Progress report for LNE19-388R
The project goal is to control cucumber downy mildew in the field using nighttime applications of UV-C. The team will conduct a laboratory study to determine optimum pre- and post-infection doses of UV-C. A trailer suitable for cucumber fields housing UV-C lamps in Northeast will be designed and built to demonstrate the efficacy of pre- and post-infection UV-C applications in the field. We will educate Northeast farmers on the practical application of UV-C treatments.
Question 1: For reducing disease severity from cucumber DM, what dosing regime is the best combination of (a) dose (amount x duration), (b) time of application (pre- or post-inoculation), and (c) interval between dosing (1 per week vs. 2 per week) for nighttime applications of UV-C? (laboratory).
Question 2: Does the efficacy of the best dosing regime increase with UV-reflective material? (laboratory).
Question 3: Is the best dosing regime as effective as conventional fungicide spraying? (field)
Question 4: Is the life-cycle benefit/cost of the UV-C system and dosing regime equal to or less than conventional fungicide spraying equipment and methods?
Due to challenges in laboratory experiments of maintaining the cucumber downy mildew inoculum, we decided it was more effective to evaluate multiple conditions in the field. Currently we are working with Jim Ward (of Ward's Berry Farm) and Sue Schuefele to design the experiment. This consists of 10 rows, 300 feet long, that will be divided up randomly into blocks and will test varying doses (from 120 - 580 Joules per meter squared, once per week), as well as with and without a UV reflective mulch. Jim explained the row structure and we discussed the possible mulches that could be used and it appeared that Mylar could be used. We are aiming to complete the experimental field design by February, so that plans for planting, building the dragon and field assessments are in place so we can move forward on those items.
Attempts to maintain and propagate downy mildew (DM) inoculum in the lab proved to be very challenging in year 1, which made exploring various combinations of UV dose, frequency, and mulch type in the lab difficult. In an effort to explore as many combinations as possible, a field study was designed to capture as many of these factors as possible. A different field layout from that described in the Year 1 narrative above was developed that consisted of twenty one 70 foot long rows. Doses of 120, 240, and 480 J·m-2 were applied both once and twice per week to plants on standard black mulch and reflective mulch. Plots were also included that received no fungicide and conventional fungicide. A document attached contains a field map of the conditions.
LRC staff worked with Jim Ward to design a tractor mounted UV applicator that was suited for use on the trial plot. The unit was designed to utilize 6 four-lamp UV fixtures mounted in a semi-circular shape and to be mounted directly to a tractor’s 3 point hitch (category 2). The electrical power to operate the lamps was provided by an on-board gasoline generator. Detailed construction drawings and assembly instructions were developed by the LRC and supplied to Jim and his staff. The construction documents and photos of the completed unit are linked below. The unit was constructed successfully on the farm during the late spring/early summer from materials supplied by the LRC.
Disease ratings were taken weekly by Sue Schuefele from July 27th through September 3rd, when all plants except those in the fungicide treated plots were severely affected by DM. Ratings were made by visually assessing the percentage of leaf area covered in DM lesions (yellowing, necrosis, sporulation) on 10 leaves per plot, then on the percentage of DM within the whole plot. In order to facilitate visual estimations, a walk-through of the whole experimental area was performed first, in order to get a sense for the overall extent of downy mildew severity and any other crop diseases or issues including bacterial wilt or fertility issues. The individual leaf inspections were then conducted using a 10X handlens and inspecting both the upper and lower leaf surfaces, in order to ensure that the researcher was looking carefully at leaf symptoms and not attributing leaf yellowing to DM without evidence of sporulation. Those ten random leaf samples further helped the researcher calibrate their eye and get a more detailed picture of disease severity. Finally, the researcher stepped back and evaluated the whole plot for percent leaf area affected by downy mildew.
The LRC plans to work with Sue Schuefele, Jim Ward, and the members of the advisory committee to develop a field trial design for the 2021 growing season based on the results of the this year’s field trial. Care will be given to ensure the trial design will have appropriate replication to perform statistical analysis on the results.
