Combating a Global Threat to a Clonal Crop: Banana Black Sigatoka Pathogen Pseudocercospora fijiensis (Synonym Mycosphaerella fijiensis) Genomes Reveal Clues for Disease Control.

Black Sigatoka or black leaf streak disease, caused by the Dothideomycete fungus Pseudocercospora fijiensis (previously: Mycosphaerella fijiensis), is the most significant foliar disease of banana worldwide. Due to the lack of effective host resistance, management of this disease requires frequent fungicide applications, which greatly increase the economic and environmental costs to produce banana. Weekly applications in most banana plantations lead to rapid evolution of fungicide-resistant strains within populations causing disease-control failures throughout the world. Given its extremely high economic importance, two strains of P. fijiensis were sequenced and assembled with the aid of a new genetic linkage map. The 74-Mb genome of P. fijiensis is massively expanded by LTR retrotransposons, making it the largest genome within the Dothideomycetes. Melting-curve assays suggest that the genomes of two closely related members of the Sigatoka disease complex, P. eumusae and P. musae, also are expanded. Electrophoretic karyotyping and analyses of molecular markers in P. fijiensis field populations showed chromosome-length polymorphisms and high genetic diversity. Genetic differentiation was also detected using neutral markers, suggesting strong selection with limited gene flow at the studied geographic scale. Frequencies of fungicide resistance in fungicide-treated plantations were much higher than those in untreated wild-type P. fijiensis populations. A homologue of the Cladosporium fulvum Avr4 effector, PfAvr4, was identified in the P. fijiensis genome. Infiltration of the purified PfAVR4 protein into leaves of the resistant banana variety Calcutta 4 resulted in a hypersensitive-like response. This result suggests that Calcutta 4 could carry an unknown resistance gene recognizing PfAVR4. Besides adding to our understanding of the overall Dothideomycete genome structures, the P. fijiensis genome will aid in developing fungicide treatment schedules to combat this pathogen and in improving the efficiency of banana breeding programs.

Analysis of the mating-type loci of co-occurring and phylogenetically related species of Ascochyta and Phoma.

Ascochyta and Phoma are fungal genera containing several important plant pathogenic species. These genera are morphologically similar, and recent molecular studies performed to unravel their phylogeny have resulted in the establishment of several new genera within the newly erected Didymellaceae family. An analysis of the structure of fungal mating-type genes can contribute to a better understanding of the taxonomic relationships of these plant pathogens, and may shed some light on their evolution and on differences in sexual strategy and pathogenicity. We analysed the mating-type loci of phylogenetically closely related Ascochyta and Phoma species (Phoma clematidina, Didymella vitalbina, Didymella clematidis, Peyronellaea pinodes and Peyronellaea pinodella) that co-occur on the same hosts, either on Clematis or Pisum. The results confirm that the mating-type genes provide the information to distinguish between the homothallic Pey. pinodes (formerly Ascochyta pinodes) and the heterothallic Pey. pinodella (formerly Phoma pinodella), and indicate the close phylogenetic relationship between these two species that are part of the disease complex responsible for Ascochyta blight on pea. Furthermore, our analysis of the mating-type genes of the fungal species responsible for causing wilt of Clematis sp. revealed that the heterothallic D. vitalbina (Phoma anamorph) is more closely related to the homothallic D. clematidis (Ascochyta anamorph) than to the heterothallic P. clematidina. Finally, our results indicate that homothallism in D. clematidis resulted from a single crossover between MAT1-1 and MAT1-2 sequences of heterothallic ancestors, whereas a single crossover event followed by an inversion of a fused MAT1/2 locus resulted in homothallism in Pey. pinodes.

Evolutionary dynamics of mating-type loci of Mycosphaerella spp. occurring on banana.

The devastating Sigatoka disease complex of banana is primarily caused by three closely related heterothallic fungi belonging to the genus Mycosphaerella: M. fijiensis, M. musicola, and M. eumusae. Previous phylogenetic work showing common ancestry led us to analyze the mating-type loci of these Mycosphaerella species occurring on banana. We reasoned that this might provide better insight into the evolutionary history of these species. PCR and chromosome-walking approaches were used to clone the mating-type loci of M. musicola and M. eumusae. Sequences were compared to the published mating-type loci of M. fijiensis and other Mycosphaerella spp., and a novel organization of the MAT loci was found. The mating-type loci of the examined Mycosphaerella species are expanded, containing two additional Mycosphaerella-specific genes in a unique genomic organization. The proteins encoded by these novel genes show a higher interspecies than intraspecies homology. Moreover, M. fijiensis, M. musicola, and M. eumusae contain two additional mating-type-like loci, containing parts of both MAT1-1-1 and MAT1-2-1. The data indicate that M. fijiensis, M. musicola, and M. eumusae share an ancestor in which a fusion event occurred between MAT1-1-1 and MAT1-2-1 sequences and in which additional genes became incorporated into the idiomorph. The new genes incorporated have since then evolved independently in the MAT1-1 and MAT1-2 loci. Thus, these data are an example of the evolutionary dynamics of fungal MAT loci in general and show the great flexibility of the MAT loci of Mycosphaerella species in particular.

