Evaluation of Tactics for Managing Resistance of Venturia inaequalis
`to Sterol Demethylation Inhibitors
`
`Wolfram Köller and W. F. Wilcox, Department of Plant Pathology, Cornell University, New York State Agricul-
`tural Experiment Station, Geneva, NY 14456
`
`ABSTRACT
`Köller, W., and Wilcox, W. F. 1999. Evaluation of tactics for managing resistance of Venturia
`inaequalis to sterol demethylation inhibitors. Plant Dis. 83:857-863.
`
`The impact on the selection and control of subpopulations of V. inaequalis resistant to the sterol
`demethylation inhibitor (DMI) fenarimol or to dodine were evaluated with respect to several
`tactics of apple scab control. Experiments were conducted in an experimental orchard with ele-
`vated levels of DMI and dodine resistance over a period of three consecutive seasons. The DMI-
`resistant subpopulation was poorly (14%) controlled at a fenarimol rate of 15 mg/liter (sprayed
`to run-off), whereas control was significantly improved (54%) at twice that rate. Mancozeb
`mixed with the low rate of fenarimol also improved the control of DMI-resistant isolates, but
`the improvement was due to the indiscriminate control of both the DMI-sensitive and -resistant
`populations provided by mancozeb. The selection of fenarimol-resistant isolates resulting from
`poor control of the resistant subpopulation by the low rate of fenarimol was equivalent whether
`fenarimol was applied singly or in mixture with mancozeb. Consequently, the use of high DMI
`rates in mixture with a protective fungicide is expected to delay the build-up of resistant sub-
`populations by limiting their increase through two separate principles of control. For dodine in
`mixture with fenarimol, it was found that each mixing partner applied alone selected both fe-
`narimol- and dodine-resistant isolates. This selection pattern was partly explained by the possi-
`bility that one of the multiple genes underlying fenarimol and dodine resistance confers resis-
`tance to both fungicides, in addition to the selection of double-resistant isolates. Regardless, a
`mixture of fenarimol with dodine each employed at a low rate controlled both the fenarimol-
`and the dodine-resistant subpopulation at least as effectively as the individual components at
`twice their mixture rate, and an accelerated selection of double-resistant isolates was not de-
`tected. In commercial orchard trials, mixtures of DMIs with either a protective fungicide or with
`dodine provided equivalent control even when levels of DMI resistance, dodine resistance, or
`both were moderately elevated. With the exception of orchards with high levels of DMI or
`dodine resistance, dodine might be an alternative to protective fungicides as a mixing partner
`with DMIs.
`
`Several fungicides introduced in the
`1980s for the control of tree fruit diseases
`act as sterol demethylation
`inhibitors
`(DMIs; 9,18). Practical resistance to DMI
`fungicides of Venturia inaequalis (Cooke)
`G. Wint., the causal agent of apple scab,
`was first documented for an experimental
`orchard in Nova Scotia, Canada, after
`DMIs had been tested at the site for more
`than 10 years (2,3,16). Subsequently,
`similar resistance to DMIs was identified
`in a commercial orchard in Michigan (16),
`and evidence for resistance was provided
`also for orchards in Europe (19). In antici-
`pation of resistance development of V.
`
`Corresponding author: W. Köller
`E-mail: wk11@cornell.edu
`
`This study was supported in part by the USDA
`(90-34103-539 and 94-37313-0678) and by the
`New York State Apple Research and Development
`Program.
`
`Accepted for publication 1 June 1999.
`
`Publication no. D-1999-0706-03R
`© 1999 The American Phytopathological Society
`
`inaequalis to DMIs, their mixtures with
`protective fungicides such as mancozeb or
`captan were suggested as an anti-resistance
`strategy (25); such mixtures are now com-
`monly used for the control of apple scab
`with DMI fungicides. Tank-mixing DMIs
`with protective fungicides also has been
`recommended to improve the control of
`fruit infections in a delayed-spray program
`developed for low-inoculum orchards and
`with baseline-sensitive populations of V.
`inaequalis (31). The merits of such mix-
`tures in delaying and managing DMI re-
`sistance have never been evaluated under
`orchard conditions. Furthermore, the reli-
`ance on purely protective fungicides as
`components of anti-resistance strategies
`might not be sustainable, because several
`of the suitable representatives remain un-
`der toxicological scrutiny in various coun-
`tries.
