Microbial Suppression of Pythium Root Rot in Soilless Systems

Cora McGehee B.S. Louisiana State University, 2015

A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science At the University of Connecticut 2018

2018

APPROVAL PAGE Master Thesis Microbial Suppression of Pythium Root Rot in Soilless Systems

Presented by Cora Shields McGehee, B.S.

Major Advisor______Rosa E. Raudales

Associate Advisor______Wade H. Elmer

Associate Advisor______Richard J. McAvoy

University of Connecticut

2018

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Acknowledgements

I am beyond grateful to my major advisor Dr. Rosa Raudales for her hard work and dedication to the lab and innovative research projects. Her guidance and patience has made me into a stronger researcher and diligent worker. I also want to thank the other members of my committee Dr.

Wade Elmer and Dr. Richard McAvoy for their time and generous advice.

I want to thank Frederick Pettit, Shelley Durocher, and Ronald Brine for their assistance in the various greenhouse projects conducted. Thank you Margery Daughtrey for supplying isolates for experiments. Special thanks to Juan Cabrera, Sohan Aziz, Steve Olenski, Joy Tosakoon, and

Carla Caballero for giving your time to help with experiments. Lastly a thank you to the office staff, Christine Strand and Nicole Gabelman for their assistance and kindness.

Special thanks to the U.S. Department of Agriculture via the Connecticut Department of

Agriculture Specialty Crop Block Grant # AG151260 for its support and funding of this work.

Thank you to friends and family who supported me over the phone during this intense intellectual pursuit.

Table of Contents Page

Abstract ...... vii Chapter 1 ...... 1 Introduction ...... 1 Literature Cited ...... 6 Chapter 2 ...... 9 Characterization of Pythium spp. Isolates Obtained in Commercial Greenhouses ...... 9 Abstract ...... 9 Introduction ...... 10 Materials and Methods ...... 12 Results ...... 17 Discussion ...... 20 Literature Cited ...... 23 Figures ...... 28 Tables...... 37 Annex ...... 43 Chapter 3 ...... 47 Efficacy of Microbial Biofungicides on Suppressing Pythium Root Rot on Hydroponic Lettuce (Lactuca sativa L.) ...... 47 Abstract ...... 47 Introduction ...... 48 Materials and Methods ...... 49 Results ...... 53 Discussion ...... 54 Conclusion ...... 56 Literature Cited ...... 57 Figures ...... 61 Tables...... 64 Chapter 4 ...... 70 Efficacy of Microbial Biofungicides against Root Rot and Damping-off of Microgreens caused by Pythium spp...... 70 Abstract ...... 70 Introduction ...... 71

Materials and Methods ...... 72 Results ...... 76 Discussion ...... 78 Conclusion ...... 78 Literature Cited ...... 81 Figures ...... 85 Tables...... 88 Chapter 5 ...... 91 Conclusions ...... 91

Abstract

Pythium spp. are the causal agents of Pythium root rot and damping-off on leafy vegetables in hydroponic systems. Infected plants have low quality and biomass resulting in significant losses to farmers. In the US, synthetic fungicides are not registered for direct application in hydroponic solutions and only a few are registered for application on edible crops in greenhouses. The efficacy of microbial biofungicides depends on the plants and environmental conditions that are conducive for the survival and activity of the microbes. The objectives of this project were to (1) isolate pathogenic Pythium spp. from commercial greenhouses in the northeastern US, (2) evaluate if recirculated nutrient solutions have the potential to inhibit Pythium spp., and (3) evaluate the efficacy of microbial biofungicides on the disease incidence and severity of Pythium root rot in hydroponic lettuce (Lactuca sativa), arugula (Eruca sativa), kale (Brassica oleracea var. sabellica), radish (Raphanus raphanistrum subsp. sativus), and mustard (Brassica juncea) microgreens in hydroponic systems. We obtained nine isolates of P. aphanidermatum, P. dissotocum, and P. graminicola. The isolates were identified using standard morphological identification keys and confirmed with sequencing of the internal transcribed spacer (ITS) regions 1 and 4 of the ribosomal DNA gene. This is the first report of P. dissotocum on lettuce in

Connecticut, and P. aphanidermatum on mustard, mizuna, Swiss chard, and wheatgrass in New

York. In a second experiment, recirculated nutrient solution from commercial farms inhibited Pythium spp. growth in vitro. In a third experiment, we tested the efficacy of the microbial biofungicides Companion® (Bacillus subtilis GB03), Triathlon BA® (Bacillus amyloliquefaciens D747), RootShield® (Trichorderma harzianum KRL-AG2), RootShield Plus®

(Trichoderma harzianum KRL-AG2 and Trichoderma virens G-41), Cease® (Bacillus subtilis QST 713), and Actinovate® (Streptomyces lydicus WYEC 108) on Pythium spp. in

vii hydroponic systems. All microgreens infected with Pythium spp. had at least 28% lower biomass, and higher disease incidence and severity compared with non-inoculated plants. Among plants treated with biofungicides, the lowest and highest root necrosis was observed with

Companion® and Triathlon BA®, respectively. However, the opposite pattern was observed with biomass. In general, leafy vegetables treated with microbial biofungicides had lower shoot (1-

58%) and root (16-61%) biomass compared with the non-inoculated control. Plants treated with Bacillus spp. products with and without Pythium spp. were bigger compared with the other biofungicides. Results from this study suggest that beneficial microbes in hydroponic systems can reduce negative symptoms caused by Pythium infection. However, in the absence of

Pythium root rot, the microbial inoculants also reduced plant biomass. Further research is required to identify native suppressive microbial inhabitants in recirculated nutrient solutions and to identify if alternative application methods of microbial biofungicides can reduce the negative effects that microbial biofungicides have on plant growth.

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Chapter 1

Introduction

Lettuce is the fourth most commonly grown crop in US greenhouses reaching 56 million dollars of total sales and encompassing over 40 hectares (USDA, 2014). In a survey conducted to

Canadian greenhouse growers, 33% of vegetable and ornamental greenhouse growers indicated that disease control was a top concern when recirculating nutrient solutions in hydroponic systems (Richard et al., 2008). This group of growers indicated that fertilizer and water savings were advantages of a closed-irrigation system. Therefore, science-based guidelines on how to proactively prevent root diseases when recirculating nutrient solutions in hydroponics is needed to reduce risk aversion from farmers and to reduce the environmental impact of discharging nutrient solutions in the environment.

Pythium Damping-off and Root Rot in Hydroponically-Grown Crops

Waterborne pathogens can be a problem in recirculating irrigation systems caused by the survival structures of the organisms and the challenge of decontaminating water sources.

Recycled irrigation water acts both as primary inoculum source and dispersal mechanism of secondary inoculum in many production systems, correlating to plant disease (Hong, 2014; Hong and Moorman, 2004; Stewart-Wade, 2011). Hydroponic systems provide a moist and nutrient rich environment for aquatic organisms, such as Pythium spp.

Pythium spp. belong to the Oomycota class, commonly known as “water molds”.

Pythium spp. form zoospores, bi-flagellated spores that allow these organisms to swim freely in solutions and actively target roots (Agrios, 2004). Fungal root pathogens (e.g. Fusarium spp. and

Thielaviopsis spp.) do not form zoospores but can also spread by means of spores and mycelial fragments carried in organic debris in irrigation systems (Stanghellini et al., 1999; Utkhede,

2005). Pythium spp. also produce oospores that allow them to survive in water for years in the absence of fresh plant material to infect, which is why they are among the most challenging pathogens to control in hydroponic systems (Deacon and Donaldson, 1993). Not all Pythium species in irrigation water are pathogenic (Moorman et al., 2002), similar to observations on

Phytophthora spp. (Parke et al., 2014). The presence of the non-pathogenic Pythium species support the idea of developing ecological management strategies in which beneficial microbial communities are enhanced, instead of aiming for sterile environments or applying draconian treatment options.

Pythium species are one of the most common causes of seed rot, root rot, and seedling damping-off, which includes pre and post emergence seedling mortality (Heffer et al., 2002).

Pythium spp. cause root rot in many plant species including lettuce in hydroponic systems

(Cooke et al., 2000). Microgreens are edible greens that are harvested at seedling stage and are particularly vulnerable to Pythium damping-off (Kaiser and Ernst, 2012). Infected plants have necrotic roots and take up less water and nutrients compared with healthy plants (Jenkins and

Averre, 1983). Hydroponically grown lettuce infected with Pythium dissotocum resulted in 54% yield reduction by weight, although there were no visible symptoms on the upper portions of the plants (Vallance et al., 2011).

Plants infected with Pythium represent a costly problem to growers. Infected areas can result in reduced crop quality and yields, increase the cost of pesticides, and increase the risk of not fulfilling market demands (Sutton et al., 2006).

Current Control Practices

Protection from Pythium is needed from the beginning of the seedling stage and throughout a major portion of the crop cycle. Once Pythium enters the system, control is very difficult and growers may be forced to destroy the whole crop (Utkhede et al., 2000). Sanitation and elimination of the sources responsible for the introduction of Pythium spp. into commercial greenhouses and monitoring environmental conditions is essential for the development of effective disease control.

Primary infection of Pythium can come from infected plant material, contaminated water sources, and debris accumulation (Sutton et al., 2006). Secondary infection can occur with the dispersal of contaminated debris and zoospores in water, or transmission by insect vectors (e.g. shore flies and fungus gnats). Oospores of P. aphanidermatum were microscopically observed in the gut of larvae, pupae, and adult shore flies, and viable oospores were excreted in frass

(Goldberg and Stanghellini, 1990). Shore flies and fungus gnats feed on algae. Therefore, populations can be controlled by preventing algae growth.

Fungicides registered for the control of root diseases on edible crops in greenhouses are limited to 18, of which eleven are microbial biofungicides (Pundt and Smith, 2017). Fungicides for the control of Pythium in greenhouse vegetable crops were not available in Canada until 1999

(Paulitz and Bélanger, 2001). Water treatment options for control of waterborne pathogens include chemicals (e.g. chlorine, chlorine dioxide, etc.) which may cause phytotoxicity to crops or are energy intensive (e.g. ozone, ultraviolet radiation). Phytotoxicity has been reported in lettuce grown in closed and soilless systems treated with as little as 0.55 mg.L-1 of chlorine

(Premuzic et al., 2007) and 8 mg.L-1 of hydrogen peroxide (Nedderhoff, 2000) with lettuce seedlings. Biosurfactants such as rhamnolipid and nitrapyrin have reduced the number of

3 zoospores in hydroponic solutions (Stangellini and Miller, 1997), yet nitrapyrin has been reported to lower plant biomass at concentrations of 12.5 mg.L-1 (Pagliaccia et al., 2007).

Traditional disease control strategies are not feasible in hydroponic systems. Therefore, non- chemical and low energy alternatives must be considered.

It is critical to understand that disease development depends on numerous environmental conditions and host susceptibility. Environmental stress such as high temperatures, low light intensity, low concentrations of dissolved oxygen, or high concentration of salts in the nutrient solution can increase the susceptibility of hydroponic plants to Pythium root rot (Sutton et al.,

2006).

In plant pathology, the term biological control agent refers to the “Purposeful utilization of introduced or resident living organisms, other than disease resistant host plants, to suppress the activities and populations of one or more plant pathogens” (Pal and Gardener, 2006).

Biological control mechanisms include antibiosis, competition, mycoparasitism, cell wall degrading enzymes, and induced resistance (Paulitz and Bélanger, 2001).

A number of microorganisms that are potentially antagonistic to root pathogens have been selected and tested in cucumbers grown in hydroponic systems such as Pseudomonas,

Trichoderma, Bacillus, and Gliocladium against Pythium aphanidermatum (Punja and Yip,

2003; Rankin and Paulitz, 1994; Utkhede and Koch, 1999). However, efficacy on lettuce and microgreens is not available.

Thesis Research Objectives

(1) Determine the pathogenicity and identification of Pythium spp. isolates from commercial greenhouse operations by measuring the incidence and severity of root rot and damping-off on lettuce seedlings in vitro.

(2) Evaluate the inhibition potential of recirculated nutrient solutions from hydroponic systems on Pythium spp. growth in vitro.

(3) Evaluate the efficacy of microbial biofungicides on the disease incidence and severity of

Pythium root rot caused by Pythium aphanidermatum and Pythium dissotocum in hydroponic lettuce and microgreens.

Literature Cited

Agrios, G. N. 2004. Plant Pathology: Fifth Ed. Elsevier Academic Press, San Diego, USA. 238 pp. Ahanger, R.A., H.A. Bhatand, N.A. Dar. 2014. Biological agents and their mechanism in plant disease management. Sciencia Acta Xaveriana. 5, 47-58 Brantner, J. R. and C.E. Windels. 1998. Variability in sensitivity to metalaxyl in vitro, pathogenicity, and control of Pythium spp. on sugar beet. Plant Disease. 82, 896-899 Cohen, Yigal and M.D. Coffey. 1986. Systemic fungicides and the control of oomycetes. Ann. Rev. Phytopathol. 24, 311-338 Cooke, D.E.L., A. Drenth, J.M. Duncan, G. Wagels, C.M. Brasier. 2000. A molecular phylogeny of Phytophthora and related oomycetes. Fungal Genetics and Biology. 30, 17-32 Deacon, J.W. and S.P. Donaldson. 1993. Molecular recognition in the homing responses of zoosporic fungi, with special reference to Pythium and Phytophthora. Mycological Research. 97, 1153-1171 Goldberg, N.P. and M.E. Stanghellini. 1990. Ingestion-egestion and aerial transmission of Pythium aphanidermatum by shore flies (Ephydrinae: Scatella stagnalis). Phytopathology. 90, 1244-1246 Heffer L.V., M.L. Powelson, K.B. Johnson. 2002. Oomycetes. The Plant Health Instructor. Retrieved on 7 June 2016 from http://www.apsnet.org/edcenter/intropp/LabExercises/Pages/Oomycetes.aspx Hong, C.X. 2014. Component analyses of irrigation water in plant disease epidemiology Ch. 11 in: Hong, C.X, G.W. Moorman, W. Wohanka, and C. Buttner (eds) Biology, detection, and management of plant pathogens in irrigation water. APS Press, St. Paul, Minneapolis, 111- 121 Hong, C. X. and G.W. Moorman. 2005. Plant pathogens in irrigation water: challenges and opportunities. Critical Reviews in Plant Sciences. 24,189-208 Jarvis, W.R., J.L. Shipp, R.B. Gardiner. 1993. Transmission of Pythium aphanidermatum to greenhouse cucumbers by the fungus gnat Bradysia impatiens (Diptera: Sciaridae). Annuals of Applied Biology. 122, 23-29 Jenkins, S.F. and C.W. Averre. 1983. Root diseases of vegetables in hydroponic culture systems in North Carolina. Plant Disease. 67, 968-970 Kaiser, C. and M. Ernst. 2012. Microgreens. University of Kentucky. Retrieved on 24 Oct. 2017 from https://www.uky.edu/Ag/CCD/introsheets/microgreens.pdf Khalil, Sammar, Hultberg, Malin, Alsanius, W. Beatrix. 2009. Effects of growing medium on the interactions between biocontrol agents and tomato root pathogens in a closed hydroponic system. The Journal of Horticultural Science and Biotechnology. 84, 489-494

Moorman, G.W., S. Kang, D.M. Geiser, S.H. Kim 2002. Identification and characterization of Pythium species associated with greenhouse floral crops in Pennsylvania. Plant Disease. 86, 1227-1231 Moorman, G.W. and S.H. Kim. 2004. Species of Pythium from greenhouses in Pennsylvania exhibit resistance to propamocarb and mefenoxam. Plant Disease. 88, 630-632 Nedderhoff, E. 2000. Hydrogen peroxide for cleaning irrigation system in: pathogen control in soilless cultures. Commercial Grower. 55, 32-34 Pagliaccia, D., D. Ferrin, M.E. Stanghellini. 2007. Chemo-biological suppression of root infecting zoosporic pathogens in recirculating hydroponic systems. Plant Soil. 299, 163-179 Pal, K.K. and B. M. Gardener. 2006 Biological control of plant pathogens. The Plant Health Instructor. 10, 1094-1117 Parke, J.L., B.J. Knaus, V.J. Fieland, C. Lewis, N.J. Grünwald. 2014. Phytophthora community structure analysis in Oregon nurseries inform systems approaches to disease management. Phytopathology. 104, 1052-1062 Paulitz, T.C., and R.R. Bélanger. 2001. Biological control in greenhouse systems. Annu. Rev. Phytopathol. 39, 103-33 Premuzic, Z., H.E. Palmucci, J. Tamborenea, M. Nakama. 2007. Chlorination: phytotoxicity and effects on the production and quality of Lactuca sativa var. Mantecosa grown in a closed, soil-less system. Intl. J. of Exptl. Bot. 76,103–117 Pundt, L. 2007. Biological control of fungus gnats. Integrated Pest Management Program at University of Connecticut. Retrieved on 8 August 2016 from ipm.uconn.edu/documents/raw2/htmL/666.php?aid=666 Pundt, L. and T. Smith. 2017. Selected fungicides and bactericides labeled for vegetable bedding plants and transplants. CT IPM UConn Extension. Retrieved on 5 February 2017 from http://ipm.uconn.edu/documents/raw2/1119/2017%20Fungicides%20and%20Bactericides%2 0Labeled%20for%20Vegetable%20Bedding%20Plants%20and%20Transplantsfeb2final.pdf Punja, Z. K. and R. Yip. 2003. Biological control of damping-off and root rot caused by Pythium aphanidermatum on greenhouse cucumbers. Can. J. Pathol. 2003. 25, 411-417 Rankin, L. and T.C. Paulitz. 1994. Evaluation of rhizosphere bacteria for biological control of Pythium root rot of greenhouse cucumbers in hydroponic culture. Plant Disease. 78, 447-451 Raudales, R.E., J.L. Parke, C.L. Guy, P.R. Fisher. 2014. Control of waterborne microbes in irrigation: a review. Agricultural Water Management. 143, 9-28 Richard, S., Y. Zheng, M. Dixon. 2008. To recycle or not to recycle? Greenh Can. (December), 22–24 Salman, M. and R. Abuamsha. 2012. Potential for integrated biological and chemical control of damping-off disease caused by Pythium ultimum in tomato. BioControl. 57, 711-718 Sanders, P. L. 1984. Failure of metalaxyl to control Pythium blight on turfgrass in Pennsylvania. Plant Disease. 68, 776-777

