Rapid Sand Filtration of Recycled IrrigationWater Controlled Pythium Root Rot ofPoinsettia in Greenhouse

Sangho Jeon1,2, Charles S. Krasnow1, Gemini D. Bhalsod1,3,

Blair R. Harlan1, Mary K. Hausbeck1, Steven I. Safferman4,

and Wei Zhang1

ADDITIONAL INDEX WORDS. activated carbon, fungicide, etridiazole, Euphorbiapulcherrima, pathogen

SUMMARY. Pythium species incite crown and root rot and can be highly destructive tofloriculture crops in greenhouses, especially when irrigation water is recycled. This studyassessed the performance of rapid filtration of recycled irrigation water for controllingpythium root rot of poinsettia (Euphorbia pulcherrima) in greenhouses. Two greenhouseexperiments investigated the effect of filter media type (sand and activated carbon), fun-gicide application (etridiazole), and pathogen inoculum source (infested growing mediaand infested irrigation water). Rapid sand filtration consistently controlled pythium rootrot of poinsettia. Significant improvements in height, weight, root rot severity, and hor-ticultural quality were observed for the plants in the sand filter treatment, compared withthe inoculated control plants. However, the activated carbon filter removed essential nu-trients from the irrigation water, resulting in plant nutrient deficiency and consequentlyleaf chlorosis, thus reducing plant weight, height, and horticultural quality. The etridia-zole application did not completely prevent root infection by Pythium aphanidermatum,but plant weight, height, and horticultural quality were not negatively affected. P. apha-nidermatum spread from infested growingmedia to healthy plants when irrigation waterwas recycledwithout filtration. Rapid sand filtration appears to have the potential to limitthe spread of P. aphanidermatum that causes root rot of greenhouse floriculture crops.

Floriculture crops in the UnitedStates have an estimated whole-sale value of $4.4 billion and

include a diverse assortment ofbedding plants, potted flowers, andcut flowers. Poinsettia contributeda wholesale value of $140 million in2015 (U.S. Department of Agricul-ture, 2016) and are one of thetop potted flowering plants in theUnited States (Dole and Wilkins,2005). In the greenhouse productionof floriculture crops, recirculating

irrigation systems have been widelyadopted to lower water usage andconserve fertilizers that can otherwisebe lost via discharge runoff (Bushet al., 2003; Hong et al., 2003;MacDonald et al., 1994; Sanogoand Moorman, 1993). This is espe-cially true for greenhouses with largewater use [1.9–3.8 million liters perday (Meador et al., 2012)]. Ebb-and-flow and flood-floor irrigationsystems typically recirculate the irri-gation water and are used to maxi-mize production area and decreaselabor costs (Ehret et al., 2001; vanDer Gaag et al., 2001). In this type ofsystem, irrigation water is pumpedfrom a water reservoir to flood thefloor or bench at a specified waterlevel for a desired duration, and thendrained back (often by gravity flow)to the reservoir for recycling in thenext irrigation event. Although recy-cling irrigation water offers manybenefits to greenhouse growers, plantpathogens can also be disseminated inthe recycled irrigation water (Ehretet al., 2001). Thus, limiting pathogentransmission in the recirculating irri-gation systems is critical to the flori-culture industry.

Pythium species and other watermolds can be highly destructive tofloriculture crops, and spread readilyin irrigation water (Goldberg et al.,1992; Hong and Moorman, 2005;Lewis Ivey and Miller, 2013;Stanghellini et al., 1996a, 1996b).Pythium root rot causes plantstunting, wilt, and death and canalso reduce horticultural qualityof infected crops (Tompkins andMiddleton, 1950). The pathogencan become established in a green-house through infested soil and dust

UnitsTo convert U.S. to SI,multiply by U.S. unit SI unit

To convert SI to U.S.,multiply by

29,574 fl oz mL 3.3814 · 10–5

29.5735 fl oz mL 0.03380.3048 ft m 3.28083.7854 gal L 0.26422.54 inch(es) cm 0.3937

25.4 inch(es) mm 0.03941 micron(s) mm 11 mmho/cm mS�cm–1 1

28.3495 oz g 0.03530.001 ppm g�L–1 10001 ppm mg�kg–1 11 ppm mg�L–1 16.8948 psi kPa 0.1450

(�F – 32) O 1.8 �F �C (�C · 1.8) + 32

Received for publication 16 Nov. 2018. Accepted forpublication 14 June 2019.

Published online 7 August 2019.

1Department of Plant, Soil and Microbial Sciences,Michigan State University, East Lansing, MI 48824

2National Institute of Agricultural Sciences, RuralDevelopment Administration, Wanju 54875, Repub-lic of Korea

3Cook County Unit, University of Illinois Extension,Arlington Heights, IL 60004

4Department of Biosystems and Agricultural Engi-neering, Michigan State University, East Lansing, MI48824

This research was supported in part by the Non-Assistance Cooperative Agreement 58-8062-5-036between the USDA-ARS Plant Protection ResearchUnit, Ithaca, NY, the Michigan State University De-partment of Plant, Soil and Microbial Sciences, E.Lansing, MI, as part of the Floriculture and NurseryResearch Initiative, the American Floral Endowment,and Michigan State University AgBioResearch. Wethank Sheila D. Linderman for proofreading andediting of the manuscript.

S.J., C.S.K., and G.D.B. are Graduate ResearchAssistants.

B.R.H. is a Research Technician.

M.K.H. is a University Distinguished Professor.

S.I.S. and W.Z. are Associate Professors.

W.Z. is the corresponding author. E-mail: weizhang@msu.edu.

This is an open access article distributed under the CCBY-NC-ND license (https://creativecommons.org/licenses/by-nc-nd/4.0/).

https://doi.org/10.21273/HORTTECH04226-18

578 • October 2019 29(5)

(Stephens et al., 1983), contaminatedseedlings, cuttings, or other plantmaterial from propagation green-houses (van Der Gaag et al., 2001).Surface water used for irrigation canalso be infested with Pythium andPhytophthora species (Bush et al.,2003). Management of Pythium spe-cies is particularly challenging forpotted plants; frequent irrigationand high moisture levels are ideal forthe reproduction and transmission ofthis pathogen (Elmer et al., 2012).The high porosity of peat pottingmedia may also facilitate the move-ment of zoospores, which are animportant type of Pythium speciesinoculum (Oh and Son, 2008). Thus,Pythium species could be rapidly dis-seminated in a greenhouse via ebb-and-flow and flood-floor productionsystems (Hoitink, 1991), leading tocrop damage and loss and requiringproactive management strategies.

Fungicide application is a com-mon and important strategy to limitpythium root rot in greenhouse pro-duction (Moorman and Kim, 2004).Currently, two of the main fungicidesused for pythium root rot control areetridiazole (Terrazole; OHP, Main-land, PA) and mefenoxam (SubdueMaxx; Syngenta Crop Protection,Greensborough, NC) (Moormanand Kim, 2004; Raabe et al.,1981). Etridiazole effectively reducedpythium root rot in poinsettia andeaster lily (Lilium longiflorum) whenapplied as a soil drench (Ascerno et al.,1981; Hausbeck and Harlan, 2013b;Raabe et al., 1981). Also, etridiazoleis one of the few commercial fungi-cides that are labeled for chemigationin ebb-and-flow and flood-floor irri-gation systems. Mefenoxam can alsolimit crop loss from pythium root rot(Moorman et al., 2002). However,resistance to mefenoxam has devel-oped in greenhouse populations ofPythium species, partly due to re-peated fungicide use (Lookabaughet al., 2015; Moorman and Kim,2004; Moorman et al., 2002). Failureto control Pythium diseases usingmefenoxam has been reported ingreenhouses (Hausbeck and Harlan,2013a; Moorman and Kim, 2004),and resistant isolates were detected insurface water used for irrigation(Carlson et al., 2004). Fungicide re-sistance has become a limiting factorin the control of pythium crown androot rot; alternative strategies (e.g.,

filtration) for pathogen control in irri-gation water are needed (Hausbeckand Zhang, 2016).

Management of pathogens inrecycled irrigation water has beena persistent challenge in greenhouseproduction. Ultraviolet radiation,heat treatment, chemical disinfection,ozonation, and filtration have beenused to remove pathogens from irri-gation water with varying degrees ofsuccess (Ehret et al., 2001; Hong andMoorman, 2005; Raudales et al.,2017). Many of these methods arecost-prohibitive to install and operatein commercial greenhouses. In con-trast, filtration is a low-cost methodthat disinfests irrigation water by thephysical removal of pathogens usinggranular porous media (e.g., sand)or membrane filters (Hong andMoorman, 2005). Membrane filtra-tion can effectively remove zoosporesif the membrane pore size is smallenough to retain the motile zoosporesthat have a pleomorphic cellmembrane(Schuerger and Hammer, 2009).Membrane filters with pore sizes of 1and 5 mm were able to remove thePythium zoospores effectively fromrecirculating irrigation water in labora-tory tests (Tu and Harwood, 2005).However, it is unknown whether thiscould be transferable to greenhousesettings. Diplanetism (where a zoo-spore encysts and releases a smallermotile zoospore) could decrease theefficacy of membrane filters (Erwinet al., 1983), although the occurrenceof diplanetism in commercial green-houses is unknown. Additional chal-lenges with membrane filters arefrequent leakage and membrane clog-ging and fouling (Ehret et al., 2001;Tu and Harwood, 2005), resulting inincreased maintenance cost and de-creased performance over time.

