Virgilio Balmas

A European Database of Fusarium graminearum and F. culmorum Trichothecene Genotypes

Fusarium species, particularly Fusarium graminearum and F. culmorum, are the main cause of trichothecene type B contamination in cereals. Data on the distribution of Fusarium trichothecene genotypes in cereals in Europe are scattered in time and space. Furthermore, a common core set of related variables (sampling method, host cultivar, previous crop, etc.) that would allow more effective analysis of factors influencing the spatial and temporal population distribution, is lacking. Consequently, based on the available data, it is difficult to identify factors influencing chemotype distribution and spread at the European level. Here we describe the results of a collaborative integrated work which aims (1) to characterize the trichothecene genotypes of strains from three Fusarium species, collected over the period 2000–2013 and (2) to enhance the standardization of epidemiological data collection. Information on host plant, country of origin, sampling location, year of sampling and previous crop of 1147 F. graminearum, 479 F. culmorum, and 3 F. cortaderiae strains obtained from 17 European countries was compiled and a map of trichothecene type B genotype distribution was plotted for each species. All information on the strains was collected in a freely accessible and updatable database (www.catalogueeu.luxmcc.lu), which will serve as a starting point for epidemiological analysis of potential spatial and temporal trichothecene genotype shifts in Europe. The analysis of the currently available European dataset showed that in F. graminearum, the predominant genotype was 15-acetyldeoxynivalenol (15-ADON) (82.9%), followed by 3-acetyldeoxynivalenol (3-ADON) (13.6%), and nivalenol (NIV) (3.5%). In F. culmorum, the prevalent genotype was 3-ADON (59.9%), while the NIV genotype accounted for the remaining 40.1%. Both, geographical and temporal patterns of trichothecene genotypes distribution were identified.

Soils of a Mediterranean hot spot of biodiversity and endemism (Sardinia, Tyrrhenian Islands) are inhabited by pan-European, invasive species of Hypocrea/Trichoderma

We have used a Mediterranean hot spot of biodiversity (the Island of Sardinia) to investigate the impact of abiotic factors on the distribution of species of the common soil fungus Trichoderma. To this end, we isolated 482 strains of Hypocrea/Trichoderma from 15 soils comprising undisturbed and disturbed environments (forest, shrub lands and undisturbed or extensively grazed grass steppes respectively). Isolates were identified at the species level by the oligonucleotide BarCode for Hypocrea/Trichoderma (TrichOKEY), sequence similarity analysis (Trichoblast) and phylogenetic inferences. The majority of the isolates were positively identified as pan-European and/or pan-global Hypocrea/Trichoderma species from sections Trichoderma and Pachybasium, comprising H. lixii/T. harzianum, T. gamsii, T. spirale, T. velutinum, T. hamatum, H. koningii/T. koningii, H. virens/T. virens, T. tomentosum, H. semiorbis, H. viridescens/T. viridescens, H. atroviridis/T. atroviride, T. asperellum, H. koningiopsis/T. koningiopsis and Trichoderma sp. Vd2. Only one isolate represented a new, undescribed species belonging to the Harzianum-Catoptron Clade. Internal transcribed spacer sequence analysis revealed only one potentially endemic internal transcribed spacer 1 allele of T. hamatum. All other species exhibited genotypes that were already found in Eurasia or in other continents. Only few cases of correlation of species occurrence with abiotic factors were recorded. The data suggest a strong reduction of native Hypocrea/Trichoderma diversity, which was replaced by extensive invasion of species from Eurasia, Africa and the Pacific Basin.

