Survey and phylogenetic analysis of culturable microbes in the oral secretions of three bark beetle species

In a recent study, we reported a previously undescribed behavior in which a bark beetle exuded oral secretions containing bacteria that have antifungal properties, and hence defend their galleries against pervasive antagonistic Hyphomycete fungi. Actinobacteria, a group known for their antibiotic properties, were the most effective against fungi that invade the spruce beetle galleries. In the present study, we describe the isolation and identification of microorganisms from oral secretions of three bark beetles (Coleoptera: Curculionidae: Scolytinae): the spruce beetle, Dendroctonus rufipennis Kirby, the mountain pine beetle, Dendroctonus ponderosae Hopkins, and the pine engraver, Ips pini Say. Bacteria isolated from these three species span the major bacterial classes α-, β-, and γ-Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria, except for D. ponderosae , which yielded no α-proteobacteria or Bacteroidetes isolates. Spruce beetles and pine engraver beetles had similar numbers of α-proteobacteria isolates, but pine engravers yielded twice as many Bacteroidetes isolates as spruce beetles. In contrast, mountain pine beetles yielded more isolates in the β- and γ-proteobacteria than spruce beetles and pine engravers. The highest percentage of Actinobacteria was obtained from spruce beetles, followed by pine engravers and mountain pine beetles. All of the fungal isolates obtained from the three beetle species were Ascomycetes. The greatest fungal diversity was obtained in spruce beetles, which had nine species, followed by pine engravers with five, and mountain pine beetles with one.     

Nature and bioactivities of endolichenic fungi in Pseudocyphellaria sp., Parmotrema sp. and Usnea sp. at Hakgala montane forest in Sri Lanka

Aim:  The aim of this study was to investigate the nature and bioactivities of endolichenic fungi in three abundant lichens, Pseudocyphellaria sp., Usnea sp. and Parmotrema sp. in the lower elevation of Hakgala montane forest in Sri Lanka.
Methods and Results:  Endolichenic fungal strains, fungi that live asymptomatically in the lichen thallus, much the same way as endophytic fungi live within healthy plant tissues, were isolated from three abundant lichen species, Pseudocyphellaria sp., Usnea sp. and Parmotrema sp., at Hakgala montane forest in Sri Lanka, using the surface sterilization method. Nine endolichenic fungal strains were isolated from Parmotrema sp. and Usnea sp. separately, while 11 endolichenic fungi were recovered from the lichen Pseudocyphellaria sp. Isolation of endolichenic fungus Chrysosporium sp. 2 was common to all three lichen species. Substrate utilization patterns and antifungal activities of eight endolichenic fungal species were evaluated and the results revealed that all the test fungi were able to produce at least one enzyme to utilize the test substrates. Nigrospora sp., Chrysosporium sp. 1 and 2 and Cladosporium sp. showed antifungal activities on growth of some selected plant pathogenic fungi.
Conclusions:  Endolichenic fungal strains (29) were isolated from the lichens Parmotrema sp., Usnea sp. and Pseudocyphellaria sp. in Sri Lanka. Chrysosporium sp. 2 was common in all three lichens. Some of these endolichenic fungal strains showed antifungal activities against common plant pathogenic fungi and they are capable of utilizing the substrates by producing specific enzymes.
Significance and Impact of the Study:  The diversity and prevalence of the endolichenic fungi have not been studied extensively and this is the first report of isolation and identification of endolichenic fungi in lichens available in Sri Lanka.     

The dietary relationships of Tribolium castaneum (Herbst) with microfungi

The ecology of Tribolium castaneum (Herbst) outside of grain storage facilities is poorly known. However, high densities of T. castaneum adults are known to infest stored cotton seeds (Gossypium hirsutum (L.)) in Queensland, Australia, despite the absence of stored food products in the immediate vicinity. Previous studies suggest that the beetles are attracted to the fungal colonies growing on the residual fibres that remain on the cotton seeds after ginning, but the specifics of this remain unclear, even as to the species of fungi involved. In the current study, 14 fungal species were isolated from stored cotton seeds collected from four sites in Queensland. The feeding preferences of the adult beetles and the developmental success of the larvae were recorded for each fungal species. Gut analyses of T. castaneum adults, after exposure in no-choice feeding tests, showed that the beetles will feed on any one of the 14 fungal species when exposed (over 14 days) to cotton seeds that had been inoculated with one particular fungal species. The developmental success of the larvae varied depending on the fungal species, with most fungal species supporting only low levels of successful development (<30%). Tests with each fungal species and the volatiles they release revealed that neither adult nor larval survival was significantly affected by the presence of any particular fungal isolate. These results indicate that T. castaneum will feed on a variety of fungal species but that none of the tested fungal species on its own provides a good larval diet for T. castaneum.     

