Ophiostoma breviusculum sp. nov. (Ophiostomatales, Ascomycota) is a new species in the Ophiostoma piceae complex associated with bark beetles infesting larch in Japan

A new species of the Ophiostoma piceae-complex was isolated from bark beetles and the insect galleries of Larix kaempferi in Japan. This species was described as Ophiostoma breviusculum. The fungus was morphologically similar to O. piceae and O. quercus. However the average length of the perithecial necks and synnemata were shorter than for O. piceae and O. quercus. Synnemata morphological characteristics also differentiated O. breviusculum from the other species of the Ophiostoma piceae-complex isolated from conifers. Mating tests demonstrated that this fungus did not produce perithecia with O. floccosum, O. piceae and O. quercus. In phylogenetic trees using rDNA ITS O. breviusculum was placed in a clade with O. canum, O. piceae and O. subalpinum, but when using beta-tubulin it was placed into a separate clade.     

Phylogenetic relationship of Ophiostoma piliferum to other sapstain fungi based on the nuclear rRNA gene

The nuclear rRNA gene of Ophiostoma piliferum was analyzed to understand its phylogenetic relationships to other sapstain fungi. Phylograms based on nucleotide sequences of the rRNA gene showed that the relationships between O. piliferum and other Ophiostoma species varied depending on the regions of the rRNA gene analyzed. Intraspecies variation in O. piliferum was found in the internal transcribed spacer regions, and the variation was related to the geographic origin of O. piliferum strains. A useful molecular marker for differentiating O. piliferum from other sapstain Ophiostoma species was generated by the HaeIII restriction fragment length polymorphism of the 26S rRNA gene.     

Development of polymorphic microsatellite markers for the tree pathogen and sapstain agent,Ophiostoma ips

Twelve pairs of simple sequence repeat markers (SSR) were developed using a single ascospore isolate of Ophiostoma ips, isolated from the bark beetle, Orthotomicus erosus, infesting Pinus elliottii in South Africa. All markers were found to be polymorphic when tested on seven isolates of O. ips collected from Austria, Chile, Israel, Mexico, South Africa, Sweden, and the USA. These will now be useful in population and phylogenetic studies on O. ips.     

Phylogeny of the Ophiostoma stenoceras-Sporothrix schenckii complex

Ophiostoma stenoceras is a well-known sapwood-colonizing fungus occurring on some coniferous and hardwood hosts in the Northern Hemisphere. In the Southern Hemisphere, the fungus has been reported only from New Zealand. The human pathogen, Sporothrix schenckii, has been suggested to be the anamorph of O. stenoceras. The aim of this study was to gain a better understanding of the phylogenetic relationship between these two species. The study also provided the opportunity to confirm the identity of some Sporothrix and O. stenoceras-like isolates recently collected from wood and soil around the world. For this purpose, the DNA sequence of internal transcribed spacer (ITS) regions of the ribosomal RNA operon was determined. Isolates of O. nigrocarpum, O. albidum, O. abietinum, O. narcissi and O. ponderosae, all morphologically similar to O. stenoceras, were included in the study. From phylogenetic analyses of the sequence data, four main clades were observed. These represented O. stenoceras, O. nigrocarpum and two separate groups containing isolates of S. schenckii. Our results confirm earlier suggestions that S. schenckii should be classified within the teleomorph genus Ophiostoma but support studies separating O. stenoceras and S. schenckii. Ophiostoma albidum and O. ponderosae should be considered synonyms of O. stenoceras. The status of O. narcissi and O. abietinum needs further clarification. The two groups within S. schenckii might represent two species, but this needs to be confirmed. This study represents the first reports of O. stenoceras from Colombia, Kenya, Uruguay and South Africa.     

Ophiostoma gemellus and Sporothrix variecibatus from mites infesting Protea infructescences in South Africa

Ophiostoma (Ophiostomatales) represents a large genus of fungi mainly known from associations with bark beetles (Curculionidae: Scolytinae) infesting conifers in the northern hemisphere. Few southern hemisphere native species are known, and the five species that consistently occur in the infructescences of Protea spp. in South Africa are ecologically unusual. Little is known about the vectors of Ophiostoma spp. from Protea infructescences, however recent studies have considered the possible role of insects and mites in the distribution of these exceptional fungi. In this study we describe a new species of Ophiostoma and a new Sporothrix spp. with affinities to Ophiostoma, both initially isolated from mites associated with Protea spp. They are described as Ophiostoma gemellus sp. nov. and Sporothrix variecibatus sp. nov. based on their morphology and comparisons of DNA sequence data of the 28S ribosomal, beta-tubulin and internal transcribed spacer (ITS1, 5.8S, ITS2) regions. DNA sequences of S. variecibatus were identical to those of a Sporothrix isolate obtained from Eucalyptus leaf litter in the same area in which S. variecibatus occurs in Protea infructescences. Results of this study add evidence to the view that mites are the vectors of Ophiostoma spp. that colonize Protea infructescences. They also show that DNA sequence comparisons are likely to reveal additional cryptic species of Ophiostoma in this unusual niche.     

