Sabrina Sarrocco

Expression of the red fluorescent protein DsRed‐Express in filamentous ascomycete fungi

The recently reported red fluorescent protein DsRed from the reef coral Discosoma sp. represents a new marker that has been codon-optimized for high expression in mammalian cells. To facilitate expression of DsRed in ascomycete fungi, we used the clone pDsRed-Express (Clontech) for constructing a plasmid vector, pPgpd-DsRed, containing the constitutive Aspergillus nidulans glyceraldehyde 3-phosphate (gpd) promoter. This vector was used for co-transformation of Penicillium paxilli, Trichoderma harzianum and Trichoderma virens (syn. Gliocladium virens) together with either pAN7-1 or gGFP, both containing a gene for hygromycin resistance for transformant selection. In addition, gGFP contains a green fluorescent protein (GFP) gene for expression in Ascomycetes. Expression of DsRed-Express was obtained in all three fungi, indicating that DsRed can be used as a highly effective vital marker in Ascomycetes. Dual marked transformants expressed both DsRed-Express and GFP in the same mycelium and were used for non-quantitative comparison of the intensity of the fluorescence using confocal laser scanning microscopy.

Biocontrol of Fusarium head blight: interactions between Trichoderma and mycotoxigenic Fusarium

Fusarium head blight (FHB) is a re-emerging wheat disease that causes extensive damage through direct losses in yield and quality due to the presence of damaged Fusarium kernels and their associated mycotoxins such as the trichothecene deoxynivalenol (DON). Biological control, including the treatment of crop residues with antagonists, in order to reduce pathogen inoculum of FHB, holds considerable promise. Ten Trichoderma isolates, previously selected for their ability to grow in the presence of DON, were preliminarily investigated as potential antagonists against Fusarium culmorum and F. graminearum mycotoxigenic strains in plate confrontation assays. The three Trichoderma isolates showing antibiosis and mycoparasitism were evaluated for their capacity to inhibit DON production by F. graminearum and F. culmorum on two natural substrates. The expression of some chitinase-encoding genes by the two best resulting Trichoderma strains, during interaction with F. culmorum and F. graminearum, was monitored. All investigated genes from chitinase subgroups A, B and the new subgroup C responded to mycoparasitic conditions and were upregulated before contact and/or when in contact with the host. T. gamsii 6085, the best antagonist, was finally used in a competition test against F. culmorum and F. graminearum on natural substrates, using a qPCR approach to evaluate its effect on the pathogens growth and DON production in haulms and rice. This test confirmed the ability of T. gamsii 6085 to antagonize the pathogens on rice. On wheat haulms, an extreme oligotrophic environment, T. gamsii 6085 seemed to develop very poorly and the growth of both the pathogens was unaffected by the presence of the antagonist.

Insights on the Evolution of Mycoparasitism from the Genome of Clonostachys rosea

Clonostachys rosea is a mycoparasitic fungus that can control several important plant diseases. Here, we report on the genome sequencing of C. rosea and a comparative genome analysis, in order to resolve the phylogenetic placement of C. rosea and to study the evolution of mycoparasitism as a fungal lifestyle. The genome of C. rosea is estimated to 58.3 Mb, and contains 14,268 predicted genes. A phylogenomic analysis shows that C. rosea clusters as sister taxon to plant pathogenic Fusarium species, with mycoparasitic/saprotrophic Trichoderma species in an ancestral position. A comparative analysis of gene family evolution reveals several distinct differences between the included mycoparasites. Clonostachys rosea contains significantly more ATP-binding cassette (ABC) transporters, polyketide synthases, cytochrome P450 monooxygenases, pectin lyases, glucose-methanol-choline oxidoreductases, and lytic polysaccharide monooxygenases compared with other fungi in the Hypocreales. Interestingly, the increase of ABC transporter gene number in C. rosea is associated with phylogenetic subgroups B (multidrug resistance proteins) and G (pleiotropic drug resistance transporters), whereas an increase in subgroup C (multidrug resistance-associated proteins) is evident in Trichoderma virens. In contrast with mycoparasitic Trichoderma species, C. rosea contains very few chitinases. Expression of six group B and group G ABC transporter genes was induced in C. rosea during exposure to the Fusarium mycotoxin zearalenone, the fungicide Boscalid or metabolites from the biocontrol bacterium Pseudomonas chlororaphis. The data suggest that tolerance toward secondary metabolites is a prominent feature in the biology of C. rosea.

