A Genome Resource for Ciborinia camelliae, the Causal Agent of Camellia Flower Blight.

Ciborinia camelliae Kohn is a camellia pathogen belonging to family Sclerotiniaceae, infecting only flowers of camellias. To better understand the virulence mechanism in this species, the draft genome sequence of the Italian strain of C. camelliae was obtained with a hybrid approach, combining Illumina HiSeq paired reads and MinIon Nanopore long-read sequencing. This combination improved significantly the existing National Center for Biotechnology Information reference genome. The assembly contiguity was implemented decreasing the contig number from 2,604 to 49. The N50 contig size increased from 31,803 to 2,726,972 bp and the completeness of assembly increased from 94.5 to 97.3% according to BUSCO analysis. This work is foundational to allow functional analysis of the infection process in this scarcely known floral pathogen. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Thermal Shift Assay as a Tool to Evaluate the Release of Breakdown Peptides from Cowpea ß-Vignin during Seed Germination.

The present work aimed to characterize the molecular relationships between structure and function of the seed storage protein ß-vignin, the vicilin storage protein of cowpea (Vigna unguiculata, l. Walp) seeds. The molecular characterization of ß-vignin was carried out firstly by assessing its thermal stability, under different conditions of pH and ionic strength, by thermal shift assay (TSA) using SYPRO Orange fluorescent dye. Secondly, its aggregation propensity was evaluated using a combination of chromatographic and electrophoretic techniques. Two forms of ß-vignin were considered: the native form purified from mature quiescent seeds, and a stable breakdown intermediate of 27 kDa produced while seeds germinate. TSA is a useful tool for determining and following over time the structural changes that occur to the protein during germination. The main result was the molecular characterization of the 27 kDa intermediate breakdown polypeptide, which, to the best of our knowledge, has never been described before. ß-vignin seems to retain its trimeric conformation despite the evident degradation of its polypeptides.

Telomere to Telomere Genome Assembly of Fusarium musae F31, Causal Agent of Crown Rot Disease of Banana.

Fusarium musae causes crown rot of banana and it is also associated to clinical fusariosis. A chromosome-level genome assembly of F. musae F31 obtained combining Nanopore long reads and Illumina paired-end reads resulted in 12 chromosomes plus one contig with overall N50 of 4.36 Mb, and is presented together with its mitochondrial genome (58,072 bp). The F31 genome includes telomeric regions for 11 of the 12 chromosomes representing one of the most complete genomes available in the Fusarium fujikuroi species complex. The high-quality assembly of the F31 genome will be a valuable resource for studying the pathogenic interactions occurring between F. musae and banana. Moreover, it represents an important resource for understanding the genome evolution in the F. fujikuroi species complex.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Valorization of Okara by Enzymatic Production of Anti-Fungal Compounds for Plant Protection.

Okara is a soybean transformation agri-food by-product, the massive production of which currently poses severe disposal issues. However, its composition is rich in seed storage proteins, which, once extracted, can represent an interesting source of bioactive peptides. Antimicrobial and antifungal proteins and peptides have been described in plant seeds; thus, okara is a valuable source of compounds, exploitable for integrated pest management. The aim of this work is to describe a rapid and economic procedure to isolate proteins from okara, and to produce an enzymatic proteolyzed product, active against fungal plant pathogens. The procedure allowed the isolation and recovery of about 30% of okara total proteins. Several proteolytic enzymes were screened to identify the proper procedure to produce antifungal compounds. Antifungal activity of the protein digested for 24 h with pancreatin against Fusarium and R. solani mycelial growth and Pseudomonas spp was assessed. A dose-response inhibitory activity was established against fungi belonging to the Fusarium genus. The exploitation of okara to produce antifungal bioactive peptides has the potential to turn this by-product into a paradigmatic example of circular economy, since a field-derived food waste is transformed into a source of valuable compounds to be used in field crops protection.

Searching molecular determinants of sensitivity differences towards four demethylase inhibitors in Fusarium graminearum field strains.