`Dodine, introduced in the late 1950s as a
`fungicide for control of apple scab, might
`serve as an alternative to conventional
`protectants in mixtures with DMIs. In ad-
`dition to providing strong protective activ-
`ity against scab infections, dodine also can
`be active when applied in pre- and post-
`
`symptom modes by preventing production
`of conidia from established scab lesions
`(1,30). However, dodine efficacy is often
`weak in typical after-infection applications
`(30). In contrast, DMIs are most active in
`an after-infection mode of application,
`whereas their protective and anti-sporulant
`activities are weak (22,26,30). Thus, mix-
`tures of DMIs and dodine might be ex-
`pected to complement the strength and
`weakness of each individual component in
`controlling scab at the various stages of
`disease development.
`The effectiveness of DMIs in mixture
`with dodine will be influenced by the sen-
`sitivity of a given V. inaequalis population
`to both of the components. The first cases
`of dodine resistance were noted in the late
`1960s, after the fungicide had been used
`extensively for approximately 10 years in
`scab control programs (7), and resistance
`became widespread during
`the 1970s
`(7,8,20,27,32). A quantitative
`test for
`measuring sensitivities of V. inaequalis
`isolates to dodine allowed us to quantify
`frequencies of dodine-resistant isolates in
`both baseline populations and populations
`from orchards with practical resistance to
`dodine (17). Monitoring of orchards in
`New York and Michigan revealed that
`dodine resistance levels (i) largely re-
`flected the dodine use histories at particular
`sites; (ii) had declined below threshold values
`in orchards with previous records of dodine
`resistance after dodine use was suspended for
`several years; and (iii) could quickly exceed
`threshold values when dodine was used as a
`single fungicide at sites where resistance
`levels were elevated (17).
`The development of DMI resistance
`within populations of V. inaequalis in
`North American apple orchards (2,3,16)
`signals the need for the development and
`implementation of effective anti-resistance
`strategies incorporated into current scab-
`control programs. The particular nature of
`resistance development to DMI fungicides
`has been described variously as directional,
`quantitative, or polygenic selection (9,11-
`13,15). In general, baseline populations of
`fungal pathogens, including V. inaequalis,
`exhibit a broad range of isolate sensitivities
`to DMIs (3,9,11-16,28); practical resis-
`tance develops when selection causes the
`frequency of the least-sensitive baseline
`isolates to increase above a threshold value
`(16). In contrast to other fungicides such as
`benomyl (12,13), however, the response of
`isolates resistant to DMIs remains dose
`
`Plant Disease / September 1999 857
`
`SYNGENTA EXHIBIT 1015
`Syngenta v. UPL, PGR2023-00017
`
`

`

`dependent (16). Consequently, a theoretical
`anti-resistance strategy would be to avoid
`the use of low application rates in order to
`maximize the control of isolates that would
`fully resist low doses of a DMI fungicide
`and, thus, would be selected more rapidly.
`In this study, the influence of DMI rates
`and of tank-mixing a DMI with either
`mancozeb or dodine was evaluated with
`respect to resistance development and ap-
`ple-scab control in both experimental and
`commercial orchard trials.
`
`MATERIALS AND METHODS
`Test orchards. Tests were conducted
`from 1992 to 1994 in an experimental ap-
`ple orchard (cv. Cortland) in Geneva, New
`York. This orchard was planted in 1967
`and had served as a site for fungicide effi-
`cacy testing until 1987. The first DMI
`fungicides were tested in 1971 and in-
`creasingly intense DMI testing continued
`until 1987. Dodine was tested extensively in
`this orchard during the 1970s. From 1988 to
`1991, the orchard served as a test site for
`insecticide and acaricides. Apple scab was
`controlled with DMIs during
`the four
`seasons preceding the tests described here.
`Tests were also conducted in six com-
`mercial orchards in 1996 in cooperation
`with participating growers. The orchards
`(cv. McIntosh) were chosen to represent a
`typical cross-section of DMI use histories
`(i.e., each had been treated with fenarimol,
`myclobutanil, or both, predominantly in
`mixture with either mancozeb or metiram,
`for at least parts of the previous seven sea-
`sons). DMI resistance was not suspected by
`any of the growers. Each test block was
`approximately 4 ha in size, and all orchards
`were located near the south shore of Lake
`Ontario, three each in the counties of Wayne
`and Orleans. The two groups of orchards
`were separated by approximately 150 km.