Responses of Tribolium castaneum to olfactory cues from cotton seeds,the fungi associated with cotton seeds,and cereals

We tested, in an olfactometer, whether or not Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) responds preferentially to the volatiles that emanate from the fungi associated with cotton [Gossypium hirsutum L. (Malvaceae)] seed over those that emanate from cereals, because cereals are usually portrayed as the primary resources of these beetles. Pairwise comparisons were conducted between cotton seed, wheat (Triticum aestivum L.), and sorghum [Sorghum bicolor (L.) Moench] (both Poaceae); volatiles were tested from intact seeds and from both water and ethanol extracts. The results demonstrate that T. castaneum is attracted more strongly to cotton seeds with its lint contaminated with fungi, than to the conventional resources of this species (i.e., wheat and sorghum). Further tests prove that it is the fungus on the lint that produces the active volatiles, because the beetles did not respond to sterilized cotton lint (i.e., without the fungi typically associated with it when cotton seed is stored). Tests with five fungal cultures (each representing an unidentified species that was isolated from the field‐collected cotton lint) were variable across the cultures, with only one of them being significantly attractive to the beetles. The others were not attractive and one may even have repulsed the beetles. The results are consistent with the beetles having a strong ecological association with fungi and suggest it would be worth investigating the ecology of T. castaneum from this perspective.     

Characterization of fungal communities associated with the bark beetle Ips typographus varies depending on detection method, location, and beetle population levels

The Eurasian spruce bark beetle Ips typographus and their fungal associates can cause severe damage to Norway spruce forests. In this paper, by using both molecular and cultural methods, we compared fungal assemblages on bark beetles from different locations, characterized by different beetle population levels. Ips typographus was trapped in the western Alps in two outbreak and in two control areas. Sequencing of clone libraries of Internal Transcribed Spacer (ITS) identified 31 fungal Operational Taxonomic Units (OTUs), while fungal isolations yielded 55 OTUs. Only three OTUs were detected by both molecular and cultural methods indicating that both methods are necessary to adequately describe fungal richness. Fungal assemblages on insects from these four and from an additional 12 study sites differed among stands in response to varying ecological conditions and to the limited spreading ability of I. typographus. Ophiostomatoid fungi showed higher diversity in outbreak areas; the pathogenic Ophiostoma polonicum was relatively uncommon, while O. bicolor was the most abundant species. This result was not unexpected, as insects were trapped not at the peak but at the end of the outbreaks and supports the hypothesis of a temporal succession among Ophiostoma species. Ubiquitous endophytes of trees or common airborne fungi were present both in outbreak and in control areas. Wood decaying basidiomycetes were almost never detected on beetles. Yeasts were detected only by molecular analysis, and the OTUs detected matched those reported elsewhere in Europe and in the world, suggesting a very long association between some yeasts and bark beetles.     

Ophiostomatoid fungi isolated from Japanese red pine and their relationships with bark beetles

We isolated ophiostomatoid fungi from bark beetles infesting Pinus densiflora and their galleries at 24 sites in Japan. Twenty-one ophiostomatoid fungi, including species of Ophiostoma, Grosmannia, Ceratocystiopsis, Leptographium, and Pesotum, were identified. Among these, 11 species were either newly recorded in Japan or were previously undescribed species. Some of these fungal species were isolated from several bark beetles, but other species were isolated from only a particular beetle species. Thus, it is suggested that some ophiostomatoid fungi have specific relationships with particular beetle species. In addition, fungus-beetle biplots from redundancy analysis (RDA) summarizing the effects of beetle ecological characteristics suggested that the association patterns between bark beetles and the associated fungi seemed to be related to the niches occupied by the beetles.     

