Four new Ophiostoma species associated with conifer- and hardwood-infesting bark and ambrosia beetles from the Czech Republic and Poland.

Fungi under the order Ophiostomatales (Ascomycota) are known to associate with various species of bark beetles (Coleoptera: Curculionidae: Scolytinae). In addition this group of fungi contains many taxa that can impart blue-stain on sapwood and some are important tree pathogens. A recent survey that focussed on the diversity of the Ophiostomatales in the forest ecosystems of the Czech Republic and Poland uncovered four putative new species. Phylogenetic analyses of four gene regions (ITS1-5.8S-ITS2 region, ß-tubulin, calmodulin, and translation elongation factor 1-α) indicated that these four species are members of the genus Ophiostoma. All four newly described species can be distinguished from each other and from closely related species based on DNA sequence comparisons, morphological characters, growth rates, and their insect associations. Based on this study four new taxa can be circumscribed and the following names are provided: Ophiostoma pityokteinis sp. nov., Ophiostoma rufum sp. nov., Ophiostoma solheimii sp. nov., and Ophiostoma taphrorychi sp. nov. O. rufum sp. nov. is a member of the Ophiostoma piceae species complex, while O. pityokteinis sp. nov. resides in a discrete lineage within Ophiostoma s. stricto. O. taphrorychi sp. nov. together with O. distortum formed a well-supported clade in Ophiostoma s. stricto close to O. pityokteinis sp. nov. O. solheimii sp. nov. groups within a currently undefined lineage A, which also includes Ophiostoma grandicarpum and Ophiostoma microsporum. This study highlights the need for more intensive surveys that should include additional countries of Central Europe, insect vectors and host tree species in order to elucidate Ophiostoma species diversity in this region.

Mycobiota associated with insect galleries in walnut with thousand cankers disease reveals a potential natural enemy against Geosmithia morbida.

Thousand Cankers Disease (TCD) affects Juglans and Pterocarya species. This disease poses not only a major threat to the nut and timber industries but also to native stands of walnut trees. Galleries created by Pityophthorus juglandis (vector) are colonized by the fungus Geosmithia morbida (causal agent of necrosis). It is unknown if other fungi colonizing these galleries might act antagonistically towards G. morbida. The objectives of this study were to: (1) characterize the fungal community associated with TCD-infected trees and (2) develop a pilot study addressing their potential antagonism towards G. morbida. We collected non-Geosmithia fungi from ten TCD-infected walnut trees from California and Tennessee. Four hundred and fifty-seven isolates, representing sixty-five Operational Taxonomic Units (99 % ITS similarity) were obtained. Fungal communities were found to be highly diverse. Ophiostoma dominated the communities associated with TCD-compromised trees from California, whereas Trichoderma dominated TCD-compromised trees in Tennessee. Six Trichoderma isolates showed varying levels of antagonism against three isolates of G. morbida, suggesting potential applications for the biological control of TCD. Furthermore, results from this study contribute to the growing knowledge about the observed differential disease development between the western and eastern USA and could overall impact our understanding of TCD etiology.

Ophiostomatoid fungi associated with mangroves in South Africa, including Ophiostoma palustre sp. nov.

Mangrove trees are continuously under stress due to environmental and/or anthropogenic pressures, which expose them to attack by pathogens, compromising their survival. Ophiostomatoid fungi cause sap stain and diseases of a wide spectrum of tree species globally. These fungi infect trees through natural, insect, animal and/or human made wounds. During routine surveys of mangrove trees in South Africa, wounds on branches and stems of Avicennia marina were regularly monitored for the presence of ophiostomatoid fungi at ten study sites in the country. The stems of four mangrove species, A. marina, Bruguiera gymnorrhiza, Rhizophora mucronata and Barringtonia racemosa were also wounded and evaluated for the appearance of these fungi. Ophiostomatoid fungi were obtained from the mangrove associate B. racemosa, but not from any of the true mangroves. Analyses of DNA sequence data for the internal transcribed spacer, ß-tubulin, calmodulin and translation elongation factor gene regions revealed that the fungi isolated from the wounds on B. racemosa belong to three species in the Ophiostomataceae, including a new taxon described here as Ophiostoma palustre sp. nov. These results suggest that the mangrove associate B. racemosa is more prone to colonization by ophiostomatoid fungi than the true mangroves.

The Ophiostoma clavatum species complex: a newly defined group in the Ophiostomatales including three novel taxa.

Two species of blue-stain fungi with similar morphologies, Ophiostoma brunneo-ciliatum and Ophiostoma clavatum, are associates of bark beetles infesting Pinus spp. in Europe. This has raised questions whether they represent distinct taxa. Absence of herbarium specimens and contaminated or mistakenly identified cultures of O. brunneo-ciliatum and O. clavatum have accentuated the uncertainty regarding their correct identification. The aim of this study was to reconsider the identity of European isolates reported as O. brunneo-ciliatum and O. clavatum by applying DNA-based identification methods, and to provide appropriate type specimens for them. Phylogenetic analyses of the ITS, ßT, TEF-1α and CAL gene sequences revealed that the investigated isolates represent a complex of seven cryptic species. The study confirmed that ITS data is insufficient to delineate species in some Ophiostoma species clusters. Lectotypes and epitypes were designated for O. clavatum and O. brunneo-ciliatum, and three new species, Ophiostoma brunneolum, Ophiostoma macroclavatum and Ophiostoma pseudocatenulatum, are described in the newly defined O. clavatum-complex. The other two species included in the complex are Ophiostoma ainoae and Ophiostoma tapionis. The results suggest co-evolution of these fungi in association with specific bark beetles. The results also confirm the identity of the fungus associated with the pine bark beetle Ips acuminatus as O. clavatum, while O. brunneo-ciliatum appears to be mainly associated with another pine bark beetle, Ips sexdentatus.