`Application and evaluation of fungi-
`cides. In the experimental orchard, fungi-
`cide treatments were arranged in a ran-
`domized complete block design with three
`replications. Individual
`treatment-blocks
`consisted of two to three trees, and the
`same trees were assigned to the same
`treatment each year. Spray solutions were
`applied dilute (approximately 2,800 li-
`ters/ha) with a handgun to the point of run-
`off. The spray program was designed to
`target the period from tight cluster through
`first cover as suggested by Wilcox et al.
`(31); however, the timings of individual
`applications were adjusted more closely to
`specific apple-scab infection periods, with
`the objective of providing sufficient dis-
`ease incidence to facilitate the monitoring
`of isolate sensitivities within each treat-
`ment regime.
`In 1992, primary infection periods de-
`termined as described previously (31) were
`recorded on 25 April; 1, 17, and 26 May;
`and 6 and 13 June. Fungicides were ap-
`plied on 5 and 21 May and 3 June. In 1993,
`very light primary infection forced us to
`
`858 Plant Disease / Vol. 83 No. 9
`
`extend the experimentation period in order
`to obtain sufficient isolate numbers for
`population profiling. In this year, infection
`periods were recorded on 22 April; 6, 19,
`23, and 31 May; 20 and 29 June; and 14
`July; fungicides were applied on 6, 17, and
`28 May; 22 June; and 13 July. In 1994,
`primary infection periods occurred on 28
`April; 8, 17, and 25 May; and 14 June;
`fungicides were applied on 3 and 19 May
`and 3 and 10 June.
`The objective of the experimental or-
`chard trials was to determine the impact of
`different DMI use strategies on the control
`of V. inaequalis subpopulations either sen-
`sitive or resistant to DMIs. Therefore, the
`evaluation of treatment efficacies was fo-
`cused on the disease incidence of terminal
`leaves as the parameter most indicative of
`disease progression over the primary scab
`season. In order to allow for lesion devel-
`opment in response to the final treatment,
`scab incidence (10 leaves each on 10 ter-
`minals per tree from the central part of
`each plot) was assessed 2 weeks after the
`last fungicide spray was applied. Diseased
`terminal leaves with actively sporulating
`lesions were sampled for isolate sensitivity
`tests at or close to the time disease inci-
`dences were evaluated. A total of 40 to 50
`isolates from individual and well-dispersed
`leaves (16,17) were tested for each treat-
`ment in each season. In order to minimize
`significant mixing of the different sub-
`populations over the course of the experi-
`ments, leaves were sampled from the inner
`parts of respective treatment blocks. The
`potential for mixing of populations was
`further reduced by the low-density nature
`of the orchard (9-m row and 5-m tree
`spacing).
`Growers cooperating in the commercial
`orchard trials in 1996 were asked to apply
`a DMI mixed with an ethylenebisdithio-
`carbamate (EBDC) to half of the test or-
`chard, and a DMI-dodine mixture to the
`other half on the same dates, using their
`own application equipment, spray prac-
`tices, and timing regimes. All growers
`started their scab-control program during
`the first week of May, at approximately the
`tight-cluster stage of blossom bud devel-
`opment as recommended previously for a
`reduced DMI spray program (31).
`Orchards 1 to 3 and 6 received four DMI
`mixture applications, while orchards 4 and
`5 received three and five applications,
`respectively. The mixtures were applied at
`intervals ranging from 5 to 15 days, with a
`mean interval of 11 days. DMIs used were
`commercial
`formulations of
`fenarimol
`(Rubigan 1EC) applied at a rate of 82 g/ha
`or of myclobutanil (Nova 40W) applied at
`a rate of 140 g/ha. EBDCs used as mixture
`components were mancozeb
`(Dithane
`75DF) or metiram (Polyram 80WP) ap-
`plied at rates of approximately 2,500 g/ha.
`Dodine (Syllit 65W) used as the alternative
`mixture component was applied at a rate of
`820 g/ha. The DMI programs were fol-
`
`lowed by cover sprays applied to the entire
`orchard, consisting of captan alone, or
`captan mixed with sulfur or thiophanate-
`methyl. Scab incidences were assessed on
`10 arbitrarily chosen trees per treatment by
`examining cluster leaves (25 clusters per
`tree), fruit (50 fruit per tree), and terminal
`leaves (10 leaves per terminal, 10 terminals
`per tree) during 25 June to 29 July.