In contrast, deep-bed filtration(e.g., sand filtration) is cost-effectivein terms of construction, operation,and maintenance. Slow filtration withgranular materials has been studied asa means to remove Pythium speciesfrom the greenhouse irrigation watersince the 1970s (Darling, 1977).However, it is not widely used incommercial U.S. greenhouses due tothe slow water flow rate [100–300L�m–2 per hour (Ehret et al., 2001)]that prohibits the movement of largevolumes of water to multiple green-house ranges in an acceptable timeperiod (Hong and Moorman, 2005).

Previous studies on the effectivenessof deep-bed filtration for remov-ing plant pathogens from irrigationwater have focused on slow sandfiltration (Ehret et al., 2001; Hongand Moorman, 2005; Lee and Oki,2013), whereas the effectiveness ofrapid filtration on pathogen removalhas not been well investigated. Addi-tional data could help to determinewhether this technique could beadopted to manage pathogens in irri-gation water in greenhouses.

The objective of this study was toinvestigate the ability of rapid filtra-tion systems to limit pythium root rotof potted poinsettia in greenhouseswith ebb-and-flow and flood-floorirrigation systems. Six small-scaleebb-and-flow recirculating irrigationsystems were constructed to simulta-neously test the effect of filter mediatype (sand and activated carbon),fungicide application (etridiazole),and inoculum source mode (infestedgrowth media vs. infested water) intwo greenhouse experiments. Poin-settia was selected as a model cropbecause of its popularity as a pottedflower, its economic importance, andthe prevalence of pythium root rotoutbreaks during its production.Pythium aphanidermatum was cho-sen because it is one of the mostprevalent Pythium species in green-houses and is more aggressive fordeveloping disease symptoms onpoinsettia than Pythium irregulare(Lookabaugh et al., 2015). This studywas intended to show a proof-of-concept to use rapid filtration systemsin removing Pythium propagulesfrom recirculating irrigation water.

Materials and methods

IRRIGATION AND FILTRATION SYS-TEMS. Six small-scale irrigation sys-tems were constructed to simulatethe ebb-and-flow and flood-floor sys-tems in greenhouse settings, eachconsisting of an ebb-and-flow bench(8 · 4 ft), an optional prefilter tank,an optional filter unit, and a holdingtank, as shown in Fig. 1. The filterunit was designed as shown in Fig. 2and was packed with either sand[99.69% silica (Granusil; UniminCorp., New Canaan, CT)] or acti-vated carbon [AC (Filtrasorb 300;Calgon Carbon Corp., Moon Town-ship, PA)]. Particle size distributionof the sand was 5.1% 297 to 420 mm,57.2% 420 to 595 mm, 36.1% 595 to

• October 2019 29(5) 579

841 mm, and 1.2% ‡841 mm. Theeffective size of AC particles was 0.8to 1.0 mm. Detailed description ofthe irrigation systems and filter unitsis provided in Supplemental MaterialsS1 and S2. The irrigation systems

allowed for automatic irrigation ofpotted plants placed in the bench viaflooding according to a pre-designatedschedule. These irrigation systems weresubsequently used in the two green-house experiments.

PLANT CULTURE AND IRRIGATION

WATER. One-month-old cuttings of‘Early Prestige Red’ poinsettia wereobtained from a local commercialgreenhouse. The cuttings were trans-planted into plastic nursery pots(6 inches diameter · 4 1/8 inchestall) filled with peat potting mixture(Suremix; Sun Gro Horticulture,Galesburg, MI). Fifteen plants wererandomly placed with about equalspacing on each of the six ebb-and-flow benches to ensure a sufficientnumber of replicates for each treat-ment. In Expt. 2, the poinsettia cut-tings were propagated from matureplants following a commercial propa-gation recommendation (Ecke et al.,2004) and planted into the nurserypots as described for the first experi-ment. The poinsettia plants were �6weeks old at the start of each exper-iment. Groundwater (pH 7.8 ± 0.2)was used as the source of irrigationwater. A 10N–1.1P–7.9Kwater-solublefertilizer (JR Peters, Allentown, PA)was added to the irrigation water,and the initial nutrient concentrationwas 125 mg�L–1 based on nitrogen.The prefilter tank (or the holdingtank in some filter-free treatments)in the irrigation systems was filledwith 120 L of the fertilized irriga-tion water. Plants were placed on

the benchtops with irrigation in op-eration for 2 d to acclimate to theexperimental condition before Pythiuminoculation.

P. aphanidermatum isolates 106and 319 were previously character-ized for their sensitivity to etridiazole(Krasnow and Hausbeck, 2017) andwere selected from the culture collec-tion of M.K. Hausbeck at MichiganState University (MSU, East Lans-ing). They were maintained on cornmeal agar (CMA) of 17 g�L–1. Beforethe study, the isolates were inoculatedonto poinsettia stems and reisolatedfrom the diseased stems to ensure viru-lence (Quesada-Ocampo andHausbeck,2010). Zoospores of P. aphaniderma-tum were produced according to a pre-viously established method (Rahimianand Banihashemi, 1979). The Pythiumisolates were grown on the V8 agarculture for 5 d. The V8 agar culturewas divided into six strips and sepa-rated into two sterile petri dishes of100 mm diameter. The petri disheswere floodedwith sterile distilled water(SDW), incubated at 30 �C for 24 h,drained, rinsed, and flooded with an-other 25mL of SDW. After incubationfor 10 h at ambient temperature (21 ±2 �C), the zoospores were releasedfrom the sporangia and transferred toa 2-L beaker half filled with SDW. Todetermine the concentration of thezoospore suspension, a 1-mL aliquotwas placed into a 1.7-mL microcen-trifuge tube, vortexed for 70 s toinduce the zoospore encystment,and then a 10-mL aliquot was pipet-ted onto a clean hemocytometerfor counting (Bright-Line; HausserScientific, Horsham, PA). The concen-tration of initially produced zoosporeswas 1.7 ± 0.9 · 104 zoospores/mL.The prepared suspensions of themotile biflagellate zoospores wereequally split into five 500-mL cappedbottles and hand-shaken vigorouslyfor 90 s to induce zoospore encyst-ment. The suspension of encystedzoospores (490 mL of 1.7 · 104

zoospores/mL) was added to theprefilter or holding tank and thor-oughly agitated with a wooden dowel.The resultant zoospore concentrationin the irrigation water was 68 ± 36zoospores/mL. During the green-house experiments, the irrigationwater was passed through the filterunit and then stored in the holdingtank before the next irrigation event.Two additional inoculations were

Fig. 1. Schematic of the small-scale ebb-and-flow irrigation system constructed ingreenhouse; 1 inch = 2.54 cm.

Fig. 2. Schematic of the filter unit: (A)12-V water pump, (B) check valve,(C) union fitting, (D) pressure sensor,(E) polyvinyl chloride (PVC) plugfitting, (F) PVC adapter fitting, (G)PVC coupling, (H) PVC end capfitting, (I) bulkhead fitting, and (J)motorized ball valve (50 cm = 19.7inches).

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RESEARCHREPORTS

made 2 and 4 weeks after the initia-tion of the experiment to increasedisease pressure. To maintain a suffi-cient volume of water during eachexperiment, the irrigation water con-taining fertilizer was added when thewater volume decreased to �70% to80% of the initial volume.

