The ecological niches of saprophytic and pathogenic wood-inhabiting filamentous fungi differ in moisture content, nutrients and defence chemicals. For such fungi to survive in and colonize their substrates and hosts, they need active transport systems that can excrete enzymes that break down complex external substrates and then import nutrients into the cells. As well, they need to modify or remove toxic host defense chemicals that have entered their cells. The wood of trees, logs and lumber has a wide range of moisture contents and a high carbon-to-nitrogen ratio . O. piceae grows more efficiently in drier pine wood than in freshly cut logs; G. clavigera, which is vectored by MPB, colonizes healthy or stressed living pine trees, which have high moisture and low oxygen contents. Neither organism degrades lignocellulosic wood fibers [1, 45]. O. piceae has to retrieve nutrients from a nutrient-poor substrate that typically contains very little nitrogen and relatively low levels of host defence chemicals. In contrast, G. clavigera has first to cope with high concentrations of defense chemicals produced by its pine host.
Recently, we reported the annotated genome sequence and transcriptomes of the pine pathogen G. clavigera. Here, we report the annotated genome and transcriptomes of the saprophyte O. piceae, the second pine wood-inhabiting ophiostomatoid fungus for which a complete genome has been sequenced. O. piceae’s genome size and the number of predicted genes and proteins were similar to those for G. clavigera and other sequenced saprophytic ascomycetes in the class Sordariomycetes (e.g. N. crassa, T. reesei). O. piceae’s predicted secretome is 10% larger than that of the pine-specific pathogen. Given its more diverse range of host trees (e.g. pines, hemlock, spruces), it is likely that the saprophyte requires more extracellular enzymes to degrade the different chemicals encountered in these substrates.
In both genomes we identified genes that are potentially involved in the biosynthesis or processing of SMs. In fungi, SMs are diverse and play a range of roles; some SMs are protective, while others are virulence factors . Both O. piceae and G. clavigera produce the SM melanin in artificial media and in their natural substrates. Fungal melanin may protect cells in harsh environments (e.g. UV radiation, extreme temperatures and toxic compounds), and may be involved in cellular development, differentiation and pathogenicity . In all conditions tested here, except with terpene treatments, O. piceae mycelia and asexual structures (i.e. synemata) were highly melanized. Scytalone dehydratase, which is a marker gene for the DHN pathway , was up-regulated in all conditions tested except with terpene treatments, and was most highly expressed in sawdust. Similarly, in G. clavigera, which is densely melanized in its pine host, scytalone dehydratase was down-regulated on CM with terpenes, but was up-regulated on other media and when monoterpenes were the only carbon source. In contrast to O. piceae, G. clavigera does not produce large numbers of asexual spores when it is actively growing on these media. It is likely that melanin protects O. piceae from the unfavourable environmental conditions that it encounters in lumber (e.g. dessication, UV), as well as being involved in cellular development. In contrast, for G. clavigera, melanin may be more important in protecting the fungus from host defense chemicals.
O. piceae and G. clavigera can grow on a variety of simple sugars that are present in phloem or in sapwood parenchyma cells , and can acquire additional sugars by degrading wood hemicelluloses [13, 14, 45]. Both fungi grow well with mannose and maltose, and can also use starch, a stored tree nutrient . For O. piceae, our data suggest that mannose uptake and the initial steps in its utilization are controlled by at least six genes that include two transporters. That none of the six were up-regulated with maltose suggests that maltose utilization involves an alternate pathway.
O. piceae and related species can use triglycerides and fatty acids in artificial media or wood; these lipids can account for up to 3% of the dry weight of sapwood . Triglycerides are hydrolyzed by extracellular lipases into fatty acids and glycerol, which are ultimately processed through ß-oxidation and glycolysis pathways . While lipase and esterase genes were present in the O. piceae genome and we noted that a lipase was expressed on sawdust, we were unable to detect up-regulated lipases on triglycerides. It is possible that on triglycerides the lipase was produced very early in growth, as shown by Gao and Breuil , who reported an optimum production of the enzyme at day 3, before the pH of the medium decreases due to the accumulation of fatty acids. Here, we collected the mycelium after seven days of growth on a solid media with triglycerides. We identified a glycerol kinase that was up-regulated for triglycerides and sawdust, which suggests that glycerol may be metabolized by the fungus. Further, we noticed that triglycerides induced a genomic cluster that contained a P450 and a reductase (described in Results). Lah et al. reported a similar genomic cluster organization and expression pattern in G. clavigera, and it is likely that the clusters have similar roles. Lah et al. suggested that the cytochrome P450 and the reductase may be specific redox partners and may play a role in the conversion of exogenous phenolics or fatty acids.
