Ophiostomatoid fungi on trees are typically bark beetle associates that cause sapstain in timber and some are pathogens. Very little is known regarding the ophiostomatoid fungi associated with bark beetles in China and the aim of this study was to identify a collection of these fungi with synnematous anamorphs. Micromorphology and DNA sequences of the internal transcribed spacer regions (ITS) of the ribosomal DNA and the partial β-tubulin gene were used for identifications. The isolates could be divided in six morphological groups. DNA sequence comparisons with published data confirmed that these groups represented six species, four in the Ophiostomatales (Sordariomycetidae) and two in the Microascales (Hypocreomycetidae). The majority of these were isolated from conifer hosts. Ophiostoma quercus, O. setosum, Pesotum fragrans (Ophiostomatales) and Graphium pseudormiticum (Microascales) were found on Tsuga dumosa infested by a Pissodes sp. In addition, O. quercus and P. fragrans were found associated with Tomicus yunnanensis on Pinus yunnanensis, P. fragrans with a Pissodes sp. on P. armandi, and O. piceae with Ips subelongatus on Larix olgensis. Only two species, O. quercus and a new species in the Graphium penicilliodes complex, described here as Graphium carbonarium sp. nov., were isolated from Pissodes galleries on Salix babylonica. These results include several new fungus-host and fungusinsect associations, and G. pseudormiticum is reported here for the first time from China.
Leptographium spp. are anamorphs of Grosmannia residing in the order Ophiostomatales. These fungi are typically associated with bark-beetles and are common causal agents of sapstain in lumber and some are important tree pathogens. In this study, Leptographium spp. associated with bark beetles collected during a survey in Jilin and Yunnan provinces of China, were identified. Identifications were achieved using comparisons of morphological characters and DNA sequence data for the ITS2-partial LSU rDNA region, as well as the β-tubulin and EF-1α gene regions. Eight unknown species of Leptographium are recognised and described from conifer and hardwood hosts, associated with beetles including Ips subelongatus, Tomicus yunnanensis, Hylurgops minor, Polygraphus verrucifrons and a Pissodes sp. Six of the new species are morphologically and phylogenetically related to species known to occur in Asia such as G. yunnanense, L. bhutanense, L. bistatum and L. sinoprocerum. The remaining two taxa are related to those in a group containing G. americana and L. abietinum, found in North America. This study also provides the first report of L. pineti on Pinus kesiya in China.
Leptographium species provide an ideal model to test the applications of a PCR microcoding system for differentiating species of other genera of ascomycetes. Leptographium species are closely related and share similar gross morphology. Probes designed for a PhyloChip for Leptographium have been transferred and tested as primers for PCR diagnostic against Leptographium species. The primers were combined with complementary universal primers to identify known and suspected undescribed species of Leptographium. The primer set was optimized for 56 species, including the three varieties of L. wageneri, then blind-tested against 10 random DNA samples. The protocols established in this study successfully identified species from the blind test as well as eight previously undescribed isolates of Leptographium. The undescribed isolates were identified as new species of Leptographium with the aid of the microcoding PCR identification system established in this study. The primers that were positive for each undescribed isolate were used to determine close relatives of these species and some of their biological characteristics. The transfer of oligonucleotides from a micro-array platform to a PCR diagnostic was successful, and the identification system is robust for both known and unknown species of Leptographium.
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