Progress report for GNE20-243
Emerging fungal plant diseases such as Fusarium wilt of tomato cause significant losses for growers in the northeast United States. Although some members of the Fusarium oxysporum Species Complex (FOSC) are responsible for this plant disease on over 100 crops, most isolates live in the soil or inhabit plants without causing disease. In fact, because of the complex evolutionary history of this group and the multiple modes of genetic exchange available, the isolates that cause Fusarium wilt can be very closely related to isolates that do not. As these isolates occupy the same ecological niche in agroecosystems, it is important to understand how these isolates relate to and impact one another. Improving this understanding helps researchers understand risks related to pathogen emergence and aid in developing better diagnostic tools. Therefore, it is crucial that we understand the evolutionary and ecological context from which pathogenicity emerges. This necessitates characterizing host adaptation among nonpathogenic isolates. Therefore, we propose to conduct a systematic sampling of FOSC isolated from specific parts of asymptomatic tomato plants and agricultural and non-agricultural soil. This sampling will allow meaningful comparisons to evaluate the genetic diversity of nonpathogenic FOSC in Pennsylvania tomato production, and identify factors that may be influencing population structure, and adaptation to tomato. Specifically, we will assess 1. Agricultural production, 2. Crop History, 3. Time, and 4. Host Variety. We will also assess current diagnostic approaches for Fusarium-associated diseases. We will identify educational needs of diagnosticians for Fusarium identification and provide resources to address those needs.
We seek to characterize nonpathogenic F. oxysporum occupying the same ecological niche as pathogenic isolates to better understand host adaptation and genetic exchange within the species complex.
Objective 1: Determine if F. oxysporum soil population is impacted by agricultural production.
Objective 2: Determine if F. oxysporum soil population is shaped by the agricultural history of the field, specifically, the crops grown in the field.
Objective 3: Determine if F. oxysporum soil population changes over the course of the growing season.
Objective 4: Determine if Fusarium wilt resistance genes impact the F. oxysporum populations asymptomatically colonizing tomato.
Objective 5: Identify current practices in Fusarium identification in plant diagnostic clinics and identify educational needs among diagnosticians.
The purpose of this project is to characterize nonpathogenic F. oxysporum occupying the same ecological niche as pathogenic isolates so as to better understand emergence of pathogenicity in this system and lay the groundwork for better diagnostic strategies. This research is important as some strains of F. oxysporum cause damaging diseases on important vegetable crops in Pennsylvania. Plant pathogenic F. oxysporum are specific to certain hosts are grouped as formae speciales (f.sp). Tomato is one of the most profitable vegetable crops grown in Pennsylvania and is impacted by Fo f. sp. lycopersici (Fol). In 2015, open-air, fresh market tomato production was valued at $33.2 million1.
Soil health and effectively managing soilborne plant pathogens is especially important in the northeast US, as many vegetable growers are transitioning to high tunnel production. Although this production technique may extend the growing season and lead to increase yields, the intensification of soil use may also lead to issues with soilborne plant pathogens such as F. oxysporum2. Growers face challenges if they find Fusarium wilt in their fields. F. oxysporum can survive in the soil for several years by colonizing degraded plant material, the roots of other crops, or by forming survival spores4–6.
Options for soil treatments exist such as steaming, solarization, and anaerobic disinfestation treatments7. These treatments involve infrastructural barriers, as they require specialized equipment, additional labor, and training. Even if growers can afford to use other effective soil treatments such as using disease-free seed and resistant tomato varieties, there is the looming possibility that certain races of Fol, will spread to new regions, or new races that overcome current genetic resistance can emerge 8. Fol can also be problematic in an agricultural setting due to the multiple ecological niches occupied by plant pathogenic F. oxysporum and their ambiguous relationship with nonpathogenic strains of F. oxysporum 4,9,10. Therefore, we need to better understand the factors contributing to emergence of this pathogen from closely related nonpathogenic relatives9. As nonpathogenic strains of FOSC are highly diverse in terms of ecological function and evolutionary background, we seek to clarify the blurry spectrum between nonpathogen and pathogen and examine how these individuals may interact.
These topics merit investigation because of the downstream impacts of understanding emergence of pathogenicity of Fusarium wilt, and on diagnostic capability. When researchers can better assess risk of pathogen emergence, we are better prepared to address the problem when it happens or implement strategies to reduce the risk. Ultimately, emerging pathogens such as those that cause Fusarium wilt, place an economic burden on vegetable growers in the northeast US as they lose part of their crops to the disease and spend time and money on management.