GREENHOUSE EXPERIMENTS.Two greenhouse experiments wereconducted in a temperature-con-trolled greenhouse that included sixexperimental treatments: 1) nonino-culated irrigation water treat-ment without filtration or pathogen(– Control), 2) inoculated irrigationwater treatment without filtration orfungicide (+ Control), 3) inoculatedirrigation water treatment with thesand filter, 4) inoculated irrigationwater treatment with the AC filter,5) inoculated irrigation water treat-ment with the fungicide (etridiazole)application in the absence of anyfilters, and 6) diseased plant treat-ment in the absence of any filters. InTreatment 5, the etridiazole (Terra-zole 35WP) was applied at the labeledrate (250 mg�L–1) into the irrigationwater in the holding tank before thefirst inoculation so that all inoculatedPythium zoospores had similar fungi-cide exposure. In the diseased planttreatment (Treatment 6), three of the15 planting pots were directly inocu-lated with the P. aphanidermatumzoospores and then placed randomlyin Expt. 1 and at the back locationnear the drainage hole of the bench inExpt. 2, as shown in SupplementalFig. 1. The encysted zoosporesuspension (50 mL) was added toa 4-cm-deep depression in the pot-ting mix 2 cm from the stem of thehealthy plant. The pots were removedfrom the bench during inoculation toavoid contamination of the benchand then replaced on the bench af-ter inoculation. This treatment wasdesigned to assess whether the path-ogen could spread among the plants ifnot directly introduced into the irri-gation water. The poinsettia plantswere irrigated twice per day at 0900and 1500 h. Expt. 1 was initiated on29 Oct. 2014 and concluded on 6Jan. 2015 (69 d in duration). Expt. 2was initiated on 2 Apr. 2015 andconcluded on 19 June 2015 (78 d induration). The initiation day was con-sidered to be that of the first inocula-tion. Due to light contaminationfrom external streetlights at night,

the plants in Expt. 1 experienced aninterruption of the dark period that isrequired to initiate flowering. Thus,in Expt. 2, the plants were coveredwith a thick black cloth at night toallow for flower initiation. The waterpressure inside the filter unit wasmonitored in real time, along withthe water temperature in the holdingtank, and air temperature and relativehumidity in the greenhouse. The ir-rigation water was sampled from theholding tanks at the beginning, mid-dle, and end of each experiment todetermine pH, electrical conductivity(EC), and nutrient concentrations.After the pH and EC measurements,water samples were filtered througha 0.45-mm membrane filter andstored in a –20 �C freezer for laternutrients analyses by the MSU Soiland Plant Nutrient Laboratory (EastLansing).

The mean (± SD) air temperatureand relative humidity were 26.8 ±2.9 �C and 30% ± 7.4% in Expt. 1,and 26.1 ± 3.3 �C and 36% ± 16.7% inExpt. 2, respectively (SupplementalFig. 2). The mean Darcy water veloc-ities through the AC and sand filterswere 19.6 ± 0.5 and 10.5 ± 2.2cm�min–1 in Expt. 1, and 18.6 ± 1.3and 8.6 ± 1.0 cm�min–1 in Expt. 2,respectively. The mean Darcy watervelocities were calculated based on3-d averages at the beginning of eachexperiment (n = 6). Thus, the twoexperiments had consistent water ve-locities through the filters. Operatingwater pressure of the AC and sandfilters were maintained at 6.9 ± 1.4and 5.7 ± 1.0 kPa, respectively (Sup-plemental Fig. 3). Because filtrationsystems with a mean water velocity of8.3 to 25 cm�min–1 are classified asrapid sand filtration (Gadgil, 1998;Huisman and Wood, 1974; WorldHealth Organization, 1996), relativeto slow sand filtration (0.17–0.5cm�min–1), these filtration systemsare classified as rapid filtration (8.6–19.6 cm�min–1) and low pressure.

The pH and EC of the irrigationwater were 7.8 ± 0.4 and 1.6 ± 0.3mS�cm–1, respectively, during the ex-perimental periods (SupplementalFigs. 4 and 5); water temperaturewas in the range typical for a green-house (Supplemental Fig. 6). Theconcentrations of macronutrients,[i.e., nitrate (NO3

–), phosphorus(P), potassium (K), calcium (Ca),magnesium (Mg), and sodium (Na)]

in the irrigation water are shown inSupplemental Table 1. The nutrientlevels (i.e., NO3

–, P, K, Ca, and Mg)in the AC filter treatment were gen-erally lower than those of other treat-ments, although the difference wasless in Expt. 2. The micronutrients[i.e., iron (Fe), copper (Cu), and zinc(Zn)] were completely removed fromthe recycled irrigation water in the ACfilter treatment (Supplemental Fig. 7).

Due to the limitation in the num-ber of constructed irrigation systemsand the greenhouse space, we couldnot include multiple irrigation systemsfor each treatment in the two green-house experiments. However, the suf-ficient number of poinsettia plants ineach treatment (15 plants) allowed forreliable assessment of plant responsesto treatment. The two experimentswere not exactly replicated due toslightly varied air temperature andrelative humidity in the greenhouseand the interruption of dark periodin Expt. 1 as described earlier. None-theless, these two experiments couldstill ensure the reliability of our con-clusions especially if they were drawnfrom the results obtained under variedexperimental conditions.

PLANT ASSESSMENTS. To assessthe performance of the filtration sys-tems in controlling pythium root rotoutbreaks, the poinsettia plants wereevaluated at the end of the experi-ments for foliar and root weight, rootrot severity, and horticultural quality.The roots were visually examined forroot necrosis in 15 plant pots usinga scale adapted from Boehm andHoitink (1992) where 1 = no symp-toms; 2 = mild root rot, less than one-third of plant roots affected; 3 =intermediate root rot, one-third totwo-thirds of plant roots affected;4 = severe root rot, more than two-thirds of plant roots affected; 5 =severe root and crown rot; and 6 =dead plant. This rating was madewithout removing the potting mixfrom the roots. In Expt. 2, the plantswere rated for their horticulturalquality (Ecke et al., 2004) based onthe appearance (e.g., color, height,and bract area) on the scale from 1(high aesthetic quality) to 5 (no aes-thetic quality) (Supplemental Fig. 8).At harvest, the roots were carefullywashed, and the fresh weight of thepoinsettia shoots and roots were mea-sured. The shoot and root sampleswere then oven-dried at 60 �C for 3 d

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and measured for their dry weight.Isolation of P. aphanidermatum fromthe roots of each plant was attempted.The roots were rinsed gently underrunning tap water to remove anyadhering potting mixture. Three wa-ter-soaked or discolored roots wereselected per plant and were surface-sterilized in 70% ethanol, blotted dry,and plated onto BARP (50 ppm ben-omyl, 100 ppm ampicillin, 30 ppmrifampicin, and 200 ppm pentachlor-onitrobenzene)-amended CMA, andincubated at 30 �C for 24 h. Apercentage of colonies were trans-ferred to CMA and confirmed as P.aphanidermatum based on sporan-gial and oospore morphology as wellas colony appearance using the key ofvan der Plaats-Niterink (1981). Thenumber of plants with the presence ofP. aphanidermatum was divided bythe total number of plants (n = 15) todetermine the root infection ratio.

Chlorophyll a and b concen-trations in the poinsettia leaveswere measured using a colorimetricmethod (Wellburn, 1994). Threepoinsettia plants were randomly se-lected, and 0.5 g of the leaf samplewas collected from more than threeleaves in themiddle part of each plant.The fresh leaf samples were processedimmediately after collection. Theweighed leaf samples were homoge-nized with the addition of 10 mL of80% acetone. The extract was centri-fuged at 2500 rpm for 5 min, andthe supernatant was diluted 10 timesby adding 80% acetone. The finalextracted solution was analyzed forthe chlorophyll concentrations bya spectrophotometer (Varian Cary50 Bio; Agilent Technologies, SantaClara, CA). To analyze the macro-and micronutrients [i.e., N, sulfur(S), Mg, Ca, Na, boron (B), Zn,manganese (Mn), Fe, Cu, and alumi-num (Al)] in the poinsettia leaves, thestems were removed and the remain-ing leaf tissues were ground beforebeing analyzed at the A & L GreatLakes Laboratories, Inc. (Fort Wayne,IN) following the standard methodsof the Association of Official Analyt-ical Chemists.

STATISTICAL ANALYSES. Statisticalanalyses of the experimental data wereperformed with R software using theleast significant difference (LSD) Rpackage for parametric tests and the‘‘coin’’ R package for a nonparametrictest such as the rating data (i.e., the

ratings of root rot severity and horti-cultural quality). Treatments werecompared by one-way analysis of var-iance (P £ 0.05). When a significant Fvalue was determined, means wereseparated by the LSD’s multiple com-parison test. Also, the Student’s t testwas used to compare paired samples.Data of preinoculated plants in thediseased plant treatment were notincluded in the statistical analyses.To compare nonparametric data,such as root rot severity and horticul-ture quality, the Kruskal-Wallis testwas used with the ‘‘coin’’ R package.Its statistical significance probabilityvalue was adjusted (P £ 0.034) byBonferroni correction to reduce therisk of committing Type I errors formultiple comparison.

ResultsPoinsettia plants in the sand fil-

ter treatment consistently showedhealthy roots similar to those of thenoninoculated control, demonstrat-ing the effectiveness of rapid sandfiltration in limiting Pythium infec-tion. In Expt. 1, the level of rootnecrosis in the inoculated controlplants was significantly (P < 0.004)more than that of the noninoculatedcontrol, sand filter, AC filter, anddiseased plant treatments (Table 1).Root necrosis in the etridiazole

treatment was the second highest. InExpt. 2, the levels of root necrosis inthe inoculated control plants, ACfilter, and etridiazole treatments weresignificantly higher (P < 0.004) thanthose of the noninoculated control,sand filter, and diseased plant treat-ments (Table 1). In Expt. 1, P. apha-nidermatum was isolated from theroots of 93%, 80%, and 50% ofthe plants for the inoculated control,etridiazole, and diseased plant treat-ments, respectively, and the pathogenwas not isolated from the roots of thenoninoculated control, AC, or sandfilter treatments (Fig. 3). The pres-ence of P. aphanidermatum in thediseased plant treatment did not re-sult in significant difference in theroot necrosis compared with that ofthe noninoculated control treatment.In Expt. 2, P. aphanidermatum wasisolated only from the roots in theinoculated control (93% incidence).However, significant root necrosiswas observed for plants in the ACfilter and etridiazole treatments, andseveral plants in the diseased planttreatment (Supplemental Fig. 9B).

