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Influence of pH and Etridiazole on time of retail sale (Hausbeck et al., 1987; Moorman, 1986). Numerous Species Pythium species cause disease on or- namental crops; however, P. ulti- mum, P. aphanidermatum,and 1 2,3 Charles S. Krasnow and Mary K. Hausbeck P. irregulare are isolated frequently from symptomatic in commer- cial production (Del Castillo Munera ADDITIONAL INDEX WORDS. , geranium, , and Hausbeck, 2016; Moorman SUMMARY. Pythium root rot (Pythium sp.) is ubiquitous in Michigan greenhouses et al., 2002). Pythium species are that produce herbaceous ornamentals, an industry worth $393 million in the state. ubiquitous in natural environments, Disease symptoms include stunting, flowering delay, root rot, and death. Fungi- and maintaining production facilities cides that are highly effective against pythium root rot are limited, and pathogen free of the pathogen is difficult. Path- resistance has been documented. The objectives of this study were to determine the ogenic Pythium species were found in sensitivity of , , and isolates from symptomatic herbaceous greenhouse ornamentals to the irrigation water (Bush et al., 2003; etridiazole and to determine the influence of pH and etridiazole on Pythium Shokes and McCarter, 1979) and mycelial growth and asexual reproduction. Isolates were tested in vitro for dust from greenhouse walk alleys sensitivity to etridiazole by growing the pathogen on amended V8-agar plates sealed (Stephens et al., 1983). Soilless pot- in plastic containers to minimize fungicide loss from the vapor phase. The majority ting medium can be conducive of isolates of all three species were sensitive to the fungicide with EC50 (effective to pythium root rot because of lim- concentration resulting in 50% inhibition of linear growth) values ranging from ited microbial activity (Bolton, 1977; m Á L1 0.10 to 5.03 g mL . Two isolates of P. irregulare had an EC90 (effective Stephens and Stebbins, 1985). How- > m Á L1 concentration resulting in 90% inhibition of linear growth) value 80 g mL . ever, reductions in seedling diseases The acidity of the medium influenced the ability of etridiazole to inhibit Pythium L and root rot after amending potting mycelial growth and asexual reproduction. Agar plates amended with 1 mgÁmL 1 etridiazole and adjusted to pH 4.5 limited the mycelial growth of two P. medium with biological controls have aphanidermatum isolates and two P. irregulare isolates by 90% and 56%, re- heightened the use of such manage- spectively, compared with amended agar at pH 6.5. Sporangial formation by P. ment tools in ornamental crop pro- aphanidermatum was less frequent on mycelial disks incubated in etridiazole- duction (Lewis and Lumsden, 2001; amended sterile distilled water (SDW) at pH 4.5 than pH 6.5 (P < 0.05). P. Thrane et al., 2000). Sanitation and aphanidermatum zoospore cyst was less sensitive to etridiazole than preventive measures remain impor- sporangia or mycelial growth; however, the influence of pH and fungicide on cyst tant to reduce inoculum levels in the < m Á L1 germination was significant (P 0.01). At 250 g mL etridiazole and solution greenhouse (Stephens et al., 1983). pH 4.5, zoospore cyst germination was inhibited 99.9% compared with 94.2% at pH Additionally, greenhouses may in- 6.5. In a greenhouse experiment, disease symptoms were observed on ‘Pinto White’ advertently purchase plantlets or geranium (Pelargonium ·hortorum) in a potting medium infested with P. aphani- dermatum and adjusted to pH 4.5 or 6.5; however, health and fresh weight cuttings that are infected but asymp- were greater in low pH potting medium. Etridiazole, applied as a drench at tomatic from propagation green- transplant, did not improve control of root rot for plants grown at low pH (P > houses (Moorman and Kim, 2004; 0.05). Fresh weight of plants grown in infested potting medium adjusted to pH 4.5 van der Gaag et al., 2001). Infected and amended with a single drench of etridiazole (100 mgÁmLL1) was reduced 20%, roots or root mucilage support the statistically similar to the untreated control. Adjusting the acidity of irrigation production of oospores that may water at the time of etridiazole application in ebb and flow and flood floor become lodged in greenhouse fix- production systems could be beneficial in pythium root rot management of certain tures and piping (Zheng et al., ornamental crops if plants have tolerance to low pH. 2000) and are a source of primary inoculum. Survival of mycelium is ythium root rot causes signifi- plants, causing wilting, stunting, also possible in the controlled green- cant losses in ornamental green- delayed flowering, and plant death. house environment (Stanghellini, Phouse production in Michigan, Catastrophic losses can occur if 1974), making Pythium an intracta- an industry worth an estimated $393 plants develop symptoms near the ble problem. Greenhouse operations million (U.S. Department of Agricul- ture, 2014). Pythium species infect roots and root hairs of ornamental Units To convert U.S. to SI, To convert SI to U.S., multiply by U.S. unit SI unit multiply by Department of Plant, Soil, and Microbial Sciences, Michigan State University, East Lansing, MI 48824 29,574 fl oz mL 3.3814 · 10–5 The authors thank Samantha Borowski for technical 29.5735 fl oz mL 0.0338 assistance. This research was partially funded through 25.4 inch(es) mm 0.0394 a Cooperative Agreement between the USDA-ARS 645.1600 inch2 mm2 0.0016 Plant Protection Research Unit, Ithaca, NY and the 1 micron(s) mm1 Michigan State University Department of Plant, Soil 28.3495 oz g 0.0353 and Microbial Sciences (#58-8062-5-036). 