Sanogo, S. and G.W. Moorman. 1993. Transmission and control of Pythium aphanidermatum in an ebb-and-flow subirrigation system. Plant Disease. 77, 287-290 Stanghellini, M. E., and R. M. Miller. 1997. Biosurfactants: their identity and potential efficacy in the biological control of zoosporic plant pathogens. Plant Disease. 81, 4-12 Stanghellini, M.E., S.L. Rasmussen, D.H. Kim, and P.A. Rorabaugh. 1999. Aerial transmission of Thielaviopsis basicola, a pathogen of corn-salad, by adult shore flies. Phytopathology. 89, 476-479 Stanghellini, M.E., S.L. Rasmussen, D.H. Kim, P.A. Rorabaugh. 1996. Efficacy of non-ionic surfactants in the control of zoospore spread of Pythium aphanidermatum in a recirculating hydroponic system. Plant Disease. 80, 422-428 Stewart-Wade, S.M. 2011. Plant pathogens in recycled irrigation water in commercial plant nurseries and greenhouses: their detection and management. Irrig Sci. 29, 267-297 Sutton, J.C., C.R. Sopher, T.N. Owen-Going, W. Liu, B. Grodzinski, J.C. Hall, R.L. Benchimol. 2006. Etiology and epidemiology of Pythium root rot in hydroponic crops: current knowledge and perspectives. Summa Phytopathologica. 32.4, 307-21 USDA Census 2014. Retrieved on 16 January 2016 from http://www.agcensus.usda.gov/ Utkhede, R. 2005. Increased growth and yield of hydroponically grown greenhouse tomato plants inoculated with arbuscular mycorrhizal fungi and Fusarium oxysporum f. sp. BioControl. 51, 393-400 Utkhede, R.S., C.A. Levesque, D. Dinh. 2000. Pythium aphanidermatum root rot in hydroponically grown lettuce and the effect of chemical and biological agents on its control. Canadian Journal of Plant Pathology. 22.2, 138-44 Vallance, J., F. Deniel, G. Le Floch, L. Guerin-Dubrana, D. Blancard, P. Rey. 2011. Pathogenic and beneficial microorganisms in soilless cultures. Gradignan, France: INRA, EDP Sciences, pp. 711-26

Chapter 2

Characterization of Pythium spp. Isolates Obtained in Commercial Greenhouses

Abstract

Pythium spp. isolated from greenhouses in the northeastern US were screened for pathogenicity in lettuce seeds in vitro. Nine isolates of P. aphanidermatum, P. dissotocum, and P. graminicola were obtained from lettuce or microgreens grown in commercial greenhouses. The isolates were identified using standard morphological identification keys and confirmed with sequencing of the internal transcribed spacer (ITS) regions 1 and 4 of the ribosomal DNA gene. This is the first report of P. dissotocum on lettuce in Connecticut (McGehee et al., 2018), and P. aphanidermatum on mustard, mizuna, Swiss chard, and wheatgrass in New York. All of the

Pythium isolates were tested for pathogenicity against lettuce seedlings in vitro. P. dissotocum

Cor1 and P. aphanidermatum Cor4 caused the highest disease incidence (96-98%) and severity of lettuce seedling rot and have the potential to infect plants in production settings. In a second experiment, recirculated nutrient solutions were collected and investigated for the inhibition of

Pythium species in vitro. Two inhibition experiments were conducted based on two separate methods but used the same experimental treatments. The inhibition tests showed that nonautoclaved recirculated nutrient solution from a hydroponic lettuce farm inhibited Pythium spp. growth in vitro between 1.6 and 27.4%, respectively. In some instances, autoclaved recirculated nutrient solution inhibited Pythium species growth. This study suggests that a combination of microbial and chemical compounds may have the potential to reduce disease incidence when using recirculated nutrient solutions. Further research is required to identify the suppressive agents in recirculated nutrient solutions and study the antagonistic properties of microbes individually or in combination with other microbes.

Introduction

Pythium spp. that cause root rot in hydroponic lettuce can result in crop losses of up to

54% (Labuschagne et al., 2002; Stanghellini and Kronland, 1986; Vallance et al., 2011). Losses up to 100% have been reported on cucumber, spinach, tomato and pepper (Table 1). Pythium seed rot, root rot, and seedling damping-off, which includes pre and post emergence seedling death, are all common diseases in greenhouse production (Heffer et al., 2002). Pythium spp. are ubiquitous in hydroponics systems. However, not all Pythium species identified in the root-zone are pathogenic (Moorman et al., 2002), similar to observations with Phytophthora spp. (Parke et al., 2014).

Pythium belongs to the order Peronosporales under the class Oomycota, which includes many plant pathogens documented in water such as Pythium, Phytophthora and the causal agents of Downy mildew. These organisms spread rapidly by producing zoospores, motile spores that move freely in the water. Oomycetes also form oospores, which are resting spores that can survive for prolonged periods of time in the absence of organic matter (Agrios, 2004) which is why they are challenging pathogens to control in hydroponic systems (Deacon and Donaldson,

1993).

The life cycle of Pythium spp. include multiple structures (Figure 1) that allow them to survive in water, soil, and organic matter. The asexual cycle of Pythium occurs as follows: the mycelium gives rise to sporangia, which can either germinate directly by producing one to several germ tubes or develop a vesicle which contains zoospores. One hundred or more zoospores are produced in each vesicle (Agrios, 2004). Then zoospores are released into the solution and they move toward the plant by chemotaxis. Once the zoospores adhere to the surface of the plant they form a cyst, and germinate by producing a germ tube. The germ tube

10 penetrates the host tissue and starts the infection process. Pythium species cause polycyclic disease cycles, therefore the dispersal of secondary inoculum is likely to occur in hydroponics.

Zoospores are one of the primary infectious propagules responsible for the spread of Pythium root rot in hydroponic systems (Stanghellini, 1996).

Recirculated irrigation water acts as a primary inoculum source and dispersal mechanism of secondary inoculum in many plant-pathosystems, correlating to plant disease (Grech and

Rijkenberg, 1992; Hong et al., 2001; Lacy et al., 1981; Van Kuik, 1992; Whiteside and Oswalt,

1973). Greenhouses in the United States are increasing the practice of reusing nutrient solutions in their operations to protect the environment and conserve water and fertilizer. Hydroponic systems for production of edible greens are mostly closed-loop irrigation systems. A greenhouse in Canada reported recirculating their nutrient solutions for more than 20 years without disease outbreaks (Tian and Zheng, 2013). In a controlled experiment in laboratory conditions, the 20- year old recirculated nutrient solution was tested for suppression of six plant pathogens, including Globisporangium ultimum (formerly Pythium ultimum) and P. intermedium. The recirculated nutrient solution suppressed G. ultimum after 10 days of incubation, and was found to contain large numbers of bacteria, filamentous fungi, and yeasts (Tian and Zheng, 2013). P. intermedium was not suppressed. These results indicate that recirculated water with a high concentration of beneficial microbes has the potential of suppressing certain Pythium spp. in a plant production system with a controlled environment.

The objectives of this project was to characterize Pythium spp. isolates obtained from commercial greenhouses by (1) identifying the Pythium isolates to the species level and determine the pathogenicity of Pythium spp. isolates by measuring the incidence and severity of root rot and damping-off on lettuce seedlings in vitro, and (2) evaluating the inhibition potential

11 of recirculated nutrient solutions obtained from commercial farms on Pythium spp. growth in vitro.

Materials and Methods

Collection of plant tissue. Nine Pythium isolates were obtained from commercial greenhouses in

Connecticut and New York in 2015, 2016, and 2017. Symptomatic plants with one or more of the following symptoms were collected: (1) seedling damping-off, (2) wilted and/or stunted plants, or (3) dark brown or necrotic roots. Plants were collected in plastic bags and stored at 4°C for one or two days. The five isolates obtained at Long Island, NY were isolated by the Cornell

University team at the Long Island Horticultural Research and Extension Center. We isolated four Pythium isolates from Connecticut and New York (Table 2).

Isolation of Pythium spp. Pythium was isolated from the roots by using Singleton’s et al. (1992) protocol. Briefly, the roots were triple washed with sterile deionized water (DiWater) and then blotted dry with paper tissue. Six root pieces (~5-mm) were transferred to three agar media: water agar, PARP, and PARP-V8 (Annex I). After one day, the hyphal tip of each isolate was transferred to a fresh water agar plate.

Identification of Pythium isolates. All Pythium isolates were identified based on morphological characteristics and DNA sequencing. Morphological identification was conducted based on the sexual structures (Dick, 1990; Moorman and May, 2012; van der Plaats-Niterink, 1981). Sexual structures such as oospores and sporangia were produced by incubating mycelial plugs of

Pythium spp. in autoclaved tall fescue leaves in sterilized deionized water under constant light for three days based on a protocol developed by Dr. Robert Wick from the University of

Massachusetts (Annex II). Then we followed the van der Plaats-Niterink (1981) key and monographs to identify Pythium species (Figure 1). The isolates obtained are listed in Table 2.

Identification was verified by sequencing the internal transcribed spacer (ITS) region ITS1 and

ITS4 of ribosomal DNA of the Pythium spp. isolates from mycelial mats (Choudhary et al.,

2016; White et al., 1990). Mycelia of nine isolates of Pythium spp. (Table 2) were grown on corn meal agar (CMA) and incubated for three days at room temperature. Two 1-mm2 pieces of media were cut, transferred to a Petri dish with 20 mL clarified-V8 broth, and then incubated for three days at room temperature in a dark drawer. After three days, the mycelial mats were rinsed twice with sterilized deionized water, and dried on sterilized filter paper for five minutes. Dry mycelium was transferred to a 2-mL microcentrifuge tube, frozen with liquid nitrogen, and pulverized to a fine powder with a TissueLyzer II machine (Qiagen, USA) for two minutes at a frequency of 30 1/s. DNA was extracted from the 150 mg of ground fine tissue powder using the commercial DNeasy Plant Mini Kit (Qiagen, USA) following manufacturer’s instructions

(Annex III).

The ITS regions (ITS1, ITS4) in ribosomal DNA of the isolates were amplified by polymerase chain reaction (PCR) using the universal primers ITS1 (5’-TCC GTA GGT GAA

CCT GCG G-3’) and ITS4 (5’-TCC TCC GCT TAT TGA TAT GC-3’) in a 20 µL PCR reaction.

The PCR mixture contained 30 ng of genomic DNA, 1 µM each of the primers ITS1 and ITS4, and 1X Taq DNA polymerase reaction buffer (Promega Corp., WI). The PCR was run in a

T100™ Thermocycler (Bio-Rad, CA). The PCR conditions were optimized and set at 94°C for 5 minutes, followed by 30 cycles of 94°C for 1 minute, 55°C for 1 minute, 72°C for 1 minute,

94°C for 1 minute 34 times, 72°C for 10 minutes, and infinite holding at 4°C. The size of the amplification product was analyzed by electrophoresis in a 0.7% agarose gel in 1X TAE [Tris-

13 acetate-ethylenediaminetetraacetate (EDTA)] buffer (Invitrogen, CA) containing 40 mM Tris- acetate and 1 mM EDTA. The gel was stained with ethidium bromide (1 µg/mL) and DNA was visualized using UV light (Figure 2). Amplicons were submitted to a commercial laboratory for sequencing. The nucleotide sequences were compared with BLAST nucleotide analysis against publicly available sequences in the GenBank online database (www.ncbi.nlm.nih.gov/BLAST).

Experiment 1.

Pathogenicity Tests. Pathogenicity, the capacity to cause disease, was measured in vitro with lettuce (Lactuca sativa cv. Spretnak) seeds based on the method described by Broders et al.,

2007 and Del Castillo Munera and Hausbeck, 2016. Pythium spp. were grown on CMA (Difco

Laboratories, MD) for four days. A 4-mm plug of actively growing mycelium was transferred to the center of an 8-cm diameter Petri plate with water agar. Lettuce seeds were disinfected by immersing them in 1% sodium hypochlorite solution for one minute and then rinsed with sterile deionized water. Eight seeds were placed on each water agar plate with the pathogen. The seeds were placed 2-cm from the margin of the plate and equidistantly from each other. The plates were incubated in the dark at 22 °C for seven days. Pathogenicity was evaluated using the following virulence scale (Broders et al., 2007; Del Castillo Munera and Hausbeck, 2016):

0=100% germination with a healthy appearance, 1=99 to 70% germination, small brown lesions on the radicle or hypocotyl, 2=69 to 30% germination, large brown lesions on the hypocotyl and radicle, and 3=29 to 0% germination, coalesced lesions covering the hypocotyl and radicle

(Figure 3). Germination was defined as a seedling with a root radicle of >1 cm long and was not visibly colonized by the pathogen (Broders et al., 2007; Del Castillo Munera and Hausbeck,

2016). Two separate experiments were conducted with two sets of Pythium species. The experiments were conducted separately because the isolates were obtained in different years.

Experiment 2.

Testing Inhibition of Nutrient Solutions from Commercial Farms. Recirculated nutrient solution was collected from two commercial farms that grow hydroponic lettuce on rafts in deep- water culture systems. The nutrient solutions were analyzed for chemical and biological characteristics.

Chemical analyses included (1) quantification of essential plant elements, pH, alkalinity, and sodium; (2) free and total chlorine levels, and (3) oxidation reduction potential (ORP). The nutrient content of the solutions was measured by induced couple plasma analysis by a commercial analytical laboratory. Free and total chlorine were measured with the Orion

AQUAfast IV Powder Chemistries in the Orion AQ4000 Advanced Colorimeter (Thermo Fisher

Scientific Inc., MA). ORP was measured with the Orion Versa Star Meter (Thermo Fisher

Scientific Inc., MA).

Biological analyses of the nutrient solutions included chemical oxygen demand (COD), total heterotrophic bacteria, and bacterial populations. COD was measured using a CODL00 kit in which the samples were digested in an Orion COD165 Thermoreactor at 150 °C for 120 minutes and then measured using an Orion AQ4000 Advanced Colorimeter (Thermo Fisher Scientific

Inc., MA). The first inhibition test was conducted with nutrient solution collected in Cheshire,

CT. The initial pH was 6.54 and the EC was 1.60 mS.cm-1. COD resulted in 45.06 mg.L-1. Free chlorine was 0.015 mg.L-1 and total chlorine was 0.045 mg.L-1. The second inhibition test used water collected in Guilford, CT. The initial pH was 6.92 and the EC was 1.79 mS.cm-1. COD was

23.45 mg.L-1. Free chlorine was 0.008 mg.L-1.and total chlorine was 0.026 mg.L-1. DNA was collected using the PowerWater DNA Isolation Kit (MoBio Laboratories Inc., CA) from the recirculated water used in the second inhibition experiment. PCR was performed using ITS1,

ITS4, and 16S forward (5’AGAGTTTGATCCTGGCTCAG-3’) and reverse (5’-

ACGGCTACCTTGTTACGACTT-3’) universal primers in order to sequence for microbial communities detected in the DNA from the recirculated water.

An in vitro assay to measure inhibition of Pythium spp. in the presence of recirculated nutrient solution was conducted based on Tian and Zheng’s (2013) protocol. Briefly, inhibition of P. aphanidermatum Kop-8 and P. dissotocum Cor1 was measured on V8 juice agar and ½ strength potato dextrose agar (PDA; Difco Laboratories, MD). A 4-mm diameter mycelial disc of a pathogen from a two-day actively growing culture was placed on one side of the 8-cm diameter

Petri plate with either potato dextrose agar or V8 juice agar. An autoclaved polyvinyl chloride

(PVC) ring (1.2-cm diameter, 0.9-cm height) was placed in the agar at the opposite side of the mycelia at an equal distance from the margin of the plate as the mycelial disc (Figure 4). Four- hundred microliters of either non-autoclaved recirculated nutrient solution, autoclaved recirculated nutrient solution, or autoclaved deionized water were pipetted inside the ring. The plates were sealed with paraffin film and incubated at ~22 °C for five days. Pathogen growth was monitored daily for five days by measuring the radius of the mycelia from the edge of the colonies in the direction toward the solution ring. The results were converted into percentage inhibition of radial growth in relation to the radial growth of the pathogen in the control plate for day five (Jinantana and Sariah, 1998; Tian and Zheng, 2013). The experiment was conducted twice, each time with six replicates per treatment for each media.

A second inhibition experiment was conducted using a modified method. The same pathogens and media described above were used. Ten milliliters of ½ PDA and V8 agar were pipetted into a

35 x 10-mm Petri plate. Once the agar solidified, a 9-mm hole was made in the middle of each plate. Then a 4-mm plug from a five day old plate of Pythium aphanidermatum Kop-8 and P.

16 dissotocum Cor1 on water agar was placed in the 9-mm hole (Figure 5). Three hundred microliters of each treatment (autoclaved deionized water, autoclaved recirculated nutrient solution, and non-autoclaved recirculated water) was pipetted into the 9-mm hole with the plug inside of it. Radial growth (mm) was measured daily.

Experimental Design and Statistical Analysis

The pathogenicity and inhibition experiments were complete randomized designs (CRD) with six experimental units, each experimental unit consisted of a Petri dish. All experiments were conducted twice. Statistical analysis was conducted using SAS Version 9.4 (SAS Institute Inc.,

NC) to establish significance of the effects of all factors at α=0.05. Data were analyzed by analysis of variance (ANOVA). Means were separated by Tukey’s studentized range HSD

(Honestly Significant Difference) separation test (α=0.05) using PROC MIXED. Severity ratings were analyzed with non-parametric analysis using the RANK and PROC GLM procedure.

Results

Identification of Pythium species. The isolates obtained were confirmed to be Pythium spp. based on morphological characteristics and sequence of the ITS region. For P. dissotocum, colonies grown on V8-juice agar had filamentous sporangia with a dendroid structure, the oogonia were between 19-23 μm in diameter and subglobose, the antheridia developed on unbranched stalks and were sessile, and the oospores were aplerotic. P. aphanidermatum had mostly terminal sporangia, the oogonia were between 19-24 µm in diameter, with an average of

21.9 µm with a globose shape and smooth surface. Antheridia were single or two per oogonium, terminal and intercalary, and were predominately monoclinous. Oospores were aplerotic with an average diameter of 17 µm and a wall up to 2 µm thick. P. graminicola had terminal or

17 intercalary sporangia consisting of irregular inflated filamentous complexes. Oogonia had an average diameter of 22 µm, was mostly terminal with a globose shape and smooth surface.

