Genome Sequence of the Biocontrol Agent Coniothyrium minitans Conio (IMI 134523).

Coniothyrium minitans (synonym, Paraphaeosphaeria minitans) is a highly specific mycoparasite of the wide host range crop pathogen Sclerotinia sclerotiorum. The capability of C. minitans to destroy the sclerotia of S. sclerotiorum has been well recognized and it is available as a widely used biocontrol product Contans WG. We present the draft genome sequence of C. minitans Conio (IMI 134523), which has previously been used in extensive studies that formed part of a registration package of the commercial product. This work provides a distinctive resource for further research into the molecular basis of mycoparasitism to harness the biocontrol potential of C. minitans.[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.

Genomics Evolutionary History and Diagnostics of the Alternaria alternata Species Group Including Apple and Asian Pear Pathotypes.

The Alternaria section alternaria (Alternaria alternata species group) represents a diverse group of saprotroph, human allergens, and plant pathogens. Alternaria taxonomy has benefited from recent phylogenetic revision but the basis of differentiation between major phylogenetic clades within the group is not yet understood. Furthermore, genomic resources have been limited for the study of host-specific pathotypes. We report near complete genomes of the apple and Asian pear pathotypes as well as draft assemblies for a further 10 isolates representing Alternaria tenuissima and Alternaria arborescens lineages. These assemblies provide the first insights into differentiation of these taxa as well as allowing the description of effector and non-effector profiles of apple and pear conditionally dispensable chromosomes (CDCs). We define the phylogenetic relationship between the isolates sequenced in this study and a further 23 Alternaria spp. based on available genomes. We determine which of these genomes represent MAT1-1-1 or MAT1-2-1 idiomorphs and designate host-specific pathotypes. We show for the first time that the apple pathotype is polyphyletic, present in both the A. arborescens and A. tenuissima lineages. Furthermore, we profile a wider set of 89 isolates for both mating type idiomorphs and toxin gene markers. Mating-type distribution indicated that gene flow has occurred since the formation of A. tenuissima and A. arborescens lineages. We also developed primers designed to AMT14, a gene from the apple pathotype toxin gene cluster with homologs in all tested pathotypes. These primers allow identification and differentiation of apple, pear, and strawberry pathotypes, providing new tools for pathogen diagnostics.

Genome Sequence of the Mycotoxigenic Crop Pathogen Fusarium proliferatum Strain ITEM 2341 from Date Palm.

Fusarium proliferatum is a widely distributed fungal pathogen associated with more than 26 crop species important in global food security. Its strong mycotoxigenic capability with potential impacts on human and animal health is well recognized. In this work, we report the draft genome sequence of F. proliferatum strain ITEM 2341, originally isolated from date palm, providing a platform for further comparative and functional genomic investigations.

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

BACKGROUND: Many 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. RESULTS: We 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. CONCLUSIONS: This study illustrates the plasticity of Colletotrichum genomes, and shows that major changes in host range are associated with relatively recent changes in gene content.

Discrete lineages within Alternaria alternata species group: Identification using new highly variable loci and support from morphological characters.

The Alternaria alternata species group is ubiquitous in the environment acting as saprotrophs, human allergens, and plant pathogens. Many morphological species have been described within the group and it is unclear whether these represent re-descriptions of the same species or discrete evolutionary taxa. Sequencing of five loci identified three major lineages within the A. alternata species group. These loci included three new phylogenetic loci (TMA22, PGS1, and REV3) identified as highly variable based on publically available genome sequence data for Dothideomycete species. Lineages were identified as A. alternata ssp. arborescens, A. alternata ssp. tenuissima, and A. alternata ssp. gaisen in accordance with the placement of reference isolates. The phylogenetic results were supported by morphological analysis, which differentiated strains in A. alternata ssp. arborescens and A. alternata ssp. tenuissima and also aligned with previous morphological species descriptions for A. arborescens and A. tenuissima. However, phylogenetic analysis placed the morphologically described species A. alternata and A. mali within the A. alternata ssp. tenuissima and did not support them as discrete taxa. As A. alternata are of phytosanitary importance, the molecular loci used in this study offer new opportunities for molecular identification of isolates by national plant protection organizations.

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.

Draft Genome Sequence of Colletotrichum acutatum Sensu Lato (Colletotrichum fioriniae).

In addition to its economic impact, Colletotrichum acutatum sensu lato is an interesting model for molecular investigations due to the diversity of host-determined specialization and reproductive lifestyles within the species complex. The pathogen Colletotrichum fioriniae forms part of this species complex and causes anthracnose in a wide range of crops and wild plants worldwide. Some members of this species have also been reported to be entomopathogenic. Here, we report the draft genome sequence of a heterothallic reference isolate of C. fioriniae (strain PJ7). This sequence provides a range of new resources that serve as a useful platform for further research in the field.

The distinctive population structure of Colletotrichum species associated with olive anthracnose in the Algarve region of Portugal reflects a host-pathogen diversity hot spot.