28,350 oz mg 3.5274 · 10–5 1Former Graduate Research Assistant 0.001 ppm gÁL–1 1,000 Á –1 2Professor 1 ppm mg L 1 1 ppm mgÁmL–1 1 3 Corresponding author. E-mail: [email protected]. 0.001 ppm mLÁL–1 1,000 doi: 10.21273/HORTTECH03633-16 (F – 32) O 1.8 F C(C · 1.8) + 32

• June 2017 27(3) 367 RESEARCH REPORTS that use flood floor or ebb and flow mitochondrial membrane between Pythium root rot continues to cause bench systems that recycle irrigation cytochromes b and c (Halos and losses in commercial greenhouses de- water are at increased risk of spread- Huisman, 1976b). The mode of ac- spite routine fungicide use (Hausbeck ing Pythium speciesasmultiple tion of etridiazole is considered mul- and Harlan, 2013; Moorman and ranges may be irrigated in the same tisite as cellular proteins may also be Kim, 2004), planting resistant culti- day and effectively disperse pathogen disrupted (Lyr, 1995). Resistance to vars (Chagnon and Belanger, 1991), propagules (Hoitink, 1991). etridiazole has not been observed in and preventive measures (Stephens The , mefenoxam [ac- Pythium species (Hausbeck and Harlan, and Stebbins, 1985), heightening tive enantiomer of metalaxyl (Subdue 2013; Jamart et al., 1988; Price and the importance of integrated disease Maxx; Syngenta Crop Protection, Fox, 1986; Raabe et al., 1981; management. Our objectives were Greensborough, NC)] and etridia- Stephens and Stebbins, 1985), al- to a) determine the sensitivity of P. zole (Terrazole; OHP, Mainland, though mechanisms for tolerance to ultimum, P. aphanidermatum, and P. PA), have been used for 40 years the fungicide are recognized (Halos irregulare isolates from Michigan to manage pythium root rot of orna- and Huisman, 1976a). Rotation of greenhouses to etridiazole; and mental crops (McCain and Byrne, fungicides with different modes of b) determine the influence of pH 1966; Moorman and Kim, 2004; action has been recommended to de- and etridiazole on P. aphaniderma- Raabe et al., 1981). Historically, lay the development of fungicide re- tum and P. irregulare growth and these fungicides have provided effec- sistance in Pythium and Phytophthora asexual reproduction. tive control of pythiaceous organ- populations (Ferrin and Rohde, isms when applied as soil drenches 1992; Hausbeck and Harlan, 2013); Materials and methods (Benson, 1979; Hausbeck and Harlan, however, there are limited numbers P. aphanidermatum (n = 9), P. 2013; Raabe et al., 1981; Stephens of additional fungicide options that irregulare (n = 14), and P. ultimum and Stebbins, 1985) or mixed into effectively control pythium root rot. (n = 14) isolates originally recovered the potting medium (McCain and Recently, labeled fungicides with ac- from symptomatic floriculture crops Byrne, 1966). Resistance to mefe- tivity toward phytophthora root rot in Michigan were selected from the noxam has developed in greenhouse (Phytophthora sp.), such as fenamidone culture collection of M.K. Hausbeck populations of Pythium and Phytoph- (Hausbeck and Harlan, 2013), have at Michigan State University (Table thora because of the site-specific been determined to be ineffective 1). The isolates were maintained on mode of action of the fungicide and against pythium root rot in green- cornmeal agar [CMA (17 gÁL–1 corn- selection pressure from the repeated house trials (Enzenbacher et al., meal)]. Molten V8 agar [163 mLÁL–1 use of this active ingredient (Del 2011; Santamaria and Uribe, 2013). V8 juice, 3 gÁL–1 calcium carbonate –1 Castillo Munera and Hausbeck, Cultural practices that reduce (CaCO3), 16 gÁL agar] was cooled 2016; Lamour et al., 2003; Moorman growth and dissemination of Pythium to 50 C and amended with etridia- et al., 2002). In Michigan, greater species are important in disease- zole (Terrazole 35 WP dissolved in than 35% of Pythium species isolates management programs (Hausbeck and SDW) at concentrations of 0, 0.1, collected from greenhouses were re- Harlan, 2013; Price and Fox, 1986; 1.0, 2.5, and 6.2 mgÁmL–1 a.i. For six sistant to mefenoxam (Del Castillo Stephens and Stebbins, 1985). Lim- isolates of P. irregulare, preliminary Munera and Hausbeck, 2016). Con- iting the duration of irrigation in ebb EC50 values were outside of this range trol failures have been reported in and flow systems reduced pythium of concentrations. These isolates were greenhouses and nurseries (Ferrin root rot of poinsettia (Euphorbia pul- additionally tested at 62 mgÁmL–1 and Rohde, 