Antheridia were mostly monoclinous and clavate (club shaped). Oospores were plerotic with a wall up to 3 µm thick. BLAST nucleotide analyses against publicly available Pythium sequences in GenBank confirmed that the isolates obtained were P. dissotocum, P. aphanidermatum, or P. graminicola with ≥91% identity match (Table 2). The amplicon size of the isolates resulted in a single product of 850 bp (Figure 2, lanes 1-9), consistent with the reports of Choudhary et al.,

2016 and White et al., 1990. Sequencing data of the Pythium species were submitted to GenBank with assigned accession numbers listed in table 2.

Pathogenicity Tests.

Experiment 1. All Pythium species tested in experiment 1 caused root rot on lettuce seedlings in vitro (Figure 6). Lettuce seedlings inoculated with P. dissotocum Cor1 and P. aphanidermatum

Cor4 had the highest disease incidence and severity of lettuce seedling rot (Figure 6A). Seedlings inoculated with P. dissotocum Cor1 and P. aphanidermatum Cor4 resulted in 98% and 96% disease incidence, respectively. P. dissotocum Cor1 and P. aphanidermatum Cor4 also resulted in the highest disease severity (Figure 6B). Pythium graminicola resulted in the lowest disease incidence and disease severity. The control resulted in 0% incidence and severity of lettuce root rot. The isolates P. dissotocum Cor1 and P. aphanidermatum Cor4 from this experiment were chosen for further studies in the greenhouse.

Experiment 2. Differences were observed by experimental run, therefore the results of this experiment are presented by run. In experimental run one, disease incidence ranged from 33% to

73% for Kop-3x and Kop-8, respectively (Figure 7A). In experimental run two, disease incidence ranged from 75% to 100% for Kop-6 and Kop-8, respectively (Figure 8A). No difference among

18 the isolates was observed for disease severity in both experimental runs (Figure 7B and 8B). The negative control had zero disease incidence and severity in both experimental runs. The isolate P. aphanidermatum Kop-8 was chosen for further experiments in the greenhouse.

Inhibition Tests. We observed interaction between all the factors in all experiments (Table 3).

Non-autoclaved recirculated nutrient solution consistently inhibited radial mycelial growth of P. aphanidermatum Kop-8 on ½ PDA and V8 juice agar after five days of incubation. Results for other combinations varied by experimental run. Inhibition was measured daily and no significance was observed until day four and five when the mycelial growth of the control came in contact with the PVC ring filled with the solution. No inhibition (0%) was observed in sterilized deionized water on any Pythium species.

In the first experimental run on day five, inhibition of P. dissotocum Cor1 was 22.1 or 15.1% using non-autoclaved nutrient solution on V8 juice agar or ½ PDA media, respectively (Table 4).

P. aphanidermatum Kop-8 was inhibited by over 17% on V8 and ½ PDA, using non-autoclaved recirculated water. P. dissotocum Cor1 was inhibited on ½ PDA using autoclaved recirculated water, and no inhibition was observed when Cor1 was grown on V8. P. aphanidermatum Kop-8 was inhibited greater on V8 juice agar than ½ PDA using autoclaved recirculated nutrient solution.

For the second experimental run the results were similar in that 0% inhibition was observed in the sterilized deionized water set as the control. P. dissotocum Cor1 was inhibited by 8.6% on

V8 juice agar and 1.6% on ½ PDA using non-autoclaved recirculated nutrient solution. Inhibition of P. aphanidermatum Kop-8 was 27.4% or 27.1 on V8 or ½ PDA, respectively using nonautoclaved recirculated water (Table 4). Inhibition of either Pythium species or media did not result in significant inhibition using autoclaved recirculated water in this experimental run. The

19 results of the bacterial counts, 16S, and ITS1 and ITS4 sequencing showed that the recirculated water contained microbial content (Figure 9). DNA samples from the recirculated water were sent to a commercial laboratory for Illumina next generation sequencing to identify the microbial populations in the water discussed below.

The second inhibition test using a separate protocol resulted in significant inhibition of P. aphanidermatum Kop-8 on V8 and ½ PDA using the recirculated water after two days of incubation. Inhibition of P. dissotocum Cor1 was 15.0% on V8 and 0% on ½ PDA using nonautoclaved recirculated nutrient solution (Table 5). P. dissotocum Cor1 exhibited a similar pattern and was inhibited on V8 but not on ½ PDA using autoclaved recirculated nutrient solution. P. aphanidermatum Kop-8 was inhibited on V8 and ½ PDA using non-autoclaved recirculated water and autoclaved recirculated nutrient solution. No inhibition (0%) was observed in sterilized deionized water on either Pythium species. Non-autoclaved recirculated nutrient solution did inhibit Pythium spp. in some instances but was not consistent. Further experiments will be conducted with altered methods to observe inhibition potential of recirculated nutrient solutions against Pythium species.

Discussion

This in vitro bioassay allowed us to identify Pythium spp. that may affect lettuce production in greenhouses. Pythium dissotocum Cor1 was collected from a hydroponic lettuce farm and consistently resulted in the highest disease incidence in lettuce seedlings in our study. P. dissotocum has been previously attributed to attacking hydroponically grown lettuce (Moller and

Hockenhull, 1997; Stanghellini and Kronland, 1986; Tortolero and Sequeira, 1978). To our knowledge, this is the first report of P. dissotocum on lettuce in Connecticut (McGehee et al.,

2018). P. dissotocum has been identified as an F isolate (filamentous sporangia) and is

20 homothallic (Lamour and Kamoun, 2009). P. dissotocum is mostly associated with subclinical infections, which means nearly or completely asymptomatic, though infection can result in significant yield losses (Stanghellini and Kronland, 1986; Favrin et al., 1988). P. aphanidermatum has also been reported as a common and destructive root-infecting pathogen of lettuce in recirculating hydroponic systems (Stanghellini and Rasmussen, 1994). Environmental conditions such as temperature may affect the survival of certain Pythium species in the water.

Higher temperatures favor P. aphanidermatum, while cooler temperatures favor P. dissotocum

(Gold and Stanghellini, 1985). P. aphanidermatum Cor4, P. dissotocum Cor1, and P. aphanidermatum Kop-8 were all selected to be used in future greenhouse research projects growing hydroponic lettuce and microgreens.

Recirculated nutrient solution collected from hydroponic lettuce farms resulted in some inhibition of P. dissotocum Cor1 and P. aphanidermatum Kop-8. Even though not dramatically, recirculated nutrient solution inhibited Pythium species growth. There were few instances where it seemed like there was a non-microbial effect that inhibited P. aphanidermatum Kop-8, but this inhibition was not consistent. These data suggest that microbial compounds have the potential to reduce disease incidence when using recirculated nutrient solutions. Non-autoclaved recirculated water resulted in the highest disease inhibition on V8 juice agar. The interaction between media,

Pythium spp., and water treatment was significant in all of the inhibition experiments (Table 3).

This suggests that environmental factors play an important role in restricting the activity of potential biological control agents (Zhao and Shamoun, 2006). The results of ITS1, ITS4, and

16S sequencing showed that the recirculated water contained fungal and bacterial microbes.

DNA samples of the recirculated water were sent to a commercial laboratory for Illumina next generation sequencing to identify the species of the inhabited microbes to explore their inhibition

21 properties against Pythium root rot. Next generation sequencing techniques will provide advanced opportunities to study the interplay between the water, the plant pathogen, and its associated microbial diversity.

Biological mechanisms that may contribute to the suppression of Pythium spp. include competition, antagonism, parasitism, antibiosis, and induction of plant systemic resistance (Pal and Gardner, 2006). Our inhibition study can only be used to identify the first four mechanisms.

In previous studies, recirculated nutrient solution inhibited Globisporangium ultimum in vitro after ten days of incubation, yet did not show inhibition of P. intermedium (Tian and Zheng,

2013). Identifying the target Pythium species for suppression may help determine which biological approach to pursue. In this study, recirculated nutrient solution demonstrated suppression of P. dissotocum and P. aphanidermatum in vitro, and will be a further study for their potential to prevent root rot in hydroponic production systems in upcoming research. There may be economic benefits on recirculation of nutrient solution. Additional research is required to identify the native suppressive microbes in recirculated nutrient solutions in order to study the antagonistic properties of microbes individually or in combination with other microbes.

Literature Cited

Agrios G.N. 2004. Plant Pathology: Fifth Ed. Elsevier Academic Press, San Diego, USA. 238 pp.

Bates, M.L. and M.E. Stanghellini. 1984. Root rot of hydroponically grown spinach caused by Pythium aphanidermatum and P. dissotocum. Plant Disease. 68, 989-991

Broders K.D., P.E. Lipps, P.A. Paul, A.E. Dorrance. 2007. Characterization of Pythium spp. associated with corn and soybean seed and seedling disease in Ohio. Plant Disease 91, 727-735

Cherif, M., J.G. Menzies, R.R. Belanger. 1994. Yield of cucumber infected with Pythium aphanidermatum when grown with soluble silicon. HortScience. 29, 896-897

Choudhary, C. E., M.L. Burgos-Garay, G.W. Moorman, C. Hong. 2016. Pythium and Phytopythium species in two Pennsylvania greenhouse irrigation water tanks. Plant Disease. 100, 926-932 Cooke, D.E.L., A. Drenth, J.M. Duncan, G. Wagels, C.M. Brasier. 2000. A molecular phylogeny of Phytophthora and related oomycetes. Fungal Genetics and Biology. 30, 17-32

Corrêa, Élida B., W. Bettiol, M.A.B. Morandi. 2010. Biological control of root rot caused by Pythium aphanidermatum and promotion of hydroponic lettuce growth with Clonostachys rosea. Tropical Plant Pathology. 35, 248-252

Deacon, J.W. and S.P. Donaldson. 1993. Molecular recognition in the homing responses of zoosporic fungi, with special reference to Pythium and Phytophthora. Mycological Research. 97, 1153-1171

Del Castillo Munera, J., and M.K. Hausbeck. 2016. Characterization of Pythium species associated with greenhouse floriculture crops in Michigan. Plant Disease. 100, 569-576

Dick, M.W. 1990. Keys to Pythium. Reading, UK. University of Reading. Print

Favrin, R.J., J.E. Rahe, B. Mauza. 1988. Pythium spp. associated with crown rot of cucumbers in British Columbia greenhouses. Plant Disease. 72, 683-687

Fortnum, B. A., J. Rideout, S.B. Martin, and D. Gooden. 2000. Nutrient solution temperature affects Pythium root rot of tobacco in greenhouse float systems. Plant Disease. 84, 289- 294

Gold, S. E., and M. E. Stanghellini. 1985. Effects of temperature on Pythium root rot of spinach grown under hydroponic conditions. Phytopathology. 75, 333-337

Grech N.M, F.H.J Rijkenberg. 1992. Injection of electrolytically generated chlorine into citrus microirrigation systems for the control of certain waterborne root pathogens. Plant Disease. 76, 457–461

Heffer Link, V., M.L. Powelson, and K.B. Johnson. 2002. Oomycetes. The Plant Health Instructor. Retrieved on 7 June 2016 from http://www.apsnet.org/edcenter/intropp/LabExercises/Pages/Oomycetes.aspx

Hong C.X., E.A. Bush, P.A. Richardson, E.L. Stromberg. 2001. The major deterrent to recycling irrigation water in nursery and greenhouse operations despite the lack of alternatives for limiting nonpoint source pollution. Proc Va Water Res Symp. 1, 72–77

Hong, C. X. and G.W. Moorman. 2005. Plant pathogens in irrigation water: challenges and opportunities. Critical Reviews in Plant Sciences. 24, 189-208

Hultberg, M., T. Alsberg, S. Khalil, B. Alsanius. 2010. Suppression of disease in tomato infected by Pythium ultimum with a biosurfactant produced by Pseudomonas koreensis. Biocontrol. 55, 435-444 Jeffers, S. N. and S.B. Martin. 1986. Comparison of two media selective for Phytophthora and Pythium species. Plant Disease. 70, 1038-1043 Jenkins, S.F. and C.W. Averre. 1983. Root diseases of vegetables in hydroponic culture systems in North Carolina. Plant Disease. 67, 968-970 Jinantana, J. and M. Sariah. 1998. Potential for biological control of Sclerotium root rot of chilli by Trichoderma spp. Pertanika J. Trop. Agric. Sci. 21, 1-10

Johnstone, M., Hai Yu, WeiZhong Liu, E. Leonardos, J. Sutton, and B. Grodzinski. 2004. Physiological changes associated with Pythium root rot in hydroponic lettuce. Acta Horticulturae. 635, 67-71

Koike, S. T., D.V. Tompkins, F. Martin, and M.L. Ramon. 2015. First report of Pythium root rot of fennel in California caused by Pythium sulcatum. Plant Disease. 99, 1645

Lacy G.H., R.C. Lambe, C.M. Berg. 1981. Iris soft rot caused by Erwinia chrysanthemi, associated with overhead irrigation and its control by chlorination. Comb. Proc. Int. Plant Propag. Soc. 31, 624–634

Lamour, K., S. Kamoun. 2009. Oomycete genetics and genomics: diversity, interactions and research tools. John Wiley & Sons Inc., New Jeresy. 217 pp.

Labuschagne, N., C. Gull, F.C. Wehner, W.J. Botha. 2002. Pythium spp. infecting hydroponically grown lettuce in South Africa. Plant Disease. 86, 1175

Labuchagne, N., C. Gull, F.C. Wehner, and W.J. Botha. 2002. Report of root rot caused by Pythium F-group on hydroponically grown celery in South Africa. Plant Disease. 86, 441

Labuschagne, N., C. Gull, F.C. Wehner, and W.J. Botha. 2003. Root rot and stunting of hydroponically grown endive, fennel, and sorrel caused by Pythium F-group in South Africa. Plant Disease. 87, 875

McGehee, C.S., R.E. Raudales, and W.H. Elmer. 2018. First report of Pythium dissotocum causing Pythium root rot on hydroponic lettuce in Connecticut. Plant Disease. Accepted.

Menzies, J. G., D. L. Ehret, and S. Stan. 1996. Effect of inoculum density of Pythium aphanidermatum on the growth and yield of cucumber plants grown in recirculating nutrient film culture. Canadian Journal Of Plant Pathology. 18, 50-54

Moller, K. and J. Hockenhull. 1997. Leaf and head rot of Chinese cabbage – a new field disease caused by Pythium tracheiphilum Matta. European Journal of Plant Pathology 103, 245– 249

Moorman, G.W., S. Kang, D.M. Geiser, S.H. Kim. 2002. Identification and characterization of Pythium species associated with greenhouse floral crops in Pennsylvania. Plant Disease. 86, 1227-1231

Moorman, G.W. and S. May. 2012. Module 3: Pythium Identification. Retrieved on 6 May 2016 from plantpath.psu.edu/pythium/module-3/pythium-characteristics-checklist

Owen-Going, T.N., C.W. Beninger, C.J. Hall, J.C. Sutton. 2011. Infection by Pythium aphanidermatum increases production of phenolics in hydroponically grown peppers and predisposes healthy plants to root rot. European Journal of Plant Pathology. 132, 341- 352 Pagliaccia, D., D. Ferrin, M.E. Stanghellini. 2007. Chemo-biological suppression of root- infecting zoosporic pathogens in recirculating hydroponic systems. Plant Soil. 299, 163- 167 Pal, K.K. and B. M. Gardener. 2006. Biological control of plant pathogens. The Plant Health Instructor. 10, 1094-1117 Pantelides, I., M. Tsolakidou, A. Chrysargyris, N. Tzortzakis. 2017. First report of root rot of hydroponically grown lettuce (Lactuca sativa) caused by a Pythium species from the cluster B2a species complex in Cyprus. Plant Disease. 101, 636 Parke, J.L., B.J. Knaus, V.J. Fieland, C. Lewis, N.J. Grünwald. 2014. Phytophthora community structure analysis in Oregon nurseries inform systems approaches to disease management. Phytopathology. 104, 1052-1062

Paulitz, T. C., T. Zhou, and L. Rankin. 1992. Selection of rhizosphere bacteria for biological control of Pythium aphanidermatum on hydroponically grown cucumber. Biological Control. 2, 226-237

Richard, S., Y. Zheng, M. Dixon. 2008. To recycle or not to recycle? Greenh Can. (December), 22–24

Rosa, F.B., L.A. Zottarelli, C.P., Teixeira, L. De Diana, A.S. Nelson. 2011. In vitro pathogenic evaluation of Pythium middletonii Sparrow and Pythium dissotocum Drechsler in lettuce. Summa Phytopathol. 37

Schwarza, D., U. Beuchbc, M. Bandteb, A. Fakhroab, C. Büttnerb, C. Obermeierbd. 2010. Spread and interaction of Pepino mosaic virus (PepMV) and Pythium aphanidermatum in a closed nutrient solution recirculation system: effects on tomato growth and yield. Plant Pathology. 59, 443-452

Shaner G., E.L. Stromberg, G.H. Lacy, K.R. Barker, T.P. Pirone. 1992. Nomenclature and concepts of pathogenicity and virulence. Annual Review of Phytopathology. 30, 47-66

Singleton, L.L., J.D. Mihail, C.M. Rush. 1992. Methods for research on soilborne phytopathogenic fungi. ed. St. Paul, Minnesota: The American Phytopathological Society, p. 41. Print

Stanghellini, M. E., D.H. Kim, J. Rakocy, K. Gloger and H. Klinton. 1998. First report of root rot of hydroponically grown lettuce caused by Pythium myriotylum in a commercial production facility. Plant Disease. 82, 831

Stanghellini, M.E. and W.C. Kronland. 1986. Yield loss in hydroponically grown lettuce attributed to subclinical infection of feeder rootlets by Pythium dissotocum. Plant Disease. 70, 1053-1056

Stanghellini, M.E. and S.L. Rasmussen. 1994. Hydroponics: a solution for zoosporic pathogens. Plant Disease. 78, 1129–1138

Stanghellini, M.E., S.L. Rasmussen, D.H. Kim and P.A. Rorabaugh. 1996. Efficacy of non-ionic surfactants in the control of zoospore spread of Pythium aphanidermatum in a recirculating hydroponic system. Plant Disease. 80, 422-428

Stanghellini, M.E., L.J. Stowell, M.L. Bates. 1984. Control of root rot of spinach caused by Pythium aphanidermatum in a recirculating hydroponic system by ultraviolet irradiation. Plant Disease. 68, 1075-1076

Stewart-Wade, S.M. 2011. Plant pathogens in recycled irrigation water in commercial plant nurseries and greenhouses: their detection and management. Irrig Sci. 29, 267-297

Tian, X. and Y. Zheng. 2013. Compost teas and reused nutrient solution suppress plant pathogens in vitro. HortScience. 48, 510-512

Tortolero O. and L. Sequeira. 1978. A vascular wilt and leaf blight disease of lettuce in Wisconsin caused by a new strain of Pythium tracheiphilum. Plant Disease Reporter. 62, 616–620

Uzuhashi, S., M. Tojo, M. Kakishima. 2010. Phylogeny of the genus Pythium and description of new genera. Mycoscience. 51, 337–365

Vallance, J., F. Deniel, G. Le Floch, L. Guerin-Dubrana, D. Blancard, P. Rey. 2011. Pathogenic and beneficial microorganisms in soilless cultures. ed. Gradignan, France: INRA, EDP Sciences, pp. 711-26

26 van der Gaag, D.J. and G. Wever. 2007. Conduciveness of different soilless growing media to Pythium root and crown rot of cucumber under near-commercial conditions. European Journal of Plant Pathology. 112, 31-41 van der Plaats-Niterink, A.J. 1981. Monograph of the genus Pythium. Studies in Mycology. 21, 242

Van Kuik, A.J. 1992. Spread of Phytophthora cinnamomi Rands in a recycling system. Mededelingen Faculteit Landbouwkundige en Toegepaste Biologische Wetenschappen Universiteit Gent. 57, 139–143

Whiteside J.O., T.W. Oswalt. 1973. Unusual brown rot outbreak in a Florida citrus grove following sprinkler irrigation with Phytophthora-infested water. Plant Disease. 57, 391– 393

White, T.J., T. Burns, S. Lee, J. Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Academic Press, Inc. 38, 317

Yang, Jian, P. D. Kharbanda, and M. Mirza. 2004. Evaluation of Paenibacillus polymyxa PKB1 for biocontrol of Pythium disease of cucumber in a hydroponic system. Acta Horticulturae. 635, 59-66

Zhao, ShiGuang, and S. F. Shamoun. 2006. Effects of culture media, temperature, pH, and light on growth, sporulation, germination, and bioherbicidal efficacy of Phoma exigua, a potential biological control agent for salal (Gaultheria shallon). Biocontrol Science and Technology. 16, 1043-1055

Figures A B

Figure 1. Morphological sexual structures of Pythium aphanidermatum isolated from a lettuce seedling observed under a compound microscope at 40x. (A) Hyphal swelling with sac-like antheridium structures. (B) Oogonium with a length of 23108.76 nm. A 20 µm bar was used for all images.

