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Abstract Background The genome of Fusarium graminearum has been sequenced and annotated previously, but correct gene annotation remains a challenge. In addition, posttranscriptional regulations, such as alternative splicing and RNA editing, are poorly understood in F. graminearum. Here we took advantage of RNA-Seq to improve gene annotations and to identify alternative splicing and RNA editing in F. graminearum. Results We identified and revised 655 incorrectly predicted gene models, including revisions of intron predictions, intron splice sites and prediction of novel introns. 231 genes were identified with two or more alternative splice variants, mostly due to intron retention. Interestingly, the expression ratios between different transcript isoforms appeared to be developmentally regulated. Surprisingly, no RNA editing was identified in F. graminearum. Moreover, 2459 novel transcriptionally active regions (nTARs) were identified and our analysis indicates that many of these could be missed genes. Finally, we identified the 5′ UTR and/or 3′ UTR sequences of 7666 genes. A number of representative novel gene models and alternatively spliced genes were validated by reverse transcription polymerase chain reaction and sequencing of the generated amplicons. Conclusions We have developed novel and efficient strategies to identify alternatively spliced genes and incorrect gene models based on RNA-Seq data. Our study identified hundreds of alternatively spliced genes in F. graminearum and for the first time indicated that alternative splicing is developmentally regulated in filamentous fungi. In addition, hundreds of incorrect predicted gene models were identified and revised and thousands of nTARs were discovered in our study, which will be helpful for the future genomic and transcriptomic studies in F. graminearum.

An abstract on the study relating to the birth-and-death evolution and horizontal transfer of a mycotoxin biosynthetic gene cluster in pathogenic fusarium species is presented.

Exploiting wheat cultivars with stable resistance to Fusarium Head blight (FHB) and toxin accumulation is a cost-effective and environmentally friendly strategy to reduce the risk of yield losses and contamination with mycotoxins. To facilitate the deployment of stable cultivar resistance, we evaluated FHB resistance and resistance to mycotoxin accumulation in 410 wheat lines bred by local breeders from four major wheat growing regions in China after natural infection at three distinct locations (Hefei, Yangzhou and Nanping). Significant differences in disease index were observed among the three locations. The disease indexes (DI's) in Nanping were the highest, followed by Yangzhou and Hefei. The distribution of DI's in Yangzhou showed the best discrimination of FHB resistance in cultivars. Growing region and cultivar had significant effect on DI and mycotoxins. Among the climate factors, relative humidity and rainfall were the key factors resulting in the severe disease. Even though most cultivars were still susceptible to FHB under the strongly conducive conditions applied, the ratio of resistant lines increased in the Upper region of the Yangtze River (UYR) and the Middle and Lower Region of the Yangtze River (MLYR) between 2015 and 2019. Deoxynivalenol (DON) was the dominant mycotoxin found in Hefei and Yangzhou, while NIV was predominant in Nanping. Disease indexes were significantly correlated with DON content in wheat grain., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yan, Chen, van der Lee, Waalwijk, van Diepeningen, Feng, Zhang and Liu.)

Fusarium graminearum is ranked among the five most destructive fungal pathogens that affect agroecosystems. It causes floral diseases in small grain cereals including wheat, barley, and oats, as well as maize and rice. We conducted a systematic review of peer-reviewed studies reporting species within the F. graminearum species complex (FGSC) and created two main data tables. The first contained summarized data from the articles including bibliographic, geographic, methodological (ID methods), host of origin and species, while the second data table contains information about the described strains such as publication, isolate code(s), host/substrate, year of isolation, geographical coordinates, species and trichothecene genotype. Analyses of the bibliographic data obtained from 123 publications from 2000 to 2021 by 498 unique authors and published in 40 journals are summarized. We describe the frequency of species and chemotypes for 16,274 strains for which geographical information was available, either provided as raw data or extracted from the publications, and sampled across six continents and 32 countries. The database and interactive interface are publicly available, allowing for searches, summarization, and mapping of strains according to several criteria including article, country, host, species and trichothecene genotype. The database will be updated as new articles are published and should be useful for guiding future surveys and exploring factors associated with species distribution such as climate and land use. Authors are encouraged to submit data at the strain level to the database, which is accessible at https://fgsc.netlify.app.

