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Melvin D. Bolton and Bart P.H.J. Thomma (eds.), Plant Fungal Pathogens: Methods and Protocols, Methods in Molecular Biology, vol. 835, DOI 10.1007/978-1-61779-501-5_23, © Springer Science+Business Media, LLC 2012

Chapter 23

Fungicide Resistance Assays for Fungal Plant Pathogens

Gary A. Secor and Viviana V. Rivera

Abstract

Fungicide resistance assays are useful to determine if a fungal pathogen has developed resistance to a fungicide used to manage the disease it causes. Laboratory assays are used to determine loss of sensitivity, or resistance, to a fungicide and can explain fungicide failures and for developing successful fungicide recommendations in the fi eld. Laboratory assays for fungicide resistance are conducted by measuring reductions in growth or spore germination of fungi in the presence of fungicide, or by molecular proce-dures. This chapter describes two techniques for measuring fungicide resistance, using the sugarbeet leaf spot fungus Cercospora beticola as a model for the protocol. Two procedures are described for fungicides from two different classes; growth reduction for triazole (sterol demethylation inhibitor; DMI) fungicides, and inhibition of spore germination for quinone outside inhibitor (QoI) fungicides.

Key words: Triazole , Strobilurin , Cercospora beticola

In vitro assays for fungicide sensitivity have been developed for a number of fungal/fungicide systems and several excellent reviews have been published ( 1– 6 ) . Such assays are useful to monitor changes in sensitivity of the fungal pathogen as fungicides are applied over time, usually years. This information is most often used to make effi cacious fungicide recommendations for disease management. For best results, sensitivity assays should be done before fungicides are registered and used commercially in the fi eld in order to establish a baseline that can be used to monitor changes in sensitivity in subsequent years. Many in vitro procedures have been developed to measure changes in fungicide sensitivity. Molecular procedures utilize PCR or real time PCR to detect spe-cifi c nucleic acid base changes that are associated with fungicide resistance ( 7 ) . In order to use molecular assays, a specifi c genetic change conferring resistance must be known and specifi c primers

1. Introduction

386 G.A. Secor and V.V. Rivera

identifi ed. Biological assays for fungicide resistance rely on changes in the growth of the fungus, such as inhibition of radial growth or reduction of spore germination, in response to exposure to the fungicide in vitro . Because fungicides have different targets site in the fungus and consequently different modes of action, the assay used may be fungicide dependent.

In this protocol, we describe two methods for monitoring fun-gicide resistance using the fungus Cercospora beticola Sacc., the cause of leaf spot of sugar beet (CLS), as a model for the procedures. This disease is endemic in all sugar beet producing areas and causes a loss in yield and sucrose due to reduced photosynthetic area by diseased leaves. The disease is controlled by crop rotation, resistant varieties and timely fungicide applications. Several fungicides from different classes, including benzimidazoles, dithiocarbamates, tin compounds, triazoles, and strobilurins have been used for managing CLS. During the past 20 years, C. beticola has developed resistance to fungicides from all of these classes and fungicide resistance is a real and eco-nomically important consideration in sugar beet production. Recent reviews of fungicide resistance in sugar beets in Europe ( 8 ) and the US ( 9 ) have been recently published. For effi cacious disease control and fungicide resistance management, the most widely used fungi-cides in both the United States and European countries for manag-ing CLS are several fungicides in the triazole and strobilurin classes. The two groups of fungicides have different metabolic targets and modes of action, and therefore require two different procedures for measuring fungicide resistance.

Triazole fungicides are ergosterol biosynthesis inhibiting fun-gicides that are divided into two groups, demethylation inhibiting fungicides and morpholines ( 10 ) . This class of fungicides inhibits mycelial growth and the in vitro bioassay uses inhibition of colony radial growth to measure resistance to fungicides.

Strobilurin fungicides are QoI inhibitors that block electron transport through the mitochondrial system ( 11 ) . This class of fun-gicides inhibits spore germination and the in vitro bioassay uses inhi-bition of spore germination to measure resistance to fungicides.

This chapter will describe two procedures, radial growth, and spore germination, used to assay fungicide resistance in C. beticola , to a triazole and strobilurin fungicide, respectively, but the proce-dures may be applicable to other biological systems that utilize control of a fungal disease by a fungicide.

