Principles of fungicide resistance management and their application to North Dakota agriculture Michael Wunsch, plant pathologist NDSU Carrington Research Extension Center
Steps in fungal disease development (A) SPORES LAND ON LEAF (B) SPORES GERMINATE ON LEAF Images adapted from Clergeot et al. 2001. PNAS 98(12):6963-6968
Steps in fungal disease development (C) FUNGUS FORMS APPRESSORIUM, PENETRATES PLANT Images adapted from Clergeot et al. 2001. PNAS 98(12):6963-6968
Steps in fungal disease development (D) FUNGUS GROWS WITHIN PLANT Image adapted from Clergeot et al. 2001. PNAS 98(12):6963-6968
Steps in fungal disease development (E) SEVERAL DAYS LATER, DISEASE SYMPTOMS APPEAR
How fungicides work Protectant fungicides: A protective barrier on leaf surface that inhibits spore germination and penetration. Image adapted from Clergeot et al. 2001. PNAS 98(12):6963-6968
How fungicides work Protectant fungicides: A protective barrier on leaf surface that inhibits spore germination and penetration. Image adapted from Clergeot et al. 2001. PNAS 98(12):6963-6968
How fungicides work Systemic fungicides: Absorbed into plants following application - Rain-fast within a few hours of application - Not sensitive to solar radiation Relative to protectants, require a less-thorough application coverage to be effective Curative: Stop or slow infections in the first 24 to 72 hours after fungal penetration.
How fungicides work Systemic fungicides: Stop or slow infections in the first 24 to 72 hours after fungal penetration. Image adapted from Clergeot et al. 2001. PNAS 98(12):6963-6968
How fungicides work Systemic fungicides: Have curative activity against new infections that are not yet showing symptoms. May exhibit limited eradicant and antisporulant activity.
How fungicides work Systemic fungicides: Locally systemic: capable of moving through a few layers of cells Translaminar movement: movement from top to bottom of leaf Xylem movement: upward movement in plants; outward movement in leaves. True systemic activity upward movement in xylem and downward movement in phloem is rare in fungicides
How fungicides work Acropetal movement: Outward movement within leaves. Xylem transport. Folicur (tebuconazole) Alto (cyproconazole) Untreated Images courtesy of Syngenta
How fungicides work Translaminar movement: Active ingredient (1) penetrates leaf cuticle, (2) moves within leaf tissue to bottom of leaf, & (3) protects top and bottom surfaces of leaf. Translaminar movement: Movement from top to bottom of leaf Image courtesy of Syngenta
Most important classes of systemic fungicides Most systemic fungicides marketed for use on field crops in western ND are in one of three major classes: 1. QoI (strobilurin; FRAC 11) azoxystrobin Quadris pyraclostrobin Headline fluoxastrobin - Evito
Most important classes of systemic fungicides Most systemic fungicides marketed for use on field crops in western ND are in one of three major classes: 1. QoI (strobilurin; FRAC 11) 2. DMI (includes imidazoles & triazoles, FRAC 3) cyproconazole Alto metconazole Caramba propiconazole Tilt, Bumper, Fitness, PropiMax, Topaz, etc. prothioconazole Proline tebuconazole Tebuzol, Monsoon, Muscle, Onset, Orius, Toledo, etc. prothioconazole + tebuconazole Prosaro
Most important classes of systemic fungicides Most systemic fungicides marketed for use on field crops in western ND are in one of three major classes: 1. QoI (strobilurin; FRAC 11) 2. DMI (includes imidazoles & triazoles, FRAC 3) 3. SDHI (carboxamides; FRAC 7) penthiopyrad Vertisan boscalid Endura
Most important classes of systemic fungicides Most systemic fungicides marketed for use on field crops in western ND are in one of three major classes: 1. QoI (strobilurin; FRAC 11) 2. DMI (includes imidazoles & triazoles, FRAC 3) 3. SDHI (carboxamides; FRAC 7) 4. Premixes of active ingredients from 2+ classes pyraclostrobin (FRAC 11) + fluxapyroxad (FRAC 7) Priaxor trifloxystrobin (FRAC 11) + propiconazole (FRAC 3) Stratego pyraclostrobin (FRAC 11) + metconazole (FRAC 3) Twinline azoxystrobin (FRAC 11) + propiconazole (FRAC 3) Quilt, Quilt Xcel
QoI fungicides (FRAC 11) QoI fungicides: Disrupt energy production by the fungus - Inhibit electron transfer in mitochondria of fungi - single-site mode of action (disruption of a single metabolic process)
QoI fungicides (FRAC 11) Efficacy can be completely lost with the change of a single nucleotide in the genetic code of the fungus. QoI fungicides bind to a specific protein in the fungal mitochondrion, blocking electron transport and thus energy production With a single nucleotide change at a specific location, the protein changes and QoI fungicides can no longer bind
QoI fungicides (FRAC 11) Efficacy can be completely lost with the change of a single nucleotide in the genetic code of the fungus. QoI fungicides bind to a specific protein in the fungal mitochondrion, blocking electron transport and thus energy production With a single nucleotide change at a specific location, the protein changes and QoI fungicides can no longer bind A very common and serious mutation: G143A Results in abrupt loss of control. Complete cross-resistance across all QoI chemistries.