1 2 3 4 5 6 7 8 9

Figure 2. DNA amplification of a polymerase chain reaction amplified region of internal transcribed spacer (ITS) of nine Pythium species collected from commercial farms using universal primers ITS1 and ITS4. The last lane contains 1-kb DNA ladder (molecular weight marker), and lanes 1-9 contain the amplified DNA from selected Pythium species. Lane no. 1) P. aphanidermatum Cor4, 2) P. aphanidermatum Cor3, 3) P. aphanidermatum Kop-3, 4) P. aphanidermatum Kop-3x, 5) P. aphanidermatum Kop-6, 6) P. aphanidrmatum Kop-8, 7) P. dissotocum Cor1, 8) P. dissotocum Cor 2, and 9) P. graminicola.

Control Pythium aphanidermatum Cor4

P. aphanidermatum Kop-3x P. aphanidermatum Kop-8

Figure 3. In vitro incidence and severity test to determine the pathogenicity potential of Pythium aphanidermatum isolates on lettuce. The Pythium isolate was placed in the middle of the water agar plate with eight lettuce seeds placed 2-cm from the edge of the plate and equally distant from each other. Control with 0% disease incidence and severity. P. aphanidermatum isolates with 0-37.5% disease incidence on lettuce seedlings grown for seven days in the dark.

Kop-8Kop-8 Autoclaved Autoclaved ½ V8PDA

Radial growth Pathogen

Figure 5. Inhibition test in vitro with a 4-mm plug of Pythium dissotocum Cor1 or P. aphanidermatum Kop-8 inside a 9-mm hole filled with the water treatment (non-autoclaved recirculated nutrient solution, autoclaved deionized water, and autoclaved recirculated water) in a 35 x 10-mm Petri-dish grown on V8 juice and ½ PDA agar (n=6, experimental unit=Petri-dish with water treatment in 9-mm hole). Images represent mycelial growth after two days of incubation. The results were converted into percentage inhibition of radial growth in relation to the radial growth of the pathogen in the control plate for day two (Jinantana and Sariah, 1998,

Tian and Zheng, 2013).

A 120 a 100 a 80 b 60 40

20 c Disease Incidence, % Incidence, Disease 0

P. dissotocum P. aphanidermatum P. graminicola Control Cor1 Cor4

B 3.5 a a 3 b 2.5 2 1.5 1 0.5 c

Disease Severity Rating Severity Disease 0

P. dissotocum P. aphanidermatum P. graminicola Control Cor1 Cor4

Figure 6. Experiment 1. (A) Disease incidence and (B) severity on lettuce seedlings caused by Pythium spp. (n=6, experimental unit=Petri-dish with 8 seeds) in vitro. Severity rating was based on scale of 0 to 3, where 0 represented 100% germination with a healthy appearance, 1 represented 99 to 70% germination, small brown lesions on the radicle or hypocotyl, 2 represented 69 to 30% germination, large brown lesions on the hypocotyl and radicle, and 3 was 29 to 0% germination, coalesced lesions covering the hypocotyl and radicle (Broders et al., 2007; Del Castillo Munera and Hausbeck, 2016). Means with the same letters are not significantly different according to Tukey’s HSD test at α=0.05. Bars represent standard error.

A 90 a 80 ab 70 ab 60 50 b 40 30

Disease Incidence, % Incidence, Disease 20 c 10 0 Kop-3 Kop-3x Kop-6 Kop-8 Control

B 3.5 a 3 a a 2.5 a 2

1.5

Disease Severity Severity Disease 1

0.5 c

0 Kop-3 Kop-3x Kop-6 Kop-8 Control

Figure 7. Experiment 2, first experimental run. (A) Disease incidence and (B) severity on lettuce seedlings caused by selected Pythium aphanidermatum isolates (n=6, experimental unit=Petri-dish with 8 seeds) in vitro. Severity rating was based on scale of 0 to 3, where 0 represented 100% germination with a healthy appearance, 1 represented 99 to 70% germination, small brown lesions on the radicle or hypocotyl, 2 represented 69 to 30% germination, large brown lesions on the hypocotyl and radicle, and 3 was 29 to 0% germination, coalesced lesions covering the hypocotyl and radicle (Broders et al., 2007; Del Castillo Munera and Hausbeck, 2016). Means with the same letters are not significantly different according to Tukey’s HSD test at α=0.05. Bars represent standard error.

A 120 a ab 100 ab b 80

40 Disease Incidence, % Incidence, Disease 20 c

0 Kop-3 Kop-3x Kop-6 Kop-8 Control

B 3.5 a a a 3 a

2.5

1.5

Disease Severity Disease 1

0.5 c

0 Kop-3 Kop-3x Kop-6 Kop-8 Control

Figure 8. Experiment 2, second experimental run. (A) Disease incidence and (B) severity on lettuce seedlings caused by selected Pythium aphanidermatum isolates (n=6, experimental unit=Petri-dish with 8 seeds) in vitro. Severity rating was based on scale of 0 to 3, where 0 represented 100% germination with a healthy appearance, 1 represented 99 to 70% germination, small brown lesions on the radicle or hypocotyl, 2 represented 69 to 30% germination, large brown lesions on the hypocotyl and radicle, and 3 was 29 to 0% germination, coalesced lesions covering the hypocotyl and radicle (Broders et al., 2007; Del Castillo Munera and Hausbeck, 2016). Means with the same letters are not significantly different according to Tukey’s HSD test at α=0.05. Bars represent standard error.

A B

1 2 3 4

Figure 9. DNA amplification of the (A) internal transcribed spacer (ITS) and (B) region of ribosomal RNA (16S) of four samples of recirculated nutrient solution from a commercial hydroponic lettuce reservoir using universal primers ITS1 and ITS4. The last lane contains 1-kb plus DNA ladder, and lanes 1-4 contain the amplified DNA from the recirculated water.

Tables

Table 1. Summary of published reports of Pythium spp. affecting crop yield in hydroponic systems up to 2017.

Crop Pythium species Crop loss Reference Lettuce P. aphanidermatum 41% reduction in shoot mass was observed in a floating Corrêa et al., 2010 system after 18 days of inoculation. P. dissotocum 23% severe stunting and poor growth in deep flow Pantelides et al., 2017 technique system. 35-54% crop yield reduction at 18°C and 12-17% crop Stanghellini and Kronland, reduction at 28 °C (92% of roots infected). 1986 35-40% growth reduction in leaf area and dry shoot weight Johnstone et al., 2004 7 days after inoculation. P. irregulare* 51% and 38% reduction of fresh shoot and root weight, Labuschagne et al., 2002 respectively. P. myriotylum 50% plant mortality of 30,000 plants died. Stanghellini et al., 1998 Cucumber P. aphanidermatum 100% mortality 63 days after inoculation. Stanghellini et al., 1996 100% mortality 32 days after inoculation. Pagliaccia et al., 2007 20% reduction in root mass 7 days after inoculation. Van der Gaag and Gerrit Wever, 2007 100% of plants were dead 10 days after inoculation. Favrin et al., 1988 50% of plants were dead 10 days after inoculation. Chérif et al., 1994 100% of plants were dead 9 days after inoculation. Jenkins and Averre, 1983 100% of plants were dead 28 days after inoculation. Menzies et al., 1996 67% reduction of root length. Paulitz et al., 1992 P. irregulare 65% of plants were dead 14 days after inoculation. Favrin et al., 1988 P. debaryanum 100% of plants were dead 14 days after inoculation. Jenkins and Averre, 1983 P. ultimum* 100% of plants were dead 22 days after inoculation. Jenkins and Averre, 1983 20.3% reduction in yield (g/plant). Yang et al., 2004 P. myriotylum 100% of plants were dead 18 days after inoculation. Jenkins and Averre, 1983 Pepper P. aphanidermatum 60% reduction of root mass after 10 days of inoculation. Owen-Going et al., 2011 Continued…

Continued from Table 1…

Table 1. Summary of published reports of Pythium spp. affecting crop yield in hydroponic systems up to 2017. Spinach P. aphanidermatum 97% of seedlings (2 weeks old) were dead 24 hours after Stanghellini et al., 1984 inoculation. 100% of plants were dead 2 days after inoculation at 30 °C. Bates and Stanghellini, 1984 100% of plants were stunted 10 days after inoculation at 20 °C. P. dissotocum 69% of plants were stunted 10 days after inoculation at 20 Bates and Stanghellini, 1984 °C. 100% of plants were stunted 5 days after inoculation at 30 °C. Tomato P. aphanidermatum 100% of plants were dead 9 days after inoculation. Jenkins and Averre, 1983 11% reduction in fruit weight. Schwarza et al., 2010

P. debaryanum 100% of plants were dead 14 days after inoculation. Jenkins and Averre, 1983 P. myriotylum 100% of plants were dead 18 days after inoculation. Jenkins and Averre, 1983 P. ultimum 100% of plants were dead 22 days after inoculation. Jenkins and Averre, 1983 31.8% reduction of fresh root weight and 10.5% reduction Hultberg et al., 2010 of fresh shoot weight. Celery Pythium F-group 70% of plants were stunted or dead after inoculation. Labuchagne et al., 2002 Endive, Fennel, Pythium F-group 40% Endive, 60% Fennel, and 7% Sorrel were stunted and Labuchagne et al., 2003 and Sorrel had root rot. Fennel P. sulcatum Disease incidence ranged from <1 to 15%. Koike et al., 2015 Tobacco P. myriotylum 100% root necrosis 21 days after inoculation at 15-30 °C. Fortnum et al., 2000 *As of 2010 P. ultimum and P. irregulare have been taxonomically assigned to the genus Globisporangium (Uzuhashi et al., 2010).

Table 2. Pythium spp. isolated from plants obtained from commercial greenhouses in

Connecticut and New York. The pathogens were identified by sequencing the internal transcribed region (ITS) region ITS1 and ITS4.

Pythium species (isolate) Crop Location Identity Match Accession (ITS1/ITS4)1 Number2 P. aphanidermatum (Cor4) Poinsettia Long Island, NY 99% KY381579.1 MG993551

P. aphanidermatum (Cor3) Mustard Brooklyn, NY 99% KY381579.1 MG993549

P. aphanidermatum (Kop-3) Mizuna Long Island, NY 99% KY381579.1 MG993545

P. aphanidermatum (Kop-3x) Mizuna Long Island, NY 99% KY381579.1 MG993550

P. aphanidermatum (Kop-6) Swisschard Long Island, NY 99% JN695785.1 MG993544

P. aphanidermatum (Kop-8) Wheatgrass Long Island, NY 99% KY381579.1 MG993547 seeds P. dissotocum (Cor1) Lettuce East Hartford, CT 99% KM061702.1 MG993548

P. dissotocum (Cor2) Lettuce Cheshire, CT 99% KM061701.1 MG993546

P. graminicola (P 1105-15) Turfgrass Storrs, CT 91% LC160346.1 MG993552

1 BLAST analysis sequence showed 91-99% similarity to the ITS gene region ITS1 and ITS4 of the isolates under the accession numbers in this column based on the GenBank database.

2 Sequences of the isolates obtained in this project were deposited in GenBank under the accession numbers in this column.

Table 3. Tests of significance for pathogen, treatment, or media for studies on inhibition of

Pythium aphanidermatum isolate Kop-8 and Pythium dissotocum isolate Cor1 (Pythium) grown on V8 juice agar and ½ potato dextrose agar (PDA) (Media) and exposed to water treatment

(Water Treatment) on day five (n=6). This ANOVA table represents data from experiment one

(run I and II) and experiment two (combined runs). P-value represents the null hypothesis of equal means. If the p-value is less than 0.05 the means between the treatments are significant.

Experiment 1 Experiment 2 Run I Run II Combined runs Effect P-value (Pr>F) P-value (Pr>F) P-value (Pr>F) Water Treatment (Wt) <.0001 <.0001 <.0001 Pythium (P) 0.0071 <.0001 <.0001 Wt*P 0.0188 <.0001 0.0148 Media (M) 0.0016 0.1978 <.0001 Wt*M 0.0728 0.0095 <.0001 M*P 0.0194 0.2390 0.2758 Wt*M*P 0.0075 0.0143 0.0045

Table 4. Experiment 1, run one and two. Inhibition of Pythium aphanidermatum isolate Kop-8 and Pythium dissotocum isolate Cor1 on V8 juice agar and ½ potato dextrose agar (PDA) based on water treatment on day five (n=6, experimental unit=Petri-dish with water treatment in PVC ring). Pathogen growth was monitored daily for five days by measuring the radius of the mycelia from the edge of the colonies in the direction toward the solution ring. The results were converted into percentage inhibition of radial growth in relation to the radial growth of the pathogen in the control plate for day five (Jinantana and Sariah, 1998, Tian and Zheng, 2013).

Pythium species Water Treatments Media Inhibition %a Run I Run II P. aphanidermatum Nonautoclaved V8 24.8 a 27.4 a P. dissotocum Nonautoclaved V8 22.2 a 27.1 a P. aphanidermatum Autoclaved V8 21.6 a 8.6 b P. aphanidermatum Nonautoclaved ½ PDA 17.2 a 2.5 c P. dissotocum Nonautoclaved ½ PDA 15.1 ab 1.6 c P. dissotocum Autoclaved ½ PDA 3.1 bc 1.3 c P. aphanidermatum Autoclaved ½ PDA 2.8 bc 1.3 c P. dissotocum Autoclaved V8 0.2 c 0.3 c P. dissotocum DI Water ½ PDA 0.0 c 0.0 c P. dissotocum DI Water V8 0.0 c 0.0 c P. aphanidermatum DI Water ½ PDA 0.0 c 0.0 c P. aphanidermatum DI Water V8 0.0 c 0.0 c a Experimental runs are presented separately. Means within a column for each weight measurement followed by the same letter are not significantly different according to Tukey’s Honestly Significant Difference (HSD) separation test (α=0.05).

Table 5. Experiment 2, combined runs. Inhibition of Pythium aphanidermatum isolate Kop-8 and Pythium dissotocum isolate Cor1 on V8 juice agar and ½ potato dextrose agar (PDA) based on water treatment on day two (n=6, experimental unit=Petri-dish with water treatment and

Pythium sp. agar plug in the middle of the Petri-dish). Pathogen growth was monitored daily for two days by measuring the radius of the mycelia from the edge of the colonies in the direction toward the edge of the plate. The results were converted into percentage inhibition of radial growth in relation to the radial growth of the pathogen in the control plate for day two (Jinantana and Sariah, 1998, Tian and Zheng, 2013).

Pythium species Water Treatments Media Inhibition %a P. aphanidermatum Nonautoclaved V8 15.0 a P. dissotocum Nonautoclaved V8 15.0 a P. aphanidermatum Autoclaved V8 14.6 a P. aphanidermatum Nonautoclaved ½ PDA 8.8 ab P. dissotocum Autoclaved V8 7.6 b P. aphanidermatum Autoclaved ½ PDA 3.3 bc P. dissotocum Autoclaved ½ PDA 0.0 c P. dissotocum Nonautoclaved ½ PDA 0.0 c P. dissotocum DI Water ½ PDA 0.0 c P. dissotocum DI Water V8 0.0 c P. aphanidermatum DI Water ½ PDA 0.0 c P. aphanidermatum DI Water V8 0.0 c aMeans within a column for each weight measurement followed by the same letter are not significantly different according to Tukey’s Honestly Significant Difference (HSD) separation test (α=0.05).

Annex ANNEX I: Agar Media used to isolate Pythium spp.

Water Agar (Source: Jeffers and Martin, 1986) 1. Add 22 g of granulated agar (Difco Laboratories, MD) to 1 liter of deionized water. 2. Autoclave at 121°C for 30 minutes. 3. Let cool to room temperature and pour into Petri-dish.