Fusarium crown rot (FCR) is one of the most important wheat diseases in northern China. The main causal agent of FCR, Fusarium pseudograminearum , can produce mycotoxins such as type B trichothecenes. Therefore, FCR could be an additional source of mycotoxin contamination during wheat production. Field inoculation experiments demonstrated that FCR disease severity strongly impacts the distribution pattern of trichothecenes in different wheat tissues. Mycotoxins were mainly observed in lower internodes, and a low amount was detected in the upper parts above the fourth internode. However, high levels of trichothecene accumulation were detected in the upper segments of wheat plants under field conditions, which would threaten the feed production. The variation of mycotoxin content among sampling sites indicated that besides disease severity, other factors like climate, irrigation, and fungicide application may influence the mycotoxin accumulation in wheat. A comprehensive survey of deoxynivalenol (DON) and its derivatives in wheat heads with FCR symptoms in natural fields was conducted at 80 sites in seven provinces in northern China. Much higher levels of mycotoxin were observed compared with inoculation experiments. The mycotoxin content varied greatly among sampling sites, but no significant differences were observed if compared at province level, which indicated the variation is mainly caused by local conditions. Trace amounts of mycotoxin appeared to be translocated to grains, which revealed that FCR infection in natural fields poses a relatively small threat to contamination of grains but a larger one to plant parts that may be used as animal feed. To our knowledge, this is the first report of trichothecene accumulation in wheat stems and heads, as well as grains after FCR infection in natural field conditions. These investigations provide novel insights into food and feed safety risk caused by FCR in northern China.

Fusarium head blight (FHB) in wheat ( Triticum aestivum L.) is caused by a consortium of mutually interacting Fusarium species. In the field, the weakly pathogenic F. poae often thrives on the infection sites of the virulent F. graminearum . In this ecological context, we investigated the efficacy of chemical and biocontrol agents against F. graminearum in wheat ears. For this purpose, one fungicide comprising prothioconazole + spiroxamine and two bacterial biocontrol strains, Streptomyces rimosus LMG 19352 and Rhodococcus sp. R-43120 were tested for their efficacy to reduce FHB symptoms and mycotoxin (deoxynivalenol, DON) production by F. graminearum in presence or absence of F. poae . Results showed that the fungicide and both actinobacterial strains reduced FHB symptoms and concomitant DON levels in wheat ears inoculated with F. graminearum . Where Streptomyces rimosus appeared to have direct antagonistic effects, Rhodococcus and the fungicide mediated suppression of F. graminearum was linked to the archetypal salicylic acid and jasmonic acid defense pathways that involve the activation of LOX1 , LOX2 and ICS . Remarkably, this chemical- and biocontrol efficacy was significantly reduced when F. poae was co-inoculated with F. graminearum . This reduced efficacy was linked to a suppression of the plant's intrinsic defense system and increased levels of DON. In conclusion, our study shows that control strategies against the virulent F. graminearum in the disease complex causing FHB are hampered by the presence of the weakly pathogenic F. poae . This study provides generic insights in the complexity of control strategies against plant diseases caused by multiple pathogens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tan, De Zutter, De Saeger, De Boevre, Tran, van der Lee, Waalwijk, Willems, Vandamme, Ameye and Audenaert.)