QoI fungicides (FRAC 11) A very common and serious mutation: G143A Results in abrupt loss of control. Complete cross-resistance across all QoI chemistries.
QoI fungicides resistance via G143A mutation QoI resistance via G143A mutation in Ascochyta rabiei: complete loss of Ascochyta disease control on chickpeas
QoI fungicides resistance via G143A mutation Sensitivity of Ascochyta rabiei to QoI fungicides, 1983-2003 azoxystrobin (Quadris) pyraclostrobin (Headline) Wise et al. 2008 Plant Disease 92:295-300
QoI fungicides resistance via G143A mutation Sensitivity of Ascochyta rabiei to QoI fungicides, 2005 azoxystrobin (Quadris) pyraclostrobin (Headline) Wise et al. 2009 Plant Disease 528-536
QoI fungicides resistance via G143A mutation Sensitivity of Ascochyta rabiei to QoI fungicides, 2006 azoxystrobin (Quadris) pyraclostrobin (Headline) Wise et al. 2009 Plant Disease 528-536
QoI fungicides (FRAC 11) With a slightly different change in the genetic code of the fungus, QoI efficacy is reduced but not lost This mutation weakens the binding of the fungicide to the target protein
QoI fungicides (FRAC 11) With a slightly different change in the genetic code of the fungus, efficacy is reduced but not lost This mutation weakens the binding of the fungicide to the target protein The most common mutation of this type: F129L Results in reduced effectiveness by fungicide. Partial cross-resistance: Some QoIs provide more control than others.
QoI fungicides resistance via F129L mutation Control of turfgrass anthracnose (Colletotrichum cereale) NORTH CAROLINA 2004 & 2005 Young et al. 2010 Plant Disease 94(6):751-757
QoI fungicides resistance via F129L mutation Control of potato early blight (Alternaria solani) CENTRAL NORTH DAKOTA 2002 & 2003 Alternaria solani population with F129L mutation Pasche & Gudmestad 2008 Crop Protection 27:427-435
QoI fungicides (FRAC 11) Another single-nucleotide change in the genetic code of the fungus reduces QoI efficacy. G137L mutation: Results in reduced sensitivity Not as common as the G143A or F129L mutations
DMI fungicides (FRAC 3) DMI fungicides: Disrupt cell wall synthesis by fungi - Inhibit the production of sterols, important components of cell membrane - single-site mode of action (disruption of a single metabolic process)
DMI fungicides (FRAC 3) Single-site mode of action: a single protein is targeted But high resistance is achieved with the additive effect of multiple mutations: 1) Mutations in the protein targeted by DMI fungicides
DMI fungicides (FRAC 3) Single-site mode of action: a single protein is targeted But high resistance is achieved with the additive effect of multiple mutations: 1) Mutations in the protein targeted by DMI fungicides 2) Increased level of expression of the target protein by fungi
DMI fungicides (FRAC 3) Single-site mode of action: a single protein is targeted But high resistance is achieved with the additive effect of multiple mutations: 1) Mutations in the protein targeted by DMI fungicides 2) Increased level of expression of the target protein by fungi 3) Dramatically increased expression of protein pumps spanning cell walls that can remove the fungicides from fungal cells
DMI fungicides (FRAC 3) Single-site mode of action: a single protein is targeted But high resistance is achieved with the additive effect of multiple mutations: 1) Mutations in the protein targeted by DMI fungicides 2) Increased level of expression of the target protein by fungi 3) Dramatically increased expression of protein pumps spanning cell walls that can remove the fungicides from fungal cells Resistance development occurs via a stepwise process:
DMI fungicides (FRAC 3) Mycosphaerella graminicola: sensitivity to fluquinconazole SOUTHERN ENGLAND, 1993-2002 registration of fluquinconazole Septoria leaf spot caused by Mycosphaerella graminicola (Septoria tritici) Photo courtesy of Dept. of Agriculture & Food, Government of Western Australia Data points represent the fungicide concentration at which 50% pathogen inhibition occurred Mavroeidi and Shaw 2006 Crop Protection 24:259-266
DMI fungicides (FRAC 3) Mycosphaerella graminicola: sensitivity to epoxiconazole EUROPE, 2007 Multiple mutations in target gene observed; all had modest effects. Stammler et al. 2008 Crop Protection 1448-1456.
DMI fungicides (FRAC 3) Mycosphaerella graminicola: sensitivity to epoxiconazole EUROPE, 2007 Greatest reductions in sensitivity: Strains that include I381V and A379G mutations Stammler et al. 2008 Crop Protection 1448-1456.
