Source: N Y AGRICULTURAL EXPT STATION submitted to
FUNGAL RESISTANCE OF VENTURIA INAEQUALIS TO ANTIFUNGAL STROBILURINS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0181851
Grant No.
(N/A)
Project No.
NYG-625506
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Apr 1, 1999
Project End Date
Sep 30, 2004
Grant Year
(N/A)
Project Director
Koeller, W.
Recipient Organization
N Y AGRICULTURAL EXPT STATION
(N/A)
GENEVA,NY 14456
Performing Department
PLANT PATHOLOGY
Non Technical Summary
(N/A)
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21240201000100%
Goals / Objectives
Determine the risk of resistance of Venturia inaequalis to the new class of strobilurin fungicides. Two parameters will be investigated. Under saprophytic growth conditions, exposure of fungal organisms to strobilurin fungicides will induce the expression of alternative respiration. This alternative pathway exerts strong antagonistic effects on the expression of full strobilurin potencies but also protects from the selection of resistant target site mutants. Evidence exists that alternative respiration is not induced during the infectious stages of host infection thus imposing the risk of selecting such arget site mutants during disease control regimes involving strobilurins. The second objective is to investigate preliminary evidence indicating that strobilurins are affected by the novel phenomenon of multiple fungicide resistance to both the new class of strobilurins and the older class of sterol demethylation inhibitors already affected by pathogen resistance.
Project Methods
The alternative oxidase gene of Venturia inaequalis will be cloned and disrupted. Respective mutants will be subjected to mutagenesis and selection toward strobilurin resistance. The strobilurin target site cytochrome b will be assessed in respective mutants with regard to the presence of mutational changes. Secondly, the expression of the alternative oxidase gene of Venturia inaequalis will be determined during the infection of apple leaves by the pathogen. Multiple fungicide resistance will be assessed by first identifying geneotypes of the pathogen displaying the respective phenomenon. These genotypes will then be subjected to an analysis of fungal multi drug resistance genes known to comprise a subgroup of the superfamily of ABC membrane transporters.

Progress 04/01/99 to 09/30/04

Outputs
The development of resistance to modern fungicides is driven by adaptive responses of fungal pathogen populations to the new environment of a new fungicide introduced. Growers have repeatedly incurred substantial crop losses caused by sudden yet unexpected outbreaks of fungicide resistance. In recognition of this unsustainable situation, a project was launched to develop an inclusive sensitivity test to determine the orchard-specific sensitivities of V. inaequalis populations causing apple scab. This test allowed us to evaluate the merits of the novel concept of site-specific management of resistance (SMOR). The SMOR concept relies on tests of orchard sensitivities to all modern fungicide options and the design of scab control programs that integrate the still active options, thus avoiding control failures and crop losses before they occur. The pilot program of offering the SMOR service to 13 growers throughout New York was successful in 2003 and 2004. Several growers had experienced poor control of scab after using DMI fungicides. In all cases, poor control was caused by emerging or established DMI resistance. Management changes guided by the SMOR test results and recommendations did restore excellent levels of scab control.

Impacts
The successful implementation of site-specific management of resistance (SMOR) as a novel service to growers will prolong the useful lifetime of modern fungicides and, at the same time, will protect growers from unexpected crop losses.

Publications

  • Koeller, W., Wilcox, W. F., and Parker, D. M. 2005. Sensitivities of Venturia inaequalis populations to anilinopyrimidines and their contribution to scab management. Plant Dis.: in press.
  • Turechek, W. W., and Koeller, W. 2004. Managing resistance of Venturia inaequalis to the strobilurin fungicides. Plant Health Progress doi: 10.1094/PHP-2004-0908-01-RS.
  • Koeller, W. 2004. Pathogens. Pages 17-19 in: Management of Pest Resistance: Strategies Using Crop Management, Biotechnology, and Pesticides. CAST Special Publication No. 24, Ames, Iowa.
  • Koeller, W., Parker, D. M., Turecheck, W. W., Avila-Adame, C., and Cronshaw, K. 2004. A two-phase resistance response of Venturia inaequalis populations to the QoI fungicides kresoxim-methyl and trifloxystrobin. Plant Dis. 88:537-544.


Progress 01/01/03 to 12/31/03

Outputs
The utility of modern fungicides in IPM programs is hampered by the repeated development of resistance. The development of resistance is driven by responses of fungal pathogen populations adapting to a new environment they compete in. It has been recognized repeatedly that growers utilizing modern fungicides under resistance risk are not aware of the status of resistance and thus, incur substantial crop losses caused by sudden yet unexpected outbreaks of resistance. In recognition, a project was launched to develop an inclusive sensitivity test to determine the orchard-specific sensitivities of V. inaequalis populations. We envision to offering this site-specific test to individual apple growers as a service to design scab management programs most appropriate for their particular orchards. The analysis of data retrieved during 2002 and 2003 were encouraging. We were able to verify that our predictions of QoI resistance development for V. inaequalis - quantitative resistance will follow the selection of immune G143A target site resistance. Furthermore, we could show that the sensitivities of populations to anilinopyrimidines were significantly decreased by the trait of multiple fungicide resistance.