PARP (pimarcin, ampicillin, rifampicin, and pentachloronitrobenzene) (Source: Jeffers and Martin, 1986) 1. Add 17 g of corn meal agar (CMA; Difco Laboratories, MD) to 1 liter of deionized water. 2. Autoclave at 121°C for 30 minutes. 3. Let cool to room temperature, or put media in a water bath at 10°C (50°F). 4. Once media is cool, add 5 mg pimaricin or 10 mg of Delvocid (50% pimaricin), 250 mg ampicillin, 10 mg rifampicin, and 100 mg PCNB or 133.3 mg Terraclor 75WP (75% PCNB). 5. Pour into Petri-dish.

PARP V8 (Source: Jeffers and Martin, 1986; Jennifer Parke, 2011) 1. Prepare clarified 20% V8 agar by centrifuging V8 juice in 50 mL tubes (Falcon, USA) at 3200 rpm for 15 minutes. Pipette the liquid supernatant from the tube and place in a glass container until 200 mL are obtained. 2. Combine 200 mL of clarified V8, 2 g CaCO3, and 15 g of granulated agar to 800 mL of deionized water. 3. Autoclave at 121 °C for 30 minutes. 4. Let cool to room temperature, or put media in a water bath at 10°C (50°F). 5. Once media is cool, add 5 mg pimaricin or 10 mg of Delvocid (50% pimaricin), 250 mg ampicillin, 10 mg rifampicin, and 100 mg PCNB or 133.3 mg Terraclor 75WP (75% PCNB).

ANNEX II: Pythium spp. Identification

Morphological identification of Pythium species involves inducing the sexual structures of the organism in order to have diagnostic characteristics. Oospores and sporangial structures were identified based on van der Plaats-Nietnak (1981) using keys and monographs.

Protocol based on Robert Wick with modifications from Moorman and May, 2012. 1. Grow Pythium isolate out on water agar for three days. 2. Autoclave tall fescue leaves for 15 minutes at 121°C. Let cool for 30 minutes. 3. Cut autoclaved fescue leaves into 2.54-cm pieces. 4. Place four fescue leaves into an empty 8-cm Petri-dish. 5. Cut two 5-mm pieces of the Pythium isolate out with a core borer from the water agar and place them in-between the fescue leaf pieces. 6. Place 8 mL of sterile deionized water into the Petri-dish. 7. Incubate the Petri-dish in room temperature (~22 °C) in constant light for 3-4 days.

ANNEX III: DNA extraction

DNA extraction (DNeasy Plant Mini Kit)

1. Disrupt root samples with liquid N2 to a fine powder (≤100 mg wet weight) using the TissueLyser II. 2. Add 400 μL Buffer AP1 and 4 μL RNase A. 3. Vortex and incubate for 10 min at 65°C. Invert the tube 2–3 times during incubation. 4. Add 130 μL Buffer P3. Mix and incubate for 5 min on ice. 5. Centrifuge the lysate for 5 min at 20,000 x g (14,000 rpm). 6. Pipet the lysate into a QIAshredder spin column placed in a 2 mL collection tube. Centrifuge for 2 min at 20,000 x g. 7. Transfer the flow-through into a new tube without disturbing the pellet if present. Add 1.5 volumes of Buffer AW1, and mix by pipetting. 8. Transfer 650 μL of the mixture into a DNeasy Mini spin column placed in a 2 mL collection tube. Centrifuge for 1 min at ≥6000 x g (≥8000 rpm). Discard the flow- through. Repeat this step with the remaining sample. 9. Place the spin column into a new 2 mL collection tube. Add 500 μL Buffer AW2, and centrifuge for 1 min at ≥6000 x g. Discard the flowthrough. 10. Add another 500 μL Buffer AW2. Centrifuge for 2 min at 20,000 x g. Note: Remove the spin column from the collection tube carefully so that the column does not come into contact with the flow-through. 11. Transfer the spin column to a new 1.5 mL or 2 mL microcentrifuge tube. 12. Add 50 μL Buffer AE for elution. Incubate for 5 min at room temperature (15–25°C). Centrifuge for 1 min at ≥6000 x g.

DNA extraction (PowerWater Pro DNA extraction kit) 1. Filter 100 mL of water samples using a 0.45 µm disposable filter funnel attached to a vacuum source. 2. Remove the 100 mL upper portion of the filter cup of the filter funnel from the catch reservoir by snapping it off. 3. Using two sets of sterile forceps, pick up the white filter membrane at opposite edges and roll the filter into a cylinder with the top side facing inward. Note: Do not tightly roll or fold the filter membrane. 4. Insert the filter into the 5 mL PowerWater® Bead Tube. 5. Add 1 mL of Solution PW1 to the PowerWater® Bead Tube. Note: Solution PW1 must be warmed to dissolve precipitates prior to use. Solution PW1 should be used while still warm. For samples containing organisms that are difficult to lyse (fungi, algae) an additional heating step can be included. 6. Secure the PowerWater® Bead Tube horizontally to a MO BIO Vortex Adapter. 7. Vortex at maximum speed for 5 minutes. 8. Centrifuge the tubes ≤ 4000 x g for 1 minute at room temperature. The speed will depend on the capability of your centrifuge.

9. Transfer all the supernatant to a clean 2 mL Collection Tube. Draw up the supernatant using a 1 mL pipette tip by placing it down into the beads. Note: Placing the pipette tip down into the beads is required. Pipette more than once to ensure removal of all supernatant. Any carryover of beads will not affect subsequent steps. Expect to recover between 600-650 µL of supernatant depending on the type of filter membrane used. 10. Centrifuge at 13,000 x g for 1 minute. 11. Avoiding the pellet, transfer the supernatant to a clean 2 mL Collection Tube. 12. Add 200 µL of Solution PW2 and vortex briefly to mix. Incubate at 4°C for 5 minutes. 13. Centrifuge the tubes at 13,000 x g for 1 minute. 14. Avoiding the pellet, transfer the supernatant to a clean 2 mL Collection Tube. 15. Add 650 µL of Solution PW3 and vortex briefly to mix. Note: Check Solution PW3 for precipitation prior to use. Warm if necessary. Solution PW3 can be used while still warm. 16. Load 650 µL of supernatant onto a Spin Filter and centrifuge at 13,000 x g for 1 minute. Discard the flow through and repeat until all the supernatant has been loaded onto the Spin Filter. Note: A total of two loads for each sample processed are required. 17. Place the Spin Filter basket into a clean 2 mL Collection Tube. 18. Shake to mix Solution PW4 before use. Add 650 µL of Solution PW4 and centrifuge at 13,000 x g for 1 minute. 19. Discard the flow through and add 650 µL of Solution PW5 and centrifuge at 13,000 x g for 1 minute. 20. Discard the flow through and centrifuge again at 13,000 x g for 2 minutes to remove residual wash. 21. Place the Spin Filter basket into a clean 2 mL Collection Tube. 22. Add 50 µL of Solution PW6 to the center of the white filter membrane. 23. Centrifuge at 13,000 x g for 1 minute. 24. Discard the Spin Filter basket. The DNA is now ready for any downstream application. Store DNA at -20° C.

Chapter 3

Efficacy of Microbial Biofungicides on Suppressing Pythium Root Rot on Hydroponic Lettuce (Lactuca sativa L)

Abstract

Pythium spp., causal agents of Pythium root rot, are accountable for lettuce yield losses in hydroponic production systems. Synthetic chemical control options are not registered for application in hydroponic solutions in the US. The objective of this project was to assess the efficacy of microbial biofungicides on root rot caused by Pythium aphanidermatum and P. dissotocum in lettuce (Lactuca sativa cv. Rex and Spretnak) seedlings. In the first experiment, seven-day old seedlings were treated with Companion® (Bacillus subtilis GB03), RootShield

Plus® (Trichoderma harzianum KRL-AG2 and Trichoderma virens G-41), Triathlon BA®

(Bacillus amyloliquefaciens D747), and Cease® (Bacillus subtilis QST 713). Two days later, the plants were inoculated with 1 x 105 zoospores of Pythium spp. Fourteen days after inoculation, we measured root necrosis, disease incidence and severity, fresh and dry shoot, and root weight.

Lettuce seedlings inoculated with Pythium spp. had 74% lower shoot and 80% lower root dry weight, and higher disease severity compared with non-inoculated plants. All plants treated with microbial biological fungicides and inoculated with Pythium spp. had higher shoot and root weight compared to the untreated control with Pythium spp. The shoot and root weight of lettuce seedlings treated with Cease®, Companion®, and RootShield Plus® did not differ statistically with or without Pythium spp. In the second experiment, Companion®, Actinovate®, and Terra

Gro® were evaluated in hydroponic lettuce trials. Companion® with P. aphanidermatum Cor4 exhibited 23.2 and 77.1% higher shoot weight than the inoculated control, respectively. Results from this study suggest that microbial biofungicides can be introduced in nutrient solutions to ameliorate damage caused by Pythium root rot. However, in the absence of Pythium root rot, the

47 microbial biofungicides also reduced plant biomass. Further research is required to identify if alternative application methods of microbial biofungicides can reduce the negative effects that microbial biofungicides have on plant growth.

Introduction

Biological control of plant diseases in greenhouses has increased because of the ease of application in controlled environments, high value of crops, and limited number of registered fungicides. Synthetic chemical fungicides for direct use in hydroponic lettuce have not been registered in the USA (EPA, 2017). They potentially cause phytotoxicity in hydroponically grown crops (Utkhede et al., 2000), and there is an increasing public concern regarding the use of synthetic chemical pesticides in water regarding the potential runoff into the environment.

Microbial biological fungicides have been evaluated in some crops as a method to control

Pythium root rot in hydroponic systems (Table 1). Biofungicides are now widely used in the ornamental greenhouse industry. Many growing media companies have amended their mixes with microbial biofungicides as a standard practice.

In plant pathology, a biological control agent refers to the “Purposeful utilization of introduced or resident living organisms, other than disease resistant host plants, to suppress the activities and populations of one or more plant pathogens” (Pal and Gardener, 2006). The mechanisms for biological controls include antibiosis, competition, mycoparasitism, cell wall degrading enzymes, and induced resistance (Paulitz and Bélanger, 2001). There are currently eleven commercial microbial biological fungicides registered against Pythium root rot in the state of

Connecticut (Pundt and Smith, 2017).

Soilless media is usually pasteurized and microbial populations that compete with pathogens are low, resulting in conditions that may favor root pathogens like Pythium spp. (Paulitz and

Bélanger, 2001). Pseudomonas, Trichoderma, Bacillus, and Gliocladium have been tested for control of P. aphanidermatum in cucumber (Punja and Yip, 2003; Rankin and Paulitz, 1994;

Utkhede and Koch, 1999). However, efficacy of these microbial biofungicides on hydroponic lettuce is not highly accessible. Disease resistant varieties of lettuce against root rot for greenhouse production are also not available. Therefore, biological control, in combination with environmental control, is a tool that can be used to prevent disease developments.

Our objective was to evaluate the effect of microbial biofungicides on the disease incidence and severity of Pythium root rot caused by P. aphanidermatum and P. dissotocum in lettuce seedlings and hydroponic lettuce.

Materials and Methods

Experiment 1.

Seedling Experiment. Lettuce (Lactuca sativa cv. Spretnak and Rex) (Johnny’s Selected Seeds,

ME) were sown in 42-mm peat pellets with one seed per pellet. Five pellets of each species per tray were maintained in 50 black, 27.9 x 54.3-cm plastic trays (Griffin Greenhouse Supplies, CT) on the greenhouse bench under a high pressure sodium light for 14 days. The experimental unit consisted of a peat pellet with one lettuce seedling. Each tray had 1 L of nutrient solution containing 5-12-26 at 31.2 mg.L-1 N and 15.5-0-0 at 88.8 mg.L-1 N. The experiment took place in a polycarbonate greenhouse with a heating set point of 18.3 °C and a ventilation set point of 26.7

°C under natural photoperiod in September and October 2017 in Storrs, CT.

The microbial biofungicides included Companion® (Bacillus subtilis GB03), Triathlon BA®

(Bacillus amyloliquefaciens D747), RootShield Plus® (Trichoderma harzianum KRL-AG2 and

Trichoderma virens G-41), and Cease® (Bacillus subtilis QST 713). These commercial products were applied following the rate and method described in the specimen label (Table 2).

Companion® and RootShield Plus® were applied directly in the nutrient solution and Triathlon

BA® was applied directly in the peat pellet (Table 2). Two days after amending the tanks with microbial biofungicides, 10 mL of P. aphanidermatum Cor4, P. aphanidermatum Kop-8, and P. dissotocum Cor1 at 1 x 105 zoospores per mL were applied directly in each peat pellet (zoospore preparation described below). The negative control consisted of applying 10 mL of sterile deionized water. All applications were made only once.

Zoospores were induced by following the protocol described by Martin (1992). All three

Pythium isolates were cultured on V8 juice agar (200 mL V8 juice, 2 g CaCO3, 2 g granulated agar, and 800 mL deionized water, autoclaved for 15 min at 121 °C). Five 4-mm plugs of three- day old Pythium sp. were transferred to an empty Petri dish, filled with 20 mL of V8-juice broth

(200 mL clarified V8 juice, 2 g CaCO3, and 800 mL DI water, autoclaved for 15 min at 121 °C), then covered with aluminum foil, and stored in a dark drawer for five days. The mycelial mats were rinsed three times with sterile pond water (autoclaved for 15 min at 121 °C). The Petri dishes were then filled with 20 mL sterile deionized water (autoclaved for 15 min at 121 °C), then placed under fluorescent light for 24 hours. The presence of sporangia was visually confirmed. The deionized water was drained into a separate flask and the the Petri dish was refilled with 20 mL of chilled (4 °C) sterile deionized water, refrigerated at 4 °C, and after two hours they were set at 22-26°C for one to two hours under light. A hemocytometer was used to count zoospores in 10 µL of each Petri dish solution, out of 57 subsamples. Each peat pellet

(Jiffy, Netherlands) was inoculated with 10 mL of 1 x 105 per mL (Heungens and Parke, 2000).

The plants were harvested and measured 14 days after pathogen infestation. The measurements included disease incidence and severity, root necrosis, stand counts, and fresh shoot and root biomass. Disease incidence was measured as percentage plants with visual wilting and damping-

50 off per peat pellet. The scale for disease severity was rated according to the following scale:

1=symptomless, 2=emerged but wilted, 3=post-emergent damping-off, and 4=pre-emergent damping-off (Boehm et. al., 1993). Each value represents the average severity rating of standing plants per peat pellet. Root disease was rated based on visual presence or absence of root necrosis and rot. Shoots were cut at the soil line and shoots and roots were weighed fresh and dry

(dried at 21.1 °C for two weeks).

The experiment was a full factorial arranged as a complete randomized design (CRD).

The factors were plant cultivar, biofungicides, and Pythium inoculation. The experimental unit consisted of a peat pellet with one lettuce seedling, there were five replicates per tray, and there were a total of five trays per treatment (n=25). The trays were randomly distributed in the greenhouse. Data were analyzed by analysis of variance (ANOVA) with SAS 9.4 (SAS Institute

Inc., NC) to establish significance of the effects of all factors (α=0.05). Means were separated by

Tukey’s studentized range HSD (Honestly Significant Difference) separation test (α=0.05) at the

95% confidence interval using PROC MIXED. Severity ratings were analyzed with non- parametric analysis using the RANK and PROC GLM procedure. Percentage of root necrosis was calculated by averaging all the yes’s and no’s per treatment and analyzed with Chi-Square.

Experiment 2.

Hydroponic Experiment. The hydroponic system was a nutrient film technique (NFT) system,

5.08-cm inches deep with six 2.54-cm square holes per channel. Lettuce ‘Rex’ plants were sown in one-squared-inch oasis cubes (CropKing Inc., OH). Each nutrient solution tank constituted a treatment. Each tank had 25 liters of nutrient solution containing 16-4-17 (JR Peters Inc., PA) at

150 mg.L-1 N. Plants were grown in a polycarbonate greenhouse with a heating set point of 21 °C

51 and a ventilation set point of 26.7 °C during the day and 24 °C at night under natural photoperiod during March through May 2015 in Storrs, CT.

Companion® (Bacillus subtilis GB03), Actinovate® (Streptomyces lydicus WYEC 108), and

TerraGrow® (Bacillus licheniformis, Bacillus subtilis, Bacillus pumilus, Bacillus amyloliquefaciens, Bacillus megaterium, Trichoderma harzianum, Trichoderma reesei) were applied directly in the nutrient solution following the specimen label instructions at 0.75 mL.L-1 for Actinovate® and 0.06 mL.L-1 for TerraGrow® and other microbial biofungicides were applied at rates previously mentioned in table 2.

P. aphanidermatum Cor4 was grown on Corn Meal Agar (CMA, Difco) for four days and a 1- cm2 piece of agar was placed at the top of each NFT channel one week after the plants were amended with microbial biofungicides.

Daily measurements of the nutrient solution consisted of pH, electrical conductivity (EC), dissolved oxygen (DO), and temperature (°C) measured with specific probes for the Orion Star

A329 Portable Meter (Thermo Fisher Scientific Inc., MA). Fresh shoot and root weight were measured at harvest.

The experiment was a full factorial in a complete randomized block design (CRBD). The blocks consisted of an individual table in the greenhouse and there were four tables, one lettuce plant consisted of an experimental unit, and there were six plants per block (n=24). The experiment was conducted twice. Statistical analysis was conducted using SAS Version 9.4 (SAS Institute

Inc., NC) to establish significance of the effects of all factors (α=0.05). Data were statistically analyzed by analysis of variance (ANOVA). Means were separated by Tukey’s studentized range

HSD (Honestly Significant Difference) separation test (α=0.05) using PROC MIXED.

Results

Experiment 1.

Seedling Experiment. Data were analyzed together for both experimental runs due to homogeneity among repeated experimental runs (P < 0.05). Interactions between all three factors

(biofungicides, cultivar, and Pythium inoculation) were significant except for disease incidence and severity (Table 3).

Lettuce cultivars ‘Rex’ and ‘Spretnak’ treated with no pathogen and no microbial biofungicides consistently resulted in higher dry shoot and root weight than all other treatments (Figure 1).