Large-scale gene discovery in the septoria tritici blotch fungus Mycosphaerella graminicola with a focus on in planta expression.

The foliar disease septoria tritici blotch, caused by the fungus Mycosphaerella graminicola, is currently the most important wheat disease in Europe. Gene expression was examined under highly different conditions, using 10 expressed sequence tag libraries generated from M. graminicola isolate IPO323 using seven in vitro and three in planta growth conditions. To identify fungal clones in the interaction libraries, we developed a selection method based on hybridization with the entire genomic DNA of M. graminicola, to selectively enrich these libraries for fungal genes. Assembly of the 27,007 expressed sequence tags resulted in 9,190 unigenes, representing 5.2 Mb of the estimated 39-Mb genome size of M. graminicola. All libraries contributed significantly to the number of unigenes, especially the in planta libraries representing different stages of pathogenesis, which covered 15% of the library-specific unigenes. Even under presymptomatic conditions (5 days postinoculation), when fungal biomass is less than 5%, this method enabled us to efficiently capture fungal genes expressed during pathogenesis. Many of these genes were uniquely expressed in planta, indicating that in planta gene expression significantly differed from in vitro expression. Examples of gene discovery included a number of cell wall-degrading enzymes, a broad set of genes involved in signal transduction (n=11) and a range of ATP-binding cassette (n=20) and major facilitator superfamily transporter genes (n=12) potentially involved in protection against antifungal compounds or the secretion of pathogenicity factors. In addition, evidence is provided for a mycovirus in M. graminicola that is highly expressed under various stress conditions, in particular, under nitrogen starvation. Our analyses provide a unique window on in vitro and in planta gene expression of M. graminicola.

The drug transporter MgMfs1 can modulate sensitivity of field strains of the fungal wheat pathogen Mycosphaerella graminicola to the strobilurin fungicide trifloxystrobin.

BACKGROUND: The major facilitator superfamily (MFS) drug transporter MgMfs1 of the wheat pathogen Mycosphaerella graminicola (Fuckel) J Schroeter is a potent multidrug transporter with high capacity to transport strobilurin fungicides in vitro. The data presented in this paper indicate that, in addition to the predominant cause of strobilurin resistance, cytochrome b G143A subsititution, MgMfs1 can play a role in sensitivity of field strains of this pathogen to trifloxystrobin. RESULTS: In a major part of field strains of M. graminicola (collected in the Netherlands in 2004) containing the cytochrome b G143A substitution, the basal level of expression of MgMfs1 was elevated as compared with sensitive strains lacking the G143A substitution. Induction of MgMfs1 expression in wild-type isolates upon treatment with trifloxystrobin at sublethal concentrations proceeded rapidly. Furthermore, in disease control experiments on wheat seedlings, disruption mutants of MgMfs1 displayed an increased sensitivity to trifloxystrobin. CONCLUSION: It is concluded that the drug transporter MgMfs1 is a determinant of strobilurin sensitivity of field strains of M. graminicola.

Control of Mycosphaerella graminicola on wheat seedlings by medical drugs known to modulate the activity of ATP-binding cassette transporters.

Medical drugs known to modulate the activity of human ATP-binding cassette (ABC) transporter proteins (modulators) were tested for the ability to potentiate the activity of the azole fungicide cyproconazole against in vitro growth of Mycosphaerella graminicola and to control disease development due to this pathogen on wheat seedlings. In vitro modulation of cyproconazole activity could be demonstrated in paper disk bioassays. Some of the active modulators (amitriptyline, flavanone, and phenothiazines) increased the accumulation of cyproconazole in M. graminicola, suggesting that they reversed cyproconazole efflux. However, synergism between cyproconazole and modulators against M. graminicola on wheat seedlings could not be shown. Despite their low in vitro toxicity to M. graminicola, some modulators (amitriptyline, loperamide, and promazine) did show significant intrinsic disease control activity in preventive and curative foliar spray tests with wheat seedlings. The results suggest that these compounds have indirect disease control activity based on modulation of fungal ABC transporters essential for virulence and 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.

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.

MgHog1 regulates dimorphism and pathogenicity in the fungal wheat pathogen Mycosphaerella graminicola.

