`
`ELSEVIER
`
`Crop Protection 21 (2002) 41-47
`
`Crop
`Protection
`
`www.elsevier.com/locate/cropro
`
`Assessment of interactions between components of fungicide mixtures
`against Monilinia fructicola
`
`K.M. Emery, H. Scherm*, A.T. Savelle
`Department of Plant Pathology, University of Georgia, Athens, GA 30602, USA
`
`Received 17 October 2000; received in revised form 5 March 2001; accepted 27 April 2001
`
`Abstract
`
`Mixtures of fungicides with different modes of action can exhibit synergism,i.e. an inhibition of pathogen growth abovethat
`expected from independentaction of the mixture components. Two-way mixtures of commercial formulations of propiconazole with
`either benomyl, captan, chlorothalonil, cyprodinil or vinclozolin were evaluated in vitro for potential synergism in inhibiting
`Monilinia fructicola, the causal agent of blossom blight and brownrot of stone fruits. Propiconazole was emphasized becauseofits
`widespread use and the recent detection of isolates of M. fructicola with reduced sensitivity to this fungicide. Experiments included
`each active ingredient at low, medium andhigh concentrationsin all possible pairwise combinations. Inhibition of radial growth of
`twoisolates of M. fructicola was notsignificantly different (P > 0.01) from that predicted by a simple model of independent action
`for any of the fungicide-concentration combinations, indicating absence of synergism between active ingredients. Results were
`similar when mixtures of propiconazole with either benomyl, chlorothalonil or cyprodinil were evaluated on peachfruit treated with
`fungicide. While fungicide mixtures are useful in delaying the development of fungicide resistance, they are unlikely to be used in
`practice unless synergistic interactions allow for applications at reduced concentrations. The absence of synergism suggestslittle
`incentive exists for favoring propiconazole-based fungicide mixtures over a rotating schedule of fungicides for control of and
`resistance management in M.fructicola. © 2002 Elsevier Science Ltd. All rights reserved.
`
`Keywords: Brown rot; Fungicide mixture; Monilinia fructicola; Peach; Prunus persica
`
`1. Introduction
`
`Control of Monilinia fructicola (G. Wint.) Honey is
`critical in peach (Prunus persica (L.) Batsch) production
`worldwide. Fungicide applications are made during
`bloom to control the blossom blight phase of the disease
`and again before harvest to prevent brown rot of the
`fruit (Byrde and Willetts, 1977; Horton et al., 2000).
`While control is generally adequate, frequent applica-
`tions are costly and repeated use of the same active
`ingredient can lead to the development of fungicide
`resistance. Indeed, reduced sensitivity toward various
`fungicides has been well documented in both field and
`laboratory populations of M. fructicola (Ritchie, 1983;
`Michailides et al., 1987; Zehr et al., 1991; Elmer and
`Gaunt, 1993; Braithwaite et al., 1995; Sanoamuang and
`Gaunt, 1995). This includes a recent report from South
`Carolina of reduced sensitivity to the demethylation-
`
`*Corresponding author. Fax: + 1-706-542-1262.
`E-mail address: scherm@uga.edu (H. Scherm).
`
`inhibiting fungicide propiconazole (Zehr et al., 1999)
`which is widely used to control the pathogen on stone
`fruits in the southeastern USA.
`The application of fungicides with different modes of
`action either on a rotating schedule or in a mixtureis a
`generally recommended resistance managementstrategy
`(Staub, 1991; Russell, 1995; Bertrand and Padgett,
`1997). Compared with a rotating schedule, fungicide
`mixtures provide the potential for synergistic interac-
`tions, which can increase control to a level above that
`expected from the sum of the individual components
`(Gisi, 1996). Because of increased control efficacy with
`mixtures that act synergistically, concentrations of the
`mixture components can be reduced, thereby reducing
`costs
`(Bertrand and Padgett,
`1997). For example,
`synergistic interactions among cymoxanil, mancozeb
`and oxadixyl against various Phytophthora species in
`vitro (Gisi et al., 1985) translated into less fungicide
`needed to control potato late blight,
`caused by
`Phytophthora infestans, when mixtures containing these
`active ingredients were used in the field (Samoucha and
`
`0261-2194/02/$-see front matter © 2002 Elsevier Science Ltd. All rights reserved.
`PII: S0261-2194(01)00062-X
`
`SYNGENTA EXHIBIT 1016
`Syngenta v. UPL, PGR2023-00017
`
`SYNGENTA EXHIBIT 1016
`Syngenta v. UPL, PGR2023-00017
`
`

