Fine-scale genetic diversity and relatedness in fungi associated with the mountain pine beetle

The mountain pine beetle (MPB; Dendroctonus ponderosae Hopkins, 1902) forms beneficial symbiotic associations with fungi. Here we explored the fine-scale spatial genetic structure of three of those fungi using single nucleotide polymorphism. We found that single mated pairs of beetles carry not only multiple fungal species, but also multiple genotypes of each species into their galleries. We observed genetic diversity at a fine spatial scale. Most of the diversity was found within and among galleries with nonsignificant diversity among trees. We observed clonal propagation almost exclusively within galleries. Ophiostoma montium (Rumbold) Arx possessed a larger expected number of multilocus genotypes and lower linkage disequilibrium than Grosmannia clavigera (Rob.-Jeffr. & R.W. Davidson) Zipfel, Z.W. de Beer & M.J. Wingf. and Leptographium longiclavatum S.W. Lee, J.J. Kim & C. Breuil. More than 80% of fungal samples were genetically unrelated, a result that parallels what has been observed in the beetles. The proportion of genetically related samples within galleries was higher in O. montium (40%) than in G. clavigera (20%) or L. longiclavatum (6%), likely the consequence of within-gallery sexual recombination in O. montium. The underlying genetic diversity reported here and the differences among fungal species could enable the symbiont community to quickly respond to new environmental conditions or changes in the host, enhancing the maintenance of this multipartite relationship and allowing the MPB to colonize new habitats.

Assessment of the impact of symbiont Ophiostomatales (Fungi) on mountain pine beetle (Coleoptera: Curculionidae) performance on a jack pine (Pinaceae) diet using a novelin vitrorearing method

A novel “rearing-tube” method was developed and used to investigate the performance of mountain pine beetle (MPB),Dendroctonus ponderosaeHopkins (Coleoptera: Curculionidae: Scolytinae), with its three main ophiostomatalean fungal symbionts,Grosmannia clavigera(Robinson-Jeffrey and Davidson) Zipfel, de Beer, and Wingfield (Ophiostomataceae),Ophiostoma montium(Rumbold) von Arx (Ophiostomataceae), andLeptographium longiclavatumLee, Kim, and Breuil (Ophiostomataceae). Transparent glass tubes filled with sterile ground jack pine (Pinus banksianaLambert; Pinaceae) phloem and sapwood (9:1 ratio) were used to rear MPB from egg to adult with each fungus under controlled environmental conditions. Mountain pine beetle mortality was higher and development longer in fungus-free controls compared to fungal treatments. Among fungal treatments, insects developed faster, constructed shorter larval galleries, and had fewer supernumerary instars withL. longiclavatum.Insect survival was not affected by fungal treatments. Hyphal extension through the rearing medium was fastest forL. longiclavatum. Phloem nitrogen was reduced significantly by the presence ofL. longiclavatum.Results support the hypothesis that ophiostomatalean symbionts provide benefits to MPB. The rearing-tube method is useful to tease apart confounding interspecific interactions between bark beetles and symbiotic fungus species.

Effects of symbiotic bacteria and tree chemistry on the growth and reproduction of bark beetle fungal symbionts

Bark beetles are associated with diverse assemblages of microorganisms, many of which affect their interactions with host plants and natural enemies. We tested how bacterial associates of three bark beetles with various types of host relationships affect growth and reproduction of their symbiotic fungi. Fungi were exposed to volatiles from bacteria in an arena that imposed physical separation but shared airspace. We also exposed fungi to vapors of the host compound, α-pinene, and to combinations of bacterial volatiles and α-pinene. Bacterial volatiles commonly stimulated growth of Leptographium procerum (W.B. Kendr.) M.J. Wingf. and Grosmannia clavigera (Rob.-Jeffr. & R.W. Davidson) Zipfel, Z.W. de Beer & Wingf., which are symbiotic fungi of Dendroctonus valens LeConte and Dendroctonus ponderosae Hopkins, respectively. They less commonly stimulated growth of Ophiostoma ips (Rumbold) Nannf., which is associated with Ips grandicollis Eichhoff. Some bacteria inhibited L. procerum, Ophiostoma montium (Rumbold) von Arx (another associate of D. ponderosae), and O. ips. Bacteria greatly stimulated spore production of symbionts of D. valens and D. ponderosae. α-Pinene strongly affected some of these relationships, causing amplification, reduction, or reversal of the interactions among the bacteria and fungi. Our results show that some bacteria associated with bark beetles directly affect fungal symbionts and interact with tree chemistry to affect fungal growth and sporulation. The strongest effects were induced by bacteria associated with beetles adapted to attacking living trees with vigorous defenses, and on fungal reproductive structures.

Detection of host habitat by parasitoids using cues associated with mycangial fungi of the mountain pine beetle, Dendroctonus ponderosae

Cues used by parasitoids to detect habitat of the mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae), were investigated by observing parasitoid attraction to logs infested with D. ponderosae, logs inoculated with one or both of the symbiotic fungi of D. ponderosae (Grosmannia clavigera (Rob.-Jeffr. & R.W. Davidson) Zipfel, Z.W. de Beer & M.J. Wingf. (Ophiostomataceae) and Ophiostoma montium (Rumbold) Arx (Ophiostomataceae)), logs containing no beetles or fungi, or empty screen cylinders. Captures of Heydenia unica Cook and Davis (Hymenoptera: Pteromalidae) and Rhopalicus pulchripennis (Crawford) (Hymenoptera: Pteromalidae) on logs with both G. clavigera and O. montium were greater than those from control treatments. These results suggest that characteristics of tree tissues simultaneously colonized by the two symbiotic fungi facilitate a detectable change in the volatile compounds released from D. ponderosae-attacked trees that may be used by parasitoids to locate hosts.

