Final report for GNE21-262
Project Information
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. 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. A survey for soilborne pathogens of high tunnel tomatoes has documented the presence of several soilborne tomato pathogens including Colletotrichum coccodes that causes black dot root rot, Pseudopyrenochaeta terretris and P. lycopersici which cause corky root rot as well as Verticillium dahlia that causes Verticillium wilt. In addition, root-knot nematodes (Meloidogyne spp.) were present in many of the high tunnels, and it was not uncommon for more than one soilborne pathogen to be present in the high tunnel. Unfortunately, there are few tools in the toolbox when it comes to management so evaluating new options under different production systems is valuable. Anaerobic soil disinfestation 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 and in the process reducing plant pathogen populations. A greenhouse experiment demonstrated that composted poultry litter combined with liquid molasses, dry molasses, or wheat midds were effective carbon sources for obtaining anaerobic conditions when flooded to field capacity. Furthermore, the treatment reduced the population of three separate isolates of Colletotrichum coccodes to zero. Although personnel changes led to the early termination of this project, ASD promises to be another potential tool for growers to use in managing soilborne pathogen populations in high tunnels across the mid-Atlantic and Northeast regions and warrants further research.
Project Objectives
This project aimed to address the gaps in our knowledge regarding the soilborne pathogens that are affecting high tunnel tomato yields and worked to optimize ASD using locally available carbon sources as a targeted soilborne disease management tactic for use in protected culture systems. This was accomplished by:
- Identifying soilborne pathogens potentially limiting yields in high tunnel tomato production systems. Despite limited information on soilborne pathogens in high tunnel system, it was hypothesized that soilborne pathogens, known to reduce marketable tomato yield, are present in high tunnels soils with a history of tomato production. Preliminary research involving the collection of soils from high tunnels and the sampling of bioassay plants confirmed the presence of potential soilborne pathogens.
- Evaluating locally available carbon sources as part of the ASD treatment for suppression of select soilborne pathogens identified as part of the first objective. It was hypothesized that local carbon sources, including agri-waste products, would generate a range of suppressiveness against the previously identified soilborne pathogens. Greenhouse trials were used to evaluate carbon sources in ASD for antagonistic effects against the most economically important soilborne pathogens identified.
The overall goal of this project was 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) could be augmented using local carbon source inputs to target and reduce soilborne disease pressure.
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.
Preliminary Research
Funding for preliminary work on this project came from the Pennsylvania Vegetable Growers Association and Pennsylvania Vegetable Marketing and Research Program and was leveraged into support from a Pennsylvania Specialty Crop Block Grant. Initial research began in 2019 and continued through the summer of 2021.
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, and soil samples were collected from 48 high tunnels. Approximately half of the tunnels sampled have been in annual tomato production for more than five years. Soilborne pathogen surveys were continued in 2021 a part of this project. It was anticipated that an additional 30-50 high tunnels would be sampled.
Genus-level nematode identification: Sub samples of each composite soil sample were sent to North Carolina Department of Agriculture and Consumer Services (NCDA&CS) Agronomic Services Division, Nematode Assay Section for nematode extraction and identification to genus level. Of the samples collected in 2019, 11 of the 13 were sent for nematode testing and eight were identified as having potentially yield limiting root-knot nematodes (RKN) populations. In 2020, 46 samples were submitted and 46% identified had damaging RKN populations.
Greenhouse bioassay: In 2019 and 2020, soils sampled from 9 and 48 of the high tunnels were bioassayed using tomatoes for the purpose of evaluating the severity of soilborne disease symptoms under controlled conditions. Fungal isolates were obtained from symptomatic tissues and identified using genus and species-specific primers. Isolates from 27 high tunnels were confirmed to be Colletrotrichum coccodes. Pseudopyrenochaeta terretris and P. lycopersici (formerly Pyrenochaeta lycopersici Types 1 and 2, respectively), were not frequently detected using the bioassay or was Verticillium dahliae.
Research
Objective 1: Identifying soilborne pathogens potentially limiting yields in high tunnel tomato production systems.
