Potential for re-emergence of wheat stem rust in the United Kingdom

This is the final version. Available on open access from Springer Nature via the DOI in this recordWheat stem rust, a devastating disease of wheat and barley caused by the fungal pathogen Puccinia graminis f. sp. tritici, was largely eradicated in Western Europe during the mid-to-late twentieth century. However, isolated outbreaks have occurred in recent years. Here we investigate whether a lack of resistance in modern European varieties, increased presence of its alternate host barberry and changes in climatic conditions could be facilitating its resurgence. We report the first wheat stem rust occurrence in the United Kingdom in nearly 60 years, with only 20% of UK wheat varieties resistant to this strain. Climate changes over the past 25 years also suggest increasingly conducive conditions for infection. Furthermore, we document the first occurrence in decades of P. graminis on barberry in the UK . Our data illustrate that wheat stem rust does occur in the UK and, when climatic conditions are conducive, could severely harm wheat and barley production.This project was funded by an institute development grant from the EI (Norwich, UK), an Industrial Partnership Award (BB/M025519/1) from the BBSRC, a European Research Council Starting Grant awarded to D.G.O.S. (number 715638), H2020 project EMPHASIS (number 634179), by the BBSRC Institute Strategic Programmes BB/J004553/1 and BB/P012574/1, the John Innes Foundation, and an African Women in Agricultural Research and Development (AWARD) fellowship to R.N.K

Fungicide-Driven Evolution and Molecular Basis of Multidrug Resistance in Field Populations of the Grey Mould Fungus Botrytis cinerea

The grey mould fungus Botrytis cinerea causes losses of commercially important fruits, vegetables and ornamentals worldwide. Fungicide treatments are effective for disease control, but bear the risk of resistance development. The major resistance mechanism in fungi is target protein modification resulting in reduced drug binding. Multiple drug resistance (MDR) caused by increased efflux activity is common in human pathogenic microbes, but rarely described for plant pathogens. Annual monitoring for fungicide resistance in field isolates from fungicide-treated vineyards in France and Germany revealed a rapidly increasing appearance of B. cinerea field populations with three distinct MDR phenotypes. All MDR strains showed increased fungicide efflux activity and overexpression of efflux transporter genes. Similar to clinical MDR isolates of Candida yeasts that are due to transcription factor mutations, all MDR1 strains were shown to harbor activating mutations in a transcription factor (Mrr1) that controls the gene encoding ABC transporter AtrB. MDR2 strains had undergone a unique rearrangement in the promoter region of the major facilitator superfamily transporter gene mfsM2, induced by insertion of a retrotransposon-derived sequence. MDR2 strains carrying the same rearranged mfsM2 allele have probably migrated from French to German wine-growing regions. The roles of atrB, mrr1 and mfsM2 were proven by the phenotypes of knock-out and overexpression mutants. As confirmed by sexual crosses, combinations of mrr1 and mfsM2 mutations lead to MDR3 strains with higher broad-spectrum resistance. An MDR3 strain was shown in field experiments to be selected against sensitive strains by fungicide treatments. Our data document for the first time the rising prevalence, spread and molecular basis of MDR populations in a major plant pathogen in agricultural environments. These populations will increase the risk of grey mould rot and hamper the effectiveness of current strategies for fungicide resistance management

Comparative Genomic Characterization of Francisella tularensis Strains Belonging to Low and High Virulence Subspecies

