Progress report for GNE21-262
This project aims to address the gaps in our knowledge regarding soilborne pathogens that are affecting high tunnel tomato yields and looks to optimize ASD using locally available carbon sources as a targeted soilborne disease management tactic for use in protected culture systems. It also looks to gain an understanding of the rate that soilborne pathogens will recolonize soils post-ASD in order to generate soilborne disease management recommendations including re-application rates for growers. This will be accomplished by:
- Identifying soilborne pathogens potentially limiting yields in high tunnel tomato production systems. Despite limited information on soilborne pathogens in high tunnel system, I hypothesize that soilborne pathogens known to detrimentally affect tomato production are present in high tunnel tomato production soils. Samples collected from high tunnels as part of the initial steps of this project will be prepared for sequencing in order to positively identify soilborne pathogens.
- Evaluate locally available carbon sources as part of the ASD treatment for targeted suppression of soilborne pathogens identified as part of the first objective. I hypothesize that local carbon sources, including agri-waste products, will generate a range of suppressiveness against the previously identified soilborne pathogens. Greenhouse pot trials will be used to evaluate carbon sources in ASD for antagonistic effects against the most economically important soilborne pathogens identified.
- Investigating the spatial and temporal recolonization of soil by economically important soilborne pathogens following ASD treatment. I hypothesize that ASD using locally available carbon sources will reduce soilborne pathogen inoculum in the soil but will not entirely eradicate the pathogen. Further, it is expected that when susceptible crops are continuously produced in the same soils, the same soilborne pathogens will again increase disease pressure to pre-treatment levels. Having an understanding of the recolonization rate of soilborne pathogens post-treatment will ensure more effectual soilborne disease management recommendations that include the re-application frequency of the soilborne disease management tactic.
The purpose of this project is to identify soilborne diseases that are negatively impacting tomato yields in high tunnel production systems and to determine ways in which anaerobic soil disinfestation (ASD) should be adjusted using local carbon source inputs to target soilborne disease.
Fresh-market tomato growers are increasingly using protected culture systems like high tunnels to extend the growing season, improve yields, meet the increasing demand for local produce, and protect against extreme weather fluctuations (Horton et al., 2014). Tomatoes are a high value crop and many growers have forgone crop rotations in favor of continuous production; some for more than 15 years. This intensification has led to higher soilborne disease pressure. Identification of soilborne diseases is difficult as symptoms are easily attributed to other factors such as nutrient deficiencies.
Chemical management of soilborne diseases is challenging due to pathogen persistence in the soil and label use patterns restricting use in enclosed production. Non-chemical management techniques such as flooding and solarization require warmer temperatures to be effective which coincides with the primary growing season in the Northeast requiring growers to forgo growing a crop (Katan, 1981). Steaming has the potential to be effective but requires the use of expensive specialized equipment that incurs additional time, fuel and labor costs.
ASD is a biological process that incorporates a labile organic amendment into the soil, covers the soil with non-permeable plastic mulch, and then saturates the soil with water. Aerobic organisms exponentially increase in response to the amendment, quickly depleting the available oxygen in the soil (Di Gioia et al., 2016). As the soil environment shifts from aerobic to anaerobic, microbial community changes elicit the production of fermentation products that are toxic or suppressive to many soilborne pathogens (Momma, 2008). Additionally, ASD creates a temporary drop in soil pH and Eh, increased soil temperatures, and the release of metal ions, all of which may contribute to the suppression of soilborne pathogens (Poret-Peterson et al, 2019). ASD has successfully been evaluated in other systems. Information regarding the tactics use in high tunnel systems is limited and does not account for the variability of high tunnel systems in the Northeast.
The identification of soilborne pathogens that affect high tunnel tomato production is an important first step in disease management and is an essential component of this project. This project will utilize molecular tools to identify soilborne pathogens in high tunnel tomato systems and then will optimize ASD for use in high tunnel systems in the Northeast using locally available labile carbon sources to target soilborne pathogens. Soilborne pathogen identification results will be used to generate disease management recommendations and will include ASD targeted carbon source information. This project will provide growers with vital information needed to effectively identify and mange soilborne diseases in high tunnel systems without the use of chemical fumigants through the targeted application of ASD and ultimately contribute to an increased understanding of soilborne disease management that will translate directly to improved yields and the long-term health of tomato production systems.
Funding for preliminary work on this project has come from the Pennsylvania Vegetable Growers Association and Pennsylvania Vegetable Marketing and Research Program and was leveraged into support from a Specialty Crop Block Grant. Initial research began in 2019 and will continue through the summer of 2021. These first steps include:
- Grower collaborator identification and sample collection: In 2019, nine grower collaborators participated in our soilborne disease survey and 13 high tunnels were sampled. In 2020, 26 grower collaborators participated in our soilborne disease survey and soil samples have been collected from 48 high tunnels. Approximately half of the tunnels sampled have been in continuous tomato production for more than five years. Disease survey’s will be conducted during the summer of 2021. It is anticipated that an additional 30-50 high tunnels will be sampled.
