Armillaria

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Apr 2, 2018 - approximately 70 known species, collectively referred to as shoestring root-rot fungi or honey mushrooms. Armillaria causes root-rot disease in ...
Current Biology

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Armillaria György Sipos1,*, James B. Anderson2,*, and László G. Nagy3,* What is Armillaria? Armillaria is a genus of plant pathogenic fungi in the phylum Basidiomycota, comprising approximately 70 known species, collectively referred to as shoestring root-rot fungi or honey mushrooms. Armillaria causes root-rot disease in a wide variety of woody hosts worldwide, including conifers and hardwoods (Figure 1). As a parasite, Armillaria is necrotrophic; it causes the plant tissues to die before colonization by the mycelium. The fungus is easily recognized by its rhizomorphs, strandlike fungal organs that push through soil in a manner analogous to that of plant roots, effectively foraging for food. Rhizomorphs can also persist in soil during conditions unfavorable for growth, allowing the fungus to wait in place for a new food source to arrive (for example, a fallen or weakened tree). Like in many other fungi, mating in Armillaria is controlled by the bifactorial sexual incompatibility system. However, the predominant vegetative phase of Armillaria is diploid, not dikaryotic as in other basidiomycetes. In nature, each individual of Armillaria originates from a mating of two haploids, and

the resultant diploid individual can be distinguished by its unique multilocus genotype. The newly established individual may colonize a territory encompassing many adjacent tree root systems. The spatial distributions of individuals can approximated by sampling rhizomorphs, fruit-bodies, or vegetative mycelia, and then genotyping these over several loci. Because the sampling is discontinuous, the exact spatial limits of an individual remain uncertain, and the territories of different individuals may overlap to some extent. Many existing individuals of Armillaria are both large and old — extending over hectares of forest floor and having come into being tens, hundreds, or even thousands of years ago. An A. ostoyae colony, known as the ‘humongous fungus’ in Oregon, USA, is among the largest and oldest terrestrial organisms known on Earth. What makes Armillaria so ubiquitous and persistent in forests? An effective dispersal strategy on two scales. First, Armillaria accomplishes longrange dispersal through abundant production of wind-born basidiospores. Furthermore, the requirements for growth of the fungus are broad and many different tree species qualify as substrates. This may be explained, at least in part, by the arsenal of plant cell-wall-degrading enzymes encoded by Armillaria genomes. Like other white rot fungi, they can decompose all

components of lignified wood, making them well-suited as both plant invaders and saprotrophs. Armillaria genomes encode a particularly high number of enzymes involved in degrading plant pectins, one of the most important components of the root cell wall and middle lamella. To accomplish long-range dispersal, the premium is on variation, and natural selection presumably acts on the high levels of genetic variability observed at the population level among individuals. In contrast, at the local level, the premium is on stability in environments that are relatively buffered from change. A significant body of research is devoted to understanding the unusual patterns of population structure, biogeography and species limits observed in Armillaria communities. What is so special about the rhizomorphs? Rhizomorphs are unique to Armillaria and its puffball-like sister group Guyanagaster, and are the primary tools used by the fungus to establish itself in forest ecosystems. Anatomically, rhizomorphs are made up of four distinct hyphal layers, the outermost of which often produces a gelatinous sheath. Like hyphae, rhizomorphs exhibit apical, polarized growth, and form an underground fungal network in the soil. Armillaria infects new trees by direct root contact via rhizomorphs. Although we don’t know the exact molecular mechanisms of the infection,

Figure 1. Characteristic signs of Armillaria root rot. Young rhizomorphs (A. ectypa, left), fruiting bodies and the decay caused by Armillaria (A. ostoyae, middle) and the spread of fungal mycelium under the bark (A. ostoyae, right) of a conifer. Photo credit: Zsolt Merenyi (left), Igor Pavlov (middle and right images).

Current Biology 28, R293–R305, April 2, 2018 © 2018 Elsevier Ltd. R297

Current Biology

Magazine some expression data point to plant cell-wall-degrading enzymes (cellulose, hemicellulose, lignin and pectindegrading CAZy enzymes). In addition, a number of factors used by plant pathogens in chemical warfare — to pierce through the bark (for example, expansins, cerato-platanins) and bypass the plant’s immune and defense systems (such as CBM50, GH75, and salicylate hydroxylase) — have been implicated. Clonal spread by rhizomorphs preserves the genotype. Although the size and age of the cell populations that constitute individuals can be enormous, a much lower-than-expected mutation rate has been documented; this may be due to a low rate of cell division in the rhizomorph tips (analogous to the quiescent center in plant root tips). Thus, along the spectrum of genetic stability, Armillaria represents the opposite of cancer. Yet, rhizomorphs are quite complex. Indeed, recent transcriptomic analyses have revealed developmental similarities between rhizomorphs and fruiting bodies, with several fruiting-bodyspecific genes (coding for hydrophobins, lectins, pore-forming toxins and certain transcription factors) highly expressed in rhizomorphs, but not in vegetative mycelia. By comparing these functionally different structures, we can get to the basics of how complex multicellularity emerges — one of the major questions in evolution. I have Armillaria mushrooms in my garden; what can I do to fight them? Not much. If you remove the mushrooms (which are edible and known as honey mushrooms), you will not change the future trajectory of the fungal infection. Armillaria may be there merely because of its opportunistic nature and may or may not be contributing to the demise of the tree host. Isolates of Armillaria species, depending also on host and environmental factors, exhibit considerable variability in virulence. However, the genes and critical biotic and abiotic factors underlying virulence have yet to be revealed. In terms of chemical control, there are two problems. Armillaria is innately resistant to many fungicides, and it is difficult or impossible to get the fungicide into the substrate where Armillaria lives. Even if there were some way to completely remove the Armillaria from R298