Additionally, providing diagnostic tools and education in Fusarium identification also contributes to reducing economic losses for growers. Diagnosticians are responsible for providing fast and accurate identification of pathogens and management recommendations for growers to respond to the specific pathogen. When diagnosticians help growers they are less likely to use unnecessary chemical treatments that add additional environmental impact.
To assess the genetic dynamics of FOSC in tomato agroecosystems in Pennsylvania, we propose to conduct a systematic sampling of FOSC isolated from specific parts of asymptomatic tomato plants, rhizosphere soil, and soil from neighboring non-agricultural soil.
We aim to isolate and characterize approximately 1,200 isolates of F. oxysporum. Previous studies investigating F. oxysporum diversity in both agricultural and non-agricultural systems have observed high diversity among isolates. To capture this diversity, we aim to isolate up to 40 isolates from each soil sample, and up to 15 isolates per plant. This similar to the approach used by Demers et al. (2015), and as we would like to draw comparisons to these results, we seek to implement comparable sampling methodologies.
All samples will be collected at Penn State’s research farm, the Russel E. Larson Agricultural Research Center.
Vegetable Field: This field has a history of varied agricultural production. Tomatoes have not been grown in this plot since 2011. Since then, squash, onions, celery, and soybean were grown in this field. Soil will be sampled in eight plots at the beginning of the tomato growing season and again from the same plots at the end of the growing season. We aim to isolate 40 isolates of F. oxysporum per soil sample. Four different varieties of tomatoes will be grown in this field. Each variety will have resistance to different races of F. oxysporum f. sp. lycopersici. Each variety will be grown in two separate plots, totaling 8 plots. Four plants will be sampled from each plot. We will aim to isolate up to 15 isolates of F. oxysporum per plant, spanning the roots, crown, and stem.
Non-agricultural soil: This area is less than 1 km away from Field 1 and has no history of agricultural production. Soil will be sampled from this location at the beginning of the growing season.
Comparisons can be made regarding the population composition of each location, tissue type, and soil type. Population genetic metrics such as Shannon’s diversity index will be calculated for each population, and differences in diversity will be tested for statistical significance using Bonferroni-adjusted P. Furthermore, genetic differentiation between populations will also be quantified using HST and Snn, which considers differences in haplotypes and nucleotide sequences, respectively. These metrics are used to determine if there are statistically significant differences in population structure between the populations we want to compare.
To address Objective 1 (Determine if F. oxysporum soil population is impacted by agricultural production), soil will be sampled from Vegetable Field, and “non-agricultural soil”. The F. oxysporum populations from these sites will be compared to determine if there is statistically significant differentiation between the populations.
To address Objective 2 (Determine if F. oxysporum soil population is shaped by the agricultural history of the field), F. oxysporum populations between the Vegetable Filed in 2020 and 2011 will be compared. The Vegetable Field was sampled for F. oxysporum in a similar fashion in 201113 after a season of tomato production. In 2020, the field had not been used for tomato production for 9 years. We can determine if there were changes to the F. oxysporum soil population over 9 years of varied agricultural production.
To address Objective 3 (Determine if F. oxysporum soil population changes over the course of the growing season), F. oxysporum isolated from soil sampled from Field 1 at the beginning of the growing season will be compared to F. oxysporum isolated from soil sampled from Field 1 at the end of the growing season.
To address Objective 4 (Determine if Fusarium wilt resistance genes impact the F. oxysporum populations asymptomatically colonizing tomato), F. oxysporum will be isolated from the root, crown, stem, and rhizosphere of four different varieties of asymptomatic tomato. At the end of the growing season, the full tomato plant will be harvested and brought to the lab for isolation. Each variety of tomato assessed in this study will contain different resistance genes to Fol, the group pathogenic to tomato. Each variety contains a different combination of resistance genes that recognize different pathogenicity genes in Fol and contribute to the race structure within Fol. If differentiation among nonpathogenic F. oxysporum populations is observed among these different tomato varieties, we may learn more about F. oxysporum host adaptation.
To address Objective 5 (Identify current practices in Fusarium identification in plant diagnostic clinics and identify educational needs among diagnosticians), we will conduct a survey through the National Plant Diagnostic Network (NPDN) to identify current practices in Fusarium identification and educational needs among diagnosticians. See Outreach Section for more details.
The following methods will be used to isolate F. oxysporum and obtain sequences in Objectives 1-4:
Whole plants and their roots will be taken at fruiting, as well as soil samples from the tomato field and from neighboring non-agricultural fields. In the lab, plant tissue will be cut into small pieces in approximately the same location of the root, crown, and stem. Tissue will be surface sterilized, and plated on Nash-Snyder media, which is selective for Fusarium spp. Samples will also be taken from the soil through dilution plating. As colonies begin to grow, they will be sub-cultured on ¼ strength potato dextrose agar.