Plant diseases are often caused by a consortium of pathogens competing with one another to gain a foothold in the infection niche. Nevertheless, studies are often limited to a single pathogen on its host. In Europe, fusarium head blight (FHB) of wheat is caused by multiple Fusarium species, including Fusarium graminearum and F. poae. Here, we combined a time series of (co)inoculations, monitored by multispectral imaging, transcriptional, and mycotoxin analyses, to study the temporal interaction between both species and wheat. Our results showed coinoculation of F. graminearum and F. poae inhibited symptom development but did not alter mycotoxin accumulation compared to a single inoculation with F. graminearum. In contrast, preinoculation of F. poae reduced both FHB symptoms and mycotoxin levels compared to a single F. graminearum infection. Interestingly, F. poae exhibited increased growth in dual infections, demonstrating that this weak pathogen takes advantage of its co-occurrence with F. graminearum. Quantitative reverse transcription PCR revealed that F. poae induces LOX and ICS gene expression in wheat. We hypothesize that the early induction of salicylic and jasmonic acid-related defences by F. poae hampers a subsequent F. graminearum infection. This study is the first to report on the defence mechanisms of the plant involved in a tripartite interaction between two species of a disease complex and their host., (© 2020 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Grape production is increasing globally and so are problems with downy mildew, one of the main constraints in grape production. Downy mildew on grape is caused by Plasmopara viticola , an obligate biotrophic pathogen belonging to the oomycetes. Control of the disease is usually performed by fungicide applications, of which carboxylic acid amide (CAA) fungicides represent one of the most widely used groups of fungicides. Our previous research showed that the extensive application of CAA fungicides can result in fungicide resistance and in China, CAA-resistant isolates of P. viticola were collected from the field in 2014. To monitor the distribution and spread of CAA fungicide resistance, we developed a TaqMan-minor groove binder (MGB) real-time PCR-based method designed on a functional mutation in the PvCesA3 gene that allows efficient identification of CAA fungicide resistant and sensitive genotypes. The assay was validated on 50 isolates using Sanger sequencing and fungicide bioassays and exploited in a comprehensive survey comprising 2,227 single-sporangiophore isolates from eight major grapevine regions in China. We demonstrate that CAA fungicide resistance in P. viticola is widespread in China. On average, 53.3% of the isolates were found to be resistant, but marked differences were found between locations with percentages of resistant isolates varying from 0.3 to 96.6%. Furthermore, the frequency of CAA-resistant isolates was found to be significantly correlated with the exposure to CAA fungicides ( P < 0.05). We further discussed the possibilities to apply the TaqMan-MGB real-time PCR assay to assess the frequency of fungicide-resistant P. viticola isolates in each region or vineyard, which would facilitate the correct choice of fungicide for grape downy mildew and resistance management strategies.

Peroxisomes are involved in a wide range of important cellular functions. Here, the role of the peroxisomal membrane protein PEX3 in the plant-pathogen and mycotoxin producer Fusarium graminearum was studied using knock-out and complemented strains. To fluorescently label peroxisomes' punctate structures, GFP and RFP fusions with the PTS1 and PTS2 localization signal were transformed into the wild type PH-1 and Δ FgPex3 knock-out strains. The GFP and RFP transformants in the Δ FgPex3 background showed a diffuse fluorescence pattern across the cytoplasm suggesting the absence of mature peroxisomes. The Δ FgPex3 strain showed a minor, non-significant reduction in growth on various sugar carbon sources. In contrast, deletion of FgPex3 affected fatty acid β-oxidation in F. graminearum and significantly reduced the utilization of fatty acids. Furthermore, the Δ FgPex3 mutant was sensitive to osmotic stressors as well as to cell wall-damaging agents. Reactive oxygen species (ROS) levels in the mutant had increased significantly, which may be linked to the reduced longevity of cultured strains. The mutant also showed reduced production of conidiospores, while sexual reproduction was completely impaired. The pathogenicity of Δ FgPex3 , especially during the process of systemic infection, was strongly reduced on both tomato and on wheat, while to production of deoxynivalenol (DON), an important factor for virulence, appeared to be unaffected., (Copyright © 2019 Kong, Zhang, Wang, van der Lee, Waalwijk, van Diepeningen, Brankovics, Xu, Xu, Chen and Feng.)

