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

2. Fungicide Resistance 101

3. 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

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

5. 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

6. 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

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

8. 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

9. 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

10. 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

11. 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

12. What Fungicides Are at Risk?

13. 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

14. 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

15. 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

16. 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

17. 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

18. 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)

19. 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)

20. 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

21. 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

22. 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

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

24. Fungicide Resistance: Two Case Studies

25. 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

26. 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

27. Powdery mildew QoI sensitive Majority QoI resistantSP 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 log to log10 2 (Figure 3.2). Since, the median EC50 of the sensitive subgroup was (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

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

29. 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

30. 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

31. 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)

32. 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

33. 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

34. Managing Resistance

35. 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

36. 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

37. 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

38. 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

39. 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

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

41. 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

42. 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

43. Any Questions?