`

`42
`
`K.M. Emery et al. | Crop Protection 21 (2002) 41-47
`
`Cohen, 1989). Similarly, the synergistic interaction of
`pyrazophos and propiconazole against the barley net
`blotch pathogen Pyrenophora teres in an in vitro assay
`correlated with enhanced disease control when the
`mixture was applied in the greenhouse (Zeun and
`Buchenauer, 1991).
`Knowledge about the general nature of interactions
`among active ingredients is important for determining
`the potential value of fungicide mixtures; however,
`no such research has been reported in relation to
`M.fructicola. Thus, the objective of this study was to
`evaluate interactions between fungicides against M.
`fructicola as a first step toward assessing the potential
`for the development of synergistic mixtures that could
`provide satisfactory disease control while also aiding in
`resistance management. Experiments werecarried out in
`vitro (on amended media) and in vivo (on treated peach
`fruit) with a focus on mixtures containing the fungicide
`propiconazole. This active ingredient was emphasized
`because of its widespread use in the southeastern USA
`and the documented risk of resistance development
`(Zehret al., 1999).
`
`2. Materials and methods
`
`2.1. Maintenance offungal cultures and production of
`inoculum
`
`Experiments were carried out with two isolates of
`M.fructicola, isolate H-211 from Georgia andisolate
`ZN-21 from South Carolina (obtained from E. I. Zehr,
`Clemson University). The effective concentrations of
`propiconazole that
`reduced growth by 50% (ECso
`values) were determined as 0.0027 and 0.0038 pg/ml
`for H-211 and ZN-21, respectively, using the in vitro
`assays described below. Theisolates were maintained on
`propiconazole-amended V-8 juice agar slants at 5°C.
`Inoculum was produced on canned peach slices
`(Nevill et al., 1978) on wire racks
`in sterile tissue
`culture boxes. Each peachslice was inoculated with an
`agar plug from a 5- to 7-day-old culture of M. fructicola
`
`and incubated at room temperature (ca. 25°C) in the
`dark for 5-7 days. To harvest conidia, peach slices
`were placed in Erlenmeyer
`flasks and washed in
`sterile distilled water for 15 min on a wrist action
`shaker. The suspension was
`filtered through two
`layers of cheesecloth, and conidia were counted with
`the aid of a hemacytometer. The concentration of the
`suspension was adjusted to range from 1.2 to 1.8 x 10°
`conidia/ml.
`
`2.2. Dose-response curves
`
`Dose-response curves were generated to identify
`concentrations of individual active ingredients that
`inhibited growth of M. fructicola by 10, 50 and 90%
`(ECio, ECsq and ECoo, respectively). Serial dilutions of
`commercial formulations of six fungicides (benomy]l,
`captan, chlorothalonil, cyprodinil, propiconazole and
`vinclozolin; Table 1) were madeinsterile distilled water.
`Aliquots of the fungicide suspensions were incorporated
`into Czapek—Dox agar buffered with 11.5 g/l TES (N-
`tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid;
`Sigma, St. Louis, Missouri) to provide eight different
`concentrations in the agar medium ranging from 0.0001
`to 1000 pg/ml. Czapek—Dox agar, a synthetic medium
`lacking aminoacids, was selected because certain amino
`acids can interfere with fungicidal action in vitro
`(Masneret al., 1994). The medium was dispensed into
`100-mm plastic petri dishes at a volume of 25-30 ml per
`dish; five dishes for each fungicide concentration were
`prepared. The medium was inoculated with 40ul of
`a conidial suspension of isolate H-211 dispensed into a
`5-mm diameter well cut in the center of each dish with
`a sterile cork borer. After 7 days at room temperature
`in the dark, colony diameters were measured across
`perpendicular axes. For each fungicide and concentra-
`tion,
`inhibition of radial growth compared with the
`untreated check (growth on non-amended medium) was
`calculated. The experiment was repeated and results
`were combined for analysis.
`Logit-transformed values of growth inhibition were
`plotted against log9-transformed fungicide concentra-
`
`Table 1
`Active ingredients used in the assessmentof interactions between components of fungicide mixtures against Monilinia fructicola
`
`Common name
`
`Formulation
`
`Manufacturer
`
`ECjo"
`
`ECs
`
`ECoo
`
`0.0774
`0.0266
`0.0091
`Du Pont
`Benlate SOWP
`Benomyl
`38.6162
`4.662
`0.5628
`Zeneca Ag Products
`Captan S5OWP
`Captan
`0.1485
`0.0356
`0.0085
`Zeneca Ag Products
`Bravo Weather Stik
`Chlorothalonil
`0.4476
`0.0536
`0.0064
`Novartis
`Vangard 75WP
`Cyprodinil
`0.0113
`0.0027
`0.0006
`Novartis
`Orbit 3.6EC
`Propiconazole
`
`
`
`
`
`Ronilan DF BASF 0.0046 0.0668Vinclozolin 0.9660
`
`*ECi9, ECs9 and ECoo are the concentrations (in g/ml) of active ingredient that reduced radial growth of isolate H-211 on fungicide-amended
`Czapek—Doxagar by 10%, 50% and 90%, respectively.
`
`