In recent years, Fusarium head blight (FHB) outbreaks have occurred much more frequently in China. The reduction of burning of the preceding crop residues is suggested to contribute to more severe epidemics as it may increase the initial inoculum. In this study, a large number of Fusarium isolates was collected from blighted wheat spikes as well as from rice stubble with perithecia originating from nine sampling sites in five provinces in Southern China. Fusarium asiaticum dominated both wheat and rice populations, although rice populations showed a higher species diversity. Chemotype analysis showed that rice is the preferred niche for NIV mycotoxin producers that were shown to be less virulent on wheat. In contrast, 3ADON producers are more prevalent on wheat and in wheat producing areas. The 3ADON producers were shown to be more virulent on wheat, revealing the selection pressure of wheat on 3ADON producers. For the first time, members of the Incarnatum -clade of Fusarium Incarnatum - Equiseti Species Complex (FIESC) were found to reproduce sexually on rice stubble. The pathogenicity of FIESC isolates on wheat proved very low and this may cause the apparent absence of this species in the main wheat producing provinces. This is the first report of the Fusarium population structure including rice stubble as well as a direct comparison with the population on wheat heads in the same fields. Our results confirm that the perithecia on rice stubble are the primary inoculum of FHB on wheat and that cropping systems affect the local Fusarium population., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Fusarium head blight is a disease caused by a complex of Fusarium species. F. poae is omnipresent throughout Europe in spite of its low virulence. In this study, we assessed a geographically diverse collection of F. poae isolates for its genetic diversity using AFLP (Amplified Fragment Length Polymorphism). Furthermore, studying the mating type locus and chromosomal insertions, we identified hallmarks of both sexual recombination and clonal spread of successful genotypes in the population. Despite the large genetic variation found, all F. poae isolates possess the nivalenol chemotype based on Tri7 sequence analysis. Nevertheless, Tri gene clusters showed two layers of genetic variability. Firstly, the Tri1 locus was highly variable with mostly synonymous mutations and mutations in introns pointing to a strong purifying selection pressure. Secondly, in a subset of isolates, the main trichothecene gene cluster was invaded by a transposable element between Tri5 and Tri6 . To investigate the impact of these variations on the phenotypic chemotype, mycotoxin production was assessed on artificial medium. Complex blends of type A and type B trichothecenes were produced but neither genetic variability in the Tri genes nor variability in the genome or geography accounted for the divergence in trichothecene production. In view of its complex chemotype, it will be of utmost interest to uncover the role of trichothecenes in virulence, spread and survival of F. poae ., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

The oomycete Phytophthora infestans was the causal agent of the Irish Great Famine and is a recurring threat to global food security. The pathogen can reproduce both sexually and asexually, with high potential to adapt to various environments and great risk to break disease resistance genes in potato. As are other oomycetes, P. infestans is regarded to be diploid during the vegetative phase of its life cycle, although some studies reported trisomy and polyploidy. Using microsatellite fingerprinting, genome-wide assessment of single nucleotide polymorphisms, nuclear DNA quantification, and microscopic counting of chromosome numbers, we assessed the ploidy level of a comprehensive selection of isolates. All progenies from sexual populations of P. infestans in nature were found to be diploid, in contrast nearly all dominant asexual lineages, including the most important pandemic clonal lineages US-1 and 13&#95;A2 were triploid. Such triploids possess significantly more allelic variation than diploids. We observed that triploid genotype can change to a diploid genome constitution when exposed to artificial stress conditions. This study reveals that fluctuations in the ploidy level may be a key factor in the adaptation process of this notorious plant destroyer and imposes an extra challenge to control this disease.

Background: Eukaryotes display remarkable genome plasticity, which can include supernumerary chromosomes that differ markedly from the core chromosomes. Despite the widespread occurrence of supernumerary chromosomes in fungi, their origin, relation to the core genome and the reason for their divergent characteristics are still largely unknown. The complexity of genome assembly due to the presence of repetitive DNA partially accounts for this., Results: Here we use single-molecule real-time (SMRT) sequencing to assemble the genome of a prominent fungal wheat pathogen, Fusarium poae, including at least one supernumerary chromosome. The core genome contains limited transposable elements (TEs) and no gene duplications, while the supernumerary genome holds up to 25 % TEs and multiple gene duplications. The core genome shows all hallmarks of repeat-induced point mutation (RIP), a defense mechanism against TEs, specific for fungi. The absence of RIP on the supernumerary genome accounts for the differences between the two (sub)genomes, and results in a functional crosstalk between them. The supernumerary genome is a reservoir for TEs that migrate to the core genome, and even large blocks of supernumerary sequence (>200 kb) have recently translocated to the core. Vice versa, the supernumerary genome acts as a refuge for genes that are duplicated from the core genome., Conclusions: For the first time, a mechanism was determined that explains the differences that exist between the core and supernumerary genome in fungi. Different biology rather than origin was shown to be responsible. A "living apart together" crosstalk exists between the core and supernumerary genome, accelerating chromosomal and organismal evolution.

