Crop Protection 121 (2019) 96–102

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Crop Protection

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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 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 ® 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 virens G-41) in a hydroponic 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 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 , 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. 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. zoospore not shown), and others' surveys (Del Castillo et al., 2016; Moorman suspensions (3 × 105 zoospores per mL) were applied to each peat et al., 2002; Stanghellini and Kronland, 1986; Sutton et al., 2006) pellet. The negative control plants received 10 mL of sterile deionized multiple species of Pythium are typically recovered from the same lo- water per peat pellet. The biofungicides and pathogens were applied cation/sample in greenhouses. Each peat pellet (Jiffy, Netherlands) had only once. a 42-mm diameter and was inoculated with 10 mL of the zoospore Tray experiment. The biofungicides and pathogen were applied as suspension. described in the hydroponic experiment. This experiment included ® Cease (Bacillus subtilis QST 713) as an additional biofungicide treat- ® 2.2. Irrigation water analysis and nutrient solution ment. Cease was applied directly in the nutrient solution at 1 mL.L−1. All applications were made only once. The nutrient solutions were prepared with municipal water with the following characteristics: pH of 7.4, electrical conductivity (EC) of 2.5. Measurements 206 μS cm−1, dissolved oxygen (DO) of 7.9 mg.L−1, oxidation reduction potential (ORP) of 721 mV, free chlorine was 0.7 mg.L−1 and total Hydroponic Experiment. Harvest was conducted four to five days chlorine was 0.7 mg.L−1. EC, pH, DO, temperature, and ORP were after pathogen inoculation (seven to eight days post-seeding). measured with specific probes for the Orion Star Meter (Thermo Fisher Measurements at harvest included damping-off incidence and severity, Scientific Inc., MA). Free and total chlorine were measured withthe root necrosis, stand counts, dry shoot and root biomass, and relative Orion AQUAfast IV Powder Chemistries in the Orion AQ4000 Advanced chlorophyll index. Disease incidence was measured as the percentage of Colorimeter (Thermo Fisher Scientific Inc., MA). The nutrient solution plants with visual damping-off or wilting per peat pellet. Disease se- was a combination of a complete fertilizer Hydroponics-Part A 5-12-26 verity was rated according to the following scale: 1 = symptomless, (JR Peters, Allentown, PA) with Ca(NO3)2 (1:1.5 ratio by mass) and the 2 = emerged but wilted, 3 = post-emergent damping-off, and 4 = pre- pH was adjusted to 5.8 for all experiments. The pH, EC, DO and tem- emergent damping-off (Boehm et al., 1993) and the mean damping-off perature of the solutions were monitored daily and the pH was adjusted rating per peat pellet was estimated. Root disease incidence was rated

