1. Colonisation of host trees by an endophytic herbivore, the spruce beetle, Dendroctonus rufipennis , is accompanied by invasion of its galleries by a number of fungal species. Four of these associated species were identified as Leptographium abietinum , Aspergillus fumigatus , Aspergillus nomius , and Trichoderma harzianum .2. Trichoderma and Aspergillus significantly reduced spruce beetle survival and reproduction in controlled assays.3. A previously undescribed behaviour was observed, in which spruce beetle adults exuded oral secretions, especially within fungus-pervaded galleries.4. These oral secretions inhibited the growth of fungi except A. nomius , and disrupted the morphology of the latter. Administration of these secretions indicated a dosedependent inhibitory effect.5. Oral secretions cultured on microbiological media yielded substantial bacterial growth.6. Filter-sterilised secretions failed to inhibit fungal growth, evidence that the bacteria are responsible for the antifungal activity.7. Nine bacterial isolates belonging to the Actinobacteria, Firmicutes, Gammaproteobacteria, and Betaproteobacteria taxa were obtained from the secretions.8. Bacterial isolates showed species-specific inhibitory activity against the four fungi antagonistic to spruce beetle. The bacterium with the strongest fungal inhibition activity was the actinomycete Micrococcus luteus .9. The production of bark beetle secretions containing bacteria that inhibit fungal growth is a novel finding. This suggests an additional level of complexity to ecological associations among bark beetles, conifers, and microorganisms, and an important adaptation for colonising subcortical tissue. Fig. 2. Adult spruce beetles in galleries invaded by Trichoderma and Aspergillus spp. The beetle shown here has confi ned itself within enlarged chambers in the distal end of the gallery.Oral secretions inhibit antagonistic fungi 641

Pathogen-induced plant responses include changes in both volatile and non-volatile secondary metabolites. To characterize the role of bacterial pathogenesis in plant volatile emissions, tobacco plants, Nicotiana tabacum L. K326, were inoculated with virulent, avirulent, and mutant strains of Pseudomonas syringae. Volatile compounds released by pathogen-inoculated tobacco plants were collected, identified, and quantified. Tobacco plants infected with the avirulent strains P. syringae pv. maculicola ES4326 (Psm ES4326) or pv. tomato DC3000 (Pst DC3000), emitted quantitatively different, but qualitatively similar volatile blends of (E)-beta-ocimene, linalool, methyl salicylate (MeSA), indole, caryophyllene, beta-elemene, alpha-farnesene, and two unidentified sesquiterpenes. Plants treated with the hrcC mutant of Pst DC3000 (hrcC, deficient in the type-III secretion system) released low levels of many of the same volatile compounds as in Psm ES4326- or Pst DC3000-infected plants, with the exception of MeSA, which occurred only in trace amounts. Interaction of the virulent pathogen P. syringae pv. tabaci (Pstb), with tobacco plants resulted in a different volatile blend, consisting of MeSA and two unidentified sesquiterpenes. Overall, maximum volatile emissions occurred within 36 h post-inoculation in all the treatments except for the Pstb infection that produced peak volatile emissions about 60 h post-inoculation. (E)-beta-Ocimene was released in a diurnal pattern with the greatest emissions during the day and reduced emissions at night. Both avirulent strains, Psm ES4326 and Pst DC3000, induced accumulation of free salicylic acid (SA) within 6 h after inoculation and conjugated SA within 60 h and 36 h respectively. In contrast, SA inductions by the virulent strain Pstb occurred much later and conjugated SA increased slowly for a longer period of time, while the hrcC mutant strain did not trigger free and conjugated SA accumulations in amounts significantly different from control plants. Jasmonic acid, known to induce plant volatile emissions, was not produced in significantly higher levels in inoculated plants compared to the control plants in any treatments, indicating that induced volatile emissions from tobacco plants in response to P. syringae are not linked to changes in jasmonic acid.

No abstract

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.