Fungicide Resistance in North America, Second Edition

Fungicide Resistance in North America S E CO N D

E D I T I O N

Edited by

Katherine L. Stevenson Margaret T. McGrath Christian A. Wyenandt

CHAPTER

4

Resistance of Plant Pathogens to Dicarboximides (FRAC Code 2) ■■ Gerd Stammler ■■ Kristin Klappach

BASF SE, Agricultural Center, Limburgerhof, Germany ■■ Andreas Mehl

Bayer CropScience AG, Crop Science Division, Monheim, Germany

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Overview and History

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Dicarboximides were introduced into markets worldwide in the mid to late 1970s. The most important compounds of this mode of action group were iprodione, vinclozolin, and procymidone. In the 1980s, chlozolinate was launched. The major target pathogens of dicarboximides are Botrytis spp., Monilinia spp., and Sclerotinia spp. Iprodione is registered for use on a broader spectrum of pathogens including Rhizoctonia solani, Monographella spp., Typhula spp., and Alternaria spp., while the other dicarboximides are restricted to the major target pathogens with some additional niches, e.g., procymidone for Stemphylium vesicarium in pears. Because of the target disease spectrum, dicarboximides are registered mainly for use on grapes, fruits, vegetables, potatoes, ornamentals, turf, and oilseed rape. Dicarboximides are used in applications to foliage, seed, and bulbs as stand-alone products or in mixtures with other pesticides.

Dicarboximides were the third major group of fungicides, after benzimidazoles and phenylamides, for which field resistance developed in agricultural crops with significant impact on field performance of the affected products. The first investigations of mechanisms of resistance, considerations of resistance risk, and resistance management strategies in the context of agriculturally used fungicides as well as the establishment of FRAC are all linked to dicarboximides.

Market Situation and Trends

The use of dicarboximides has decreased over time until recently. Chlozolinate is not available anymore, and because vinclozolin and procymidone are not reregistered in the European Union, global sales also declined for these compounds. While total sales of the three dicarboximides declined for several years, they have stabilized over the last few years. Currently, the most important compound is iprodione, which is still registered for use on a wide range of crops especially grapes, fruits, vegetables, ornamentals, and turf but also in canola and as a seed treatment in cereals.

Dicarboximides and in particular iprodione are still regarded as valuable tools in disease and resistance management of target pathogens that have developed (multiple) fungicide resistances, such as Botrytis spp. or Alternaria spp.

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Mode of Action and Mechanism of Resistance

The mode of action of dicarboximides has been widely investigated, and a variety of possible modes of action have been suggested (Edlich & Lyr, 1995; Yamaguchi & Fujimura, 2005). The most common theory was that dicarboximides induce membrane 41

42 PA R T I I / Fungicide Modes of Action and Mechanisms of Resistance

lipid peroxidation triggered by active oxygen species within susceptible fungal cells, but later studies suggest a link with an osmoregulatory pathway.

Dicarboximides interfere with the osmotic signal transduction pathway consisting of histidine kinase and mitogen-activated protein kinase cascades (Cui et al., 2002, 2004; Leroux et al., 2002; Oshima et al., 2002, 2006). It has been suggested that dicarboximides bind to the “coiled-coil region” of a putative osmosensing histidine kinase orthologous to os-1 in Neurospora crassa (Cui et al., 2002, 2004), because mutations in this region of the os-1 were found to cause resistance to dicarboximides. Therefore, the histidine kinase os-1 can be proposed as the target protein of dicarboximides and that inhibition of this enzyme represents their mode of action. This is in accordance with the information given by FRAC, both by the Dicarboximide Expert Forum of FRAC (www.frac.info) and on the FRAC published poster and with the description of the mode of action on the FRAC webpage.

Mechanisms of resistance have been investigated with laboratory-induced mutants and field isolates of different fungal species, mainly B. cinerea.

Generally, most dicarboximide-resistant laboratory mutants of B. cinerea, N. crassa, and Alternaria spp. are hypersensitive to osmotic stress compared with their parental strains, acquire high levels of dicarboximide resistance, and are cross-resistant to aromatic hydrocarbons and phenylpyrrols (Beever, 1983; Cui et al., 2004; Grindle & Temple, 1982; Ma & Michailides, 2004; Ochiai et al., 2002). Dicarboximide-resistant field isolates of B. cinerea lack, or have only slightly higher, osmotic sensitivity compared with sensitive isolates and are not cross-resistant to phenylpyrrols (Gehmann et al., 1990; Leroux et al., 1992). These findings suggest that the mechanisms of resistance of field isolates differ from those of laboratory mutants. From B. cinerea and Alternaria spp., the genes homologous to the os-1 gene of N. crassa were cloned and sequenced.