Selective acquisition of novel mating type and vegetative incompatibility genes via interspecies gene transfer in the globally invading eukaryote Ophiostoma novo-ulmi

The Dutch elm disease fungus Ophiostoma novo-ulmi, which has destroyed billions of elm trees worldwide, originally invaded Europe as a series of clonal populations with a single mating type (MAT-2) and a single vegetative incompatibility (vic) type. The populations then rapidly became diverse with the appearance of the MAT-1 type and many vegetative incompatibility types. Here, we have investigated the mechanism using isolates from sites in Portugal at which the rapid evolution of O. novo-ulmi populations from clonality to heterogeneity was well established. We show by genetic mapping of vic and MAT loci with AFLP markers and by sequence analysis of MAT loci that this diversification was due to selective acquisition by O. novo-ulmi of the MAT-1 and vic loci from another species, Ophiostoma ulmi. A global survey showed that interspecies transfer of the MAT-1 locus occurred on many occasions as O. novo-ulmi spread across the world. We discuss the possibility that fixation of the MAT-1 and vic loci occurred in response to spread of deleterious viruses in the originally clonal populations. The process demonstrates the potential of interspecies gene transfer for facilitating rapid adaptation of invasive organisms to a new environment.     

Two Ophiostoma species associated with bark beetles in wave-regenerated Abies veitchii forests in Japan

Two species of Ophiostoma were isolated from four bark beetles (i.e., Cryphalus montatus, C. piceae, Dryocoetes hectographus, and Polygraphus proximus) infesting Abies veitchii and from their galleries in the wave-regenerated forests in the central part of the main island (Honshu) of Japan. One of them is described here as a new species, Ophiostoma subalpinum, and the other is identified as O. davidsonii, newly reported in Japan. Ophiostoma subalpinum is characterized by short ostiolar hyphae, oblong or allantoid ascospores enclosed in a thin, hyaline sheath, and a Pesotum anamorph. Received: June 4, 2001 / Accepted: December 25, 2001     

Two new Ophiostoma species with Sporothrix anamorphs from Austria and Azerbaijan

The genus Ophiostoma includes numerous species of primarily insect-vectored, wood-staining fungi. Several anamorph genera that differ in their micronematous or macronematous conidiogenous cells have been associated with Ophiostoma species. Among the former group, Sporothrix is associated with many species and is characterized by conidiogenous cells that arise laterally or terminally from any place on the hyphae and produce nonseptate conidia on sympodially developing denticles. The purpose of this study was to characterize ophiostomatoid isolates with Sporothrix anamorphs recently collected in Austria and Azerbaijan. The isolates were characterized based on comparisons of rDNA and β-tubulin sequence data. Morphology, growth in culture, and sexual reproductive mode were also considered. Phylogenetic analyses of the combined sequence data showed that the isolates formed two distinct groups, one including isolates from Austria and the other isolates from Austria and Azerbaijan. Growth at 25 C and morphology revealed some differences between the two groups, and supported the view that they represent two new species, which we describe here as Ophiostoma fusiforme sp. nov. and Ophiostoma lunatum sp. nov. Both these groups phylogenetically were related to, but distinct from, Ophiostoma stenoceras.     

Evolutionary Relationships of the Dutch Elm Disease Fungus Ophiostoma novo-ulmi to Other Ophiostoma Species Investigated by Restriction Fragment Length Polymorphism Analysis of the rDNA Region

Restriction fragment length polymorphisms (RFLPs) in the ribosomal RNA gene (rDNA) region were used to assess relationships between the Dutch elm disease fungi Ophiostoma novo-ulmi and Ophiostoma ulmi , the recently described Himalayan Dutch elm disease pathogen, Ophiostoma himal-ulmi , the morphologically similar sapstain fungi, Ophiostoma piceae and Ophiostoma quercus , and several Ophiostoma species from hardwood trees, including Ophiostoma stenoceras and Ophiostoma proliferum . A distance matrix and cluster analysis indicated that the rDNA region of O. himal-ulmi is more closely related to those of O. novo-ulmi and O. ulmi than to those of O. piceae and O. quercus and is more distantly related to O. stenoceras and the other Ophiostoma species, which formed a separate clade. The rDNA region of O. quercus was found to be at least as closely related to that of O. novo-ulmi and O. ulmi as it is to that of O. piceae . The implications of these results for the evolution of the Dutch elm disease fungi are discussed.     