Use of a non-homologous end-joining-deficient strain (delta-ku70) of the biocontrol fungus Trichoderma virens to investigate the function of the laccase gene lcc1 in sclerotia degradation.

The aim of this study was to apply a generated Δtku70 strain with increased homologous recombination efficiency from the mycoparasitic fungus Trichoderma virens for studying the involvement of laccases in the degradation of sclerotia of plant pathogenic fungi. Inactivation of the non-homologous end-joining pathway has become a successful tool in filamentous fungi to overcome poor targeting efficiencies for genetic engineering. Here, we applied this principle to the biocontrol fungus T. virens, strain I10, by deleting its tku70 gene. This strain was subsequently used to delete the laccase gene lcc1, which we found to be expressed after interaction of T. virens with sclerotia of the plant pathogenic fungi Botrytis cinerea and Sclerotinia sclerotiorum. Lcc1 was strongly upregulated at early colonization of B. cinerea sclerotia and steadily induced during colonization of S. sclerotiorum sclerotia. The Δtku70Δlcc1 mutant was altered in its ability to degrade the sclerotia of B. cinerea and S. sclerotiorum. Interestingly, while the decaying ability for B. cinerea sclerotia was significantly decreased, that to degrade S. sclerotiorum sclerotia was even enhanced, suggesting the operation of different mechanisms in the mycoparasitism of these two types of sclerotia by the laccase LCC1.

Draft Whole-Genome Sequence of the Biocontrol Agent Trichoderma harzianum T6776

ABSTRACT Trichoderma harzianum T6776 is a promising beneficial isolate whose effects consist of growth promotion, positive response of photosynthetic activity, hormonal signaling, and carbon partitioning in tomato, coupled with biocontrol of plant pathogens. Here, we present the first genome assembly of T6776, providing a useful platform for the scientific community.

Gene family expansions and contractions are associated with host range in plant pathogens of the genus Colletotrichum.

BackgroundMany species belonging to the genus Colletotrichum cause anthracnose disease on a wide range of plant species. In addition to their economic impact, the genus Colletotrichum is a useful model for the study of the evolution of host specificity, speciation and reproductive behaviors. Genome projects of Colletotrichum species have already opened a new era for studying the evolution of pathogenesis in fungi.ResultsWe sequenced and annotated the genomes of four strains in the Colletotrichum acutatum species complex (CAsc), a clade of broad host range pathogens within the genus. The four CAsc proteomes and secretomes along with those representing an additional 13 species (six Colletotrichum spp. and seven other Sordariomycetes) were classified into protein families using a variety of tools. Hierarchical clustering of gene family and functional domain assignments, and phylogenetic analyses revealed lineage specific losses of carbohydrate-active enzymes (CAZymes) and proteases encoding genes in Colletotrichum species that have narrow host range as well as duplications of these families in the CAsc. We also found a lineage specific expansion of necrosis and ethylene-inducing peptide 1 (Nep1)-like protein (NLPs) families within the CAsc.ConclusionsThis study illustrates the plasticity of Colletotrichum genomes, and shows that major changes in host range are associated with relatively recent changes in gene content.

