Mutual interactions between an invasive bark beetle and its associated fungi

Interactions between invasive insects and their fungal associates have important effects on the behavior, reproductive success, population dynamics and evolution of the organisms involved. The red turpentine beetle (RTB), Dendroctonus valens LeConte (Coleoptera: Scolytinae), an invasive forest pest in China, is closely associated with fungi. By carrying fungi on specialized structures in the exoskeleton, RTB inoculates fungi in the phloem of pines (when females dig galleries for egg laying and when males join them for mating). After eggs hatch, larvae gregariously feed on the phloem colonized by the fungi. We examined the effects of five isolates of RTB associated fungi (two from North America, Leptographium terebrantis and L. procerum, and three from China, Ophiostoma minus, L. sinoprocerum and L. procerum) on larval feeding activity, development and mortality. We also studied the effects of volatile chemicals produced in the beetle hindgut on fungal growth. Ophiostoma minus impaired feeding activity and reduced weight in RTB larvae. Leptographium sinoprocerum, L. terebrantis and L. procerum did not dramatically influence larval feeding and development compared to fungi-free controls. Larval mortality was not influenced by any of the tested fungi. Hindgut volatiles of RTB larvae, verbenol, myrtenol and myrtenal, inhibited growth rate of all the fungi. Our results not only show that D. valens associated fungus, O. minus, can be detrimental to its larvae; but, most importantly, they also show that these notorious beetles have an outstanding adaptive response evidenced by the ability to produce volatiles that inhibit growth of harmful fungus.     

Distinguishing Ophiostoma ips and Ophiostoma montium,two bark beetle-associated sapstain fungi

Two synonymous sapstain species, Ophiostoma montium and Ophiostoma ips, which are vectored by Dendroctonus ponderosae and various bark beetles, respectively, were differentiated into separate species using growth and molecular characteristics. Analysis of 32 isolates of the two species from different countries showed that O. ips was able to grow at 35 degrees C while O. montium was not. This growth-based differentiation was strongly supported by sequence data for the internal transcribed spacer (ITS), 5.8S and partial 28S rDNA, and the beta-tubulin genes. The beta-tubulin gene sequence data indicate that the two species can easily be differentiated with a single polymerase chain reaction (PCR) assay.     

Ophiostomatoid fungi associated with the northern spruce engraver, <Emphasis Type="Italic">Ips perturbatus</Emphasis>, in western Canada

A number of ophiostomatoid fungi were isolated from the spruce-infesting bark beetle, Ips perturbatus and its galleries collected from felled spruce trees and logs in northern BC and the Yukon Territory. Isolates were identified to species using morphological characteristics, nuclear ribosomal DNA and partial β-tubulin gene sequences. Thirteen morphological and phylogenetic species were identified among the isolates. Leptographium fruticetum, Leptographium abietinum, Ophiostoma bicolor, Ophiostoma manitobense, O. piceaperdum, and eight undescribed species of the genus Ophiostoma and the anamorph genera Leptographium, Hyalorhinocladiella, Ambrosiella and Graphium. A number of these species, i.e. L. fruticetum, Hyalorhinocladiella sp. 2, O. bicolor and O. manitobense, were isolated repeatedly from I. perturbatus, while others, i.e. Graphium sp. 1 and O. piceaperdum, seemed to be␣sporadic associates. Among all the isolates, L. fruticetum had the highest relative dominance in this survey. A high frequency of occurrence of this species with the beetle may indicate a specific relationship between the two partners.     

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.     

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.     

Leptographium pruni, sp. nov. from bark beetle-infested Prunus jamasakura in Japan

Leptographium species are anamorphs of Ophiostoma, commonly isolated from conifer. There are, however, a small number of these fungi that have been collected from angiosperm hosts. In this study, we describe Leptographium pruni, sp. nov. isolated from the bark of Prunus jamasakura infested by the bark beetle Polygraphus ssiori. This new species is unusual in having a distinct Sporothrix synanamorph with ramoconidia. No evidence of a teleomorph was found, but a high level of tolerance to the antibiotic cycloheximide and the presence of a Sporothrix synanamorph suggest that L. pruni is an Ophiostoma anamorph. Analysis of sequence data for the domain 1 region of the LSUrDNA operon also supports the phylogenetic relationship of L. pruni with Ophiostoma. In addition, sequence data suggest that L. pruni is related to other species of Leptographium rather than Pesotum species with distinct Sporothrix synanamorphs.     

