Evaluation of biological fungicides to control diseases of spinach in winter high tunnels

Final Report for ONE15-231

Project Type: Partnership
Funds awarded in 2015: $14,982.00
Projected End Date: 12/31/2017
Region: Northeast
State: Massachusetts
Project Leader:
Katie Campbell-Nelson
Cornell Cooperative Extension, Columbia and Greene Counties
Co-Leaders:
Susan Scheufele
UMass Extension Vegetable Program
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Project Information

Summary:

UMass Extension and Queen’s Greens commercial vegetable farm partnered to evaluate efficacy of OMRI-approved biofungicides in controlling diseases of spinach grown through the winter of 2015 to 2016. A lab assay was conducted in which the biocontrol organisms were cultured at different temperatures, to assess their activity at the low temperatures found in winter high tunnel soils, and a field study was conducted to evaluate commercially available biofungicide products. In the lab study we found that the organisms grew slowly or not at all at 50°F, though Streptomyces griseoviridis Strain K61 (Mycostop) grew best at that temperature and was the only organism capable of growth at 42°F. The two strains of Trichoderma present in Rootshield Plus grew slowly at 50°F and not at all at 42°F.

In the field study, we were not able to distinguish any significant differences in germination, stand, vigor, or yield across treatments. We did see a decrease in plant number and vigor at the second timepoint and then a rebound—this may have been due to post-emergence damping off. Plant number at the second timepoint (20 Oct) was significant, with all treatments except Rootshield Plus performing better than the untreated control and Mycostop G performing the best. This may indicate that the materials tested, except for Rootshield, provided some protection against post-emergence damping off. We also include a summary of the cost of each treatment.

While our methods could certainly be fine-tuned to be more discriminating, we feel that the results here accurately reflect what we observed in this tunnel–that none of the treatments had any noticeable effect on stand, vigor, or yield.  That said, Mycostop performed best in the lab study and in the field study, improving early season plant stand significantly relative to the control. Based on our findings, we feel that applications are most effective when soil is at or above 50°F, when beneficial microbes (and pathogens) are more active, so the number of applications made during the colder winter months can be reduced, further lowering costs.  The partner grower in this study has incorporated our findings into her production system and has been using Rootshield Plus at seeding and has also begun seeding less densely to combat damping off and improve stand establishment.

The results of these studies were shared with growers through the Vegetable Notes newsletter, on the UMass Extension Vegetable Program’s website, and at the Frozen Ground Conference in VT and the NOFA-MA Winter Conference.Figure-1

Introduction:

Across the Northeast, growers are struggling to meet the ever-increasing demand for fresh produce year-round.  High-tunnels are being used more-and-more to increase production of spinach and other greens for harvest all winter long.  This environment presents many challenges, with disease management being often identified by growers as a critical research need. We have observed that damping-off is a particularly important disease, causing reduced germination that often requires growers to re-seed, potentially missing the narrow window for successful establishment of a winter spinach crop and thus drastically reducing yields.  Seedling blight caused by Rhizoctonia solani and leaf spots such as Cercospora and Cladosporium build up in tunnels where spinach is grown year after year, reducing marketable yield and quality.  In this study, UMass Extension partnered with Queen’s Greens to evaluate efficacy of biopesticides to improve germination, reduce disease severity, and improve yields in winter-grown spinach.

To date there have been few studies on the efficacy of biopesticides to control damping-off and seedling diseases in spinach, and those that have been reported show inconsistent results. One research group from Washington State University investigated efficacy of biofungicides to control damping-off in spinach and found largely inconsistent results (Cummings et al., 2009). In one of these studies, biofungicides with active ingredients Bacillus subtilis, Streptomyces lydicus, S. griseoviridis and Gliocladium virens significantly reduced damping-off (Cummings et al., 2008b) but the same products tested in another set of studies did not improve emergence or reduce damping-off (Cummings et al., 2007a). In one study, they found that spinach treated with Micro 108 seed inoculant (Streptomyces lydicus) in combination with a soil drench of Actinovate Ag (S. lydicus) resulted in significantly lower incidence of damping-of and higher final emergence than the non-treated control or any other biological pesticides tested. Overall, these studies show that some of these materials do have the potential to increase emergence and reduce disease, but more data is necessary to support the use of these particular materials, and there are still many other products that have not yet been tested.

