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

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

Summary

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.

In 2016, we completed the field trial which was started in 2015, and completed the lab assay in duplicate. In the field study we evaluated four biopesticides alone or in a rotation with a contact, foliar-applied biopesticide in a commercial high tunnel. In the lab assay, the effects of low temperature on growth of biocontrol organisms used in the field trial were studied.  Project goals and results were summarized for a group of winter growers at the Frozen Ground Conference in Lake Morey, VT on July 25^th and 26^th. Results will also be shared with growers via newsletters, factsheets, and at the NOFA-MA Winter Conference in January 2017.

Objectives/Performance Targets

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.

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 time-point. 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 Figure 2). 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 (see Figure 3).

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. 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, we continued making treatment applications as described in Table 1 and measured changes in 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.

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

Had our rating system incorporated more replication within plots as opposed to whole-plot measures we may have picked up more subtle treatment differences, however, we do 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. Future research on spinach disease management would certainly be useful and should be focused in the following areas: repeating the study to confirm results; expand the field study to include more products; determine effects of biofungicides on foliar diseases, which were not observed in this study.

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 sp. we isolated from spinach roots was causing the yellowing symptoms observed. These tests demonstrated that the Fusarium sp. 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 remain very perplexed by this issue, which seems to be associated with soil, and are currently investigating the possible impacts of salt buildup in the surface of the soil on spinach growth and nitrogen availability in cold soils.

2016 Annual Report Addendum

Accomplishments/Milestones

January – April 2016: The farmer will be responsible for maintaining the crop in terms of insect pest management, weed management, irrigation, and fertility for the duration of the experiment. The spinach crop will be monitored by UMass Extension staff for pre- and post-emergence damping-off and other diseases. This milestone was completed as anticipated.

January – April 2016: Collection of yield data will occur at two to five time-points throughout the growing period, depending on crop growth and the number of cuttings achieved. The first harvest is yet to occur but we are on target to achieve this milestone as anticipated.

April – May 2016: Data will be analyzed for statistically significant treatment differences. This milestone has been achieved.

Summer 2016: Written outreach materials including an article for Vegetable Notes and a factsheet for the UMass Extension Vegetable Program website will be drafted over the summer of 2016 for publication in August or September of 2016, to coincide with planting of the next years’ winter spinach crop. This milestone was completed now and an article was published in the December issue of Veg Notes (Vol 28:25), a little behind schedule but still relevant as growers will be noticing stand issues and diseases now.

August 2016: Results will be presented by UMass Extension staff and the farmer at the Frozen Ground Winter Grower’s Conference in August 2016 if possible. This milestone was completed on time; Katie Campbell-Nelson shared a presentation of our findings with growers and Extension staff present.

October 2016: Results will be presented by UMass Extension staff at the regional meeting of the American Phytopathological Society. This milestone was not performed. After consulting with peers I determined that this research would not be as well-suited to this meeting as I’d hoped. No SARE funding was used for travel to this meeting, as the results were not presented there.

November 2016: Results of this study were shared with Extension and Ag Research colleagues through the NE-IPM Center’s Online Conference on November 9, 2016.

January 2017: Results will be presented by UMass Extension staff and the grower at the NOFA-MA Winter Conference in January 2017. We are on schedule to achieve this milestone as anticipated.

February 2017: A final report will be written collaboratively by the farmer and UMass Extension staff. We are on schedule to achieve this milestone as anticipated.

Collaborators: