Understanding Emergence of Vegetable Vascular Wilt Disease from an Ecological Perspective

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. Collections will take place at the Russel E. Larson Agricultural Research Center in Pennsylvania Furnace, PA and at the University of Florida Gulf Coast Research and Education Center in Wimauma, FL.

We aim to isolate and characterize approximately 3,750 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 sampling is 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 (Table 1).

Table 1: Sampling Strategy

Vegetable production Fields in PA: These fields have a history of varied agricultural production. Tomatoes have not been grown in these plots since 2011. Since then, squash, onions, celery, and soybean were grown in these fields. 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 four separate plots, totaling 16 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 (Table 1). The trial will be repeated in 2021, with the inclusion of 5 additional tomato lines with varying Fol resistance genes (Table 2).

Table 2: Summary of Fol resistance in tomato lines

Vegetable production Fields in FL: In 2021, 4 of the tomato lines included in the 2021 PA field trial will be included in a similar field trial in Wimauma, FL.

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 end of the growing season.

Comparisons can be made regarding the F. oxysporum population composition of each location, tissue type, and soil type. Population diversity 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 Fields, 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 associated with tomato is shaped by the geographic location of the field), F. oxysporum populations between the PA Vegetable Fileds in 2011, 2020 and 2021 and the FL Vegetable Field in 2021 will be compared. The fields will be 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 and endophyte populations over 9 years of varied agricultural production. We can also determine if there are differences in F. oxysporum populations between years at the same location (2020 vs 2021) and also if there are differences in F. oxysporum populations in PA vs. FL.

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 the PA Vegetable Field at the beginning of the growing season 2020 will be compared to F. oxysporum isolated from soil sampled from the PA Vegetable field at the end of the growing season in 2020. This will be repeated in the PA Vegetable field in 2021.

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 in the PA Vegetable Field in 2020, and nine different varieties of asymptomatic tomato in the PA Vegetable Field and FL Vegetable Field in 2021. At the end of the growing seasons, full tomato plants 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 (Table 2). If differentiation among nonpathogenic F. oxysporum populations is observed among these different tomato varieties, we may learn more about F. oxysporum adaptation to tomato.

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 1⁄4 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 1⁄4 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.