Managing Fungicide Resistance Anne DeMarsay Maryland Wine & Grape Industry Meeting February 28, 2009

Fungicide Resistance 101

What Is Fungicide Resistance? Resistance = a stable, heritable trait that results in a reduction in sensitivity to a fungicide by an individual fungus Practical resistance = labeled rates of a fungicide no longer provide commercially acceptable control of a disease

Why Monitor Resistance? Resistance  control failures  crop losses Use of ineffective fungicides may increase resistant strains of fungi

How Does Resistance Occur? Origin: rare genetic mutation(s) that alter the target site in the fungus to block the action of the fungicide Natural selection: fungicide causes selection of the fittest (resistant) individuals

Fungicides select resistant individuals Blue ovals indicate fungal spores, intensity of blue color denotes resistance to a fungicide The blue layer that is “animated in” represents a fungicide spray that kills most spores that are a lighter color (sensitive to the dose applied). The one light-blue oval that remains visible denotes an “escape”, a spore that survives in a protected location or outside the sprayed area A. B. Baudoin, VPI & SU

After the fungicide “wears off”... The surviving resistant (dark-blue) spores become a much larger proportion of the population A. B. Baudoin, VPI & SU

How Does Resistance Occur? Origin: rare genetic mutations that alter the target site(s) in the fungus to block the action of the fungicide Natural selection: fungicide causes selection of the fittest (resistant) individuals Resistant individuals are more likely to survive to reproduce When the fungus reproduces, resistant individuals pass on the mutation

Types of Resistance Qualitative resistance: sudden loss of control Resistance results from a single mutation in one gene Quantitative resistance: gradual reduction in control Resistance results from mutations in several genes that interact

Target site mutation Stepwise, small changes One-step, large changes Same information as the preceding and following slide, but Powerpoint animation. Intensity of the red color of the fungicide molecule denotes the degree of efficacy. A. B. Baudoin, VPI & SU

Resistance to More Than One Fungicide Cross resistance: when a pathogen resistant to one fungicide exhibits resistance to other fungicides in the same chemical class, even without exposure Multiple resistance: when a pathogen independently develops resistance to fungicides in different chemical classes

What Fungicides Are at Risk?

Fungicide categories Systemic Penetrant Protectant A. B. Baudoin, VPI & SU Systemic Penetrant Same information as next slide, but Powerpoint version of animations Three categories. Penetration into the tissues of the plant occurs with both penetrant and systemic fungicides, but to different extents. In the rest of the slides, “penetrant” is taken to include systemic fungicides. These categories are are not absolute; they intergrade Protectant

Contrasts: protectant vs. penetrant Protectant fungicides tend to... have a multi-site mode of action have few problems with resistant strains of target fungi Penetrant fungicides tend to... have a single-site mode of action allow target fungi to develop strains with resistance to the fungicide (risk varies) The terms “single-site” and “multi-site” are described and illustrated in later slides. A. B. Baudoin, VPI & SU

Multi-site inhibitor A. B. Baudoin, VPI & SU The blue oval represents a fungal spore The darker structures represent specific enzymes or ot other possible fungicide target sites Red flashes indicate a multi-site fungicide that can destroy (land on) a variety of different target sites A. B. Baudoin, VPI & SU 6

Protectants cannot penetrate plant tissue A. B. Baudoin, VPI & SU The shape on the right represents plant tissue. It contains some of the same target sites as the fungal spore, but the cuticle protects the plant tissue from penetration by the fungicide Cuticle 6

Penetrants and systemics DO penetrate plant tissue. Target site differs: single-site A penetrant or systemic fungicide penetrates into plant tissue (per definition), and therefore cannot have target sites that are found in plant tissue. They usually have a single site or a very few sites of action, denoted by the yellow shape, which DIFFERS in the fungus and the plant. Cuticle A. B. Baudoin, VPI & SU 6

Fungicides at Risk Most newer fungicides are single-site inhibitors (penetrant or systemic) Single-site inhibitors are more prone to resistance development (qualitative and quantitative)