Lettuce ‘Spretnak’ inoculated with Pythium spp. resulted in a higher disease incidence and severity in the shoots than any other treatment (Table 4). Lettuce ‘Rex’ and ‘Spretnak’ inoculated with Pythium spp. had lower shoot and root dry weight greater by 74% than non- inoculated plants, respectively. All plants treated with microbial biofungicides and inoculated with Pythium species resulted in higher dry weight compared to the inoculated control. Lettuce varieties treated with Cease®, Companion®, and RootShield Plus® consistently resulted in the same dry shoot and root weight with and without Pythium spp. (Figure 1). Triathlon BA® did not differ in dry weight in ‘Rex’ regardless of the presence or absence of Pythium spp. Lettuce ‘Rex’ and ‘Spretnak’ inoculated with Pythium spp. resulted in higher disease severity compared to the non-inoculated control. Root necrosis was 100% for all lettuce species inoculated with Pythium spp. alone. Lettuce ‘Rex’ treated with Cease® reduced root necrosis by 10% compared to the inoculated control and other treatments (Table 4).

Experiment 2.

Hydroponic Experiment. Differences were observed by experimental run, therefore the results of this experiment are presented separately. Interactions between biofungicide and Pythium were

53 statistically significant for run one and two with p-values 0.0219 and <0.0001. In experimental run one, lettuce treated with TerraGrow® resulted in 18.7% more shoot weight compared to the control and was significantly greater than the positive control and Actinovate® (Figure 2A).

Lettuce shoots treated with Companion® amended with and without P. aphanidermatum was not significantly different from each other. Treatments with Actinovate® and the controls treated with and without P. aphanidermatum showed no significant difference in shoot weight. In experimental run two, lettuce amended with Companion® inoculated with and without P. aphanidermatum resulted in 16.7% more shoot weight than the positive and negative control

(Figure 2B). Lettuce grown with Actinovate® inoculated with and without the pathogen were not statistically different from each other but both were higher than the inoculated control.

TerraGrow® was only evaluated during the first run of this experiment and may be repeated for future studies.

In a third experiment conducted evaluating microbial biofungicides on hydroponic lettuce

(methods not shown), fresh shoot and root weights of lettuce treated with and without P. aphanidermatum were not significantly different from each other in both runs (Figure 3).

Interactions between biofungicide and Pythium were significant according to the ANOVA table for experimental run one and two with a p-value of <.0001 and 0.0061. However, DNA was extracted from plant roots of each treatment, and polymerase chain reaction (PCR) was conducted and showed no presence of P. aphanidermatum in the plant roots of all treatments.

RootShield® and Companion® treatments exhibited a greater pH and dissolved oxygen concentration than other treatments (data not shown). RootShield® resulted in the highest nitrate concentration than any other treatments (Table 5).

Discussion

There have been several studies showing that biological fungicides can control disease caused by Pythium species (Table 1). The mechanisms of action are known to vary by species, strain and cultivation medium (Akpa et al., 2001). Some strains of Bacillus subtilis can secrete antibiotics which can directly inhibit the growth of fungal, oomycete, and bacterial plant pathogens (Asaka and Shoda, 1996; Gebhardt et al., 2002; Joshi and Gardener, 2005; McKeen et al., 1986; Toure et al., 2004). In this study, B. subtilis exhibited inhibition against Pythium species in lettuce but the exact biological mechanisms are still uncertain.

Some biological control agents have been reported to cause damage to plants without disease present. In preliminary trials, plants treated with Actinovate® had lower fresh shoot and root biomass by 75% and 52.3%, respectively, without the influence of P. aphanidermatum.

Actinovate® clogged the NFT system on day two, four, and seven after transplanting, and consistently clogged the system once reapplied. Other studies have shown that Streptomyces lydicus products such as Actino-Iron® predisposed plants to disease and reduced biomass

(Pasura, 2008). For example, Trichoderma psudokoningii strain To10 was used to treat Pythium ultimum in pea and resulted in damaged root systems and an overall decrease in root weight

(Naseby et al., 2000). Current research suggests that plants are able to influence the composition and activation of their rhizosphere microbiome through exudation of compounds which allows plant roots to select for specific microorganisms to thrive in the rhizosphere (Berendsen et al.,

2012). Bacillus subtilis secretes surfactin and forms a stable and extensive biofilm when it colonizes a root system which has been shown to be crucial for disease suppression (Bais et al.,

2004).

Lettuce seedlings showed greater susceptibility to Pythium spp. than mature plants in hydroponics systems. These experiments indicate that cultivars can be susceptible at the seedling

55 stage, but the effect may not sustain all the way to harvest. There is still further research to be done on the complex interactions among plants, plant pathogens, and beneficial microbes.

Conclusion

Growers facing Pythium root rot would benefit of microbial biological fungicides as a preventative in lettuce seedlings since there are still currently no synthetic chemical fungicides registered against this disease for hydroponic lettuce. This current study has shown that microbial biofungicides reduce the shoot and root weight of lettuce seedlings. Lettuce seedlings treated with microbial biofungicides also resulted in higher shoot and root weight than the positive control suggesting suppressive potential against Pythium root rot. Growers are less likely to get reduced growth and root necrosis caused by Pythium spp. when applying products with Bacillus spp. as a preventative depending on the lettuce variety. Growers should start with small trials of these microbial biofungicides to see how fungicide rates, the crop, and environmental conditions alter the efficacy of these products. More research needs to be conducted on how environmental conditions have an effect on disease severity and infection while using microbial biofungicides.

Literature Cited

Ahanger, R.A., H.A. Bhatand, N.A. Dar. 2014. Biological agents and their mechanism in plant disease management. Sciencia Acta Xaveriana. 5, 47-58

Akpa, E., P. Jacques, B. Wathlet, M. Paquot, R. Fuchs, H. Budzikiewiez, P. Thonart. 2001. Influence of culture conditions on lipopeptide production by Bacillus subtilis. Appl. Biochem. Biotechnol. 91-93, 551-561

Asaka, O., M. Shoda. 1996. Biocontrol of Rhizoctonia solani damping off of tomato with Bacillus subtilis RB14. Appl. Environ. Microbiol. 62, 4081-4085

Bais, H.P., R. Fall, J.M. Vivanco. 2004. Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production. Plant Physiol. 134, 307-319.

Berendsen, R. L., C. M. J. Pieterse, and P. A. H. M. Bakker. 2012. The rhizosphere microbiome and plant health. Trends In Plant Science. 17, 478-486

Boehm, M.J., L.V. Madden, H.A.J. Hoitink. 1993. Effect of organic matter decomposition level on bacterial species diversity and composition in relationship to Pythium damping-off severity. Applied and Environmental Microbiology. 59, 4171-4179

Burhanuddin, B., and M. Kusum. 2005. Evaluation of native isolates of biocontrol agents and neem based formulations for management of damping-off in tomato. Indian Journal Of Plant Protection. 33, 102-104

Corrêa, É. B., W. Bettiol, M.A.B. Morandi. 2010. Biological control of root rot caused by Pythium aphanidermatum and promotion of hydroponic lettuce growth with Clonostachys rosea. Tropical Plant Pathology. 35, 248-252

Ehret, D., B. Alsanius, W. Wohanka, J. Menzies, and R. Utkhede. 2001. Disinfection of recirculating nutrient solutions in greenhouse horticulture. Agronomie. 21, 323-339

El-Tarabily, K. A., G. E. S. J. Hardy, and K. Sivasithamparam. 2010. Performance of three endophytic actinomycetes in relation to plant growth promotion and biological control of Pythium aphanidermatum, a pathogen of cucumber under commercial field production conditions in the United Arab Emirates. European Journal Of Plant Pathology. 128, 527- 539

Environmental Protection Agency (EPA). 2017. Registered pesticides. Retrieved 1 March 2017 from https://www.epa.gov/pesticides

Gebhardt, K., J. Schimana, J. Muller, H.P. Fiedler, H.G. Kallenborn, M. Holzenkampfer, P. Krastel, A. Zeeck, J. Vater, A. Holtzel, D.G. Schmid, J. Rheinheimer, K. Dettner. 2002.

Screening for biologically active metabolites with endosymbiotic bacilli isolated from arthropods. FEMS Microbiol. Lett. 217, 199-205

Heungens, K., J.L. Parke. 2000. Zoospore homing and infection events: effects of the biocontrol bacterium Burkholderia cepacia AMMDR1 on two oomycete pathogens of pea (Pisum sativum L.). Applied and Environmental Microbiology. 66, 5192-5200

Joshi, R., B.B. McSpadden Gardener. 2005. Identification and characterization of novel genetic markers associated with biological control activities in Bacillus subtilis. Phytopathology. 96, 145-154

Khalil, S., M. Hultberg, B.W. Alsanius. 2009. Effects of growing medium on the interactions between biocontrol agents and tomato root pathogens in a closed hydroponic system. The Journal of Horticultural Science and Biotechnology. 84, 489-494 Khare, A., B. K. Singh, and R. S. Upadhyay. 2010. Biological control of Pythium aphanidermatum causing damping-off of mustard by mutants of Trichoderma viride 1433. International Journal Of Agricultural Technology. 6, 231-243 Lemanceau, P., P. A. H. M. Bakker, W.J. de Kogel, C. Alabouvette, B. Schippers. 1993. Antagonistic effect of nonpathogenic Fusarium oxysporum Fo47 and Pseudobactin 358 upon pathogenic Fusarium oxysporum f. sp. dianthi. Applied and Environmental Microbiology. 59, 74-82

Martin, F. 1992. Pythium in: Singleton, L.L., J. Mihail, C.N. Rush (eds) Methods for research on soilborne phytopathogenic fungi. APS Press St. Paul, MN. Pg.39-49

McKeen, C.D., C.C. Rielly, P.L. Pusey. 1986. Production and partial characterization of antifungal substances antagonistic to Monilinia fructicola from Bacillus subtilis. Phytopathology. 76, 136-139

Mendes, R., P. Garbeva, J.M. Raaijmakers. 2013. The rhizosphere microbiome: significance of plant beneficial, plant pathogenic, and human pathogenic microorganisms. Microbiol Rev. 37, 634-663 Muthukumar, A., A. Eswaran, and Sanjeevkumar. 2008. Biological control of Pythium aphanidermatum (Edson). Fitz. Mysore Journal Of Agricultural Sciences. 42, 20-25 Nakkeeran, S., K. Kavitha, G. Chandrasekar, P. Renukadevi, and W. G. D. Fernando. 2006. Induction of plant defence compounds by Pseudomonas chlororaphis PA23 and Bacillus subtilis BSCBE4 in controlling damping-off of hot pepper caused by Pythium aphanidermatum. Biocontrol Science And Technology. 16, 403-416 Naseby, D.C., P.J.A., J.M. Lynch. 2000. Effect of biocontrol strains of Trichoderma on plant growth, Pythium ultimum populations, soil microbial communities and soil enzyme activities. Journal of Applied Microbiology. 88, 161-169 Pal, K.K. and B. McSpadden Gardener. 2006 Biological control of plant pathogens. The Plant Health Instructor. 10, 1094-1117

Pasura, A. 2008. Biological control of Pythium root rot in soilless potting mixes. University of Connecticut. Paulitz, T. C., and R. R. Belanger. 2001. Biological control in greenhouse systems. Annu. Rev. Phytopathol. 2001. 39, 103-33 Paulitz, T. C., T. Zhou, and L. Rankin. 1992. Selection of rhizosphere bacteria for biological control of Pythium aphanidermatum on hydroponically grown cucumber. Biological Control. 2, 226-237 Pill, W. G., C. M. Collins, N. Gregory, and T. A. Evans. 2011. Application method and rate of Trichoderma species as a biological control against Pythium aphanidermatum (Edson) Fitzp. in the production of microgreen table beets (Beta vulgaris L.). Scientia Horticulturae. 129,914-918 Postma, J., L. H. Stevens, G. L. Wiegers, E. Davelaar, and E. H. Nijhuis. 2009. Biological control of Pythium aphanidermatum in cucumber with a combined application of Lysobacter enzymogenes strain 3.1T8 and chitosan. Biological Control. 48, 301-309 Pundt, L. and T. Smith. 2017. Selected fungicides and bactericides labeled for vegetable bedding plants and transplants. CT IPM UConn Extension. Retrieved on 5 February 2017 from http://ipm.uconn.edu/documents/raw2/1119/2017%20Fungicides%20and%20Bactericide s%20Labeled%20for%20Vegetable%20Bedding%20Plants%20and%20Transplantsfeb2fi nal.pdf Punja, Z. K. and R.Yip. 2003. Biological control of damping-off and root rot caused by Pythium aphanidermatum on greenhouse cucumbers. Can. J. Pathol. 25, 411-417 Rankin, L. and T.C. Paulitz. 1994. Evaluation of rhizosphere bacteria for biological control of Pythium root rot of greenhouse cucumbers in hydroponic culture. Plant Disease. 78, 447- 451

Rose, S., R. Yip, and Z. K. Punja. 2004. Biological control of Fusarium and Pythium root rots on greenhouse cucumbers grown in rockwool. Acta Horticulturae. 635, 73-78

Stanghellini, M.E. and W.C. Kronland. 1986. Yield loss in hydroponically grown lettuce attributed to subclinical infection of feeder rootlets by Pythium dissotocum. Plant Disease. 70, 1053-1056 Toure, Y., M. Ongena, P. Jacques, A. Guiro, P. Thonart. 2004. Role of lipopeptides produced by Bacillus subtilis GA1 in the reduction of grey mould disease caused by Botrytis cinerea on apple. J. Appl. Microbiol. 96, 1151-1160 Truong, H. X., M. D. Salinas, A. S. Obien, and R. C. Carasi. 1988. Biological control of Sclerotium rolfsii and Pythium aphanidermatum damping-off on tobacco with Trichoderma harzianum culture. Brighton Crop Protection Conference. Pests and Diseases. 3, 1189-1194 Utkhede, R.S., C.A. Levesque, and D. Dinh. 2000. Pythium aphanidermatum root rot in hydroponically grown lettuce and the effect of chemical and biological agents on its control. Canadian Journal of Plant Pathology. 22.2, 138-44

Walker, E.L. and E.L. Connolly. 2008. Time to pump iron: iron-deficiency-signaling mechanisms of higher plants. Curr Opin Plant Biol. 11, 530-535 White, T.J., T. Burns, S. Lee, J. Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Academic Press, Inc. 38, 3

Tables Table 1. Published reports on the efficacy of beneficial microbes to control disease caused by Pythium aphanidermatum. Crop Biological Control(s) Efficacy References Lettuce Clonostachys rosea Reduced root rot Corrêa et al., 2010 incidence by 28.6% and 42.8% when applied two or three times, respectively. Bacillus velezensis Reduced root necrosis by Kanjanamaneesathian 26%. et al., 2013 Bacillus subtilis and B. subtilis increased Utkhede et al., 2000 Trichoderma harzianum yields by 21% T. harzianum decreased root rot by 23.5%. Pseudomonas spp. Increased shoot and root Cipriano et al., 2013 (MP1, Ps864C) biomass. Cucumber Pseudomonas corrugata Plants treated with Pc13 Rankin and Paulitz, (Pc13 and Pc35) or Pf15 produced 88% 1994 Pseudomonas fluorescens more marketable fruit. (Pf15, Pf16, and Pf27) Gliocladium catenulatum Reduced plant mortality, Punja and Utkhede, JI446 while increasing height 2003 and mass. Lysobacter enzymogenes Reduced disease on Postma et al., 2009 3.1T8 and chitosan seedlings by 74%. Gliocladium Reduced disease severity Rose et al., 2004 catenulatum strain J1446 by 86.9%. Actinoplanes A. campanuatus, M. El-Tarabily et al., 2010 campanulatus, chalcea, and S. spiralis Micromonospora chalcea reduced plant mortality and Streptomyces by 68% , 50.8% and spiralis 73.2%, respectively. Rhizobacteria isolate 15 Increased dry weight and Paulitz et al., 1992 root volume by 20.1% and 27.6%, respectively.

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Continued from Table 1 Crop Biological Control(s) Efficacy of Biocontrol References

Tomato Bacillus subtilis isolate Ch- B. subtilis and Burhanuddin and G-D4-1 and Streptomyces sp. Streptomyces sp. Kusum, 2005 Cot-coi-b-1 increased yield by 17% and 41.2% and reduced damping off by 73.3% and 70%, respectively. Microgreen table Trichoderma harzianum T. harzianum reduced Pill et al., 2011 beets damping-off by 36%.

Hot Pepper Bacillus subtilis BSCBE4 B. subtilis and P. Nakkeeran et al., 2006 and Pseudomonas chloroaphis reduced chlororaphis strain PA23 damping-off by 65 and 57%, or more respectively. Mustard Trichoderma viride mutant T. viride m6 reduced Khare et al., 2010 m6 (Tv m6) and Trichoderma pre- and post-emergence viride mutant m13 (Tv m13) damping-off by 77.2% and 83.6%, respectively. Tv m13 reduced pre emergence and post emergence damping-off by 72.5% and 80.7%, respectively. Chili Pepper Trichoderma sp. and Trichoderma sp. reduced Muthukumar et al., Psudomonas fluorescens Pythium populations (102 2008 cfu/g) by 29.4%. P. fluorescens reduced populations by 20.6%. Combined they reduced populations by 38.2%. Tobacco Trichoderma harzianum, T. harzianum, T. Truong et al., 1988 Trichoderma hamatum, and hamatum and T. Trichoderma aureoviridae aureoviridae increased survival by 67.4%, 69.3% and 48.1%, respectively.

Table 2. Microbial biofungicide products used in 2015, 2016, and 2017 experimental trials. Microbesa Product Manufacturer Rate Bacillus subtilis Companion® Growth Products 0.98 mL.L-1 GB03c Ltd., White Plains, NY Trichoderma RootShield Plus® BioWorks Inc., 1.56 g.L-1 harzianum strain T- Fairport, NY 22 (KRL-AG2)e

Bacillus Triathlon BA® OHP, Inc., 2.5 mL.L-1 amyloliquefaciens Mainland, PA strain D747e

Bacillus TerraGrow® BioSafe Systems, 0.06 mL.L-1 licheniformis, LLC Bacillus subtilis, Bacillus pumilus, Bacillus amyloliquefaciens, Bacillus megaterium, Trichoderma harzianum, Trichoderma reeseid

Streptomyces lydicus Actinovate® Novozymes BioAg 0.75 mL.L-1 WYEC108b Inc., Brookfield WI Bacillus subtilis Cease® BioWorks In., 1 mL.L-1 QST713e Fairport, NY a Commercial products mentioned in this research are mainly for the purpose of providing specific information. Results shown in this research are based on experimental conditions for research purposes and may not be guaranteed as recommendations. b Applied in 2015 and 2016 trials. c Applied in 2015, 2016, and 2017 trials. d Applied only in 2015 trial. e Applied only in 2017 trial.