97 C.S. McGehee, et al. Crop Protection 121 (2019) 96–102 based on categorical data of visual presence or absence of root necrosis necrosis compared with the other biofungicides. Plants treated with ® ® and rot. Root necrosis was calculated by averaging the presence or Companion and RootShield Plus resulted in lower root necrosis in- absence of necrosis per treatment, statistical analysis was conducted cidence than the inoculated control. Plants inoculated with Pythium using Chi-Square. Shoots were cut at the soil line and shoots and roots spp. and with no biofungicides were smaller by 28% or more compared were weighed fresh and dry (dried at 21.1 °C for two weeks). Relative with non-inoculated plants (Figs. 1 and 2). Disease severity and in- chlorophyll index was measured using a SPAD 502 Plus chlorophyll cidence was in general low (Table 2), but biomass was reduced within meter (Spectrum Technologies, Inc., IL). most treatments inoculated with Pythium spp. (Figs. 1 and 2). Tray experiment. Harvest was conducted five to six days after Arugula inoculated with Pythium spp. and treated with Triathlon ® pathogen inoculation (nine to ten days old post-seeding). Measurements BA had the highest root necrosis; however, no differences were ob- at harvest included disease incidence and severity, root necrosis, stand served in disease incidence and severity. Arugula and radish inoculated ® counts, and fresh shoot and root biomass. These measurements were with Pythium spp. and treated with Triathlon BA had higher dry shoot made as described above. weight than the untreated inoculated plants (with Pythium spp. and no ® biofungicides) (Figs. 1 and 2). Radish treated with RootShield Plus and 2.6. Experimental design and data analysis Pythium spp. had greater shoot weight than the untreated inoculated plants in the second experimental run. Arugula and radish untreated Hydroponic Experiment. The experiment was a full factorial or- non-inoculated control had the highest dry root weight than all other ® ganized in a randomized complete block design (RCBD). The factors treatments. Mustard treated with Triathlon BA and inoculated with included plant species (four levels), biofungicides (four levels), and Pythium spp. was the only treatment combination that had higher dry Pythium inoculation (two levels). The experimental unit consisted of a root weight compared with the untreated inoculated control. However, ® peat pellet with fifteen seeds. Each block had three replicates and there kale treated with Triathlon BA and no Pythium had the same dry root were four blocks in the experiment (n = 12). The data were analyzed weight compared with the untreated inoculated control. ® using SAS Version 9.4 (SAS Institute Inc., NC) to establish significance Dissolved oxygen was higher in solutions treated with Triathlon BA of the effects of all factors (α = 0.05) by analysis of variance (ANOVA). (8.1 mg.L−1) or untreated (8.1 mg.L−1) compared with solutions ® ® Severity ratings were analyzed with non-parametric analysis using the treated with RootShield Plus (7.6 mg.L−1) and Companion RANK and PROC GLM procedure. Means were separated by Tukey's (7.5 mg.L−1). No differences were observed in any other nutrient so- studentized range HSD (Honestly Significant Difference) separation test lution parameters. (α = 0.05). Percentage of root necrosis was calculated by averaging all the yes's and no's per treatment, and statistical analysis was conducted using Chi-Square. 3.2. Tray experiment Tray Experiment. The experiment was a full factorial arranged as a complete randomized design (CRD). The factors were plant species The interaction between biofungicide and pathogen was significant (four levels), biofungicides (five levels), and Pythium inoculation (two for all measurements (Table 3). Arugula and mustard plants inoculated levels). The experimental unit consisted of a peat pellet with fifteen with Pythium spp. resulted in higher root necrosis compared with the seedlings, there were five replicates per tray, and there were a total of non-inoculated control (Table 4). Visual disease incidence and severity ® five trays (n = 25). The trays were randomly distributed in the green- in this experiment were very low. Mustard plants treated with Cease house. Statistical analysis was conducted as described above. were the only plants where root necrosis was lower than the untreated inoculated plants. Arugula and mustard inoculated with Pythium spp. 3. Results had 74.4% reduction of shoot dry weight. Arugula and mustard treated ® with Cease , with and without Pythium spp., consistently resulted in 3.1. Hydroponic experiment over 59% more fresh shoot and root weight compared to the untreated inoculated control. Arugula and mustard inoculated with Pythium spp. Data were analyzed separately for each experimental run because had lower shoot and root dry weight by 63% and 75% compared with there was heterogeneity (α = 0.05) between experimental runs for all untreated non-inoculated plants, respectively (Fig. 3). All plants treated measurements, except disease incidence and severity. Interaction be- with biofungicides and Pythium spp. had higher shoot and root growth tween biofungicides and pathogen were significant for all measure- compared with the untreated inoculated plants. Arugula and mustard ® ments (Table 1). treated with Cease consistently did not differ in dry shoot and root All plant species inoculated with Pythium spp. developed symptoms weight with and without Pythium spp. Arugula and mustard treated ® (Table 2). Root necrosis was 100% for all microgreen species inoculated with Cease and Pythium spp. also consistently resulted in higher dry ® ® with Pythium spp. and untreated with biofungicides. Across species, shoot weight than RootShield Plus and Companion inoculated with ® plants treated with Triathlon BA and Pythium spp. had the highest root Pythium spp.

