Sensitivity of Venturia inaequalis Populations to Anilinopyrimidine Fungicides and Their Contribution to Scab Management in New York.

Sensitivities of Venturia inaequalis isolates to the anilinopyrimidine fungicides (APs) pyrimethanil and cyprodinil were determined for nine populations by measuring the growth of colonies formed from germinating conidia derived from single scab lesions. At the discriminatory pyrimethanil dose of 0.2 µg ml-1, the mean relative growth range measured for eight V. inaequalis populations (n = 39 to 74) never treated with AP fungicides varied from 18.1 to 48.2, translating into an approximately sixfold difference in mean baseline sensitivities. For the composite of all 469 isolates tested, sensitivities to pyrimethanil and to the sterol demethylation inhibitor (DMI) myclobutanil were significantly correlated. When isolates were organized into subpopulations based on their sensitivities to an individual fungicide, sensitivities to both fungicides declined in parallel through the highly and moderately sensitive spectra of subpopulations, but they diverged for isolates in subpopulations least sensitive to either fungicide. The result suggested that at least one of the multiple genes conferring DMI resistance also lowered the sensitivity to AP fungicides. The relative contribution of AP fungicides to scab management was evaluated at an experimental orchard representative of the Great Lakes region of the United States. Frequencies of DMI-resistant isolates of V. inaequalis had progressed to the stage of practical resistance at the site, and the sensitivity to pyrimethanil was similar to several commercial orchard populations never treated with APs. For management programs at the experimental site involving the AP fungicides cyprodinil and pyrimethanil and conducted from 1996 to 2000, the level of fruit and terminal leaf scab control was inferior to that of nonspecific protectants such as mancozeb or captan. For the control of scab on cluster leaves, the efficacy of AP fungicides equaled the performance of nonspecific protectants. This modest contribution of AP fungicides to scab management might have been caused by a lack of the extended cool temperature conditions that were conducive to AP performance in northern Europe in previous studies, and/or by the reduced sensitivity to AP fungicides in this DMI-resistant V. inaequalis population.

A Two-Phase Resistance Response of Venturia inaequalis Populations to the QoI Fungicides Kresoxim-Methyl and Trifloxystrobin.

The class of fungicides acting as respiration inhibitors by binding to the Qo center of cyto-chrome b (QoIs) are in wide use for the management of apple scab caused by Venturia inaequalis. In order to assess responses of V. inaequalis populations to treatments with QoIs, sensitivities of isolates were determined for germinating conidia or for mycelial colonies developing from germinating conidia. Under both test conditions, inhibitory potencies of kresoxim-methyl and trifloxystrobin were largely equivalent. V. inaequalis populations treated with QoIs in a commercial and an experimental orchard both responded with significant shifts toward declining QoI sensitivities. However, the population responses were quantitative in nature, and highly resistant isolates indicative of a cytochrome b target site mutation were not detected. V. inaequalis populations from both orchards investigated also were fully resistant to sterol de-methylation-inhibiting fungicides (DMIs) such as fenarimol and myclobutanil, but isolate sensitivities to QoIs and DMIs were largely unrelated. Performance tests with kresoxim-methyl and trifloxystrobin at the experimental orchard diagnosed as DMI-resistant revealed that the quantitative shift toward declining QoI sensitivities did not constitute the status of practical QoI resistance. In contrast to these quantitative responses, emergence of qualitative QoI resistance was documented for V. inaequalis in an orchard in North Germany, which had been treated intensively with a total of 25 QoI applications over four consecutive seasons. Isolates retrieved from the orchard were highly resistant to both kresoxim-methyl and trifloxystrobin and were characterized as G143A cytochrome b mutants. The results indicated that the paths of QoI resistance can be both quantitative and qualitative in nature. A similar phenomenon has not been described before. Circumstantial evidence suggests that the quantitative phase of V. inaequalis population responses to QoIs might be succeeded by a quantitative selection of highly resistant G143A target-site mutants.