1992; Moorman and cherrima), geranium, and chrysanthe- etridiazole. A 7-mm diameter colo- Kim, 2004) and may become more mum (Chrysanthemum morifolium) nized agar plug from the margin of common if resistant isolates are acci- compared with standard irrigation a 2- to 4-d-old CMA culture was dently moved among greenhouses practices (Elmer et al., 2012). Poin- placed in the center of an amended with prefinished plants and propaga- settia grown in a Pythium-infested plate, and the plates were incubated tive material (Moorman et al., 2002). potting medium adjusted to pH 4.0– in the dark at ambient temperature Greenhouses that recycle irrigation 4.5 remained healthy compared with (21 ± 1 C) in a plastic chamber that water face additional challenges as plants grown in an infested potting was sealed to minimize the loss of etri- resistant isolates may be selected medium at pH levels >5.5 (Bateman, diazole to the vapor phase (Ioannou and when mefenoxam is applied repeat- 1962; Bolton, 1980). Substrate pH Grogan, 1984). Radial growth on edly (Ferrin and Rohde, 1992) and can also influence the effectiveness two axes was measured 3 d post in- sublethal fungicide residues that leach of certain biocides. Chlorine, a disin- oculation. There was one plate per from pots into recirculating irrigation festant widely used in greenhouse etridiazole concentration for each iso- water can enhance disease (Garzon production facilities, was the most late, and the experiment was con- et al., 2011). effective in reducing Streptococcus ducted three times. Growth inhibition Etridiazole is a lipophilic fun- speciesgrowthinanacidicsolution (percent) was calculated by compar- gicide that contains a heterocyclic (Shannon et al., 1965). The influ- ing radial growth on amended agar nitrogen ring and has specificity to- ence of solution pH on fungicides plates with radial growth on non- ward (Lyr, 1995; Vuik used to control Pythium species in amended control plates. Amended et al., 1990). Etridiazole disrupts ornamental production is not fully plateswereused1to2dafterpre- the lipid structure of cell membranes understood; however, pH is known paring the medium. (Radzuhn and Lyr, 1984) and in- to affect fungicide efficacy (Smith P. aphanidermatum isolates 106 hibits respiration by binding to the et al., 1946; Wolfe et al., 1976). and 319 and P. irregulare isolates 125

368 • June 2017 27(3) Table 1. Isolates of P. aphanidermatum, P. irregulare, and P. ultimum obtained formation and zoospore cyst germi- from infected ornamental plants in Michigan, and evaluated in vitro for nation, SDW was buffered with 20 sensitivity to etridiazole. mL citric acid and dibasic phosphate, Isolate no.z Pythium species Hosty Locationx adjusted to pH 4.5 or 6.5 with NaOH · or HCl, and amended with etridiazole 106 P. aphanidermatum Geranium (P. hortorum)Bat 0, 0.1, 0.5, and 2.5 mgÁmL–1.An 670 P. aphanidermatum Geranium G agar slice (5 mm diameter, 0.5– 672 P. aphanidermatum Geranium G 1.0 mm thickness) was removed from 283 P. aphanidermatum Poinsettia (E. pulcherrima)Dthe surface of a 5-mm diameter plug 288 P. aphanidermatum Poinsettia D of a 2-d-old CMA culture of P. apha- 290 P. aphanidermatum Poinsettia D nidermatum (106 and 319), placed 292 P. aphanidermatum Poinsettia D into a 60-mm petri dish containing 302 P. aphanidermatum Poinsettia D 6 mL of etridiazole-amended solution, 319 P. aphanidermatum Poinsettia D and incubated at 30 C in the dark for 108 P. irregulare Geranium H 12 h. The agar slice was removed with 115 P. irregulare Geranium D forceps to a glass microscope slide and 125 P. irregulare Geranium D rated for the density of lobate and 464 P. irregulare Geranium C inflated sporangia at three random 468 P. irregulare Geranium C 1-mm2 focal planes per slice with 667 P. irregulare Geranium G a compound microscope (100·) us- 458 P. irregulare Geranium A ing a 0–2 scale, where 0 = no sporu- 49 P. irregulare Poinsettia E lation, 1 = light sporulation (1–5 94 P. irregulare Poinsettia B sporangia/mm2), and 2 = heavy spor- 98 P. irregulare Poinsettia B ulation (6–20 sporangia/mm2). Two 306 P. irregulare Poinsettia D agar slices were evaluated per plate 308 P. irregulare Poinsettia D with two plates per pH · concentra- 147 P. irregulare Snapdragon ()D tion; the experiment was conducted 149 P. irregulare Snapdragon D three times. 19 P. ultimum Poinsettia E Zoospores of P. aphaniderma- 26 P. ultimum Poinsettia E tum (319) were produced according 46 P. ultimum Poinsettia E to the method of Rahimian and 50 P. ultimum Poinsettia E Banihashemi (1979). A 5-d-old V8-agar 52 P. ultimum Poinsettia F culture was divided into six strips and 56 P. ultimum Poinsettia F separated into two sterile 100-mm 65 P. ultimum Poinsettia F diameter petri dishes. The dishes were 69 P. ultimum Poinsettia F flooded with SDW, incubated at 76 P. ultimum Poinsettia F 30 C for 24 h, drained, rinsed, and 80 P. ultimum Poinsettia F flooded with SDW. After incubation 422 P. ultimum Poinsettia F for 10 h, zoospores were enumerated 424 P. ultimum Poinsettia F with a hemocytometer. Motile zoo- 433 P. ultimum Poinsettia F spores (1 · 104 in 1 mL) were added 439 P. ultimum Poinsettia F to 1 mL of solution adjusted to a pH Total 37 of 4.5 or 6.5 containing etridiazole at z Isolates selected from the culture collection of M.K. Hausbeck at Michigan State University. m Á –1 yHost plant of original isolation. 