Demethylase inhibitors (DMIs) also referred to as azoles or triazoles are currently the main fungicides used for controlling Fusarium diseases and associated toxins in cereals. DMIs also represent an important class of fungicides used in the medical domain. The level of sensitivity of a set of F. graminearum strains (n = 23), collected over the period 1994-2010 in Luxembourg, Germany, Canada, USA, Italy and Belgium against three DMIs (cyproconazole, propiconazole, tebuconazole) used in agriculture and one DMI used in medicine (tioconazole) was assessed using a microplate test. Median molar EC50 values varied 113-fold among DMIs and on average 11-fold within DMIs with cyproconazole and tebuconazole being the least and the most effective ones, respectively. The EC50 values of the two DMIs registered for use against Fusarium species on cereals (propiconazole and tebuconazole) were significantly correlated (r = 0.597**), while no evidence for cross-resistance was obtained for other fungicide combinations. Haplotypes for CYP51A and CYP51C were defined based on snps determining amino acid variations in the two genes. EC50 values of strains with the CYP51A haplotype A0 and the CYP51C haplotype D1 varied greatly for the agricultural DMIs tebuconazole, propiconazole and cyproconazole, but not for the medical DMI tioconazole. None of the mutations and snps that were previously reported to be associated with resistance towards propiconazole was unambiguously related with resistance to tioconazole, because the mutations and snps were found in strains with low as well as with high EC50 values. Our results show that (1) DMI sensitivity of F. graminearum mycelium has been largely stable between 1994 and 2010, (2) effects of snps on sensitivity towards one DMI detected in one set of strains cannot be extrapolated to other DMIs and sets of strains and (3) F. graminearum strains responded differently to DMIs used in agriculture and to a representative of a medical DMI with no evidence for cross-resistance.

Critical Assessment of Streptomyces spp. Able to Control Toxigenic Fusaria in Cereals: A Literature and Patent Review.

Mycotoxins produced by Fusarium species on cereals represent a major concern for food safety worldwide. Fusarium toxins that are currently under regulation for their content in food include trichothecenes, fumonisins, and zearalenone. Biological control of Fusarium spp. has been widely explored with the aim of limiting disease occurrence, but few efforts have focused so far on limiting toxin accumulation in grains. The bacterial genus Streptomyces is responsible for the production of numerous drug molecules and represents a huge resource for the discovery of new molecules. Streptomyces spp. are also efficient plant colonizers and able to employ different mechanisms of control against toxigenic fungi on cereals. This review describes the outcomes of research using Streptomyces strains and/or their derived molecules to limit toxin production and/or contamination of Fusarium species in cereals. Both the scientific and patent literature were analyzed, starting from the year 2000, and we highlight promising results as well as the current pitfalls and limitations of this approach.

A Threshold-Based Weather Model for Predicting Stripe Rust Infection in Winter Wheat.

Wheat stripe rust (caused by Puccinia striiformis f. sp. tritici) is a major threat in most wheat growing regions worldwide, which potentially causes substantial yield losses when environmental conditions are favorable. Data from 1999 to 2015 for three representative wheat-growing sites in Luxembourg were used to develop a threshold-based weather model for predicting wheat stripe rust. First, the range of favorable weather conditions using a Monte Carlo simulation method based on the Dennis model were characterized. Then, the optimum combined favorable weather variables (air temperature, relative humidity, and rainfall) during the most critical infection period (May-June) was identified and was used to develop the model. Uninterrupted hours with such favorable weather conditions over each dekad (i.e., 10-day period) during May-June were also considered when building the model. Results showed that a combination of relative humidity >92% and 4°C < temperature < 16°C for a minimum of 4 continuous hours, associated with rainfall ≤0.1 mm (with the dekad having these conditions for 5 to 20% of the time), were optimum to the development of a wheat stripe rust epidemic. The model accurately predicted infection events: probabilities of detection were ≥0.90 and false alarm ratios were ≤0.38 on average, and critical success indexes ranged from 0.63 to 1. The method is potentially applicable to studies of other economically important fungal diseases of other crops or in different geographical locations. If weather forecasts are available, the threshold-based weather model can be integrated into an operational warning system to guide fungicide applications.

2D difference gel electrophoresis reference map of a Fusarium graminearum nivalenol producing strain.

Fusarium graminearum is widely studied as a model for toxin production among plant pathogenic fungi. A 2D DIGE reference map for the nivalenol-producing strain 453 was established. Based on a whole protein extract, all reproducible spots were systematically picked and analyzed by MALDI-TOF/TOF, leading to the identification of 1102 protein species. The obtained map contributes to the annotation of the genome by identifying previously nondescribed hypothetical proteins and will serve as a reference for future studies aiming at deciphering F. graminearum biology and chemotype diversity.