1. Water agar medium: distilled water 1 L, 15 g agar. Mix agar and water and autoclave 15 min. Pour the molten agar into Petri dishes (see Note 1).

2. Materials

38723 Fungicide Resistance Assays for Fungal Plant Pathogens

2. Clarifi ed V8 medium (CV8): V8 juice (see Fig. 1 , Note 2), 500 mL, 1.5 g calcium carbonate Add the calcium carbonate to the V8 juice and stir to dissolve the CaCO 3 . Clarify the V8 juice by centrifuging the mixture at 1,700 × g for 10 min in 250-mL bottles using a GSA rotor. The supernatant fl uid is used for CV8 medium preparation. Discard the pellet. To pre-pare the medium combine 100 mL of the clarifi ed V8 superna-tant, 900 mL of distilled water and 15 g agar (see Note 3). Sterilize by autoclaving for 20 min (see Note 4).

Ampillin can be added to CV8 during the initial isolation to reduce bacterial contamination, but is not necessary for sub-sequent transfers to CV8 medium after a pure culture has been established. Ampicillin is added after the medium has cooled to 50°C at fi nal concentration of 0.2 g/L.

3. Salicylhydroxamic acid (SHAM): Prepare a stock solution of SHAM by adding 400 mg SHAM to 4,000 μ L methanol and shake under hot water to dissolve; it dissolves slowly. Add 500 μ L to 500 mL water agar cooled to 55°C, which gives a fi nal concentration of 100 mg/L.

All procedures are conducted at room temperature using basic sterile techniques in a clean environment. All testing is conducted using the technical grade active ingredient of each fungicide, not

3. Methods

Fig. 1. V8 juice can.

388 G.A. Secor and V.V. Rivera

the formulated commercial fungicide in order to eliminate potential interference of fungicide activity or fungal growth due to compounds present in commercial fungicide formulations. The term μ g/mL is equivalent to parts per million (ppm).

Sugar beet leaves with Cercospora leaf spot (CLS) are collected and processed immediately to insure viability of spores. From each fi eld sample, C. beticola spores are collected from a minimum of fi ve spots per leaf from fi ve leaves per fi eld. The spores are collected by applying 20–30 μ L of sterile distilled water to a Cercospora spot using a microliter pipette and fl ushed several times to dislodge spores. The spores from the fi ve spots/leaf are pooled in a 1.5-mL centrifuge tube and a composite of 120 μ L of the pooled spore suspension is transferred to a Petri plate containing water agar plus ampicillin (0.2 g/L). This plate is used as a source of germinated single spore subcultures for subsequent testing of triazole resis-tance by radial growth and strobilurin resistance by spore germina-tion. Spore germination time is approximately 16 h.

For triazole fungicide sensitivity testing, a radial growth procedure for C. beticola is used because these fungicides inhibit mycelial growth. Sensitive isolates have reduced growth in the presence of fungicide compared to sensitive isolates in the presence of fungi-cide on artifi cial media. Radial growth is easy to measure, since most fungi naturally grow radially in artifi cial media.

1. Select a single spore subculture from the original non-amended media and transfer to CV8 medium.

2. Incubate the culture at 20°C in a continuous light regime until the colony covering about 60% most of the plate is produced (ca. 15 days for C. beticola ).

3. Remove an agar plug 4 mm in diameter from the active growth area of the colony and place in the center of a set of Petri dishes containing non-amended CV8 medium and CV8 media amended with serial tenfold dilutions of a technical grade triazole fungicide active ingredient from 0.001 to 1.0 μ g/mL (see Note 5). A sepa-rate test is conducted for each triazole fungicide.

4. Incubate the dishes for 15 days in the dark at a temperature of 20°C.

5. Evaluate growth by making two perpendicular measurements across the colonies and averaging the diameters.

6. This data is converted to a percent reduction of growth by comparing the average colony diameter data on amended media to the average colony diameter data on non-amended water agar medium. This growth reduction data is used to calculate an EC 50 value for each isolate; EC 50 is the effective concentration of fungicide that reduces radial growth by 50%

3.1. Collection of Cercospora Isolates for Testing

3.2. Inhibition of Radial Growth Procedure for Triazole Resistance Testing

38923 Fungicide Resistance Assays for Fungal Plant Pathogens

compared to the growth on non-amended media. Higher EC 50 values indicate reduced sensitivity, and possible resistance, to the fungicide.