0, 50, 100, and 250 g mL in a xIsolates with the same letter designation were collected from the same greenhouse. 2-mL screwcap vial and incubated for 10 min. The vial was vortexed briefly, and a 100-mL aliquot was removed and 147 were used to determine the diameter agar plug of P. aphanider- and added to a flask containing influence of pH and etridiazole on matum or P. irregulare from an ac- 100 mL SDW. The suspension was Pythium growth inhibition. To de- tively growing CMA culture was immediately filtered through 2.5-mm termine radial growth inhibition, the transferred to the center of each plate, pore-size quantitative filter paper (GE pH of the agar medium was adjusted and the plates were incubated at Healthcare, Pittsburgh, PA), and the with sterile 1 N sodium hydroxide ambient temperature (21 ± 1 C) in filter paper was plated onto BARPR (NaOH) and 1 N hydrochloric acid a sealed chamber under constant fluo- (50 ppm benomyl, 100 ppm ampicil- (HCl) before autoclaving to obtain rescent light for 3 d. Colony diameter lin, 30 ppm rifampicin, 200 ppm CMA at pH 4.5 and 6.5. Adjusting was measured on two axes, and the pentachloronitrobenzene, and 10 ppm the pH of CMA to 7 before auto- percentage of growth inhibition was rose bengal)-amended CMA. After claving was necessary to obtain a pH calculated as described. There were 1 d, the filter paper was removed, of 6.5 (C.S. Krasnow, unpublished two replicate plates per fungicide and the colonies were enumerated. data). Etridiazole was incorporated concentration and pH, and the exper- Two vials per pH · concentration into molten CMA to achieve final iment was conducted three times. were used with three replicates per concentrations of 0, 0.5, 1.0, 2.0, To determine the influence of vial, and the experiment was con- 4.0, and 8.0 mgÁmL–1.A5-mm pH and etridiazole on sporangial ducted twice.

• June 2017 27(3) 369 RESEARCH REPORTS

A greenhouse experiment to as- and stunting; 4 = necrotic lower concentration and pH as fixed effects. sess the influence of potting medium leaves, stunting, and necrotic stem There was a significant pH · fungicide pH and etridiazole on pythium root tissue (black leg) at the soil line; and concentration interaction (P < 0.05) rot of geranium was conducted. 5 = plant death. Plant height and so a slice statement was used to test ‘Pinto White’ geranium was seeded width were measured at planting simple main effects. Residual data were into 288-cell plug flats and grown for and at the conclusion of the experi- analyzed to ensure heteroscedasticity. 4 weeks in a research greenhouse at ment, and plant volume was deter- Michigan State University. Soilless- mined using the shape of a cylinder to Results peat potting mixture (Suremix Mich- approximate plant size (Enzenbacher Based on mycelial growth, P. igan Grower Products, Galesburg, et al., 2015). Plants were excised at irregulare was not as sensitive to etri- MI) was adjusted to pH 4.0 to 4.5 the soil line, and the aboveground diazole as P. aphanidermatum and P. and 6.5 to 7.0 by periodically adding plant fresh weight was recorded. ultimum (Table 2). Mean EC50 values –1 sulfuric acid (1% H2SO4) or potas- About 10% of inoculated plants were were 2.64, 0.97, and 0.58 mgÁmL sium hydroxide (1% KOH) for 3 randomly selected, the roots rinsed, etridiazole for P. irregulare, P. apha- weeks before the experiment to per- and three water-soaked and symp- nidermatum,andP. ultimum, respec- mit pH equilibration. Potting mix- tomatic roots per plant isolated onto tively. Two P. irregulare isolates ture samples were taken weekly, and BARPR-amended CMA and con- exhibited a notably reduced sensitivity the acidity was measured (1 potting firmed as P. aphanidermatum based to etridiazole compared with the other medium:2 SDW) using a glass elec- on sporangial morphology (van der isolates, with EC90 values of 134.0 and trode pH meter (Mettler-Toledo, Plaats-Niterink, 1981). The trial was 84.8 mgÁmL–1 (data not shown). The Columbus, OH). The potting mix- arranged as a completely randomized mean slope values for mycelial growth ture was autoclaved for 45 min at design with 10 plants per treatment inhibition of P. irregulare, P. aphani- 121 C immediately before the exper- and was conducted once. dermatum,andP. ultimum were iment. Millet (Pennisetum glaucum) SAS (version 9.4; SAS Institute, 2.90, 2.36, and 2.34, respectively inoculum (Quesada-Ocampo and Cary, NC) was used to analyze data (data not shown). Hausbeck, 2010) was prepared by from the study. Mycelial growth- Linear mycelial growth of autoclaving millet seed (100 g), dis- inhibition data were analyzed with lin- pythium was influenced by the pH tilled water (72 mL), and L-asparagine ear regression in PROC REG. The