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Fusarium is one of the most important phytopathogenic fungi of agricultural and human concern. More than 300 species have been described, many of which are pathogenic to important crops, flowers, forest trees, animals, and humans. Species belonging to this genus have been detected in all environments: grassland, desert, littoral, agricultural, alpine zones, aquatic, man-made, and hospitals. Despite the importance of molecular techniques for the identification of a fungal species, morphological criteria still have an important role, including for Fusarium species, for which morphological identification of species requires adequate training and experience. In this paper, we present FusaHelp, a computer-based, user-friendly tool for the morphological identification of common Fusarium species, based on the wide experience of the authors who have devoted most of their scientific careers to the identification and characterization of these species. The web-location of FusaHelp (https://www.fusahelp.com) will greatly facilitate morphological identification and is intended to provide support for all those people who work with this important genus and need a quick clue on the identification, even incomplete, of the Fusarium species that they are working with. more...

Monosporascus root rot and vine decline of melon (MRRVD) is a destructive disease complex mainly occurring in semiarid cultivation areas. In the last decade, in the melon producing area of Sardinia (Italy), yield reductions up to 100% were recorded due to the occurrence of MRRVD. The present study aimed to undertake a two-year survey of fungal pathogens associated with MRRVD grown as a monocrop in two locations in Central Sardinia, Sinis, and Sassu, and to investigate the possible role of soil fungal diversity in the disease development. Melon plants were affected by similar symptoms but colonized by a set of soil-borne fungal pathogens different between surveyed sites and cropping seasons, including Plectosphaerella melonis, P. cucumerina, Fusarium solani, Macrophomina phaseolina and Monosporascus cannonballus. Olpidium bornovanus and O. virulentus were isolated using bait plants and detected by NGS analysis. Bait plants had a general decrease in biomass and yellowing of foliage. The presence of most of the isolated pathogens was also confirmed by the NGS analysis of the soil microbiome. Our results confirm that among the fungal complex implicated in the occurrence of RRVD in Sardinia, O. bornovanus along with O. virulentus likely assume a key role in the development of the disease alone and possibly in association with other pathogens. Differences in the pathogenic fungal spectrum here recorded in symptomatic roots may be associated with agricultural practices, soil physicochemical characteristics, and fungal community composition and function profile. more...

International audience; Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. Previously (Geiser et al. 2013; Phytopathology 103:400-408. 2013), the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani Species Complex (FSSC). Subsequently, this concept was challenged by one research group (Lombard et al. 2015 Studies in Mycology 80: 189-245) who proposed dividing Fusarium into seven genera, including the FSSC as the genus Neocosmospora, with subsequent justification based on claims that the Geiser et al. (2013) concept of Fusarium is polyphyletic (Sandoval-Denis et al. 2018; Persoonia 41:109-129). Here we test this claim, and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species recently described as Neocosmospora were recombined in Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural and practical taxonomic option available. more...

This study aimed to explore the incidence of Fusarium fungi and their mycotoxins in marketed feed in Qatar. Samples of mixed-grain-cereal, maize, wheat, millet, barley, oat and soybean were tested for the presence of zearalenone (ZEN), fumonisins, T2/HT2 and deoxynevalenol (DON)mycotoxins. PCR-based identification of the Fusarium isolates was followed by investigation into genes involved in trichothecenes (tri gene cluster), fumonisins (fum gene cluster)and ZEN (pks gene)biosynthesis. In-vitro mycotoxin production potential of the isolates was compared with their molecular profiles. Mixed-grain cereals showed highest contamination with Fusarium, followed by maize and wheat. The incidence of ZEN was highest in mixed grain samples (87.5%)followed by maize (50%), millet (42.85%)and wheat (40%). In case of DON; 62.5%, 30%, 40%, 14.28%, 50% and 66.67% of the tested mixed cereal grain, maize, wheat, millet, barley and soybean samples were positive. Based on PCR results, F. verticillioides showed highest frequency distribution (34%), followed by F. graminearum (16%), F. oxysporum (15%), F. proliferatum (13%), F. culmorum (8%), F. solani (7%), F. subglutinans (4%)and F. avenaceum (3%)in the feed cereals. All the isolates of F. verticillioides presented both fum1 and fum13 genes, and produced fumonisin mycotoxins in-vitro. All the isolates of F. graminearum and F. culmorum had the tri5 and tri6 genes and resulted in DON accumulation in rice media. In addition to DON, all the F. culmorum isolates produced ZEN in artificially contaminated media, in line with their molecular profile of having tri5, tri6 and pks13 genes. The findings of present study confirmed the occurrence of toxigenic Fusarium and their mycotoxins in animal feed, all below the maximum limit set by EU. Further, the in-vitro mycotoxin production potential of the isolates was in agreement with their tri, fum and pks genetic profiles. - 2019 Elsevier Ltd This publication was made possible by the NPRP Grant '8-392-4-003 ' from the Qatar National Research Fund (a member of the Qatar Foundation ). The statements made herein are solely the responsibility of the authors. Appendix A Scopus more...