Sporothrix brunneoviolacea and Sporothrix dimorphospora, two new members of the Ophiostoma stenoceras-Sporothrix schenckii complex

Sporothrix inflata is a saprobic member of the Ophiostoma stenoceras-Sporothrix schenckii species complex, reported mainly from soil. Ophiostoma bragantinum, an ascomycete described from Brazil, has been proposed as its possible teleomorph. Previous studies revealed that Sporothrix inflata is phenotypically and genetically variable, suggesting the existence of cryptic species. During a continued survey on the biodiversity of microfungi from different countries, seven isolates morphologically similar to S. inflata were obtained from soil samples collected in Spain and USA. In this study their phenotypic features and phylogenetic relationships were assessed. DNA sequence data of two nuclear loci revealed that these isolates correspond to two unnamed clades in S. inflata s.l., one of which also included the type strain of Humicola dimorphospora, a species that traditionally has been considered a synonym of S. inflata. These two groups are proposed herein as Sporothrix brunneoviolacea sp. nov. and Sporothrix dimorphospora comb. nov. S. brunneoviolacea is characterized phenotypically by the production of a diffusible violet-brown pigment in culture and mostly globose, pigmented, lateral blastoconidia. On the other hand S. dimorphospora lacks diffusible pigments and shows mostly subglobose to obovoid pigmented lateral blastoconidia. In contrast to the type strain of S. inflata S. brunneoviolacea and S. dimorphospora assimilate raffinose. The phylogenetic analysis suggested that the proposed anamorph-teleomorph connection between S. inflata and O. bragantinum might not be correct.     

High intercontinental migration rates and population admixture in the sapstain fungus Ophiostoma ips

Ophiostoma ips is a common fungal associate of various conifer-infesting bark beetles in their native ranges and has been introduced into non-native pine plantations in the Southern Hemisphere. In this study, we used 10 microsatellite markers to investigate the population biology of O. ips in native (Cuba, France, Morocco and USA) and non-native (Australia, Chile and South Africa) areas to characterize host specificity, reproductive behaviour, and the potential origin as well as patterns of spread of the fungus and its insect vectors. The markers resolved a total of 41 alleles and 75 haplotypes. Higher genetic diversity was found in the native populations than in the introduced populations. Based on the origin of the insect vectors, the populations of O. ips in Australia would be expected to reflect a North American origin, and those in Chile and South Africa to reflect a European origin. However, most alleles observed in the native European population were also found in the native North American population; only the allele frequencies among the populations varied. This admixture made it impossible to confirm the origin of the introduced Southern Hemisphere (SH) populations of O. ips. There was also no evidence for specificity of the fungus to particular bark beetle vectors or hosts. Although O. ips is thought to be mainly self-fertilizing, evidence for recombination was found in the four native populations surveyed. The higher genetic diversity in the North American than in the European population suggests that North America could be the possible source region of O. ips.     

Development and characterization of polymorphic markers for the sap-stain fungus Ophiostoma quercus

Eight polymorphic markers were developed from South African isolates of Ophiostoma quercus. The genome was screened for repeat regions using the fast isolation by amplified fragment length polymorphism of sequences containing repeats protocol and 20 de novo primer pairs flanking putative microsatellite regions were designed. Eight loci were optimized and their polymorphisms evaluated by sequencing. The repeat and flanking regions were highly polymorphic containing both indels and base-pair substitutions revealing a total of 46 alleles in 14 isolates and an average heterozygosity of 0.68. Substantial sequence variability makes these markers useful for genotyping populations in order to calculate diversity and monitor global movement of O. quercus.     