`

`K.M. Emery et al. | Crop Protection 21 (2002) 41-47
`
`43
`
`for each fungicide
`tion and dose-response curves
`were generated by fitting linear regression equations.
`Regressions were
`statistically significant
`(P<0.01)
`for all fungicides, with correlation coefficients ranging
`from 0.825 to 0.965 (data not shown). Based on
`parameter estimates obtained from the regression
`equations, ECj9, ECs) and ECog values were determined
`(Table 1).
`
`days, lesion diameters were measured to the closest mm
`with a tape measure and expressed as a proportion
`of the fruit circumference. Relative inhibition com-
`pared with the inoculated check (treated with sterile
`distilled water) was calculated for each fungicide—
`concentration—isolate combination. Each combination
`wastested at least three times, each with eight fruit per
`combination.
`
`2.3. Evaluation offungicide mixtures in vitro
`
`2.5. Data analysis
`
`Interactions between the components of the fungicide
`mixtures were evaluated with the Gowing equation
`(Gowing, 1960; Levy et al., 1986; Kosman and Cohen,
`1996):
`
`Cexp = Ci + Cx(1 — Cj),
`
`(1)
`
`Two-way fungicide mixtures consisting of propicona-
`zole with either benomyl, captan, chlorothalonil, cypro-
`dinil or vinclozolin (Table 1) were evaluated with both
`isolates. Experiments included each component of the
`mixture at its ECy (no fungicide), ECj9, ECso and ECo9
`concentration (to simulate interactions at low, medium
`and high levels) in all possible pairwise combinations,
`yielding a total of 16 combinations per fungicide pair
`and isolate. The EC values for each fungicide were
`as determined with isolate H-211 (Table 1). Fungicide
`mixtures were made in sterile distilled water and
`incorporated into Czapek—Dox agar which was pre-
`pared, inoculated and assessed for inhibition of radial
`growth as described above. Relative inhibition com-
`pared with the untreated check (non-amended medium)
`was calculated for each fungicide-concentration—isolate
`combination. Each combination wastested at least three
`times.
`
`2.4. Evaluation offungicide mixtures in vivo
`
`where Cgxp is the expected level of inhibition with
`the mixture when the components act independently and
`C; and C) are the actual levels of inhibition observed
`when each component
`is applied alone. If observed
`inhibition with the mixture, Cops, is equal to Cexp, the
`components exhibit
`independent action.
`If Cop,
`is
`greater or less than C.xp, the mixture components act
`synergistically or antagonistically, respectively.
`For each fungicide—concentration-isolate combina-
`tion, AC,
`the difference between Cop; and Cap, was
`calculated. Using the
`repeats of
`the experiments
`as
`replications,
`t-tests were applied to determine
`whether AC deviated significantly from zero. All
`tests were carried out at P=0.01 because a large
`number of
`significance
`tests was made,
`thereby
`increasing the probability of declaring significance
`A subset of active ingredients (propiconazole in
`by chance alone. The analysis was conducted with the
`mixtures with either
`benomyl,
`chlorothalonil
`or
`Statistical Analysis System (SAS_Institute, Cary,
`cyprodinil) was
`selected for postharvest
`testing on
`North Carolina).
`firm-ripe peach fruit cv.
`‘Blake’
`that
`received no
`preharvest fungicide applications. Fruit were surface-
`sterilized in a solution of 0.5% sodium hypochlorite
`(NaOCl)
`for 2 min and allowed to dry overnight.
`Two-way mixtures of
`fungicides were prepared in
`sterile distilled water with each component at 0.5%,
`2.5%,
`and 5% of
`its
`standard field application
`rate
`(0.13 ml/l, 0.60g/l, 0.67ml/l
`and 0.28¢/l of
`active ingredient for propiconazole, benomyl, chlor-
`othalonil and cyprodinil, respectively). This range of
`concentrations resulted in negligible (0.5% rate)
`to
`almost complete (5% rate) inhibition of M. fructicola
`in preliminary tests on peach fruit. Fruits were
`dipped individually in these
`suspensions
`for 30s
`before
`placement
`on
`plywood
`racks
`previously
`disinfested with 0.5% NaOCl. Each fruit was inoculated
`with a 30-pl drop of conidial suspension prepared
`from isolate H-211 or ZN-21 placed on the uninjured
`cheek surface. The racks holding inoculated fruit
`were covered with a plastic sheet
`to maintain high
`humidity and kept at room temperature. After 5 or 6
`
`3. Results
`
`3.1. Evaluation offungicide mixtures in vitro
`
`The twoisolates of M. fructicola reacted similarly to
`increasing concentrations of propiconazole alone or in
`combination with other
`fungicides.
`Isolate ZN-21
`showed lowerlevels of inhibition, particularly at low
`and medium concentrations of the mixture components.
`The general response of the two isolates to fungicide
`mixtures,
`in terms of relative growth inhibition,
`is
`illustrated in Fig. 1 using the combination of propico-
`nazole and benomy]las an example.
`AC was negative for 65 of 90 fungicide—concentra-
`tion-isolate combinations evaluated in vitro (Fig. 2),i.e.
`growth inhibition observed with the mixture was
`generally less than that expected from Eq. (1). This
`pattern was similar for both isolates. However, AC did
`
`