of etridiazole-amended CMA. At –1 (0.08 mg) in mushroom bags (RJG EC50 and EC90 were interpolated CMA pH 4.5 and 1 mgÁmL etridia- Sales, Port Richey, FL) twice con- from growth inhibition data. Growth zole, there was a >96% reduction of secutively, and adding seven 7-mm inhibition data were transformed to mycelial growth of P. aphaniderma- agar plugs colonized by P. aphanider- probits, and etridiazole concentration tum isolates compared with the non- matum. Infested millet seed was was log-transformed before analysis amended controls (Table 3); growth grown under constant fluorescent to straighten the dosage–response inhibition was <70% of the controls at light for 3 to 4 weeks and mixed curve. The data for the influence of CMA pH 6.5. Radial growth after weekly before use. Isolates 106 and pH and etridiazole on mycelial growth 3 d on pH 4.5 CMA at 1 mgÁmL–1 319 were used to infest millet singly inhibition, sporangial production, and etridiazole was <5 mm for P. aphani- and were mixed 1:1 (v/v) immedi- inhibition of zoospore cyst germi- dermatum isolates and <25 mm for ately before the experiment. The ge- nation are presented as inhibition P. irregulare isolates, compared with ranium seedlings were transplanted relative to the nonamended control >60 mm for the controls at both pH into 3-inch diameter pots containing plates, and differences were sepa- levels (Table 3). The slope of the potting mixture adjusted to pH 4.0 to rated with Fisher’s protected least dosage–response curve for mycelial 4.5 or 6.5 to 7.0 and 3 gÁL–1 Pythium- significant difference test (LSD; P = growth on the log-probit scale was infested millet, an inoculum density 0.05) using PROC MIXED. Normal- steeper at pH 4.5 than pH 6.5 for P. selected based on previous research ity of residual data was assessed using aphanidermatum and P. irregulare on root rot symptom development in PROC UNIVARIATE. Data from isolates (Fig. 1), indicating greater geranium (Hausbeck et al., 1989). each experiment were pooled before inhibition at low pH. Control pots received 3 gÁL–1 steril- analysis as assumptions for homoge- Etridiazole inhibited P. aphani- ized millet seeded with sterile V8- neity of variance were met. Data on dermatum sporangial production agar plugs. Etridiazole was applied at plant fresh weight, volume, and dis- more effectively at low pH (P < transplantingasan80mL/plant ease severity rating were analyzed us- 0.01). Sporangia were absent on my- drench at 0, 10, 50, and 100 mgÁmL–1, ing PROC MIXED with etridiazole celial disks immersed in a solution at followed by an 80-mL drench of water to improve etridiazole move- Table 2. Mean concentration resulting in 50% and 90% inhibition of linear ment into the potting medium. The growth (EC50 and EC90, respectively) for Pythium species grown on etridiazole- greenhouse day/night temperatures amended V8 agar. were 27/26 C; supplemental light- EC z EC ing was provided by sodium lamps for 50 90 16 h per day. Plants were rated for Pythium species Mean ± SD Range Mean ± SD Range disease severity 4 weeks post inocula- P. aphanidermatum 0.97 ± 0.276 0.55–1.36 6.89 ± 3.81 2.68–14.67 tion using a 1–5 scale; 1 = healthy; 2 = P. irregulare 2.64 ± 1.16 0.36–5.03 25.20 ± 37.33 6.29–134.02 minor chlorosis and stunting; 3 = P. ultimum 0.58 ± 0.86 0.10–3.29 7.03 ± 4.70 3.59–20.50 z chlorotic or necrotic lower leaves EC50 and EC90 values interpolated from regression equations describing the dosage–response relationship.

370 • June 2017 27(3) Table 3. Influence of solution pH and etridiazole on radial growth of P. Phytophthora species (Benson, 1979; aphanidermatum and P. irregulare isolates on corn meal agar. Ioannou and Grogan, 1984; Jamart x et al., 1988). P. parasitica and P. Etridiazole concn Radial growth (mm) Isolatez Pythium species (mgÁmLL1)y pH 4.5w pH 6.5 cinnamomi mycelial growth and spo- rangial formation had EC50 values 0.0 64.0 82.0* <1.0 mgÁmL–1 etridiazole (Benson, 0.5 23.8 — 1979; Ioannou and Grogan, 1984). 106 P. aphanidermatum 1.0 2.2 25.7* Price and Fox (1986) reported similar 2.0 0.0 8.4* etridiazole sensitivity for a P. irregu- 4.0 0.0 6.7* lare isolate from Australian soil with 8.0 — 0.0 –1 an EC50 value <1.0 mgÁmL and 86% 0.0 77.7 82.0 mycelial growth inhibition at 10 0.5 31.4 — mgÁmL–1. An isolate of P. irregulare 319 P. aphanidermatum 1.0 2.5 26.5* from ivy (Hedera sp.) had EC50 values 2.0 0.0 9.8* of 3.44 and 0.28 mgÁmL–1 for mycelial 4.0 0.0 6.2* growth and oospore formation, re- 8.0 — 0.0 spectively, on etridiazole-amended 0.0 81.1 82.0 CMA (Jamart et al., 1988). The 0.5 53.5 — growth of some P. irregulare isolates 125 P. irregulare 1.0 8.5 19.5* in the current study at >50 mgÁmL–1 2.0 0.2 9.8* etridiazole and detection of EC90 4.0 0.0 6.1* values of 84.8 and 134.0 mgÁmL–1 8.0 — 0.8 for two isolates of this species suggest 0.0 82.0 82.0 that there may be diversity in etridia- 0.5 70.3 — zole sensitivity of P. irregulare from 147 P. irregulare 1.0 24.7 56.7* Michigan greenhouses. A limited 2.0 0.0 30.8* number of P. irregulare isolates were 4.0 0.0 7.9* tested in