Phylogeny and evolution of mating-type genes from Pyrenophora teres, the causal agent of barley “net blotch” disease

The main aim of this study was to test the patterns of sequence divergence and haplotype structure at the MAT locus of Pyrenophora teres, the causal agent of barley ‘net blotch’ disease. P. teres is a heterothallic ascomycete that co-occurs in two symptomatological forms, the net form (NF) and the spot form (SF). The mating-type genes MAT1-1-1 and MAT1-2-1 were sequenced from 22 NF isolates (12 MAT1-1-1 and 10 MAT1-2-1 sequences) and 17 SF isolates (10 MAT1-1-1 and seven MAT1-2-1 sequences) collected from Sardinian barley landrace populations and worldwide. On the basis of a parsimony network analysis, the two forms of P. teres are phylogenetically separated. More than 85% of the total nucleotide variation was found between formae speciales. The two forms do not share any polymorphisms. Six diagnostic nucleotide polymorphisms were found in the MAT1-1-1 intron (1) and in the MAT1-1-1 (3) and MAT1-2-1 (2) exons. Three diagnostic non-synonymous mutations were found, one in MAT1-1-1 and two in MAT1-2-1. For comparison with P. teres sequence data, the mating-type genes from Pyrenophora graminea were also isolated and sequenced. Divergence between P. graminea and P. teres is of a similar magnitude to that between NF and SF of P. teres. The MAT genes of P. graminea were closer to those of SF than to NF, with the MAT1-2-1 SF peptide not different from the MAT1-2-1 peptide of P. graminea. Overall, these data suggest long genetic isolation between the two forms of P. teres and that hybridization is rare or absent under field conditions, with each form having some particular niche specialization. This indicates that research on resistance to P. teres should consider the two forms separately, as different species.

Molecular phylogenetic diversity of dermatologic and other human pathogenic fusarial isolates from hospitals in northern and central Italy.

ABSTRACT Fifty-eight fusaria isolated from 50 Italian patients between 2004 and 2007 were subject to multilocus DNA sequence typing to characterize the spectrum of species and circulating sequence types (STs) associated with dermatological infections, especially onychomycoses and paronychia, and other fusarioses in northern and central Italy. Sequence typing revealed that the isolates were nearly evenly divided among the Fusarium solani species complex (FSSC; n = 18), the F. oxysporum species complex (FOSC; n = 20), and the Gibberella (Fusarium) fujikuroi species complex (GFSC; n = 20). The three-locus typing scheme used for members of the FSSC identified 18 novel STs distributed among six phylogenetically distinct species, yielding an index of discrimination of 1.0. Phylogenetic analysis of the FOSC two-locus data set identified nine STs, including four which were novel, and nine isolates of ST 33, the previously described widespread clonal lineage. With the inclusion of eight epidemiologically unrelated ST 33 isolates, the FOSC typing scheme scored a discrimination index of 0.787. The two-locus GFSC typing scheme, which was primarily designed to identify species, received the lowest discrimination index, with a score of 0.492. The GFSC scheme, however, was used to successfully identify 17 isolates as F. verticillioides, 2 as F. sacchari, and 1 as F. guttiforme. This is the first report that F. guttiforme causes a human mycotic infection, which was supported by detailed morphological analysis. In addition, the results of a pathogenicity experiment revealed that the human isolate of F. guttiforme was able to induce fusariosis of pineapple, heretofore its only known host.

Multilocus phylogenetics show high levels of endemic fusaria inhabiting Sardinian soils (Tyrrhenian Islands)

The Mediterranean island of Sardinia is well known for high levels of vascular plant diversity and endemism, but little is known about its microbial diversity. Under the hypothesis that Fusarium species would show similarly high diversity, we estimated variability in Fusarium species composition among 10 sites around the island. Markers previously adopted for multilocus sequence typing (MLST) were used to determine multilocus DNA sequence haplotypes for 263 Fusarium isolates. In addition portions of the translation elongation factor 1-alpha and second largest RNA polymerase subunit genes were sequenced for all isolates. The intergenic spacer (IGS) region of the nuclear ribosomal RNA gene repeat was sequenced for members of the F. oxysporum species complex (FOSC), and a portion of the nuclear ribosomal RNA gene repeat comprising the internal transcribed spacer (ITS) and part of the large nuclear ribosomal RNA subunit was sequenced for members of the F. solani species complex (FSSC). Seventy-three multilocus haplotypes were identified among the 263 isolates typed, of which 48 represented FOSC and FSSC. Thirty-seven of 48 FOSC two-locus and FSSC three-locus haplotypes had not been observed previously. The 38 non-FOSC/FSSC fusaria comprised 25 haplotypes distributed among 10 species, five of which appear to represent novel, phylogenetically distinct species. In general newly discovered haplotypes were restricted to one or a few sites. All FSSC isolates represented new haplotypes in phylogenetic species FSSC 5 and 9, which differ from the phylogenetic species dominant in soils worldwide. No obvious correlations were found between haplotype diversity and geospatial or habitat distribution. Overall these results indicate a high degree of Fusarium genetic diversity on multiple geographic scales within Sardinia. These results contrast with recent work showing that common, cosmopolitan species dominate Sardinia’s Trichoderma biodiversity. All data are available for access and viewing from the FUSARIUM-ID database.