`

`44
`
`
`
`
`
`Relativeinhibitionofradialgrowth
`
`1.0
`
`c 0.0026 pg/ml propiconazole
`
`~
`
`0.6
`
`0.4
`
`MW
`
`4
`
`|
`
`4
`
`4
`
`SS
`
`40+
`
`4 0.0113 pg/ml propiconazole
`
`0.6
`0.4
`02
`0.0
`
`l
`y)
`y
`Z
`y
`.
`
`iY
`]
`y
`L
`
`Isolate
`
`Fig. 1. Effect of two-way mixtures of propiconazole and benomy] at
`various concentrations on relative inhibition of radial growth of two
`isolates (H-211 and ZN-21) of Monilinia fructicola on fungicide-
`amended Czapek—Dox medium. Fungicide concentrations correspond
`to ECi9, ECs9 and ECoo values for isolate H-211 determinedin vitro.
`Values are meansand standard errors of four experiments.
`
`not differ significantly from zero (P > 0.01) for any of
`the combinations,
`indicating independent action be-
`tween the mixture componentsin all cases.
`
`K.M. Emery et al. | Crop Protection 21 (2002) 41-47
`
`a_ No propiconazole
`
`[-_] No benomy!
`£Z7) 0.0091 jg/ml benomyl
`[4) 0.0266 g/m! benomy!
`HE (0.0774 y1g/m! benomy!
`
`b 0.0006 jig/ml propiconazole P47) I
`
`
`3.2. Evaluation offungicide mixtures in vivo
`
`Inhibition of M. fructicola on peach fruit ranged from
`0% to 100% for the various fungicide—concentration—
`isolate combinations (data not shown). Asin thein vitro
`experiments, a trend toward negative values of AC was
`apparent (Fig. 3), with 45 of 54 fungicide—-concentra-
`tion-isolate combinations showing less inhibition of M.
`fructicola than that predicted by Eq. (1). For isolate ZN-
`21, this trend was most pronounced at low concentra-
`tions for all fungicides (Fig. 3A); for isolate H-211, it
`only occurred for mixtures containing cyprodinil at low
`concentrations. However,nosignificant antagonism was
`detected at P = 0.01. Similarly, no significant synergism
`was detected in these experiments.
`
`4. Discussion
`
`Active ingredients in two-way mixtures of propicona-
`zole with other
`fungicides against M. fructicola in
`culture and on peach fruit generally acted indepen-
`dently,
`i.e. inhibition achieved with the mixtures was
`equal to that of the sum of the individual components of
`the mixture. This result is consistent with the hypothesis
`that each active ingredient inhibits a fixed proportion of
`the residual pathogen growth notinhibited by the other.
`Similar results have been reported in other pathosys-
`tems. For example, Couch and Smith (1991) evaluated a
`range of fungicides for interactions against Pythium
`aphanidermatum on perennial ryegrass and observed
`mostly independent action. In contrast, other studies
`that involved screening of various active ingredients
`either in vitro or in vivo reported a greater incidence of
`synergistic (Gisi et al., 1985) or antagonistic (Buche-
`nauer, 1980) interactions. It should be noted, however,
`that most previous studies used predetermined thresh-
`olds of “synergy ratios” (calculated as Cops/Cexp) to
