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.     

Leptographium terebrantis sp. nov. associated with dendroctonus terebrans in loblolly pine

ALeptographium fungus associated with the black turpentine beetleDendroctonus terebrans (Olivier) is described for the first time. The nameLeptographium terebrantis sp. nov. is proposed.

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Zusammenfassung Der Pilz,Leptographium, der mit dem schwarzen Terpentinkäfer,Dendroctonus terebrans (Olivier) vergesellschaftet ist, wird das erste Mal beschrieben. Für den Pilz wird die BenennungLeptographium terebrantis vorgeschlagen.

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The authors are research entomologist and biological aide (microbiology) with the Forest Insect Research Project, at the Southern Forest Experiment Station, USDA Forest Service, Pineville, Louisiana.     

Corticioid basidiomycetes associated with bark beetles,including seven new Entomocorticium species from North America and Cylindrobasidium ipidophilum,comb. nov

Antonie van Leeuwenhoek - Seven new Entomocorticium species (Peniophoraceae) are described based on morphology and phylogenetic analyses. Along with the type species (E. dendroctoni),...     

Leptographium tereforme sp. nov. and other Ophiostomatales isolated from the root-feeding bark beetle Hylurgus ligniperda in California

The redhaired pine bark beetle Hylurgus ligniperda (F.) is native to Europe but was discovered in Los Angeles, California, in 2003. This root-and stump-feeding beetle is a common vector of Ophiostomatales, which are potential tree pathogens or causes of blue stain of conifer sapwood. In this study Ophiostomatales were isolated on a cycloheximide-amended medium from 118 adult H. ligniperda collected from infested logs of Pinus halepensis and P. pinea at two sites in California. In total eight species of Ophiostomatales were identified and seven species that occasionally were isolated were unidentified. The most frequently isolated species were Ophiostoma ips and Grosmannia galeiforme, which were isolated respectively from 31% and 23% of the 118 beetles. The other species isolated included O. piceae (isolated from 9% of the beetles), O. querci (8%) and Leptographium tereforme sp. nov. (6%). Grosmannia huntii, L. serpens, three Sporothrix species, O. floccosum, O. stenoceras, two unidentified Hyalorhinocladiella sp. and a sterile fungus each were isolated from fewer then 5% of beetles. Most of the identified species already were known in USA and have been found in association with H. ligniperda in other countries. However the new species, L. tereforme, and G. galeiforme were recorded from USA for the first time, and this is the first report of L. serpens from western North America.     

Leptographium wushanense sp. nov., associated with Tomicus armandii on Pinus armandii in Southwestern China

Leptographium wushanense sp. nov., which is associated with Tomicus armandii, is described based on molecular sequence data and morphological characters. The phylogenetic analyses based on ITS2-partial LSU rDNA region, β-tubulin and elongation factor 1-α genes showed that L. wushanense formed a well-supported clade and was closely related to L. conjunctum and L. yunnanense, and then nested within L. lundbergii complex. The species is characterized that its conidiophores were longer than those of L. yunnanense and shorter than those of L. conjunctum, while its conidia were larger than those of L. conjunctum and smaller than those of L. yunnanense. Furthermore, the colonial growth of L. wushanense is faster than those of L. conjunctum and L. yunnanense at 25 °C on 2% MEA. Both molecular data and morphological comparison confirmed the placement of the new species in genus of Leptographium.     

Leptographium pini-densiflorae sp. nov. from Japanese red pine

ALeptographium species was isolated from deadPinus densiflora at six sites in Japan. The fungus is morphologically most similar toL. lundbergii but could be distinguished from that species by its short stipes, primary branches of conidiophores, and conidia with a rounded to sub-truncate base. In addition, the colony morphology, growth rate and tolerance to the antibiotic cycloheximide of theLeptographium species andL. lundbergii differed markedly. Here we describe the fungus as a new species,Leptographium pini-densiflorae. Contribution No. 144, Laboratory of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

Leptographium sinoprocerum sp. nov., an undescribed species associated with Pinus tabuliformis-Dendroctonus valens in northern China

During a study of ophiostomatoid fungi associated with the invasive pest Dendroctonus valens in the Pinus tabuliformis ecosystem in northern China, a multigenic (ITS2-LSU, beta-tubulin and EF1-alpha) phylogenetic analysis and examination of morphological features revealed in addition to Leptographium procerum the occurrence of an undescribed species. The new species, Leptographium sinoprocerum, belongs to the L. procerum-L. profanum clade. Both L. procerum and L. sinoprocerum are similar to each other and occur sympatrically in the ecosystem studied. Nevertheless L. sinoprocerum can be distinguished from L. procerum by shorter conidiophore stipes arising from both submerged and aerial hyphae, slightly more oblong conidia, a granular ornamentation on the submerged hyphae and dark olivaceous colonies on MEA.     