The use of yeast-derived volatile organic compounds (VOCs) represents a promising strategy for the biological control of various plant pathogens, including mycotoxin-producing fungi. Previous studies demonstrated the efficacy of the low-fermenting yeast Candida intermedia isolate 253 in reducing growth, sporulation, and ochratoxin A biosynthesis by Aspergillus carbonarius MPVA566. This study aimed to investigate whether the inhibitory effect of the yeast volatilome is solely attributable to 2-phenylethanol, its major component, or if a synergistic effect of all volatilome components is required to achieve an effective control of the fungal growth and metabolism. Microbiological methods, HPLC measurements and a UPLC-MS/MS approach were used to investigate the metabolic profile of A. carbonarius MPVA566 at different growing conditions: standard incubation (control), exposed to C. intermedia 253 volatilome, and incubation in the presence of 2-phenylethanol. Both yeast volatilome and 2-phenylethanol succeeded in the macroscopic inhibition of the radial mycelial growth, along with a significant reduction of ochratoxin A production. Functional classification of the fungal proteome identified in the diverse growing conditions revealed a different impact of both yeast VOCs and 2-phenylethanol exposure on the fungal proteome. Yeast VOCs target an array of metabolic routes of fungal system biology, including a marked reduction in protein biosynthesis, proliferative activity, mitochondrial metabolism, and particularly in detoxification of toxic substances. Exposure to 2-phenylethanol only partially mimicked the metabolic effects observed by the whole yeast volatilome, with protein biosynthesis and proliferative activity being reduced when compared with the control samples, but still far from the VOCs-exposed condition. This study represents the first investigation on the effects of yeast-derived volatilome and 2-phenylethanol on the metabolism of a mycotoxigenic fungus by means of proteomics analysis. Chemical compounds studied or used in this article: 2-Phenylethanol (PubChem CID: 6054); ochratoxin-A (PubChem CID: 442530); sodium dodecyl sulfate (PubChem CID: 3423265); dithiothreitol (PubChem CID: 446094); phenylmethylsulfonyl fluoride (PubChem CID: 4784); iodoacetamide (PubChem CID: 3727); ammonium bicarbonate (PubChem CID: 14013); acetic acid (PubChem CID: 176); and acetonitrile (PubChem CID: 6342). - 2019 The Authors This publication was made possible by NPRP grant # 8-392-4-003 from the Qatar National Research Fund (a member of Qatar Foundation). The findings achieved herein are solely the responsibility of the authors. Scopus more...

Environmental conditions in Sardinia (Tyrrhenian Islands) are conducive to fusarium root rot (FRR) and fusarium head blight (FHB). A monitoring survey on wheat was carried out from 2001 to 2013, investigating relations among these diseases and their causal agents. FHB was more frequently encountered in the most recent years while FRR was constantly present throughout the monitored period. By assessing the population composition of the causal agents as well as their genetic chemotypes and EF-1α polymorphisms, the study examined whether the two diseases could be differentially associated to a species or a population. Fusarium culmorum chemotypes caused both diseases and were detected at different abundances (88% 3-ADON, 12% NIV). Fusarium graminearum (15-ADON genetic chemotype) appeared only recently (2013) and in few areas as the causal agent of FHB. In F. culmorum, two haplotypes were identified based on an SNP mutation located 34 bp after the first exon of the EF-1α partial sequence (60% adenine, 40% thymine); the two populations did not segregate with the chemotype but the A-haplotype was significantly associated with FRR in the Sardinian data set (P = 0·001), suggesting a possible fitness advantage of the A-haplotype in the establishment of FRR that was neither dependent on the sampling location nor the sampling year. The SNP determining the Sardinian haplotype is distributed worldwide. The question whether the A-haplotype segregates with characters facilitating FRR establishment will require further validation on a specifically sampled international data set. more...