The plants in Expt. 1 did notproperly develop red bracts due to theinterruption of the dark period bylight contamination (SupplementalFig. 10A) and thus could not beevaluated for aesthetic quality. In

Table 1. Ratings of root rot severity and horticultural quality of poinsettia plantsin Expts. 1 and 2 (n = 15).z

Treatmentz

Expt. 1y Expt. 2y

Root rot severity Root rot severity Horticultural quality

[mean ± SD (1–6 scale)]x [mean ± SD (1–5 scale)]w

–Control 1.7 ± 1.0 av 1.1 ± 0.4 av 1.4 ± 0.6 av

+Control 3.8 ± 1.0 b 3.2 ± 0.9 b 3.0 ± 0.8 bAC filter 1.4 ± 0.5 a 3.2 ± 0.6 b 4.7 ± 0.5 cSand filter 1.7 ± 0.9 a 1.2 ± 0.4 a 1.7 ± 0.5 aDiseased plant 1.8 ± 1.1 a 1.5 ± 1.2 a 2.3 ± 0.8 abEtridiazole 2.5 ± 1.2 ab 3.0 ± 0.7 b 1.9 ± 0.6 az–Control = noninoculated irrigation water treatment without filtration or pathogen; +Control = inoculatedirrigation water treatment without filtration or fungicide; AC filter = inoculated irrigation water treatment with theactivated carbon filter; Sand filter = inoculated irrigation water treatment with the sand filter; Diseased plant =diseased plant treatment in the absence of any filters; Etridiazole = inoculated irrigation water treatment with thefungicide (etridiazole) application in the absence of any filters. In the diseased plant treatment, the infested plantingpots were placed randomly in Expt. 1, but at the back location near the drainage of the bench in Expt. 2.yExpts. 1 and 2 were performed to assess the effectiveness of rapid filtration and fungicide application to controlpythium root rot of poinsettia in greenhouse. Irrigation water in constructed ebb-and-flow recirculating irrigationsystems was inoculated with encysted Pythium aphanidermatum zoospores at 68 ± 36 zoospores/mL (2011.0 ±1064.6 zoospores/fl oz) at the initiation and then 2 and 4 weeks afterward in each experiment. Each Expt. 2included six treatments; sand and activated filters were used to remove Pythium zoospores from infested irrigationwater. Expt. 1 experienced the interruption of the dark period.xRoot rot severity was rated as per the following scale: 1 = no symptoms; 2 = mild root rot, less than one-third ofplant roots affected; 3 = intermediate root rot, one-third to two-thirds of plant roots affected; 4 = severe root rot,more than two-thirds of plant roots affected; 5 = severe root and crown rot; and 6 = dead plant.wHorticultural quality was rated according to the appearance (e.g., color, height, and bract area) on the scale from1 (high aesthetic quality) to 5 (no aesthetic quality).vMeans within a column with different letters are significantly different based on the Kruskal-Wallis withBonferroni corrected test (P < 0.004).

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Expt. 2, the inoculated control plantshad an inferior appearance with a hor-ticultural quality rating of 3.0, andwere not marketable (Table 1, Sup-plemental Fig. 10B). The plants sub-jected to the AC filter treatment hadthe poorest aesthetic quality witha horticultural rating of 4.7, whereasthe plants in the remainder of thetreatments had a more similar horti-cultural quality [1.4–2.3 (Table 1)].

In Expt. 1, the sand filter, etri-diazole, diseased plant, and nonino-culated control treatments hadsignificantly higher plant height, fo-liar fresh weight, and root dryweight than those of the inoculatedcontrol plants [P < 0.05 (Fig. 4)].However, plants in the AC filtertreatment had significantly lowerfresh foliar weight and root dryweight than those of the noninocu-lated control [P < 0.05 (Fig. 4)]. InExpt. 2, the sand filter, etridiazole,diseased plant, and noninoculatedcontrol treatments had significantlyhigher plant height, and foliar freshand dry weight than those of the

inoculated control and AC filtertreatment [(P < 0.05 (Fig. 5)].

Chlorosis of young leaves wasobserved for plants in the AC filtertreatment in both experiments (Sup-plemental Fig. 11). Chlorophyll a andb concentrations for the medium-sized leaves after harvest were signifi-cantly lower in the AC filter treatmentthan those of other treatments (Table2). The leaves had higher levels of Sand Ca and lower levels of B, Zn, Fe,and Cu in the AC filter treatment,relative to the noninoculated control(Table 3). There was no significantdifference in the concentrations ofN, P, K, Mg, Na, Mn, and Al in theleaves from theACfilter treatment andthe noninoculated control treatments(Table 3).

DiscussionOur results demonstrated that

poinsettia plants under rapid sandfiltration were consistently similar tothe non-inoculated control, regard-ing the presence of P. aphaniderma-tum, root necrosis, plant height and

weight, and horticultural quality (Ta-ble 1, Figs. 3–5, Supplemental Fig.10). Thus, rapid sand filtration effec-tively limited pythium root rot andmaintained poinsettia plant quality,likely by removing P. aphaniderma-tum zoospores from the irrigationwater. Many laboratory and green-house experiments have consistentlyshown that sand filters can effectivelyremove pythiaceous zoospores fromwater (D�eniel et al., 2004; Jeon et al.,2016; Lee and Oki, 2013; Ufer et al.,2008; van Os et al., 1998; Vankuik,1994). Similar results have also beenfound with slow sand filtration underexperimental conditions (Darling,1977; D�eniel et al., 2004; Lee andOki, 2013; van Os et al., 1998,2000). The slow sand filtration wasinitially developed for wastewatertreatment through biological pro-cesses; a biological layer termedSchmutzdecke is the most critical fac-tor for purification (Huisman andWood, 1974). However, others sug-gested that the main mechanismsto remove pythiaceous zoospores

Fig. 3. Representative roots of poinsettia at the end of Expt. 1 (69 d after inoculation with Pythium aphanidermatumzoospores). Expt. 1 was performed to assess the effectiveness of rapid filtration and fungicide application to control pythiumroot rot of poinsettia in greenhouse, including six treatments: –Control = noninoculated irrigation water treatment withoutfiltration or pathogen; DControl = inoculated irrigation water treatment without filtration or fungicide; Sand filter =inoculated irrigation water treatment with the sand filter; AC filter = inoculated irrigation water treatment with the activatedcarbon filter; Etridiazole = inoculated irrigation water treatment with the fungicide (etridiazole) application in the absence ofany filters; Diseased plant = diseased plant treatment in the absence of any filters. Irrigation water in constructed ebb-and-flowrecirculating irrigation systems was inoculated with encysted P. aphanidermatum zoospores at 68 ± 36 zoospores/mL (2011.0 ±1064.6 zoospores/fl oz) at the initiation of Expt. 2 and then 2 and 4 weeks afterward. Sand and activated filters were used toremove P. aphanidermatum zoospores from infested irrigation water. In the diseased plant treatment of Expt. 1, the infestedplanting pots were placed randomly. Expt. 1 experienced the interruption of the dark period. IR is the infection ratio of P.aphanidermatum in roots.

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primarily include surface attachment,pore straining, and adhesive interac-tions of the zoospores in porousmedia (D�eniel et al., 2004; Jeonet al., 2016; van Os et al., 1998,2000). Thus, the physicochemicallycontrolled rapid filtration can be a vi-able alternative for treating recycledirrigation water in the greenhouse.Slow sand filtration is often not suit-able for greenhouse production dueto its low water flow rate and the largearea required for the filtration system(Wohanka and Helle, 1996). In thisstudy, the flow velocity of the rapidsand filter was �40–50 times that intypical slow sand filters and wouldthus meet the water demand in com-mercial greenhouses with a small in-stallation area.