The dimorphic ascomycete pathogen Mycosphaerella graminicola switches from a yeastlike form to an infectious filamentous form that penetrates the host foliage through stomata. We examined the biological function of the mitogen-activated protein kinase-encoding gene MgHog1 in M. graminicola. Interestingly, MgHog1 mutants were unable to switch to filamentous growth on water agar that mimics the nutritionally poor conditions on the foliar surface and, hence, exclusively developed by a yeastlike budding process. Consequently, due to impaired initiation of infectious germ tubes, as revealed by detailed in planta cytological analyses, the MgHog1 mutants failed to infect wheat leaves. We, therefore, conclude that MgHog1 is a new pathogenicity factor involved in the regulation of dimorphism in M. graminicola. Furthermore, MgHog1 mutants are osmosensitive, resistant to phenylpyrrole and dicarboximide fungicides, and do not melanize.

Impact of fungal drug transporters on fungicide sensitivity, multidrug resistance and virulence.

Drug transporters are membrane proteins that provide protection for organisms against natural toxic products and fungicides. In plant pathogens, drug transporters function in baseline sensitivity to fungicides, multidrug resistance (MDR) and virulence on host plants. This paper describes drug transporters of the filamentous fungi Aspergillus nidulans (Eidam) Winter, Botrytis cinerea Pers and Mycosphaerella graminicola (Fückel) Schroter that function in fungicide sensitivity and resistance. The fungi possess ATP-binding cassette (ABC) drug transporters that mediate MDR to fungicides in laboratory mutants. Similar mutants are not pronounced in field resistance to most classes of fungicide but may play a role in resistance to azoles. MDR may also explain historical cases of resistance to aromatic hydrocarbon fungicides and dodine. In clinical situations, MDR development in Candida albicans (Robin) Berkhout mediated by ABC transporters in patients suffering from candidiasis is common after prolonged treatment with azoles. Factors that can explain this striking difference between agricultural and clinical situations are discussed. Attention is also paid to the risk of MDR development in plant pathogens in the future. Finally, the paper describes the impact of fungal drug transporters on drug discovery.

The ABC transporter MgAtr4 is a virulence factor of Mycosphaerella graminicola that affects colonization of substomatal cavities in wheat leaves.

The role in virulence of the ATP-binding cassette (ABC) transporters MgAtr1, MgAtr2, MgAtr3, MgAtr4, and MgAtr5 from Mycosphaerella graminicola was analyzed by gene disruption or replacement on seedlings of the susceptible wheat cultivar Obelisk. Disruption strains of MgAtr1 and MgAtr2 and replacement strains of MgAtr3 and MgAtr5 displayed the same phenotype as control strains, while virulence of the MgAtr4 disruption strains was significantly reduced. This reduction in virulence was independent of the wheat cultivar used. Histopathological analysis of the infection process revealed that MgAtr4 disruption strains colonize substomatal cavities less efficiently and display reduced intercellular growth in the apoplast of wheat leaves. In vitro growth experiments in different media showed no fitness penalty associated with the disruption of MgAtr4. Expression analysis demonstrated that transcripts of the constitutively expressed gene CYP51 encoding the fungal-specific cytochrome P450 sterol 14alpha-demethylase from M. graminicola were not detectable in interaction RNA from wheat infected with MgAtr4 disruption strains, thus confirming the reduced intercellular growth of these strains. The results indicate that MgAtr4 is a virulence factor of M. graminicola during pathogenesis on wheat and may function in protection against fungitoxic compounds present around the substomatal cavities of wheat leaves. MgAtr4 is the first virulence factor cloned from this important plant pathogen.

ABC transporters and azole susceptibility in laboratory strains of the wheat pathogen Mycosphaerella graminicola.

Laboratory strains of Mycosphaerella graminicola with decreased susceptibilities to the azole antifungal agent cyproconazole showed a multidrug resistance phenotype by exhibiting cross-resistance to an unrelated chemical, cycloheximide or rhodamine 6G, or both. Decreased azole susceptibility was found to be associated with either decreased or increased levels of accumulation of cyproconazole. No specific relationship could be observed between azole susceptibility and the expression of ATP-binding cassette (ABC) transporter genes MgAtr1 to MgAtr5 and the sterol P450 14alpha-demethylase gene, CYP51. ABC transporter MgAtr1 was identified as a determinant in azole susceptibility since heterologous expression of the protein reduced the azole susceptibility of Saccharomyces cerevisiae and disruption of MgAtr1 in one specific M. graminicola laboratory strain with constitutive MgAtr1 overexpression restored the level of susceptibility to cyproconazole to wild-type levels. However, the level of accumulation in the mutant with an MgAtr1 disruption did not revert to the wild-type level. We propose that variations in azole susceptibility in laboratory strains of M. graminicola are mediated by multiple mechanisms.