Among the few studies carried out in spinach, none were performed in winter production systems where soil temperature and moisture are low. Understanding how the winter growing environment might affect efficacy of biopesticides is an important question we will address in the proposed study.

Through our SARE-funded project “Expanding winter harvest and sales for New England vegetable crops,” we have visited many winter spinach tunnels and have often observed poor stands caused by damping-off, seedling blight caused by Rhizoctonia solani, and various leaf diseases. Growers managing winter spinach organically have little recourse for combating disease in tunnels. Crop rotation is difficult, as there are limited crops that can be grown in tunnels and very few that can be grown in winter, meaning that spinach is grown in the same spot year after year. Growers have been experimenting with using biopesticides, but without the resources to adequately evaluate impact they remain unsure of which products to choose and how to use them. By partnering with a local grower who has expertise in growing the crop, is willing to provide tunnel space to do a replicated trial, and where spinach diseases have been confirmed, we can complete a rigorous analysis of several products and share the results with growers across the region. Expected impacts of the proposed study are: increased knowledge among growers regarding the use of biopesticides for winter spinach production, and in the years following this study, improved yields in high-tunnel winter spinach, increased sales, and greater economic viability of businesses with winter greens production.

The goal of this study is to determine the efficacy of biological pesticides to control important soil-borne diseases and increase yield in overwintered, high-tunnel greens production systems. We will evaluate three to five OMRI-approved, soil-applied biopesticides compared to an untreated control. The study will be conducted in a commercial high tunnel in partnership with a farmer who has grown winter spinach for fresh market sales for over five years. Additionally, a supplementary assay will be performed in the laboratory growth chamber in order to determine the effect of low temperature on growth rate of various commercial biocontrol organisms. The objective of this study is to generate information that will help growers across the Northeast produce higher yields and higher quality greens throughout the winter by using reduced-risk pesticides to control common seedling diseases.

Works Cited

Cummings, J.A., du Toit, L.J., and Miles, C.A. 2008a. “Evaluation of seed and drench treatments for organic management of soilborne diseases of spinach in western WA, 2007a.” Plant Disease Management Reports. 2:V134.

Cummings, J.A., du Toit, L.J., and Miles, C.A. 2008b. “Evaluation of seed and drench treatments for organic management of soilborne diseases of spinach in Sequim, WA, 2007.” Plant Disease Management Reports. 2:V133.

Cummings, J.A., Miles, C.A., and du Toit, L.J. 2009. “Greenhouse evaluation of seed and drench treatments for organic management of soilborne pathogens of spinach.” Plant Disease. 93:1281-1292.

Hazzard, Ruth. 2014. “‘Frozen Ground’ Winter Growing Conference August 10-11, 2014: Research needs that came up in discussion.” Published on VT Veg and Berry Growers Association, Conference Proceedings, UVM Extension. http://www.uvm.edu/vtvegandberry/WinterGrowing2014/Research_Needs.pdf.

