NRRL 2314) was chosen because it was isolated from soil in the northeastern region (Maryland), making it local and potentially relevant to other parts of the region if found effective.
Three soil samples (500g) will be collected from three locations on the farm in consultation with farm partners. Soil samples will be diluted and plated on Trichoderma selective medium (TSM); isolated strains will be purified by three consecutive rounds on Potato Dextrose agar (PDA) for each colony. Trichoderma isolates will be further evaluated by colony morphology and microscopy.
All Trichoderma spp. used in this study will be maintained on standard Potato Dextrose Agar (PDA) or as spore stocks in 25% glycerol at -80C. Stains will be validated prior to experiments using Sanger sequencing of the ITS 1/4 region and Tef1 gene 32.
1.2 Experimental approach for adverse temperature and water activity trials.
A two-factor complete factorial design with temperatures ranging (T)(25 - 40°C) and two water activities (Aw)(0.90 - 0.98) was set up to evaluate the synergistic effect of T*Aw combinations on the development of Trichoderma strains PDA supplemented with 2% (w/v) corn flour)(PDC). Higher ranges of temperature and water activities were decided on to reflect extreme conditions that could be met with droughts and climate change. The ongoing experiment tests Group 1 strains mentioned in 1.1, which were chosen to be evaluated first because they showed biocontrol efficacy in our preliminary studies. Growth rates and fungal development of Trichoderma strains selected from 1.1. will be evaluated under T*Aw combinations. Briefly, to achieve different Aw levels, glycerol will added to PDC as previously established 33. Equal spore concentrations (10^6 spores/ml) of each Trichoderma strain will be center inoculated and placed under the appropriate T*Aw combination for up to 7 days. Growth as a function of colony diameter will be measured daily and reported as growth rate. Spore counts will be obtained as a measure of the degree of sporulation. ATP activity of fungal colonies will be measured as an indicator of fungal biomass. The conidial spore concentration will be measured with a hemocytometer, and the ATP activity will be evaluated from the dry biomass using a biochemical ATP assay.
Each biological treatment will contain triplicate plates (n=3) and be repeated twice. Technical triplicates will be conducted for spore counts and ATP assay. Statistical analysis of the factorial design will be performed using MINITAB, and the effects of the combinations of Aw and T on growth rate, spore count, and ATP activity will be evaluated. A two-way interaction ANOVA (T*Aw) will be carried out. Two Trichoderma strains will be selected based on growth rates and fungal development (i.e., the most resilient strains across T*Aw experiments) for use in Obj. 2 and 3.
Objective 2: Evaluate the synergistic effects of T*Aw combinations on Trichoderma- Aspergillus interaction on laboratory growth medium using the three ecological relevant models. (Graphical representation of the models' setup attached)
Methods common to objectives 2.1-2.3: A two-factor complete factorial design with two temperature levels (T)(30,35°C) and two water activity levels (Aw) (0.95 and 0.98) will be conducted. The synergistic effect of T*Aw combinations on the efficacy of the two selected Trichoderma strains from Obj.1 will be tested. Potato Dextrose Agar supplemented with 2% (w/v) corn flour will be used as a growth medium (PDC). Glycerol will be added to the media to achieve the necessary Aw in the used PDC plates. Each biological treatment will contain triplicate plates (n=3) and be repeated twice (two independent biological experiments). All experiments will be tested using Aspergillus flavus NRRL 3357, a major producer of aflatoxin in field and laboratory studies 34 . Aflatoxin levels will be measured using Ultra Performance Liquid Chromatography (UPLC). All methods proposed have been conducted under university-approved safety protocol (IBC #BIO202300088) for safe handling and disposal of aflatoxin-producing strains and aflatoxin-containing samples.
Aflatoxin levels and growth inhibition of Aspergillus flavus NRRL 3357 by Trichoderma strains under T*Aw combinations will be calculated as: % IM= [(Mc-Mt)/Mc] x100. Where IM is the percentage inhibition of the measurement (A. flavus growth or aflatoxin levels), Mc is the measurement in the control plate (A. flavus only), and Mt is the measurement in treatment (A. flavus exposed to Trichoderma). The statistical effect of T*Aw combinations on A. flavus growth and aflatoxin levels by each Trichoderma strain will be evaluated by ANOVA using R studio.
2.1 Assess the impact of T*Aw combinations on the inhibitory effect of Trichoderma volatiles on Aspergillus growth and aflatoxin levels.