Year 3 (2021):
All images and tables referenced in the following narrative may be found: Tables and Figures (Methods) 2022
The focus of the 2021 field trial was to compare the DM control efficacy of the best UV-C only condition from the year 1 field study, the grower’s conventional fungicide program, and a combination of both treatment types. Further analysis of the 2020 field trial data showed that although the 120 J·m⁻² dose applied once weekly (with reflective mulch) was empirically the most effective treatment, the same treatment was not as effective with black mulch. Collapsing across mulch type, 480 J·m⁻² had slightly (albeit not statistically significantly) higher effectiveness than the other doses, and application frequency of twice weekly was slightly more effective than once weekly. Accordingly, this dose/frequency combination was selected.
The treatments used were: (1) UV-C only (480 J·m-2) twice weekly, (2) weekly conventional fungicide, (3) fungicide weekly plus UV-C twice weekly, and (4) fungicide every other week plus UV-C twice weekly. The third treatment was included to determine if the addition of UV-C to the conventional weekly fungicide program would offer additional control beyond the conventional fungicide program alone. The fourth condition, added at the suggestion of Sue Schuefele (project cooperator), was included to determine if DM control could be maintained by adding UV-C treatments while reducing the number of conventional fungicide applications. The conventional fungicide treatments for DM applied during the 2021 field trial are summarized in Table 1. Each condition was replicated twice on both black and UV reflective mulch, for a total of four replications for each treatment. The last 4.5 m (15 feet) of one row was devoted to a control condition with no fungicide or UV-C treatment. The layout for this field study is shown in Figure 1. The cucumber variety Raider F1 (Harris Seeds) was used again for the second year of the trial.
Each row was divided into ten sections (as shown in Figure 1) and assessments of percentage foliar DM severity were made within each of the 10 sections to increase the sample size within each row. Assessments were performed visually within a square quadrant with 61 cm (24 inch) sides, placed randomly within each of the ten-foot row sections, using the same methodology used in the first year. The top and bottom sides of leaves within the quadrat were inspected to verify that the symptoms were consistent with DM in the same manner as 2020
Assessments were performed by a farm staff member trained to positively identify cucurbit DM by cooperative extension agents. A total of 5 disease assessments were performed between August 3, 2021 and September 9, 2021.
The below file summarizes the status of experiments, analysis and plans as of December. As described above, we are currently in the process of finalizing plans for spring and summer.
The whole plot data is then used to calculate area under the disease progress curve (AUDPC). AUDPC was calculated from 27 Jul to 3 Sept according to the formula : Σni=1[(Ri+1 + Ri)/2] [ti+1 – ti], where R = disease severity rating (% of leaf surface affected) at the ith observation, ti = time (days) since the previous rating at the ith observation, and n = total number of observations). Values were calculated based on the average percent disease severity across the plot.
Data plots are included in the following document: 2020 Data Figures Only
Due to the lack of replication, no data statistical analysis was completed. The data show that, while some treatments may have had lower disease severity at the earlier timepoints, none of the UV treatments performed as well as the fungicide treated plots on either the black or silver mulch and all were >90% infected, similar to the untreated control, by September 3rd.
Year 3 (2021):
All images and tables referenced in the following narrative may be found: Tables and Figures (Results) 2022
During this year, the sets of data from the 2020 and 2021 field trials were analyzed in two ways. First, analysis of variance (ANOVA) was performed using the foliar disease severity ratings to determine which independent variables had a reliable effect on disease severity. Second, the area under the disease progress stairs (AUDPS) method (Simko and Piepho, 2012) was used to provide a composite index of the relative impact of each treatment and control condition on disease progression throughout the assessment period in each year.
2020 (Year 1) Field Data
Figure 2 shows the observed foliar disease severity values for each treatment and control condition, when black mulch was used, and Figure 3 shows the corresponding data for reflective mulch. Each point in Figures 2 and 3 is a single observation for the once-weekly doses or the average of two observations for the twice-weekly doses. The conventional fungicide program in 2020 was only applied with the black mulch, so that condition only appears in Figure 2.
A four-way analysis of variance (ANOVA) was performed on the foliar disease severity data comprising a balanced experimental design with the type of mulch, the UV-C dose, the dosing frequency, and the date of assessment as independent factors. The mulch type had a statistically significant effect (F1,45=15.3, p<0.05) on disease severity, as did the date of assessment (F5,45=1184, p<0.05). There was also a statistically significant interaction (F5,45=8.89, p<0.05) between the mulch type and the date of assessment on disease severity. This can be observed by the fact that the disease severity values for the two mulch types were similar for the earliest and latest assessment dates but differed around day 20.