`In order to test the levels of resistance of
`the respective V. inaequalis populations to
`both DMIs and dodine at the start of the
`season, growers were asked to leave sev-
`eral trees at the orchard corners unsprayed
`until the scab incidence reached a level
`suitable for sampling. Diseased cluster
`leaves were arbitrarily sampled from these
`trees on a single date for each orchard
`during 28 May through 11 July.
`Sensitivity tests. Procedures employed
`for the isolation and propagation of mono-
`conidial isolates of V. inaequalis and for
`testing their sensitivities to fungicides have
`been described in detail (17,28). Briefly,
`conidia originating from distinct scab le-
`sions were germinated on water agar
`amended with antibiotics, and single ger-
`minating conidia were transferred to potato
`dextrose agar (PDA). Fungicide sensitivi-
`ties of mycelia developing from single
`conidia were determined by comparing the
`colony diameters of mycelia developing on
`PDA or on PDA amended with a fungicide.
`Isolate sensitivities were expressed as
`relative growth (RG) of mycelial colonies
`at discriminatory doses of the respective
`fungicides, which were 0.05 µg ml–1 for
`fenarimol, 0.1 µg ml–1 for myclobutanil,
`and 0.2 µg ml–1 for dodine (14,16,17). RG
`was defined as mean colony diameter on
`PDA amended with the discriminatory
`dose per colony diameter on unamended
`PDA × 100. Fenarimol (technical grade)
`was obtained from Dow Agrosciences
`(Indianapolis, IN); myclobutanil (technical
`grade) from Rohm and Haas (Philadelphia,
`PA), and dodine (analytical standard) was
`from Cyanamid (Princeton, NJ). Fungi-
`cides used in orchard trials were commer-
`cial formulations as specified.
`Data analysis. Sensitivities of V. inae-
`qualis isolates to DMIs and dodine were
`analyzed as described previously (16,17).
`Categorical sensitivity data were compared
`according to the log linear model of SYS-
`TAT (version 5.2; Systat, Inc., Evanston,
`IL), with numbers of isolates grouped into
`the categories sensitive (S) and resistant
`(R). Isolates with RG values >80 deter-
`mined for fenarimol were classified DMI-
`resistant (16), whereas isolates with RG
`values >90 determined for dodine were
`classified resistant to that fungicides (17).
`Mean RG values were compared with
`the nonparametric Kolmogorov-Smirnov
`test of SYSTAT version 5.2. Comparisons
`of mean RG values were restricted to iso-
`lates classified as sensitive to fenarimol or
`dodine. The objective was to detect any
`potential impact of scab control on the
`
`

`

`selection of least-sensitive isolates classi-
`fied as DMI- or dodine-sensitive according
`to the criteria described previously (16,17).
`Additive, synergistic, or antagonistic
`interactions between mixture components
`were analyzed by applying the formula Exp
`= X + Y – XY/100 described by Richter
`(24), with Exp as the percentage of control
`expected from additive effects of the two
`components when tested in mixture, and
`with X and Y as the percentages of control
`provided by each of the two components
`independently. Synergism is indicated if
`the percentage of control observed with the
`mixture (Obs) is higher than the expected
`additive effect; antagonism is indicated if
`the observed control is lower than ex-
`pected.
`
`RESULTS
`Sensitivities of V. inaequalis isolates to
`fenarimol and dodine. The frequency of
`fenarimol-resistant isolates (RG > 80)
`sampled from leaves of non-treated trees in
`1992 was 7% and significantly higher (P =
`0.05) than the frequency of 1.7% deter-
`mined for typical baseline populations
`(16); the frequency of dodine-resistant
`isolates (RG > 90) was 7% and also sig-
`nificantly higher (P = 0.02) than the base-
`line value (17). By the third year of our
`tests, resistance levels determined for iso-
`lates sampled from non-treated trees were
`14% for fenarimol and 8% for dodine.
`Neither increase was significant (P = 0.3
`and 0.9, respectively); therefore, all sensi-
`tivity data for
`isolates sampled from
`nontreated trees over the three-year period
`
`Fig. 1. Distribution of fenarimol and dodine
`sensitivities of Venturia inaequalis populations.