Ophiostoma species (Ascomycetes: Ophiostomatales) associated with bark beetles (Coleoptera: Scolytinae) colonizing Pinus radiata in northern Spain

Bark beetles (Coleoptera: Scolytinae) are known to be associated with fungi, especially species of Ophiostoma sensu lato and Ceratocystis. However, very little is known about these fungi in Spain. In this study, we examined the fungi associated with 13 bark beetle species and one weevil (Coleoptera: Entiminae) infesting Pinus radiata in the Basque Country of northern Spain. This study included an examination of 1323 bark beetles or their galleries in P. radiata. Isolations yielded a total of 920 cultures, which included 16 species of Ophiostoma sensu lato or their asexual states. These 16 species included 69 associations between fungi and bark beetles and weevils that have not previously been recorded. The most commonly encountered fungal associates of the bark beetles were Ophiostoma ips, Leptographium guttulatum, Ophiostoma stenoceras, and Ophiostoma piceae. In most cases, the niche of colonization had a significant effect on the abundance and composition of colonizing fungi. This confirms that resource overlap between species is reduced by partial spatial segregation. Interaction between niche and time seldom had a significant effect, which suggests that spatial colonization patterns are rarely flexible throughout timber degradation. The differences in common associates among the bark beetle species could be linked to the different niches that these beetles occupy.     

Assessing RNAi frequency and efficiency in Ophiostoma floccosum and O. piceae

Ophiostoma species are an economically important group of saprophytic and pathogenic fungi that grow in trees or wood. Ophiostoma like O. piceae and O. floccosum produce melanin, a pigment that stains lumber and logs. We used such species as model organisms for characterizing the molecular mechanisms in fungal melanin production. Because homologous recombination is rare in the Ophiostoma, identifying gene function in this group is challenging. We addressed this by assessing RNA interference (RNAi) as an alternative to gene replacement. For this, we built different inverted repeat transgene (IRT) constructs to down-regulate the polyketide synthase (PKS1) gene of the melanin pathway in O. piceae and O. floccosum. Transformation with IRT-PKS reduced mRNA levels for the PKS1 gene, and consequently decreased pigmentation in transformants. We showed that the PKS1 RNAi efficiency was proportional to the length of the dsRNA expressed from IRT constructs. These results indicated that RNAi is an appropriate tool for functional analysis of genes in Ophiostoma.     

Fungal radiation in the Cape Floristic Region: an analysis based on Gondwanamyces and Ophiostoma

The Cape Floristic Region (CFR) displays high levels of plant diversity and endemism, and has received focused botanical systematic attention. In contrast, fungal diversity patterns and co-evolutionary processes in this region have barely been investigated. Here we reconstruct molecular phylogenies using the ITS and beta-tubulin gene regions of the ophiostomatoid fungi Gondwanamyces and Ophiostoma associated with southern African Protea species. Results indicate that they evolved in close association with Protea. In contrast to Protea, Ophiostoma species migrated to the CFR from tropical and subtropical Africa, where they underwent subsequent radiation. In both Gondwanamyces and Ophiostoma vector arthropods probably facilitated long-distance migration and shorter-distance dispersal. Although ecological parameters shaped most associations between ophiostomatoid fungi and Protea, there is congruence between fungal-host-associations and the systematic classification of Protea. These results confirm that the entire biotic environment must be considered in order to understand diversity and evolution in the CFR as a whole.     

Ophiostoma novo-ulmi sp. nov., causative agent of current Dutch elm disease pandemics

The aggressive subgroup of the Dutch elm disease pathogen Ophiostoma ulmi (Buism.) Nannf. syn. Ceratocystis ulmi (Buism.) Moreau is named as a new species, O. novo-ulmi, and is thereby separated from the old non-aggressive subgroup, which is retained as O. ulmi. O. novo-ulmi differs from O. ulmi in colony morphology, growth rate, optimum temperature for growth, perithecial neck length, pathogenicity to elm, bark colonising ability, cerato-ulmin protein production, synnemetal and protoperithecial production, mating type frequency, protein and isozyme polymorphisms, mitochondrial DNA and nuclear DNA polymorphisms, and mitochondrial DNA size. In addition, a strong unidirectional fertility barrier operates between the two species, while their hybrids show remarkable variation, poor fitness, and many are infertile. These aspects are summarised. New information on perithecial dimensions is presented. O. ulmi is redefined and a neotype designated. The status of the Eurasian and North American races of O. novo-ulmi is currently under investigation.Abbreviations EAN Eurasian race - NAN North American race     

Isolation, characterization, and localization of beta-tubulin genomic clones of three Drosophila montium subgroup species.

Genomic libraries were constructed from three Drosophila species, namely Drosophila auraria, Drosophila serrata, and Drosophila kikkawai, belonging to the Drosophila montium subgroup of the Drosophila melanogaster species group. Clones containing beta-tubulin specific sequences were isolated, characterized by restriction endonuclease digestions and Southern hybridizations, and mapped by in situ hybridization on the polytene chromosomes of the species studied. The distribution of the beta-tubulin loci was found to be similar in D. montium species and D. melanogaster.