Qualitatively, the curves in Figure 2 also illustrate the large difference found in 2020 between the conventional fungicide treatment conditions and the control and UV-C treatment conditions. Disease severity remained under 20% under the fungicide condition for all observation periods, whereas it approached 90%-100% for all other conditions by the last observation period. Generally, the differences among the control and UV-C treatment conditions were small, although the untreated control condition tended to have greater disease severity values than the UV-C conditions.
AUDPS values (Simko and Piepho, 2012) were calculated for each condition representing each treatment type (or control), the frequency of application (for the UV-C treatment conditions) and type of mulch. These values are shown in Figure 4. Qualitatively, Figure 4 shows the much lower AUDPS value for the conventional fungicide condition than for all other conditions. It can also be seen that the AUDPS values are usually (with one exception for 120 J·m⁻² applied twice weekly) lower for the reflective than for the black mulch.
A one-way ANOVA for each treatment condition in Figure 4 was performed to identify whether there were statistically significant differences among the treatment conditions, and there were (F14,10=16.2, p<0.05). Tukey’s post hoc tests were carried out among each treatment to identify which conditions differed from the others. It was found that the conventional fungicide treatment (with black mulch) was statistically significantly (t=5.07 to 13.2, p<0.05) different from all other conditions. No other conditions differed from one another after adjustment for multiple pairwise comparisons.
Considering only the UV treatment groups, the AUDPS values could be analyzed using a three-way ANOVA with the UV-C dose, the dosing frequency and the type of mulch as independent variables. This ANOVA revealed a statistically significant main effect of mulch type (F1,7=9.80, p<0.05), but no other main effects nor interactions among the variables. Because the AUDPS values collapse across the date of assessment, this analysis result is consistent with the ANOVA on the disease severity values.
2021 (Year 2) Field Data
Figures 5 and 6 show the progression of disease for each of the control and/or treatment conditions as a function of time (day after assumed infection as described previously). There are two primary qualitative differences between the data in these figures for 2021 and the corresponding data in Figures 2 and 3 for 2020. First, there appears to be a greater separation among the conditions in terms of the days that the disease begins to take hold in the plants, especially between the untreated control condition (which exhibited greater than 50% foliar disease severity by day 21, and the other conditions which exhibited less than 20% disease severity on the same day. Second, the disease severity for the fungicide treatment conditions approached 80% by the end of data collection whereas in 2020, disease severity was held to less than 20% with the application of fungicide. (Possibly, disease severity in 2020 for the fungicide treatment condition would have eventually increased to nearly 100%.)
For the four treatment conditions (i.e., fungicide, UV, UV plus fungicide, and UV plus EOW fungicide) for which both types of mulch were used, a three-way ANOVA was performed on the disease severity values, with treatment, mulch type and date of assessment as independent factors. The section number of each row was included in the analysis as a covariate factor to identify whether there were any systematic differences within each row; there were not. The treatment (F3,761=118, p<0.05) and the date of assessment (F4,761=1958, p<0.05) had statistically significant main effects on disease severity, and there was also a statistically significant interaction between treatment and assessment date (F12,761=52.5, p<0.05). This can be observed in Figures 5 and 6 where the disease severity was similar across all treatments for the first and last treatment dates, with the most variation among treatments for the intermediate dates. Unlike 2020, the type of mulch did not have a statistically significant (F1,761=0.98, p>0.05) effect on disease progression.
Mean AUDPS values (Simko and Piepho, 2012) for each treatment and mulch condition were calculated and are shown in Figure 7. A one-way ANOVA was performed to assess differences among the conditions, which were statistically significant (F8,149=45.2, p<0.05), with Tukey’s tests to assess pairwise comparisons (summarized in Table 4). In general, there were no significant differences (p>0.05) in AUDPS between mulch types for the same condition. All conditions except for the UV-only conditions differed significantly (p<0.05) from the untreated control condition (which only used black mulch). The combination of fungicide and UV-C treatment with the black mulch was statistically significantly different (p<0.05) from the fungicide-only treatment with the same mulch type, suggesting a small impact of UV-C treatment in conjunction with fungicide.