Host specificity of ambrosia and bark beetles (Col., Curculionidae: Scolytinae and Platypodinae) in a New Guinea rainforest

Abstract.  1. Bark and ambrosia beetles are crucial for woody biomass decomposition in tropical forests worldwide. Despite that, quantitative data on their host specificity are scarce.
2. Bark and ambrosia beetles (Scolytinae and Platypodinae) were reared from 13 species of tropical trees representing 11 families from all major lineages of dicotyledonous plants. Standardised samples of beetle-infested twigs, branches, trunks, and roots were taken from three individuals of each tree species growing in a lowland tropical rainforest in Papua New Guinea.
3. A total of 81 742 beetles from 74 species were reared, 67 of them identified. Local species richness of bark and ambrosia beetles was estimated at 80–92 species.
4. Ambrosia beetles were broad generalists as 95% of species did not show any preference for a particular host species or clade. Similarity of ambrosia beetle communities from different tree species was not correlated with phylogenetic distances between tree species. Similarity of ambrosia beetle communities from individual conspecific trees was not higher than that from heterospecific trees and different parts of the trees hosted similar ambrosia beetle communities, as only a few species preferred particular tree parts.
5. In contrast, phloeophagous bark beetles showed strict specificity to host plant genus or family. However, this guild was poor in species (12 species) and restricted to only three plant families (Moraceae, Myristicaceae, Sapindaceae).
6. Local diversity of both bark and ambrosia beetles is not driven by the local diversity of trees in tropical forests, since ambrosia beetles display no host specificity and bark beetles are species poor and restricted to a few plant families.     

Fungal Diversity of Norway Spruce Litter: Effects of Site Conditions and Premature Leaf Fall Caused By Bark Beetle Outbreak

Fungi play an important role in leaf litter decomposition due to their ability to break down the lignocellulose matrix, which other organisms are unable to digest. However, little is known regarding the factors affecting components of fungal diversity. Here, we quantified richness of internal fungi in relation to litter nutrient and phenolic concentrations, sampling season (spring or fall), and premature leaf shedding due to low precipitation and infestation of bark beetles (mainly Ips typographus and Ips duplicatus). The study was conducted in 37-year-old Norway spruce [Picea abies (L.) Karst.] stands, with three plots each in mixed forest (MF) and coniferous forest (CF) site conditions in south-central Poland. Fifty-four species of sporulating fungi were identified in 2,330 freshly fallen needles sampled during 2003-2005, including 45 species in MF and 31 in CF. The significantly higher number of species in MF was likely related to moister conditions at that site. Among isolated fungi, 22% (12 species) were identified as endophytes of Norway spruce in prior studies. During spring of 2005, we found less than half the number of isolates and fungal species at each forest site as compared to fall for the two prior years. This pattern was observed in typical soil fungi (e.g., Penicillium daleae, Penicillium purpurogenum) and endophytes/epiphytes (e.g., Aureobasidium pullulans, Alternaria alternata, Cladosporium spp., and Lophodermium piceae). Premature shedding of needles was the most likely cause of this decline because it shortened the time period for fungi to infect green needles while on the tree. For all sites and sampling periods, richness of internal fungi was strongly and positively related to the age of freshly fallen litter (assessed using needle Ca concentration as a needle age tracer) and was also negatively related to litter phenolic concentration. Richness of internal fungi in freshly fallen litter may be adversely affected by low soil moisture status, natural inhibitors slowing fungal colonization (e.g., phenolics) and biotic (e.g., insect infestation) and abiotic (e.g., drought) factors that shorten leaf life span.     