Grower collaborator identification and sample collection: In 2021, composite soil samples were collected from 28 high tunnels on 17 farms across Pennsylvania representing the southeast, central, and western regions of the state. Of these high tunnels, five had been previously sampled from one of the farms.
Genus-level nematode identification: Subsamples of each composite soil sample were sent to North Carolina Department of Agriculture and Consumer Services (NCDA&CS) Agronomic Services Division, Nematode Assay Section for nematode extraction and identification at the genus level as described previously. NCDA&CS extracts and quantified the populations of root-knot, dagger, lesion, spiral, ring, stubby root, lance, sting, soybean cyst and pin nematodes in soil (number/500 cc soil). The lab also assigned an action code based on the next planned crop and nematode population extracted. The code was as follows: A = no expected harm to crop production, B = possible damage; consider chemical treatment, C = chemical treatment recommended, D = use of nematode -resistant variety recommended, and E = rotate with nonhost crop(s).
Greenhouse bioassay: At the end of the 2021 high tunnel survey soil collection period (June – October), a greenhouse bioassay was conducted from 26 Oct 2021until 11 Jan 2022 (11 weeks) using the soils collected from 22 high tunnels. Each soil sample was divided and into 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). Therefore, each soil sample consisted of 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, the above soil portion of the tomato plants were removed, leaving a 2-4-inch stem, and the roots were washed dislodging any visibly adhered soil. Roots were evaluated for soilborne disease symptom severity using taproot rot and root-knot nematode severity rating (described below). Microbiological Isolations from the symptomatic root and stem tissue were made and the obtained fungal cultures were single spored to obtain pure isolates.
Bioassay disease severity assessment: Following completion of the bioassay, taproot rot and root-knot nematode ratings were recorded for each plant. Taproot rot ratings were 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 severity ratings were 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).
Fungal isolate identification: DNA was extracted from fungal isolates collected each year of the survey (2019-2021) using a Dneasy Plant Mini Kit (Qiagen, Germantown, MD). The manufacturer protocol was used without modification. DNA supernatants underwent 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 were 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.
Direct soil PCR pathogen detection: In addition, DNA was extracted from a 0.25g subsample using a Qiagen DNeasy PowerSoil Kit following the manufacturer’s protocol. Primer sources were the following: Pyrenochaeta lycopersici (Infantino and Pucci, 2005); Verticillium dahliae (Inderbitzin et al., 2013) and Colletotrichum coccodes (Cullen et al, 2002). All amplified PCR products obtained using the Pseudopyrenochaeta terretris, P. lycopersici and Verticillium primers were sequencing while only select isolates of the C. coccodes were sequenced for confirmation.
Objective 2: Evaluation of locally available carbon sources as part of the ASD treatment for reducing or inhibiting the viability of Colletrotrichum coccodes microsclerotial inoculum.
Colletrotrichum coccodes (black dot root rot) inoculum and microcosm preparation: Three isolates were selected from those collected as part of objective 1. The isolates were grown for 10 days on SNA agar plates containing three 70 mm diameter disks of filter paper to promote the formation of microsclerotia. The day before the greenhouse experiment was set-up, the number of microsclerotia on the upper and lower surface of each filter paper were quantified and recorded. Two SNA agar plates per isolate were used to determine the initial population. All three filter paper pieces per plate were transferred into a 2.0 mL lysing tube and filled with one 5 mm glass bead, 1 mL sterile water and then vortexed for 30 sec. The sample was then run in a Tissue Lyser, centrifuged and the neat solution serial diluted to estimate the population. After 72 hr the colonies were counted, and CFU/ml calculated. The remaining plates were used to inoculate the microcosms [VWR culture cell inserts (#62406-163) sealed with nanofabric (GVS #1239558, PCTE 0.03 µm)] what were filled with 1.0 g sterile potting media and sealed with silicone. Each microcosm was labeled with a permanent marker.
