Contrasting levels of genetic predictability in the evolution of resistance to major classes of fungicides.

The evolution of resistance has been seen across all major classes of xenobiotics, including antimicrobial drugs and agricultural pesticides. This repeated emergence of resistance is a case of phenotypic parallel evolution, but often the parallelism extends to the molecular level too, with multiple species gaining the same mutation in response to the same chemical treatment. We review the degree of repeatability in target-site resistance mutations affecting different classes of site-specific agricultural fungicides used in crop protection, comparing the extent to which resistance in different pathogen species has evolved via the same or different mutations. For all major fungicide target sites, substantial levels of molecular parallel evolution can be seen, with at least one mutation recurring in over 50% of species. Target-site mutations appear to be most repeatable in cytochrome b, target site of quinone-outside inhibitor fungicides, and least predictable for CYP51, target site of the azoles. Intermediate levels of repeatability are seen for the MBC target site ß-tubulin, and the SDHI target site succinate dehydrogenase. Repeatability may be lower where there are selective trade-offs between resistance and pleiotropic fitness penalties, or differing levels of cross-resistance across members of a fungicide class; or where single mutations confer only partial resistance, and epistatic interactions between multiple mutations result in a rugged fitness landscape. This affects the predictive power of in vitro mutation studies, and has practical implications for resistance monitoring strategies and diagnostic methods.

First application of loop-mediated isothermal amplification (LAMP) assays for rapid identification of mating type in the heterothallic fungus Aspergillus fumigatus.

BACKGROUND: Loop-mediated isothermal amplification (LAMP) assays, which operate at a single temperature and require no postreaction processing, have been described for rapid species-specific detection of numerous fungi. The technology has much less commonly been applied to identification of other key genetic traits such as fungicide resistance, and has not yet been applied to mating-type determination in any fungus. OBJECTIVES: To develop first LAMP assays for mating-type identification in a fungus, in this instance with the saprophytic mould and human opportunistic pathogen Aspergillus fumigatus, a heterothallic ascomycete requiring isolates of opposite mating type (MAT1-1, MAT1-2) for sexual reproduction. METHODS: New LAMP primer sets, targeted to MAT gene sequences, were screened against 34 A fumigatus isolates (of known mating type) from diverse clinical, environmental and geographic sources to establish whether they could distinguish MAT1-1 or MAT1-2 genotypes. RESULTS AND CONCLUSIONS: The new assays, operating at a single temperature of 65°C, correctly identified the mating type of A fumigatus isolates in <20 minutes, and thus have numerous research and practical applications. Similar MAT LAMP assays could now be developed for other fungi of agricultural, environmental, industrial and/or medical importance.

Paralog re-emergence: a novel, historically contingent mechanism in the evolution of antimicrobial resistance.

Evolution of resistance to drugs and pesticides poses a serious threat to human health and agricultural production. CYP51 encodes the target site of azole fungicides, widely used clinically and in agriculture. Azole resistance can evolve due to point mutations or overexpression of CYP51, and previous studies have shown that fungicide-resistant alleles have arisen by de novo mutation. Paralogs CYP51A and CYP51B are found in filamentous ascomycetes, but CYP51A has been lost from multiple lineages. Here, we show that in the barley pathogen Rhynchosporium commune, re-emergence of CYP51A constitutes a novel mechanism for the evolution of resistance to azoles. Pyrosequencing analysis of historical barley leaf samples from a unique long-term experiment from 1892 to 2008 indicates that the majority of the R. commune population lacked CYP51A until 1985, after which the frequency of CYP51A rapidly increased. Functional analysis demonstrates that CYP51A retains the same substrate as CYP51B, but with different transcriptional regulation. Phylogenetic analyses show that the origin of CYP51A far predates azole use, and newly sequenced Rhynchosporium genomes show CYP51A persisting in the R. commune lineage rather than being regained by horizontal gene transfer; therefore, CYP51A re-emergence provides an example of adaptation to novel compounds by selection from standing genetic variation.

The evolution of fungicide resistance.