Mutations in the os-1 were identified in dicarboximideresistant field isolates of B. cinerea, leading to amino acid exchanges I365S, I365N, I365R, V368F, Q369H, T447S, and Q369P + N373S (Cui et al., 2004; Grabke & Stammler, 2015; Grabke et al., 2014; Leroux et al., 2002, Oshima et al., 2002, 2006). In field isolates of the genus Alternaria, mutations in the os-1-homologous gene can also be found in A. alternata (Dry et al., 2004) but not in A. arborescens (Ma & Michailides, 2004).

Enhanced activities of catalase (Choi et al., 1997; Steel & Nair, 1991) and superoxide dismutase (Choi et al., 1997) as defense mechanisms against

active oxygen species have also been proposed as mechanisms of resistance in B. cinerea. An additional mechanism that reduces sensitivity to dicarboximides has been identified for B. cinerea. It is the so-called “multidrug resistance phenotype” (MDR), which was initially described by Leroux et al. (1999, 2002) and subsequently elucidated by Kretschmer et al.

(2009). Different MDR types (MDR 1, 2, and 3) have been identified by their sensitivity patterns, and progress has been made in elucidating the molecular mechanisms (Leroch et al., 2011, 2013; Mernke et al., 2011). Mechanisms other than target site mutations have in common that the resulting resistance factors are much lower than those caused by target site mutations (Fig. 4.1). For MDR types, mean resistance factors for iprodione are 2–5 according to the study and method of Leroux and Walker (2013) and our own data (Fig. 4.1; microtiter test with MDR1 isolate). Different phenotypes described as sensitive, partially resistant, and highly resistant have also been described, but the underlying mechanisms for these reduced sensitivities have not been identified (Weber, 2011). In greenhouse studies, it has been determined that resistance mechanisms other than target site mutations may be less relevant for in vivo efficacy of registered field rates of iprodione and that

FI G . 4.1. Efficacy of iprodione in a greenhouse trial with bell pepper infected with Botrytis cinerea isolates with different levels of sensitivity. One-day preventive application. Left to right: untreated control, 167 g a.i./ha, 500 g a.i./ha, and 1,500 g a.i./ha. Top row, sensitive isolate; middle row, MDR1 but wild type in OS1 isolates; and bottom row, an MDR3 combined with the I365S in an OS1 isolate. (Courtesy G. Stammler—© APS)

C H A P T E R 4 / Resistance of Plant Pathogens to Dicarboximides (FRAC Code 2) 43

dicarboximides might still contribute to field control in MDR phenotypes (Fig. 4.1; efficacy on MDR1 isolate).

Data on dicarboximide-resistant isolates of B. cinerea from a German trial site in 2013 with a history of intensive fungicide use indicate that the combination of target site mutations with the MDR phenotype led to the lowest sensitivity, or, in other words, isolates with the highest EC 50 values had not only one of the abovementioned target site mutations, but also the MDR phenotype (Grabke & Stammler, 2015).

Cross-resistance covers all dicarboximide fungicides (iprodione, vinclozolin, procymidone, and chlozolinate), which has been shown, for example, in studies by Sztejnberg and Jones (1978) for dicarboximide-resistant isolates of Monilinia fructicola. Dicarboximide-resistant field isolates of B. cinerea are also cross-resistant to the aromatic hydrocarbon group of fungicides (e.g., quintozene, chloroneb, and dicloran) (Leroux et al., 1999). Crossresistance of dicarboximides and phenylpyrrols (e.g., fludioxonil) was restricted mainly to laboratory mutants. Only single cases of cross-resistance between dicarboximides and fludioxonil have been reported for field isolates of Alternaria alternata (Dry et al., 2004) and A. brassicicola (Iacomi-Vasilescu et al., 2004). However, these are single findings with minor practical relevance and no such cases have been reported for B. cinerea.

The studies of B. cinerea by Weber (2011) and our own sensitivity data (Table 4.1) confirm the lack of cross-resistance of dicarboximides to any other fungicides currently registered for control of B. cinerea.