ASD treatments, experimental design, and set-up: The ASD treatments consisted of 1) standard ASD treatment with 63.4 ml liquid molasses (Golden Barrel Blackstrap Molasses, Good Food Inc., Honeybrook, PA) and 27.4 g composted chicken litter (CPL, Kreher’s 5-4-3 with 9% calcium, FedCo. Seeds) per 7.33 L pot (C900, Nursery Supply, Inc., Chambersburg, PA); 2) ASD with wheat midds (Snavely’s Mill, Lititz, PA) at 63.8 g and CPL at 27.4 g/pot; 3) ASD with dry molasses (Prairie Pride) at 68.4 g and 27.4 g CPL/pot; and 4) field soil control without ASD. The experiment was set-up as a randomized complete block design with four replicates and enough pots to destructively harvest on days 1, 3, 7, 14, and 28 for a total of 64 pots. The control pots were not destructively sampled until the end of the experiment on day 28.
In each 7.33 L pot, the carbon sources were individually mixed with Hagerstown silt loam soil collected from Penn State’s Russell E. Larson Agricultural Research Center, Pine Grove Mills, PA and screened for large rocks and clodes. Each pot was placed in a 8.52 L solid pot (C1000, Nursery Supply, Inc., Chambersburg, PA) to create a pot-in-pot arrangement that would prevent water from draining during the ASD treatment. Each pot set-up was filled with approx. 10 cm of soil, temperature sensor (HOBO Pendant Temperature/Light Data Logger, Onset Brands) was placed horizontally in the pot for those harvested on day 1, 3, and 7. Pots harvested on day 14 also included IRIS tubes (IRIStuve.com, West Laffayette, IN) for assessing reductive soil conditions. Those harvested on day 28 included the pathogen microcosms placed approx. 7.6 cm from the top of the pot as well as ORP sensors to continually measure soil redox potential (mV) throughout the course of the experiment. Each pot was sealed with totally impermeable film (TIF; VaporSafe, Raven, Sioux Falls, SD) to limit gas exchange and sealed with a large rubber band. The pots were filled to field capacity using drip emitters.
Data collection: On days 1, 3, and 7, pots were destructive sampled by removing the TIF and collecting four soil cores using a soil probe, placing them in a bag and 15 ml was used for analyzing soil pH and EC and the remainder was frozen at -20°C for later processing. Each 15 ml sample was placed in a 50 ml centrifuge tube mixed with 30 ml diH20, shaken at 100 rpm for 30 min and filtered through a Whatman grade 1 filter before reading pH and EC. The TIF was replaced and resealed with a rubber band.
On day 21, the interior pots were removed from the solid pots and permitted to drain. On day 28, the remaining pots were harvested. The temperature sensors were recovered from all pots, IRIS tubes removed from 14 and 28-day pots and microcosms and ORP sensors recovered from both day 28 and control pots.
IRIS tubes were assessed for reductive soil conditions by quantifying paint removal following ASD treatment using the protocol of Rabenhorst (2012) using a transparent grid around the tube and marking the number of grid squares where the paint has been removed.
Colletotrichum coccodes microsclerotial survival was assessed by transferring the potting media and filter paper inoculum from each microcosm into a sterile lysing tube filled with approx. 30, 1.0 mm glass beads and 1 ml sterile MQ H20, vortex (Vortex Genie 2) at max speed for 20 min. Contents of the tube were transferred to a glass tube with 8 ml sterile H20, vortexed again and then serial dilution plated onto ½ strength PDA+ agar plates. The population was assessed as previously described.
Objective 1: In 2021, composite soil samples were collected from 28 high tunnels on 17 farms across Pennsylvania representing the southeast, central, and western regions of the state. Soils from 17 high tunnels were assessed for select soilborne pathogens using direct soil PCR. Of these, 94% were positive for Colletrotricum coccodes, 65% were positive for one or both Pyrenochaeta species, and 87% (n=14 of 16) were positive for Verticillium dahlia. In cases where fungal isolates were able to be cultured from the bioassay tomato plants, the results confirmed the presence of pathogenic isolates of C. coccodes and species of Pseudopyrenochaeta. Verticillium spp. was unable to be cultured from the bioassay plants despite being detected in the soil. This is not surprising given the slow growing nature of the pathogen in culture. More selective techniques to isolate this pathogen should be employed in future bioassay experiments.