Fungicides are widely used in developed agricultural systems to control disease and safeguard crop yield and quality. Over time, however, resistance to many of the most effective fungicides has emerged and spread in pathogen populations, compromising disease control. This review describes the development of resistance using case histories based on four important diseases of temperate cereal crops: eyespot (Oculimacula yallundae and Oculimacula acuformis), Septoria tritici blotch (Zymoseptoria tritici), powdery mildew (Blumeria graminis), and Fusarium ear blight (a complex of Fusarium and Microdochium spp). The sequential emergence of variant genotypes of these pathogens with reduced sensitivity to the most active single-site fungicides, methyl benzimidazole carbamates, demethylation inhibitors, quinone outside inhibitors, and succinate dehydrogenase inhibitors illustrates an ongoing evolutionary process in response to the introduction and use of different chemical classes. Analysis of the molecular mechanisms and genetic basis of resistance has provided more rapid and precise methods for detecting and monitoring the incidence of resistance in field populations, but when or where resistance will occur remains difficult to predict. The extent to which the predictability of resistance evolution can be improved by laboratory mutagenesis studies and fitness measurements, comparison between pathogens, and reconstruction of evolutionary pathways is discussed. Risk models based on fungal life cycles, fungicide properties, and exposure to the fungicide are now being refined to take account of additional traits associated with the rate of pathogen evolution. Experimental data on the selection of specific mutations or resistant genotypes in pathogen populations in response to fungicide treatments can be used in models evaluating the most effective strategies for reducing or preventing resistance. Resistance management based on robust scientific evidence is vital to prolong the effective life of fungicides and safeguard their future use in crop protection.

Assessing the predictability of fungicide resistance evolution through in vitro selection.

Plant pathogens are highly adaptable, and have evolved to overcome control measures including multiple classes of fungicides. More effective management requires a thorough understanding of the evolutionary drivers leading to resistance. Experimental evolution can be used to investigate evolutionary processes over a compressed timescale. For fungicide resistance, applications include predicting resistance ahead of its emergence in the field, testing potential outcomes under multiple different fungicide usage scenarios or comparing resistance management strategies. This review considers different experimental approaches to in vitro selection, and their suitability for addressing different questions relating to fungicide resistance. When aiming to predict the evolution of new variants, mutational supply is especially important. When assessing the relative fitness of different variants under fungicide selection, growth conditions such as temperature may affect the results as well as fungicide choice and dose. Other considerations include population size, transfer interval, competition between genotypes and pathogen reproductive mode. However, resistance evolution in field populations has proven to be less repeatable for some fungicide classes than others. Therefore, even with optimal experimental design, in some cases the most accurate prediction from experimental evolution may be that the exact evolutionary trajectory of resistance will be unpredictable.

Digest: Plant-pathogen coevolution extends to shifts in plant breeding systems.

Plants and their pathogens are in a coevolutionary arms race. Some pathogens, such as anther smuts, use their host plants' pollinators for spore dispersal. In the plant Dianthus pavonius, gynodioecy (having female and hermaphroditic plants) has evolved to reduce flowering duration and therefore limit exposure to anther smut pathogens. Bruns et al. (2019) show that this shift in breeding system has evolved as a disease escape mechanism.

The evolutionary origins of pesticide resistance.

Durable crop protection is an essential component of current and future food security. However, the effectiveness of pesticides is threatened by the evolution of resistant pathogens, weeds and insect pests. Pesticides are mostly novel synthetic compounds, and yet target species are often able to evolve resistance soon after a new compound is introduced. Therefore, pesticide resistance provides an interesting case of rapid evolution under strong selective pressures, which can be used to address fundamental questions concerning the evolutionary origins of adaptations to novel conditions. We ask: (i) whether this adaptive potential originates mainly from de novo mutations or from standing variation; (ii) which pre-existing traits could form the basis of resistance adaptations; and (iii) whether recurrence of resistance mechanisms among species results from interbreeding and horizontal gene transfer or from independent parallel evolution. We compare and contrast the three major pesticide groups: insecticides, herbicides and fungicides. Whilst resistance to these three agrochemical classes is to some extent united by the common evolutionary forces at play, there are also important differences. Fungicide resistance appears to evolve, in most cases, by de novo point mutations in the target-site encoding genes; herbicide resistance often evolves through selection of polygenic metabolic resistance from standing variation; and insecticide resistance evolves through a combination of standing variation and de novo mutations in the target site or major metabolic resistance genes. This has practical implications for resistance risk assessment and management, and lessons learnt from pesticide resistance should be applied in the deployment of novel, non-chemical pest-control methods.

Digest: Plants adapt under attack: genotypic selection and phenotypic plasticity under herbivore pressure.

Plant species adapt to changing environmental conditions through phenotypic plasticity and natural selection. Agrawal et al. (2018) found that dandelions responded to the presence of insect pests by producing higher levels of defensive compounds. This defensive response resulted both from phenotypic plasticity, with individual plants' defenses triggered by insect attack, and from evolution by natural selection acting on genetic variation in the plant population.

Worldwide emergence of resistance to antifungal drugs challenges human health and food security.