Target-specific PCR primers can detect and differentiate ophiostomatoid fungi from microbial communities associated with the mountain pine beetle Dendroctonus ponderosae

The aim of this study was to develop DNA probes that could identify the major fungal species associated with mountain pine beetles (MPB). The beetles are closely associated with fungal species that include ophiostomatoid fungi that can be difficult to differentiate morphologically. The most frequently isolated associates are the pine pathogens Grosmannia clavigera and Leptographium longiclavatum, the less pathogenic Ophiostoma montium, and an undescribed Ceratocystiopsis species (Cop. sp.). Because growing, isolating and extracting DNA from fungi vectored by MPB can be time and labour intensive, we designed three rDNA primer sets that specifically amplify short rDNA amplicons from O. montium, Cop. sp. and the pine Leptographium clade. We also designed two primer sets on a gene of unknown function that can differentiate G. clavigera and L. longiclavatum. We tested the primers on 76 fungal isolates that included MPB associates. The primers reliably identified their targets from DNA obtained from pure fungal cultures, pulverized beetles, beetle galleries, and tree phloem inoculated with G. clavigera. The primers will facilitate large-scale work on the ecology of the MPB-fungal-lodgepole pine ecosystem, as well as phytosanitary/quarantine sample screening.     

Ectomycorrhizal fungal communities in late-successional Swedish boreal forests, and their composition following wildfire

This study was conducted to evaluate the effects of wildfires on ectomycorrhizal (EM) fungal communities in Scots pine ( Pinus sylvestris ) stands. Below- and above-ground communities were analysed in terms of species richness and evenness by examining mycorrhizas and sporocarps in a chronosequence of burned stands in comparison with adjacent unburned late-successional stands. The internal transcribed spacer (ITS)-region (rDNA) of mycobionts from single mycorrhizas was digested with three restriction enzymes and compared with an ITS–restriction fragment length polymorphism (RFLP) reference database of EM sporocarps. Spatial variation seemed to be more prominent than the effects of fire on the EM fungal species composition. Most of the common species tended to be found in all sites, suggesting that EM fungal communities show a high degree of continuity following low-intensity wildfires. Species richness was not affected by fire, whereas the evenness of species distributions of mycorrhizas was lower in the burned stands. The diversity of EM fungi was relatively high considering that there were only three EM tree species present in the stands. In total, 135 EM taxa were identified on the basis of their RFLP patterns; 66 species were recorded as sporocarps, but only 11 of these were also recorded as mycorrhizas. The species composition of the below-ground community of EM fungi did not reflect that of the sporocarps produced. EM fungal species present in our ITS–RFLP reference database accounted for 54–99% of the total sporocarp production in the stands, but only 0–32% of the mycorrhizal abundance.     

Metabarcoding of fungal communities associated with bark beetles

Many species of fungi are closely allied with bark beetles, including many tree pathogens, but their species richness and patterns of distribution remain largely unknown. We established a protocol for metabarcoding of fungal communities directly from total genomic DNA extracted from individual beetles, showing that the ITS3/4 primer pair selectively amplifies the fungal ITS. Using three specimens of bark beetle from different species, we assess the fungal diversity associated with these specimens and the repeatability of these estimates in PCRs conducted with different primer tags. The combined replicates produced 727 fungal Operational Taxonomic Units (OTUs) for the specimen of Hylastes ater, 435 OTUs for Tomicus piniperda, and 294 OTUs for Trypodendron lineatum, while individual PCR reactions produced on average only 229, 54, and 31 OTUs for the three specimens, respectively. Yet, communities from PCR replicates were very similar in pairwise comparisons, in particular when considering species abundance, but differed greatly among the three beetle specimens. Different primer tags or the inclusion of amplicons in separate libraries did not impact the species composition. The ITS2 sequences were identified with the Lowest Common Ancestor approach and correspond to diverse lineages of fungi, including Ophiostomaceae and Leotiomycetes widely found to be tree pathogens. We conclude that Illumina MiSeq metabarcoding reliably captures fungal diversity associated with bark beetles, although numerous PCR replicates are recommended for an exhaustive sample. Direct PCR from beetle DNA extractions provides a rapid method for future surveys of fungal species diversity and their associations with bark beetles and environmental variables.     