Impact of alternative respiration and target-site mutations on responses of germinating conidia of Magnaporthe grisea to Qo-inhibiting fungicides.

Qo-inhibiting fungicides act as respiration inhibitors by binding to the Qo center of cytochrome b. Sensitivities of fungi to Qo inhibitors can be influenced by the induction of alternative respiration or by mutational changes of the cytochrome b target site. Previous studies on both mechanisms in Magnaporthe grisea (Hebert) Barr were focused on the mycelial stage of the pathogen. The present study describes the expression and impact of both resistance mechanisms during the stage of conidia germination. In the absence of a host, alternative respiration provided a >500-fold rescue from azoxystrobin during the germination of conidia derived from four wild-type isolates of M. grisea. This rescue potential during conidia gemination was substantially more pronounced than for mycelial growth. However, the pronounced effectiveness of alternative respiration during conidia germination was not apparent when barley leaves were protected with azoxystrobin prior to inoculation with conidia. In a comparison of a wild-type strain and an alternative respiration-deficient mutant, azoxystrobin efficacies in suppressing symptom development differed by a factor of two, with full disease control achieved for both genotypes at 1 microg ml(-1) azoxystrobin. In contrast, conidia derived from two QoI-resistant target site mutants were highly resistant to azoxystrobin and trifloxystrobin and fully capable of infecting leaf surfaces protected with 10 microg ml(-1) of azoxystrobin. Both target-site mutants had emerged spontaneously in the presence of high azoxystrobin doses when residual mycelial growth was supported by alternative respiration. The effective silencing of alternative respiration in protective applications of Qo-inhibiting fungicides might constitute a strategy of slowing the emergence of highly resistant target site mutants.

Characterization of spontaneous mutants of Magnaporthe grisea expressing stable resistance to the Qo-inhibiting fungicide azoxystrobin.

The class of Qo-inhibiting fungicides (QoIs) act as respiration inhibitors by binding to the Qo center of cytochrome b. The longevity of these fungicides has been challenged by the selection of fungal sub-populations resisting high doses of QoI fungicides, with a G143A amino acid exchange in the cytochrome b target site identified as the most common cause of resistance. In contrast, the mechanism of alternative respiration, as another mechanism of fungal QoI resistance, has thus far not been affiliated with practical resistance. In the present study, azoxystrobin-resistant mutants of Magnaporthe grisea were generated and characterized. Emergence of these spontaneous mutants was facilitated when resting melanized mycelia were allowed to escape full inhibition by azoxystrobin. This escape was related to the intactness of alternative respiration, indicating that residual expression of this rescue mechanism was involved in the spontaneous emergence of target-site mutants. The two mutants characterized resisted high doses of the QoI, azoxystrobin, with resistance factors exceeding 1,000. Two different mutations of the cytochrome b gene were identified as exchanges of guanine, leading to a G143A or a G143S amino acid exchange. Resistance of both target-site mutants remained stable during four consecutive disease cycles in the absence of azoxystrobin. Several parameters tested to measure fitness penalties inherent to the mutational changes revealed that the G143A mutant was not compromised. In contrast, the conidia production of the G143S mutant was significantly lower under both saprophytic and pathogenic conditions of reproduction.

Characterization of Colletotrichum graminicola Isolates Resistant to Strobilurin-Related QoI Fungicides.

