Management of resistance to the fungicide fenpropimorph in Erysiphe graminis f.sp tritici

In the last three decades, plant disease control has become heavily dependent on fungicides. This practice increased yield significantly but had also negative side-effects on the environment. In many countries, integrated control programs have been initiated in order to reduce pesticide use and their side-effects (Chapter 2). In addition, modern systemic fungicides have been discovered with a high selective and potent activity. These compounds can be applied in relatively low dosages and are less harmful to the environment than conventional fungicides. A problem of these modern fungicides is resistance development in their target organisms since they have a site-specific mode of action. This risk also applies for the morpholine fungicide fenpropimorph, the active ingredient of Corbel, in the control of Erysiphe graminis f.sp. tritici . This pathogen is the causal agent of wheat powdery mildew, which is one of the most common and severe foliar diseases of wheat in Europe.

In the context of integrated control, farmers started using fenpropimorph in cereals at dosages lower than recommended. Adopted approaches are the use of split applications which is defined as a treatment in which the recommended dosage is divided over a number of applications, and the use of reduced dosages. To study the effect of these strategies on resistance development, a four-year-field trial was conducted in the south of the Netherlands. In the first three years (1992-1994), the effect of split applications of Corbel (active ingredient fenpropimorph) and of Tilt Top (active ingredients fenpropimorph and propiconazole) on sensitivity of E. graminis f.sp. tritici was investigated (Chapter 3). In 1995, the effect of reduced dosages of Corbel was studied (Chapter 4). The sensitivity of isolates collected was assessed in foliar spray tests. A change in sensitivity to fenpropimorph of the mildew population in plots treated with applications at the recommended dosage (2×1.0 l ha ^-1) was not observed, except for 1995 when a minor change was noticed. In contrast, powdery mildew isolates from plots treated with split applications (5×0.4 l ha ^-1) displayed a significant decrease in sensitivity. Reduced dosages (2×0.25 l ha ^-1) may also increase the rate of resistance development. Since maintenance of sensitivity of the pathogen population is valued highly, the split application strategy and the use of reduced dosages is not recommended for wheat powdery mildew control. Another disadvantage of the application of low dosages is reduced disease control of other foliar pathogens.

A parameter influencing resistance development in fungi to fungicides is the fitness of resistant subpopulations (Chapter 5). Fitness is a rather complex characteristic with many parameters involved. If resistance is correlated with reduced fitness, it may significantly slow down resistance development. Therefore, the competitive ability and survival in winter of isolates of E. graminis f.sp. tritici with wild-type and reduced sensitivity to fenpropimorph was investigated. In addition, a broad range of fitness components, such as germination, latent period, colony forming ability and production of conidia were studied. Under crowded infection conditions, isolates with reduced sensitivity showed a lower competitive ability than wild-type isolates. The lower competitive ability could not be ascribed to changes in germination, latent period, colony forming ability or production of conidia. Hence, reduction in competitive ability seems to be a complex change in phenotype which cannot easily be assessed by characterization of individual fitness components.

Use of mildew isolates containing fungicide-sensitivity and virulence markers made it possible to study the survival of E. graminis f.sp. tritici in winter in field trials (Chapter 5). Results revealed that isolates with a reduced sensitivity to fenpropimorph survived the winter. The competitive ability of these isolates seemed to be slightly lower than that of a mildew population which migrated naturally into the experimental field. The data indicate that in practice, in periods without selection pressure, the frequency of isolates with reduced sensitivity to fenpropimorph may decrease.

Changes in sterol composition may play a role in the reduced sensitivity of E. graminis f.sp. tritici to fenpropimorph (Chapter 6). Therefore, sterols were extracted from mildew conidia and analyzed by gas chromatography and mass spectrometry. Isolates of E. graminis f.sp. tritici with a wild-type and reduced sensitivity to fenpropimorph had the same sterol composition, viz. ergosta-5,24(28)-dien-3-ol (±90%) and episterol (±10%). Following treatment with fenpropimorph, the content of episterol increased in conidia of all isolates tested while that of ergosta-5,24(28)-dien-3-ol decreased. These results suggest that fenpropimorph, under the test conditions used, did not inhibit activity of sterol-reductase or-isomerase but rather interfered with the final part of the demethyl sterol synthesis. However, modifications in this part of the pathway are probably not responsible for the decreased sensitivity of the pathogen to the fungicide.

Genetic studies on resistance of E. graminis f.sp. tritici are not feasible since crossings cannot be made under controlled laboratory conditions. As an alternative, Aspergillus niger and Aspergillus nidulans were used as model fungi (Chapter 7). Treatment of conidia with UV-light did not result in the detection of fenpropimorph-resistant mutants of A. nidulans . In contrast, resistance to fenpropimorph was readily induced in A. niger . The degree of resistance varied between 2 and 8. Dominance tests showed that resistance to fenpropimorph is recessive. Genetic analysis of fenpropimorph-resistance in this fungus was carried out by means of the parasexual cycle. Resistance to fenpropimorph involved two genes, located on linkage group II.

Biochemical studies on the mechanism of resistance to fenpropimorph in E. graminis f.sp. tritici are virtually impossible because of the obligate character of the pathogen. Therefore, the genetically characterized mutants of A. niger described in Chapter 7 were used to study effects of morpholine fungicides on cell-free sterol synthesis and on fenpropimorph-accumulation in mycelium (Chapter 8). Cell-free extracts of A. niger actively synthesized C4-demethyl sterols from [2- ¹⁴C]mevalonate. Sterol analyses by means of thin-layer chromatography and high-performance liquid chromatography displayed that the main sterol formed in cell-free assays from wild-type isolates was ergosterol. Inhibition of sterol biosynthesis in cell-free bioassays by the morpholine fungicides fenpropimorph and tridemorph, the morpholine-related fungicide fenpropidin and the triazole propiconazole was investigated. Results suggest that, in sensitive isolates, fenpropimorph and fenpropidin inhibited activity of both sterol-isomerase and sterol-reductase. Tridemorph proved to be a relatively strong inhibitor of sterol7-isomerase activity only. In cell-free bioassays from resistant isolates fenpropimorph and fenpropidin predominantly inhibited activity of sterol-isomerase while activity of sterol-reductase remained less affected. Tridemorph inhibited sterol biosynthesis from sensitive and resistant isolates in a similar way. Therefore, the biochemical mechanism of resistance to fenpropimorph in the isolates tested is probably related to reduced affinity of sterol-reductase to the fungicide. Accumulation of fenpropimorph by wild-type and resistant isolates did not differ significantly, indicating that differential efflux is not responsible for resistance to fenpropimorph.

It is concluded that the results presented provide a better understanding of a possible evolution of resistance to morpholine fungicides in E. graminis f.sp. tritici and other plant pathogens. The results also support the relevance of strategies aimed to counteract development of resistance to morpholine fungicides.