The production of volatile organic compounds (VOC) is one of the mechanisms used by Trichoderma to inhibit pathogens. Here, we will assess the production and efficacy of Trichoderma VOC under adverse T*Aw combinations. The center of a PDC plate will be inoculated with Trichoderma and a separate PDC plate with Aspergillus flavus. Spore concentrations will be constant (5 uL of 10*6 spores/mL). The Plate with Aspergillus flavus will be placed on top of a Trichoderma plate, sealed with two layers of Parafilm, and incubated at the appropriate temperature. Separate plates of Trichoderma species and Aspergillus flavus will be sandwiched with PDC plates inoculated with water (5 μΙ) for control. The colony diameter of A. flavus will be measured daily for seven days, and the overall percentage of growth inhibition caused by Trichoderma volatiles will be calculated on day seven.
The inhibitory effect of the Trichoderma strains on AfB1(the most potent aflatoxin type) production by A. flavus will also be measured at the end of the seven days. Aflatoxin levels will be measured by the excision of identical (cut at a regular distance from the inoculation point along the colony radius) 8 mm mycelial disks from the treated and control A. flavus plates and placed in a test tube. A sample (approximately 1g) of the disks will be weighed and mixed with 5 ml of the extraction solution (methanol: water, 80:20 v/v). The mixture will then be vortexed, centrifuged, and filtered to obtain the aflatoxin extract. Aflatoxin extracts with standards will then be sent to the HUCK metabolomics core facility for quantification using UPLC.
2.2 Assess the impact of the T*Aw combinations on the inhibitory effect of Trichoderma non-volatile metabolites on Aspergillus growth and aflatoxin levels.
Production of non-VOC (secreted metabolites) is another mechanism by which Trichoderma interacts with other organisms in the environment. Here, we will evaluate how the production and efficacy of Trichoderma non-VOCs are affected by the different T*Aw combinations while inhibiting Aspergillus flavus growth and aflatoxin production.
A cellophane membrane (Ultraclear Roll Cellophane for gel dryers, Idea Scientific Company, USA) will be added to fit on the top of the appropriate Aw PDC plate entirely and left to dry for a day. Cellophane membranes will avoid the invasion of the mycelium on the growth media while allowing the Trichoderma-secreted metabolites (non-VOCs) to diffuse into the media. A Trichoderma isolate will be center inoculated onto a PDC plate with cellophane. Spore concentrations will be constant (5 uL of 10*6 spores/mL) for treatments and 5 uL water for controls. These plates will then be incubated at the temperatures of interest for 48 hours to allow metabolite diffusion (preliminary studies show that Trichoderma metabolites that inhibit Aspergillus growth and toxin production peak at 48 h. At 48 h, the cellophane will be removed, and Aspergillus flavus will be center inoculated onto the PDC plate at the same spore concentration. The plates will then be incubated at their respective temperatures for seven days. The colony diameter of A. flavus will be measured daily for seven days, and the overall percentage of growth inhibition caused by Trichoderma non-VOCs will be calculated on day seven.
To evaluate the inhibitory effect of the Trichoderma isolates on AfB1(the most potent aflatoxin type) production by A. flavus. Aflatoxin B1 will be extracted from plates at the end of the seven days, and its levels will be quantified using UPLC (as detailed in 2.1). Aflatoxin levels and growth inhibition of A. flavus by Trichoderma isolates will be calculated and statistically analyzed, as described above in methods common for 2.1-2.3.
2.3 Assess the impact of the T*Aw combinations on the antagonistic effect of Trichoderma isolates against A. flavus.
Competition for space and nutrients is the third possible mechanism of Trichoderma spp. use to inhibit plant pathogens, and they will be evaluated here for A. flavus inhibition. Five μΙ of spore suspension containing 1 x 10^6 spore/mL in sterile distilled water of fungi will be point inoculated 1 cm from the edge of the plate at one side with Trichoderma spp. and at the opposite side with A. flavus. Separate plates with five μΙ of sterile deionized (DI) water will be point inoculated on one side while Trichoderma or A. flavus is added on the other side for control. All co-culture and control plates will be incubated at their respective temperatures of interest for seven days. The radial growth of both fungi will be measured daily. A. flavus radial growth percentage inhibition by Trichoderma isolates under the studied T*Aw combinations will be calculated and statistically analyzed as described above in methods common for 2.1-2.3.
Objective 3: Evaluate the synergistic effects of adverse T*Aw on the efficacy of Trichoderma isolates in reducing levels of aflatoxin B1 in corn grains.
Preharvest utilization of biological agents for aflatoxin control involves applying them as grain coatings to mitigate potential contamination in the field. However, their effectiveness may be influenced by changing climate conditions. Here, we will evaluate how T*Aw affects the efficacy of Trichoderma in protecting corn grains against aflatoxin contamination.
Corn grains will be sourced from a PA farmer (see letter attached). The grain batches will then be gamma-irradiated to kill the natural microbiome without inhibiting the germinability of the kernels. Three representative samples of each batch will be used to evaluate the Aw of the grains, and the necessary amount of DI water to attain Aw (0.95 and 0.98) will be determined in a preliminary study and confirmed before each run using the AquaLab 4TE (Decagon, USA). For each Trichoderma isolate, 10 g of corn from each Aw batch will be placed in a sterile conical flask covered with cotton plugs to allow gaseous exchange. A 50:50 inoculum solution of Trichoderma and A. flavus mix will be used for the biocontrol efficacy study (ratio found effective for other A. flavus biocontrol).