Large Shift in Symbiont Assemblage in the Invasive Red Turpentine Beetle

Changes in symbiont assemblages can affect the success and impact of invasive species, and may provide knowledge regarding the invasion histories of their vectors. Bark beetle symbioses are ideal systems to study changes in symbiont assemblages resulting from invasions. The red turpentine beetle (Dendroctonus valens) is a bark beetle species that recently invaded China from its native range in North America. It is associated with ophiostomatalean fungi in both locations, although the fungi have previously been well-surveyed only in China. We surveyed the ophiostomatalean fungi associated with D. valens in eastern and western North America, and identified the fungal species using multi-gene phylogenies. From the 307 collected isolates (147 in eastern North America and 160 in western North America), we identified 20 species: 11 in eastern North America and 13 in western North America. Four species were shared between eastern North America and western North America, one species (Ophiostoma floccosum) was shared between western North America and China, and three species (Grosmannia koreana, Leptographium procerum, and Ophiostoma abietinum) were shared between eastern North America and China. Ophiostoma floccosum and O. abietinum have worldwide distributions, and were rarely isolated from D. valens. However, G. koreana and L. procerum are primarily limited to Asia and North America respectively. Leptographium procerum, which is thought to be native to North America, represented >45% of the symbionts of D. valens in eastern North America and China, suggesting D. valens may have been introduced to China from eastern North America. These results are surprising, as previous population genetics studies on D. valens based on the cytochrome oxidase I gene have suggested that the insect was introduced into China from western North America.     

Leptographium piriforme sp. nov., from a taxonomically diverse collection of arthropods collected in an aspen-dominated forest in western Canada

During a survey of fungi associated with arthropods collected in a southern boreal mixed-wood forest in Alberta we obtained 29 isolates of a unique species of Leptographium. This species displayed a distinct combination of characteristics, including curved conidia on short-stipitate conidiophores, a secondary micronematous conidial state, stalked pear-shaped cells and an optimal growth rate at 35 C, and is described as Leptographium piriforme sp. nov. The isolates were most similar morphologically to L. crassivaginatum, but ITS sequence comparisons indicate that our isolates cannot be assigned to this or any other sequenced species in the genus. Initial observations on the pear-shaped cells in feeding experiments with Sancassania berlesei show that these structures may act as a nutritional incentive for visiting arthropods. Most arthropods carrying this new species were caught in traps baited with dung which, in light of its optimum growth temperature, suggests a coprophilous phase in the life cycle of this species. Additional isolates from woody species typical of the survey area might clarify whether Leptographium piriforme in its forest habitat occurs as a plant pathogen or saprobe.     

Interactions of Hylastes species (Coleoptera: Scolytidae) with Leptographium species associated with loblolly pine decline

Hylastes spp. (Coleoptera: Scolytidae) were evaluated as potential vectors of Leptographium spp. fungi. Bark beetles were trapped from stands ofloblolly pine, Pinus taeda L., exhibiting a range of decline symptoms in central Alabama. Under controlled conditions, field-collected adult Hylastes salebrosus Eichoff (Coleoptera: Scolytidae) and Hylastes tenuis Eichoff (Coleoptera: Scolytidae), which had been surface-sterilized and inoculated with Leptographium terebrantis Barras & Perry and Leptographium serpens (Goid.) Wingfield, transmitted the fungi into 100% of wounded and unwounded loblolly root sections with which they were confined. None of the sterilized and uninoculated beetles transmitted any Leptographium spp. to roots. Significantly more H. salebrosus and H. tenuis brood emerged from roots infected with Leptographium species than from sterile roots, indicating an enhancement of Hylastes reproduction.     