Nitrogen-fixing bacteria associated with bark beetles

Nitrogen-fixingEnterobacter agglomerans andEnterobacter spp. were consistently isolated from the bark beetleDendroctonus terebrans. Large populations of nitrogen-fixing bacteria were found with the beetle, although no in situ acetylene reduction was demonstrated. The constant occurrence of nitrogenfixing bacteria withD. terebrans suggests a symbiotic relationship. Nitrogen-fixing bacteria were also isolated from the bark beetlesDendroctonus frontalis andIps avulsus.Mention of trade names is not an endorsement by the U.S. Department of Agriculture.     

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.     

Isolation and characterization of Beauveria spp. associated with exotic bark beetles in New Zealand Pinus radiata plantation forests

A survey of entomopathogenic fungi associated with Hylastes ater and Hylurgus ligniperda was undertaken in six Pinus radiata forest sites in New Zealand. In each site soil, bark and frass associated with beetle galleries were sampled, as well as a selection of live beetles, larvae and mycosed cadavers. The density of Beauveria spp. (as measured by colony forming units) varied both within and between the sites and substrates sampled. The highest CFU density detected was 4 × 10⁶ CFU/g dry frass, which was collected from a stump at the Mahurangi site in the North Island. A number of isolates were selected and characterized using PCR amplification of a terminal region of the EF1-α fragment. This analysis revealed that two Beauveria species, Beauveria caledonica and Beauveria bassiana, were commonly associated with bark beetle habitat. B. caledonica was the only species isolated from mycosed beetles, and was also recovered from soils, bark and frass, though not live insects. B. bassiana was also found in soils, bark, frass and live insects; however, it was not isolated from mycosed cadavers in this study. It is speculated that B. caledonica is the common ‘natural’ pathogen of bark beetles in their breeding habitat. A third species, Beauveria malawiensis, was isolated once from soil. This is the first report of this species from New Zealand. Thirteen isolates (selected from all substrates sampled and live beetles) were selected for laboratory bioassays. Under laboratory conditions all isolates were pathogenic to H. ater and H. ligniperda adults; there was little difference among the isolates in terms of efficacy despite the paucity of B. bassiana infection observed in the field. The mechanisms behind the lack of field infection observed for B. bassiana are not known, but are likely to be linked to limiting environmental factors in the breeding sites. These results indicate potential risks associated with the selection of strains for use in biological control programmes solely on the basis of laboratory bioassays, especially if the mode of action and biology of the species used are not fully understood. The importance of selecting strains which are environmentally competent is evident. Overall, however, this study demonstrates that entomopathogenic fungi can play a significant role in the regulation of H. ater and H. ligniperda populations in pine plantations.     

Infections with the ascomycete fungus Metschnikowia typographi sp.nov. in the bark beetles Ips typographus and Ips amitinus (Coleoptera, Scolytidae)

The ascomycete fungus Metschnikowia typographi sp.nov. is described. It infects the spruce bark beetles Ips typographus L. and Ips amitinus Eichl. Masses of vegetative cells and navicular asci (I. typographus 13-17 x 2 microns; I. amitinus 17-22 x 2 microns) were found in cells of the midgut epithelium and in the body cavity of infected beetles. Each ascus contains two needle-shaped ascospores flattened in the central part, 0.5-1.5 x 0.3 x 13-15 microns and pointed at both ends. The parasitic species of Metschnikowia, M. bicuspidata, M. artemiae, M. unicuspidata, M. wickerhami and M. typographi are discussed as a special group of the genus characterized by morphological characters.     

Morphological and ecological similarities: wood-boring beetles associated with novel xylose-fermenting yeasts, Spathaspora passalidarum gen. sp. nov. and Candida jeffriesii sp. nov.

Ascomycete yeasts that both ferment and assimilate xylose were reported previously as associates of insects living in woody substrates. Most notable have been reports of Pichia stipitis-like yeasts that are widely associated with the wood-boring beetle, Odontotaenius disjunctus (Coleoptera: Passalidae), in the eastern United States. Our continuing investigation of insect gut yeasts has lead to the discovery of two new xylose-fermenting yeasts that phylogenetic analysis places as sister taxa. The beetle hosts, O. disjunctus and Phrenapates bennetti (Coleoptera: Tenebrionidae), are similar in habitat and appearance, and the presence of similar gut yeasts is an additional common feature between them. Here we describe the new yeast genus Spathaspora, the type species S. passalidarum, and its sister taxon Candida jeffriesii and discuss their natural history, including a comparison with Pichia stipitis, another member of a guild of xylose-fermenting yeasts with similar metabolic traits. In addition a morphologically distinct yeast ascospore type is described for Spathaspora.     