FRAC Class/Code Risk Common name Trade name Benzimidazoles (1) High thiophanate-methyl Topsin-M Phenylamides (4) mefenoxam Ridomil Strobilurins (QoI) (11) azoxystrobin, kresoxim-methyl, pyraclostrobin, trifloxystrobin Abound, Sovran, Pristine (+boscalid), Flint Dicarboximides (2) Med– High iprodione Rovral Sterol biosynthesis inhibitors (SBI) (3) Med fenarimol, myclobutanil, tebuconazole, triflumizole Rubigan, Rally, Elite, Procure Carboximides (7) boscalid Endura, Pristine (+ pyraclostrobin) Anilinopyrimidines (9) cyprodinil, pyrimethanil Vangard, Scala Quinolines (13) quinoxyfen Quintec Hydroxyanilid (17) fenhexamid Elevate, CaptEvate (+ captan)

HOW much more? Depends on... Single-site inhibitors are much more prone to resistance development than multi-site inhibitors HOW much more? Depends on... Fungicide and its target site Characteristics of target fungus and disease A. B. Baudoin, VPI & SU

HOW much more? Factor #1: fungicide and target site Nature of target site, how essential? Nature of changes possible How easily does site mutate? Are changes detrimental to organism? A. B. Baudoin, VPI & SU

Factor #2: Characteristics of target fungus and disease How fast does fungus reproduce? How easily does it spread? How much variability in population? A. B. Baudoin, VPI & SU (Information equivalent to that in following slide) Left: Rhizoctonia, a fungus which often produces few or no spores, and which spreads relatively slowly Right: powdery mildew, producing abundant spores, which are easily and widely dispersed by wind Variation in fungus populations not illustrated (Photo credits as indicated) H. Couch J. Verreet

Fungicide/target site risk A. B. Baudoin, VPI & SU Hot zone! High Benzimidazoles Strobilurins Mancozeb Chlorothalonil Powdery mildew Rhizoctonia Low Low Pathogen risk High

Fungicide Resistance: Two Case Studies

Case #1: Strobilurins (QoIs) Used to control grape powdery mildew (PM) and grape downy mildew (DM) First sold in 1996; in widespread use by 1998 Azoxystrobin (Abound) labeled for grapes in 1997 Others introduced between 2001 and 2008 High risk of resistance development Mode of action: single-site inhibitor of energy production in fungal mitochondria Qualitative resistance (one mutation): sudden Cross resistance among all strobilurins

Case #1: Strobilurins (QoIs) Resistant grape PM strains detected In NY and PA in 2002 (1st cases in U.S.) In VA, MD, NC, and PA in 2005–07 In Europe in 2006 Resistant grape DM strains detected In Europe in 2000 In VA in 2005 (1st case in U.S.) In MD, NC, and PA in 2005–07

Powdery mildew QoI sensitive Majority QoI resistant SP B Be Su Li JW Sh SC Bx HC Ha NK Ar RO JR BR GL Vi CM MR CO AM LV MV GR IV AV Re CR WF PR Majority QoI resistant QoI sensitive Powdery mildew Figure 3.1 shows the locations from which the E. necator isolates were collected and their reaction to azoxystrobin. The frequency distribution of the log-EC50 values of 154 PM isolates was broad and continuous ranging from log10 -2.5 to log10 2 (Figure 3.2). Since, the median EC50 of the sensitive subgroup was 0.007 (µg/ml) we believe that 1 (µg/ml) is a valid discriminatory dose to classify whether an isolate is QoI-resistant or QoI-sensitive. Out of the 154 E. necator isolates, 28 (18%) were QoI-sensitive and 126 isolates (82%) were QoI-resistant. The EC50 values ranging from 1-10 was classified as intermediate resistance while EC50 values greater than 10 was classified as high resistance. Seventy-nine of the QoI-resistant (63%) isolates were highly resistant and 47 isolates (37%) had intermediate resistance (Fig 3.2). Resistant isolates were collected from all geographic areas sampled (Figure 3.1) indicating that QoI resistance is widespread in the Mid-Atlantic US region. Map: J. F Colcol, A. B. Baudoin 27