Tularemia is a geographically widespread, severely debilitating, and occasionally lethal disease in humans. It is caused by infection by a gram-negative bacterium, Francisella tularensis. In order to better understand its potency as an etiological agent as well as its potential as a biological weapon, we have completed draft assemblies and report the first complete genomic characterization of five strains belonging to the following different Francisella subspecies (subsp.): the F. tularensis subsp. tularensis FSC033, F. tularensis subsp. holarctica FSC257 and FSC022, and F. tularensis subsp. novicida GA99-3548 and GA99-3549 strains. Here, we report the sequencing of these strains and comparative genomic analysis with recently available public Francisella sequences, including the rare F. tularensis subsp. mediasiatica FSC147 strain isolate from the Central Asian Region. We report evidence for the occurrence of large-scale rearrangement events in strains of the holarctica subspecies, supporting previous proposals that further phylogenetic subdivisions of the Type B clade are likely. We also find a significant enrichment of disrupted or absent ORFs proximal to predicted breakpoints in the FSC022 strain, including a genetic component of the Type I restriction-modification defense system. Many of the pseudogenes identified are also disrupted in the closely related rarely human pathogenic F. tularensis subsp. mediasiatica FSC147 strain, including modulator of drug activity B (mdaB) (FTT0961), which encodes a known NADPH quinone reductase involved in oxidative stress resistance. We have also identified genes exhibiting sequence similarity to effectors of the Type III (T3SS) and components of the Type IV secretion systems (T4SS). One of the genes, msrA2 (FTT1797c), is disrupted in F. tularensis subsp. mediasiatica and has recently been shown to mediate bacterial pathogen survival in host organisms. Our findings suggest that in addition to the duplication of the Francisella Pathogenicity Island, and acquisition of individual loci, adaptation by gene loss in the more recently emerged tularensis, holarctica, and mediasiatica subspecies occurred and was distinct from evolutionary events that differentiated these subspecies, and the novicida subspecies, from a common ancestor. Our findings are applicable to future studies focused on variations in Francisella subspecies pathogenesis, and of broader interest to studies of genomic pathoadaptation in bacteria

Drug transporters of the fungal wheat pathogen Mycosphaerella graminicola

Dit proefschirft levert een belangrijke bijdrage aan de wetenschap dat drug transporters een significant rol spelen bij gevoeligheid en (multi)drug-resistentie van schimmels tegen fungiciden. Bij M. graminicola kan de transporter MgMfs1 bijdragen aan een normale fitness van strobilurine-resistente veld-isolaten. Dit inzicht is nieuw en draagt bij tot een beter inzicht in de overlevingskansen van fungiciden-resistente populaties van het pathogeen. Het is niet uitgesloten dat vergelijkbare drug transporters een rol spelen bij resistentie tegen andere specifiek werkende middelen en in andere plantenpathogenen. De rol van de ABC-transporter MgAtr7 met een nog niet eerder beschreven functie in ijzerevenwicht dient nader te worden onderzocht. Het vermogen van niet-fungitoxische modulatoren om plantenziekten te bestrijden is ook niet eerder gepubliceerd. Tot nu toe zijn slechts enkele middelen met een indirecte werking toegelaten voor de bestrijdingvan plantenziekten. Mogelijk kan het uitgevoerde onderzoek dienen als leidraad bij de ontwikkeling van nieuwe ziektewerende middelen met een indirecte werkin