Metschnikowia noctiluminum sp. nov., Metschnikowia corniflorae sp. nov., and Candida chrysomelidarum sp. nov., isolated from green lacewings and beetles

Fourteen yeast isolates belonging to the Metschnikowia clade were isolated from the digestive tracts of lacewings (Neuroptera: Chrysopidae), soldier beetles and leaf beetles (Coleoptera: Cantharidae and Chrysomelidae), and a caddisfly (Trichoptera: Hydropsychidae). The insect hosts were associated with sugary substances of plants, a typical habitat for yeasts in this clade. Based on DNA sequence comparisons and phenetic characters, the yeasts were identified as Candida picachoensis, Candida pimensis, and four undescribed taxa. Among the undescribed taxa, three yeasts were distinguished from one another and from other described taxa by nucleotide differences in the ribosomal DNA repeat, which were sufficient to consider them as new species. Two of the novel yeast species are described as Metschnikowia noctiluminum (NRRL Y-27753^T) and M. corniflorae spp. nov. (NRRL Y-27750^T) based in part on production of needle-shaped ascospores, which are found in most Metschnikowia species. Sexual reproduction was not observed in the third new yeast, Candida chrysomelidarum sp. nov. (NRRL Y-27749^T). A fourth isolate, NRRL Y-27752, was not significantly distinct from Metschnikowia viticola and Candida kofuensis to be described as a new species. Phylogenetic analysis of the D1/D2 loop sequences placed M. noctiluminum within the M. viticola clade, while C. chrysomelidarum was a sister taxon of Candida rancensis. Metschnikowia corniflorae was phylogenetically distinct from other new species and fell outside of the large-spored Metschnikowia group.     

The Nocardia salmonicida clade, including descriptions of Nocardia cummidelens sp. nov., Nocardia fluminea sp. nov. and Nocardia soli sp. nov.

Large numbers of strains selectively isolated from soil, water and deteriorating vulcanised natural rubber pipe rings were provisionally assigned to the genus Nocardia. Twenty-eight representative isolates were found to have chemical and morphological properties typical of nocardiae. These organisms formed a monophyletic clade in the 16S rDNA tree together with Nocardia salmonicida. Three of the strains, isolates S1, W30 and R89, were distinguished from one another and from representatives of the validly described species of Nocardia using genotypic and phenotypic data. These organisms were considered to merit species status and were named Nocardia cummidelens sp. nov., Nocardia fluminea sp. nov. and Nocardia soli sp. nov. respectively. Additional comparative studies are needed to resolve the finer taxonomic relationships of the remaining isolates assigned to the Nocardia salmonicida clade and to further unravel the extent of nocardial diversity in artificial and natural ecosystems.     

Genetic relationships among Leptographium terebrantis and the mycangial fungi of three western Dendroctonus bark beetles

Morphology, mitochondrial DNA (mtDNA) restriction fragment polymorphisms (RFLPs) and nuclear DNA (nDNA) fingerprinting were used to clarify relationships among the morphologically similar Ophiostoma and Leptographium species associated with mycangia of three Dendroctonus bark beetles (Ophiostoma clavigerum associated with both D. ponderosae and D. jeffreyi, and L. pyrinum associated with D. adjunctus), as well as a closely related nonmycangial bark beetle associate (L. terebrantis). Most isolates of O. clavigerum form long (40-70 μm), septate conidia, while all isolates of L. terebrantis and L. pyrinum form conidia less than 17.0 μm in length. The conidia of L. pyrinum are pyriform, with truncate bases, while the conidia of the other species form only slightly truncate bases. Conidial masses of L. terebrantis are creamy yellow, while the conidial masses of the other species are white. Nuclear DNA fingerprints resulting from probing PstI restrictions with the oligonucleotide probe (CAC)(5) and HaeIII and MspI restrictions of mtDNA, exhibited three major clusters. In the dendrogram developed from mtDNA RFLPs, the L. pyrinum isolates formed one cluster, while the majority of O. clavigerum isolates, including all D. jeffreyi isolates, formed another. A third cluster was composed of all L. terebrantis isolates, as well as several O. clavigerum isolates from D. ponderosae. The inclusion of some O. clavigerum isolates in the L. terebrantis cluster suggests that horizontal transfer of mtDNA has occurred among these fungi. The nDNA dendrogram also exhibited three clusters, and most isolates of L. pyrinum, L. terebrantis and O. clavigerum grouped separately; however, one isolate of O. clavigerum grouped with the L. terebrantis isolates, while one isolate of L. terebrantis grouped with O. clavigerum. No genetic markers were found that distinguished between O. clavigerum associated with D. ponderosae and O. clavigerum associated with D. jeffreyi. Ophiostoma clavigerum might be a recently diverged morphological variant of L. terebrantis, with special adaptations for grazing by young adults of D. jeffreyi and D. ponderosae. The anamorph of O. clavigerum, Graphiocladiella clavigerum, is transferred to Leptographium.