`determine whether mixtures acted synergistically or
`antagonistically and generally did not include formal
`tests to confirm that observed deviations from indepen-
`dent action werestatistically significant.
`In the present study, trends toward antagonism were
`apparent, although not significant at P=0.01,
`for
`mixtures of propiconazole with the two contact fungi-
`cides captan or chlorothalonil evaluated in vitro at
`medium and high concentrations (Fig. 2). Similar trends
`toward antagonism in vitro were reviewed by De Waard
`and Gisi
`(1995) and Scardavi
`(1966)
`for various
`fungicide mixtures tested against a range of plant
`pathogenic fungi. Couch and Smith (1991) observed
`only one case of significant antagonism when screening
`a large numberof fungicide mixtures for their effect on
`P. aphanidermatum in vivo. Interestingly, this occurred
`in a mixture used commercially for more than 25 years.
`
`

`

`K.M. Emery et al. | Crop Protection 21 (2002) 41-47
`
`45
`
`Isolate H-211
`
`Isolate ZN-21
`
`a 0.0006 g/ml propiconazole
`
`0.0
`
`-0.2
`
`0.4
`
`oe
`
`a
`
`|
`
`iT
`
`To
`
`Concentration of 2nd (__] Low
`mixture component: [5] Medium
`MB High
`
`
`
`
`5 b 0.0026 pg/ml propiconazole
`
`0.0
`
`| l
`
`AC
`
`c 0.0113 pg/ml propiconazole
`
`Fig. 2. In vitro interactions between fungicides in two-way mixtures of propiconazole with other active ingredients with respect to inhibition of two
`isolates (H-211 and ZN-21) of Monilinia fructicola. Interactions are expressed as differences (AC) between observed inhibition (reduction of radial
`growth on Czapek—Dox medium amended with the fungicide mixture relative to the non-amended check) and predicted inhibition assuming
`independent action of the components of the mixture (see Eq. (1)). Low, medium and high fungicide concentrations correspond, respectively, to
`ECj0, ECs59 and ECoo values for isolate H-211 determined in vitro. Values are means and standarderrors of at least three experiments.
`
`For the three fungicide mixtures included in both
`the in vitro and in vivo experiments in the present
`study, results were similar in that all interactions were
`of an independent nature. In contrast, Grabski and
`Gisi (1987) and Zeun and Buchenauer (1991) noted
`more pronounced activity in vivo than in vitro when
`fungicide mixtures were evaluated against P. infestans or
`P. teres.
`
`in studies with fungicide
`interest
`Of particular
`mixtures are effects on isolates of pathogenic fungi with
`reduced sensitivity to one of the mixture components
`(Grabski and Gisi, 1985, 1987; Samoucha and Cohen,
`1988; Couch and Smith, 1991; Gisi, 1996). For example,
`Grabski and Gisi (1987) noted that the degree to which
`an isolate of P.
`infestans was resistant to a fungicide
`influenced strongly its reaction to mixtures containing
`
`