Excluding the untreated control condition, a two-way ANOVA was performed to assess how the treatment condition and mulch type, and the interaction between them, affected AUDPS. The section from 1 to 10 was included in this analysis as a covariate to identify whether there were any systematic differences across each of the treatment rows; there was not. There was a statistically significant (F3,149=110, p<0.05) main effect of treatment, but the mulch type did not exhibit a statistically significant main effect (p>0.05). There was a significant interaction (F3,149=2.72, p<0.05) between treatment condition and mulch type on AUDPS; this is seen in Figure 7 where the black mulch resulted in somewhat higher AUDPS for the fungicide treatment condition, but lower for the UV-only treatment. Aside from the two-way interaction between the treatment and mulch type, this analysis of the AUDPS values was consistent with the ANOVA on the disease severity values in identifying significant differences among the treatments but not between the two types of mulch in 2021.
All research conclusions will be completed in the final report.
Education & Outreach Activities and Participation Summary
We have not yet reached this stage of the project, but do have some materials in development from prior projects that we will be able to utilize and build upon. This includes the website that we maintain, which will contain copies of the build plans and educational materials used for in-person presentations: https://www.lrc.rpi.edu/programs/plants/plants_home.html
In addition to maintaining the materials included on the lightandplanthealth.org website, LRC researchers also authored an article featuring the research involving use of UV to combat plant disease. The article specifically mentions this research project and contains a quote from Jim Ward, the cooperating farmer, and was published in January, 2021 in Country Folks Grower magazine. A copy of this article is attached here: Country Folks Grower UV Article - 2021
Year 3 (2021):
In addition to the Country Folks Grower article published in January, 2021 (previously reported), Nick Skinner participated in a one hour podcast “Shining UV Light on Plant Pathogen Management” with a panel including a farmer and two extension agents. Many topics including the history of germicidal UV use, modes of action, pathogens and crops that have been researched, UV safety, and ongoing research, including this project, were covered. Support of the NE SARE was acknowledged. The podcast's audience is individuals involved in commercial horticulture and has reached 118 listeners at the time of this report, and can be found at the following site: https://enych.cce.cornell.edu/podcasts.php
A field-day highlighting the research project and providing attendees with information about the use of UV as a plant pathogen control technique was held on August 18, 2021 at Ward’s Berry Farm in Sharon, MA. A total of 14 stakeholders (not including the organizers) attended. A summary of the field day event may be found in the attached files: Field Day Announcement, Field Day Summary
Additional outreach activities currently planned for 2022:
The research results from this project and another project focused on UV powdery mildew control will be presented at the 2022 Eastern NY Fruit & Vegetable Conference organized by Cornell Cooperative Extension on February 16, 2022. Additionally, the team prepared materials to submit for presentation at the 2022 American Phytopathological Society (APS) annual meeting once the solicitation opens in February, 2022.
The team has prepared a manuscript and plans to pursue publication in a peer-reviewed journal in the coming months to further disseminate the research results as well.
We are currently in the process of planning field trials. Outreach activities will begin only after we have obtained field trial results. In the meantime, we are actively participating in workshops to educate farmers on use of UV light for squash powdery mildew and strawberry powdery mildew, but these workshops are not under this sponsorship. We have discussed the value of using UV light for cucumber downy mildew mainly with our farmer collaborators to include Jim Ward, Amy Hepworth, Larry Eckhardt, as well as our extension agent collaborator Sue Schuefele and advisory board members Dr. Meg McGrath and Dr. David Gadoury.
Year 3 (2021):
The key areas focused on during the 2021 outreach activities were: history of germicidal UV use, modes of action, pathogens and crops that have been researched, UV safety, ongoing research, how to construct a UV applicator attachment, and the print resources available to producers and extension staff/educators.
Based on the research conducted to date, a commercial producer from eastern Massachusetts said he thought "that a combination of varietal resistance and the UV-C could really work."
In reference to the UV control of downy and powdery mildews, a commercial producer from Massachusetts attending the field day said, "Wonderful and very informative. Love this potential for commercial organic growing."