Multigene phylogenies and morphological characterization of five new Ophiostoma spp. associated with spruce-infesting bark beetles in China.

Ophiostoma spp. (Ophiostomatales, Ascomycota) are well-known fungi associated with bark beetles (Coleoptera: Scolytinae). Some of these are serious tree pathogens, while the majority is blue-stain agents of timber. In recent years, various bark beetle species have been attacking spruce forests in Qinghai province, China, causing significant damage. A preliminary survey was done to explore the diversity of the ophiostomatoid fungal associates of these beetles. The aims of the present study were to identify and characterize new Ophiostoma spp. associated with spruce-infesting bark beetles in Qinghai Province, and to resolve phylogenetic relationships of Ophiostoma spp. related to the Chinese isolates, using multigene phylogenetic analyses. Results obtained from four gene regions (ribosomal internal transcribed spacer regions, ß-tubulin, calmodulin, translation elongation factor-1α) revealed five new Ophiostoma spp. from Qinghai. These included O. nitidus sp. nov., O. micans sp. nov., and O. qinghaiense sp. nov. in a newly defined O. piceae complex. The other two new species, O. poligraphi sp. nov. and O. shangrilae sp. nov., grouped in the O. brunneo-ciliatum complex. Based on DNA sequence and morphological comparisons, we also show that O. arduennense and O. torulosum are synonyms of O. distortum, while O. setosum is a synonym of O. cupulatum.

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.

High-resolution DNA melt-curve analysis for cost-effective mass screening of pairwise species interactions.

Ecological studies of pairwise interactions are constrained by the methods available for rapid species identification of the interacting organisms. The resolution of data required to characterize species interaction networks at multiple spatio-temporal scales can be intensive, and therefore laborious and costly to collect. We explore the utility of high-resolution DNA melt-curve analysis (HRM) as a rapid species identification method. An approach was developed to identify organisms at the pairwise interaction level, with particular application to cryptic species interactions that are traditionally difficult to study. Here, we selected a challenging application; to identify the presence/absence of pathogenic fungi (Sporothrix inflata, Ophiostoma nigrocarpum and Ophiostoma galeiforme) transported by bark beetle vectors (Hylastes ater and Hylurgus ligniperda). The technique was able to distinguish between different species of DNA within a single, pooled sample. In test applications, HRM was effective in the mass screening and identification of pathogenic fungal species carried by many individual bark beetle vectors (n = 455 beetles screened) across large geographic scales. For two of the fungal species, there was no difference in the frequency of association with either of their vectors, but for the third fungal species there was a shift in vector-pathogen associations across locations. This technique allows rapid, mass screening and characterization of species interactions at a fraction of the time and cost of traditional methods. It is anticipated that this method can be readily applied to explore other cryptic species interactions, or other studies requiring rapid generation of large data sets and/or high-throughput efficiency.

The relative abundance of mountain pine beetle fungal associates through the beetle life cycle in pine trees.

The mountain pine beetle (MPB) is a native bark beetle of western North America that attacks pine tree species, particularly lodgepole pine. It is closely associated with the ophiostomatoid ascomycetes Grosmannia clavigera, Leptographium longiclavatum, Ophiostoma montium, and Ceratocystiopsis sp.1, with which it is symbiotically associated. To develop a better understanding of interactions between beetles, fungi, and host trees, we used target-specific DNA primers with qPCR to assess the changes in fungal associate abundance over the stages of the MPB life cycle that occur in galleries under the bark of pine trees. Multivariate analysis of covariance identified statistically significant changes in the relative abundance of the fungi over the life cycle of the MPB. Univariate analysis of covariance identified a statistically significant increase in the abundance of Ceratocystiopsis sp.1 through the beetle life cycle, and pair-wise analysis showed that this increase occurs after the larval stage. In contrast, the abundance of O. montium and Leptographium species (G. clavigera, L. longiclavatum) did not change significantly through the MPB life cycle. From these results, the only fungus showing a significant increase in relative abundance has not been formally described and has been largely ignored by other MPB studies. Although our results were from only one site, in previous studies we have shown that the fungi described were all present in at least ten sites in British Columbia. We suggest that the role of Ceratocystiopsis sp.1 in the MPB system should be explored, particularly its potential as a source of nutrients for teneral adults.

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.

Fatal Ophiostoma piceae infection in a patient with acute lymphoblastic leukaemia.

We report to our knowledge the first case of human infection with Ophiostoma piceae. This Sporothrix schenckii-related fungus caused disseminated infection involving the lung and the brain in a patient with lymphoblastic lymphoma. The case emphasizes the significance of molecular techniques for identification of rare fungi in the clinical microbiology laboratory.