DMI fungicides (FRAC 3) The mutations with greatest effect on sensitivity to epoxiconazole had no measurable effect on fungicide performance in field FRANCE, 2007 - Septoria leaf blight Stammler et al. 2008 Crop Protection 1448-1456.
DMI fungicides (FRAC 3) EUROPE: As of 2008, reductions in the sensitivity of Mycosphaerella graminicola to DMI fungicides have plateaued. Field performance of fungicides largely unaffected. But only mutations in the target gene have been observed. Septoria leaf spot caused by Mycosphaerella graminicola (Septoria tritici) Photo courtesy of Dept. of Agriculture & Food, Government of Western Australia
DMI fungicides (FRAC 3) EUROPE: As of 2008, reductions in the sensitivity of Mycosphaerella graminicola to DMI fungicides have plateaued. Field performance of fungicides largely unaffected. But only mutations in the target gene have been observed. Septoria leaf spot caused by Mycosphaerella graminicola (Septoria tritici) Increased expression of the target gene not yet observed. Photo courtesy of Dept. of Agriculture & Food, Government of Western Australia
DMI fungicides (FRAC 3) EUROPE: As of 2008, reductions in the sensitivity of Mycosphaerella graminicola to DMI fungicides have plateaued. Field performance of fungicides largely unaffected. But only mutations in the target gene have been observed. Septoria leaf spot caused by Mycosphaerella graminicola (Septoria tritici) Photo courtesy of Dept. of Agriculture & Food, Government of Western Australia Increased expression of the target gene not yet observed. Increased expression of cell membrane protein pumps that remove fungicides from fungal cells not yet observed.
DMI fungicides (FRAC 3) Ascochyta rabiei: sensitivity to prothioconazole (Proline) PRIOR TO REGISTRATION OF PROLINE Wise et al. 2011
DMI fungicides (FRAC 3) Ascochyta rabiei: sensitivity to prothioconazole (Proline) 2007 Wise et al. 2011
DMI fungicides (FRAC 3) Ascochyta rabiei: sensitivity to prothioconazole (Proline) 2008 Wise et al. 2011
DMI fungicides (FRAC 3) Ascochyta rabiei: sensitivity to prothioconazole (Proline) 2009 Wise et al. 2011
DMI fungicides (FRAC 3) Has field performance of Proline been affected by shifts in sensitivity? Within-column means followed by different letters are significantly different (P < 0.05; Tukey multiple comparison procedure). 2009 Fungicide application timing: A = June 23; prior to bloom and 3 days after the first appearance of disease symptoms B = July 6 C = July 18 D = July 29 E = Aug. 10 NIS denotes non-ionic surfactant applied at 0.125% (v/v)
DMI fungicides (FRAC 3) Has field performance of Proline been affected by shifts in sensitivity? NDSU Williston REC - Nesson Valley Irrigation Research Site CDC Frontier chickpeas Fungicide application timing: A = June 27 (bloom initiation; Ascochyta at trace levels) B = July 10 C = July 20 D = August 2 Within-column means followed by different letters are significantly different (P < 0.05; Tukey multiple comparison procedure)
DMI fungicides (FRAC 3) Has field performance of Proline been affected by shifts in sensitivity? Within-column means followed by different letters are significantly different (P < 0.05; Tukey multiple comparison procedure) NDSU Williston REC Nesson Valley Irrigation Research Site CDC Frontier chickpeas Fungicide application timing: A = June 27 (bloom initiation; Ascochyta at trace levels) B = July 10 C = July 20 D = August 2
DMI fungicides (FRAC 3) Has field performance of Proline been affected by shifts in sensitivity of Ascochyta rabiei to prothioconazole? We don t know for certain. The current situation might be very similar to Septoria leaf spot of wheat in Europe. The risk of additional mutations that significantly reduce efficacy remains high.
SDHI fungicides (FRAC 7) SDHI fungicides: Disrupt energy production by the fungus - Like the QoIs, bind to a disrupt a biochemical process within mitchondria - Different target within mitochondrion than the QoIs - single-site mode of action (disruption of a single metabolic process)
SDHI fungicides (FRAC 7) Like the QoIs: Abrupt loss of control Different from the QoIs: Cross-resistance across all SDHI chemistries often does not occur
Minimizing the risk that fungicide resistance develops 1. Rotate fungicides that have different modes of action. 2. Adopt cropping practices that reduce disease pressure: Reduce the number of fungal individuals exposed to selection. 3. Avoid unnecessary fungicide applications. 4. Make timely applications of fungicides. 5. Apply the full labeled rate.
Online resources Fungicide efficacy reports: Side-by-side comparisons of registered products Disease diagnosis and management guides: Up-to-date management recommendations
Online resources www.ag.ndsu.edu/carringtonrec Or Google NDSU Carrington Research Extension Center Click on the plant pathology link
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