`(A) Fenarimol sensitivity distribution in base-
`line populations (hatched bars; n = 748) (16)
`and in the experimental test orchard (closed
`bars; n = 142). (B) Dodine sensitivity distribu-
`tion in baseline populations (hatched bars; n =
`232) (17) and distribution in the experimental
`test orchard (closed bars; n = 142).
`
`were combined to reflect the orchard
`population prior to treatments in individual
`plots. The sensitivity distributions of these
`isolates were significantly different from
`the distributions described for baseline
`populations (Fig. 1). In addition to signifi-
`cantly higher frequencies of resistant iso-
`lates, the mean RG value of the fenarimol-
`sensitive subpopulation had increased from
`41 in baseline populations (16) to 53 (P <
`0.01); the mean RG value of the dodine-
`sensitive subpopulation was 55 compared to
`41 in baseline populations (P < 0.01; 17).
`Relative efficacy of apple-scab con-
`trol. Fenarimol and mancozeb applied
`alone at half of the rates typically recom-
`mended for commercial control of scab (15
`and 900 mg/liter, respectively) provided
`the least control in all three seasons,
`whereas the combination of the two fungi-
`cides was significantly more effective
`(Table 1). The interactive effects of the two
`mixture components (Obs – Exp = –5, +9,
`and +5 for 1992, 1993, and 1994, respec-
`tively) suggested that effects were largely
`additive. Fenarimol alone applied at twice
`the rate utilized in the mixture provided
`control equivalent to the half-rate mixture
`with mancozeb (Table 1). Although inci-
`dences of scab on non-treated trees ranged
`from 23.5 to 85.6% during the three years
`of testing, levels of control achieved with
`the various treatments were uniform, with
`the exceptions of fenarimol applied at the
`low rate and of dodine (Table 1).
`The control of scab achieved with
`dodine at a rate of 290 mg/liter, which
`reflects the low range of the rate typically
`recommended, was similar to the high rate
`of fenarimol, although seasonal variations
`were high (Table 1). The mixture of fe-
`narimol and dodine was as effective as the
`single components applied at twice their
`mixture rates. Furthermore, the level of
`control achieved with this mixture was
`very consistent over the three years of
`testing (Table 1).
`Impact of scab-control tactics on de-
`velopment of resistance to fenarimol and
`
`dodine. All treatments significantly in-
`creased the frequencies of fenarimol-re-
`sistant isolates relative to the population of
`isolates sampled from non-treated trees
`(Table 2). Differences among treatments in
`the frequencies of fenarimol-resistant iso-
`lates were not significant (P > 0.32). Mean
`RG values of fenarimol-sensitive isolates
`surviving the spray regimes increased sig-
`nificantly only for the low rate of fenari-
`mol applied alone or in mixture with man-
`cozeb (Table 2). Thus, isolates belonging
`to the least-sensitive part of the DMI-sen-
`sitive subpopulation (16) were selected by
`these treatments but not by the low rate of
`fenarimol mixed with dodine or the high
`rate of fenarimol applied singly.
`All treatment regimes except the fe-
`narimol-mancozeb mixture
`resulted
`in
`significantly higher frequencies of dodine-
`resistant isolates (Table 2). The selection
`of dodine-resistant isolates was signifi-
`cantly more pronounced for the dodine-
`only treatment than for any other treatment
`(P £
` 0.05), with the exception of fenarimol
`applied at the high rate (P = 0.14). A
`higher mean RG value for the dodine-sen-
`sitive population was detected only when
`dodine was applied as the single fungicide
`(Table 2), indicating that dodine at the rate
`it was used in our trials also selected iso-
`lates belonging to the least-sensitive spec-
`trum of the population normally classified
`as dodine-sensitive (17).
`Differential levels of control achieved
`for subpopulations sensitive or resistant
`to fenarimol or dodine. In order to evalu-
`ate the significance of differential levels of
`control achieved for either fungicide-re-
`sistant or -sensitive isolates (16), the inci-
`dences of apple scab recorded in each of
`the three years for non-treated trees and for
`trees subjected to the various treatments
`(Table 1) and the corresponding frequen-
`cies of fenarimol- and dodine-resistant
`isolates (Table 2) were utilized to sepa-
`rately calculate the percentages of scab
`control achieved for the sensitive and re-
`sistant subpopulations (Table 3).
