Geosmithia-Ophiostoma: a New Fungus-Fungus Association.

In Europe as in North America, elms are devastated by Dutch elm disease (DED), caused by the alien ascomycete Ophiostoma novo-ulmi. Pathogen dispersal and transmission are ensured by local species of bark beetles, which established a novel association with the fungus. Elm bark beetles also transport the Geosmithia fungi genus that is found in scolytids' galleries colonized by O. novo-ulmi. Widespread horizontal gene transfer between O. novo-ulmi and Geosmithia was recently observed. In order to define the relation between these two fungi in the DED pathosystem, O. novo-ulmi and Geosmithia species from elm, including a GFP-tagged strain, were grown in dual culture and mycelial interactions were observed by light and fluorescence microscopy. Growth and sporulation of O. novo-ulmi in the absence or presence of Geosmithia were compared. The impact of Geosmithia on DED severity was tested in vivo by co-inoculating Geosmithia and O. novo-ulmi in elms. A close and stable relation was observed between the two fungi, which may be classified as mycoparasitism by Geosmithia on O. novo-ulmi. These results prove the existence of a new component in the complex of organisms involved in DED, which might be capable of reducing the disease impact.

Simultaneous induction of jasmonic acid and disease-responsive genes signifies tolerance of American elm to Dutch elm disease.

Dutch elm disease (DED), caused by three fungal species in the genus Ophiostoma, is the most devastating disease of both native European and North American elm trees. Although many tolerant cultivars have been identified and released, the tolerance mechanisms are not well understood and true resistance has not yet been achieved. Here we show that the expression of disease-responsive genes in reactions leading to tolerance or susceptibility is significantly differentiated within the first 144 hours post-inoculation (hpi). Analysis of the levels of endogenous plant defense molecules such as jasmonic acid (JA) and salicylic acid (SA) in tolerant and susceptible American elm saplings suggested SA and methyl-jasmonate as potential defense response elicitors, which was further confirmed by field observations. However, the tolerant phenotype can be best characterized by a concurrent induction of JA and disease-responsive genes at 96 hpi. Molecular investigations indicated that the expression of fungal genes (i.e. cerato ulmin) was also modulated by endogenous SA and JA and this response was unique among aggressive and non-aggressive fungal strains. The present study not only provides better understanding of tolerance mechanisms to DED, but also represents a first, verified template for examining simultaneous transcriptomic changes during American elm-fungus interactions.

Novel ophiostomatalean fungi from galleries of Cyrtogenius africus (Scolytinae) infesting dying Euphorbia ingens.

Euphorbia ingens trees have been dying in large numbers in the Limpopo Province of South Africa for approximately 15 years. The ambrosia beetle Cyrtogenius africus is often found infesting diseased and dying trees. The aim of this study was to identify the ophiostomatoid fungi occurring in the galleries of C. africus. Logs infested with this beetle were collected from the KwaZulu-Natal, Limpopo, Mpumalanga, and North West Provinces of South Africa. Fungi belonging to the Ophiostomatales were identified based on morphology and comparison of sequence data for the ß-tubulin, ITS1-5.8S-ITS2 and LSU gene regions. A novel species of Ophiostoma and a novel genus in the Ophiostomatales were identified. Inoculation studies with these fungi produced lesions in the branches of healthy E. ingens trees.

The Role of Pathogen-Secreted Proteins in Fungal Vascular Wilt Diseases.

A limited number of fungi can cause wilting disease in plants through colonization of the vascular system, the most well-known being Verticillium dahliae and Fusarium oxysporum. Like all pathogenic microorganisms, vascular wilt fungi secrete proteins during host colonization. Whole-genome sequencing and proteomics screens have identified many of these proteins, including small, usually cysteine-rich proteins, necrosis-inducing proteins and enzymes. Gene deletion experiments have provided evidence that some of these proteins are required for pathogenicity, while the role of other secreted proteins remains enigmatic. On the other hand, the plant immune system can recognize some secreted proteins or their actions, resulting in disease resistance. We give an overview of proteins currently known to be secreted by vascular wilt fungi and discuss their role in pathogenicity and plant immunity.

RNAseq Analysis Highlights Specific Transcriptome Signatures of Yeast and Mycelial Growth Phases in the Dutch Elm Disease Fungus Ophiostoma novo-ulmi.

Fungal dimorphism is a complex trait and our understanding of the ability of fungi to display different growth morphologies is limited to a small number of model species. Here we study a highly aggressive dimorphic fungus, the ascomycete Ophiostoma novo-ulmi, which is a model in plant pathology and the causal agent of Dutch elm disease. The two growth phases that this fungus displays, i.e., a yeast phase and mycelial phase, are thought to be involved in key steps of disease development. We used RNAseq to investigate the genome-wide gene expression profiles that are associated with yeast and mycelial growth phases in vitro. Our results show a clear molecular distinction between yeast and mycelial phase gene expression profiles. Almost 12% of the gene content is differentially expressed between the two phases, which reveals specific functions related to each growth phase. We compared O. novo-ulmi transcriptome profiles with those of two model dimorphic fungi, Candida albicans and Histoplasma capsulatum. Few orthologs showed similar expression regulation between the two growth phases, which suggests that, globally, the genes associated with these two life forms are poorly conserved. This poor conservation underscores the importance of developing specific tools for emerging model species that are distantly related to the classical ones. Taken together, our results provide insights into transcriptome regulation and molecular specificity in O. novo-ulmi and offer a new perspective for understanding fungal dimorphism.

Functional annotation of the Ophiostoma novo-ulmi genome: insights into the phytopathogenicity of the fungal agent of Dutch elm disease.