To isolate F. oxysporum from these substrates, soil and plant parts will be plated on ¼-strength potato dextrose agar. Sub culturing will be necessary in order to obtain pure, single-spore isolates. Isolates displaying morphology consistent with F. oxysporum will be further prepared for long-term storage as well as DNA extraction. Isolates will be stored in glycerol and added to the Fusarium Research Center’s culture collection. To prepare tissue for DNA extraction, isolates will be grown in ¼ strength potato dextrose broth. These methods require substantial petri dishes and microcentrifuge tubes.
DNA will be extracted using CTAB, ammonium acetate, and isopropanol. CTAB and ammonium acetate are readily available in our labs, as they are inexpensive and needed in small quantities for many projects. Genomic DNA will be needed for sequencing.
The sequence types of each sample will be then determined by using Polymerase Chain Reaction (PCR) to amplify the translation elongation factor 1- (TEF) region, a common barcoding region for Fusarium24. This will allow us to make meaningful comparisons between each sampling location and describe population dynamics of this agroecosystem. Furthermore, as other studies use TEF to assess F. oxysporum diversity, we can compare our results to other published studies from around the world, giving our results a global context.
Methodologies were started to address Objectives 1-4 as follows: The Vegetable Field was set up for the field experiment in May. Four tomato varieties with differing resistance to Fusarium oxysporum f. sp. lycopersici (Fol) races were planted and kept in the greenhouse for 5 weeks, then transplanted into the field. Soil was sampled from each plot before transplanting for the first time point. The varieties were arranged in a randomized design so each variety was represented across the span of the field. There were four replicated treatments, with each treatment being a “block” of 8 plants of a specific variety.
When fruit reached maturity (12 weeks after transplanting), 4 whole plants were harvested from each block, including main stem, crown, and roots. Soil was also sampled from each block. There was some leaf spot disease, so disease was also rated for each plant. Whole plants were kept in individual bags and stored in a cold room (4 C) until they were brought to the lab for endophyte isolation.
In the lab, each harvested plant was thoroughly rinsed with water, surface sterilized for 2 minutes in a 2% solution of commercial bleach, then rinsed with water again. The above-ground part whole was measured, and 10 1 cm stem pieces were harvested at even intervals up the stem. 1 cm section of the crown was harvested, as well as 10 1 cm sections of the root system. Each plant part was individually wrapped in labeled paper towel, and kept in an envelope labeled by plant ID and kept at room temperature until they were plated.
Plant parts were plated on labeled Nash-Snyder media, which is selective for Fusarium growth. Plates were kept in a dark incubator set to at 25 C and monitored daily for fungal growth. Fungal growth that could potentially be F. oxysporum, based on gross morphology, were subcultured to 1/4 strength Potato Dextrose Agar (PDA) to obtain a pure culture and observe morphology microscopically.
Currently, sub cultures are being catalogued, maintained, and examined microscopically to obtain a final collection of F. oxysporum isolated from asymptomatic tomato plants.
Preliminary findings from soil isolation trials indicated that our proposed media, Nash-Snyder media, may not be as effective for preventing the growth of other fungi present in the soil. We are assessing if a different selective media, Komada’s media, is more effective for isolating F. oxysporum from the soil.
Efforts were initiated to address Objective 5. We have had several planning meetings with collaborator, Dr. Sara May, director of the Penn State University Plant Disease Clinic. We have drafted questions we want to include in the survey to be distributed to the National Plant Diagnostic Network (NPDN). We submitted a data request for all reports of Fusarium-associated diseases on specialty crops that have been entered in the NPDN’s national data repository. This will give us valuable background information on trends of Fusarium-associated diseases on specialty crops based on geographic location, how often a species-level identification is made, and methods diagnosticians use to determine causal agent. Additionally, we have started the IRB process, with all team members taking required training on research involving human subjects. Finally, we have initiated contact with the NPDN’s Professional Development Committee and will be attending a future committee meeting where we will present our survey goals and recruit volunteers to provide input on our survey draft.
As culture preparation is ongoing, there are no research conclusions for Objectives 1-4 to report at this time.
As the NPDN survey is still being developed, there are no research conclusions for Objective 5 to report at this time.