`
`Table 1. Control of apple scab with single fungicides and fungicide mixtures in experimental orchard
`trials
`
`Treatmentx
`
`Rate (mg a.i./liter)
`
`1992
`
`Incidencew
`1993
`
`1994 Mean control (%)y
`
`53.3
`29.0
`15.3
`11.3
`…
`14.0
`11.3
`6.7
`…
`
`23.5
`12.9
`14.4
`6.0
`…
`3.9
`1.8
`4.1
`…
`
`85.6
`35.6
`44.5
`13.8
`…
`15.0
`31.0
`13.1
`…
`
`None
`…
`Mancozeb
`900
`Fenarimol
`15
`Fenarimol
`15
`+ Mancozeb
`+900
`Fenarimol
`30
`Dodine
`290
`Fenarimol
`15
`+ Dodine
`+145
`w Mean percentage of terminal leaves infected.
`x Mancozeb, fenarimol, and dodine were applied as Dithane 75DF, Rubigan 1EC, and Syllit 65 WP,
`respectively.
`y Standard deviations are given in parentheses.
`z Means not followed by a common letter are significantly different (P £
`range test).
`
`…
`50 (7) az
`53 (17) a
`79 (5) b
`…
`80 (5) b
`78 (14) ab
`85 (2) b
`…
`
` 0.05, Duncan’s multiple
`
`Plant Disease / September 1999 859
`
`

`

`Fenarimol applied alone at the low rate
`provided the least and least-uniform con-
`trol of the fenarimol-resistant subpopula-
`tion (Table 3). Although control was 42%
`in 1992, no control was provided in 1993
`and 1994. Applying fenarimol at twice the
`rate substantially (P = 0.07) improved
`control of the resistant population (Table
`3). Differences were most pronounced for
`1993 and 1994, during which mean control
`of the fenarimol-resistant subpopulation
`was 62% versus 0% for the high and low
`fenarimol rate, respectively.
`Control of the resistant subpopulation
`was also substantially (P = 0.07) improved
`when fenarimol at the low rate was applied
`in mixture with mancozeb (Table 3). How-
`ever, this improved level of control was
`due to the indiscriminate contribution pro-
`vided by mancozeb. Eliminating this man-
`cozeb contribution revealed that control of
`the fenarimol-resistant subpopulation pro-
`vided by the low rate of fenarimol was
`equally poor (P = 0.87) whether the fungi-
`
`cide was applied alone or in mixture with
`mancozeb (Table 3). Likewise, the control of
`the sensitive population provided by the low
`rate of fenarimol contained in the mixture or
`applied alone was equivalent (P = 0.59),
`indicating that the relative contribution of
`mancozeb was additive for both the DMI-
`resistant and -sensitive subpopulations. The
`most effective and most consistent control of
`the fenarimol-resistant subpopulation was
`achieved with the low-rate mixture of
`fenarimol and dodine (Table 3).
`Because selection of dodine-resistant
`isolates was most pronounced for dodine
`applied singly (Table 2), poor control of
`the dodine-resistant subpopulation was also
`expected to be most pronounced for this
`treatment. The data reflected this expecta-
`tion, but the differential control of the
`dodine-sensitive and -resistant subpopula-
`tions were of low statistical significance
`(Table 3) due to the variable control of the
`dodine-resistant population provided by
`dodine applied alone (percentages of con-
`
`trol were 43, 78, and 3 in 1992, 1993, and
`1994, respectively). The best and most
`consistent control of the dodine-resistant
`subpopulation was achieved with
`the
`dodine plus fenarimol mixture (Table 3),
`very similar to the consistently high level
`of control of the fenarimol-resistant popu-
`lation provided by the same mixture.
`Although differences were not always of
`high statistical significance due to the
`sometimes large variations over the three
`test seasons, control of the fenarimol-re-
`sistant subpopulation was consistently
`lower for all treatments, including dodine
`applied alone (Table 3). Conversely, the
`inferior control of the dodine-resistant
`population with fenarimol also was ob-
`served (Table 3).