USDA, 2012. “More Communities Warm Up to Winter Markets.” USDA Newsroom. News Release # 0352.12 Last modified: 12/6/2012. http://www.usda.gov/wps/portal/usda/usdahome?contentid=2012/12/0352.xml

Works-Cited

Project Objectives:

Our specific objectives were to: a) determine if there is a lower temperature limit past which the biocontrol organisms become inactivated and other control strategies should be used, or if certain biocontrol organisms are more cold tolerant and would thus be better suited for use in winter production systems; and b) if any of the products evaluated can significantly increase crop yield and quality. Towards these objectives, UMass staff and the partner farmer will:

  • conduct a laboratory assay to grow biocontrol organisms at different temperatures
  • conduct a field trial to test efficacy of several biological and/or biorational fungicides
  • summarize results of both studies and share with growers via print, web, and in-person outreach materials and presentations

Cooperators

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Research

Materials and methods:

For the lab experiment, each of the biocontrol organisms in the products being evaluated in the field experiment plus some additional products were subjected to a growth assay performed at the University of Massachusetts by UMass Extension staff. UMass Extension staff cultured each of the organisms on solid or liquid growth media in temperature and humidity controlled growth chambers. Once these isolations were successfully carried out under optimal growth conditions, the cultures were then grown at a range of temperatures from 21° C to 0° C, in order to determine the lower range for growth of these organisms. Plates were assessed every 4-7 days and the diameter of growth of the colony was recorded for fungal cultures and number of colonies on serial dilution plates for bacterial cultures. UMass Extension staff performed all lab work including culturing organisms, collecting weekly growth data, analyzing data, performing the appropriate statistical tests, and interpreting results.

The field experiment was conducted at Queen’s Greens commercial vegetable farm in Amherst, MA in a field with Raynham silt loam soil.  A Rimol Nor’Easter (Rimol Greenhouses, Hooksett, NH) high tunnel measuring 200 x 300 ft., was amended with 3000 lb./A dolomitic limestone, 1000 lb. feathermeal, and 50 lb. of potassium sulfate on 26 Sept and roto-tilled in to incorporate. ‘Raccoon’ spinach seeds (Johnny’s Selected Seeds, ME) were direct-seeded at approximately 0.5 in. in-row spacing into beds on 5 ft. centers, with 11 rows per bed and rows spaced approximately 2 in. apart. A randomized complete block design was used with six treatments plus an untreated control, each replicated four times, in plots consisting of 20-ft of bed with 5-ft buffers in between plots.

All treatments (see Table-1 for products and rates) were applied as a soil drench over the row at seeding, with follow-up applications made as soil drenches over the row according to rates and intervals specified by the manufacturers. All drench applications were made using a CO2 pressurized backpack sprayer delivering 200 gal/A at 50 psi through one TeeJet Floodjet nozzle (TK-7.5).  The first treatments were made on 05 Oct and were watered in lightly using the overhead irrigation sprinklers in the tunnel on 06 Oct. Plants were also irrigated on 11 Oct, 19 Oct, 24 Oct, 02 Nov, 17 Nov, and 15 Dec.  Germination was first observed on 09 Oct and by 13 Oct most plots had germinated and plant stand was rated by estimating percent of plot area germinated (0-100%). Weekly ratings of plant stand and plot vigor and follow-up treatment applications were made all winter, through 03 Mar 2016. Marketable yield was measured by measuring wet weight of the crop harvested from the whole plot at the first cutting. Replicates A and B were harvested on 17 January and the remaining two replicates were harvested on 24 January, in order to harvest only what the grower could sell during the following week. The growers determined what was marketable and not, leaving unmarketable spinach unharvested in the tunnel. Environmental data including air temperature, light intensity and soil temperature (2 in. depth) were recorded every 2 hr by Hobo Weather Stations and/or data loggers (Onset Computer, Bourne, MA) from 03 Oct to 03 Mar. All data were analyzed using SAS 9.4 and means were compared using Fisher’s LSD (α = 0.05).

Research results and discussion:

For Objective A, we successfully isolated pure cultures of all of the biofungicides being tested in the field study (Rootshield, Actinovate Ag, Mycostop G, Double Nickel) plus three additional biofungicides (Serenade Max, Sonata ASO, Taegro) so that we had a total of seven products (8 organisms) to evaluate in the lab assay.  Most of the biofungicides evaluated were bacteria while one, Rootshield Plus, consisted of a mix of two fungal species. Since they grow differently, we used different methods for culturing and quantifying growth of bacteria and fungi, with bacteria being cultured in potato dextrose (PD) broth and fungi being cultured on solid media or potato dextrose agar (PDA) plates.

