Endemic Jeffrey Pine Beetle Associates: Beetle/Mite Fungal Dissemination Strategies and Interactions That May Influence Beetle Population Levels

Fungal and mite associates may drive changes in bark beetle populations, and mechanisms constraining beetle irruptions may be hidden in endemic populations. We characterized common fungi of endemic-level Jeffrey pine beetle (JPB) in western USA and analyzed their dissemination by JPB (maxillae and fecal pellet) and fungivorous mites to identify if endogenous regulation drove the population. We hypothesized that: (1) as in near-endemic mountain pine beetle populations, JPB’s mutualistic fungus would either be less abundant in endemic than in non-endemic populations or that another fungus may be more prevalent; (2) JPB primarily transports its mutualistic fungus, while its fungivorous mites primarily transport another fungus, and (3) based on the prevalence of yeasts in bark beetle symbioses, that a mutualistic interaction with blue-stain fungi present in that system may exist. Grosmannia clavigera was the most frequent JPB symbiont; however, the new here reported antagonist, Ophiostoma minus, was second in frequency. As hypothesized, JPB mostly carried its mutualist fungus while another fungus (i.e., antagonistic) was mainly carried by mites, but no fungal transport was obligate. Furthermore, we found a novel mutualistic interaction between the yeast Kuraishia molischiana and G. clavigera which fostered a growth advantage at temperatures associated with beetle colonization.

Molecular assays to detect the presence and viability of Phytophthora ramorum and Grosmannia clavigera

Abstract/KeywordsTo determine if living microorganisms of phytosanitary concern are present in wood after eradication treatment and to evaluate the efficacy of such treatments, the method of choice is to grow microbes in petri dishes for subsequent identification. However, some plant pathogens are difficult or impossible to grow in axenic cultures. A molecular methodology capable of detecting living fungi and fungus-like organisms in situ can provide a solution. RNA represents the transcription of genes and can therefore only be produced by living organisms, providing a proxy for viability. We designed and used RNA-based molecular diagnostic assays targeting genes essential to vital processes and assessed their presence in wood colonized by fungi and oomycetes through reverse transcription and real-time polymerase chain reaction (PCR). A stability analysis was conducted by comparing the ratio of mRNA to gDNA over time following heat treatment of wood infected with the Oomycete Phytophthora ramorum and the fungus Grosmannia clavigera. The real-time PCR results indicated that the DNA remained stable over a period of 10 days post treatment in heat-treated wood samples, whereas mRNA could not be detected after 24 hours for P. ramorum or 96 hours for G. clavigera. Therefore, this method provides a reliable way to evaluate the viability of these pathogens and test the effectiveness of existing and emerging wood treatments. This can have important phytosanitary impacts on assessing both timber and non-timber forest products of commercial value in international wood trade.

Effects of Grosmannia clavigera and Leptographium longiclavatum on Western White Pine Seedlings and the Fungicidal Activity of Alamo®, Arbotect®, and TREE-äge®

Bark beetles carry a number of associated organisms that are transferred to the host tree upon attack that are thought to play a role in tree decline. To assess the pathogenicity to western white pine (WWP; Pinus monticola) of fungi carried by the mountain pine beetle (MPB; Dendroctonus ponderosae), and to evaluate the potential for systemic prophylactic treatments for reducing fungal impacts, experiments were conducted with WWP seedlings to meet three objectives: 1) evaluate pathogenicity of two MPB-associated blue-stain fungi; 2) evaluate phytotoxicity of tree injection products; 3) evaluate the anti-fungal activity of tree injection products, in vitro and in vivo, toward the associated blue-staining fungi. To evaluate pathogenicity, seedlings were inoculated with Grosmannia clavigera or Leptographium longiclavatum, common fungal associates of MPB. Seedling mortality at four months after inoculation was 50% with L. longiclavatum and 90% with G. clavigera, both significantly higher than controls and thereby demonstrating pathogenicity. Phytotoxic effects of TREE-äge®, Alamo®, and Arbotect® were evaluated by stem injection; no phytotoxic effects were observed. Anti-fungal properties of the same three products were evaluated in vitro against G. clavigera, where Alamo was most active. Co-inoculation of G. clavigera and L. longiclavatum into seedlings after a stem injection of Alamo showed significantly less mortality and lesion formation than either species alone. Results support the hypothesis that MPB blue-stain associates, particularly G. clavigera, promote death of WWP when attacked by MPB. These findings suggest that the administration of a fungicide with insecticide for tree protection against bark beetles may be advantageous.

Gene Discovery for Enzymes Involved in Limonene Modification or Utilization by the Mountain Pine Beetle-Associated Pathogen Grosmannia clavigera

ABSTRACTTo successfully colonize and eventually kill pine trees,Grosmannia clavigera(Gs cryptic species), the main fungal pathogen associated with the mountain pine beetle (Dendroctonus ponderosae), has developed multiple mechanisms to overcome host tree chemical defenses, of which terpenoids are a major component. In addition to a monoterpene efflux system mediated by a recently discovered ABC transporter, Gs has genes that are highly induced by monoterpenes and that encode enzymes that modify or utilize monoterpenes [especially (+)-limonene]. We showed that pine-inhabiting Ophiostomale fungi are tolerant to monoterpenes, but only a few, including Gs, are known to utilize monoterpenes as a carbon source. Gas chromatography-mass spectrometry (GC-MS) revealed that Gs can modify (+)-limonene through various oxygenation pathways, producing carvone,p-mentha-2,8-dienol, perillyl alcohol, and isopiperitenol. It can also degrade (+)-limonene through the C-1-oxygenated pathway, producing limonene-1,2-diol as the most abundant intermediate. Transcriptome sequencing (RNA-seq) data indicated that Gs may utilize limonene 1,2-diol through beta-oxidation and then valine and tricarboxylic acid (TCA) metabolic pathways. The data also suggested that at least two gene clusters, located in genome contigs 108 and 161, were highly induced by monoterpenes and may be involved in monoterpene degradation processes. Further, gene knockouts indicated that limonene degradation required two distinct Baeyer-Villiger monooxygenases (BVMOs), an epoxide hydrolase and an enoyl coenzyme A (enoyl-CoA) hydratase. Our work provides information on enzyme-mediated limonene utilization or modification and a more comprehensive understanding of the interaction between an economically important fungal pathogen and its host's defense chemicals.