Evaluation of PCR-DGGE methodology to monitor fungal communities on grapes

Aims:  Some fungi present on the surface of grapes may have a negative effect on the quality of wine. The aim of this study was to evaluate PCR-denaturing gradient gel electrophoresis (PCR-DGGE), for the establishment of fungal community profiles from grapes, in order to monitor fungi potentially involved in wine defects.
Methods and Results:  A fragment of the β-tubulin gene was amplified from filamentous fungi and yeasts described from grapes and analysed using two different denaturing gradient gels to constitute a reference database. The use of β-tubulin sequences instead of ITS rDNA in PCR-DGGE showed a progress in the discrimination of these fungal species but comigration problems were still observed. The technique was then applied on grape samples. The profiles counted up to 10 bands of which half corresponded to species which were not recorded in the reference database.
Conclusion:  PCR-DGGE represents a useful tool to compare environmental samples for the study of the dynamics of fungal communities, but comigrations represent a limit in its use to describe the species present.
Significance and Impact of the Study:  A better knowledge of the fungal diversity on grapes, particularly species responsible for wine defect, is necessary to develop accurate molecular detection tools.     

Species diversity of polyporoid and corticioid fungi in northern hardwood forests with differing management histories

Effects of forest management on fungal diversity were investigated by sampling fruit bodies of polyporoid and corticioid fungi in forest stands that have different management histories. Fruit bodies were sampled in 15 northern hardwood stands in northern Wisconsin and the upper peninsula of Michigan. Sampling was conducted in five old-growth stands, five uneven-age stands, three even-age unthinned stands and two even-age thinned stands. Plots 100 m x 60 m were established and 3000 m2 within each plot was sampled during the summers of 1996 and 1997. A total of 255 polyporoid and corticioid morphological species were identified, 46 (18%) of which could not be assigned to a described species. Species accumulation curves for sites and management classes differed from straight lines, although variability from year to year suggests that more than 2 y of sampling are needed to characterize annual variation. Mean species richness and diversity index values did not vary significantly by management class, although mean richness on large diameter wood (> or = 15 cm diam) varied with moderate significance. Richness values on small diameter debris varied significantly by year, indicating that a large part of year-to-year variability in total species richness is due to small diameter debris. Ten species had abundance levels that varied by management class. Two of these species. Changes in the diversity and species composition of the wood-inhabiting fungal community could have significant implications for the diversity, health and productivity of forest ecosystems.     

Diversity and pathogenicity of ophiostomatoid fungi associated with <Emphasis Type="Italic">Tetropium</Emphasis> species colonizing <Emphasis Type="Italic">Picea abies</Emphasis> in Poland

The ophiostomatoid fungi associated with cerambycid beetles Tetropium spp. (their symbiotic vectors) colonizing Norway spruce in Poland (six species collected) were isolated. The virulence of representative isolates was evaluated through inoculations using 2-year-old Norway spruce seedlings. A total of 1325 isolates (Ophiostoma piceae, O. tetropii, O. minus, Grosmannia piceiperda, G. cucullata, and five other less frequent taxa) were obtained. Tetropium castaneum and T. fuscum were vectors of similar spectra of ophiostomatoid fungi although some differences in fungal frequency between these Tetropium spp. were found. Among the fungal associates of the Tetropium spp. collected only G. piceiperda was pathogenic, which suggests that it can play a role in the death of spruce trees following attack by Tetropium spp.     

Fungi inhabiting knotwood of Pinus sylvestris infected by Porodaedalea pini

Abundance and diversity of fungi in naturally formed knots of Pinus sylvestris affected by Porodaedalea pini were investigated. Samples were taken from trees that were (i) affected, with internal heartwood decay and no conks, (ii) affected, with internal heartwood decay and conks and (iii) controls. The Illumina sequencing technology was used for amplification of DNA, sequencing and analysis. In total, 566,279 raw sequences were obtained from six samples. Sequences included 74% of culturable and 8.4% of non‐culturable fungi and 17.6% of organisms with no reference sequences in NCBI. Abundance of organisms in knotwood, measured as number of OTUs, ranged from 36,272 (29,506 for fungi) to 178,535 (177,484 for fungi) and differed significantly between two trees in a stand and between stands. The highest and lowest average number of fungal OTUs occurred in infected trees with no conks and in trees with conks, respectively. Number of taxa ranged from 171 to 415 and often differed significantly between two trees in one stand and between stands. Greatest diversity occurred in control trees. The number of fungal taxa shared by two trees in one stand was 67–152 and that shared by two stands was 51–141. The majority of fungi were Ascomycota. Those most common in pines affected by P. pini were Coniochaeta hoffmannii and C. fodinicola (19.65%–59.92%). Infundichalara microchona, Leotiomycetes spp. and Rhinocladiella atrovirens were also present. Another common species, Lecanora conizaeoides, occurred most often in control trees (0.30%–8.82%). Porodaedalea pini was detected only sporadically. Non‐culturable fungi were most frequent in the control trees. The greater average abundance and smaller average diversity of fungi in knots of trees infected by P. pini suggest that the pathogen successfully competes with some fungal species and does not inhibit the growth of survivors. Some fungi detected may contribute to production of natural biocides.     