Bacterial Assays: Flasks of PD broth were sterilized and inoculated with a one milliliter aliquot of bacterial suspension at a concentration of approximately 1×106 cells/mL. Each organism was grown in triplicate at each of the three temperatures studied, 6°, 10°, and 24° Celsius. To quantify growth of bacterial cultures in PD broth, we made serial dilutions of each flask, plated each dilution out on solid PDA, and counted bacterial colonies (colony forming units, cfu) at each timepoint. This assay was run twice in order to improve confidence in results observed.

In the first replication, all the organisms had grown exponentially overnight at 24°C, while at colder temperatures  (both 10° and 6°C) Mycostop grew more quickly than other organisms (see Figures-2-3). After 72 hours, most of the organisms were growing at 10°C but only Mycostop and Actinovate Ag were growing at 6°C. After 7 days, some cultures had crashed while Mycostop and Actinovate Ag were the only ones still growing at 10°C and only Mycostop was still active at 6°C.

In the second replication, we only repeated the 24°C and 10°C incubations since nothing had grown at 6°C. All the cultures grew well at both temperatures, but after three days Sonata and Serenade crashed (see Figure). After seven days, Mycostop and Taegro had the most growth at 10°C, although Actinovate Ag, Double Nickel, and Serenade did continue growing as well, although more slowly.

Fungal Assay: To quantify growth of fungal isolates we transferred 7mm agar plugs from actively growing culture plates to fresh PDA plates and measured the width of the growth ring at each timepoint. At 24°C, the two Trichoderma spp. grew to 90mm (filling the plate) within three days, while at 6°C, neither of the fungi grew at all even after 11 days. At 10°C, both species grew slowly to a maximum width of about 5.0 cm for isolates A and B and 2.0 cm for isolate C after 11 days (see Figure 4). Isolates A and B consistently grew better at low temperature than did isolate C, indicating that isolates A and B are likely to be Trichoderma harzianum strain T-22 while isolate C is likely to be Trichoderma virens strain G-41, which grows more slowly at low temperature.

For objective B, all treatment applications were made as described in Table 1 and we collected data on plant vigor until the experiment ended on 03 March. Biofungicides were applied just after seeding on 05 October, 2016 and then weekly, biweekly, or less frequently depending on manufacturer instructions (see Table-1). We also continued to scout for other diseases such as Cercospora and Cladosporium leaf spots, but these were not observed. Marketable yield was measured by measuring wet weight of the crop harvested from the whole plot at the first cutting. Replicates A and B were harvested on 17 January and the remaining two replicates were harvested on 24 January, in order to harvest only what the grower could sell during the following week. The growers determined what was marketable and not, leaving unmarketable spinach unharvested in the tunnel. We attempted to quantify yield at subsequent harvest (e.g. second and third cuttings) but the patchwork nature of harvesting for different markets made it impossible to continue harvesting rep by rep. We continued to spray and rate for disease until 03 March at which time the plots were determined to be too irregular in growth stage after having been cut and regrown once or twice.

Unfortunately, we were not able to distinguish any significant differences in germination, stand, vigor, or yield across treatments (see Figure-5 and Figure-6). For some reason, we did see plant number and vigor decrease at the second timepoint and then rebound—this may have been due to post-emergence damping off. Plant number at the second timepoint (20 Oct) was significant, with all treatments except Rootshield Plus performing better than the untreated control and Mycostop G performing the best (see Figure-5).

While our methods could certainly be fine-tuned to be more discriminating, we feel that the results here accurately reflect what we observed in this tunnel–that none of the treatments had any noticeable effect on stand, vigor, or yield. That said, Mycostop performed best in the lab study and in the field study, improving early season plant stand significantly relative to the control.