Impacts
The successful initiation of our site-specific sensitivity tests enabling growers to design sensitivity-based scab management programs constitutes a new approach to the management of fungicide (and pesticide) resistance management.

Publications

  • Avila-Adame, C., and Koeller, W. 2003. Insertional mutagenesis of Magnaporthe grisea toward decreased responsiveness of alternative respiration to inhibition by azoxystrobin. J. Gen. Plant Pathol. 69: 126-130.
  • Avila-Adame, C., Olaya, G., and Koeller, W. 2003. Characterization ofColletotrichum graminicola isolates resistant to strobilurin-related QoI fungicides. Plant Dis. 87: 1426-1432.
  • Avila-Adame, C., and Koeller, W. 2003. Characterization of spontaneous mutants of Magnaporthe grisea expressing stable resistance to the Qo inhibiting fungicide azoxystrobin. Curr. Genet. 42: 332-338.
  • Avila-Adame, C., and Koeller, W. 2003. Impact of alternative respiration and target-site mutantions on responses of germinating conidia of Magnaporthe grisea to QoI-inhibiting fungicides. Pest Managem. Sci 59:303-309.
  • Koeller, W. 2003. Mode of action of sterol-inhibiting fungicides. Pages 628-640 in: The Encyclopedia of Agrochemicals. Plimmer, J. R., ed., John Wiley, New York.


Progress 01/01/02 to 12/31/02

Outputs
The utility of modern fungicides is hampered by the development of resistance. Two resistance mechanisms to the new class of QoI fungicides, which is highly prone to the development of resistance, were investigated for Magnaporthe grisea. The impact of alternative respiration as one of the known mechanisms of QoI resistance was investigated by employing SHAM as an inhibitor of alternative oxidase and by disrupting the alternative oxidase gene MgAOX. The results demonstrated that alternative respiration was a highly effective mechanism of rescuing germinating conidia and mycelia from the action of the QoI azoxystrobin under in vitro conditions of development. However, the mechanism was silenced completely when conidia germinated on the surface of the barley host protected with azoxystrobin prior to infection. In contrast, the rescue mechanism was not entirely silenced, when azoxystrobin was applied subsequent to infection. Under such post-infection conditions, residual development of disease symptoms progressed. Residual mycelial growth in the presence of a QoI was also sufficient to generate spontaneous G143A target site mutations in the presence of azoxystrobin. Therefore, post-infection applications of QoI fungicides might not only have selected but also generated highly resistant target site mutants. The rapid selection of QoI-resistant target site mutants has also been identified for Colletotrichum graminicola infecting turf grasses. Here, sequence analysis of the mitochondrial cytochrome b gene indicated that particular SNPs might functionally stabilize a G143A mutation, indicating for the first time that particular mutations not directly involved in inhibitor binding might predispose a target site to a mutation toward fungicide resistance. In contrast to C. graminicola, the selection of highly resistant G143A mutants of Venturia inaequalis was preceded by a quantitative shift of populations toward decreased isolate sensitivities. In this and most likely other cases, such initial quantitative population responses allowing residual development of the pathogen have contributed to the selection and potentially even generation of highly QoI-resistant target site mutants. This intermediate quantitative response can be slowed by applying QoI fungicides in a protective mode and at increased application rates, as documented for the class of DMI fungicides characteristic for quantitative population responses.

Impacts
The modern class of QoI fungicides, which allow management of a broad spectrum of fungal diseases, has been hampered by the occasionally rapid development of resistance. Our research has prepared the ground for a preventative anti-resistance strategy prolonging the usefulness of these fungicides.

Publications

  • Avila-Adame, C., and Koeller, W. 2002. Disruption of the alternative oxidase gene in Magnaporthe grisea and its impact on host infection. Mol. Plant-Microbe Interact. 8:122-130.
  • Olaya, G., Avila-Adame, C., and Koeller, W. 2002. Resistance of Colletotrichum graminicola to QoI inhibitors. Phytopathology 92:S61.