Drug transporters of the fungal wheat pathogen Mycosphaerella graminicola

Wheat is the most important cereal crop in the world. It occupies 17% of the cultivated land and is the main source of food for 35% of the world population. InIran, wheat is the most important agricultural crop and bread as its main product provides over 50% of daily consumed calories.The ascomycetousfungus Mycosphaerella graminicola(Fuckel) J. Schroeter isthe causal agent of septoria tritici leaf blotch disease of wheat.The fungus has gradually emerged as one of the most damagingfoliarpathogensespeciallyin areas with high rainfall during the growing season.Septoria tritici leaf blotch diseaseis mainly managed by usingresistant cultivars, cultural practices, and chemical control.Extensive application of different classes of fungicidesto control the disease has imposed selection pressure on this pathogen leading to evolutionof fungicide-resistant strains.The objective of this thesis was the analysis of major facilitator superfamily (MFS) and ATP-binding cassette (ABC) drug efflux transporters of M. graminicola for their role in fungicide sensitivity, multidrug resistance (MDR) development and virulence on wheat. The thesis also describes a novel type of ABC transporter with a role in iron homeostasis. Inhibitors of drug transporters that function as virulence factors may act as indirect disease control agents and therefore, the potency of medical modulators of ABC drug transporters to control septoria leaf blotch on wheat seedlings was investigated.Chapter 1 presents a general overview on the global importance of wheat, septoria leaf blotch, M. graminicola , and disease management. Special attention is given to chemical control of the disease, fungicide resistance, and the role of drug transporters in fungicide sensitivity and resistance.Chapter 2 gives a review on MFS transporters with special emphasis on drug transporters and their relevance in plant pathogenic fungi. This chapter provides an overview of common characteristics of MFS transporters, including theirclassification, physical characteristics, and themechanisms of transport. Furthermore, the role of several MFS transporters from plant pathogenic fungi in secretion of various types of antifungal compounds, MDR and virulence are reviewed. Finally, the impact of fungal MFS drug transporters on fungicide baseline sensitivity, MDR, and drug discovery is discussed.The chapters 3>and 4 focus on the identification and functional analysis of theMFS transporter gene MgMfs1 , the first MFS transporter gene cloned from M. graminicola with high homology to fungal MFS transporters within the DHA2 family, involved in mycotoxin secretion and MDR. The results described in chapter 3 indicate that MgMfs1 is not involved in virulence on wheat. However, heterologous expression of this gene in yeast and the phenotypic characterization of MgMfs1 disruption mutants of M. graminicola demonstrate that in vitro MgMfs1 can act as a very potent multidrug transporter, capable totransport a wide range of substrates such as fungal toxins, plant metabolites, and fungicides, particularly strobilurins. In Chapter 4, it is demonstrated that MgMfs1plays a significant role in both in vitro and in plantasensitivity ofM. graminicola to thestrobilurin fungicide trifloxystrobin. The efflux of strobilurin fungicides inMgMfs1 disruption mutants is reduced, and in disease control experiments on wheat seedlings these disruption mutants exhibit an increased sensitivity to trifloxystrobin. Chapter 4 also demonstrates that, besides the dominant G143A target site mutation in the cytochrome b gene, overexpression of MgMfs1 may act as an additional protection mechanism in strobilurin-resistant field strains. MgMfs1 overexpression is probably required for proper maintenance of membrane functioning and normal fitness of these strains in the presence of strobilurins.Chapter 5 describes the molecular cloning and functional characterization of MgAtr7, an ABC transporter gene withhigh homology to fungal ABC transporters involved in azole sensitivity that seems to be unique for M. graminicola and Fusarium graminearum . The encoded protein MgAtr7 is a novelhybrid type of ABC transporterwith the [NBF-TM] 2 configuration which contains a motif characteristic for a dityrosine / pyoverdine biosynthesis protein at the N-terminus, and is the first member of a new class of fungal ABC transporters harboring both a transporter and a biosynthetic moiety. Functional analyses revealed that the gene is involved neither in fungicide sensitivity nor in virulence on wheat, but functions in maintenance of iron homeostasis.Chapter 6 describes the ability of medical drugs known to modulate the activity of ABC transporters, to potentiate the activity of the azole fungicide cyproconazole against in vitro growth of M. graminicola and to control disease development of the pathogen on wheat seedlings. It is shown that some of these compounds can synergize cyproconazole activity in vitro and inhibit efflux activity of the fungicide from fungal cells. However, synergistic interactions between the modulators and cyproconazole were not observed in planta . Some of the compounds have virtually no toxic activity to M. graminicolain vitro , but do show a significant disease control activity on wheat seedlings in preventive and curative foliar spray experiments. The results suggest that these compounds have an indirect disease control activity based on modulation of fungal ABC transporters essential for virulence. Such modulators may constitute a new class of disease control agents. , the function of the ABC transporter MgAtr7 in iron-siderophore transport, and the potential of modulators of ABC transporters to act as lead products in the development of disease control agents.In conclusion, the data presented in this thesis show that the MFS drug transporter MgMfs1 from M. graminicola plays an important role in fungicide sensitivity and resistance, specifically in relation to the strobilurin fungicides. A new type of ABC drug transporter with a new function in iron homeostasis was discovered. In addition, we demonstrated that medical drugs known as modulators of ABC transporters can have an indirect disease control activity and may constitute a new class of disease control agents

MgAtr7, a new type of ABC transporter from Mycosphaerella graminicola involved in iron homeostasis