Differences between the succinate dehydrogenase sequences of isopyrazam sensitive Zymoseptoria tritici and insensitive Fusarium graminearum strains.

Forty-one Zymoseptoria tritici strains isolated in Luxembourg between 2009 and 2010 were highly sensitive towards the new succinate dehydrogenase inhibitor (SDHI) isopyrazam, with concentrations inhibiting fungal growth by 50% (EC50) ranging from 0.0281 to 4.53µM, whereas 41 Fusarium graminearum strains isolated in Europe and Northern America between 1969 and 2009 were insensitive with the average rate of inhibition converging towards 28% with increasing isopyrazam concentration. Seven isolates of both species covering the range of isopyrazam sensitivities observed in the present study were selected for the sequencing of the subunits B, C and D of the succinate dehydrogenase (sdh) gene. Predicted sdh amino acid sequences of subunits B, C and D were identical among F. graminearum strains. By comparing with fungal strains where resistance towards SDHIs was previously reported, three variations were unique to F. graminearum; B-D130N located in the iron-sulfur cluster [2Fe-2S], B-A275T located in the [3Fe-4S] cluster and an additional S at amino acid position 83-84 of sdhC, probably modifying structurally the ubiquinone binding site and therefore the biological activity of the fungicide. No variation was found among the Z. tritici strains in subunits B and D. Two variations were observed within the subunit C sequences of Z. tritici strains: C-N33T and C-N34T. The difference in EC50 values between Z. tritici strains with the NN and TT configuration was non-significant at P=0.289. Two outliers in the Z. tritici group with significantly higher EC50 values that were not related to mutations in the sdhB, sdhC, or sdhD were detected. The role of isopyrazam for the control of F. graminearum and Z. tritici in Luxembourg is discussed.

Molecular Characterization of Ciborinia camelliae Kohn Shows Intraspecific Variability and Suggests Transcontinental Movement of the Pathogen.

Ciborinia camelliae Kohn is the causal agent of camellia flower blight. The fungus infects only the flowers of camellias. C. camelliae isolates obtained from symptomatic samples, collected in 13 different localities worldwide, were characterized by Multi-Locus Sequence Typing (MLST) using the following: (i) a nuclear ribosomal DNA internal transcribed spacer; (ii) subunit 2 of ß-tubulin (ß-TUB II), (iii) elongation factor 1-α (EF1α); and (iv) glycerol-3-phosphate dehydrogenase (GPDH). The variability of the strains was assessed using a universally primed-polymerase chain reaction (UP-PCR) with six universal primers. Gene sequence comparison showed high similarity among all the European strains and highlighted the diversity of the New Zealand and Chinese representative strains. The profiles obtained by UP-PCR confirmed the significant diversity of extra-European strains and identified subgroups within the European population. The presence of shared genetic profiles obtained from strains isolated in different countries (New Zealand and France) suggests the movement of strains from one location to another, which is probably due to the exchange of infected plant material. Moreover, our study shows the overall high intraspecific variability of C. camelliae, which is likely due to the sexual reproduction of the fungus, suggesting the risk of emergence of new pathotypes adapting to novel camellia varieties.

Two distinct Fusarium graminearum populations colonized European wheat in the past two decades.

Fusarium graminearum is the main causal agent of Fusarium head blight (FHB) disease in wheat in Europe. To reveal population structure and to pinpoint genetic targets of selection we studied genomes of 96 strains of F. graminearum using population genomics. Bayesian and phylogenomic analyses indicated that the F. graminearum emergence in Europe could be linked to two independently evolving populations termed here as East European (EE) and West European (WE) population. The EE strains are primarily prevalent in Eastern Europe, but to a lesser extent also in western and southern areas. In contrast, the WE population appears to be endemic to Western Europe. Both populations evolved in response to population-specific selection forces, resulting in distinct localized adaptations that allowed them to migrate into their environmental niche. The detection of positive selection in genes with protein/zinc ion binding domains, transcription factors and in genes encoding proteins involved in transmembrane transport highlights their important role in driving evolutionary novelty that allow F. graminearum to increase adaptation to the host and/or environment. F. graminearum also maintained distinct sets of accessory genes showing population-specific conservation. Among them, genes involved in host invasion and virulence such as those encoding proteins with high homology to tannase/feruloyl esterase and genes encoding proteins with functions related to oxidation-reduction were mostly found in the WE population. Our findings shed light on genetic features related to microevolutionary divergence of F. graminearum and reveal relevant genes for further functional research aiming at better control of this pathogen.