In Tunisia, late blight caused by Phytophthora infestans is a serious threat to potato and tomato. The Mediterranean weather conditions can be conducive to infection in all seasons and the host crops, tomato and potato, are grown year round. Potato is planted and harvested in two to four overlapping intervals from August to June and tomato is grown both in open fields and in greenhouses. The consequences of these agricultural practices and the massive import of seed potato on the genetic variation of P. infestans are largely unknown. We conducted a survey in which 165 P. infestans isolates, collected from five subregions in Tunisia between 2006 and 2008, on which we studied genotypic diversity through nuclear (simple-sequence repeat [SSR]) markers and combined this with a previous study on their mitochondrial haplotypes (mtDNA). The phylogenetic analysis revealed the presence of a major clonal lineage (NA-01, A1 mating type, mitochondrial haplotype Ia). Isolates belonging to this clonal lineage were found in all regions and showed a relatively simple virulence pattern on a potato differential set carrying different Solanum demissum resistance genes. Apart from isolates belonging to this NA-01 clonal lineage, a group of isolates was found that showed a high genetic diversity, comprising both mating types and a more complex race structure that was found in the regions where late blight on potato was more difficult to control. The population on potato and tomato seems to be under different selection pressures. Isolates collected from tomato showed a low genetic diversity even though potato isolates collected simultaneously from the same location showed a high genetic diversity. Based on the SSR profile comparison, we could demonstrate that the four major clonal lineages found in the Netherlands and also in other European countries could not be found in Tunisia. Despite the massive import of potato seed from Europe, the P. infestans population in Tunisia was found to be clearly distinct.

Background: Genome comparisons between closely related species often show non-conserved regions across chromosomes. Some of them are located in specific regions of chromosomes and some are even confined to one or more entire chromosomes. The origin and biological relevance of these non-conserved regions are still largely unknown. Here we used the genome of Fusarium graminearum to elucidate the significance of non-conserved regions., Results: The genome of F. graminearum harbours thirteen non-conserved regions dispersed over all of the four chromosomes. Using RNA-Seq data from the mycelium of F. graminearum, we found weakly expressed regions on all of the four chromosomes that exactly matched with non-conserved regions. Comparison of gene expression between two different developmental stages (conidia and mycelium) showed that the expression of genes in conserved regions is stable, while gene expression in non-conserved regions is much more influenced by developmental stage. In addition, genes involved in the production of secondary metabolites and secreted proteins are enriched in non-conserved regions, suggesting that these regions could also be important for adaptations to new environments, including adaptation to new hosts. Finally, we found evidence that non-conserved regions are generated by sequestration of genes from multiple locations. Gene relocations may lead to clustering of genes with similar expression patterns or similar biological functions, which was clearly exemplified by the PKS2 gene cluster., Conclusions: Our results showed that chromosomes can be functionally divided into conserved and non-conserved regions, and both could have specific and distinct roles in genome evolution and regulation of gene expression.

Fumonisins are a family of carcinogenic secondary metabolites produced by members of the Fusarium fujikuroi species complex (FFSC) and rare strains of Fusarium oxysporum. In Fusarium, fumonisin biosynthetic genes (FUM) are clustered, and the cluster is uniform in gene organization. Here, sequence analyses indicated that the cluster exists in five different genomic contexts, defining five cluster types. In FUM gene genealogies, evolutionary relationships between fusaria with different cluster types were largely incongruent with species relationships inferred from primary-metabolism (PM) gene genealogies, and FUM cluster types are not trans-specific. In addition, synonymous site divergence analyses indicated that three FUM cluster types predate diversification of FFSC. The data are not consistent with balancing selection or interspecific hybridization, but they are consistent with two competing hypotheses: (i) multiple horizontal transfers of the cluster from unknown donors to FFSC recipients and (ii) cluster duplication and loss (birth and death). Furthermore, low levels of FUM gene divergence in F. bulbicola, an FFSC species, and F. oxysporum provide evidence for horizontal transfer of the cluster from the former, or a closely related species, to the latter. Thus, uniform gene organization within the FUM cluster belies a complex evolutionary history that has not always paralleled the evolution of Fusarium., (Published 2013. This article is a U.S. Government work and is in the public domain in the USA.)