The ABC transporter-encoding gene MgAtr7 from the wheat pathogen Mycosphaerella graminicola was cloned based upon its high homology to ABC transporters involved in azole-fungicide sensitivity. Genomic and cDNA sequences indicated that the N-terminus of this ABC transporter contains a motif characteristic for a dityrosine/pyoverdine biosynthesis protein. This makes MgAtr7 the first member of a new class of fungal ABC transporters harboring both a transporter and a biosynthetic moiety. A homologue of MgAtr7 containing the same biosynthetic moiety was only found in the Fusarium graminearum genome and not in any other fungal genome examined so far. The gene structure of both orthologous transporters is highly conserved and the genomic area surrounding the ABC transporter exhibits micro-synteny between M. graminicola and F. graminearum. Functional analyses revealed that MgAtr7 is neither required for virulence nor involved in fungicide sensitivity but indicated a role in maintenance of iron homeostasis

MgAtr7, a new type of ABC transporter from Mycosphaerella graminicola involved in iron homeostasis

The ABC transporter-encoding gene MgAtr7 from the wheat pathogen Mycosphaerella graminicola was cloned based upon its high homology to ABC transporters involved in azole-fungicide sensitivity. Genomic and cDNA sequences indicated that the N-terminus of this ABC transporter contains a motif characteristic for a dityrosine/pyoverdine biosynthesis protein. This makes MgAtr7 the first member of a new class of fungal ABC transporters harboring both a transporter and a biosynthetic moiety. A homologue of MgAtr7 containing the same biosynthetic moiety was only found in the Fusarium graminearum genome and not in any other fungal genome examined so far. The gene structure of both orthologous transporters is highly conserved and the genomic area surrounding the ABC transporter exhibits micro-synteny between M. graminicola and F. graminearum. Functional analyses revealed that MgAtr7 is neither required for virulence nor involved in fungicide sensitivity but indicated a role in maintenance of iron homeostasis

MgMfs1, a major facilitator superfamily transporter from the fungal wheat pathogen Mycosphaerella graminicola, is a strong protectant against natural toxic compounds and fungicides

MgMfs1, a major facilitator superfamily (MFS) gene from the wheat pathogenic fungus Mycosphaerella graminicola, was identified in expressed sequence tag (EST) libraries. The encoded protein has high homology to members of the drug:H+ antiporter efflux family of MFS transporters with 14 predicted transmembrane spanners (DHA14), implicated in mycotoxin secretion and multidrug resistance. Heterologous expression of MgMfs1 in a hypersensitive Saccharomyces cerevisiae strain resulted in a strong decrease in sensitivity of this organism to a broad range of unrelated synthetic and natural toxic compounds. The sensitivity of MgMfs1 disruption mutants of M. graminicola to most of these compounds was similar when compared to the wild-type but the sensitivity to strobilurin fungicides and the mycotoxin cercosporin was increased. Virulence of the disruption mutants on wheat seedlings was not affected. The results indicate that MgMfs1 is a true multidrug transporter that can function as a determinant of pathogen sensitivity and resistance to fungal toxins and fungicides

MgMfs1, a major facilitator superfamily transporter from the fungal wheat pathogen Mycosphaerella graminicola, is a strong protectant against natural toxic compounds and fungicides

MgMfs1, a major facilitator superfamily (MFS) gene from the wheat pathogenic fungus Mycosphaerella graminicola, was identified in expressed sequence tag (EST) libraries. The encoded protein has high homology to members of the drug:H+ antiporter efflux family of MFS transporters with 14 predicted transmembrane spanners (DHA14), implicated in mycotoxin secretion and multidrug resistance. Heterologous expression of MgMfs1 in a hypersensitive Saccharomyces cerevisiae strain resulted in a strong decrease in sensitivity of this organism to a broad range of unrelated synthetic and natural toxic compounds. The sensitivity of MgMfs1 disruption mutants of M. graminicola to most of these compounds was similar when compared to the wild-type but the sensitivity to strobilurin fungicides and the mycotoxin cercosporin was increased. Virulence of the disruption mutants on wheat seedlings was not affected. The results indicate that MgMfs1 is a true multidrug transporter that can function as a determinant of pathogen sensitivity and resistance to fungal toxins and fungicides