Iprodione-sensitive isolates with resistance to QoIs, fenhexamid, succinate dehydrogenase inhibitors, and anilinopyrimidines have been identified as have isolates with dicarboximide resistance and sensitivity to other modes of action. Isolates with resistance to up to six different modes of action have been found (Grabke & Stammler, 2015; Leroch et al., 2013; Weber, 2011), but these phenotypes are based on an accumulation of independent resistance mechanisms, mainly mutations in the different genes of the fungicide target proteins. This multiple resistance has to be differentiated from cross-resistance. Since multiple resistance is common, especially in B. cinerea, the availability of different tools for disease control is mandatory to reduce selection pressure on the limited number of modes of action that remain effective against this pathogen.

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Examples of Resistance and Sensitivity Monitoring Data

Methods for monitoring dicarboximide sensitivity are based on inhibition by different concentrations of dicarboximides on radial mycelial growth on agar, microtiter assays, conidial germination, or germ tube elongation (Table 4.2). Pyrosequencing also is an efficient method for detection of mutated isolates in a population. Most sensitivity monitoring studies have been carried out for B. cinerea and Sclerotinia

TAB LE 4.1. Fungicide sensitivity of three isolates of Botrytis cinerea sensitive (s) and three resistant (r) to iprodione.

Data show lack of cross-resistance of iprodione with other modes of action and that multiple resistance can occur.a,b Isolate ID

Iprodione

Fenhexamid

Pyraclostrobin

Pyrimethanil

Fludioxonil

Boscalid

63559

1.41 (s)

0.09

0.13

>30

0.126

0.19

63233

0.59 (s)

0.03

>10

0.38

0.016

>10

63688

0.45 (s)

>10

>10

0.54

0.005

>10

63684

>10 (r)

0.12

>10

>30

0.264

0.28

63692

>10 (r)

6.98

>10

>30

0.071

0.12

63707

>10 (r)

5.84

0.07

N.d.

N.d.

0.12

a Courtesy

G. Stammler—© APS. are EC50 values determined by microtiter tests. Medium was YBA (Stammler & Speakman, 2006) for all compounds except pyrimethanil, which was tested with GG medium as described in the FRAC Monitoring Method folder (www.frac.info).

N.d. = not determined.

b Values

44 PA R T I I / Fungicide Modes of Action and Mechanisms of Resistance

sclerotiorum. A literature research on these two pathogens, combined with our own unpublished data, is summarized in the following paragraphs.

B. cinerea and B. squamosa Isolates from different hosts and regions were investigated. Sensitivity studies since 2003 were considered. Results from outside North America are included for comparison.

No or low frequency of resistance (<10%) has been found in more than 1,300 isolates from pears in the

United States (0%; Lennox & Spotts, 2003), from grapes in Germany in 2006, 2007, 2008, and 2009 (6.1, 2.8, 1.1, and 3.3%, respectively; Leroch et al., 2011), from kiwi in Greece in 2008–2009 (9%; Bardas et al., 2010), and from vegetables in Germany (0%, n = 11; G. Stammler et al., unpublished data).

Low to moderate frequency (<20% of tested isolates) of resistance has been reported from strawberries and blackberries in the United States (20%, n = 245; Grabke et al., 2014), from onions in Canada in 2003 (11% in B. squamosa; Tremblay et al., 2003), from vegetables in Greece in 2007 (18%; Myresiotis et al.,

TAB LE 4. 2. Methods used to monitor fungicide sensitivity in pathogensa Species

Methods

Source

Botrytis cinerea

Spore germination, mycelial growth

Grabke et al., 2014; Latorre et al., 1994; Stammler & Speakman, 2006

Mycelial growth

Bardas et al., 2010; Kretschmer et al., 2009; LaMondia & Douglas, 1997; Lennox & Spotts, 2003; Myresiotis et al., 2007; Pappas, 1997

Germ tube elongation

Leroux et al., 1999; Leroux & Walker, 2013; Weber, 2011; Weber & Hahn, 2011

Pyrosequencing for codons 365, 370, 371

Grabke & Stammler, 2015

PCR-RFLP, allele-specific PCR, sequencing

Grabke et al., 2014; Oshima et al., 2006

Monilinia laxa

Spore germination, mycelial growth

Guizzardi et al., 1995

Monilinia fructicola

Mycelial growth

Ma et al., 2006; Penrose & Senn, 1995; Yoshimura et al., 2004

Clarireedia spp.