Table 1 Hydroponic Experiment. Effect of biofungicide (B), Pythium inoculation (P), plant species and their interaction on dry shoot weight (DSW), dry root weight (DRW), disease incidence (DI), and disease severity average (DS) for experimental runs I and II based on analysis of variance.

Treatments DSW DRW DI DS

I II I II I & II

Biofungicide (B) < .0001 0.1866 0.0016 0.0003 < .0001 < .0001 Pythium (P) < .0001 < .0001 < .0001 < .0001 < .0001 < .0001 B*P 0.0061 < .0001 < .0001 < .0001 < .0001 < .0001 Species (Sp) < .0001 < .0001 < .0001 < .0001 < .0001 < .0001 B*Sp 0.1332 0.0504 0.0104 0.0215 0.0682 0.1215 P*Sp < .0001 < .0001 0.0398 0.0924 < .0001 < .0001 B*P*Sp 0.019 0.0002 0.1365 0.1175 0.0682 0.1215

98 C.S. McGehee, et al. Crop Protection 121 (2019) 96–102

Table 2 Disease incidence, severity, and root necrosis caused by Pythium spp. on microgreens when treated with biofungicides in a nutrient film technique production system (n = 12).

4. Discussion to affect net growth. However, Koohakan et al. (2004) recovered dif- ferent levels of plant pathogen genera by hydroponic system, while In this project, we observed positive and negative effects of bio- total bacterial levels were sustained. Hence, further research is needed fungicides on microgreen brassicas when applied in nutrient solutions to compare how production systems affect disease levels and efficacy of to irrigate microgreens in greenhouses. Our results indicated a general biofungicides. trend in which plants treated with biofungicides were bigger (Figs. 1–3) Understanding the mechanisms of these organisms will help hy- and had less Pythium-related symptoms (Tables 1–4) compared with pothesize why biofungicides are inconsistent in promoting or reducing untreated plants. However, applying biofungicides in combination with plant growth. Specific microorganisms are able to protect the plant or without Pythium spp., in general, reduced plant biomass. The positive either directly or indirectly against pathogens and their efficacy is and negative effects observed in this project highlight the importance of largely influenced by the total microbiome and its interactions that evaluating the compatibility of organic amendments with different affect plant health (Berendsen et al., 2012). Many beneficial soil-borne agricultural practices and crops. microorganisms such as Bacillus and Trichoderma spp. activate defense The organism's ability to survive and multiply is context dependent mechanisms in shoots and reproductive parts of the plant (Bonanomi (Boehm et al., 1993; Bonanomi et al., 2018) which may affect beneficial et al., 2018). Beneficial bacteria and fungi act, in part, by triggering microbes and pathogens in different agricultural settings. We did not induced systemic resistance (ISR). Studies have shown that the bio- compare disease levels or plant growth between production systems synthesis of indole-3-acetic acid (IAA) in the plant growth-promoting (tray and nutrient film technique) because we conducted the experi- rhizobacterium (PGPR) B. amyloliquefaciens affects its ability to pro- ments at different times of the year and spaces. However, we observed mote plant growth (Choudhary and Johri, 2009; Idris et al., 2007; that in the tray experiment (Fig. 3), all the plants treated with bio- Kamilova et al., 2006). B. subtilis was demonstrated to produce auxin as fungicides whether they were inoculated with Pythium spp. or not, were the likely mechanism behind the increase in seedling growth of wheat bigger than the negative control (without pathogen, no biofungicide). plants (Egorshina et al., 2011). Bacillus subtilis GB03 is known to sti- Whereas plant size in the NFT system varied by plant-microbe combi- mulate phytohormones, which trigger the plant's systemic acquired nations. Our experiments cannot discriminate whether the production resistance (SAR) to disease. SAR can be triggered by exposing the plant systems had an effect on the beneficial microbes, the pathogens, orboth to virulent, avirulent, or non-pathogenic microbes (Choudhary and

99 C.S. McGehee, et al. Crop Protection 121 (2019) 96–102

Fig. 1. Hydroponic Experiment- First Experimental Run. Dry weights of microgreens in response to biofungicides and Pythium spp. inoculation (‘+’ = treatment with Pythium spp.; ‘-’ = treatment without pathogen) in a hydroponic system (n = 12). These graphs represent dry shoot weight (DSW) and dry root weight (DRW) of microgreens seven days after seeding. Means within a graph (by measurement type) followed by the same letter are not different according to Tukey HSD (P = 0.05). Error bars represent standard error.