7. Percent growth is calculated for each dilution is using the fol-lowing formula: (average colony diameter with fungicide)/(average colony diameter without fungicide) times 100 (see Table 1 ). Percent growth reduction is then calculated by sub-tracting percent growth from 100 (see Table 1 ).

8. Plot logarithmic fungicide concentration vs. reduction of col-ony growth. For mathematical reasons, fungicide concentra-tions of zero (the non-amended medium) cannot be used to construct a graph using a logarithmic scale. Therefore, it is necessary to assign a value to the non-amended control fungi-cide concentration of tenfold lower than the lowest fungicide dilution used in the assay. In Fig. 1 , this assigned value is the 0.0001 μ g/mL.

9. On the resultant curve, fi nd the fungicide concentration point on the curve where growth is reduced by 50% (see Fig. 2 ), or more precisely by regression curve fi tting.

For strobilurin fungicide sensitivity testing, it is necessary to use a procedure that measures spore germination because these fungi-cides act by inhibiting spore germination. Resistant isolates have higher spore germination rates compared to sensitive isolates in the presence of fungicide on artifi cial media.

1. Transfer a subculture from the original non-amended medium to non-amended CV8 medium and incubate in conditions to induce sporulation (see Note 6).

2. Spores are induced from a 15-day-old C. beticola culture. Add 2 mL of sterile distilled water to the plate and gently scrape the

3.3. Reduction of Spore Germination Procedure for Strobilurin Resistance Testing

Table 1 Colony radial growth measurements of a C. beticola isolate growing on media amended with fi ve concentrations of tetraconazole (0.0–1.0 m g/mL)

0 0.001 0.01 0.1 1 EC 50

Diameter 1 38 38 35 21 7

Diameter 2 37 35 34 18 9

Average diameter 37.5 36.5 34.5 19.5 8

% of growth 100 97.3 92 52 21.3

% Growth reduction 0 2.7 8 48 78.7 0.159

390 G.A. Secor and V.V. Rivera

mycelium using the edge of a microscope slide. Transfer about 500 μ L of the aqueous phase onto CV8 medium and spread the solution using a bent glass rod, avoiding transfer of large fragments of mycelium. Allow the plate to dry in the hood and incubate the plate at room temperature under continuous light using an equal mixture of cool white fl uorescent light and plant growth black light bulbs. Spores will be visible after 3 days and spores should be harvested after 5 days for testing.

3. Add 3 mL of sterile distilled water containing to the plate and gently shake the plate to dislodge the spores.

4. Transfer a 120 μ L aliquot of the spore suspension to non-amended water agar medium and a water agar series amended with serial tenfold dilutions of technical grade strobilurin fun-gicide from 0.001 to 1.0 μ g/mL and containing salicylhy-droxamic acid (SHAM) at a concentration of 100 μ g/mL (see Note 7).

5. Incubate the Petri dishes at room temperature. 6. Studies in our lab have demonstrated that C. beticola spores

reach >80% germination in about 16 h; therefore, view germina-tion of 100 spores at random 16 h after plating (see Note 8 ).

7. Calculate a percent germination for each fungicide concentra-tion in the series.

8. Calculate percent growth reduction by subtracting percent growth from 100 (see Fig. 1 ).

Fig. 2. Plot of percent growth reduction ( Y axis) against fungicide concentration ( X axis) using data from Table 1 to calculate an EC 50 value.

39123 Fungicide Resistance Assays for Fungal Plant Pathogens

9. Plot logarithmic fungicide concentration vs. reduction of colony growth. For mathematical reasons, fungicide concen-trations of zero (the non-amended medium) cannot be used to construct a graph using a logarithmic scale. Therefore, it is necessary to assign a value to the non-amended control fungicide concentration of tenfold lower than the lowest fungicide dilution used in the assay. In Fig. 2 , this assigned value is the 0.0001 μ g/mL.