- Nematode identification assay: Sub samples of each collected composite soil sample were sent to North Carolina Department of Agriculture and Consumer Services (NCDA&CS) Agronomic Services Division, Nematode Assay Section for nematode testing. Of the samples collected in 2019, 11 of the 13 were sent for nematode testing. Eight of the samples were identified as having root-knot nematodes (RKN). In 2020, 46 samples were submitted with 21 samples being identified as having RKN in densities that warrant treatment. Samples collected during the summer of 2021 will undergo the same testing through NCDA&CS.
- 2019-2020 Greenhouse bioassay: In 2019 and 2020, soils sampled from nine and 48 of the high tunnels, respectively were used in bioassays for the purpose of evaluating symptoms of soilborne disease in tomatoes grown in the collected soil samples. From the 2019 samples, a total of 24 fungal isolates were obtained and are in the process of being prepared for DNA sequencing. Cultures obtained from the 2020 bioassay plants are currently undergoing several subculturing steps in order to obtain pure cultures for DNA sequencing.
- 2021 Nematode identification assay: Sub samples of each collected composite soil sample were sent to North Carolina Department of Agriculture and Consumer Services (NCDA&CS) Agronomic Services Division, Nematode Assay Section for nematode testing. Soil samples were collected from 18 unique high tunnels (12 unique farms) and 23 high tunnels in total (18 farms in total). All samples were sent to NCDA&CS for nematode testing. Sixteen of the 23 samples collected in 2021 were identified as having root-knot nematodes (RKN).
- 2021 Greenhouse bioassay: At the end of the 2021 high tunnel survey soil collection period (June – October), a greenhouse bioassay was conducted from October 26, 2021 until January 11, 2022 (11 weeks) with the soil collected from 22 high tunnels. Collected soil from each high tunnel was divided and placed in two sterilized Deepots (D20T-20 tray, D40H, Stuewe & Sons, Tangent, OR) that had been pre-filled with approximately 60mL of rinsed drainage rock (KolorScape, Oldcastle, Atlanta, GA). Each collected soil was used to generate two biological replicates. The soil was moistened, and cv. Mountain Fresh Plus tomato seeds were directly sown into each Deepot. These were maintained in the research greenhouses on the Penn State main campus (ASI110, University Park, PA) and fertilized every two weeks with a water-soluble fertilizer with a nutrient analysis of 24-8-16 (Miracle-Gro Water-Soluble All-Purpose Plant Food, Marysville, OH). After 11 weeks (January 11, 2022) the above soil portion of the tomato plants were removed, leaving a 2-4-inch stem, and the soil carefully washed away from the roots. Roots were evaluated for soilborne disease signs and symptoms through taproot rot and root-knot nematode ratings. Isolation of fungal pathogens from symptomatic root tissue is underway for the purpose of obtaining a pure isolate.
- Sequencing of fungal isolates: DNA extractions from fungal isolates collected each year of the survey (2019-2021) are being conducted using a DNeasy Plant Mini Kit (Qiagen, Germantown, MD). The manufacturer protocol is used without modification. DNA supernatants undergo PCR using the master mix Go Taq Green (Promega Corp., Madison, WI) with either targeted diagnostic primer pairs for known soilborne pathogens or generalist fungal primers (ITS) for unknown isolates. Generated PCR products from unknown isolates will be submitted to the Eurofins Genomics (Louisville, KY) for Sanger Sequencing and then compared to other fungal organisms through BLAST in the NCBI GenBank database to obtain an identification.
- A series of greenhouse pot trials will be used to evaluate the efficacy of carbon source selection in ASD against select soilborne pathogens identified in the first objective using locally available carbon sources. A two factorial greenhouse trial will be used to evaluate the effects of carbon source selection on the rate of survival of the top three most economically significant soilborne pathogens identified as the first objective. Field soil will be collected from Penn State’s Russell E. Larson Agricultural Research Center at Rock Springs and selected carbon sources will be thoroughly incorporated into each soil. Each soil-carbon source mixture will be placed in 7.33L black pots (C900, Nursery Supplies, Inc., Chambersburg, PA) with drainage holes in the bottom. Inoculum satchels consisting of each soilborne pathogen to be tested, will have predetermined propagule amounts prepared on a mineral or inert organic substrate, placed in a 25-micron nylon mesh satchel, and then will be buried three inches (7 cm) deep in each container. The containers will then be flooded with a volume of sterilized water, allowed to drain for five minutes, and sealed with totally impermeable film (TIF; VaporSafe, Raven, Sioux Falls, SD) on the top. The covered containers will then be placed inside another 7.33L black pot that does not have drainage holes to ensure anaerobic conditions are achieved. Containers will be incubated in a greenhouse for three weeks (see Figure 12). Reductive soil conditions will be monitored using IRIS tubes (Indicator of reduction in soils; IRIStuve.com, West Laffayette, IN). After three weeks, TIF will be removed, and pots will be allowed to air for 3-5 days. The inoculum satchel will be removed from the pot and surviving propagules will be counted/measured. Each carbon source/pathogen combination will be replicated four times and repeated twice. Pathogen propagule survival will be compared to the pre-treatment inoculum satchel propagule counts. The PCR-based detection of soilborne pathogens from the soil will be combined with the results of the bioassay. Descriptive statistics will be used to characterize the prevalence of identified pathogens in PA high tunnel production systems. Ordinal data will be collected and evaluated using descriptive statistics. Odds ratios may be determined using fungal incidence and root rot ratings with associated carbon sources.