Table 3. ANOVA table of the seedling experiment. Fresh shoot weight (FSW), fresh root weight

(FRW), dry shoot weight (DSW), dry root weight (DRW), disease incidence (DI), disease severity average (DS) were measured in two lettuce cultivars (Cultivar) in response to biofungicides application and Pythium spp. inoculation, n=25. P-value represents the null hypothesis of equal means. If the p < 0.05 the means between the treatments are significant.

Treatments FSW FRW DSW DRW DI DS Biofungicide (B) <.0001 <.0001 <.0001 <.0001 0.0004 0.0008 Pythium (P) <.0001 <.0001 <.0001 <.0001 0.0232 0.0295 Cultivar (Cv) <.0001 0.0591 <.0001 0.3379 0.6494 0.2760 B * P <.0001 <.0001 <.0001 <.0001 0.0004 0.0008 B * Cv <.0001 0.2632 <.0001 0.1086 0.9347 0.3145 P* Cv 0.6367 0.3674 0.6197 0.1478 0.6494 0.2760 B * P * Cv 0.0003 0.0070 0.0041 <.0001 0.9347 0.3145

Table 4. Seedling Experiment. Effect of microbial biofungicides on disease caused by Pythium spp. on lettuce seedlings grown in black plastic trays, n=25. Disease incidence was measured as percentage plants with foliar visual symptoms (wilting, stunting) per peat pellet. Disease severity was estimated based on a scale of 1-4, where 4 is the highest severity. Root necrosis was assessed visually.

Root Lettuce Biofungicide Pythium Incidence (%)a,b Severity average a,b,c Necrosis cultivar (%)a,b,d - 0.0 1.0 0 c Untreated + 0.0 1.0 100 a - 0.0 1.0 0 c Companion + 0.0 1.0 100 a Rex - 0.0 1.0 0 c RootShield Plus + 0.0 1.0 100 a - 0.0 1.0 0 c Triathlon BA + 0.0 1.0 98 a - 0.0 1.0 0 c Cease + 0.0 1.0 90 b - 0.0 b 1.0 b 0 b Untreated + 0.1 a 1.1 a 100 a - 0.0 b 1.0 b 0 b Companion + 0.0 b 1.0 b 100 a Spretnak - 0.0 b 1.0 b 0 b RootShield Plus + 0.0 b 1.0 b 100 a - 0.0 b 1.0 b 0 b Triathlon BA + 0.0 b 1.0 b 96 a - 0.0 b 1.0 b 0 b Cease + 0.0 b 1.0 b 98 a a Means within a column within each crop followed by the same letter are not significantly different according to Tukey’s HSD separation test (α=0.05). b Experimental runs one and two were analyzed together. c The scale for disease severity was rated according to the following scale: 1=symptomless, 2=emerged but wilted, 3=post-emergent damping-off, and 4=pre-emergent damping-off (Boehm et. al., 1993). d Root disease was rated based on visual presence or absence of root necrosis 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.

Table 5. Hydroponic Experiment. Total nitrate concentration (mg.L-1) in the nutrient solution of hydroponic lettuce treated with microbial biological fungicides with and without P. aphanidermatum Cor4. Nitrate was measured weekly with a LAQUA Twin Nitrate Ion Meter.

Data represents experimental run one and two of experiment three, n=24.

Week Week (1st Experimental Run)a (2nd Experimental Run) Biofungicide Pythium 1 2 3 1 2 3 - 134c 150b 153c 140bc 110b 134b Untreated + 125c 140b 143c 115c 113b 125b Companion - 139bc 135b 143c 118c 120b 139b + 133c 145b 148c 136bc 120b 133b - 235a 218a 220a 178ab 180a 235a RootShield + 185b 203a 205ab 195a 193a 185a - 121c 153b 158bc 115c 115b 121b Triathlon BA + 140bc 150b 158bc 113c 120b 140b a Means within a column for each weight measurement followed by the same letter are not significantly different according to Tukey’s HSD separation test (α=0.05).

Chapter 4 (To be submitted to Crop Protection)

Efficacy of Microbial Biofungicides against Root Rot and Damping-off of Microgreens

caused by Pythium spp.

Abstract

Pythium spp. are the causal agents of Pythium root rot and damping-off on microgreens in hydroponic systems. Synthetic chemical fungicides are not registered for application in hydroponic solutions and few are registered for application on edibles in greenhouses. The objective of this project was to assess the efficacy of microbial biofungicides on Pythium root rot caused by Pythium aphanidermatum and Pythium dissotocum on microgreens in hydroponic systems. In the first experiment, arugula (Eruca 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 BA®

(Bacillus amyloliquefaciens D747), or RootShield Plus® (Trichoderma harzianum KRL-AG2 and Trichoderma virens G-41). Two days after treatment, the plants were inoculated with 1 x 105 zoospores of Pythium spp. After seven days, we measured root necrosis, disease incidence and severity, fresh and dry shoot and root weight. All plants infected with Pythium spp. were smaller by 28% or more compared with non-inoculated plants. Overall disease severity and incidence was low, but biomass was lower in all treatments inoculated 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 necrosis. In the second experiment, arugula and mustard were treated with the same microbial biofungicides as the first experiment but Cease® (Bacillus subtilis QST 713) was added as an additional treatment. Arugula and mustard inoculated with Pythium spp. resulted in more than 74.4%

70 reduction of shoot and root weight. Arugula and mustard treated with Cease®, with and without

Pythium spp., consistently resulted in over 59% more fresh shoot and root weight compared to the inoculated control. Results from this experiment suggest that microbial biofungicides can be introduced in nutrient solutions to prevent Pythium root rot and damping-off in microgreens.

1. Introduction

Microgreens are edible plants that are smaller than baby greens and more mature than sprouts (USDA, 2014), typically harvested seven to 21 days after germination when the cotyledons or the first true leaves have expanded. Microgreens are produced at high density, which is known to increase the incidence of Pythium damping-off in seedlings (Burdon and

Chilvers, 1975). The combination of young, succulent tissue, and high production density makes microgreens a vulnerable production system for root rot.

Pythium spp. are the causal agents of Pythium root rot and damping-off in seedlings

(Paulitz et al., 2001). Pythium aphanidermatum has been reported to cause rot in brassica species

(Ebenezar et al., 1996; Tanina et al., 2003; Tojo et al., 2005) and was reported to cause up to

98% mortality in seedlings (Lim and See, 1983). Fungicides registered for the control of root diseases in edible crops in greenhouses are limited to 18, of which 11 are microbial biofungicides

(Pundt and Smith, 2017). Many Pythium aphanidermatum and Pythium dissotocum isolates have developed resistance against the synthetic chemical fungicides mefenoxam and propamocarb

(Broders et al., 2007; Moorman et al., 2002). The risk of fungicide resistance and limited availability of synthetic chemistries increases the need to implement preventative control for hydroponically-grown crops. Mustard plants treated with Trichoderma viride had 77.2% and

83.6% lower incidence of pre and post-emergence damping-off compared with the untreated control (Khare et al., 2010). Lettuce yields were 21% higher when treated with Bacillus subtilis

71 and had 23.5% less root rot incidence when treated with Trichoderma harzianum (Utkhede et al.,

2000).

Our objective was to evaluate the effect of microbial biofungicides on the disease incidence and severity of Pythium root rot and damping-off caused by Pythium dissotocum and

Pythium aphanidermatum in microgreens of the brassica family.

2. Materials and Methods

2.1. Pythium inoculum preparation

Pythium spp. isolates used in this experiment were obtained from greenhouses in the northeastern

US. All isolates were tested for pathogenicity in vitro (Chapter 2). P. aphanidermatum Cor4, P. aphanidermatum Kop-8, and P. dissotocum Cor1 were the isolates used in this project. The isolates were identified using the ITS regions 1 and 4 as described by White (1990) and registered in GenBank under accession numbers MG993551, MG993547, and MG993548.

Zoospores were induced by following the protocol described by Martin (1992). Five 4-mm plugs of three-day old Pythium sp. grown on V8-juice agar were transferred to an empty Petri dish and then filled with 20 mL of clarified-V8 broth. The plates were incubated in the dark for five days.

The mycelial mats were rinsed three times with sterile pond water. The Petri dishes were then filled with 20 mL of sterile deionized water. The plates were incubated under fluorescent light for 24 hours. Sporangia formation was confirmed visually and then the solution was drained. The

Petri dishes were refilled with 20 mL of chilled sterile deionized water and incubated for two hours at 4 °C. Then Petri dishes were set at 22-26 °C for 1 to 2 hours under light. A dilution was made to deliver 1 x 105 zoospores per mL of inoculation. Each peat pellet (Jiffy, Netherlands) was inoculated with 10 mL of 1 x 105 zoospore suspension (Heungens and Parke, 2000).

2.2 Irrigation water analysis

The nutrient solutions were prepared with municipal water with the following characteristics: pH of 7.4, electrical conductivity (EC) of 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 chlorine was 0.7 mg.L-1. EC, pH, DO, temperature, and ORP were measured with specific probes for the

Orion Star Meter (Thermo Fisher Scientific Inc., MA). Free and total chlorine were measured with the Orion AQUAfast IV Powder Chemistries in the Orion AQ4000 Advanced Colorimeter

(Thermo Fisher Scientific Inc., MA).

2.3 Growing system

Hydroponic experiment. The hydroponic setup was a closed-loop nutrient film technique

(NFT) system. The NFT channels were 1.83-m long by 10.16-cm wide and with six 5.08-cm holes. Every two channels were connected to a ten-gallon nutrient solution tank. Mustard

(Brassica juncea L. Czern cv. Green Wave), kale (Brassica oleracea L. var. Red Russian), arugula (Eruca sativa Mill.), and radish (Raphanus raphanistrum subsp. sativus L. cv. Hong Vit) were sown in 42-mm peat pellets. Fifteen seeds of each plant species were sown per pellet which represented an experimental unit. There were three pellets of each plant species per treatment combination in each block, and there were a total of four blocks. The pellets were irrigated with clear water for the first three days. The seedlings were maintained in a tray on a greenhouse bench under a high pressure sodium light for 14 hours per day. The seedlings were hand-irrigated with a nutrient solution of a final electrical conductivity (EC) of 0.33 the day before they were transplanted. The pellets were transferred into the hydroponic channels when the cotyledons were visible. The nutrient solution had an EC of 1.0 µS.cm-1 and a pH of 5.8. The solutions were adjusted daily to maintain the target pH and EC. The nutrient solution was a combination of a

73 complete fertilizer 5-12-26 with CaNO3 (1:1.5 ratio by mass). Plants were grown in Connecticut in a polycarbonate greenhouse with a heating set point of 18.3 °C and a ventilation set point of

26.7 °C under natural photoperiod in June and July 2017.

Tray experiment. In a separate experiment, mustard and arugula were sown as described above.

We put 15 seeds per pellet, five pellets of each species per tray were maintained in black plastic trays on the greenhouse bench under a high pressure sodium light for seven days. The experimental unit consisted of a peat pellet with fifteen seedlings. The greenhouse heating and cooling set points were the same as the previous experiment. The experiments were conducted in

August and September 2017.

2.4 Biofungicide and pathogen applications

Hydroponic experiment. Companion® (Bacillus subtilis GB03), Triathlon BA® (Bacillus amyloliquefaciens D747), and RootShield Plus® (Trichoderma harzianum KRL-AG2 and

Trichoderma virens G-41) were applied following bases on specimen label instructions at 0.98 mL.L-1 for Companion® and 1.56 g.L-1 for RootShield Plus®. Companion® and Rootshield Plus® were applied directly in the nutrient solution, and Triathlon BA® was applied directly in the peat pellet, per label recommendation at 2.5 mL.L-1. Two days after amending the tanks with microbial biofungicides, 10 mL of Pythium spp. inoculant at 1 x 105 zoospores per mL were applied directly in each peat pellet. The negative control consisted of applying 10 mL of sterile deionized water. All applications were made only once.

Tray experiment. Microbial biofungicides and pathogen applications were conducted as described in the hydroponic experiment. This experiment had Cease® (Bacillus subtilis QST 713) as an additional microbial biofungicide treatment. Cease® was applied directly in the nutrient solution at 1 mL.L-1. All applications were made only once.

2.5 Measurements

Hydroponic Experiment. Daily measurements of the solution included pH, electrical conductivity (EC), dissolved oxygen (DO), and temperature. Harvest was conducted four to five days after pathogen inoculation, at which time the seedlings were seven to eight days old.

Measurements at harvest included disease incidence and severity, root necrosis, stand counts, fresh shoot and root biomass, and relative amount of chlorophyll (SPAD). Disease incidence was measured as percentage plants with foliar visual symptoms (wilting, stunting) per peat pellet.

The scale for disease severity was rated according to the following scale: 1=symptomless,

2=emerged but wilted, 3=post-emergent damping-off, and 4=pre-emergent damping-off (Boehm et. al., 1993). Each value represents the average severity rating of standing plants per peat pellet.

Root disease was rated based on visual presence or absence of root necrosis 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. Shoots were cut at the soil line and shoots and roots were weighed fresh and dry (dried at 21.1 °C for two weeks). Relative greenness was measured using a SPAD 502 Plus chlorophyll meter (Spectrum Technologies, Inc., IL).

Tray experiment. Harvest was conducted five to six days after pathogen inoculation, at which time the seedlings were nine to ten days old. Arugula and mustard were harvested together randomly during a five day time period. Measurements at harvest included disease incidence and severity, root necrosis, stand counts, and fresh shoot and root biomass. These measurements were made as described above.

2.6 Experimental design

Hydroponic Experiment. The experiment was a full factorial organized in a randomized complete block design (RCBD). The factors included plant species, biofungicides, and Pythium

75 inoculation. The experimental unit consisted of a pellet with fifteen seeds. Each block had three replicates. There were four blocks in the experiment (n=12). Both experiments were analyzed using SAS Version 9.4 (SAS Institute Inc., NC) to establish significance of the effects of all factors (α=0.05) by analysis of variance (ANOVA). Severity ratings were analyzed with non- parametric analysis using the RANK and PROC GLM procedure. Means were separated by

Tukey’s studentized range HSD (Honestly Significant Difference) separation test (α=0.05) at the

95% confidence interval using PROC MIXED.

Tray Experiment. The experiment was a full factorial arranged as a complete randomized design (CRD). The factors were plant species, biofungicides, and Pythium inoculation. The experimental unit consisted of a peat pellet with fifteen seedlings, there were five replicates per tray, and there were a total of five trays (n=25). The trays were randomly distributed in the greenhouse. Both experiments were analyzed using SAS Version 9.4 (SAS Institute Inc., NC) to establish significance of the effects of all factors (α=0.05) by analysis of variance (ANOVA).

Severity ratings were analyzed with non-parametric analysis using the RANK and PROC GLM procedure. Means were separated by Tukey’s studentized range HSD (Honestly Significant

Difference) separation test (α=0.05) at the 95% confidence interval using PROC MIXED.

3. Results

3.1. Hydroponic Experiment

Data were analyzed separately for each experimental run because there was heterogeneity between runs (P > 0.05) for all measurements, except disease incidence and severity. Interaction between treatment and pathogen were significant for all measurements (Table 1). All plant species inoculated with Pythium spp. presented some disease incidence and severity, and root necrosis, compared with plants with no Pythium (Table 2). Root necrosis was 100% for all

76 microgreen species inoculated with Pythium spp. and untreated with microbial biofungicides.

Across species, plants treated with Triathlon BA® and Pythium spp. had the highest root necrosis compared with the other microbial biofungicides. Whereas plants treated with Companion® and

RootShield Plus® resulted in lower root necrosis incidence. All plants inoculated with Pythium spp. were smaller by 28% or more compared with non-inoculated plants (Figure 1 and 2).

Disease severity and incidence was low, but biomass was reduced within all treatments inoculated with Pythium spp. Arugula inoculated with Pythium spp. and treated with Triathlon

BA® resulted in 8% lower disease incidence compared to the control, although Triathlon BA® with Pythium spp. resulted in high root necrosis. Arugula and radish treated with Triathlon BA® and inoculated with Pythium spp. had higher dry shoot weight than the inoculated control.

Radish treated with RootShiled Plus® and Pythium spp. had greater shoot weight than the inoculated control in the second experimental run. Mustard treated with Triathlon BA® and inoculated with Pythium spp. was the only microbial biofungicide that had higher dry root weight compared with the inoculated control.

Dissolved oxygen was higher in solutions treated with Triathlon BA® (8.1 mg.L-1) or untreated

(8.1 mg.L-1) compared with solutions treated with RootShield Plus® (7.6 mg.L-1) and

Companion® (7.5 mg.L-1). No differences were observed in any other nutrient solution parameters.

3.2. Tray Experiment

Interaction between treatment and pathogen was significant for all measurements (Table 3).

Arugula and mustard plants inoculated with Pythium spp. resulted in higher root necrosis compared with the non-inoculated control (Table 4). Overall disease incidence and severity in this experiment were very low. Root necrosis was only reduced in mustard treated with Cease®.

Arugula and mustard infected with Pythium spp. had lower shoot and root dry weight by 63-75% compared with non-inoculated plants, respectively (Figure 3). All plants treated with microbial biofungicides and Pythium spp. had higher shoot and root growth compared to the inoculated control. Arugula and mustard treated with Cease® consistently had no difference in dry shoot and root weight with and without Pythium spp.

4. Discussion

Microbial biofungicides have shown potential for the suppression of Pythium root rot. This is especially important in the absence of registered synthetic fungicides in hydroponics and an increased interest in sustainable practices. When microbial biofungicides were applied as a preventative treatment with and without Pythium spp. they exhibited a significant reduction in biomass compared to the control. Understanding the mechanisms of these organisms will help hypothesize why microbial biofungicides are inconsistent in promoting or reducing plant growth.