Fusarium culmorum, a fungal pathogen of small grain cereals, produces 4-deoxynivalenol and its acetylated derivatives that may cause toxicoses on humans or animals consuming contaminated food or feed. Natural and natural-like compounds belonging to phenol and hydroxylated biphenyl structural classes were tested in vitro to determine their activity on vegetative growth and trichothecene biosynthesis by F. culmorum. Most of the compounds tested at 1.5 or 1.0 mM reduced 3-acetyl-4-deoxynivalenol production by over 70% compared to the control, without affecting fungal growth significantly. Furthermore, several compounds retained their ability to inhibit toxin in vitro production at the lowest concentrations of 0.5 and 0.25 mM. Magnolol 27 showed fungicidal activity even at 0.1 mM. No linear correlation was observed between antioxidant properties of the compounds and their ability to inhibit fungal growth and mycotoxigenic capacity. A guaiacyl unit in the structure may play a key role in trichothecene inhibition. © 2014 American Chemical Society. more...

Fourteen spice samples were collected from local markets in Doha, Qatar, during 2012, and were surveyed for the presence of potentially harmful mycoflora and for contamination with aflatoxins B1, B2, G1, and G2 by high-performance liquid chromatography (HPLC). Among the tested spice samples, chili powder showed the highest presence of fungal propagules, while ginger, curry and garlic samples did not present any fungal contamination. A total of 120 isolates, mostly belonging to Aspergillus and Penicillium genera, were collected and 33 representative species were identified by amplification and sequencing of the internal transcribed spacer (ITS) region. Aspergillus flavus, Aspergillus nomius and Aspergillus niger were the most dominant. Thirty-seven Aspergillus strains were screened for their potential to produce aflatoxins using biochemical and molecular tools: only 9 A. flavus strains showed both fluorescence and amplification with all the three primers targeting aflP, aflM and aflR genes. Aflatoxins were detected in five spices (black pepper, chili, tandoori masala. turmeric and garam masala), and with the exception of garam masala, the tested samples of turmeric, black pepper, tandoori masala and chili powder exceeded B1 and/or total aflatoxin maximum levels. Our results demonstrate the potential for mycotoxin biosynthesis by fungi contaminating imported spice products. NPRP grant # NPRP 4-259-2-083 from the Qatar National Research Fund (a member of Qatar Foundation) more...

An RNA silencing construct was used to alter mycotoxin production in the plant pathogenic fungus Fusarium culmorum , the incitant of crown and foot rot on wheat. The transformation of a wild-type strain and its nitrate reductase-deficient mutant with inverted repeat transgenes (IRTs) containing sequences corresponding to the trichothecene regulatory gene TRI6 was achieved using hygromycin B resistance as a selectable marker. Southern analysis revealed a variety of integration patterns of the TRI6 IRT. One transformant underwent homologous recombination with deletion of the endogenous TRI6 gene, whereas, in another transformant, the TRI6 IRT was not integrated into the genome. The TRI6 IRT did not alter the physiological characteristics, such as spore production, pigmentation or growth rate, on solid media. In most transformants, a high TRI6 amplification signal was detected by quantitative reverse transcriptionpolymerase chain reaction, corresponding to a TRI6 -hybridizing smear of degraded fragments by Northern analysis, whereas TRI5 expression decreased compared with the respective nontransformed strain. Four transformants showed increased TRI5 expression, which was correlated with a dramatic (up to 28-fold) augmentation of deoxynivalenol production. Pathogenicity assays on durum wheat seedlings confirmed that impairment of deoxynivalenol production in the TRI6 IRT transformants correlated with a loss of virulence, with decreased disease indices ranging from 40% to 80% in nine silenced strains, whereas the overproducing transformants displayed higher virulence compared with the wild-type. more...

Endo-polygalacturonase (PG) may be a critical virulence factor secreted by several fungi upon plant invasion. The single-copy gene encoding PG in Fusarium verticillioides and in eight other species of the Gibberella fujikuroi complex (F. sacchari, F. fujikuroi, F. proliferatum, F. subglutinans, F. thapsinum, F. nygamai, F. circinatum, and F. anthophilum) was functionally analyzed in this paper. Both the nucleotide and amino acid sequences were highly similar among the 12 strains of F. verticillioides analyzed, as well as among those from the G. fujikuroi complex. The PGs were not inhibited by the polygalacturonase-inhibiting proteins (PGIPs) from the monocot asparagus and leek plants, but were inhibited to variable extents by bean PGIP. PGs from F. verticillioides, F. nygamai and one strain of F. proliferatum were barely inhibited. Residue 97 within PG was demonstrated to contribute to the different levels of inhibition. Together these findings provide new insights into the structural and functional relationships between the PG from the species of the G. fujikuroi complex and the plant PGIP. more...