- A successive grow-out test will be performed to determine recolonization rates of previously identified soilborne pathogens. High tunnels that are identified as part of objective one, as having soils that are infested with soilborne pathogens, will have fresh soil samples collected. The soil will be tested for pH and EC then homogenized and pressed through a sieve to remove debris and rocks. A small sub-sample (1-2g) from each homogenized soil will undergo qPCR to determine the baseline propagule rates of soilborne pathogens of interest. Soil DNA extractions will be prepared using the MoBio PowerSoil DNA Isolation Kit (Mo Bio Laboratories, Inc., Carlsbad, CA). Real-time PCR of samples will follow protocols developed for each pathogen. Field soil collected from the Rock Springs research farm will be used as controls (both treated and untreated). After baseline testing (pH, EH, and qPCR), each soil will undergo the ASD treatment protocol previously described in objective two using the carbon source(s) that was most successful at reducing soil inoculum (quantified with qPCR). Four biological replicates of each treatment will be laid out in a completely randomized block design. After the ASD treatment each soil will be planted in tomato (cv. Mountain Fresh Plus) for four 16-week successive grow outs to mimic continuous tomato production. At the end of each 16-week grow out, the tomato roots will be evaluated for signs and symptoms of soilborne pathogens. Each soil will be retained for further successive grow outs, but a small subsample of soil will be collected at the end of each grow out and undergo qPCR to track changes in select pathogen populations. If naturally infested native high tunnel soils are not available for use due to the estimated volume required, natural field soils will be inoculated with known inoculum quantities of soilborne pathogens and/or root-knot nematodes identified as part of objective one. Ordinal and survey (binary) data will be collected and evaluated using descriptive statistics. Correlative analyses based on pathogen severity and duration of continuous production will be assessed. The correlated structure of the experimental data will be assessed using a repeated-measures analysis.
Qualitative Disease Assessment:
- Following completion of the bioassay as part of objective one, taproot rot and root-knot nematode ratings will be completed for each tomato root mass. Taproot rot ratings will be conducted using an ordinal 1-5 scale developed by Testen and Miller (2017): 1 = taproot healthy; 2 = one to two small lesions or slight discoloration on taproot; 3 = multiple lesions covering less than 50% of the taproot; 4 = multiple lesions covering more than 50% of the taproot; 5 = taproot completely rotted or missing. Root-knot incidence ratings will be conducted using the Root Gall Evaluation Chart developed by Bridge and Page (1980): 0 = no galls; 1 = few, small galls, not easily found; 2 = small galls, not in main roots; 3 = some big galls, not in main roots; 4 = mostly big galls, not in main roots; 5 = galls in 50% of the roots, some main roots affected; 6 = main roots clearly affected; 7 = most of main roots with galls; 8 = all main roots with galls, few secondary roots without galls; 9 = all roots very affected, plant close to death; 10 = all roots very affected, no radicular system, plant usually dead (modified from Piedra-Buena et al., 2011).
Education & Outreach Activities and Participation Summary
I will work with grower collaborators to evaluate and optimize ASD under field conditions based on the findings generated as part of objectives two and three. Disease management recommendations will be generated for grower use, including carbon source recommendations to target specific pathogens. Extension fact sheets and other publications will be developed or updated for each soilborne pathogen identified as contributing to disease in high tunnel tomato production systems with specific management recommendations included for each. Information regarding the emergence of soilborne disease complexes in PA will be included if warranted and specific recommendations made. Twilight meetings and field days will be organized at on-farm demonstration sites with grower collaborators and Penn State Extension to illustrate the application of ASD in high tunnels, share information, and address concerns. The results of my proposed research will also be shared through poster and oral presentations at professional, industry and grower meetings. I will share my findings at the Mid-Atlantic Fruit and Vegetable Convention in 2022 and 2023. Approximately 2,200 people attend the convention as it attracts a diverse audience and is recognized as one of the top grower meetings in the region. I will also present my results at the Northeastern American Phytopathological Society (APS) regional meeting and the APS Annual national meeting in 2022 and 2023 in order to share my work with researchers from around the country and beyond.