previous studies, and base- 8.0 — 0.3 line threshold values for etridiazole zIsolates selected from the culture collection of M.K. Hausbeck at Michigan State University. sensitivity were not established y1 mgÁmL–1 = 1 ppm. xRadial diameter on pH-adjusted corn meal agar (CMA) amended with etridiazole measured on two axes per plate (Jamart et al., 1988; Price and Fox, after 3 d growth. Two plates/pH/concentration were used, and the experiment was conducted 3·;1mm= 1986). Including Pythium isolates 0.0394 inch. collected from ornamental crops in wCMA pH adjusted with 1 N hydrochloric acid or sodium hydroxide before autoclaving and amending with etridiazole. multiple production regions and *Indicates significant difference between pH treatments at P < 0.05, according to Fisher’s protected LSD test. years in future in vitro fungicide — = not tested. screening would provide meaningful comparisons for Michigan isolates. Even though etridiazole is one of pH 4.5 containing 2.5 mgÁmL–1 etri- data not shown). Plants grown in the primary fungicides used in nursery diazole while sporangia developed in potting medium adjusted to pH 4.5 and ornamental crop production to a solution at pH 6.5 (Table 4). Zoo- or 6.5 and treated with etridiazole did manage root rot caused by Pythium spores of P. aphanidermatum were not differ in root rot symptoms or fresh and Phytophthora species (Benson, less sensitive to etridiazole than spo- weight at any fungicide concentration 1979; Hausbeck and Harlan, 2013; rangia and mycelial growth. At 100 [P > 0.05 (Table 6)]. However, plants McCain and Byrne, 1966; Raabe mgÁmL–1 etridiazole, there was 37.7% grown in infested potting medium at et al., 1981), the level of control and 2.9% inhibition of zoospore cyst pH4.5andtreatedwithetridiazole can be variable. A single drench of germination at pH 4.5 and pH 6.5, had significantly greater plant volume etridiazole at 222 mgÁmL–1 did not respectively (Table 5). Cyst germina- than treated plants at pH 6.5 [P < 0.05 provide control of phytophthora tion was inhibited 99.9% in a pH 4.5 (data not shown)]. There was no effect root rot (P. cinnamomi)ofazalea solution containing 250 mgÁmL–1 etri- of potting medium pH on plant health (Rhododendron obtusum), although diazole, but inhibition was less (about for the noninoculated control plants four drenches at that rate or a single 94%) in a solution containing the same (data not shown). drench at 444 mgÁmL–1 effectively concentration etridiazole adjusted to limited the disease (Benson, 1979). pH 6.5 (P < 0.05). Discussion Etridiazole prevented phytophthora The potting medium pH had a Etridiazole has provided effec- root rot (P. nicotianae)oftomato significant effect on geranium health tive control of pythium root rot in (Solanum lycopersicum) when incor- in the greenhouse experiment [P < greenhouse production. Most of the porated into potting soil at 250 0.001 (Table 6)]. Plants grown in Pythium isolates tested in this study mgÁmL–1 in a greenhouse study infested potting medium at pH 4.5 were sensitive to etridiazole in vitro (Ioannou and Grogan, 1984), and remained healthy (average disease se- and displayed reduced mycelial growth a single drench of etridiazole at 100 verity rating 1.7) but were stunted with at concentrations <1.0 mgÁmL–1.The mgÁmL–1 reduced root rot of larkspur chlorotic foliage when grown at pH inhibition levels observed were similar (Delphinium sp.) to <5% (Bloch et al., 6.5 (average disease severity rating 3.5, to results with other Pythium and 1976). Etridiazole limited symptoms

• June 2017 27(3) 371 RESEARCH REPORTS of geranium root rot at 50 and 100 diseased root tissue when fungicide ionization of the toxophore (Smith mgÁmL–1 in this Michigan study; how- and zoospore or mycelial inoculum et al., 1946). Captan, a fungicide used ever, applying etridiazole at a greater were added consecutively to the recir- to control pythium root rot on some rate (labeled rate = 92–262 mgÁmL–1) culating irrigation water (Sanogo and crops in greenhouses, hydrolyzes to may improve disease control. Moorman, 1993; S. Jeon and C.S. nonfungitoxic compounds at alkaline In greenhouse experiments us- Krasnow, unpublished data). The pH (Wolfe et al., 1976), and Spergon ing ebb and flow systems, control of relatively low sensitivity of P. aphani- (tetra-chloro-p-benzoquinone) seed pythium root rot by etridiazole has dermatum zoospores to etridiazole treatment was least inhibitory to Rhi- been variable (Jamart et al., 1988; observed in the current study sug- zoctonia solani in alkaline soil as a re- Sanogo and Moorman, 1993), and gests that these propagules may not sult of chemical conversion of the Pythium species were recovered from be killed when infested recirculating fungicide (Kelman, 1947). Uptake irrigation water is treated with the of 2,5-dimercapto-1,3,4-thiadiazole fungicide. Etridiazole is not mobile in homologs by Monolinia fructicola the potting medium and soil (Helling conidia was 2· greater at pH 1.5 than et al., 1974; King and Zentmyer, at pH 6.0 (Somers, 1958). The fun- 