Impact of Enniatin and Deoxynivalenol Co-Occurrence on Plant, Microbial, Insect, Animal and Human Systems: Current Knowledge and Future Perspectives.

Fusarium mycotoxins commonly contaminate agricultural products resulting in a serious threat to both animal and human health. The co-occurrence of different mycotoxins in the same cereal field is very common, so the risks as well as the functional and ecological effects of mycotoxins cannot always be predicted by focusing only on the effect of the single contaminants. Enniatins (ENNs) are among the most frequently detected emerging mycotoxins, while deoxynivalenol (DON) is probably the most common contaminant of cereal grains worldwide. The purpose of this review is to provide an overview of the simultaneous exposure to these mycotoxins, with emphasis on the combined effects in multiple organisms. Our literature analysis shows that just a few studies on ENN-DON toxicity are available, suggesting the complexity of mycotoxin interactions, which include synergistic, antagonistic, and additive effects. Both ENNs and DON modulate drug efflux transporters, therefore this specific ability deserves to be explored to better understand their complex biological role. Additionally, future studies should investigate the interaction mechanisms of mycotoxin co-occurrence on different model organisms, using concentrations closer to real exposures.

Wheat Seed Coating with Streptomyces sp. Strain DEF39 Spores Protects against Fusarium Head Blight.

Streptomycetes are promising candidates for the biological control of Fusarium Head Blight (FHB) in wheat. Studies involving the use of streptomycetes as biological control agents (BCAs) have been limited to the application when the wheat plant is developed, close to the infection on the spike during flowering. Here, we tested the effects of seed treatment with the Streptomyces sp. DEF39 spores before sowing on FHB symptoms' development. The seed treatment protected the plant from infection by Fusarium graminearum by 49% (p = 0.04). We traced Streptomyces sp. DEF39 in plant organs using strain-specific primers here developed, showing that the streptomycete acts as an endophyte, colonizing the plant tissues up to the spike as well as the roots. This work suggests that it is possible to use a streptomycete as a seed coating BCA, able to partially protect wheat from FHB disease.

Exploring Mitogenomes Diversity of Fusarium musae from Banana Fruits and Human Patients.

Fusarium musae has recently been described as a cross-kingdom pathogen causing post-harvest disease in bananas and systemic and superficial infection in humans. The taxonomic identity of fungal cross-kingdom pathogens is essential for confirming the identification of the species on distant infected hosts. Understanding the level of variability within the species is essential to decipher the population homogeneity infecting human and plant hosts. In order to verify that F. musae strains isolated from fruits and patients are part of a common population and to estimate their overall diversity, we assembled, annotated and explored the diversity of the mitogenomes of 18 F. musae strains obtained from banana fruits and human patients. The mitogenomes showed a high level of similarity among strains with different hosts' origins, with sizes ranging from 56,493 to 59,256 bp. All contained 27 tRNA genes and 14 protein-coding genes, rps3 protein, and small and large ribosomal subunits (rns and rnl). Variations in the number of endonucleases were detected. A comparison of mitochondrial endonucleases distribution with a diverse set of Fusarium mitogenomes allowed us to specifically discriminate F. musae from its sister species F. verticillioides and the other Fusarium species. Despite the diversity in F. musae mitochondria, strains from bananas and strains from human patients group together, indirectly confirming F. musae as a cross-kingdom pathogen.

Whole-genome single nucleotide polymorphism analysis for typing the pandemic pathogen Fusarium graminearum sensu stricto.