The recent rate of emergence of pathogenic fungi that are resistant to the limited number of commonly used antifungal agents is unprecedented. The azoles, for example, are used not only for human and animal health care and crop protection but also in antifouling coatings and timber preservation. The ubiquity and multiple uses of azoles have hastened the independent evolution of resistance in many environments. One consequence is an increasing risk in human health care from naturally occurring opportunistic fungal pathogens that have acquired resistance to this broad class of chemicals. To avoid a global collapse in our ability to control fungal infections and to avoid critical failures in medicine and food security, we must improve our stewardship of extant chemicals, promote new antifungal discovery, and leverage emerging technologies for alternative solutions.

Dose-dependent selection drives lineage replacement during the experimental evolution of SDHI fungicide resistance in Zymoseptoria tritici.

Fungicide resistance is a constant threat to agricultural production worldwide. Molecular mechanisms of fungicide resistance have been studied extensively in the wheat pathogen Zymoseptoria tritici. However, less is known about the evolutionary processes driving resistance development. In vitro evolutionary studies give the opportunity to investigate this. Here, we examine the adaptation of Z. tritici to fluxapyroxad, a succinate dehydrogenase (Sdh) inhibitor. Replicate populations of Z. tritici derived from the sensitive isolate IPO323 were exposed to increasing concentrations of fluxapyroxad with or without UV mutagenesis. After ten increases in fungicide concentration, sensitivity had decreased dramatically, with replicate populations showing similar phenotypic trajectories. Sequencing the Sdh subunit B, C, and D encoding genes identified seven mutations associated with resistance to fluxapyroxad. Mutation frequency over time was measured with a pyrosequencing assay, revealing sequential lineage replacement in the UV-mutagenized populations but not in the untreated populations. Repeating selection from set time-points with different fungicide concentrations revealed that haplotype replacement of Sdh variants was driven by dose-dependent selection as fungicide concentration changed, and was not mutation-limited. These findings suggest that fungicide field applications may select for highly insensitive Sdh variants with higher resistance factors if the fungicide concentration is increased to achieve a better disease control. However, in the absence or presence of lower fungicide concentrations, the spread of these strains might be restricted if the underlying Sdh mutations carry fitness penalties.

Azole sensitivity in Leptosphaeria pathogens of oilseed rape: the role of lanosterol 14α-demethylase.

Lanosterol 14-α demethylase is a key enzyme intermediating the biosynthesis of ergosterol in fungi, and the target of azole fungicides. Studies have suggested that Leptosphaeria maculans and L. biglobosa, the causal agents of phoma stem canker on oilseed rape, differ in their sensitivity to some azoles, which could be driving pathogen frequency change in crops. Here we used CYP51 protein modelling and heterologous expression to determine whether there are interspecific differences at the target-site level. Moreover, we provide an example of intrinsic sensitivity differences exhibited by both Leptosphaeria spp. in vitro and in planta. Comparison of homologous protein models identified highly conserved residues, particularly at the azole binding site, and heterologous expression of LmCYP51B and LbCYP51B, with fungicide sensitivity testing of the transformants, suggests that both proteins are similarly sensitive to azole fungicides flusilazole, prothioconazole-desthio and tebuconazole. Fungicide sensitivity testing on isolates shows that they sometimes have a minor difference in sensitivity in vitro and in planta. These results suggest that azole fungicides remain a useful component of integrated phoma stem canker control in the UK due to their effectiveness on both Leptosphaeria spp. Other factors, such as varietal resistance or climate, may be driving observed frequency changes between species.

Predicting Resistance by Mutagenesis: Lessons from 45 Years of MBC Resistance.

When a new fungicide class is introduced, it is useful to anticipate the resistance risk in advance, attempting to predict both risk level and potential mechanisms. One tool for the prediction of resistance risk is laboratory selection for resistance, with the mutational supply increased through UV or chemical mutagenesis. This enables resistance to emerge more rapidly than in the field, but may produce mutations that would not emerge under field conditions. The methyl benzimidazole carbamates (MBCs) were the first systemic single-site agricultural fungicides, and the first fungicides affected by rapid evolution of target-site resistance. MBC resistance has now been reported in over 90 plant pathogens in the field, and laboratory mutants have been studied in nearly 30 species. The most common field mutations, including ß-tubulin E198A/K/G, F200Y and L240F, have all been identified in laboratory mutants. However, of 28 mutations identified in laboratory mutants, only nine have been reported in the field. Therefore, the predictive value of mutagenesis studies would be increased by understanding which mutations are likely to emerge in the field. Our review of the literature indicates that mutations with high resistance factors, and those found in multiple species, are more likely to be reported in the field. However, there are many exceptions, possibly due to fitness penalties. Whether a mutation occurred in the same species appears less relevant, perhaps because ß-tubulin is highly conserved so functional constraints are similar across all species. Predictability of mutations in other target sites will depend on the level and conservation of constraints.