Mycelial growth

Burpee, 1997; Liu et al., 2009; Stammler et al., 2007

Sclerotinia minor

Mycelial growth

Davet & Martin, 1993

Alternaria brassicicola

Germ tube elongation, mycelial growth

Huang & Levy, 1995

Spore germination, germ tube elongation, mycelial growth

Iacomi-Vasilescu et al., 2004

Alternaria brassicae

Mycelial growth

Iacomi-Vasilescu et al., 2004

Alternaria japonica

Mycelial growth

Iacomi-Vasilescu et al., 2004

Rhizoctonia solani

Mycelial growth

Campion et al., 2003; Csinos & Stephenson, 1999

Botryosphaeria dothidea

Mycelial growth

Ma et al., 2001

Botrytis squamosa

Mycelial growth

Tremblay et al., 2003

a Courtesy

G. Stammler—© APS.

C H A P T E R 4 / Resistance of Plant Pathogens to Dicarboximides (FRAC Code 2) 45

2007), from grapes in Israel (16%; Korolev et al., 2011), from grapes in France and Germany in 2005 (20%, n = 65, and 14%, n = 21, respectively; G. Stammler et al., unpublished data), and from strawberries in Germany in 2005 and 2012 (17%, n = 12, and 15%, n = 175, respectively; G. Stammler et al., unpublished data).

Moderate to high levels (21–50%) of resistant isolates were reported for isolates from small fruits in Germany for 2008–2010 (25%; Weber & Hahn, 2011), in another study in 2010 in Germany (29%; Weber & Entrop, 2011), from strawberries in Germany in 2009 and 2013 (33%, n = 24, and 28%, n = 39, respectively; G. Stammler et al., unpublished data) and from strawberries in Germany in an additional study in 2013 (40%, n = 199; Grabke & Stammler, 2015), and from Japan in a survey in 2005 in which 53% were evaluated as dicarboximide resistant (Oshima et al., 2006).

This sensitivity data as a whole indicates that dicarboximide resistance in B. cinerea is established in vine-growing areas and occurs in other crops like strawberries. However, in most cases, populations are not dominated by resistant individuals but rather the majority is sensitive. This could be explained by the fact that when use of dicarboximides stops, the frequency of resistant strains tends to decrease (Leroux & Clerjeau, 1985; Northover, 1988; Pak et al., 1990). In general, dicarboximide-resistant strains of B. cinerea showed fitness penalties in temperature tolerance and aggressiveness and were less competitive in dual inoculation tests (Beever et al., 1989; Gullino et al., 1982; Katan, 1982; Pommer & Lorenz, 1995; Raposo et al., 2000).

S. sclerotiorum Studies were carried out with isolates from oil seed rape. No resistant isolates were found in Canada in monitoring studies in 2009 (n = 17; G. Stammler et al., unpublished data), in Europe in 2012 (n = 188; G. Stammler et al., unpublished data), and in China in 2007–2008 (Liu et al., 2009). Isolates of S. sclerotiorum collected from vegetables in Europe in 2012 (n = 29; G. Stammler et al., unpublished data) were also found to be sensitive to iprodione. This sensitivity data as a whole documents that S. sclerotiorum has not developed resistance to dicarboximides.

Other Species In other fungal species, including Clarireedia spp. (formerly Sclerotinia homoeocarpa) in turf and Monilinia spp. in fruits, resistance to dicarboximides has been

found sporadically and has not been associated with practical problems controlling disease (www.frac.info; Burpee, 1997; Csinos & Stephenson, 1999; Guizzardi et al., 1995; Hubbart et al., 1997; Ma et al., 2001; Penrose & Senn, 1995; Yoshimura et al., 2004).

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Recommended Uses for Resistance Management

Dicarboximides are classified by FRAC in general as medium to high risk compounds (www.frac. info) according to the principles described in FRAC Monographs 1 and 2 (Brent & Hollomon, 2007a, 2007b).

The pathogen resistance risk depends on the biology of the fungus (e.g., number of generations per season, spore dispersal mechanisms, and spore production capacity) and the occurrence of the disease (frequency and control of disease). On the basis of these factors and the history of fungicide resistance development in a species, a risk classification can be made. The risk is high for B. cinerea, medium for Monilinia spp. and S. sclerotiorum, and low for R. solani (www.frac.info) (Fig. 4.2).