In the AC filter treatment therewas no presence ofP. aphanidermatum

zoospores (Fig. 3). However, theplant weight and height under theAC filter treatment were, in general,significantly reduced compared withthe noninoculated and sand filtertreatments (Figs. 4 and 5). BecauseP. aphanidermatum was not detectedin the relatively healthy-appearingroots in the AC filter treatment ofExpt. 1 (Fig. 3), the leaf chlorosisand stunted growth may haveresulted from abiotic factors. As theAC removed essential micronu-trients (e.g., Fe, Cu, Mn, and Zn)from the irrigation water (Supple-mental Fig. 7), and the concentra-tions of Fe, Cu, and B in the plantleaves (Table 3) were in the defi-cient range (Campbell, 2000), leafchlorosis was likely a result of micro-nutrient deficiency. The chlorosis ofyoung leaves indicates Fe deficiency

(Hochmuth et al., 2004; McCauleyet al., 2009). Similarly, kiwifruit(Actinidia sp.) showed the Fe de-ficiency symptoms when a soil wasamended with a wood-based bio-char (Sorrenti et al., 2016), a car-bon-rich porous media sharing somesimilar properties to those of the AC(Chen et al., 2011; Hale et al., 2011).Additionally, interplays between thenutrient absorption by poinsettiaroots and nutrient removal by theAC filter were also observed. Nutrientdeficiency in Expt. 2 may have causedsevere root necrosis for plants in theAC filter treatment, which mightin turn result in insufficient macronu-trient absorption and subsequentlyhigher nutrient levels in irrigationwater than those in Expt. 1. In gen-eral, the removal of nutrients by theACmay prevent its adoption for water

Fig. 4. Poinsettia height (A), foliar fresh weight (B), foliar dry weight (C), and root dry weight (D) in Expt. 1. Expt. 1 wasperformed to assess the effectiveness of rapid filtration and fungicide application to control pythium root rot of poinsettia ingreenhouse, including six treatments: –Control = noninoculated irrigation water treatment without filtration or pathogen;DControl = inoculated irrigation water treatment without filtration or fungicide; Sand filter = inoculated irrigation watertreatment with the sand filter; AC filter = inoculated irrigation water treatment with the activated carbon filter; Etridiazole =inoculated irrigation water treatment with the fungicide (etridiazole) application in the absence of any filters; Diseased plant =diseased plant treatment in the absence of any filters. Irrigation water in constructed ebb-and-flow recirculating irrigationsystems was inoculated with encysted Pythium aphanidermatum zoospores at 68 ± 36 zoospores/mL (2011.0 ± 1064.6zoospores/fl oz) at the initiation of Expt. 2 and then 2 and 4weeks afterward. Sand and activated filters were used to remove P.aphanidermatum zoospores from infested irrigation water. In the diseased plant treatment of Expt. 1, the infested plantingpots were placed randomly. Expt. 1 experienced the interruption of the dark period. The different lower case letters abovecolumns indicate the significant difference in means at P < 0.05 by least significant difference test; 1 cm = 0.3937 inch, 1 g =0.0353 oz.

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treatments in commercial greenhouses(Runia, 1993). Despite its effective-ness in controlling plant pathogens,AC filters would be impractical forcommercial poinsettia productiondue to poor plant quality and nutri-ent deficiency, unless fertilizers couldbe properly applied through meansother than fertigation in irrigationwater.

In the etridiazole treatmentacross both experiments, poinsettiashad similar weight and height tothose in the noninoculated control(Figs. 4 and 5). However, root in-fection was not prevented by fungi-cide application. P. aphanidermatumwas isolated from roots exhibiting anintermediate level of root rot severityin Expt. 1 (Fig. 3; Table 1). In Expt.2, the plant roots also displayed an

intermediate level of root rot severity(Table 1), but no P. aphanidermatumwas isolated, probably due to a lowlevel of P. aphanidermatum nearthe detection limit of the isolationmethod. In a previous study usingthe ebb-and-flood system, when etri-diazole was incorporated into thegrowing medium, necrosis was re-duced at the stem base of cucumbers(Cucumis sativus), whereas P. apha-nidermatum recovery from rootswas not decreased (Sanogo andMoorman, 1993). In another studyusing a hydroponic system, etridia-zole applied in the recirculating irri-gation water reduced root rot of ivy(Hedera sp.) but not as effectively asthe fungicide mefenoxam (Jamartet al., 1988). Thus, the rate of etri-diazole in the irrigation water may

not be adequate in controlling infec-tion, due to the presence of P. apha-nidermatum in the poinsettia roots inExpt. 1. The labeled rate of etridia-zole did not cause 100% mortality tozoospores of sensitive isolates of P.aphanidermatum in vitro (Krasnowand Hausbeck, 2017). Additionally,application of sublethal doses of fun-gicides may influence sensitivity ofPythium species to fungicides andexacerbate disease symptoms (Garz�onet al., 2011). Plants may not beadequately protected if the fungicideconcentration is below the thresholdnecessary to prevent disease. Symp-toms of root rot occurred with etri-diazole-treated water in the absenceof P. aphanidermatum in Expt. 2(Supplemental Fig. 9B), suggestingthat the recommended dosage of this

Figure 5. Poinsettia height (A), foliar freshweight (B), and foliar dryweight (C) in Expt. 2. Expt. 2 was performed to assess theeffectiveness of rapid filtration and fungicide application to control pythium root rot of poinsettia in greenhouse, including sixtreatments: –Control = non-inoculated irrigation water treatment without filtration or pathogen; DControl = inoculatedirrigation water treatment without filtration or fungicide; Sand = inoculated irrigation water treatment with the sand filter;AC = inoculated irrigation water treatment with the activated carbon filter; Etridiazole = inoculated irrigation water treatmentwith the fungicide (etridiazole) application in the absence of any filters; Diseased plant = diseased plant treatment in the absenceof any filters. Irrigation water in constructed ebb-and-flow recirculating irrigation systems was inoculated with encystedPythium aphanidermatum zoospores at 68 ± 36 zoospores/mL (2011.0 ± 1064.6 zoospores/fl oz) at the initiation of Expt. 2and then 2 and 4 weeks afterward. Sand and activated filters were used to remove P. aphanidermatum zoospores from infestedirrigation water. In the diseased plant treatment of Expt. 2, the infested planting pots were placed at the back location near thedrainage of the bench. The different lower case letters above columns indicate the significant difference inmeans at P < 0.05 byleast significant difference test; 1 cm = 0.3937 inch, 1 g = 0.0353 oz.

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fungicide may cause phytotoxicity inroots that resemble root rot symp-toms (Dumroese et al., 1990). Futurestudies are needed to confirm theobservations in this proof-of-conceptstudy and to further explore underly-ing mechanisms.

To demonstrate pathogen trans-mission from growth media to plant,we randomly placed three infestedpots among 12 healthy plant pots.In Expt. 1, P. aphanidermatum frominfested pots was transferred tohealthy plants (Fig. 3). The infectiondid not result in a significant differ-ence in the average root rot severityor plant height and weight (Table 1;Fig. 4), likely due to low migrationof zoospores from inoculated potsto irrigation water, limited sporan-gial and zoospore production frominfected plants, or the masking effectfrom other healthy plants (Supple-mental Fig. 1). Some of the infected

plants exhibited root rot symptomsby the end of the experiment (Sup-plemental Fig. 9A). Transmission ofP. aphanidermatum from infestedgrowth media to healthy plantsappeared less efficient than infestingthe irrigation water, and has beenobserved by other researchers(Stanghellini et al., 1996b). Thepathogen did not spread to healthyplants when infested pots were placedat the back near the drain (Supple-mental Fig. 1A). It is possible that thereleased zoospores from the infectedroots could be drained away with theirrigation water, and then becomeattached to the surfaces of pipes andwalls during encystment (Gubleret al., 1989). It is unlikely that thezoospores would be transportedagainst the flow direction. Thus, zoo-spores were possibly transported byirrigation water to infect the plants inExpt. 1, as the infested plants were

located along the flow direction in themiddle of the bench (SupplementalFig. 1). Future studies should be di-rected to explore the effectiveness ofcontrol practices such as filtration andfungicide application for preventingPythium diseases of plants when in-oculum is applied to growing mediaand plants rather than to irrigationwater.

It was noted that after�1monthfrom the start of Expt. 2, a significantwater flow reduction was observed inthe sand filter treatment over time,likely due to clogging of the sandfilter by debris or biofilm (Vankuik,1994). The clogging can usually beremediated easily by backwash thatis often performed in typical filtra-tion operations (Tu and Harwood,2005). Recently, Kim et al. (2015)reported that a pungent oil of freshginger (Zingiber officinale), 6 gin-gerol, reduced Pseudomonas aeruginosa

Table 3. Macro- and micronutrients in the leaves of poinsettia in activated carbon filter and negative control treatments ofExpt. 2 (n = 3).z

TreatmentsyMacronutrients [mean ± SD (%)]

Nitrogen Sulfur Phosphorous Potassium Magnesium Calcium Sodium

AC filter 3.07 ± 0.21 ax 0.33 ± 0.01 a 0.52 ± 0.12 a 4.52 ± 0.51 a 0.97 ± 0.19 a 2.04 ± 0.17 a 0.030 ± 0.01 a–Control 3.39 ± 0.02 a 0.29 ± 0.01 b 0.41 ± 0.02 a 3.57 ± 0.04 a 0.68 ± 0.06 a 1.55 ± 0.03 b 0.013 ± 0.00 a

Micronutrients [mean ± SD (mg�kgL1)]w

Boron Zinc Manganese Iron Copper Aluminum

AC filter 14.7 ± 4.7 a 31.3 ± 4.2 a 129.7 ± 16.7 a 44.0 ± 8.3 a 2.0 ± 0.8 a 41.7 ± 13.3 a–Control 41.7 ± 2.9 b 59.3 ± 3.3 b 95.7 ± 7.6 a 74.0 ± 3.3 b 5.0 ± 0.8 b 39.3 ± 5.4 azExpt. 2 was performed to assess the effectiveness of rapid filtration and fungicide application to control pythium root rot of poinsettia in greenhouse. Irrigation water inconstructed ebb-and-flow recirculating irrigation systems was inoculated with encysted Pythium aphanidermatum zoospores at 68 ± 36 zoospores/mL (2011.0 ± 1064.6zoospores/fl oz) at the initiation of Expt. 2 and then 2 and 4 weeks afterward.y–Control = noninoculated irrigation water treatment without filtration or pathogen; AC filter = inoculated irrigation water treatment with the activated carbon filter used toremove Pythium zoospores from irrigation water.xStudent’s t test, and different letters in the same column indicate a significant difference at P < 0.05.w1 mg�kg–1 = 1 ppm.