Recent improvements in microbiology and molecular epidemiology were largely stimulated by whole- genome sequencing (WGS), which provides an unprecedented resolution in discriminating highly related genetic backgrounds. WGS is becoming the method of choice in epidemiology of fungal diseases, but its application is still in a pioneer stage, mainly due to the limited number of available genomes. Fungal pathogens often belong to complexes composed of numerous cryptic species. Detecting cryptic diversity is fundamental to understand the dynamics and the evolutionary relationships underlying disease outbreaks. In this study, we explore the value of whole-genome SNP analyses in identification of the pandemic pathogen Fusarium graminearum sensu stricto (F.g.). This species is responsible for cereal diseases and negatively impacts grain production worldwide. The fungus belongs to the monophyletic fungal complex referred to as F. graminearum species complex including at least sixteen cryptic species, a few among them may be involved in cereal diseases in certain agricultural areas. We analyzed WGS data from a collection of 99 F.g. strains and 33 strains representing all known cryptic species belonging to the FGSC complex. As a first step, we performed a phylogenomic analysis to reveal species-specific clustering. A RAxML maximum likelihood tree grouped all analyzed strains of F.g. into a single clade, supporting the clustering-based identification approach. Although, phylogenetic reconstructions are essential in detecting cryptic species, a phylogenomic tree does not fulfill the criteria for rapid and cost-effective approach for identification of fungi, due to the time-consuming nature of the analysis. As an alternative, analysis of WGS information by mapping sequence data from individual strains against reference genomes may provide useful markers for the rapid identification of fungi. We provide a robust framework for typing F.g. through the web-based PhaME workflow available at EDGE bioinformatics. The method was validated through multiple comparisons of assembly genomes to F.g. reference strain PH-1. We showed that the difference between intra- and interspecies variability was at least two times higher than intraspecific variation facilitating successful typing of F.g. This is the first study which employs WGS data for typing plant pathogenic fusaria.

Biological characterization and in vitro fungicide screenings of a new causal agent of wheat Fusarium head blight in Tibet, China.

Wheat (Triticum aestivum L.) is an important cereal crop, widely grown throughout the temperate zones, and also suitable for cultivation at higher elevations. Fusarium head blight (FHB) is a highly destructive disease of wheat throughout the globe. In July 2020, serious wheat FHB symptoms were observed in open fields located in Linzhi City, southeast of Tibet, China. The causal agent was identified as Fusarium avenaceum (Fr.) Sacc. by amplification and sequencing of the internal transcribed spacer (ITS) region, translation elongation factor 1-alpha (EF-1α) gene, and RNA polymerase II subunit (RPB-2) gene, as well as by morphological characterization. Koch's postulates were confirmed by a pathogenicity test on healthy spikes, including re-isolation and identification. To our knowledge, this is the first report of F. avenaceum causing FHB on wheat in Tibet, China. Moreover, to determine pathogen characteristics that may be useful for future disease management, the utilization of different carbon and nitrogen resources, temperature, light, and ultraviolet (UV) irradiation on mycelium growth and conidia germination were studied. Soluble starch and peptone were the best carbon, and nitrogen source for the pathogen respectively. The optimal temperatures for the pathogen's mycelium growth and conidia germination were 15-20°C, matching the average temperature during the growing season in Linzhi (Tibet). Meanwhile, alternating 8-h light and 16-h dark was shown to be conducive to mycelia growth, and complete darkness facilitated conidia germination. In addition, UV Irradiation of 48 MJ/cm2, approximately 100 times of the local condition, did not inhibit the germination of conidia. Furthermore, in vitro screening of effective fungicides was conducted. Among the seven tested pesticides, carbendazim showed the best inhibition rate, with an EC50 (concentration for 50% of maximal effect) value of 2.1 mg/L. Propiconazole also showed sufficient inhibitory effects against F. avenaceum, with an EC50 value of 2.6 mg/L. The study provides insights into the newly identified causal agent of wheat FHB in Tibet, China, as well as first pathogen characteristics and promising candidate substances for its management.

The transcription factor FgStuAp influences spore development, pathogenicity, and secondary metabolism in Fusarium graminearum.