Soil (2 in. depth) temperature in the high tunnel plots 03 Oct to 03 Mar 2016 averaged minimum of 42.2 and maximum of 56.5°F, and air temperature averaged minimum of 32.8 and maximum of 59.9°F (Figure-7). The soil temperature fluctuated a lot over the course of the day, but the average soil temperature dipped below 50°F on November 8, 2015 and, except for a few warm spikes, stayed there until March 9, 2016.

One interesting observation in this study was a widespread and patchy yellowing of older leaves. Plants with symptoms showed leaf yellowing starting from the margins and affecting older leaves with no associated wilt or vascular discoloration. We did a root extraction to look for plant pathogenic nematodes but found none and cultured from root and hypocotyl tissue several times and consistently isolated a fungus which we determined was a Fusarium species based on spore morphology. A Fusarium wilt of spinach is known and in fact causes severe problems for spinach seed growers in WA. We conducted greenhouse assays on live spinach seedlings using the Fusarium isolate we collected from Queen’s Greens and compared it to a reference isolate from WA of Fusarium oxysporum f.sp. spinicae that we got from Lindsey du Toit at UWA, in order to carry out Koch’s postulates and determine if the Fusarium we isolated from spinach roots was causing the yellowing symptoms observed. These tests demonstrated that the Fusarium we isolated was not causing yellowing symptoms, and the pathogenic isolate from WA caused different symptoms than those that we were seeing at Queen’s Greens, including wilt and vascular discoloration. We are currently investigating the possible impacts of salt buildup in the surface of the soil on spinach growth.

Research conclusions:

We did not conduct any impact surveys. We anticipate the impacts of this study will be to increase understanding of damping off as a cause of poor stand, and that some growers will experiment on their farms with biofungicides at seeding and perhaps through the early fall.

Participation Summary
1 Farmer participating in research

Education & Outreach Activities and Participation Summary

Participation Summary:

Education/outreach description:

This work was summarized and published in the December 8, 2016 issue of Vegetable Notes. The article was also published on our website and is accessible here: https://ag.umass.edu/vegetable/outreach-project/evaluation-of-biological-fungicides-to-control-diseases-of-spinach-in

The results of the field trial were also published in Plant Disease Management Reports (in press).

Results were presented by UMass Extension staff and the farmer at the Frozen Ground Winter Grower’s Conference in August 2016, and by UMass Extension staff and the grower at the NOFA-MA Winter Conference in January 2017. Though we had only about 6 attendees at the NOFA conference, those present were very interested and engaged participants and a good discussion of winter spinach production challenges, pest management, and use of biofungicides was had.

Project Outcomes

Project outcomes:

We did document the cost of each of the treatments applied and those findings are summarized in Table-1. The cost of treatments listed in Table 1 incorporates the rate used as well as the number of applications. In the case of Mycostop, we feel a much lower rate could be used to bring down the cost per application. With other fungicides, information from the manufacturers was available to advise on the best rate to use, but in the case of Mycostop, we were not able to get more specific guidance from the company and so we used the highest labeled rate. Based on our findings, we feel that applications are most effective when soil is at or above 50°F, when beneficial microbes (and pathogens) are more active, so the number of applications made during the colder winter months can be reduced, further lowering costs. Therefore, in our final report we have also included the cost per application, as we feel one or two applications at seeding should suffice for reducing effects of damping off and improving stand.

Farmer Adoption

The partner grower in this study has incorporated our findings into her production system and has been using Rootshield Plus at seeding and has also begun seeding less densely to combat damping off and improve stand establishment.

Assessment of Project Approach and Areas of Further Study:

Areas needing additional study

Future research on spinach disease management would certainly be useful and should be focused in the following areas: repeating the study to confirm results; expanding the field study to include more products; and determining effects of biofungicides on foliar diseases in the regular season, which were not observed in this study.

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.