Interaction Chemistry Volatile And Diffusible Organic Compounds

When two fungi grow in close proximity, changes in both mycelial morphology and secondary compound chemistry occur, resulting in the formation of characteristic 'barrages' and colour changes in the mycelia of both species. These changes are mediated by up-regulation of genes involved in antagonism (Iakovlev et al., 2004; C.A. Eyre, L. Boddy and H.J. Rogers, unpublished), resulting in production of stress compounds, enzymes and low molecular weight secondary metabolites, in the hyphae and into the surrounding environment. The production of such metabolites by one species may have profound effects on other species in the vicinity, mediating antagonism at a distance or following contact, or leading to attraction or stimulation of growth.

Fungi colonizing wood of both conifer (e.g. Woodward et al., 1993; Woods, 1996) and angiosperm (Heilmann-Clausen and Boddy, 2005) trees secrete secondary metabolites in the absence of other fungi, which under natural conditions may function, at least in part, to protect occupied territory from potential invaders. These chemicals may have differential effects on species attempting to colonize, some antagonists being totally inhibited, the growth of others being slowed to a greater or lesser extent, whereas some are apparently unaffected and growth of others stimulated. Moreover, the composition of the secondary metabolites produced by a given species may alter when the presence of a potential competitor is detected (Griffith et al., 1994b, 1994c; Woods, 1996; Hynes et al, 2007). Some chemicals exert their effects indirectly, e.g. by lowering pH, whilst others can be considered to be antibiotics.

Although the production of antibiotic compounds by fungi has long been known (e.g. Fleming, 1929; Brian, 1951), several major questions must be addressed before the function of these compounds in natural environments can be corroborated. With few exceptions, it remains unclear whether such compounds occur in the natural environment in sufficient quantities to impact upon the growth of potential competitors. Other questions include: when and where are the compounds produced, and are they sufficiently persistent in the niche to impact significantly on other organisms?

Certainly, some antimicrobial compounds are persistent; methylbenzoates produced in vivo by the brown rot basidiomycete Sparassis crispa are present in occupied wood in large quantities, and persist for many years, even following death of the host tree (Woodward et al., 1993). Such high concentrations represent a significant biosynthetic commitment by the fungus, implying considerable benefit to the producing organism. Lipid-soluble antifungal compounds were detected in 85% of fungi isolated from stumps of Sitka spruce (Picea sitchensis) when grown in defined liquid medium (Woods, 1996). Activity of these compounds against other fungi varied. For example, three compounds from liquid malt extract cultures of Stereum sanguinolentum were antifungal to the bioassay species Cladosporium cucumerinum on bioassay-lead thin-layer chromatography plates, but only one of these compounds inhibited mycelial growth of Hypholoma fasciculare, Heterobasidion annosum and Resinicium bicolor. The same compounds were inactive against Truncatella sp., Melanotus proteus and S. sanguinolentum itself (Figure 2). Active compounds with similar, though not identical, UV spectra were

Figure 2 Antifungal activity of organic-phase extracts from 28-day-old 2% malt extract liquid cultures of Stereum sanguinolentum towards (a) S. sanguinolentum, (b) Truncatella sp., (c) Melanotus proteus, (d) Hypholoma fasciculare, (e) Heterobasidion annosum and (f) Resinicium bicolor. Antibiotic assay discs were soaked in antifungal compounds 1-3 obtained from Cladosporium cucumerinum bioassay-lead preparative TLC plates developed in hexane:ethyl acetate (1:1, v/v). Assay disc C lacked extracts.

Figure 2 Antifungal activity of organic-phase extracts from 28-day-old 2% malt extract liquid cultures of Stereum sanguinolentum towards (a) S. sanguinolentum, (b) Truncatella sp., (c) Melanotus proteus, (d) Hypholoma fasciculare, (e) Heterobasidion annosum and (f) Resinicium bicolor. Antibiotic assay discs were soaked in antifungal compounds 1-3 obtained from Cladosporium cucumerinum bioassay-lead preparative TLC plates developed in hexane:ethyl acetate (1:1, v/v). Assay disc C lacked extracts.

also produced by H. annosum, S. sanguinolentum, M. proteus and R. bicolor in stumps of Sitka spruce inoculated with individual species. Moreover, additional active compounds were produced during interactions between pairs of fungal species inoculated into stumps.

Beech (Fagus sylvatica) wood decayed by a range of fungi contained diffusible metabolites with differential effects on a range of Asco- and Basidiomycota (Heilmann-Clausen and Boddy, 2005). Stereum hirsutum-decayed wood reduced growth of most of the fungi tested, whereas that colonized by Fomes fomentarius stimulated many species. These results suggested that the strategies utilized by decay fungi in defending an occupied resource may vary considerably in vivo: S. hirsutum appeared to utilize an antibiosis mechanism in defence; the strategy used by F. fomentarius, however, was unknown.

In addition to diffusible secondary metabolites, fungi also produce volatile organic compounds (VOCs; Wheatley, 2002), some of which may impact on potential competitors. VOCs can induce changes in the behaviour of potential competitors at a distance from the producing organism. Both soil and wood are highly porous materials, and the concentrations of VOCs in the lumen of a colonized xylem vessel or in soil pore space may be very high in the absence of rapid diffusion. In in vitro interactions between R. bicolor and H. fasciculare (Figure 1c), changes were noted in the VOCs produced, principally sesquiterpenes, with time (Hynes et al., 2007). These changes appeared to be related temporally with pigment production and changes in mycelial morphology. Similar work on the effect of microbially produced VOCs on growth of Serpula lacrymans suggested an impact on protein synthesis (Humphris et al., 2002).

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