For a comprehensive survey of the structure and dynamics of the Dutch Phytophthora infestans population, 652 P. infestans isolates were collected from commercial potato fields in the Netherlands during the 10-year period 2000-2009. Genotyping was performed using 12 highly informative microsatellite markers and mitochondrial haplotypes. In addition, for each isolate, the mating type was determined. STRUCTURE analysis grouped the 322 identified genotypes in three clusters. Cluster 1 consists of a single clonal lineage NL-001, known as "Blue&#95;13"; all isolates in this cluster have the A2 mating type and the Ia mitochondrial haplotype. Clusters 2 and 3 display a more elaborate substructure containing many unique genotypes. In Cluster 3, several distinct clonal lineages were also identified. This survey witnesses that the Dutch population underwent dramatic changes in the 10 years under study. The most notable change was the emergence and spread of A2 mating type strain NL-001 (or "Blue&#95;13"). The results emphasize the importance of the sexual cycle in generating genetic diversity and the importance of the asexual cycle as the propagation and dispersal mechanism for successful genotypes. Isolates were also screened for absence of the Avrblb1/ipiO class I gene, which is indicative for virulence on Rpi-blb1. This is also the first report of Rpi-blb1 breakers in the Netherlands. Superimposing the virulence screening on the SSR genetic backbone indicates that lack the Avrblb1/ipiO class I gene only occurred in sexual progeny. So far, the asexual spread of the virulent isolates identified has been limited.

A large number of Fusarium isolates was collected from blighted wheat spikes originating from 175 sampling sites, covering 15 provinces in China. Species and trichothecene chemotype determination by multilocus genotyping (MLGT) indicated that F. graminearum s. str. with the 15-acetyl deoxynivalenol (15ADON) chemotype and F. asiaticum with either the nivalenol (NIV) or the 3-acetyl deoxynivalenol (3ADON) chemotype were the dominant causal agents. Bayesian model-based clustering with allele data obtained with 12 variable number of tandem repeats (VNTR) markers, detected three genetic clusters that also show distinct chemotypes. High levels of population genetic differentiation and low levels of effective number of migrants were observed between these three clusters. Additional genotypic analyses revealed that F. graminearum s. str. and F. asiaticum are sympatric. In addition, composition analysis of these clusters indicated a biased gene flow from 3ADON to NIV producers in F. asiaticum. In phenotypic analyses, F. asiaticum that produce 3ADON revealed significant advantages over F. asiaticum that produce NIV in pathogenicity, growth rate, fecundity, conidial length, trichothecene accumulation and resistance to benzimidazole. These results suggest that natural selection drives the spread of a more vigorous, more toxigenic pathogen population which also shows higher levels of fungicide resistance.

Zn(2)Cys(6) transcription factors are unique to fungi and have been reported to be involved in different regulatory functions. Here, we characterized EBR1 (enhanced branching 1), a novel Zn(2)Cys(6) transcription factor of Fusarium graminearum. Knocking out EBR1 in F. graminearum PH-1 caused reduction of both radial growth and virulence. The conidia of knock-out strain PH-1?ebr1 germinated faster than those of wild-type PH-1, but the conidiation of the mutant was significantly reduced. Detailed analysis showed that the reduced radial growth might be due to reduced apical dominance of the hyphal tip, leading to increased hyphal branching. Inoculation assays on wheat heads with a green fluorescent protein (GFP)-labeled PH-1?ebr1 mutant showed that it was unable to penetrate the rachis of the spikelets. Protein fusion with GFP showed that EBR1 is localized in the nucleus of both conidia and hyphae. Knocking out the orthologous gene FOXG&#95;05408 in F. oxysporum f. sp. lycopersici caused a much weaker phenotype than the PH-1?ebr1 mutant, which may be due to the presence of multiple orthologous genes in this fungus. Transformation of FOXG&#95;05408 into PH-1?ebr1 restored the mutant phenotype. Similar to EBR1, FOXG&#95;05408 is localized in the nucleus of F. oxysporum f. sp. lycopersici. Possible functions of EBR1 and its relation with other fungal transcription factors are discussed.