Root-knot nematodes populations were detected in 73% (n=22) high tunnels uniquely sampled in 2021. Of these populations were high enough to recommend chemical treatment in seven of the tunnels and in another nine high tunnels populations could cause possible damage and chemical treatment should be considered. Other nematodes detected but not considered problematic for tomato include dagger, lesion, spiral and stunt nematode and to a much lesser extent ring, lance, sting, and pin nematode were detected in only one or two high tunnels.
Objective 2: Based on both the IRIS tubes and ORP sensor data, all ASD treatments regardless of carbon sources went anaerobic. Only the no ASD controls remained aerobic as expected. Three isolates of Colletrotrichum coccodes were selected from the objective 1 survey based on being from a unique high tunnel in each of 2019, 2020, and 2021. Average microsclerotia counts on the filter paper disk did not differ between the isolates (77.7, 71.6, and 60.6 microsclerotia) and the CFUs/ml were 2.8E+04, 1.32E+04, and 1.81E+04 respectively prior to the ASD treatment. Following ASD treatment, regardless of the original carbon source or the C. coccodes isolates, the treatment rendered the no viable fungal biomass in the microcosms. Only the no ASD control microcosms contained viable spores although their populations were reduced (7.5E+02, 4.5E+02, and 7.3E+02 CFU/ml).
A survey of fresh-market tomato production high tunnels across Pennsylvania has documented the presence of several soilborne tomato pathogens including Colletotrichum coccodes that causes black dot root rot, Pseudopyrenochaeta terretris and P. lycopersici which cause corky root rot as well as Verticillium dahlia that causes Verticillium wilt. In addition, root-knot nematodes (Meloidogyne spp.) were present in most high tunnels. It is not uncommon for more than one to be present. In fact in the high tunnels were all three fungal pathogens were assayed, 13 of the 14 high tunnels had at least two of the three soilborne pathogens present and of those 10 also had root-knot nematode populations high enough to recommend considering treatment due to potential crop loss.
Although more research is needed, anaerobic soil disinfestation regardless of the carbon source mixed with the composted poultry litter (liquid molasses, dry molasses, or wheat midds) reduced the viability of microsclerotial inoculum in the microcosms to zero. Unfortunately, due to the early termination of the project, the ASD greenhouse experiment was not repeated for the other soilborne pathogens identified through the survey work.
Education & Outreach Activities and Participation Summary
Participation Summary:
Results of this research were presented at several vegetable grower winter and summer twilight meetings in Lancaster and Berks, Co, PA as well as the Mid-Atlantic Fruit and Vegetable Convention in 2022 and 2023 and as part of a Penn State Extension Vegetable and Small Fruit Pesticide Webinar in Feb 2022. In total, over 500 growers, extension and ag industry personnel were reached through these events. The project was also briefly mentioned as part of a larger presentation on the importance of soil health on disease management of vegetable crops a the 2022 Colorado Fruit and Vegetable Growers Association.
Project Outcomes
Due to the early termination of this project, the formal assessment of project outcomes was not determined. The new knowledge gained from the survey of tomato high tunnels for soilborne pathogens and the preliminary data demonstrating the efficacy of anaerobic soil disinfestation for managing Pennsylvania isolates of Colletotrichum coccodes is promising and warrants additional research. The dissemination of of the survey results, in particular, have prompted more inquiries about how to assay their soils for soilborne pathogens which provides anecdotal evidence for increased grower awareness soilborne pathogens as potential yield constraints. Increased knowledge and awareness is the first promising step to implementing practices such as anaerobic soil disinfestation to mitigating production constraints.
The knowledge gained from the survey has been beneficial in communicating with growers about the challenges that lack of crop rotation creating in intensive production systems like high tunnels. Prior to this research, root-knot nematodes had not been considered a significant concern in vegetable production in Pennsylvania now growers are becoming increasingly aware and additional research is being conducted to identify effectively nematode management strategies. These results also made a small contribution to a large USDA-NIFA OREI project being led by Dr. Francesco Di Gioia at Penn State looking at the use of ASD to enhance and advance the sustainability of organic specialty crop production systems.