Crop Protection 121 (2019) 96–102 Contents lists available at ScienceDirect Crop Protection journal homepage: www.elsevier.com/locate/cropro Efficacy of biofungicides against root rot and damping-off of microgreens T caused by Pythium spp. ∗ Cora S. McGeheea, Rosa E. Raudalesa, , Wade H. Elmerb, Richard J. McAvoya a Department of Plant Science and Landscape Architecture, University of Connecticut, 1376 Storrs Rd. Storrs, CT, 06269, USA b Department of Plant Pathology and Ecology, The Connecticut Agricultural Experiment Station, 123 Huntington Street, New Haven, CT, 06504, USA ARTICLE INFO ABSTRACT Keywords: Pythium spp. are the causal agents of Pythium root rot and damping-off on microgreens. The objective of this Biocontrol project was to assess the efficacy of biofungicides on Pythium root rot and damping-off causedby Pythium Root pathogen aphanidermatum and Pythium dissotocum on microgreens in greenhouses. In the first experiment, arugula (Eruca Greenhouse sativa Mill.), kale (Brassica oleracea var. sabellica L.), radish (Raphanus raphanistrum subsp. sativus L.), and Hydroponics ® mustard (Brassica juncea L. Czern) microgreens were treated with Companion (Bacillus subtilis GB03), Triathlon Water ® ® BA (Bacillus amyloliquefaciens D747), or RootShield Plus (Trichoderma harzianum KRL-AG2 and Trichoderma Oomycete virens G-41) in a hydroponic nutrient film technique system. Two days after treatment, the plants werein- oculated with 3 × 105 zoospores of Pythium spp. After seven days, we measured root necrosis, damping-off incidence and severity, and plant biomass. All plants infected with Pythium spp. were smaller by 28% or more compared with non-inoculated plants. Overall disease was low, but biomass was lower in all treatments in- ® oculated with Pythium spp. Arugula infected with Pythium spp. and treated with Triathlon BA resulted in 8% ® lower disease incidence compared with the positive control, yet Triathlon BA resulted in the highest root ne- crosis. On a separate experiment, arugula and mustard were grown in propagation trays, irrigated manually, and ® treated with the biofungicides mentioned above or Cease (Bacillus subtilis QST 713). Arugula and mustard plants inoculated with Pythium spp. had 74.4% reduction of shoot dry weight. Arugula and mustard treated with ® Cease , with and without Pythium spp., resulted in ≥59% more biomass compared with the untreated inoculated control. In the tray experiment, all the infected plants treated with biofungicides had more biomass than plants with no biofungicides. Results from this experiment suggest that microbial biofungicides can be introduced in nutrient solutions in nutrient film technique or applied in the irrigation to prevent Pythium root rotand damping-off in brassica microgreens. However, biofungicides can reduce plant biomass and growers mayneedto extend production time to achieve target yields. 1. Introduction (Stanghellini and Rasmussen, 1994; Sutton et al., 2006). The combi- nation of young and succulent tissue, high production density, and re- Microgreens are young seedlings of vegetables and herbs produced circulated-nutrient solutions makes microgreen production vulnerable at high density and harvested when the cotyledons or the first true to soil-borne pathogens. leaves have expanded, typically seven to 21 days after sowing. Brassicas The unpredictability of biofungicides to control soilborne plant are grown as microgreens at densities greater than 2000 seeds per pathogens is a barrier for adoption in commercial agriculture germination-tray (approximately 1441 cm2). Brassicas are sensitive to (Bonanomi et al., 2018). However, the limited availability of synthetic Pythium root rot, which can result in high seedling mortality (> 98%) chemistries and risk of fungicide resistance increases the need to further (Ebenezar et al., 1996; Lim and See, 1983; Tanina et al., 2003; Tojo evaluate the efficacy of non-chemical options for disease control. Inthe et al., 2005). Burdon and Chilvers (1975) observed that as plant density United States (U.S.), only five synthetic chemicals are registered for increased the dispersion rate and incidence of Pythium damping-off on root-rot control applications on edible crops in greenhouses, compared brassica seedlings also increased. In closed-loop hydroponic systems with 19 biofungicides (US-EPA, 2018). Pythium aphanidermatum and (e.g. nutrient-film technique) the nutrient solution can be a source and Pythium dissotocum isolates have already developed resistance to syn- dispersal mechanism of pathogens, particularly oomycete pathogens thetic-chemical fungicides, like mefenoxam and propamocarb (Broders ∗ Corresponding author. E-mail address: [email protected] (R.E. Raudales). https://doi.org/10.1016/j.cropro.2018.12.007 Received 12 June 2018; Received in revised form 10 December 2018; Accepted 12 December 2018 Available online 03 April 2019 0261-2194/ © 2018 Elsevier Ltd. All rights reserved. C.S. McGehee, et al. Crop Protection 121 (2019) 96–102 et al., 2007; Moorman et al., 2002). Biofungicides represent a sustain- to maintain 5.8. able alternative for