Education & Outreach Activities and Participation Summary
Our major outreach goals aim to strengthen infrastructure that provide valuable support to growers dealing with Fusariumdiseases in the northeast US. The National Plant Diagnostic Network (NPDN) is a consortium of plant diagnostic clinics across academia, state and federal governments, and industry. Its purpose is to facilitate information and resource sharing so reports of new diseases can be communicated to those charged with responding to such outbreaks. Plant disease clinics provide the most important first step in determining the most appropriate management response to a certain disease. Having an accurate and fast diagnosis ensures that growers are responding to a disease using the right tools at the right time. This prevents growers from spending money on unnecessary treatments or putting avoidable chemical input in the environment. Acting fast and appropriately can minimize losses and maximize profits for growers. As Fusarium diseases can be challenging to diagnose, there is a need for continued education on best practices for diagnosticians. Determining current practices and educational needs is the first necessary step in adapting and developing workshops, trainings, and other resources. Therefore, we propose to conduct a survey for members of the NPDN to determine current practices and educational needs in Fusarium diagnostics. This information is important to Fusarium experts, such as Dr. David Geiser, who regularly offer practical workshops on Fusarium.
Another resource used by diagnosticians are “Disease Notes”, short, peer-reviewed articles published in Plant Diseasethat report first occurrences of plant pathogens on a new host or in a new geographic region. These reports are crucial for documenting and cataloging distribution and impact of plant pathogens. Diagnosticians use information from these reports when making diagnoses and assessing risk and appropriate management recommendation. Diagnosticians are also frequent authors of these reports. Diseases associated with Fusarium are notoriously difficult to describe, particularly when it comes to establishing an isolate as the causal agent of the observed disease16. Differentiating secondary colonizers from true pathogens can be challenging, and oftentimes, multiple species may be involved in the disease. Furthermore, there are specific morphological characteristics and molecular protocols that will provide the best information for an identification, but with changing identification tools, taxonomy, and nomenclature rules, all of this information may not be clear to diagnosticians16,24. Therefore, we believe a short article providing guidelines for writing Disease Notes on Fusarium-associated diseases will be very helpful to the community. All participants will work on this article together, each providing expertise from a different perspective. This article will be published through Phytopathology News, the official newsletter for the American Phytopathological Society (APS). This newsletter often features informative articles such as this to the plant pathology community.
Additionally, the findings of this research project will be communicated to different audiences, with the emphasis of the results contextualized for each audience. The results will be presented to a grower-focused audience at the 2022 Mid-Atlantic Fruit and Vegetable Convention. The results will also be presented to an academic audience at the 12thInternational Mycological Congress in July of 2022.
As I do not have results for the objectives, I have not made formal presentations on this project for academic or grower conferences at this time. However, I did give a presentation on my research objectives, methodologies, and future steps for Objectives 1-5 during a lab meeting with four fungal biology labs. As several new graduate students were present, it was a helpful learning experience for them to learn about methodologies in fungal isolation and survey design.
As mentioned in progress made for Objective 5, We have had several planning meetings with collaborator, Dr. Sara May, director of the Penn State University Plant Disease Clinic. We have drafted questions we want to include in the survey to be distributed to the National Plant Diagnostic Network (NPDN). We submitted a data request for all reports of Fusarium-associated diseases on specialty crops that have been entered in the NPDN’s national data repository. This will give us valuable background information on trends of Fusarium-associated diseases on specialty crops based on geographic location, how often a species-level identification is made, and methods diagnosticians use to determine causal agent. Additionally, we have started the IRB process, with all team members taking required training on research involving human subjects. Finally, we have initiated contact with the NPDN’s Professional Development Committee and will be attending a future committee meeting where we will present our survey goals and recruit volunteers to provide input on our survey draft.
We are still in the preliminary stages of this study, but we are hoping the results of Objectives 1-4 will contribute to future sustainability efforts as we hope to learn more about population dynamics and evolutionary relationships between members of the Fusarium oxysporum Species Complex (FOSC). As this group contains endophytes, economically important plant pathogens, and biological control agents, it is important to know factors shaping these populations, specifically in vegetable production. Objective 5 will contribute to agricultural sustainability because we hope to provide resources that will aid in professional development goals of the National Plant Diagnostic Network (NPDN), thus strengthening this crucial service.
We are still in the preliminary stages of this study, but we are hoping the results of Objectives 1-4 will help us learn more about the population dynamics of an ecologically important group of fungi that can have a negative or positive impact on plant health. Additionally, through Objective 5, we hope to learn more about how Fusarium researchers can improve training materials to help plant diagnosticians as they serve growers.
As we do not have results at this time, I do not have the data to determine which of our approaches will be key to our project’s success.