Fusarium graminearum is an important plant-pathogenic fungus and the major cause of cereal head blight. Here, we report the functional analysis of FgStuA, the gene for a transcription factor with homology to key developmental regulators in fungi. The deletion mutant was greatly reduced in pathogenicity on wheat heads and in production of secondary metabolites. Spore production was significantly impaired in ΔFgStuA, which did not develop perithecia and sexual ascospores, and lacked conidiophores and phialides, leading to delayed production of aberrant macroconidia. FgStuAp appears to act as a global regulator that may affect many diverse aspects of the life cycle of F. graminearum. Transcriptome analysis shows that thousands of genes are differentially expressed in the mutant during asexual sporulation and infection of wheat heads and under conditions that induce secondary metabolites, including many that could account for the mutant phenotypes observed. The primary regulatory targets of FgStuAp are likely genes involved in cell-cycle control, and the predicted FgStuAp sequence has an APSES domain, with homology to helix-loop-helix proteins involved in cell-cycle regulation. The Aspergillus StuAp response element (A/TCGCGT/ANA/C) was found highly enriched in the promoter sequences of cell-cycle genes, which was upregulated in the ΔFgStuA deletion mutant.

Npc1 is involved in sterol trafficking in the filamentous fungus Fusarium graminearum.

The ortholog of the human gene NPC1 was identified in the plant pathogenic, filamentous fungus Fusarium graminearum by shared amino acid sequence, protein domain structure and cellular localization of the mature fungal protein. The FusariumNpc1 gene shares 34% amino acid sequence identity and 51% similarity to the human gene, has similar domain structure and is constitutively expressed, although up-regulated in ungerminated macroconidia and ascospores. GFP-tagged Npc1p localizes to the fungal vacuolar membrane. Cultures derived from a Δnpc1 mutant strain contain significantly more ergosterol than cultures of the wildtype. Staining with the fluorescent, sterol binding dye filipin, shows that ergosterol accumulates in vacuoles of the Δnpc1 mutant but not the wildtype strain. The Δnpc1 mutant has a temperature dependent reduction in growth and greater sensitivity to the ergosterol synthesis inhibiting fungicide tebuconazole compared with the wildtype strain or the mutant complemented with wildtype Npc1. The mutant also is significantly reduced in pathogenicity to wheat. Our results are consistent with the interpretation that Npc1p is important for normal transport of ergosterol from the vacuole and is essential for proper membrane function under particular environmental conditions.

Grapevine Powdery Mildew: Fungicides for Its Management and Advances in Molecular Detection of Markers Associated with Resistance.

Grapevine powdery mildew is a principal fungal disease of grapevine worldwide. Even though it usually does not cause plant death directly, heavy infections can lead to extensive yield losses, and even low levels of the disease can negatively affect the quality of the wine. Therefore, intensive spraying programs are commonly applied to control the disease, which often leads to the emergence and spread of powdery mildew strains resistant to different fungicides. In this review, we describe major fungicide classes used for grapevine powdery mildew management and the most common single nucleotide mutations in target genes known to confer resistance to different classes of fungicides. We searched the current literature to review the development of novel molecular methods for quick detection and monitoring of resistance to commonly used single-site fungicides against Erysiphe necator. We analyze and compare the developed methods. From our investigation it became evident that this research topic has been strongly neglected and we hope that effective molecular methods will be developed also for resistance monitoring in biotroph pathogens.

Fusarium musae from Diseased Bananas and Human Patients: Susceptibility to Fungicides Used in Clinical and Agricultural Settings.

Fusarium musae belongs to the Fusarium fujikuroi species complex. It causes crown rot disease in banana but also keratitis and skin infections as well as systemic infections in immunocompromised patients. Antifungal treatments in clinical and agricultural settings rely mostly on molecules belonging to the azole class. Given the potential risk of pathogen spread from food to clinical settings, the goal of the work was to define the level of susceptibility to different azoles of a worldwide population of F. musae. Eight fungicides used in agriculture and five antifungals used in clinical settings (4 azoles and amphotericin B) were tested using the CLSI (Clinical and Laboratory Standards Institute) protocol methodology on 19 F. musae strains collected from both infected patients and bananas. The level of susceptibility to the different active molecules was not dependent on the source of isolation with the exception of fenbuconazole and difenoconazole which had a higher efficiency on banana-isolated strains. Minimal inhibitory concentrations (MICs) of the different molecules ranged from 0.12-0.25 mg/L for prochloraz to more than 16 mg/L for tetraconazole and fenbuconazole. Compared to the F. verticillioides, F. musae MICs were higher suggesting the importance of monitoring the potential future spread of this species also in clinical settings.