One hundred μΙ of the mixed inoculum and individual fungal isolates for control were added to the 10 g of maize, targeting 100 spores/g. The grain samples will then be placed at their respective temperatures for 10 days. After 10 days, grains will be blended. Aflatoxin extraction will be done by mixing 2g of ground corn from treated and A. flavus control samples with 8 ml of the extraction solution (methanol: water, 80:20 v/v). The mixture will then be vortexed, centrifuged, and filtered to obtain the aflatoxin extract. Aflatoxin extracts with standards will then be sent to the HUCK metabolomics core facility for quantification using Ultra Performance Liquid Chromatography (UPLC). Percentage Inhibition of aflatoxin levels in A. flavus by Trichoderma isolates will be calculated and statistically analyzed, as described above in methods common for 2.1-2.3.
No results have yet been generated on the aims since the project took some time to launch.
Education & Outreach Activities and Participation Summary
Participation Summary:
In an effort to understand the gravity of mold and mycotoxin problems in the field for farmers in PA, I visited the plant pathology research farm with Dr. Murrillo-Williams to examine mold in corn fields under various fertilizer treatments. This field visit confirmed high mold contamination presence in the corn field with observed multiple fungal genera, including Fusarium, Penicillium, Giberella, and Cladosporium. This initial consultation confirmed our rationale and will be used as a basis during our detailed needs assessment with farmers. For the outreach component of the study, we will first understand corn growers’ mycotoxins and their mitigation strategies’ concerns and then develop need-specific informational materials on mycotoxins and the use of biologicals as a mitigation strategy to be used in multiple outreach activities.
The first step to providing relevant information is directly listening to the concerned parties and establishing the type of knowledge gaps and needs they might have. I will conduct field visits and field day workshops with Dr. Murrillo-Williams (see letter) for the first summer of the project. During field visits and workshops, I will talk to corn growers and document reports of mycotoxin prevalence. In addition, I am also interested in understanding farmer concerns, attitudes, and knowledge gaps surrounding the use of biologicals as a mycotoxin mitigation strategy.
Moreover, the dairy industry leads the northeastern agricultural sector; in PA, it generates an estimated $8.3 Billion and creates 15400 direct jobs 1. Pennsylvania has ranked in the top 5 states for organic dairy cow production, supported by the increased demand for organic dairy products, paying three times the price for organic than conventional corn 29,35. This has increased the number of growers practicing organic farming. These farmers have limited mitigation strategies for the control of plant pathogens, creating a need for diverse approaches, including the use of biologicals for mycotoxin concerns. Using Dr. Murrillo-Williams' network through the Penn State extension, I will also attend the organic study circles with organic cereal growers to document their mycotoxin concerns, attitudes, and knowledge needs concerning using biologicals as a mitigation strategy.
Based on what I will have learned from the first summer of need evaluation, I will develop needs-specific information using appropriate strategies to disseminate the research result and address mycotoxin and biologicals knowledge gaps. These materials will include videos, presentation decks, and fact sheets developed using lay language. The videos and presentation decks will explain the general concepts of relevant mycotoxin contaminations (aflatoxin and vomitoxin), factors favoring their levels, contamination detection, and biologicals as a climate-smart and resilient mitigation strategy. The information in the material will be formatted to reflect the concerns and knowledge gaps learned from early discussions with farmers and to inspire new knowledge-based attitudes and behavior changes that are good for farmers' economies and the environment. Fact sheets will also be developed to summarize the slides and video content. Dr. Murillo-Williams and her network at Penn State Extension will then use all these materials during their corn grower education workshop, individual field visits, and field days, accordingly. My mentor, Dr. Wee, will also share the slides to explain grain mycotoxins and an example of their mitigation strategies during the Food Science Department’s “Mycology short course.”
I will complete this task, building on my experience with extension and outreach education materials gathered throughout the last two years of my education. For instance, I am currently working on an independent study, collaborating with the Global Teach Ag Network (GTAN) team at Penn State on a U.S. Department of Education-funded project, where I am developing two modules on international agriculture and development topics to be added to the GTAN collaborative educator’s curriculum. I have created two videos on mycotoxins and the 4Cs of food safety, introducing these concepts to Ag teachers and how they affect food security. I also took a project development and delivery course, in which I learned to develop stakeholder’s appropriate extension materials.
Finally, to share the results of my project with a broader audience, I will publish a minimum of one open-access manuscript in a peer-reviewed journal and present my findings at scientific conferences such as the Gordon Research Conference on Mycotoxin and Phycotoxins.