Anthracnose (Colletotrichum spp.) is an important disease of olive fruits. Diversity and biogeographic relationships of the olive anthracnose pathogens in the Algarve (Portugal) were investigated, along with host association patterns and disease levels during 2004-2007, to test the hypothesis that this region is a host-pathogen diversity hot spot. Diverse Colletotrichum acutatum and Colletotrichum gloeosporioides populations were identified based on rRNA-internal transcribed spacer and partial beta-tubulin 2 gene sequences of 95 isolates. Spatial and temporal variations in the occurrence of the eight genetic entities of the pathogens were linked to olive biogeography. Disease occurrence patterns suggest that C. acutatum populations are more stable pathogens, while C. gloeosporioides populations appear to be more influenced by favourable conditions. Three unique C. acutatum populations were identified, but none of the eight populations were dominant, with the most frequent type representing only 27%. Thus, the population structure of olive anthracnose pathogens in the Algarve is distinct from other parts of Portugal and other world locations, where only one or two genetic entities are dominant. This pattern and level of genetic diversity in a restricted area, where oleaster (wild olive tree), ancient landraces and modern cultivars of olive occur in close proximity, suggests the Algarve as a centre of diversity of the anthracnose pathogens and corroborates recent work suggesting western Mediterranean as an important centre of olive diversity and domestication.

Disruption of the Coniothyrium minitans PIF1 DNA helicase gene impairs growth and capacity for sclerotial mycoparasitism.

A non-mycoparasitic restriction enzyme-mediated DNA integration (REMI) mutant of Coniothyrium minitans (R2427) contains two tandem plasmid copies integrated towards the 3' end of an ORF. The predicted polypeptide (845 aa) exhibits high similarity with DNA-helicase proteins from other filamentous fungi and yeasts that play a role in mitochondrial DNA maintenance and repair. Disruption of the C. minitans PIF1 DNA helicase gene results in altered morphology, reduced growth rates and a concomitant loss in ability to mycoparasitize sclerotia of Sclerotinia sclerotiorum. In infection bioassays, R2427 exhibited sparse mycelial growth on the surface of live sclerotia, but no mycelia were detected inside the sclerotia. Conversely, R2427 readily colonized autoclaved sclerotia. Complementation of the mutant with wild-type PIF1 restored normal mycelial growth and mycoparasitic capability, confirming a functional role in the host-pathogen interaction. The C. minitans PIF1 DNA helicase may maintain mitochondrial stability in response to reactive oxygen species, either produced endogenously within the mycoparasite, or exogenously from the sclerotial host.

Analysis of cDNA transcripts from Coniothyrium minitans reveals a diverse array of genes involved in key processes during sclerotial mycoparasitism.

Coniothyrium minitans colonises and destroys the sclerotia of Sclerotinia sclerotiorum in nature exhibiting ecologically obligate mycoparasitism as its spores remain dormant in soil and only grow actively in the presence of the sclerotia. Molecular mechanisms underlying sclerotial mycoparasitism are poorly defined. We identified 251 unisequences representing genes preferentially expressed by C. minitans during sclerotial mycoparasitism, substantially increasing the molecular knowledge of this commercially important biocontrol agent. Genes associated with signalling and cellular communication, degradation of host cell walls and energy reserves, nutrient utilisation, detoxification and stress response were identified suggesting that C. minitans employs a number of key processes during host colonisation. Several of these genes are novel to fungal-fungal interactions (e.g. PTH11-like GPCR and the ETP gene cluster). Secretin receptor-like GPCR and the TGF-beta signalling system have not yet been characterised in filamentous fungi. This study provides the basis for in-depth gene function analysis in sclerotial mycoparasitism.

A novel Arabidopsis-Colletotrichum pathosystem for the molecular dissection of plant-fungal interactions.

The ability of a Colletotrichum sp., originally isolated from Brassica campestris, to infect Arabidopsis thaliana was examined. Sequence analysis of the internal transcribed spacer (ITS)1, 5.8S RNA gene and ITS2 regions of ribosomal (r)DNA showed the pathogen to be Colletotrichum destructivum. The host range was broad, including many cruciferous plants and some legumes. At 25 degrees C, all A. thaliana accessions tested were susceptible to the Brassica isolates of C. destructivum; however, at 15 degrees C, the accession Ws-2 showed a temperature-dependant resistance, in which single epidermal cells underwent a rapid hypersensitive response. Legume isolates of C. destructivum were unable to infect A. thaliana and induced deposition of callose papillae at sites of attempted penetration. In compatible interactions, C. destructivum showed a two-stage, hemibiotrophic infection process. The initial biotrophic phase was associated with large, intracellular primary hyphae and was confined to one epidermal cell; whereas, in the subsequent necrotrophic phase, narrow secondary hyphae extensively colonized the tissue and conidia were produced in acervuli. An efficient transformation system was established for C. destructivum, using Agrobacterium-mediated transfer of DNA. The ability to genetically manipulate both partners in the interaction is an important advantage, and the Arabidopsis-Colletotrichum pathosystem should provide a valuable new model for dissecting plant-fungal interactions.