1979), and fungicide effectiveness in gicide homologs were almost com- flood floor and ebb and flow pro- pletely unionized (99.9%) at low pH, duction systems may be affected only which might have improved cellular if the roots in the lower region of pots penetration and pathogen inhibi- are protected when etridiazole is ap- tion (Lukens, 1971; Somers, 1958). plied in the irrigation water. Conceiv- Low pH may indirectly affect inhibi- ably, zoospores or cysts present in tion by affecting fungicide solubil- fungicide-treated irrigation water ity (Buchenauer and Erwin, 1972). could enter pots from the base during Benomyl and benzimidiazole were irrigation and move with capillary more effective at reducing verticil- force to roots and potting medium lium wilt (Verticillium dahliae)of with an ineffective concentration of cotton (Gossypium hirsutum)when fungicide. Additionally, etridiazole applied as a drench in acidic solution concentration in recirculating irriga- than at alkaline pH levels (Buchenauer tion water is likely diluted by the and Erwin, 1972). Greater water sol- periodic addition of untreated water ubility of these fungicides at low pH to holding tanks (Themann et al., increased uptake by cotton plants, 2002) or by volatilization (Ioannou improving disease control. However, and Grogan, 1984) and may affect the effect of benomyl drenches on disease control during the 1-month cylindrocladium root and petiole rot Fig. 1. Dosage–response curves for labeled application interval. of lily (Spathiphyllum sp.) was not (A) Pythium irregulare isolates 125 Inhibition of radial growth and influenced by the potting medium s D ( ) and 147 ( ) and (B) P. asexual reproduction of Pythium spe- pH [pH 3.8–7.0 (Chase and Poole, aphanidermatum isolates 106 (s) D cies by etridiazole was influenced by 1987)]. Additionally, the pH of V8 and 319 ( ) on etridiazole-amended pH in this study; however, the mech- agar medium (pH 4.5–8.0) did not corn meal agar adjusted to pH 4.5 (shaded) or 6.5 (open). Radial anisms responsible for inhibition at influence mefenoxam, dimethomorph, growth was determined on two axes low pH were not determined in or fluopicolide inhibition of Phytoph- of each petri plate after 3 d. Each data the study. Solution pH may directly thora capsici mycelial growth (C.S. point is the mean of six replicate affect the stability of a fungicide Krasnow, unpublished data). Addi- plates; 1 mgÁmLL1 = 1 ppm. (Wolfe et al., 1976) or the degree of tional testing is necessary to deter- mine the effect of pH on other fungicides and biological control Table 4. Influence of solution pH and etridiazole on P. aphanidermatum agents labeled for pythium root rot sporangial formation for each of the two isolates. control in greenhouses. Sporangial formation (0–2 scale)y Growing plants in potting me- x dium maintained at low pH has been Etridiazole concn Isolate 106 Isolate 319 L recommended to control pythium (mgÁmL 1)z pH 4.5 pH 6.5 pH 4.5 pH 6.5 root rot of poinsettia (Bateman, 0 1.8 2.0* 1.5 2.0* 1962; Bolton, 1980) and in nursery 0.1 1.6 1.2 1.6 2.0* production to control Phytophthora 0.5 0.3 1.0 0.8 1.9* species (Blaker and Macdonald, 2.5 0.0 0.1 0.0 0.1 1983). In a hydroponic growing sys- z1 mgÁmL–1 = 1 ppm. tem, greater control of pythium root yDensity of sporangia at three random 1-mm2 (0.0016 inch2) focal planes of a 5-mm (0.2 inch) mycelial disk soaked in pH-adjusted solution amended with etridiazole at each concentration estimated using a 0–2 scale, where 0 = no rot of tomatoes was realized when the sporulation, 1 = light sporulation (1–5 sporangia/mm2), and 2 = heavy sporulation (6–20 sporangia/mm2). Values nutrient solution was maintained at represent the mean of six mycelial disks; 1 /mm2 = 645.1600 sporangia/inch2. pH 4.5–5.0 compared with pH 6.0– xIsolate 106 and 319 refer to isolate designation from the culture collection of M.K. Hausbeck at Michigan State University. 6.5 as a result of reduced zoospore *Indicates significant difference between pH treatments at P < 0.05, according to Fisher’s protected LSD test. motility and attachment to roots;

372 • June 2017 27(3) Table 5. Influence of solution pH and etridiazole on P. aphanidermatum Bolton, A. 1980. Effects of temperature zoospore cyst germination inhibition. and pH of soilless media on root rot of