QoI sensitive Majority QoI resistant Map: J. F Colcol, A. B. Baudoin

Case #1: Strobilurins (QoIs) When resistance was first detected in Virginia Vineyards with resistant PM/DM had been sprayed from 0 to 17 times with strobilurins Sprays per season averaged 2 to 3.4 FRAC recommends 3 sprays max. per year; labels allow 4 sprays Current resistance management guidelines not adequate to prevent RAPID development of resistance

Case #2: Sterol Biosynthesis Inhibitors (SBIs) In use since 1982 for grape PM 1982: triadimefon (Bayleton) 1989: fenarimol (Rubigan), myclobutanil (Nova/Rally) 1990s: tebuconazole (Elite), triflumizole (Procure) Medium risk of resistance Mode of action: single-site inhibitor of ergosterol synthesis Quantitative resistance (several mutations in interacting genes): gradual loss of sensitivity Partial cross resistance

Case #2: Sterol Biosynthesis Inhibitors (SBIs) Loss of sensitivity in grape PM strains detected In CA in 1985–86 (triadimefon) In NY in 1995 (triadimefon, fenarimol, myclobutanil) In Canada in 1999–2000 (myclobutanil) In VA, MD, NC, and PA in 2005–07 (triadimefon, fenarimol, myclobutanil, tebuconazole, triflumizole)

Case #2: Sterol Biosynthesis Inhibitors (SBIs) Mid-Atlantic samples of grape PM Greater loss of sensitivity to myclobutanil (Nova/Rally) and tebuconazole (Elite) than other SBIs Multiple resistance: isolates that were resistant to strobilurins were more resistant to SBIs A possible reason: correlated selection pressure

Case #2: Sterol Biosynthesis Inhibitors (SBIs) Spray history at Upper Marlboro, MD farm Vineyard planted in 2001 Reduced sensitivity to SBIs detected in 2006 Myclobutanil (Nova) applied 11 times 2003–07 Sprays per season: 1 or 2; once 3 FRAC recommends 4 sprays max. per year; Nova/Rally label allows ~5 sprays Current resistance management guidelines not adequate to prevent loss of sensitivity

Managing Resistance

Other Reasons for Control Failures Poor timing Starting protectant applications too late Spray intervals too long Missed sprays Spotty coverage Sprayer not calibrated or nozzles not adjusted Inadequate spray volume for canopy

Coverage Variable More uniform A. B. Baudoin, VPI & SU Mosaic of microsites with greater or smaller fungicide dosages (intensity of blue color) Variable More uniform

Other Reasons for Control Failures Wrong rate of fungicide Rate too low for the disease—read the label! In a tank mix, use at least the minimum rate on the label for each fungicide Canopy too dense Too windy during spraying

Delaying Resistance Development Practice integrated disease management Plant disease-resistant varieties Use cultural controls first (good canopy management, sanitation) Use protectant fungicides to prevent disease buildup Spray effectively Calibrate your sprayer every year. Check coverage with water-sensitive spray cards Use appropriate spray intervals and volume for the fungicide and time of the season

Area of poor coverage Good coverage A. B. Baudoin, VPI & SU …because partially resistant strains would not be eliminated in areas of insufficient coverage A. B. Baudoin, VPI & SU

After the fungicide “wears off”... … and has an advantage in repopulating the area A. B. Baudoin, VPI & SU Poor coverage Better coverage

Delaying Resistance Development Plan your spray program Limit the number of applications of high- and medium-risk fungicides Use the right rate for the disease Rotate among different classes of fungicides Tank-mix fungicides from different classes that are effective against the same disease Add 2–5 lb sulfur to a strobilurin or SBI spray for powdery mildew Add captan or a phosphorous acid to Pristine for downy mildew

Summary Resistance is the evolutionary response of a fungus to a threat to its survival: the fungicide Fungicides that act at a single site are more prone to resistance development How quickly resistance will develop, and how much control will be lost, depend on both the fungicide and the target fungus Resistance to most newer fungicides will occur, but we can prolong their effective life by careful use

Any Questions?