`Interdependence of DMI and dodine
`resistance. The possibility that the selec-
`tion of fenarimol-resistant
`isolates by
`dodine and vice versa (Tables 2 and 3)
`might be explained by the selection of
`isolates double-resistant to both inhibitors
`
`Table 2. Levels of resistance of Venturia inaequalis populations controlled with fenarimol, dodine or fungicide mixtures
`
`Fungicide
`
`Isolate values
`
`Checkv
`
`Fenarimol (15)
`
`Fenarimol (30)
`
`Treatmentsu
`Fenarimol (15) +
`mancozeb (900)
`
`Dodine (290)
`
`Fenarimol (15) +
`dodine (145)
`
`Fenarimol
`
`Dodine
`
`149
`26.1
`<0.01
`52.5
`0.38
`149
`19.5
`0.01
`55.0
`0.98
`
`150
`26.0
`<0.01
`55.8
`0.03
`150
`10.7
`0.67
`52.8
`0.60
`
`150
`23.3
`<0.01
`53.2
`0.40
`150
`26.7
`<0.01
`59.7
`0.01
`
`150
`21.3
`0.02
`53.5
`0.25
`150
`16.7
`0.05
`55.8
`0.63
`
`n
`142
`133
`Rw
`11.3
`22.6
`Px
`…
`0.01
`Mean RG (sen)y
`52.7
`55.4
`Pz
`…
`0.04
`n
`142
`128
`Rw
`9.9
`17.2
`Px
`…
`0.05
`Mean RG (sen)y
`55.0
`55.7
`Pz
`…
`0.71
`u Rates of fungicides (g a.i./1,000 liters) are given in parentheses.
`v Isolates collected from nontreated trees.
`w Frequencies of isolates (%) classified as resistant (R) to fenarimol (relative growth [RG] > 80) or dodine (RG > 90).
`x Comparison of counts of isolates classified as sensitive (S) or R obtained from nontreated trees with respective counts for isolates obtained from trees
`treated as specified (log linear model).
`y Mean RG values of isolates sensitive to fenarimol (RG £
` 80) or dodine (RG £
` 90).
`z Comparison of mean RG values determined for the sensitive population of isolates retrieved from nontreated trees with respective means for isolates
`retrieved from trees treated as specified (Kolmogorov-Smirnov test).
`
`Table 3. Control of Venturia inaequalis subpopulations sensitive (S) or resistant (R) to fenarimol or dodine
`
`Mean control (%)v
`
`Treatment
`
`Rate (mg a.i./liter)
`
`Fenarimol
`R
`
`Pw
`
`S
`
`Dodine
`R
`
`Pw
`
`S
`59 (14)x
`Fenarimol
`15
`14 (24)
`0.05
`57 (16)
`20 (27)
`0.13
`Fenarimol
`30
`83 ( 4)
`54 (14)
`0.10
`82 ( 5)
`60 (10)
`0.02
`Fenarimol
`15
`…
`…
`…
`…
`…
`…
`+ Mancozeb (mixture)y
`900
`83 ( 4)
`52 (11)
`0.01
`79 ( 5)
`78 ( 4)
`0.74
`Fenarimol
`15
`…
`…
`…
`…
`…
`…
`+ Mancozeb (fenarimol)z
`900
`64 ( 4)
`17 ( 8)
`0.01
`62 ( 4)
`27 ( 9)
`0.004
`Dodine
`290
`82 (12)
`56 (30)
`0.23
`83 (12)
`39 (38)
`0.14
`Fenarimol
`15
`…
`…
`…
`…
`…
`…
`+ Dodine
`145
`87 ( 7)
`72 ( 5)
`0.02
`86 ( 2)
`74 ( 5)
`0.01
`v The levels of control (relative to the untreated check) achieved for sensitive and resistant subpopulations were calculated from disease incidences re-
`corded for the various treatments in each year and averaged frequencies of sensitive or resistant isolates determined for isolates obtained from the re-
`spective treatments.
`w Significance of differences between mean percentages of control achieved for sensitive and resistant subpopulations (analysis of variance).
`x Standard deviations are given in parentheses.
`y Calculated from the disease control and resistance frequency for trees treated with the mixture.
`z Disease control was calculated by comparing incidences of scab on trees treated with the mixture relative to incidences on trees treated with mancozeb
`alone. Frequencies of sensitive and resistant isolates in the mancozeb-only treatment were assumed to be the same as for non-treated trees.