a site, it may well come back! On the other hand, in natural forest stands, Armillaria species parasitize only the weak and damaged trees, and in return, they reintroduce sequestered carbon from dead wood back into the soil. Because only white rot fungi are able to efficiently decompose lignin, Armillaria species — along with other white rotters from the Agaricomycetes — fulfill an important role in the ecosystem. What do Armillaria genome projects tell us? There are several completed and ongoing genome-sequencing projects for Armillaria. Six draft genomes have been published to date: those for A. cepistipes, A. gallica, A. fuscipes, A. mellea, A. ostoyae and A. solidipes. Analyses of these revealed that the Armillaria genomes have expanded relative to those of related saprotrophic fungi. The expansion was driven by protein coding genes, in contrast to most plant pathogens, in which genome expansion is driven by transposable elements. These genomes also revealed a rich repertoire of pathogenicity-related genes and plant cell-wall-degrading enzymes in Armillaria species, as well as many lineage-specific genes that are expressed during rhizomorph and fruiting-body development. Further projects are under way to obtain a greater understanding of genome evolution in these organisms and will serve as important resources for understanding their biology. Where do we go from here? Two of the big unanswered questions concerning Armillaria include the molecular details of the interaction between Armillaria and host plants and the identity and nature of fungal virulence determinants and host susceptibility. Both virulence and susceptibility vary greatly across individuals of both the fungi and their host plants, and are further influenced by the local (micro)habitats, including biotic and abiotic factors. For example, preliminary data indicate that the infection trajectory depends on the soil microbiota and population structure of the coexisting Armillaria community, although understanding how exactly these influence disease is a future challenge. We only scratched the surface of how infection happens:

Current Biology 28, R293–R305, April 2, 2018

Armillaria represents an independent origin of pathogenicity, so whether the established principles of plant invasion learnt from other plant pathogens apply to Armillaria remains to be understood. Fortunately, the genomic resources, genetic-manipulation techniques and in vitro infection assays for Armillaria are coming along, and will help us to unravel the secrets of this enigmatic group of fungi and develop strategies against the disease they cause. Where can I find out more? Aanen, D.K. (2014). Developmental Biology. How a long-lived fungus keeps mutations in check. Science 346, 922–923. Anderson, J.B., and Catona, S. (2014). Genomewide mutation dynamic within a long-lived individual of Armillaria gallica. Mycologia 106, 642–648. Baumgartner, K., Coetzee, M.P., and Hoffmeister, D. (2011). Secrets of the subterranean pathosystem of Armillaria. Mol. Plant. Pathol. 12, 515–534. Collins, C., Keane, T.M., Turner, D.J., O’Keeffe, G., Fitzpatrick, D.A., and Doyle, S. (2013). Genomic and proteomic dissection of the ubiquitous plant pathogen, Armillaria mellea: toward a new infection model system. J. Proteome Res. 12, 2552–2570. Ford, K.L., Baumgartner, K., Henricot, B., Bailey, A.M., and Foster, G.D. (2015). A reliable in vitro fruiting system for Armillaria mellea for evaluation of Agrobacterium tumefaciens transformation vectors. Fungal Biol. 119, 859–869. Ford, K.L., Baumgartner, K., Henricot, B., Bailey, A.M., and Foster, G.D. (2016). A native promoter and inclusion of an intron is necessary for efficient expression of GFP or mRFP in Armillaria mellea. Sci. Rep. 6, 29226. Koch, R.A., Wilson, A.W., Séné, O., Henkel, T.W., and Aime, M.C. (2017). Resolved phylogeny and biogeography of the root pathogen Armillaria and its gasteroid relative, Guyanagaster. BMC Evol. Biol. 17, 33. Prospero, S., Lung-Escarmant, B., and Dutech, C. (2008). Genetic structure of an expanding Armillaria root rot fungus (Armillaria ostoyae) population in a managed pine forest in southwestern France. Mol. Ecol. 17, 3366–3378. Prospero, S., Holdenrieder, O., and Rigling, D. (2004). Comparison of the virulence of Armillaria cepistipes and Armillaria ostoyae on four Norway spruce provenances. For. Pathol. 34, 1–14. Sipos, G., Prasanna, A.N., Walter, M.C., O’Connor, E., Bálint, B., Krizsán, K., Kiss, B., Hess, J., Varga, T., Slot, J., et al. (2017). Genome expansion and lineage-specific genetic innovations in the forest pathogenic fungi Armillaria. Nat. Ecol. Evol. 1, 1931–1941. Smith, M.L., Bruhn, J.N., and Anderson, J.B. (1992). The fungus Armillaria bulbosa is among the largest and oldest living organisms. Nature 356, 428–431. 1

Fungal Genomics and Bioinformatics Group, Research Center for Forestry and Wood Industry, University of Sopron, Sopron, 9400 Hungary. 2Department of Biology, University of Toronto, Mississauga, ON L5L 1C6 Canada. 3Synthetic and Systems Biology Unit, Biological Research Center, Hungarian Academy of Sciences, Szeged, 6726, Hungary. *E-mail: [email protected] (G.S.), [email protected] (J.B.A.), [email protected] (L.G.N.)