Fig. 2. Hydroponic Experiment- Second Experimental Run. Dry weights of microgreens in response to biofungicides and Pythium spp. inoculation (‘+’ = treatment with Pythium spp.; ‘-’ = treatment without pathogen) in a hydroponic system (n = 12). These graphs represent dry shoot weight (DSW) and dry root weight (DRW) of microgreens seven days after seeding. Means within a graph (by measurement type) followed by the same letter are not different according to Tukey HSD (P = 0.05). Error bars represent standard error.

100 C.S. McGehee, et al. Crop Protection 121 (2019) 96–102

Table 3 ANOVA for the Tray Experiment. Dry shoot weight (DSW), dry root weight (DRW), disease incidence (DI), disease severity average (DS) were measured in two microgreen species (Species) in response to biofungicides application and Pythium spp. inoculation (n = 25).

Treatments DSW DRW DI DS

Biofungicide (B) < .0001 < .0001 < .0001 < .0001 Pythium (P) < .0001 < .0001 < .0001 < .0001 B*P < .0001 < .0001 < .0001 < .0001 Species (Sp) < .0001 0.1621 0.2597 0.2278 B*Sp 0.4030 0.0060 0.1231 0.1351 P*Sp 0.1423 0.5590 0.2597 0.2278 B*P*Sp 0.3295 0.6177 0.1231 0.1351

Table 4 Disease incidence, severity, and root necrosis caused by Pythium spp. on mi- crogreens when treated with biofungicides in propagation trays (Tray Experiment, n = 25).

Species Biofungicide Pythiuma Damping-off Damping-off Root Incidence Severityb,c Necrosis (%)b (%)b,d