Altered trichothecene biosynthesis in TRI6-silenced transformants of Fusarium culmorum influences the severity of crown and foot rot on durum wheat seedlings

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.

Molecular characterisation of vegetative compatibility groups in Fusarium oxysporum f. sp. radicis-lycopersici and f. sp. lycopersici by random amplification of polymorphic DNA and microsatellite-primed PCR

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.

Identification of potential marker genes for Trichoderma harzianum strains with high antagonistic potential against Rhizoctonia solani by a rapid subtraction hybridization approach.

A rapid subtraction hybridization approach was used to isolate genes differentially expressed during mycelial contact between Trichoderma harzianum (Hypocrea lixii) and Rhizoctonia solani, and could serve as marker genes for selection of superior biocontrol strains. Putatively positive clones were evaluated by transcription analysis during mycelial contact with R. solani versus growth on glucose, and for their differential transcription between two strains with either strong or poor biocontrol capability before, at, and after contact with R. solani. Besides four clones, which had similarity to putative but as yet uncharacterized proteins, they comprised ribosomal proteins, proteins involved in transcriptional switch and regulation, amino acid and energy catabolism, multidrug resistance, and degradation of proteins and glucans. Transcription of three clones was evaluated in five T. harzianum strains under confrontation conditions with R. solani. Two clones—acetyl-xylane esterase AXE1 and endoglucanase Cel61b—showed significant upregulation during in vivo confrontation of a T. harzianum strain that successively demonstrated a very high antagonistic capability towards R. solani, while expression was progressively lower in a series of T. harzianum strains with intermediate to poor antagonistic activity. These clones are promising candidates for use as markers in the screening of improved T. harzianum biocontrol strains.

Development of reliable molecular markers to detect non-pathogenic binucleate Rhizoctonia isolates (AG-G) using PCR

Non-pathogenic binucleate Rhizoctonia species (BNR) belonging to the anastomosis group AG-G are commonly associated with members of the Rhizoctonia solani complex. They provide effective protection to young bean seedlings against root rot caused by R. solani AG-4. Both fungi are morphologically similar and it is difficult to differentiate between them without using laborious conventional techniques. RAPD assays were carried out on a large range of isolates of binucleate Rhizoctonia species to identify markers common to all AG-G isolates. Two fragments of 1368 bp and 882 bp were isolated, cloned and used to probe Southern blots of DNA from: AG-G isolates; isolates from other AGs of binucleate and multinucleate Rhizoctonia species; various heterogeneous pathogens known to infect bean plants; and co-inoculated bean plants with BNR AG-G and R. solani AG-4. The fragments hybridized only to DNA from AG-G isolates. Both fragments were nucleotide sequenced and two pairs of SCAR (sequence characterized amplified region) primers (BR1a F/R and BR1b F/R) were generated for use in PCR. Two fragments of anticipated size were generated following PCR of all isolates of AG-G and not from any range of other fungal species associated with root and leaf diseases of beans. The SCAR primers were also used to detect AG-G isolates in DNA extracted from bean and soil samples co-inoculated with binucleate and multinucleate Rhizoctonia species. The assays were capable of detecting as little as 2.6 pg of fungal DNA in extracts of soil samples. This system offers the potential to determine the presence of AG-G isolates in infected soil and plant samples.

Natural and Natural-like Phenolic Inhibitors of Type B Trichothecene in Vitro Production by the Wheat (Triticum sp.) Pathogen Fusarium culmorum

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.