`

`|
`|
`
`
`46
`
`K.M. Emery et al. | Crop Protection 21 (2002) 41-47
`
`Isolate H-211
`
`Isolate ZN-21
`
`a_ Propiconazole, low conc.
`
`0.4
`
`0.2
`
`4
`
`ma
`
`
`
`=
`
`Ey
`
`4
`
`|
`|
`|
`|
`|
`|
`
`
`
`Concentration of 2nd
`mixture component:
`[J Low
`[5 Medium
`MB High
`
`0.0
`
`-0.2
`
`0.4
`
`-0.6
`
`0.2
`
`00
`.
`
`-0.2
`
`-0.4
`
`0.2
`
`oO
`a
`
`
`+
`+ +
`
`|
`
`1
`
`LT wo
`
`CI ]
`
`|
`
`4
`
`|
`
`4
`
`|
`
`b Propiconazole, medium conc.
`
`CS
`
`ey
`
`CJ
`
`=
`
`al
`
`|
`|
`
`||
`
`|
`|
`
`t
`
`t
`
`=
`
`=
`
`a
`
`||
`
`|
`|
`|
`|
`
`||
`
`|
`
`||
`
`+
`
`=
`
`t
`c Propiconazole, high conc.
`
`os
`
`0.0
`
`co
`
`-0.2
`
`-0.4
`
`
`+
`+
`+
`
`|||
`
`||
`
`\
`Ss
`s
`°
`
`»
`es
`se
`e
`oS
`
`o
`Ss
`<
`S
`o
`
`)
`Ss
`Ss
`S
`¥
`
`>
`Ss
`e
`&
`Rey
`o
`
`a
`ss
`O
`é
`Oo
`
`Fig. 3. In vivo interactions between fungicides in two-way mixtures of propiconazole with other active ingredients with respect to inhibition of two
`isolates (H-211 and ZN-21) of Monilinia fructicola. Interactions are expressed as differences (AC) between observed inhibition (reduction in lesion
`diameter caused by M.fructicola on peach fruit dipped in the fungicide mixture relative to the untreated check) and predicted inhibition assuming
`independent action of the components of the mixture (see Eq. (1)). Low, medium and high fungicide concentrations correspond, respectively, to
`0.5%, 2.5% and 5% of the compounds’ standard field application rates. Values are means andstandarderrorsofat least three experiments.
`
`the most
`the full concentration of
`than that of
`that fungicide. In contrast, Couch and Smith (1991)
`efficacious component used alone (Couch and Smith,
`determined that mixtures of metalaxyl and mancozeb
`1991). Because of the lack of synergistic effects observed
`were equally effective against metalaxyl-sensitive and
`in this
`study,
`the level of control achieved with
`resistant
`isolates of P. aphanidermatum. Differential
`
`
`responses to fungicide mixtures in relation to fungicide not_providepropiconazole-based mixtures may
`sensitivity were not investigated in the present study
`sufficient benefit to offset the added costs of using two
`active ingredients instead of one. Thus,
`there would
`because of the similarity in propiconazole-sensitivity of
`the two isolates of M.fructicola.
`be little incentive for favoring fungicide mixtures over a
`the control potential
`In the absence of synergism,
`rotating schedule of fungicides for control of and
`of a fungicide mixture at reduced concentrationsis less
`resistance management in M. fructicola, assuming both
`
`
`
`i '|a: |
`
`

`

`K.M. Emery et al. | Crop Protection 21 (2002) 41-47
`
`47
`
`strategies are equally successful in delaying resistance
`development.
`
`Acknowledgements
`
`Funded in part by the USDA-CSREESPest Manage-
`ment Alternatives Program (grant no. 97-34365-5034)
`and the Georgia Agricultural Commodity Commission
`for Peaches. We thank J. Lance and L. Chenault for
`technical assistance.
`
`References
`
`Bertrand, P.F., Padgett, G.B., 1997. Fungicide resistance manage-
`ment. Bulletin 1132, University of Georgia, College of Agricultural
`and Environmental Sciences, Cooperative Extension Service,
`Athens.
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