disease control in commercial settings; however, the efficacy of these products depends on the compatibility with theen- 2.3. Growing system vironmental conditions (e.g. rhizosphere of specific crops, production systems, etc.) (Bonanomi et al., 2018; Boehm et al., 1993). While many Hydroponic experiment. The hydroponic setup was a closed-loop biofungicides are labeled for applications in irrigation systems and nutrient film technique (NFT) system. The NFT channels were 1.83-m previous reports have shown a benefit in applying beneficial organisms long by 10.16-cm wide and with six 5.08-cm diameter holes (Crop King, in hydroponic systems (Utkhede et al., 2000) or the growing-substrate Lodi, OH). Every two channels were connected to a ten-gallon tank with of Beta vulgaris L. microgreens (Pill et al., 2011), the effect of bio- nutrient solution. Mustard (Brassica juncea L. Czern cv. Green Wave), fungicides on plant growth and Pythium root rot and damping-off on kale (Brassica oleracea L. var. Red Russian), arugula (Eruca sativa Mill.), brassica microgreens has not been evaluated. and radish (Raphanus raphanistrum subsp. sativus L. cv. Hong Vit) were Our objective was to evaluate the effect of microbial biofungicides sown in 42-mm peat pellets. Fifteen seeds of each plant species were on plant quality and the disease incidence and severity of Pythium root sown per pellet, which represented an experimental unit. There were rot and damping-off caused by P. dissotocum and P. aphanidermatum on three pellets of each plant species per treatment combination in each microgreens in the brassica family. block, and there were a total of four blocks (n = 12). The pellets were irrigated with clear water for the first three days. The seedlings were 2. Materials and methods maintained in a tray on a greenhouse bench under high pressure sodium lights for 14 h per day. The seedlings were hand-irrigated with a nu- 2.1. Pythium inoculum preparation trient solution with an EC of 330 μS cm−1 the day before they were transferred into the hydroponic channels. The nutrient solution had an Pythium spp. isolates used in this experiment were obtained from EC of 1000 μS cm−1. The plants were grown in Connecticut in a poly- greenhouses in the northeastern U.S. All the isolates were tested for carbonate greenhouse with a heating set point of 18.3 °C and a venti- pathogenicity using in vitro seedling assays (data not shown). P. apha- lation set point of 26.7 °C under natural photoperiod in June and July nidermatum Cor4, P. aphanidermatum Kop-8, and P. dissotocum Cor1 2017. were the isolates used in this project. The isolates were identified using Tray experiment. In a separate experiment, mustard and arugula the ITS sequence amplified with primers ITS1 and ITS4 as described by were sown as described above. We placed five peat pellets (each pellet White (1990) and registered in GenBank under accession numbers with 15 seeds) of each species in propagation trays on the greenhouse MG993551, MG993547, and MG993548. Zoospores were induced by bench under high pressure sodium lights for seven days. The experi- following the protocol described by Martin (1992) and Heungens and mental unit consisted of a peat pellet with 15 seedlings replicated five Parke (2000). Pythium spp. were grown on V8-juice agar, after three times (n = 5). The plants were irrigated with the same solution as de- days five 4-mm plugs were transferred to an empty Petri dish andthen scribed above. The greenhouse heating and cooling set points were the filled with 20 mL of clarified-V8 broth. The plates were incubated inthe same as the previous experiment. The experiments were conducted dark for five days. The mycelial mats were rinsed three times with twice between August and September 2017. sterile outdoor-pond water. The Petri dishes were then filled with 20 mL of sterile deionized water. The plates were incubated under fluorescent 2.4. Biofungicide and pathogen applications light for 24 h. Sporangia formation was confirmed visually and then the ® solution was drained. The Petri dishes were refilled with 20 mL of Hydroponic experiment. Companion (Bacillus subtilis GB03), ® ® chilled-sterile deionized water and incubated for 2 h at 4 °C. Then Petri Triathlon BA (Bacillus amyloliquefaciens D747), and RootShield Plus dishes were set at 22–26 °C for one to 2 h under constant fluorescent (Trichoderma harzianum KRL-AG2 and Trichoderma virens G-41) were light. We prepared separate inoculums for each isolate, adjusted the applied following the manufacturer label instructions at 0.98 mL.L−1, ® concentration to 1 × 105 zoospores per mL, and then combined the 2.5 mL.L−1, and 1.56 g.L−1, respectively. Companion and Rootshield ® ® three inoculums into a single solution. The plants were inoculated with Plus were applied directly in the nutrient solution and Triathlon BA a solution that contained the three isolates at 3 × 105 zoospores per was applied directly to the peat pellet, per label recommendation. Two mL. We inoculated with both species because in our own sampling (data days after applying the biofungicides, 10 mL of Pythium spp.