Distinguishing homokaryons and heterokaryons in Phellinus sulphurascens using pairing tests and ITS polymorphisms

Phellinus sulphurascens Pilát causes laminated root rot of coniferous species in both western North America (WNA) and Asia. Accurate somatic incompatibility tests for mapping population structures have been difficult to conduct for P. sulphurascens because no single, unambiguous criterion has allowed differentiation of homokaryotic and heterokaryotic isolates. In a population study of P. sulphurascens in WNA, two types of ITS sequences were found in the single spore and vegetative isolates. All single spore isolates (SSIs) had either ITS type-1 or type-2 whereas some vegetative isolates had both ITS types. The segregation pattern for inheritance of ITS, which we observed in SSIs from eight basidiocarps, suggested that each ITS type occurred in a different nucleus and that each basidiospore inherited only one ITS type. In four SSIs from Russia and eight heterokaryotic isolates from Japan, nine different ITS types, referred to as type-3 to -11, were detected. A variety of pairing tests conducted between known Asian and WNA homokaryon and heterokaryon isolates did not always give consistent results with respect to fungal mat morphologies and formation of demarcation lines. However, the ITS types that occurred after pairing tests did follow consistent patterns. Thus, using ITS polymorphisms and pairing tests between Asian tester isolates and 49 vegetative isolates from WNA, we were able to accurately distinguish between homokaryotic and heterokaryotic isolates.     

Interleukin-23 is critical for full-blown expression of a non-autoimmune destructive arthritis and regulates interleukin-17A and RORγt in γδ T cells

Introduction  

Interleukin (IL)-23 is essential for the development of various experimental autoimmune models. However, the role of IL-23 in non-autoimmune experimental arthritis remains unclear. Here, we examined the role of IL-23 in the non-autoimmune antigen-induced arthritis (AIA) model. In addition, the regulatory potential of IL-23 in IL-17A and retinoic acid-related orphan receptor gamma t (RORγt) expression in CD4⁺ and TCRγδ⁺ T cells was evaluated systemically as well as at the site of inflammation.     

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.     

A new Leptographium species associated with the northern spruce engraver, Ips perturbatus, in western Canada

An undescribed Leptographium species was isolated from the spruce-infesting bark beetle Ips perturbatus collected from felled spruce trees and logs in northern British Columbia and Yukon Territory. Morphologically, this fungus is similar to L. abietinum and L. hughesii but differed in a number of characteristics (e.g. the arrangement of its conidiophores). The fungus grew optimally at 25 C on 2% malt-extract agar and showed a high level of tolerance to cycloheximide. Comparison of rDNA and beta-tubulin gene sequences also confirmed that this Leptographium species represents an undescribed taxon. Thus we described it as a new species, Leptographium fruticetum sp. nov.     

HistoChoice® as an alternative to formalin fixation of undecalcified bone specimens

We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice®. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice® resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice® produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visuralized more easily in HistoChoice® fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice® fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice® fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice®. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice® yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling.     

Proliferative activity in the cerebellar external granular layer evaluated by bromodeoxyuridine labeling

We have optimised an indirect immunoperoxidase technique demonstrating bromodeoxyuridine (BrdU) incorporation into dividing cells for cerebellar tissue sections of four-day-old rats injected with this marker. This permits confident identification of granule-cell precursors engaged in DNA synthesis in the external granular layer of the developing cerebellum. Preservation of BrdU immunoreactivity is attained using methanol/acetic acid fixation and different pretreatments before immunostaining, while unlabeled nuclei can be recognized clearly after Feulgen or hematoxylin counterstaining. We established conditions to ensure satisfactory BrdU uptake without affecting cell-cycle progression during the postlabeling time period. The dose of BrdU employed provides saturation S-phase labeling from at least 1 h after BrdU delivery. Various kinetic parameters and phase durations have been determined in experiments involving a single injection or cumulative labeling sequences, and the cycle time was calculated based on two models of generative behavior: steady-state and exponential growth. The working hypothesis of steadystate kinetics can be adopted successfully if the existence of neuroblasts with different proliferation rates is taken into account.     

Monoclonal antibodies to Gliocladium roseum, a potential biological control fungus of sap-staining fungi in wood.

Immunological probes were developed to discriminate between a potential biological control fungus and sap-staining fungi present in wood. This paper describes the production of monoclonal antibodies to isolated cell wall fragments of the biological control fungus Gliocladium roseum. Two monoclonals, designated 6A5 and 3F12, were characterized. Their specificity was assessed by ELISA, by immunogold silver staining light microscopy, by immunogold electron microscopy, and by immunoblotting. Monoclonal 6A5 specifically recognized G. roseum and closely related species and did not react with any of 21 sap-staining fungi tested. Monoclonal 3F12 recognized most of the biological control fungi tested and also showed reactivity with two of the 21 sap-staining fungi. Both monoclonals appeared to recognize carbohydrate epitopes of the cell wall in G. roseum. Although the antibodies were produced against the cell wall of fungus grown in liquid culture, they also detected specific fungi in wood and, therefore, can be used for studies of wood colonization by fungi and for investigations of the interactions between different fungi growing on wood.     

A comparison of two xylanases from the thermophilic fungi Thielavia terrestris and Thermoascus crustaceus

Two thermophilic xylanases (xylanase II from Thielavia terrestris 255B and the 32-kDa xylanase from Thermoascus crustaceus 235E) were studied to determine if they had different and complementary modes of action when they hydrolysed various types of xylans. Partial amino acid sequencing showed that these two enzymes belonged to different families of -1,4-glycanases. Xylanase II achieved faster solubilization of insoluble xylan whereas the 32-kDa xylanase was more effective in producing xylose and short xylooligomers. An assessment of the combined hydrolytic action of the two xylanases did not reveal any co-operative action. The sugars released when the two thermophilic xylanases were used together were almost identical to those released when the 32-kDa xylanase acted alone. The two xylanases were able to remove about 12% of the xylan remaining in an aspen kraft pulp. This indicated that either one of these thermophilic enzymes may be useful for enhancing the bleaching of kraft pulps. Correspondence to: J. N. Saddler