Broadscale Specificity in a Bark Beetle–Fungal Symbiosis: a Spatio-temporal Analysis of the Mycangial Fungi of the Western Pine Beetle

Whether and how mutualisms are maintained through ecological and evolutionary time is a seldom studied aspect of bark beetle–fungal symbioses. All bark beetles are associated with fungi and some species have evolved structures for transporting their symbiotic partners. However, the fungal assemblages and specificity in these symbioses are not well known. To determine the distribution of fungi associated with the mycangia of the western pine beetle (Dendroctonus brevicomis), we collected beetles from across the insect’s geographic range including multiple genetically distinct populations. Two fungi, Entomocorticium sp. B and Ceratocystiopsis brevicomi, were isolated from the mycangia of beetles from all locations. Repeated sampling at two sites in Montana found that Entomocorticium sp. B was the most prevalent fungus throughout the beetle’s flight season, and that females carrying that fungus were on average larger than females carrying C. brevicomi. We present evidence that throughout the flight season, over broad geographic distances, and among genetically distinct populations of beetle, the western pine beetle is associated with the same two species of fungi. In addition, we provide evidence that one fungal species is associated with larger adult beetles and therefore might provide greater benefit during beetle development. The importance and maintenance of this bark beetle–fungus interaction is discussed.     

Ectomycorrhizal fungal succession in mixed temperate forests

Ectomycorrhizal (ECM) fungal communities of Douglas-fir (Pseudotsuga menziesii) and paper birch (Betula papyrifera) were studied along a chronosequence of forest development after stand-replacing disturbance. Previous studies of ECM succession did not use molecular techniques for fungal identification or lacked replication, and none examined different host species. Four age classes of mixed forests were sampled: 5-, 26-, 65-, and 100-yr-old, including wildfire-origin stands from all four classes and stands of clearcut origin from the youngest two classes. Morphotyping and DNA sequences were used to identify fungi on ECM root tips. ECM fungal diversities were lower in 5-yr-old than in older stands on Douglas-fir, but were similar among age classes on paper birch. Host-specific fungi dominated in 5-yr-old stands, but host generalists were dominant in the oldest two age classes. ECM fungal community compositions were similar in 65- and 100-yr-old stands but differed among all other pairs of age classes. Within the age range studied, site-level ECM fungal diversity reached a plateau by the 26-yr-old age class, while community composition stabilized by the 65-yr-old class. Simple categories such as 'early stage', 'multi stage', and 'late stage' were insufficient to describe fungal species' successional patterns. Rather, ECM fungal succession may be best described in the context of stand development.     

Antimicrobial endophytic fungal assemblages inhabiting bark of <Emphasis Type="Italic">Taxus baccata</Emphasis> L. of Indo-Burma mega biodiversity hotspot

Fungal endophytes were isolated from inner bark of Taxus baccata L., an important source of potent anticancer drug taxol. Bark samples were collected from two locations of Arunachal Pradesh, India, part of the Indo-Burma mega biodiversity hotspot, during two seasons i.e. monsoon and winter. Altogether 77 fungal strains representing 18 genera were isolated from T. baccata bark during the present investigation. The colonizing frequency was recorded as 38.5% and the fungal community comprised of 78% of Hyphomycetes, 5.2% of Coelomycetes, 2.6% of Zygomycetes and Ascomycetes and 9.1% of sterile mycelia. Most common and frequently isolated genera were Fusarium, Penicillium and Aspergillus. Simpson and Shannon diversity indices indicated higher species diversity during monsoon than during winter seasons irrespective of the locations. The two locations harbored 5 to 37 endophyte species and the similarity index was low during winter and high during monsoon. Ethyl acetate extract of fermentation cultures of these fungi were tested for their antimicrobial activity against a panel of human pathogenic Gram-positive and Gram-negative bacteria and fungi. Fifteen fungal isolates out of the isolated strains displayed antimicrobial activity. An endophytic fungus, identified as Fusarium sp. displayed significant antimicrobial activity against all the test pathogens.     