In Vitro Interactions Between Yeasts and Bacteria and the Fungal Symbionts of the Mountain Pine Beetle (Dendroctonus ponderosae)

Multi-trophic interactions between prokaryotes, unicellular eukaryotes, and ecologically intertwined metazoans are presumably common in nature, yet rarely described. The mountain pine beetle, Dendroctonus ponderosae, is associated with two filamentous fungi, Grosmannia clavigera and Ophiostoma montium. Other microbes, including yeasts and bacteria, are also present in the phloem, but it is not known whether they interact with the symbiotic fungi or the host beetle. To test whether such interactions occur, we performed a suite of in vitro assays. Overall, relative yield of O. montium grown with microbes isolated from larval galleries was significantly greater than when the fungus was grown alone. Conversely, the yield of G. clavigera grown with these same microbes was less than or equal to when it was grown alone, suggesting that O. montium, and at least some microbes in larval galleries, have a mutualistic or commensal relationship, while G. clavigera and those same microbes have an antagonistic relationship. A bacterium isolated from phloem not colonized by beetles was found to inhibit growth of both G. clavigera and O. montium and appears to be an antagonist to both fungi. Our results suggest that bacteria and yeasts likely influence the distribution of mycangial fungi in the host tree, which, in turn, may affect the fitness of D. ponderosae.     

Three new species of Ophiostomatales from <Emphasis Type="Italic">Nothofagus</Emphasis> in Patagonia

The Ophiostomatales (Ascomycota) include mainly insect and mite-associated fungi, the majority of which are found on trees. Very little is known regarding the occurrence or diversity of these fungi in South America. The aim of this study was to consider their occurrence on native Nothofagus trees in the Patagonian Andes of Argentina. Isolates were collected in national parks and provincial reserves in Patagonia between 2009 and 2011. These were grouped based on morphology, and 22 representative isolates were included in phylogenetic analyses based on sequence data of multiple loci (LSU, ITS, beta-tubulin and translation elongation factor-1 alpha genes). The isolates could be assigned to ten different taxa, and included eight species of Ophiostoma s. l., one species of Leptographium, and one species in the Sporothrix lignivora complex. Three of the species are described as new, including Ophiostoma patagonicum, Leptographium gestamen, and Sporothrix cabralii. Ophiostoma quercus and O. noveae-zelandiae are reported for the first time from Argentina, and we show that the latter species is distinct from O. pluriannulatum, in contrast to a previous suggestion that they represent the same taxon.     

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.     

Fungal associates of the lodgepole pine beetle, <Emphasis Type="Italic">Dendroctonus murrayanae</Emphasis>

Bark beetles are well known vectors of ophiostomatoid fungi including species of Ophiostoma, Grosmannia and Ceratocystis. In this study, the most common ophiostomatoid fungi associated with the lodgepole pine beetle, Dendroctonus murrayanae, were characterized. Pre-emergent and post-attack adult beetles were collected from lodgepole pines at four sites in British Columbia, Canada. Fungi were isolated from these beetles and identified using a combination of morphology and DNA sequence comparisons of five gene regions. In all four populations, Grosmannia aurea was the most common associate (74–100% of all beetles) followed closely by Ophiostoma abietinum (29–75%). Other fungi isolated, in order of their relative prevalence with individual beetles were an undescribed Leptographium sp. (0–13%), Ophiostoma ips (0–15%), Ophiostoma piliferum (0–11%), a Pesotum sp. (0–11%) and Ophiostoma floccosum (0–1%). Comparisons of the DNA sequences of Leptographium strains isolated in this study, with ex-type isolates of G. aurea, Grosmannia robusta, Leptographium longiclavatum, and Leptographium terebrantis, as well as with sequences from GenBank, revealed a novel lineage within the Grosmannia clavigera complex. This lineage included some of the D. murrayane isolates as well as several isolates from previous studies referred to as L. terebrantis. However, the monophyly of this lineage is not well supported and a more comprehensive study will be needed to resolve its taxonomic status as one or more novel taxa.     

Differentiation of mitochondrial subgroups in the Verticillium lecanii species complex

Presumptive mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs) were obtained from 54 fungal isolates identified as Verticillium lecanii, V. psalliotae or ' V. lecanii -like'. Analysis of the mtDNA RFLPs showed 20 different patterns, indicating considerable genetic variation within the V. lecanii species complex. There was no direct correlation between host and mtDNA pattern, or between mtDNA patterns and previously described isoenzyme-defined specific groups. Isolates from tropical and subtropical areas showed considerable variation in genotypes, while isolates from temperate regions appeared less variable.     