Molecular Diversity of Anthracnose Pathogen Populations Associated with UK Strawberry Production Suggests Multiple Introductions of Three Different Colletotrichum Species

Fragaria × ananassa (common name: strawberry) is a globally cultivated hybrid species belonging to Rosaceae family. Colletotrichum acutatum sensu lato (s.l.) is considered to be the second most economically important pathogen worldwide affecting strawberries. A collection of 148 Colletotrichum spp. isolates including 67 C. acutatum s.l. isolates associated with the phytosanitary history of UK strawberry production were used to characterize multi-locus genetic variation of this pathogen in the UK, relative to additional reference isolates that represent a worldwide sampling of the diversity of the fungus. The evidence indicates that three different species C. nymphaeae, C. godetiae and C. fioriniae are associated with strawberry production in the UK, which correspond to previously designated genetic groups A2, A4 and A3, respectively. Among these species, 12 distinct haplotypes were identified suggesting multiple introductions into the country. A subset of isolates was also used to compare aggressiveness in causing disease on strawberry plants and fruits. Isolates belonging to C. nymphaeae, C. godetiae and C. fioriniae representative of the UK anthracnose pathogen populations showed variation in their aggressiveness. Among the three species, C. nymphaeae and C. fioriniae appeared to be more aggressive compared to C. godetiae. This study highlights the genetic and pathogenic heterogeneity of the C. acutatum s.l. populations introduced into the UK linked to strawberry production.

A comparative study of Neogymnomyces virgineus, a new keratinolytic species from dung, and its relationships with the Onygenales

Isolations of onygenalean fungi were made recently from different dung samples from Italy. A striking snow-white species with gymnothecial ascomata, developed in damp chamber on dormouse dung collected in a cave, was subjected to keratinolytic tests and morphological, cultural, and phylogenetic studies. The keratinolytic ability of this species, associated with a Chrysosporium anamorph and a sexual state of appendiculate reticuloperidia and oblate ascospores, allows it to be accomodated in Onygenaceae. White ascomata, blunt or subcapitate peridial appendages, pitted ascospores, and tuberculate conidia suggest it to be a new Neogymnomyces, and this was confirmed by parsimony analyses of LSU and ITS nrDNA sequences. Following recent phylogenetic analyses, the morphological and physiological features of order Onygenales and its families are re–examined and discussed. After the introduction of a new species, Neogymnomyces is reviewed and compared with all other genera in Onygenaceae. The Chrysosporium imperfect state of Neogymnomyces virgineus is described and compared to the anamorph of N. demonbreunii. It is also compared to the atypical Chrysosporium merdarium and to several other Chrysosporium species with echinulate to verrucose–tuberculate conidia, isolated from guano, dung, and nitrogen–rich soils in caves. The onygenalean fungi isolated from any kind of dung are discussed and their facultative coprophily ascribed to variable faecal contents of keratin or other degradable substances. A key to the families and genera of the Onygenales is provided.

Draft Whole-Genome Sequence of Trichoderma gamsii T6085, a Promising Biocontrol Agent of Fusarium Head Blight on Wheat

ABSTRACT Trichoderma gamsii T6085 is a promising beneficial isolate whose effects consist of growth inhibition of the main agents causing Fusarium head blight, reduction of mycotoxin accumulation, competition for wheat debris, and reduction of the disease in both the lab and the field. Here, we present the first genome assembly of a T. gamsii isolate, providing a useful platform for the scientific community.

Rodentomyces, a new hypocrealean genus from Italy

An ascomycete with non stromatic, narrowly ovoidal or subpyriform, pale yellow to luteous perithecia, a Nectria-like centrum, yellowish-brown reticulate ascospores, and a Trichothecium-like anamorph has been isolated from small rodent dung in Italy. Based on morphological, cultural and molecular (nrLSU and ITS sequences) data, a new genus, Rodentomyces, and a new species, R. reticulatus, are therefore introduced to accommodate this fungus. Rodentomyces represents a hypocrealean genus in the Nectriaceae. Within this family, and based on molecular data, R. reticulatus is placed in the group including Calonectria, Leuconectria, Nectricladiella, Neonectria and Nectria mariannaeae. Several coprophilous genera are recorded in Hypocreales almost evenly distributed among the main families of this order.