y poinsettia caused by Pythium aphani- Zoospore cyst germination inhibition (%) dermatum. Can. J. Plant Pathol. 2:83–85. Etridiazole concn (mgÁmLL1)z pH 4.5 pH 6.5 Buchenauer, H. and D. Erwin. 1972. 50 7.2 0.0 Control of verticillium wilt of cotton by 100 37.7 2.9* spraying with acidic solutions of benomyl, 250 99.9 94.2* methyl 2-benzimidazole carbamate, and z1 mgÁmL–1 = 1 ppm. thiabendazole. J. Phytopathol. 75:124–139. yZoospore cyst germination inhibition (percent) of P. aphanidermatum (319) zoospores after the addition to fungicide-amended solution adjusted to pH 4.5 or pH 6.5, filtering the zoospores on 2.5 mm (micron) filter paper, Bush, E.A., C. Hong, and E.L. Stromberg. plating on benomyl, pentachloronitrobenzene, ampicillin, rifampicin, and rose bengal–amended corn meal agar 2003. Fluctuations of Phytophthora and and determining the reduction in cfu per milliliter compared with the nonamended control plates. *Indicates significant difference between pH treatments at P < 0.05, according to Fisher’s protected LSD test. Pythium spp. in components of a recy- cling irrigation system. Plant Dis. 87:1500– 1506. Table 6. Influence of potting medium pH and etridiazole on pythium root rot of Chagnon, M. and R. Belanger. 1991. geranium. Tolerance in greenhouse geraniums to Reduction in plant fresh wt (%)y Pythium ultimum. Plant Dis. 75:820–823. m Á L1 z x Etridiazole concn ( g mL ) pH 4.5 pH 6.5 Chase, A. and R. Poole. 1987. Effects of 0 22.0 70.8* potting medium pH and air temperature 10 30.0 43.8 on severity of cylindrocladium root and 50 26.0 22.9 petiole rot of Spathiphyllum sp. Plant Dis. 71:509–511. 100 20.0 25.0 z1 mgÁmL–1 = 1 ppm. Del Castillo Munera, J. and M.K. yReduction (percent) in fresh weight compared with the untreated noninoculated control plants. Etridiazole applied Hausbeck. 2016. Characterization of as a transplant drench [80 mL (2.71 fl oz) per plant] followed by an 80-mL drench of water to improve fungicide Pythium species associated with green- movement into the potting medium. Means represent a single greenhouse experiment with 10 plants/treatment. xPotting medium pH adjusted with sulfuric acid or potassium hydroxide for 3 weeks before the experiment. house floriculture crops in Michigan. Plant *Indicates significant difference between pH treatments at P < 0.05, according to Fisher’s protected LSD test. Dis. 100:569–576. Elmer, W., M. Gent, and R. McAvoy. plant health and yield were unaffected without adversely affecting plant 2012. Partial saturation under ebb and in uninfested nutrient solution main- health. Tolerance of greenhouse orna- flow irrigation suppresses pythium root tained at a low pH (Huang and Tu, mentals to extended periods of low pH rot of ornamentals. Crop Prot. 33:29–33. 1998). Geranium plant health did not should be tested further. Enzenbacher, T., R. Naegele, and M. appear to be affected by low pH Hausbeck. 2015. Susceptibility of green- potting medium in the present study, Literature cited house ornamentals to Phytophthora capsici similar to poinsettia that remained and P. tropicalis. Plant Dis. 99:1808–1815. Bateman, D.F. 1962. Relation of soil pH healthy when grown in potting Enzenbacher, T.B., H. Hausbeck, and B. to development of poinsettia root rot. medium adjusted to pH 4.0–4.5 Harlan. 2011. Evaluation of registered Phytopathology 52:559–566. (Bateman 1962; Bolton 1980). How- and experimental fungicides for control of ever, Biernbaum et al. (1988) reported Benson, D.M. 1979. Efficacy and in pythium root rot of geranium, 2010. iron and manganese toxicity in her- vitro activity of two systemic acylala- Plant Dis. Mgt. Rpt. 5:OT001. baceous ornamentals grown at low nines and ethazole for control of Phy- Ferrin, D. and R. Rohde. 1992. In vivo ex- pH, and soilless potting me- tophthora cinnamomi root rot of azalea pression of resistance to metalaxyl by a nurs- dium is typically maintained at (Rhododendron obtusum). Phytopathol- ery isolate of Phytophthora parasitica from pH 5.5–6.0 in commercial green- ogy 69:174–178. Catharanthus roseus. Plant Dis. 76:82–84. houses to optimize nutrient up- Biernbaum, J.A., W.H. Carlson, C. take (Biernbaum et al., 1988; Garzon, C.D., J.E. Molineros, J.M. Shoemaker, and R. Heins. 1988. Low pH Yanez, F.J. Flores, M. del Mar Jimenez- Warncke and Krauskopf, 1983). causes iron and manganese toxicity. In this study, the growth of all Gasco, and G.W. Moorman. 2011. Sub- Greenhouse Grower 6(3):92–93, 96–97. lethal doses of mefenoxam enhance three Pythium species was reduced by Blaker, N.S. and J.D. Macdonald. 1983. pythium damping-off of geranium. Plant etridiazole at an acidic pH. The in- Dis. 95:1233–1238. fluence of low pH and etridiazole on Influence of container medium pH on spo- Pythium species highlights the impor- rangium formation, zoospore release, and Halos, P.M. and O. Huisman. 1976a. infection of rhododendron by Phytophthora Mechanism of tolerance of Pythium species tance of monitoring water acidity cinnamomi. Plant Dis. 67:259–263. when etridiazole is applied with irri- to ethazol. Phytopathology 66:152–157. gation water in flood floor or ebb and Bloch, E.D., R.D. Raabe, and J.H. Halos, P.M. and O. Huisman. 1976b. flow production systems. Etridiazole Hurlimann. 1976. Control of pythium Inhibition of respiration in Pythium may provide insufficient control of root rot of larkspur. Plant Dis. Rptr. spp. by ethazol. Phytopathology 66:158– root rot when applied in irrigation 60:600–601. 164. water maintained near a neutral pH. Bolton, A. 1977. The severity of root rot Hausbeck, H. and B. Harlan. 2013. Improvements in integrated Pythium and persistence of in Pythium root rot in the greenhouse. 8 Sept. management may be realized if irriga- geranium cuttings grown in soilless mix- 2016. .

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