Many beneficial soil-borne microorganisms such as Bacillus and Trichoderma spp. have been found to activate defense mechanisms in shoots and reproductive parts of the plant. Induced systemic resistance (ISR) is a state in which the immune system of the plant is informed to accelerate activation for defense (Berendsen et al., 2012). Beneficial bacteria and fungi act in part by triggering the plant ISR towards pathogenic bacteria. Studies have shown that the biosynthesis of indole-3-acetic acid (IAA) in the plant growth-promoting rhizobacteria (PGPR) of B. amyloliquefaciens affects its ability to promote plant growth (Choudhary and Johri, 2009;

Idris et al., 2007; Kamilova et al., 2006). B. subtilis was demonstrated to produce auxin as the likely mechanism behind the increase in seedling growth of wheat plants (Egorshina et al.,

2011). Bacillus subtilis GB03 is known to stimulate phytohormones, which trigger the plant's systemic acquired resistance (SAR) to disease. SAR can be triggered by exposing the plant to

78 virulent, avirulent, or non-pathogenic microbes (Choudhary and Johri, 2009), which may allocate energy from the plants growth-promoting activity. Specific microorganisms are able to protect the plant either directly or indirectly against pathogens and their efficacy is largely influenced by the total microbiome and its interactions that affect plant health (Berendsen et al., 2012). Our research suggests that plants grown in hydroponic systems are vulnerable to Pythium root rot and damping-off. While closed hydroponic systems allow savings of water and nutrient use, the recirculation of water increases the risk of pathogen dispersal (Hosseinzadeh et al., 2017).

Pythium spp. in this study had a greater effect on the reduction of plant biomass and root necrosis than visual disease incidence and severity. Pythium root rot has been known to be a potential threat to plant biomass production in lettuce and various studies have reported 23-54% reduction in growth caused by P. aphanidermatum in hydroponic production (Corrêa et al., 2010;

Johnstone et al., 2004; Pantelides et al., 2017; Stanghellini and Kronland, 1986). This can cause a problem for growers since the symptoms of the disease are not noticeable from the shoots and it is often treated too late. Plants treated with Cease® and Triathlon BA® exhibited the least amount of biomass reduction with and without Pythium spp. suggesting that these two products provided the best protection against Pythium root rot. Recent studies have shown the antagonistic properties of Bacillus species against damping-off caused by P. aphanidermatum

(Jeyaseelan et al., 2012; Utkhede et al., 2000). More specifically, a strain of B. amyloliquefaciens has proven to effectively inhibit P. aphanidermatum more than other fungal pathogens and microorganisms (Zouari et al., 2016). Although research provides evidence for these selected beneficial microorganisms to reduce disease and inhibit growth of pathogens it is still dependent on the host plant and environmental conditions. Differences were found in the ability of wheat cultivars to accumulate naturally occurring antibiotic 2,4-diacetylphloroglucinol (DAPG)-

79 producing Pseudomonas spp. resulting in inconsistent disease suppression (Berendsen et al.,

2012). These results suggest that plant genotype can affect the accumulation of microorganisms that assist the plant to defend itself against pathogen attack. Further research needs to be conducted to know more about how different species and cultivars of microgreens perform with microbial biofungicides. Results from this greenhouse experiment suggest that microbial biofungicides can be introduced in nutrient solutions to prevent disease development. Additional research may be conducted to identify specific microbes in nutrient solutions and how other environmental factors can be altered to promote beneficial microbes.

5. Conclusion

In this study, microbial biofungicides reduced plant biomass in the absence of disease, but none of the products affected visual quality of the crop. In general when the pathogen was present, the microbial biofungicides protected the crop from extreme damage. Therefore, growers facing the risk of Pythium root rot in hydroponics can use microbial biofungicides as preventative mechanisms and sanitize all production areas if an outbreak has occurred. Growers are less likely to get root necrosis on mustard from Pythium spp. if using Cease®, Companion®, or RootShield

Plus® as preventative fungicides. Infection can be more costly than prevention so these alternative options are available to avoid yield losses. However, we must conduct further research to identify sustainable practices that protect plants biotic and abiotic stresses without affecting crop yield and the environment.

Literature Cited

Berendsen, R. L., C. M. J. Pieterse, and P. A. H. M. Bakker. 2012. The rhizosphere microbiome and plant health. Trends In Plant Science. 17, 478-486. doi: 10.1016/j.tplants.2012.04.001

Broders K.D., P.E. Lipps, P.A. Paul, A.E. Dorrance. 2007. Characterization of Pythium spp. associated with corn and soybean seed and seedling disease in Ohio. Plant Disease 91, 727-735. doi:10.1094/PDIS-91-6-0727

Burdon, J. J., G.A. Chilvers, G. A. 1975. Epidemiology of damping-off disease (Pythium irregulare) in relation to density of Lepidium sativum seedlings. Annals of Applied Biology. 81, 135-143. doi: 10.1111/j.1744-7348.1975.tb00530.x

Choudhary, D.K. and B.N. Johri 2009. Interactions of Bacillus spp. and plants-with special reference to induced systemic resistance. Microbiological Research. 164, 493-513. doi:10.1016/j.micres.2008.08.007

Ebenezar, E. G., D. Alice, and K. Sivaprakasam. 1996. Biological control of damping off disease in mustard caused by Pythium aphanidermatum. Journal Of Ecobiology. 8, 55-57

Egorshina, A.A., R.M. Khairullin, A.R. Sakhabutdinova, M.A. Luk’yantsev. 2011. Involvement of phytohormones in the development of interaction between what seedlings and endophytic Bacillus subtilis strain 11BM. Russ. J. Plant Physiol. 59, 134-140. doi:10.1134/S1021443711050062

Hosseinzadeh, S., Y. Verheust, G. Bonarrigo, and S. van Hulle. 2017. Closed hydroponic systems: operational parameters, root exudates occurrence and related water treatment. Reviews In Environmental Science And Bio/Technology. 16, 59-79. doi:10.1007/s11157- 016-9418-6

Idris E.E.S, D.J. Iglesias, M. Talon, R. Borriss. 2007. Tryptophanedependent production of indole-3-actic acid (IAA) affects level of plant growth-promotion by Bacillus amyloliquefaciens FZB42. Mol Plant-Microbe Interact. 20, 619-626. doi: 10.1094/MPMI-20-6-0619

Jeyaseelan, E.C., S. Tharmila, K. Niranjan. 2012. Antagonistic activity of Trichoderma spp. and Bacillus spp. against Pythium aphanidermatum isolated from tomato damping off. Archives of Applied Science Research. 4, 1623-1627

Johnstone, M., Hai Yu, WeiZhong Liu, E. Leonardos, J. Sutton, and B. Grodzinski. 2004. Physiological changes associated with Pythium root rot in hydroponic lettuce. Acta Horticulturae. 635, 67-71

Kamilova F., L.V. Kravchenko, A.I. Shaposhnikov, T. Azarova, N. Makarova, B. Lugtenberg. 2006. Organic acids, sugars, and L-tryptophane in exudates of vegetables growing on stonewool and their effects on activities of rhizosphere bacteria. Mol Plant-Microbe Interact. 19, 250-256. doi:10.1094/MPMI-19-0250

Khare, A., B. K. Singh, and R. S. Upadhyay. 2010. Biological control of Pythium aphanidermatum causing damping-off of mustard by mutants of Trichoderma viride 1433. International Journal Of Agricultural Technology. 6, 231-243

Lim, G., G.K. See. 1983. Damping-off of brassica seedlings by Pythium aphanidermatum. Int. J. Tropical Plant Diseases. 1, 65-68

Martin, F. 1992. Pythium in: Singleton, L.L., J. Mihail, C.N. Rush, Methods for research on soilborne phytopathogenic fungi. APS Press St. Paul, MN. 39-49 pp.

Moorman, G.W., S. Kang, D.M. Geiser, S.H. Kim. 2002. Identification and characterization of Pythium species associated with greenhouse floral crops in Pennsylvania. Plant Disease. 86, 1227-1231. doi:10.1094/PDIS.2002.86.11.1227

Pantelides, I., M. Tsolakidou, A. Chrysargyris, N. Tzortzakis. 2017. First report of root rot of hydroponically grown lettuce (Lactuca sativa) caused by a Pythium species from the cluster B2a species complex in Cyprus. Plant Disease. 101, 636

Paulitz, T. C., H. C. Huang, and J. A. Gracia-Garza. 2001. Pythium spp., damping-off, root and crown rot (Pythiaceae). doi:10.1079/9780851995274.0478

Pundt, L. and T. Smith. 2017. Selected Fungicides and Bactericides Labeled for Vegetable Bedding Plants and Transplants. CT IPM UConn Extension. Retrieved on 5 February 2017 from http://ipm.uconn.edu/documents/raw2/1119/2017%20Fungicides%20and%20Bactericide s%20Labeled%20for%20Vegetable%20Bedding%20Plants%20and%20Transplantsfeb2fi nal.pdf

Tanina, K., M. Tojo, H. Date, H. Nasu, and S. Kasuyama. 2004. Pythium rot of chingensai (Brassica campestris L. chinensis group) caused by Pythium ultimum var. ultimum and Pythium aphanidermatum. Journal Of General Plant Pathology. 70, 188-191. doi:10.1007/s10327-004-0109-8

Tojo, M., T. Shigematsu, H. Morita, YingJie Li, T. Matsumoto, and S. T. Ohki. 2005. Pythium rot of Chinese cabbage (Brassica rapa L. subsp. pekinensis) caused by Pythium aphanidermatum. Journal Of General Plant Pathology. 71, 384-386. doi:10.1007/s10327-005-0218-z

USDA. 2014. Specialty greens pack a nutritional punch. AgResearch Magazine. Retrieved 18 October 2017 from https://agresearchmag.ars.usda.gov/2014/jan/greens/

Utkhede, R.S., C.A. Levesque, and D. Dinh. 2000. Pythium aphanidermatum root rot in hydroponically grown lettuce and the effect of chemical and biological agents on its control. Canadian Journal of Plant Pathology. 22.2, 138-44

White, T.J., T. Burns, S. Lee, J. Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Academic Press, Inc. 38, 317

Zouari, I., L. Jlaiel, S. Tounsi, and M. Trigui. 2016. Biocontrol activity of the endophytic Bacillus amyloliquefaciens strain CEIZ-11 against Pythium aphanidermatum and purification of its bioactive compounds. Biological Control. 100, 54-62. doi:10.1016/j.biocontrol.2016.05.012

Table 1. ANOVA table for fresh shoot weight (FSW), fresh root weight (FRW), dry shoot

weight (DSW), dry root weight (DRW), disease incidence (DI), and disease severity average

(DS) were measured in four microgreen species (Species) in response to biofungicides

application and Pythium spp. inoculation for both experimental runs (I and II) in experiment one

(Hydroponic Experiment, n=12). P-value represents the null hypothesis of equal means. If the

P< 0.05 the means between the treatments are significant.

Experiment FSW DSW FRW DRW DI DS

Treatments I II I II I II I II I & II Biofungicide (B) <.0001 0.0005 <.0001 0.1866 <.0001 <.0001 0.0016 0.0003 <.0001 <.0001 Pythium (P) <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 B*P 0.0374 <.0001 0.0061 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 Species (Sp) <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 B*Sp 0.0231 <.0001 0.1332 0.0504 0.0017 0.2128 0.0104 0.0215 0.0682 0.1215 P*Sp <.0001 0.0003 <.0001 <.0001 <.0001 <.0001 0.0398 0.0924 <.0001 <.0001 B*P*Sp 0.0357 0.0431 0.019 0.0002 0.1539 0.0556 0.1365 0.1175 0.0682 0.1215

Table 2. Disease incidence and severity caused by Pythium spp. on microgreens when treated with microbial biofungicides in a hydroponic system (Hydroponic Experiment, n=12). Species Biofungicide Pythium Incidence (%)a Severitya,c Root Necrosis (%)a,b,d Arugula Untreated - 0.0 c 1.0 c 0 c 0 d Untreated + 8.9 a 1.2 a 100 a 100 a Companion - 0.0 c 1.0 c 0 c 0 d Companion + 4.2 b 1.1 b 58 b 50 bc Rootshield Plus - 0.0 c 1.0 c 0 c 0 d RootShield Plus + 2.1 bc 1.0 bc 75 ab 33 cd Triathlon BA - 0.0 c 1.0 c 0 c 0 d Triathlon BA + 0.9 bc 1.0 bc 92 ab 92 ab Mustard Untreated - 0.0 1.0 0 b 0 b Untreated + 2.4 1.1 100 a 100 a Companion - 0.0 1.0 0 b 0 b Companion + 0.6 1.0 25 b 25 b Rootshield Plus - 0.0 1.0 0 b 0 b RootShield Plus + 0.6 1.0 25 b 25 b Triathlon BA - 0.0 1.0 0 b 0 b Triathlon BA + 0.9 1.0 67 a 75 a Kale Untreated - 0.0 b 1.0 b 0 c 0 b Untreated + 6.0 a 1.1 a 100 a 100 a Companion - 0.0 b 1.0 b 0 c 0 b Companion + 1.8 b 1.0 b 42 b 17 b Rootshield Plus - 0.0 b 1.0 b 0 c 0 b RootShield Plus + 3.0 ab 1.1 ab 42 b 67 a Triathlon BA - 0.0 b 1.0 b 0 c 0 b Triathlon BA + 3.3 ab 1.1 ab 100 a 83 a Radish Untreated - 0.0 b 1.0 b 0 c 0 c Untreated + 8.0 a 1.2 a 100 a 100 a Companion - 0.0 b 1.0 b 0 c 0 c Companion + 3.3 b 1.1 b 42 b 75 ab Rootshield Plus - 0.0 b 1.0 b 0.0 c 0 c RootShield Plus + 2.1 b 1.0 b 50 b 42 bc Triathlon BA - 0.0 b 1.0 b 0 c 0 c Triathlon BA + 3.6 b 1.1 b 100 a 67 ab a Means within a column within each crop followed by the same letter are not significantly different according to Tukey’s HSD separation test (α=0.05). b Experimental runs one and two were analyzed separately. The column at the left represents run one and the column at the right represents run two. c The scale for disease severity was rated according to the following scale: 1=symptomless, 2=emerged but wilted, 3=post-emergent damping-off, and 4=pre-emergent damping-off (Boehm et. al., 1993). d Root necrosis was rated based on visual presence or absence of root necrosis 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. 88

Table 3. ANOVA table for fresh shoot weight (FSW), fresh root weight (FRW), 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 evaluated for experiment two (Tray Experiment, n=25). P-value represents the null hypothesis of equal means. If the p-value is less than 0.05 the means between the treatments are significant.

Treatments FSW FRW DSW DRW DI DS Biofungicide (B) <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 Pythium (P) <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 B*P <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 Species (Sp) <.0001 0.457 <.0001 0.1621 0.2597 0.2278 B*Sp 0.2735 0.4731 0.4030 0.0060 0.1231 0.1351 P*Sp 0.0401 0.3585 0.1423 0.5590 0.2597 0.2278 B*P*Sp 0.1163 0.2427 0.3295 0.6177 0.1231 0.1351

Table 4. Effect of microbial biofungicides on disease caused by Pythium spp. on microgreens grown in a black plastic tray (Tray Experiment, n=25). Disease incidence was measured as percentage plants with foliar visual symptoms (wilting, stunting) per peat pellet (n=14). Disease severity was estimated based on scale of 1-4, where 4 is the highest severity. Root necrosis was assessed visually and analyzed using Chi-Square test.

Root Incidence Species Biofungicide Pythium Severitya,b,c Necrosis (%)a,b (%)a,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 Mustard Untreated - 0.0 b 1.0 b 0 c Untreated + 0.02 a 1.1 a 100 a Companion - 0.0 b 1.0 b 0 c Companion + 0.0 b 1.0 b 98 a RootShield Plus - 0.0 b 1.0 b 0 c RootShield Plus + 0.0 b 1.0 b 94 a Triathlon BA - 0.0 b 1.0 b 0 c Triathlon BA + 0.0 b 1.0 b 88 a Cease - 0.0 b 1.0 b 0 c Cease + 0.0 b 1.0 b 72 b a Means within a column within each crop followed by the same letter are not significantly different according to Tukey’s HSD separation test (α=0.05). b Experimental runs one and two were analyzed together. c The scale for disease severity was rated according to the following scale: 1=symptomless, 2=emerged but wilted, 3=post-emergent damping-off, and 4=pre-emergent damping-off (Boehm et. al., 1993). d Root disease was rated based on visual presence or absence of root necrosis 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.

Chapter 5 Conclusion The purpose of this research was to identify pathogenic Pythium spp. isolates from commercial greenhouse operations and then evaluate the potential of controlling Pythium root rot caused by Pythium spp. on hydroponic leafy greens when using recirculated nutrient solutions or microbial biofungicides. Identifying Pythium spp. infecting greenhouse crops and evaluating different microbial biofungicides against them on several crop species will help prioritize grower’s economic inputs against Pythium root rot.

Pythium isolates that caused the highest disease incidence and severity in lettuce seeds were identified as P. dissotocum isolate Cor1, P. aphanidermatum Kop-8, and P. aphanidermatum Cor4. These isolates were registered in GenBank under accession numbers

MG993551, MG993547, and MG993548. This is the first published report of P. dissotocum being detected on lettuce in Connecticut (McGehee et al., 2018). Additional research needs to be conducted on surveying Pythium species present in commercial hydroponic systems around New

England to identify which species are most prevalent.

The in vitro inhibition tests showed that recirculated nutrient solutions collected from commercial hydroponic farms have the potential to suppress Pythium species. The suppression could be attributed to microbial or chemical agents. We are in the process of identifying the microbial populations found in these solutions and determine if known microbial antagonists are present or if a general suppression might be causing the suppression. Further research is required to identify specific native suppressive microbial inhabitants and study the antagonistic properties of the microbes and how they relate to suppressing root pathogens and other parameters of plant health and quality.

Leafy vegetables treated with microbial biofungicides exhibited a significant reduction in biomass compared to the control when applied without Pythium spp. The biological products were not consistent in their effect on the incidence and severity of root rot disease in the presence of Pythium spp. Quantitative-PCR may give a better understanding of biofungicide and Pythium spp. abundance in the plant roots. We did not compare pure microbial isolates with the commercial product. Therefore, we cannot conclude whether microbes (the active ingredient) or other ingredients in the products reduce plant growth. We hypothesize the microbes might activate plant defense mechanisms and therefore resources are diverted from growth. Additional research needs to be conducted in order to identify microbial biofungicides that have do not affect commercial traits while protection crops from disease in hydroponics.

Plants grown in hydroponic systems are more vulnerable to Pythium root rot and damping-off. Pathogenic Pythium spp. were present in all the samples we collected from hydroponic farms. Therefore, root rot management is crucial in hydroponic systems. We discovered registered microbial biofungicides can have negative effects on plant growth, but can also reduce the negative effects of Pythium infection. Selecting an efficient microbial biofungicide may be the best prevention strategy available against Pythium aphanidermatum and

Pythium dissotocum in hydroponic leafy vegetables, yet more science-based guidelines are needed.