Arugula Untreated – 0.0 b 1.0 b 0 b Untreated + 0.03 a 1.1 a 100 a Companion – 0.0 b 1.0 b 0 b Companion + 0.0 b 1.0 b 96 a RootShield Plus – 0.0 b 1.0 b 0 b RootShield Plus + 0.0 b 1.0 b 100 a Triathlon BA – 0.0 b 1.0 b 0 b Triathlon BA + 0.0 b 1.0 b 90 a Cease – 0.0 b 1.0 b 0 b Cease + 0.0 b 1.0 b 88 a Fig. 3. Tray Experiment. Dry weights of microgreens in response to biofungi- Mustard Untreated – 0.0 b 1.0 b 0 c cides and Pythium spp. inoculation (‘+’ = treatment with Pythium spp.; Untreated + 0.02 a 1.1 a 100 a ‘-’ = treatment without pathogen) in propagation trays (n = 25). Graphs re- Companion – 0.0 b 1.0 b 0 c Companion + 0.0 b 1.0 b 98 a present the mean dry shoot weight (DSW) and mean dry root weight (DRW) of RootShield Plus – 0.0 b 1.0 b 0 c microgreens seven days after seeding. The experiment was replicated twice. RootShield Plus + 0.0 b 1.0 b 94 a Means within a graph (by measurement type) followed by the same letter are Triathlon BA – 0.0 b 1.0 b 0 c not different according to Tukey HSD (P = 0.05). Error bars represent standard Triathlon BA + 0.0 b 1.0 b 88 a error. Cease – 0.0 b 1.0 b 0 c Cease + 0.0 b 1.0 b 72 b aboveground and yield loss caused by Pythium spp. may occur without ® a ‘+’ = treatment with Pythium spp.; ‘-’ = treatment without pathogen. their awareness. In this project, plants treated with Cease and b ® Means within a column within each crop followed by the same letter are Triathlon BA exhibited the highest biomass, other than the control not significantly different according to Tukey's HSD separation test(P = 0.05). with and without Pythium spp., suggesting that these products provide c Scale of damping-off severity based on Boehm et al. (1993). 1 = symp- protection against Pythium root rot in a greenhouse setting. However, tomless, 2 = emerged but wilted, 3 = post-emergent damping-off, and 4 = pre- we cannot generalize that Bacillus spp. adapt better to greenhouse set- emergent damping-off. ® tings because plants treated with Companion , which active ingredient d Root disease was rated based on visual presence or absence of root necrosis is Bacillus subtilis GB03, did not respond in a similar way. This suggests and rot. Percentage of root necrosis was calculated by averaging all the yes's and no's per treatment, and statistical analysis was conducted using Chi-Square that the specific isolate or manufacturing of the product may influence test. its efficacy. Although research provides evidence for these selected beneficial microorganisms to inhibit growth of pathogens and reduce disease Johri, 2009). All these activities require reallocation of energy from the (Jeyaseelan et al., 2012; Utkhede et al., 2000; Zouari et al., 2016) their growth to activate defense mechanisms and might explain why we efficacy may depend on the rhizosphere chemical and biological para- observed growth reduction with the application of biofungicides. meters (Bonanomi et al., 2018), application method (Pill et al., 2011), While closed-loop hydroponic systems allow savings of water and and timing of the application (both pathogen or biofungicides). In our nutrient use, the recirculation of water increases the risk of pathogen study, we observed that arugula was more susceptible than kale to dispersal (Hosseinzadeh et al., 2017; Stanghellini and Rasmussen, Pythium spp. Plant genotype can affect the accumulation of micro- 1994). Pythium spp. in this study had a greater effect on the reduction of organisms that activate plant defenses or be conducive or unfavorable plant biomass than on root necrosis, damping-off, or wilt incidence and to general suppression (Bonanomi et al., 2018). Different cultivars, severity. Hydroponically-grown lettuce infected with P. aphani- within a plant species, harbor naturally occurring beneficial bacteria at dermatum (Corrêa et al., 2010; Johnstone et al., 2004; Pantelides et al., different concentrations and vary in disease suppression (Berendsen 2017) or P. dissotocum (Stanghellini and Kronland, 1986) had biomass et al., 2012). Further research needs to be conducted to understand how reduction levels of 23–54% compared with the controls. Similar to our different species, application time, and cultivars of microgreens per- project, Stanghellini and Kronland (1986) reported significant yield form with microbial biofungicides. Results from these experiments reductions and little-to-no visible symptoms. This can cause a problem suggest that microbial biofungicides can be introduced in nutrient so- for growers since the disease symptoms may not be noticeable lutions to prevent disease development with plant growth reduction as

101 C.S. McGehee, et al. Crop Protection 121 (2019) 96–102 a tradeoff. Therefore, in practice farmers would need to adjust cropping 134–140. https://doi.org/10.1134/S1021443711050062. time to achieve target biomass levels. In addition, a cost/benefit study Heungens, K., Parke, J.L., 2000. Zoospore homing and infection events: effects of the biocontrol bacterium Burkholderia cepacia AMMDR1 on two oomycete pathogens of may prove valuable to assess the use of microbial fungicide in a high pea (Pisum sativum L.). Appl. Environ. Microbiol. 66, 5192–5200. https://doi.org/10. risk area to suppress disease despite the loss in biomass. Additional 1128/AEM.66.12.5192-5200.2000. research may also be conducted to identify native microbes in nutrient Hosseinzadeh, S., Verheust, Y., Bonarrigo, G., van Hulle, S., 2017. Closed hydroponic systems: operational parameters, root exudates occurrence and related water treat- solutions that are better adapted in these production systems and crops. ment. 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