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.     

Ophiostomatoid fungi including two new fungal species associated with pine root-feeding beetles in northern Spain

Many bark beetles live in a symbiosis with ophiostomatoid fungi but very little is known regarding these fungi in Spain. In this study, we considered the fungi associated with nine bark beetle species and one weevil infesting two native tree species (Pinus sylvestris and Pinus nigra) and one non-native (Pinus radiata) in Cantabria (Northern Spain). This included examination of 239 bark beetles or their galleries. Isolations yielded a total of 110 cultures that included 11 fungal species (five species of Leptographium sensu lato including Leptographium absconditum sp. nov., five species of Ophiostoma sensu lato including Ophiostoma cantabriense sp. nov, and one species of Graphilbum). The most commonly encountered fungal associates of the bark beetles were Grosmannia olivacea, Leptographium procerum, and Ophiostoma canum. The aggressiveness of the collected fungal species was evaluated using inoculations on two-year-old P. radiata seedlings. Leptographium wingfieldii, Leptographium guttulatum, and Ophiostoma ips were the only species capable of causing significant lesions.     

Evolutionary Dynamics of the mS952 Intron: A Novel Mitochondrial Group II Intron Encoding a LAGLIDADG Homing Endonuclease Gene

Examination of the mitochondrial small subunit ribosomal RNA (rns) gene of five species of the fungal genus Leptographium revealed that the gene has been invaded at least once at position 952 by a group II intron encoding a LAGLIDADG homing endonuclease gene. Phylogenetic analyses of the intron and homing endonuclease sequences indicated that each element in Leptographium species forms a single clade and is closely related to the group II intron/homing endonuclease gene composite element previously reported at position 952 of the mitochondrial rns gene of Cordyceps species and of Cryphonectria parasitica. The results of an intron survey of the mt rns gene of Leptographium species superimposed onto the phylogenetic analysis of the host organisms suggest that the composite element was transmitted vertically in Leptographium lundbergii. However, its stochastic distribution among strains of L. wingfieldii, L. terebrantis, and L. truncatum suggests that it has been horizontally transmitted by lateral gene transfer among these species, although the random presence of the intron may reflect multiple random loss events. A model is proposed describing the initial invasion of the group II intron in the rns gene of L. lundbergii by a LAGLIDADG homing endonuclease gene and subsequent evolution of this gene to recognize a novel DNA target site, which may now promote the mobility of the intron and homing endonuclease gene as a composite element.     

Characterization of diversity in Colletotrichum acutatum sensu lato by sequence analysis of two gene introns, mtDNA and intron RFLPs, and mating compatibility

A diverse collection of isolates identified as Colletotrichum acutatum, including a range of fruit-rot and foliar pathogens, was examined for mtDNA RFLPs and RFLPs and sequence variation of a 900-bp intron of the glutamine synthetase (GS) gene and a 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase (GPDH) gene. RFLPs of mtDNA, RFLPs of the 900-bp GS intron and sequence analysis of each intron identified the same seven distinct molecular groups, or clades, within C. acutatum sensu lato. Sequence analysis produced highly concordant tree topologies with definitive phylogenetic relationships within and between the clades. The clades might represent phylogenetically distinct species within C. acutatum sensu lato. Mating tests also were conducted to assess sexual compatibility with tester isolates known to outcross to form the teleomorph Glomerella acutata. Mating compatibility was identified within one clade, C, and between two phylogenetically distinct clades, C and J4. The C clade represented isolates from a wide range of hosts and geographic origins. J4 clade contained isolates from Australia or New Zealand recovered from fruit rot and pine seedlings with terminal crook disease. That isolates in two phylogenetically distinct clades were capable of mating suggests that genetic isolation occurred before reproductive isolation. No other isolates were sexually compatible with the mating testers, which also were in groups C and J4. Certain clades identified by mtDNA and intron analysis (D1, J3 and J6) appeared to represent relatively host-limited populations. Other clades (C1, F1 and J4) contained isolates from a wide range of hosts. Isolates described as C. acutatum f. sp. pineum were clearly polyphyletic.