Table 2. Chlorophyll a and b concentrations in leaves of greenhouse poinsettia in Expts. 1 and 2.

TreatmentszExpt. 1y Expt. 2y

Chlorophyll a (mg�kgL1)x Chlorophyll b (mg�kgL1) Chlorophyll a (mg�kgL1) Chlorophyll b (mg�kgL1)

–Control 854 bcw 1526 bc 324 ab 2208 ab+Control 982 a 1788 a 263 bc 1821 bcAC filter 330 d 520 d 81 d 508 dSand filter 783 c 1361 c 260 c 1772 cDiseased plant 959 a 1613 b 302 abc 2037 abcEtridiazole 922 ab 1538 b 347 a 2345 az–Control = noninoculated irrigation water treatment without filtration or pathogen; +Control = inoculated irrigation water treatment without filtration or fungicide; ACfilter = inoculated irrigation water treatment with the activated carbon filter; Sand filter = inoculated irrigation water treatment with the sand filter; Diseased plant = diseasedplant treatment in the absence of any filters; Etridiazole = inoculated irrigation water treatment with the fungicide (etridiazole) application in the absence of any filters. In thediseased plant treatment, the infested planting pots were placed randomly in Expt. 1, but at the back location near the drainage of the bench in Expt. 2.yExpts. 1 and 2were performed to assess the effectiveness of rapid filtration and fungicide application to control pythium root rot of poinsettia in greenhouse. Irrigation water inconstructed ebb-and-flow recirculating irrigation systems was inoculated with encysted Pythium aphanidermatum zoospores at 68 ± 36 zoospores/mL (2011.0 ± 1064.6zoospores/fl oz) at the initiation and then 2 and 4 weeks afterward in each experiment. Each Expt. 2 included six treatments; sand and activated filters were used to removePythium zoospores from infested irrigation water. Expt. 1 experienced the interruption of the dark period.x1 mg�kg–1 = 1 ppm.wLeast significant difference test was performed and different letters within a column indicate a significant difference in means at P < 0.05.

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biofilm formation up to 53% by inhib-iting quorum sensing-regulated viru-lence behaviors. In addition, severalquorum-sensing inhibitors includingRNAIII-inhibiting peptide, usnicacid, and a natural secondary metab-olite of lichen could also inhibit bio-film formation (Algburi et al., 2017).However, no study has been con-ducted on whether the quorum sens-ing inhibitors can maintain the waterflow rate in a sand filter. Thus, tomaintain the proper water flow rate,backwash was conducted once perweek after 1 month and then everyother day during the last 3 weeks ofExpt. 2. No significant reduction ofwater flow rate was found in Expt. 1,so backwash was only performed twotimes during the final month of theexperiment. Because performing thebackwash sustained the desired waterflow rate in our sand filters, usingrapid sand filtration may be an optionto limit Pythium species in recycledirrigation water in commercial green-houses due to its low cost to install,maintain, and operate (Ehret et al.,2001). It is possible that some of thefiltered zoospores would be flushedout to the prefilter tank and thenfiltered again in subsequent filtrationevents. Alternatively, backwash watercould be bypassed and collected sep-arately for disposal.

ConclusionThe findings of this study

had several important implicationsfor controlling Pythium diseases ingreenhouse floriculture crops. Theinfection and disease symptoms inpoinsettia plants by P. aphaniderma-tum were effectively controlled bythe sand and AC filters duringrapid filtration with low water pres-sure. Rapid sand filtration maintainedpoinsettia quality comparable to thatof the noninoculated control. How-ever, the AC filter removed essentialnutrients from the irrigation waterand caused Fe deficiency symptomsin poinsettia plants. Thus, rapid sandfiltration could be a viable optionfor minimizing Pythium outbreaksthrough recycled irrigation water,whereas the use of rapid AC filtrationfor recirculating irrigation systemsseems to be impractical, unless nutri-ents can be applied separately otherthan through irrigation water. Theapplication of etridiazole did notcompletely prevent Pythium infection

(e.g., root rots), but plant quality interms of weight, height, and horticul-tural quality was not compromised.Hence, etridiazole application is stilluseful to control Pythium outbreaks,but proper application rate of etridia-zole needs to be studied to avoidpotential phytotoxicity. When poin-settia plants were randomly infectedby P. aphanidermatum, the pathogenspread among plants in the absence ofany treatment, suggesting the need ofproactive measures to control patho-gen transmission either by fungicideapplication or filtration, which war-rants further studies. As a proof-of-concept study, the present workwas limited in scope regarding thenumber of replications, operationconditions, and pathogen exposurescenarios. Future work should focuson more replications under realisticfield conditions and on assessing thelongevity of the system performanceby optimizing filter media and oper-ation parameters (e.g., water velocityand chemistry). For instance, filterdesign can be improved by incorpo-rating anticlogging mechanisms suchas a deeper coarse layer at the inlet ora removable screen for dislodgingaccumulated debris. Sand grain sizecould also be optimized to improvewater flow while maintaining thezoospore removal efficiency. Finally,irrigation frequency and duration andbackwash scheduling may be opti-mized to ensure continuous perfor-mance of the filtration system.Overall,this study suggests that rapid sandfiltration of irrigation water can effec-tively reduce crop disease outbreaks ingreenhouses, thus potentially lower-ing use of fungicides and promotingcrop and environmental health.

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Stanghellini, M., D. Kim, S. Rasmussen,and P.A. Rorabaugh. 1996a. Control ofroot rot of peppers caused by Phytophthoracapsici with a nonionic surfactant. PlantDis. 80(10):1113–1116.

Stanghellini, M., S. Rasmussen, D. Kim,and P.A. Rorabaugh. 1996b. Efficacy ofnonionic surfactants in the control ofzoospore spread of Pythium aphani-dermatum in a recirculating hydroponicsystem. Plant Dis. 80(4):422–428.

Stephens, C., L. Herr, A. Schmitthenner,and C. Powell. 1983. Sources of Rhizoc-tonia solani and Pythium spp. in a beddingplant greenhouse. Plant Dis. 67(3):272–275.

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RESEARCHREPORTS

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Supplemental Material 1.Construction of ebb-and-flowirrigation systems

To test the effectiveness of fil-tration units in controlling diseaseoutbreaks in greenhouse-grown poin-settias, six self-contained ebb-and-flowirrigation systems (including op-tional filtration units) were con-structed (Fig. 1). A typical irrigationsystem consisted of a 8 · 4 ft (2.4 ·1.2 m) black plastic ebb-and-flowbench (Hummert, St. Louis, MO),an optional filtration unit, two 130-L(34.3-gal) holding tanks, two 12V-centrifugal water pumps, two checkvalves, two auto valves, twowater-levelsensors, and a timer. Only one hold-ing tank was included in the non-inoculated and inoculated controltreatments in Expt. 1, and in theinoculated control and ‘‘diseasedplant’’ treatments in Expt. 2. Other-wise, the prefilter tank and the hold-ing tank were directly connected forthe nonfiltration treatments. Theirrigation water was drawn from theprefilter tank by one water pump,passed through a check valve, thefilter unit, an auto valve [i.e., 0.5-inch (1.27 cm) motorized ball valve(modelMV-2-20-12V-R01-1;MISOL,Jiaxing, China)], and stored in theholding tank until a prescheduled ir-rigation time controlled by a timer.At the time of irrigation, the irriga-tion water in the holding tank waspumped into the ebb-and-flow benchvia a check valve until reaching a de-sired watering height [i.e., 1–1.5inches (2.54–3.81 cm) or 10 min ofpumping time]. The two check valveswere installed to prevent backflow.One check valve was next to the pumpconnected to the prefilter tank, andthe other next to the pump connectedto the holding tank (Fig. 1). Theirrigation water in the bench was keptfor a desired irrigation period beforebeing drained back to the prefiltertank by opening an auto valve [i.e., the0.75-inch (1.905 cm) motorized ballvalve]. Two magnetic float water-level

sensors (model a11062100ux0008,Uxcell, Kwai Fong,Hong Kong) wereinstalled in the prefilter tank andholding tank, respectively. The wa-ter-level sensor in the prefilter tankdetects the irrigation water drainedfrom the bench, and the water pumpis automatically turned on to deliverthe water to the inlet of the filter unit.The second water-level sensor turnsoff the water pump connected to theholding tank, when the water levelreaches the minimum level so as toprevent air entry into the pump.