Progress 01/01/01 to 12/31/01

Outputs
The utility of modern fungicides, which have to be preserved in IPM programs, are hampered by the development of resistance. For the apple scab fungus Venturia inaequalis, three rounds of resistance have been established in the past, first to dodine, and then to the benzimidazoles and the DMIs. An analysis of monitoring data indicated the possibility that respective phenotypes had been selected for the trait of fungicide resistance but also for the trait of mutability. This phenomenon could potentially lead to an accelerated speed of resistance development to new fungicides. Two resistance mechanisms to the new class of QoI fungicides were investigated for Magnaporthe grisea. Alternative respiration as one of the mechanisms of QoI resistance was investigated by disrupting the alternative oxidase gene MgAOX. Respective transformants were not impaired in pathogenicity and virulence, but they became significantly more sensitive to the QoI azoxystrobin in post-infection applications to infected barley leaves. Residual mycelial growth provided by the expression of alternative respiration was also found to be involved in the spontaneous and frequent emergence of cytochrome b target site mutants. In addition to the G143A mutation, which was first identified for a laboratory mutant of Venturia inaequalis, a novel G143A mutant was identified in Magnaporthe gisea. The latter mutant, however, was impaired in the production of conidia, while the G143A mutant was not impaired in any fitness parameter investigated. Alternative respiration was highly effective in protecting conidia germinating conidia from azoxystrobin action in the absence of a host. This rescue potential was not expressed in the infection of host surfaces protected with a QoI. Protective applications and the avoidance of post-infection treatments appear to constitute an anti-resistance strategy.

Impacts
The results will guide the development of sustainable anti-resistance strategies.

Publications

  • Koeller, W., Avila-Adame, C., Olaya, G., and Zheng, D., 2001. Resistance to strobilurin fungicides. Pages 215-229 in: Agrochemical Resistance - Extent, Mechanism, and Detection. J. M. Clark and I. Yamaguchi, eds. American Chemical Society, Washington, DC.
  • Koeller, W., and Wilcox, W.F., 2001. Evidence for the predisposition of fungicide-resistant isolates of Venturia inaequalis to a preferential selection for resistance to other fungicides. Phytopathology 91:776-781.
  • Koeller, W., 2001. Fungicide Resistance. Pages 483-488 in: The Encyclopedia of Plant Pathology. Maloy, O.C., and Murray, T.D., eds., John Wiley, New York.


Progress 01/01/00 to 12/31/00

Outputs
The new class of strobilurin fungicides now referred to as Qo inhibitors has experienced rapid resistance development for several pathogens. Severe cases of resistance have not yet been reported for the apple scab fungus Venturia inaequalis, most likely because a target site mutation is accompanied by a fitness penalty. This positive perspective does not exclude that other mechanisms of resistance will not cause future problems. A three-year orchard study suggested that the Qo inhibitor kresoxim-methyl is partially cross resistant to the entirely different class of DMI fungicides, which has to be preserved in IPM programs of apple scab control. Partial cross resistance was expressed by the selection of DMI resistant subpopulations in response to kresoxim-methyl treatments. However, this selection process was effectively managed at a high kresoxim-methyl rate. The mechanistical reasons for this unexpected partial cross resistance with impact on anti-resistance strategies remains unknown. Analysis of isolate sensitivity data retrieved over the past decade indicates that isolates already resistant to one fungicide respond faster to the selection pressure exerted by an unrelated fungicide. This new phenomenon of hypermutability might be involved in partial cross resistance among unrelated classes of modern fungicides.

Impacts
(N/A)

Publications

  • Zheng, D., Olaya, G., and Koeller, W. 2000. Characterization of laboratory mutants of Venturia inaequalis resistant to the strobilurin-related fungicide kresoxim-methyl. Curr. Genet. 38:148-155.
  • Koeller, W., and Wilcox, W.F. 2000. Interactive effects of dodine and the DMI fungicide fenarimol in the control of apple scab. Plant Dis. 84:863-870.


Progress 01/01/99 to 12/31/99

Outputs
In anticipation of the new class of strobilurins as commercial fungicides active against apple scab, work on the assessment of strobilurin resistance risks were initiated. The distribution of isolate sensitivities to the strobilurin fungicide kresoxim-methyl in Venturia inaequalis populations is broad and continuous in character. This broad baseline sensitivity distribution is partly explained by the induction of alternative respiration, a mechanism not necessarily relevant to scab control under orchard conditions. Under laboratory conditions, a target site mutant could be generated under mutagenic conditions with a G143A amino acid exchange for the cytochrome b target site of strobilurins. However, mitochondria carrying this mutation replicated more slowly than wild-type mitochondria and declined in frequencies in the absence of a strobilurin inhibitor.

Impacts
(N/A)

Publications

  • Olaya, G., and Koeller, W. 1999. Baseline sensitivities of Venturia inaequalis populations to the strobilurin fungicide kresoxim-methyl. Plant Dis. 83:274-278.
  • Olaya, G., and Koeller, W. 1999. Diversity of kresoxim-methyl sensitivities in baseline populations of Venturia inaequalis. Pestic. Sci. 55: 1083-1088.
  • Koeller, W. 1999. Chemical approaches to managing plant pathogens. Pages 337-376 in: Handbook of Pest Management. J. R. Ruberson, ed., Marcel Dekker, New York.