Combined Risk The combined risk for dicarboximides and the pathogens is visualized in Figure 4.2 (modified from Brent & Hollomon, 2007b). It is assessed as medium for dicarboximides and R. solani (score 3) and for Monilinia spp. (score 6) and high for B. cinerea (score 9). An alternative model has been suggested by Brent and Hollomon (2007a) and published in the EPPO guidelines (EPPO, 2015) (Fig. 4.3). In this scheme, the position of the target pathogens and dicarboximides can be assessed in a somewhat more differentiated way, which results in the dicarboximides being seen as less risky than QoIs and benzimidazoles, whereas they are similar in Figure 4.2. When the data from the analyses in Figures 4.2 and 4.3 are taken together, it is apparent that the inherent risk of resistance developing to dicarboximides is high for B. cinerea, medium for Monilinia spp. and Sclerotinia spp., and low to medium for Rhizoctonia solani. These classifications are confirmed by the history of resistance development in the different pathogens against dicarboximides; after more than 30 years of use, dicarboximide resistance with practical field

46 PA R T I I / Fungicide Modes of Action and Mechanisms of Resistance

relevance has been observed only for B. cinerea, while for Monilinia spp. or Sclerotinia spp. only single cases without any practical relevance have been reported, and no resistance cases are known for R. solani.

In the case of B. cinerea and dicarboximides, the need for modification in how these fungicides are used is dictated by the fact that resistance risk assessment has indicated a high risk under

unrestricted use. The objective of resistance management strategies is the reduction of selection pressure to avoid or delay the buildup of resistance.

This can be achieved by good agricultural practices that lead to less infection pressure (e.g., phytosanitary procedures; cultivation of less susceptible varieties; appropriate crop management that is unfavorable for pathogen infection and development such as

FI G . 4. 2. Combined risk analysis. Score of risk classes: 0.5–2 = low risk, 3–6 = medium risk, and 9 = high risk.

Combined resistance risk can be assessed as dicarboximides × B. cinerea; dicarboximides × Monilinia spp., Clarireedia spp.; dicarboximides × Sclerotinia sclerotiorum; and dicarboximides × Rhizoctonia solani. Relevant fungicide group (dicarboximides) and relevant target pathogens are underlined. (Risk analysis adapted from Brent & Hollomon, 2007b; risk classification of fungal species from www.frac.info, status 2015. Figure courtesy G. Stammler—© APS)

C H A P T E R 4 / Resistance of Plant Pathogens to Dicarboximides (FRAC Code 2) 47

picking and discarding diseased fruits during harvest; and well managed irrigation schemes).

Forecasting systems may help to optimize and reduce fungicidal treatments and therefore selection pressure.

Limiting the number of sprays is also an important factor in delaying the buildup of resistant pathogen populations. Another tool is the use of fungicide mixtures (Hobbelen et al., 2013, 2014; van den Bosch et al., 2014). A combination of iprodione with another fungicidal mode of action that is also active against the target pathogens, in particular B. cinerea, is an appropriate disease and resistance management strategy. Since pathogen populations are smaller at disease onset than when disease already is established

in the crop, selection pressure is less when preventive applications are used rather than curative or eradicative spray schemes. Therefore, dicarboximides should be applied in a preventive manner following the recommendations on the label. Applications should be well timed (van den Berg et al., 2013) and the appropriate dose used.

The FRAC expert forum formulated the following use recommendations for control of B. cinerea: ■■

Minimize the selection pressure by minimizing the number of applications. As a guide, do not apply more than two to three applications per crop per season.

FI G . 4. 3. Scheme for visualizing the combined resistance risk. (Adapted from Brent & Hollomon, 2007a; EPPO, 2015.

Figure courtesy G. Stammler—© APS)

48 PA R T I I / Fungicide Modes of Action and Mechanisms of Resistance

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Restrict applications to those times when infection pressure from Botrytis spp. is high.

Maintain regular prolonged times without exposure to dicarboximides.

Where resistance is well established, use dicarboximides in combinations with other fungicides to stabilize control of Botrytis spp., but their application must follow the same rules as for dicarboximides alone.

Summary and Conclusions

Dicarboximides were introduced into the market in the late 1970s. The major target diseases of this fungicide group are those caused by B. cinerea, Monilinia spp., and Sclerotinia spp. Resistance to dicarboximides developed within a few years after market introduction and intensive use because of the lack of effective alternatives. Studies on the genetic background of the resistance showed that point mutations in the histidine kinase gene are related to dicarboximide resistance. Decreased fitness has been found for both lab-derived and field strains resistant to dicarboximides. The use of dicarboximides has decreased over time because of the introduction to the market of modern selective botryticides and fungicides with new modes of action. However, this group of fungicides is still regarded as a valuable tool in different crops against the target pathogens, particularly against B. cinerea isolates with multiple resistances to other active ingredients.

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