O. piceae retrieves and metabolizes diverse nutrients that are present in low concentrations in sawdust, particularly nitrogen sources, while removing or modifying diverse toxic compounds like terpenes, and aromatic compounds that include simple phenolics. While the fungus grows more slowly on sawdust, diverse transporters were up-regulated. Some of these are involved in acquiring nutrients like sugars and nitrogen, while others, like ABC or MFS transporters, are known to contribute to drug resistance or chemical modification or detoxification .
While small amounts of simple sugars are available in sapwood, O. piceae can retrieve additional sugars by degrading pine hemicellulose [13, 45]. Fleet et al.  reported that mannose was the most depleted sugar in logs and lumber inoculated with Ophiostoma species. In our data, the genes up-regulated on sawdust also included glycoside hydrolases (e.g. two xylanases and one pectinase), which are involved in degrading hemicellulose and pectin. As well, the fungus can retrieve quinic acid through a quinate permease, and can utilize this carbon source by processing it through the quinate/shikamate pathway, which was up-regulated on sawdust. Further, in artificial media O. piceae can readily use inorganic or organic nitrogen. However, in pine sapwood nitrogen is found mainly as amino acids and proteins, and at very low concentrations (~0.05% of the wood dry weight) . We have shown that O. piceae and related species have to produce proteases in order to retrieve organic nitrogen from wood . In the current work, an amino acid permease, and urea and ammonium transporters were up-regulated on sawdust. Urea can be used as a source of nitrogen by many fungi, and it can be efficiently converted into ammonium by a urease enzyme . However, while ammonium is present in trace amounts in pine lumber [53, 54], urea has not been reported in wood.
Mono- and diterpenes are well known biocides for microorganisms, including fungi, and for beetle vectors [21, 55]. Our data show that on artificial media O. piceae tolerates monoterpenes but does not use them as a carbon source. It is not found in living trees, which have the highest terpene concentrations. However, it is able to remain viable for extended periods in the presence of monoterpenes, and likely in the presence of diterpenes, which can account for ~0.4% of pine sapwood dry weight . Here, we show that monoterpenes affected the macroscopic morphology of O. piceae’s mycelia, and inhibited its production of synemata and asexual spores. Further, in the saprophyte, monoterpene/diterpene treatments rapidly up-regulated expression of genes involved in oxidative and reductive processes, as well as transmembrane transport, suggesting that the fungus’ primary response involves protecting itself from these chemicals. During these processes, an ABC transporter (OPP_06758), which is homologous to the G. clavigera efflux transporter characterized by Wang et al. , was highly expressed.
We have shown that this G. clavigera ABC-G transporter is expressed in young trees and that the transporter excretes monoterpenes . As we have not yet demonstrated this function for the homologous gene in O. piceae, at this time we can only suggest that this unique transporter may play a similar role in the saprophyte by allowing it to survive in toxic mixtures of terpenes. When O. piceae is treated with terpenes on rich media, there is an initial growth delay, after which the fungus resumes its growth. In this growth phase, while genes providing most of the primary protective biological functions were active, genes involved in degrading hydrophobic compounds were up-regulated. This suggests that, like G. clavigera, O. piceae may be able to modify terpenes into less toxic compounds. However, while G. clavigera has a gene cluster that specifically responds to terpenes and is potentially involved in metabolizing terpenes , in O. piceae we found no such gene cluster. Only five of the 30 genes in this G. clavigera cluster had homologs in O. piceae, and these five genes were dispersed through the O. piceae genome. In ongoing work we are characterizing O. piceae genes that are involved in modifying terpenes.