We examined the local adaptation patterns in a system comprising several interconnected heterogeneous plant populations from which populations of two phylogenetically closely related pathogens were also sampled. The host is Hordeum vulgare (cultivated barley); the pathogens are Pyrenophora teres f. teres (net form) and Pyrenophora teres f. maculata (spot form), the causal agents of barley net blotch. We integrated two approaches, the comparison between the population structures of the host and the pathogens and a cross-inoculation test. We demonstrated that two closely related pathogens with very similar niche specialisation and life-styles can give rise to different co-evolutionary outcomes on the same host. Indeed, we detected local adaptation for the net form of the pathogen but not for the spot form. We also provided evidence that an a-priori well-known resistance quantitative-trait-locus on barley chromosome 6H is involved in the co-evolutionary ‘arms race’ between the plant and the net-form pathogen. Moreover, data suggested latitudinal clines of host resistance and that different ecological conditions can result in differential selective pressures at different sites. Our data are of interest for on-farm conservation of plant genetic resources, as also in establishing efficient breeding programs and strategies for deployment of resistance genes of P. teres. more...

Fusarium semitectum is a widespread species often isolated from plants with complex disease and also known to be toxigenic. Twenty-seven isolates of F. semitectum obtained from wilted plants of Medicago sativa grown in northern Italy were analysed by Amplified Fragment Length Polymorphisms (AFLP). The isolates were divided in two distinct clusters, coded A and B. Investigations were carried out on morphology, pathogenicity on alfalfa seedlings and toxin production of 18 isolates representative of the two AFLP clusters. Isolates from cluster A showed fast growth and pale peach colour of the colonies on potato-dextrose-agar, whereas those from cluster B showed slow growth and orange pigmentation, changing to brown with age. Length of 5-septate macro-conidia was higher in isolates from cluster A and the frequency of classes of conidia septation was statistically different between the two clusters. All isolates from cluster A were pathogenic to alfalfa, whereas those from cluster B were mostly non-pathogenic. The mycotoxigenic profiles were distinct between the two clusters and similar within the same cluster: isolates from cluster A produced zearalenone, alone or together with diacetoxyscirpenol, whereas isolates from cluster B produced deoxyfusapyrone. The results showed that there is a good correspondence between the AFLP clustering and the distinction of F. semitectum isolates from alfalfa into two groups based on morphology, pathogenicity and toxin-production. more...

Pyrenophora teres f. sp. teres mating-type genes (MAT-1: 1190 bp; MAT-2: 1055 bp) have been identified. Their predicted proteins, measuring 379 and 333 amino acids, respectively, are similar to those of other Pleosporales, such as Pleospora sp., Cochliobolus sp., Alternaria alternata, Leptosphaeria maculans, and Phaeosphaeria nodorum. The structure of the MAT locus is discussed in comparison with those of other fungi. A mating-type PCR assay has also been developed; with this assay we have analyzed 150 isolates that were collected from 6 Sardinian barley landrace populations. Of these, 68 were P. teres f. sp. teres (net form; NF) and 82 were P. teres f. sp. maculata (spot form; SF). Within each mating type, the NF and SF amplification products were of the same length and were highly similar in sequence. The 2 mating types were present in both the NF and the SF populations at the field level, indicating that they have all maintained the potential for sexual reproduction. Despite the 2 forms being sympatric in 5 fields, no intermediate isolates were detected with amplified fragment length polymorphism (AFLP) analysis. These results suggest that the 2 forms are genetically isolated under the field conditions. In all of the samples of P. teres, the ratio of the 2 mating types was consistently in accord with the 1:1 null hypothesis. This ratio is expected when segregation distortion and clonal selection among mating types are absent or asexual reproduction is rare. Overall, sexual reproduction appears to be the major process that equalizes the frequencies of the 2 mating types within populations.Key words: Pyrenophora teres, mating-types, AFLPs, sexual reproduction, selection, barley. more...

Thirty six isolates of Phoma tracheiphila from Italy, the causal agent of the “mal secco” disease on Citrus species, were characterised by different molecular tools in comparison with representative isolates of other phytopathogenic Phoma species. These included analysis of the distribution of RAPD and microsatellite markers and sequencing of the internal transcribed spacer (ITS) region of the nuclear rRNA genes. The results obtained with 12 RAPD primers (92 markers) and 7 microsatellite primers (56 markers) suggest that Italian isolates of P. tracheiphila are genetically homogeneous, leading to identical patterns upon amplification with all the tested primers. Accordingly, ITSI-5.8S-ITS2 sequences were highly conserved (98–100% identity along a 544-characters alignment) among all the isolates of P. tracheiphila. A neighbor-joining analysis of ITS sequences of P. tracheiphila in comparison with those of other Phoma species, as well as with alignable sequences from anamorphic and teleomorphic taxa retrieved in BLAST searches, revealed a close relationship between P. tracheiphila and Leptosphaeria congesta. A pair of P. tracheiphila-specific primers was designed on the consensus sequence (555 residues) obtained from the alignment of the newly generated P. tracheiphila ITS sequences. A PCR-based specific assay coupled to electrophoretic separation of amplicons made it possible to detect P. tracheiphila in naturally infected Citrus wood tissue collected from both symptomatic and symptomless plants. The limit of detection was 10 pg of genomic DNA and 5 fg of the ITS target sequence. more...