The filter unit design is describedin detail next. Activated carbon (AC)and sand were used as filter media.Operating water pressure of the ACand sand filters was maintained at6.9 ± 1.4 and 5.7 ± 1.0 kPa (1.00 ±0.20 and 0.83 ± 0.15 psi), respec-tively. Water pressure was measuredby a pressure transducer at the topof the filters and recorded in a data-logger (model MCR-4V; T&DCorp., Matsumoto, Japan). All ofthe irrigation systems were sterilizedbefore each experiment with a solu-tion of greater than 30% of householdbleach [i.e., 6.15% sodium hypochlo-rite (NaClO) solution] was applied inthe benches and tanks using sprayers.Any adhering grime or algae was re-moved using scrub brushes. Then 10L (2.6 gal) of 5% bleach solution wasadded to the prefilter tank andallowed to recirculate a couple oftimes in the absence of a filter unit.After cleaning, the systems were thor-oughly rinsed several times with tapwater and air-dried for several days.

Supplemental Material 2. Filterunit design

Low-pressure sand and activatedcarbon (AC) filters were constructedfor the greenhouse experiments (Fig.2). Each filter unit was made with a 6-inch (15.2 cm)-diameter PVC pipe of50 cm (19.7 inches) in length. Thebottom of each filter column wassealed with an end cap fitting, andthe top of each filter was assembled

with a coupling, an adapter fitting,and a plug fitting in order. Two typesof filter media [i.e., sand (99.69%silica, Granusil; Unimin Corp., NewCanaan, CT) and AC (Filtrasorb 300;CalgonCarbon, Moon Township,PA), were used. The particle sizedistribution of the sand was 5.1%297 to 420 mm, 57.2% 420 to 595mm, 36.1% 595 to 841 mm, and 1.2%‡841 mm (1 mm = 1 micron). Theeffective size of AC particles was 0.8to 1.0 mm (0.03–0.04 inch). A 3-cm(1.2 inch) layer of coarser sand (500–841 mm) was placed at the bottom-most and uppermost layers in thesand filter and bottommost layer inthe AC filter to filter out large debrisand minimize clogging. The totaldepth of filter media was 50 cm in-cluding the coarser sand layers. Allfilter media were used directly with-out washing. To support the filtermedia and allow for free drainage offiltered water, two screens each withdifferent size openings [i.e., 0.5 · 0.5inch (1.27 cm) and 0.25 · 0.25 inch(0.64 cm)] were prepared and bent tobe fixed onto about the 2-cm (0.8inch) length of the 6-inch PVC pipeusing 12 screws and then mountedinside the end of the bottom cap. Astainless steel screenwith 100 · 100mmopening size was placed on the screens.The top of the filter media was alsocovered with the 100 · 100-mm and0.5 · 0.5-inch screens similar mannerto the bottom part to filter large debrisand allow for an even distribution ofwater flow (Fig. 2). The bottom endcapwasdrilled andfittedwith a0.5-inchpolypropylene bulkhead tank fitting(TF050; Banjo, Crawfordsville, IN) toconnect the outlet pipe with a unionfitting (Mueller/B&K, Collierville,TN) and a0.5-inchmotorizedball valve(MISOL, Jiaxing, China). The motor-ized ball valve was open during theoperation of the pump connected tothe prefilter tank. The pressure sen-sors were installed at the inlet of eachfilter. All of the components wereassembled and sealed to make water-tight columns.

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Supplemental Fig. 1. Location of preinoculated plants (red), infected plants (yellow), and healthy plants (green) at the end ofthe diseased plant treatment in Expts. 1 (A) and 2 (B). Expts. 1 and 2 were performed to assess the effectiveness of rapidfiltration and fungicide application to control pythium root rot of poinsettia in greenhouse. Irrigation water in constructedebb-and-flow recirculating irrigation systems was inoculated with encysted Pythium aphanidermatum zoospores at 68 ± 36zoospores/mL (2011.0 ± 1064.6 zoospores/fl oz) at the initiation and then 2 and 4 weeks afterward in each experiment. Theinfested planting potswere placed randomly in Expt. 1, but at the back location near the drainage of the bench inExpt. 2. Expt.1 experienced the interruption of the dark period. The number in the column is the root rot severity. A positive plant wasidentified by the isolation of P. aphanidermatum.

Supplemental Fig. 2. Air temperature and relative humidity in Expts. 1 (A) and 2 (B). Expts. 1 and 2 were performed to assessthe effectiveness of rapid filtration and fungicide application to control pythium root rot of poinsettia in greenhouse.Irrigation water in constructed ebb-and-flow recirculating irrigation systems was inoculated with encysted Pythiumaphanidermatum zoospores at 68 ± 36 zoospores/mL (2011.0 ± 1064.6 zoospores/fl oz) at the initiation and then 2 and 4weeks afterward in each experiment; (�C · 1.8) D 32 = �F.

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Supplemental Fig. 3.Water pressure in the activated carbon (AC) and sand filters in Expts. 1 (A) and 2 (B). Expts. 1 and 2wereperformed to assess the effectiveness of rapid filtration and fungicide application to control pythium root rot of poinsettia ingreenhouse. Irrigation water in constructed ebb-and-flow recirculating irrigation systems was inoculated with encystedPythium aphanidermatum zoospores at 68 ± 36 zoospores/mL (2011.0 ± 1064.6 zoospores/fl oz) at the initiation and then 2and 4weeks afterward in each experiment. Sand = inoculated irrigationwater treatment with the sand filter; Activated carbon =inoculated irrigation water treatment with the activated carbon filter. Sand and activated filters were used to remove P.aphanidermatum zoospores from infested irrigation water; 1 kPa = 0.1450 psi.

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Supplemental Fig. 4. pH of irrigation water measured in Expts. 1 (A) and 2 (B). Expts. 1 and 2 were performed to assess theeffectiveness of rapid filtration and fungicide application to control pythium root rot of poinsettia in greenhouse, including sixtreatments: LControl = noninoculated irrigation water treatment without filtration or pathogen; DControl = inoculatedirrigation water treatment without filtration or fungicide; AC = inoculated irrigation water treatment with the activatedcarbon filter; Sand = inoculated irrigation water treatment with the sand filter; Diseased plant = diseased plant treatment in theabsence of any filters; Etridiazole = inoculated irrigation water treatment with the fungicide (etridiazole) application in theabsence of any filters. Irrigation water in constructed ebb-and-flow recirculating irrigation systems was inoculated withencysted Pythium aphanidermatum zoospores at 68 ± 36 zoospores/mL (2011.0 ± 1064.6 zoospores/fl oz) at the initiationand then 2 and 4 weeks afterward in each experiment. Sand and activated filters were used to remove P. aphanidermatumzoospores from infested irrigation water. The infested planting pots were placed randomly in Expt. 1, but at the back locationnear the drainage of the bench in Expt. 2.

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Supplemental Fig. 5. Electrical conduction (EC) of irrigation water measured in Expts. 1 (A) and 2 (B). Expts. 1 and 2 wereperformed to assess the effectiveness of rapid filtration and fungicide application to control pythium root rot of poinsettia ingreenhouse, including six treatments: LControl = noninoculated irrigation water treatment without filtration or pathogen;DControl = inoculated irrigation water treatment without filtration or fungicide; AC = inoculated irrigation water treatmentwith the activated carbon filter; Sand = inoculated irrigation water treatment with the sand filter; Diseased plant = diseasedplant treatment in the absence of any filters; Etridiazole = inoculated irrigationwater treatmentwith the fungicide (etridiazole)application in the absence of any filters. Irrigation water in constructed ebb-and-flow recirculating irrigation systems wasinoculated with encysted Pythium aphanidermatum zoospores at 68 ± 36 zoospores/mL (2011.0 ± 1064.6 zoospores/fl oz) atthe initiation and then 2 and 4 weeks afterward in each experiment. Sand and activated filters were used to remove P.aphanidermatum zoospores from infested irrigation water. The infested planting pots were placed randomly in Expt. 1 but atthe back location near the drainage of the bench in Expt. 2; 1 mS�cmL1 = 1 mmho/cm.