We searched the genome of Mycosphaerella fijiensis for molecular markers that would allow population genetics analysis of this plant pathogen. M. fijiensis, the causal agent of banana leaf streak disease, also known as black Sigatoka, is the most devastating pathogen attacking bananas (Musa spp). Recently, the entire genome sequence of M. fijiensis became available. We screened this database for VNTR markers. Forty-two primer pairs were selected for validation, based on repeat type and length and the number of repeat units. Five VNTR markers showing multiple alleles were validated with a reference set of isolates from different parts of the world and a population from a banana plantation in Costa Rica. Polymorphism information content values varied from 0.6414 to 0.7544 for the reference set and from 0.0400 and 0.7373 for the population set. Eighty percent of the polymorphism information content values were above 0.60, indicating that the markers are highly informative. These markers allowed robust scoring of agarose gels and proved to be useful for variability and population genetics studies. In conclusion, the strategy we developed to identify and validate VNTR markers is an efficient means to incorporate markers that can be used for fungicide resistance management and to develop breeding strategies to control banana black leaf streak disease. This is the first report of VNTR-minisatellites from the M. fijiensis genome sequence.

A strategy to control the devastating late blight disease is providing potato cultivars with genes that are effective in resistance to a broad spectrum of Phytophthora infestans isolates. Thus far, most late blight resistance (R) genes that were introgressed in potato were quickly defeated. In contrast, the Rpi-blb1 gene originating from Solanum bulbocastanum has performed as an exclusive broad-spectrum R gene for many years. Recently, the RXLR effector family ipiO was identified to contain Avr-blb1. Monitoring the genetic diversity of the ipiO family in a large set of isolates of P. infestans and related species resulted in 16 ipiO variants in three distinct classes. Class I and class II but not class III ipiO variants induce cell death when coinfiltrated with Rpi-blb1 in Nicotiana benthamiana. Class I is highly diverse and is represented in all analyzed P. infestans isolates except two Mexican P. infestans isolates, and these were found virulent on Rpi-blb1 plants. In its C-terminal domain, IPI-O contains a W motif that is essential for triggering Rpi-blb1-mediated cell death and is under positive selection. This study shows that profiling the variation of Avr-blb1 within a P. infestans population is instrumental for predicting the effectiveness of Rpi-blb1-mediated resistance in potato.

SUMMARY In eukaryotes, a family of serine/threonine protein kinases known as mitogen-activated protein kinases (MAPKs) is involved in the transduction of a variety of extracellular signals and in the regulation of growth and development. We identified a MAPK-encoding gene in Mycosphaerella graminicola strain IPO323 with high homology to the orthologous Fus3 gene of Saccharomyces cerevisiae and designated it MgFus3. Early colony development of the MgFus3 mutants during in vitro growth was similar to those of the wild-type and ectopic controls, but at the later stages of growth MgFus3 mutants did not become melanized, showed altered polarized growth and were unable to produce aerial mycelia. The MgFus3 mutants were non-pathogenic, and detailed microscopic analyses revealed that they failed to colonize the mesophyll tissue owing to the inability to penetrate stomata. Unlike the wild-type strain, MgFus3 mutants were unable to differentiate pycnidia on plant-derived media. Thus, in addition to the crucial role of MgFus3 in the regulation of penetration, it may also be involved in regulating asexual fructification. Hence, MgFus3 can be regarded as a multifunctional pathogenicity factor of M. graminicola.

Among expressed sequence tag libraries of Mycosphaerella graminicola isolate IPO323, we identified a full-length cDNA clone with high homology to the mitogen-activated protein (MAP) kinase Slt2 in Saccharomyces cerevisiae. This MAP kinase consists of a 1242-bp open reading frame, and encodes a 414-amino-acid protein. We designated this homolog MgSlt2, generated MgSlt2 knockout strains in M. graminicola isolate IPO323, and found several altered phenotypes in vitro as well as in planta. In yeast glucose broth, MgSlt2 disruptants showed a defective polarized growth in the tip cells upon aging, causing substantial local enlargements culminating in large swollen cells containing two to four nuclei. The MgSlt2 disruptants showed a significantly increased sensitivity to several fungicides, including miconazole (2x), bifonazole (>4x), imazalil (5x), and cyproconazole (10x), and were hypersensitive to glucanase. Unlike the wild type, MgSlt2 disruptants did not produce aerial mycelia and did not melanize on potato dextrose agar. Although cytological analysis in planta showed normal penetration of wheat stomata by the germ tubes of the MgSlt2 disruptants, subsequently formed hyphal filaments frequently were unable to branch out and establish invasive growth resulting in highly reduced virulence, and prevented pycnidia formation. Therefore, we conclude that MgSlt2 is a new pathogenicity factor in M. graminicola.