10. On the resultant curve, fi nd the fungicide concentration point on the curve where growth is reduced by 50%, or more pre-cisely by regression curve fi tting.

Higher EC 50 values indicate reduced sensitivity and possible resistance to the fungicide.

1. Any agar will work for the protocol regardless of purity. Add approximately 15 mL/dish. The volume of agar added is not critical, and with experience the volume can be estimated while pouring. No nutrients are provided by the water agar. Use of disposable plastic dishes facilitates testing.

2. The original V8 is made mainly from tomatoes and the juices from seven additional vegetables, specifi cally: beets, celery, car-rots, lettuce, parsley, watercress, and spinach. A photo is included as many countries outside the US do not recognize V8 juice.

3. Any agar will work for this purpose. 4. Autoclave 10–15 min longer if several liters are prepared

simultaneously. 5. Because the technical formulations of the fungicide active

ingredients used for resistance testing have limited solubility in water, they must fi rst be dissolved in solvent for dilution and addition to the agar medium. We prepare a stock solution at 10 μ g/mL of each technical ingredient to use as a stock solu-tion for subsequent dilutions. For triazole and strobilurin fun-gicides, acetone is the choice. The only precaution is to add the dissolved fungicide to the media when it is about 55°C, by injecting the fungicide plus solvent into the media using a pipette to prevent evaporation of the acetone.

6. Conditions for inducing sporulation, viz. light quality, light duration, light intensity, temperature, growth medium compo-sition, etc, vary among fungi; consequently it is necessary to know a procedure for spore production in order to conduct this procedure.

4. Notes

392 G.A. Secor and V.V. Rivera

7. Salicylhydroxamic acid (SHAM) is added to prevent alternate oxidation pathways in the fungus to overcome resistance and give false resistance results ( 12 ) .

8. C. beticola spores are hyaline (clear) and needle-shaped which makes them diffi cult to see through a stereoscope; the light must be adjusted to diffract the light to enhance visibility. Spores are considered germinated when the mycelial growth from the spore (conidium) is at least double of the length of the spore. Percent germination is calculated by the number of spores germinated divided by the number of spores viewed multiplied by 100.

References

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2. Brent, K J, Holloman, D W (2007) Fungicide resistance: The assessment of risk. FRAC Monograph 2, 2nd Ed. FRAC, Brussels, Belgium

3. Delp, CJ (1988) Fungicide resistance in North America. APS Press, St. Paul, MN

4. Russell, PE (2003 ) Sensitivity baselines in fun-gicide resistance research and management. FRAC Monograph 3, Crop Life International, Brussels, Belgium

5. Staub, T, Sozzi, D (1984) Fungicide resistance. Plant Dis. 68:1026–1031

6. van den Bosch, F, Gilligan, CA (2008) Models of fungicide resistance dynamics. Ann. Rev. Phytopathol. 46:123–147

7. Pasche, JS et al (2004) Shift in sensitivity of Alternaria solani response to QoI fungicides. Plant Disease 88:181–187

8. Karaoglandis, GS, Ioannidis PM (2010) Fungicide resistance of Cercospora beticola in Europe. Pp 189–211 in Cercospora leaf spot of sugar beet and related species. Eds. Lartey et al . APS Press. St. Paul, MN

9. Secor, GA et al (2010) Monitoring fungicide sensitivity of Cercospora beticola of sugar beet for disease management decisions. Plant Disease 94: 1272–1282

10. Ioannidis, PM, Karaoglanidis, GS (2000) Resistance of Cercospora beticola Sacc. to fungi-cides. In Advances in Sugar beet Research Vol. 2: Cercospora beticola Sacc. Biology, agronomic infl uence and control measures in sugar beet 2000. Pp. 123–145. Inter. Inst. Beet Res. Brussels

11. Bartlett, DW et al ( 2002) The strobilurin fun-gicides. Pest Mgmt. Sci. 58:649–662

12. Ziogas, BN et al (1997) Alternative Respiration: a Biochemical Mechanism of Resistance to Azoxystrobin (ICIA 5504) in Septoria tritici Pestic. Sci. 50:28–34