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Supplemental Fig. 6. Irrigation water temperature in the holding tank for Expts. 1 (A) and 2 (B). Expts. 1 and 2 wereperformed to assess the effectiveness of rapid filtration and fungicide application to control pythium root rot of poinsettia ingreenhouse, including six treatments: LControl = noninoculated irrigation water treatment without filtration or pathogen;DControl = inoculated irrigation water treatment without filtration or fungicide; AC = inoculated irrigation water treatmentwith the activated carbon filter; Sand = inoculated irrigation water treatment with the sand filter; Diseased plant = diseasedplant treatment in the absence of any filters; Etridiazole = inoculated irrigationwater treatmentwith the fungicide (etridiazole)application in the absence of any filters. Irrigation water in constructed ebb-and-flow recirculating irrigation systems wasinoculated with encysted Pythium aphanidermatum zoospores at 68 ± 36 zoospores/mL (2011.0 ± 1064.6 zoospores/fl oz) atthe initiation and then 2 and 4 weeks afterward in each experiment. Sand and activated filters were used to remove P.aphanidermatum zoospores from infested irrigation water. The infested planting pots were placed randomly in Expt. 1, but atthe back location near the drainage of the bench in Expt. 2; (�C · 1.8) D 32 = �F.

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Supplemental Fig. 7. Micronutrient concentrations [iron (Fe), copper (Cu), manganese (Mn), and zinc (Zn)] in the irrigationwater in the activated carbon (AC) filter and noninoculated control treatments during Expt. 2. Expt. 2 was performed to assessthe effectiveness of rapid filtration and fungicide application to control pythium root rot of poinsettia in greenhouse.Irrigation water in constructed ebb-and-flow recirculating irrigation systems was inoculated with encysted Pythiumaphanidermatum zoospores at 68 ± 36 zoospores/mL (2011.0 ± 1064.6 zoospores/fl oz) at the initiation of the experimentand then 2 and 4 weeks afterward; 1 ppm = mg�LL1.

Supplemental Fig. 8.Horticultural rating scale in Expt. 2 (#1 = high aesthetic quality, and #5 = no aesthetic value). Expt. 2 wasperformed to assess the effectiveness of rapid filtration and fungicide application to control pythium root rot of poinsettia ingreenhouse. Irrigation water in constructed ebb-and-flow recirculating irrigation systems was inoculated with encystedPythium aphanidermatum zoospores at 68 ± 36 zoospores/mL (2011.0 ± 1064.6 zoospores/fl oz) at the initiation of theexperiment and then 2 and 4 weeks afterward.

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Supplemental Fig. 9. Poinsettia roots for evaluating root necrosis in Expts. 1 (A) and 2 (B). Expts. 1 and 2 were performed toassess the effectiveness of rapid filtration and fungicide application to control pythium root rot of poinsettia in greenhouse,including six treatments: LControl = noninoculated irrigation water treatment without filtration or pathogen; DControl =inoculated irrigation water treatment without filtration or fungicide; AC = inoculated irrigation water treatment with theactivated carbon filter; Sand = inoculated irrigation water treatment with the sand filter; Diseased plant = diseased planttreatment in the absence of any filters; Etridiazole = inoculated irrigation water treatment with the fungicide (etridiazole)application in the absence of any filters. Irrigation water in constructed ebb-and-flow recirculating irrigation systems wasinoculated with encysted Pythium aphanidermatum zoospores at 68 ± 36 zoospores/mL (2011.0 ± 1064.6 zoospores/fl oz) atthe initiation and then 2 and 4 weeks afterward in each experiment. Sand and activated filters were used to remove P.aphanidermatum zoospores from infested irrigation water. The infested planting pots were placed randomly in Expt. 1, but atthe back location near the drainage of the bench in Expt. 2. White roots indicate a healthy root system, and dark roots anddispersed growing media are symptoms of root rot. The number is the root rot severity rating. Root rot severity was rated perthe following scale: 1 = no symptoms; 2 =mild root rot, less than one-third of plant roots affected; 3 = intermediate root rot,one-third to two-thirds of plant roots affected; 4 = severe root rot, more than two-thirds of plant roots affected; 5 = severe rootand crown rot; and 6 = dead plant.

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Supplemental Fig. 10. Poinsettia plants, representative of the range of plant quality, at the end of Expt. 1 (A) (69 d afterinoculation with Pythium aphanidermatum zoospores) and Expt. 2 (B) (78 d after inoculation with P. aphanidermatumzoospores). Expts. 1 and 2 were performed to assess the effectiveness of rapid filtration and fungicide application to controlpythium root rot of poinsettia in greenhouse, including six treatments:LControl = noninoculated irrigation water treatmentwithout filtration or pathogen; DControl = inoculated irrigation water treatment without filtration or fungicide; AC =inoculated irrigation water treatment with the activated carbon filter; Sand = inoculated irrigation water treatment with thesand filter; Diseased plant = diseased plant treatment in the absence of any filters; Etridiazole = inoculated irrigation watertreatment with the fungicide (etridiazole) application in the absence of any filters. Irrigation water in constructed ebb-and-flow recirculating irrigation systems was inoculated with encysted P. aphanidermatum zoospores at 68 ± 36 zoospores/mL(2011.0 ± 1064.6 zoospores/fl oz) at the initiation and then 2 and 4 weeks afterward in each experiment. Sand and activatedfilters were used to remove P. aphanidermatum zoospores from infested irrigation water. The infested planting pots wereplaced randomly in Expt. 1 but at the back location near the drainage of the bench in Expt. 2.

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Supplemental Fig. 11. Color of leaves comparison between noninoculated control and activated carbon (AC) filter treatmentsin Expts. 1 (A) and 2 (B). Expts. 1 and 2 were performed to assess the effectiveness of rapid filtration and fungicide applicationto control pythium root rot of poinsettia in greenhouse. Irrigation water in constructed ebb-and-flow recirculating irrigationsystems was inoculated with encysted Pythium aphanidermatum zoospores at 68 ± 36 zoospores/mL (2011.0 ± 1064.6zoospores/fl oz) at the initiation and then 2 and 4 weeks afterward in each experiment.

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Supplemental Table 1. Nutrient concentrations in the irrigation water during Expts. 1 and 2.

Treatmentsz

Nutrients (mg�LL1)y

Nitrate Phosphorus Potassium (K) Calcium Magnesium Sodium

Expt. 1 (d)x

0 22 69 0 22 69 0 22 69 0 22 69 0 22 69 0 22 69

–Control 92 128 172 7.7 9.7 8.8 111 117 135 95 123 150 38 47 59 16 21 29+Control 88 128 263 7.5 6.8 8.8 100 109 149 95 123 177 41 60 73 16 22 38Sand 91 117 150 6.2 7 8.6 91 96 109 95 123 136 35 45 53 16 21 26Activated carbon 41 53 95 2.6 2.7 2.4 100 91 99 55 95 109 36 35 44 16 18 22Etridiazole 83 88 147 6.2 6.6 5.3 94 110 126 95 109 150 39 45 83 21 24 35Diseased plant 85 116 151 6.6 5.6 14.5 94 107 110 95 123 123 36 48 51 16 20 23Ground water 0 n.a. n.a. 0.4 n.a. n.a. 2 n.a. n.a. 109 n.a. n.a. 36 n.a n.a. 17 n.a. n.a.

TreatmentsyExpt. 2 (d)x

5 38 78 5 38 78 5 38 78 5 38 78 5 38 78 5 38 78

–Control 87 154 154 5.5 6.2 6.4 96 122 125 123 150 150 44 56 57 26 30 24+Control 89 154 150 7.7 9.2 5.2 103 132 143 123 150 164 41 53 62 26 30 27Sand 62 100 63 4 3.5 2.3 91 97 54 123 123 109 42 48 44 25 26 19Activated carbon 36 72 94 1.8 2.2 2.6 94 109 111 68 123 136 41 51 54 25 29 27Etridiazole 83 98 80 6.4 2.8 4.4 103 123 100 123 109 109 45 54 47 31 36 27Diseased plant 88 163 163 7 4.7 3.7 106 147 147 123 164 164 45 62 63 25 31 28Ground water 0 n.a. n.a. 0.1 n.a. n.a. 2 n.a. n.a. 95 n.a. n.a. 35 n.a. n.a. 15 n.a. n.a.z–Control = noninoculated irrigation water treatment without filtration or pathogen; +Control = inoculated irrigation water treatment without filtration or fungicide; Sand =inoculated irrigation water treatment with the sand filter; Activated carbon = inoculated irrigation water treatment with the activated carbon filter; Etridiazole = inoculatedirrigation water treatment with the fungicide (etridiazole) application in the absence of any filters; Diseased plant = diseased plant treatment in the absence of any filters. In thediseased plant treatment, the infested planting pots were placed randomly in Expt. 1, but at the back location near the drainage of the bench in Expt. 2.y1 mg�L–1 = 1 ppm.xNumbers of days from the start of the experiments. Expts. 1 and 2 were performed to assess the effectiveness of rapid filtration and fungicide application to control pythiumroot rot of poinsettia in greenhouse. Irrigation water in constructed ebb-and-flow recirculating irrigation systems was inoculated with encysted Pythium aphanidermatumzoospores at 68 ± 36 zoospores/mL (2011.0 ± 1064.6 zoospores/fl oz) at the initiation and then 2 and 4 weeks afterward in each experiment. Each experiment included sixtreatments; sand and activated filters were used to remove P. aphanidermatum zoospores from infested irrigation water. Expt. 1 experienced the interruption of the dark period.

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