The ascomycete fungus Ophiostoma novo-ulmi is responsible for the pandemic of Dutch elm disease that has been ravaging Europe and North America for 50 years. We proceeded to annotate the genome of the O. novo-ulmi strain H327 that was sequenced in 2012. The 31.784-Mb nuclear genome (50.1% GC) is organized into 8 chromosomes containing a total of 8,640 protein-coding genes that we validated with RNA sequencing analysis. Approximately 53% of these genes have their closest match to Grosmannia clavigera kw1407, followed by 36% in other close Sordariomycetes, 5% in other Pezizomycotina, and surprisingly few (5%) orphans. A relatively small portion (∼3.4%) of the genome is occupied by repeat sequences; however, the mechanism of repeat-induced point mutation appears active in this genome. Approximately 76% of the proteins could be assigned functions using Gene Ontology analysis; we identified 311 carbohydrate-active enzymes, 48 cytochrome P450s, and 1,731 proteins potentially involved in pathogen-host interaction, along with 7 clusters of fungal secondary metabolites. Complementary mating-type locus sequencing, mating tests, and culturing in the presence of elm terpenes were conducted. Our analysis identified a specific genetic arsenal impacting the sexual and vegetative growth, phytopathogenicity, and signaling/plant-defense-degradation relationship between O. novo-ulmi and its elm host and insect vectors.

Establishment of invasive and non-invasive reporter systems to investigate American elm-Ophiostoma novo-ulmi interactions.

Dutch elm disease (DED), caused by ascomycete fungi in the Ophiostoma genus, is the most devastating disease of American elm (Ulmus americana) trees. Cerato ulmin (CU), a hydrophobin secreted by the fungus, has been implicated in the development of DED, but its role in fungal pathogenicity and virulence remains uncertain and controversial. Here, we describe reporter systems based on the CU promoter and three reporter proteins (GFP, GUS and LUC), developed as research tools for quantitative and qualitative studies of DED in vitro, in vivo and in planta. A strain of the aggressive species Ophiostoma novo-ulmi was transformed with the reporter constructs using Agrobacterium-mediated transformation and the fungal transformants, namely M75-GFP, M75-GUS and M75-LUC, were examined for mitotic stability after repeated subcultures. The intensity of GFP fluorescence was strong in M75-GFP spores and hyphae, allowing microscopic investigations of spore structure, fungal morphogenesis and fungal development. The interaction of M75-GFP and U. americana callus cells was explored with scanning laser confocal microscopy facilitating qualitative studies on fungal strategies for the invasion and penetration of elm cells. M75-GUS was generated to provide an invasive, yet quantitative approach to study fungal-plant interactions in vitro and in planta. The generation of M75-LUC transformants was aimed at providing a non-destructive quantitative approach to study the role of CU in vivo. The sensitivity, low background signal and linearity of LUC assays all predict a very reliable approach to investigate and re-test previously claimed roles of this CU in fungal pathogenicity. These reporter systems provide new tools to investigate plant-pathogen interactions in this complex pathosystem and may aid in better understanding the development of DED.

Identification of transcripts up-regulated in asexual and sexual fruiting bodies of the Dutch elm disease pathogen Ophiostoma novo-ulmi.

Suppression subtractive hybridization cDNA libraries were prepared from asexual synnemata (S-lib) and sexual perithecia (P-lib) fruiting bodies of the Dutch elm disease pathogen Ophiostoma novo-ulmi subsp. novo-ulmi isolate H327 (mating-type MAT1-1) consisting of 630 and 401 cDNA clones, respectively. Both libraries were differentially screened in duplicate with forward and reverse subtracted probes. Up-regulated S-lib transcripts included those with homologies to phosphoenolpyruvate carboxykinase and aquaporin. Up-regulated P-lib transcripts included those with homologies to aspartyl proteinase, DNA lyase 2, and part of a mating-type (MAT) protein containing a DNA-binding domain of the high-mobility group (HMG) type. Phylogenetic analyses of HMG domains present within the putative O. novo-ulmi MAT protein and within MAT1-1-3 and MAT1-2-1 proteins of other ascomycete fungi identified the O. novo-ulmi protein as a homologue of the MAT1-1-3 protein, which represents part of the so far uncharacterized O. novo-ulmi MAT1-1 idiomorph. Reverse transcription - quantitative real-time PCR indicated up-regulation of the MAT1-1-3 homologue in O. novo-ulmi perithecia and synnemata. The present work identifies, for the first time, proteins involved in the formation of asexual and sexual fruiting bodies in O. novo-ulmi and should be of interest to researchers concerned with reproduction, mating type, and sexuality of filamentous ascomycete fungi.

Fungal colonization and host defense reactions in Ulmus americana callus cultures inoculated with Ophiostoma novo-ulmi.

The host-pathogen interaction leading to Dutch elm disease was analyzed using histo- and cyto-chemical tests in an in vitro system. Friable and hard susceptible Ulmus americana callus cultures were inoculated with the highly aggressive pathogen Ophiostoma novo-ulmi. Inoculated callus tissues were compared with water-treated callus tissues and studied with light microscopy (LM), transmission-electron microscopy (TEM), and scanning-electron microscopy (SEM). New aspects of this interaction are described. These include the histological observation, for the first time in plant callus cultures, of suberin with its typical lamellar structure in TEM and the intracellular presence of O. novo-ulmi. Expression of the phenylalanine ammonia lyase gene, monitored by real-time quantitative polymerase chain reaction, was correlated with the accumulation of suberin, phenols, and lignin in infected callus cultures. This study validates the potential use of the in vitro system for genomic analyses aimed at identifying genes expressed during the interaction in the Dutch elm disease pathosystem.