Random amplification of polymorphic DNA (RAPD-PCR) analysis was conducted on 48 isolates of Fusarium oxysporum f. sp. radicis-lycopersici (F.o.r.l.) from different geographic regions, representing all known vegetative compatibility groups (VCGs) except VCG 0097 and VCG 0099 and on eight isolates of F.oxysporum f. sp. lycopersici (F.o.l.), representing VCGs 0030, 0031, 0032 and 0033. Upon UPGMA (unweighted pair-group method with arithmetic averages) analysis of 86 RAPD-PCR markers generated by 16 informative primers and 44 markers obtained with eight microsatellite primers, a close relatedness was evident for F.o.r.l. isolates in VCGs 0090, 0092, 0096, and, to a lesser extent, for those in VCG 0093. Representatives of VCG 0091 formed a distinct group, while F.o.r.l. isolates in VCGs 0094 and 0098 were not distinguishable by the tested markers, most of which were also shared by F.o.l. isolates belonging to VCGs 0031 and 0033. F.o.l. isolates in VCGs 0030 and 0032 shared most of the molecular markers. The correlation between RAPD-PCR and microsatellite genetic distance was highly significant (R2 = 0.77; P by Mantel test < 0.001). The molecular variability observed in both formae speciales is discussed in relation to the development of F.o.r.l.- and F.o.l.-specific diagnostic tools. more...

The inverse PCR technique was applied to clone genomic DNA flanking insertion sites of sequences homologous to the transposable element Fot1 in the genome of a new pathogenic isolate of Fusarium oxysporum obtained from wilted Argyranthemum frutescens (Paris daisy). Based on the genomic flanking regions, a primer was designed which when paired to a second primer matching the Fot1 sequence allowed detection of this pathogen by PCR. The primer pair Mg5/Mg6 could specifically identify nine tested isolates of F. oxysporum from A. frutescens, when fungal genomic DNA was used as template. Moreover, the primer pair Mg5/Mg6 allowed successful detection of the pathogen in stem and root tissue from asymptomatic plants that were artificially inoculated with a representative isolate of F. oxysporum from A. frutescens. more...

An RNA silencing construct was used to alter mycotoxin production in the plant pathogenic fungus Fusarium culmorum, the incitant of crown and foot rot on wheat. The transformation of a wild‐type strain and its nitrate reductase‐deficient mutant with inverted repeat transgenes (IRTs) containing sequences corresponding to the trichothecene regulatory gene TRI6 was achieved using hygromycin B resistance as a selectable marker. Southern analysis revealed a variety of integration patterns of the TRI6 IRT. One transformant underwent homologous recombination with deletion of the endogenous TRI6 gene, whereas, in another transformant, the TRI6 IRT was not integrated into the genome. The TRI6 IRT did not alter the physiological characteristics, such as spore production, pigmentation or growth rate, on solid media. In most transformants, a high TRI6 amplification signal was detected by quantitative reverse transcription‐polymerase chain reaction, corresponding to a TRI6‐hybridizing smear of degraded fragments by Northern analysis, whereas TRI5 expression decreased compared with the respective nontransformed strain. Four transformants showed increased TRI5 expression, which was correlated with a dramatic (up to 28‐fold) augmentation of deoxynivalenol production. Pathogenicity assays on durum wheat seedlings confirmed that impairment of deoxynivalenol production in the TRI6 IRT transformants correlated with a loss of virulence, with decreased disease indices ranging from 40% to 80% in nine silenced strains, whereas the overproducing transformants displayed higher virulence compared with the wild‐type. more...

The comparative pathogenicity of Fusarium graminearum Group 1 and Fusarium crookwellense to the durum wheat cv. Yallaroi was studied in the greenhouse. F. graminearum Group 1 was shown to cause more severe above ground symptoms of crow rot and a higher incidence of infection than F. crookwellense at all the sampling dates. F. crookwellense did, however, cause some root and crow necrosis. more...