Isolates of Colletotrichum graminicola were collected from annual bluegrass or bent grass turf in Japan and the United States, and their sensitivities to QoI fungicides (QoIs) as well as their cytochrome b sequences were characterized. Five isolates sampled from turf treated repeatedly with azoxystrobin were highly QoI resistant under both in vivo and in vitro test conditions. The nucleotide sequences of a large cytochrome b gene segment involving the binding site of QoIs were fully homologous for all resistant isolates and contained the G143A target site mutation known to confer QoI resistance in other pathogens. QoI-sensitive isolates collected prior to treatments with QoIs were more diverse with regard to their cytochrome b gene sequences and their phenotype responses to QoIs. All wild-type isolates retained a glycine in position 143 of cytochrome b. Three of the four QoI-sensitive isolates were, in addition, distinguished by leucines in positions 95, 130, and 141, which were exchanged to threonine in all resistant but also in one of the sensitive isolates. In addition to a more pronounced divergence of cytochrome b sequences, the sensitive wild-type isolates also were diverse with regard to the induction of alternative respiration in response to QoI action, as indicated by comparisons of QoI sensitivities displayed in the absence or presence of the alternative oxidase inhibitor salicylhydroxamic acid. These different phenotype responses expressed under in vitro test conditions had no or only a slight impact on anthracnose control in protective applications of azoxystrobin. Isolate responses in vitro were very similar for trifloxystrobin, indicating cross-resistance among the class of QoIs. Our results imply that C. graminicola falls into the class of pathogens with a potential for rapid selection of highly QoI-resistant phenotypes. Frequent monitoring of population sensitivities will be required to determine the status of population responses toward practical QoI resistance.

Disruption of the alternative oxidase gene in Magnaporthe grisea and its impact on host infection.

Plants and numerous fungi including Magnaporthe grisea protect mitochondria from interference by respiration inhibitors by expressing alternative oxidase, the enzymatic core of alternative respiration. The alternative oxidase gene AOXMg of M. grisea was disrupted. Several lines of evidence suggested that the disruption of AOXMg was sufficient to completely curb the expression of alternative respiration. In the infection of barley leaves, several AOXMg-minus and, thus, alternative respiration-deficient mutants of M. grisea retained their pathogenicity without significant impairment of virulence. However, differences between the wild-type strain and an AOXMg-minus mutant were apparent under oxidative stress conditions generated by the treatment of infected barley leaves with the commercial respiration inhibitor azoxystrobin. Symptom development was effectively suppressed on leaves infected with the alternative respiration-deficient mutant, while lesions on leaves infected with the wild-type strain continued to develop at much higher inhibitor doses. However, respective lesions rarely developed to the stage of full maturity. The results did not conform to a previous model implying that expression of alternative respiration is silenced during pathogenesis by the presence of constitutive plant antioxidants. Rather, alternative respiration provided protection from azoxystrobin during both saprophytic and infectious stages of the pathogen. The nature of similar oxidative stress conditions in the ecology of M. grisea remains an open question.

Evaluation of Tactics for Managing Resistance of Venturia inaequalis to Sterol Demethylation Inhibitors.

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 elevated 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 subpopulations 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 possibility that one of the multiple genes underlying fenarimol and dodine resistance confers resistance 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 detected. 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.

Baseline Sensitivities of Venturia inaequalis Populations to the Strobilurin Fungicide Kresoxim-methyl.

The efficacies of the new strobilurin fungicide kresoxim-methyl for the protection of apple leaves from infection by baseline populations of Venturia inaequalis were uniform across five major apple growing regions in North America. The mean ED50 value determined for 25 populations was 0.35 µg ml-1, with values ranging from 0.11 µg ml-1 to 0.75 µg ml-1. The mean level of scab control achieved at the kresoxim-methyl dose of 4 µg ml-1 was 93%. For one of the five orchards sampled in each region, kresoxim-methyl sensitivities of germinating conidia were determined. Sensitivities of 250 isolates were broadly distributed, with ED50 values ranging from 0.003 µg ml-1 to 0.14 µg ml-1 and a mean of 0.02 µg ml-1. This broad range of in vitro sensitivities was not reflected for the in vivo efficacy of kresoxim-methyl in the protection of apple leaves from scab infections. The discrepancy between in vivo and in vitro sensitivities implies that in vivo tests are more useful for the monitoring of kresoxim-methyl sensitivities of orchard populations. Because it can be expected that only isolates resistant under both test conditions will be prone to future selection, such isolates will contribute to increased frequencies of the least sensitive isolates described in this baseline study. Testing of in vitro isolate sensitivities will, therefore, provide an additional tool in the monitoring of kresoxim-methyl resistance development.