`
`860 Plant Disease / Vol. 83 No. 9
`
`

`

`was analyzed for all isolate sensitivities
`determined in 1993 and 1994. In these two
`seasons, sensitivities to myclobutanil were
`tested in addition to fenarimol in order to
`provide comparative data for a second
`DMI fungicide.
`The fenarimol-sensitive subpopulation
`contained a significantly lower frequency
`of dodine-resistant isolates than the fe-
`narimol-resistant subpopulation (Table 4).
`Although of slightly lower statistical sig-
`nificance, the same relationship applied to
`myclobutanil (Table 4). The result of this
`analysis indicated that resistance of V. in-
`aequalis to DMIs and dodine was not an
`entirely independent trait, as would have
`been expected if double resistance arose
`from a random distribution of distinctly
`different resistance genes within a popula-
`tion. Applying the mixture of fenarimol and
`dodine could have been expected to acceler-
`ate the selection of double-resistant isolates.
`This expectation was not confirmed. The
`mean frequency of fenarimol-resistant iso-
`lates obtained from treatments with the
`mixture was 18.6% and that of dodine re-
`sistance was 12.7%. If fenarimol and dodine
`resistance were independent traits, the ex-
`pected frequency of double-resistant isolates
`would be 2.4%. The frequency determined
`was 2.7% and, thus, not different from the
`expected frequency considering the sample
`size of 150 isolates tested.
`
`In addition, the comparison of fenarimol
`and myclobutanil sensitivities (Table 4)
`confirmed the high degree of cross-resis-
`tance described before (14,16). Although
`myclobutanil had not been applied in the
`orchard, frequencies of myclobutanil-re-
`sistant isolates were as high as fenarimol
`frequencies (Table 4).
`Evaluation of DMI mixtures with
`mancozeb and dodine under commercial
`orchard conditions. Frequencies of resis-
`tance to fenarimol and myclobutanil for
`isolates retrieved from nontreated trees in
`six commercial orchards ranged from
`baseline to significantly higher than base-
`line (Table 5). However, all orchard popu-
`lations were significantly (P < 0.01) less
`resistant than the threshold of practical
`resistance determined previously (16). For
`dodine, resistance was baseline at one of
`the orchards; at all other sites, resistance
`levels were significantly higher than base-
`line (Table 5). With the exception of or-
`chard 4, all other levels of dodine resis-
`tance were also significantly (P < 0.01)
`lower than the threshold described for
`dodine (17; Table 5).
`When a DMI was mixed with either
`mancozeb or dodine, scab control was
`equivalent or significantly higher with
`dodine as the mixing partner (Table 6).
`Scab control was not compromised at the
`two sites with elevated frequencies of iso-
`
`Table 4. Relationship between resistance of Venturia inaequalis isolates to two sterol demethylation
`inhibitor (DMI) fungicides and to dodine
`
`Fenarimolx
`
`Myclobutanil
`
`S
`
`R
`
`S
`
`R
`
`16.3
`472
`
`24.8
`165
`
`0.02
`
`Dodine Ry
`n
`Pz
`x S = sensitive, R = resistant.
`y Frequency (%) of dodine-resistant isolates (relative growth [RG] > 90) in populations either S or R
`(RG > 80) to the DMIs fenarimol or myclobutanil.
`z Comparison of counts of dodine-resistant isolates classified as S or R to the DMIs fenarimol or
`myclobutanil (log-linear model).
`
`16.6
`477
`
`23.5
`162
`
`0.06
`
`lates resistant to both fenarimol and dodine
`(orchards 5 and 6, Table 6). The different
`levels of control observed among orchards
`appeared to be more closely related to
`disease management practices rather than
`to respective levels of resistance at these
`sites. For example, the poor control of scab
`obtained in orchard 4 with myclobutanil
`employed as the DMI was associated with
`a V. inaequalis population whose frequency
`of myclobutanil-resistant isolates was not
`different from baseline (Table 5). Although
`the frequency of dodine-resistant isolates
`was relatively high in this orchard (Table
`5), scab control was equally poor whether
`mancozeb or dodine was used as the mix-
`ing partner, providing further evidence that
`poor control of apple-scab development
`was due to inadequate management prac-
`tices rather than fungicide resistance.
`
`DISCUSSION
`Strategies for delaying and managing
`fungicide resistance hav