Patterns of fungal diversity in New Zealand Nothofagus forests

The development of protocols for the conservation of fungi requires knowledge of the factors controlling their distribution, diversity, and community composition. Here we compare patterns of variation in fungal communities across New Zealand's Nothofagus forests, reportedly the most myco-diverse in New Zealand and hence potentially key to effective conservation of fungi in New Zealand. Diversity of leaf endophytic fungi, as assessed by culturing on agar plates, is assessed for three Nothofagus sp. growing in mixed stands from four sites. Host species was found to have a greater influence on fungal community assemblage than site. The leaf endophyte communities associated with Nothofagus solandri and Nothofagus fusca (both Nothofagus subgenus Fuscopora), were more similar to each other than either were to the community associated with Nothofagus menziesii (Nothofagus subgenus Lophozonia). The broad taxonomic groups isolated, identified on the basis of internal transcribed spacer (ITS) sequences, were similar to those found in similar studies from other parts of the world, and from an earlier study on the endophyte diversity in four podocarp species from New Zealand, but there were few matches at species level. Average levels of endophyte species diversity associated with single Nothofagus species and single podocarp species were similar, despite historical literature and collection data recording more than twice as many fungal species on average from the Nothofagus species. The significance of these findings to fungal conservation is discussed.     

Ophiostomatoid fungi associated with hardwood-infesting bark and ambrosia beetles in Poland: Taxonomic diversity and vector specificity

Fungi in the orders Ophiostomatales and Microascales (Ascomycota), often designated as ophiostomatoid fungi, are frequent associates of scolytine bark and ambrosia beetles that colonize hardwood and coniferous trees. Several species, e.g., Ophiostoma novo-ulmi, are economically damaging pathogens of trees. Because little is known regarding the ophiostomatoid fungi in Europe, we have explored the diversity of these fungi associated with hardwood-infesting beetles in Poland. This study aims to clarify the associations between fungi in the genera Ambrosiella, Graphium (Microascales), Graphilbum, Leptographium, Ophiostoma and Sporothrix (Ophiostomatales) and their beetle vectors in hardwood ecosystems. Samples associated with 18 bark and ambrosia beetle species were collected from 11 stands in Poland. Fungi were isolated from adult beetles and galleries. Isolates were identified based on morphology, DNA sequence comparisons for five gene regions (ITS, LSU, ßT, TEF 1-α, and CAL) and phylogenetic analyses. In total, 36 distinct taxa were identified, including 24 known and 12 currently unknown species. Several associations between fungi and bark and ambrosia beetles were recorded for the first time. In addition, associations between Dryocoetes alni, D. villosus, Hylesinus crenatus, Ernoporus tiliae, Pteleobius vittatus and ophiostomatoid fungi were reported for the first time, and Sporothrix eucastanea was reported for the first time outside of the USA. Among the species of Ophiostomatales, 14 species were in Ophiostoma s. l., two species were in Graphilbum, nine species were in Sporothrix, and seven species were in Leptographium s. l. Among the species of Microascales, three species were in Graphium, and one was in Ambrosiella. Twenty taxa were present on the beetles and in the galleries, twelve only on beetles, and four only in galleries. Bark and ambrosia beetles from hardwoods appear to be regular vectors, with ophiostomatoid fungi present in all the beetle species. Most ophiostomatoid species had a distinct level of vector/host specificity, although Ophiostoma quercus, the most frequently encountered species, also had the greatest range of beetle vectors and tree hosts. Plant pathogenic O. novo-ulmi was found mainly in association with elm-infesting bark beetles (Scolytus multistriatus, S. scolytus, and P. vittatus) and occasionally with H. crenatus on Fraxinus excelsior and with Scolytus intricatus on Quercus robur.