Ophiostomatoid and basidiomycetous fungi associated with green, red, and grey lodgepole pines after mountain pine beetle (Dendroctonus ponderosae) infestation

The mountain pine beetle (MPB) is a major concern for lodgepole pine (Pinus contorta var. latifolia Engelm.) forests in British Columbia, Canada. MPB and the ophiostomatoid staining fungi for which they serve as vector have a close, mutualistic relationship. In this work, we determined which fungi colonized MPB-killed standing trees with green, red, and grey crowns and quantified how rapidly the fungi stained and reduced the moisture content of sapwood. Green trees were mainly colonized by Ophiostoma clavigerum (Robinson-Jeffrey & Davidson) Harrington, Ophiostoma montium (Rumbold) von Arx, Ophiostoma nigrocarpum (Davidson) De Hoog, Ophiostoma minutum (Olchow. & Reid) Hausner, and unknown Leptographium species. In red and grey pines (2 and 3 years after the original MPB attack, respectively), the frequency of the original fungal colonizers decreased, and other sapstaining fungal species were encountered. Among basidiomycetous fungi, decay fungi were rarely present in green trees but were isolated more frequently in red and grey trees. The frequency and the type of decay fungi isolated varied between harvesting sites.

A comparison of mycangial and phoretic fungi of individual mountain pine beetles

Two ophiostomatoid fungi, Ophiostoma clavigerum (Robinson-Jeffrey & Davidson) Harrington and Ophiostoma montium (Rumbold) von Arx, are known to be associated with the mycangia of the mountain pine beetle, Dendroctonus ponderosae Hopkins. However, virtually nothing is known regarding the phoretic fungi carried on the external surface of the exoskeleton of this beetle. In this study, I compared the phoretic fungi of individual D. ponderosae with the fungi carried in their mycangia. As many beetles carried ophiostomatoid fungi on the exoskeleton as in the mycangia; however, the species of ophiostomatoid fungus carried phoretically on an individual beetle was not always the same as was carried in its mycangia. Ophiostoma montium was isolated more often from exoskeletal surfaces than from mycangia, while the reverse was true for O. clavigerum. It appears that O. clavigerum is highly adapted for mycangial dissemination, while O. montium is adapted to phoretic as well as mycangial dissemination. Ophiostoma ips (Rumbold) Nannf. was phoretic on two beetles, indicating that cross-contamination with fungi from cohabiting Ips spp. may sometimes occur. Several non-ophiostomatoid fungi were isolated from exoskeletal surfaces, but none consistently so. All non-ophiostomatoid fungi isolated were common saprophytes often found in beetle-killed trees. Yeasts were also common and were isolated more often from the exoskeleton than from mycangia.

Growth and virulence of mountain pine beetle associated blue-stain fungi, Ophiostoma clavigerum and Ophiostoma montium

The mountain pine beetle (Dendroctonus ponderosae) is commonly associated with the blue-stain fungi Ophiostoma clavigerum and Ophiostoma montium. Ophiostoma clavigerum is the primary invader of sapwood after beetle infestation and is thought to be the most virulent of the two fungi. Growth of these fungi was studied under oxygen-deficient conditions on malt agar in test tubes and Petri dishes. In addition, growth was studied in phloem and sapwood of young living shore pines (Pinus contorta var. contorta) and western white pines (Pinus monticola) inoculated with fungus in low densities (eight inoculations per tree). In test tubes with limited oxygen O. clavigerum grew for a longer time than O. montium. Both fungi are fast growing on malt agar (maximum growth 4.4-9.0 mm/day), but O. clavigerum grew better at temperatures below 25°C. The rapid growth and the ability to tolerate low oxygen levels may be important adaptations for O. clavigerum as the primary invader of fresh sapwood. However, although O. clavigerum grew better in the phloem of both tree species, there were no differences between the two fungi in their ability to colonize the sapwood of the inoculated trees.Key words: blue-stain fungi, Dendroctonus ponderosae, growth rate, oxygen deficiency, virulence.

Early stages of blue-stain fungus invasion of lodgepole pine sapwood following mountain pine beetle attack

Invasion of lodgepole pine sapwood by blue-stain fungi was followed for 7 weeks after infestation by the mountain pine beetle, Dendroctonus ponderosae. During this period all sapwood was heavily stained blue and blue-stain fungi were always isolated close to the front of visible occlusion. Ophiostoma clavigerum and Ophiostoma montium were commonly isolated, both of which are known to be carried in the mycangia of the mountain pine beetle. Ophiostoma montium was most frequently isolated, but when both fungi were present O. clavigerum was always at the leading edge of fungal penetration. On average O. montium trailed 7.3 mm behind O. clavigerum. Other microorganisms were seldom isolated. Key words: lodgepole pine, Dendroctonus ponderosae, fungal succession, blue-stain fungi.