Spores of ectomycorrhizal fungi: ecological strategies for germination and dormancy

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Some plant seeds germinate immediately following dispersal if provided with favorable water, oxygen, temperature and light conditions, whereas others do not germinate even when environmental conditions are suitable. Such dormancy strategies are widespread in many temperate plant species to prevent the germination of seeds during unfavorable seasons such as winter. The seeds of some plant species can undergo dormant periods exceeding 1000 yr (Shen-Miller et al., 1995) while waiting for rare chances of regeneration. The traits of seed germination/dormancy are directly relevant to plant life and have therefore been studied extensively in various scientific fields, from molecular genetics to ecology (Finch-Savage & Leubner-Metzger, 2006, Holdsworth et al., 2008).

‘... only a small proportion of spores (1–8% at maximum estimates) were initially receptive to host roots, and receptive spores increased year by year as a result of the release of spore dormancy.’

Fungal spores are functionally analogous to plant seeds in many respects. Spores are dispersed from mature individuals, germinate in new places and grow into mature individuals. Thus, spore germination and dormancy traits have a substantial influence on fungal life. However, few published studies have examined the germination and dormancy of fungal spores. This is especially true for ectomycorrhizal fungi (EMF), which are indispensable symbionts to major trees in many forests. Specifically, it is unclear whether EMF spores become dormant and how long they remain viable. In this issue of New Phytologist, Bruns et al. (pp. 463–470) report an important step towards answering these fundamental questions by providing evidence that spores of some Rhizopogon species remained viable for at least 4 yr in field soil and, more surprisingly, that the infectivity of spores increased with time.

The importance of basidiospores in EMF ecology

Most EMF communities are dominated by basidiomycetes, which include many mushrooms, puffballs, boletes, chanterelles and earth stars. Recent molecular ecological studies revealed that EMF populations are usually composed of many genetically different individuals of the same species, or genets. Moreover, genets of many EMF are relatively small in size (e.g. Redecker et al., 2001), and in some EMF species, rapid genet turnover is apparent (e.g. Guidot et al., 2004). These studies strongly indicate the importance of basidiospores (hereafter referred to as spores) for the development and maintenance of EMF populations because spore-mediated regeneration is the only way to produce new genets. Spores also play critical roles in the development of EMF communities because new EMF species are mostly delivered to new habitats in the form of spores. This is especially true in primary successional areas where no other inoculum sources are available (e.g. Ashkannejhad & Horton, 2006).

Interactions between EMF spores and host roots

Despite growing evidence indicating the importance of spores in EMF ecology, there is a lack of information regarding EMF spore germination. This is partly a result of the difficulty in managing EMF spore germination. Spores of most EMF species do not germinate on ordinary synthetic media, on which saprophytic fungal spores easily germinate (Fries, 1987 and references therein). In the presence of host roots, however, spore germination is significantly improved for some EMF (Fig. 1). In particular, pioneer EMF, which appear early in undeveloped habitats, usually respond to host roots very well. For example, the maximum spore-germination rates of Rhizopogon, Laccaria, Hebeloma and Inocybe can exceed 50% in the presence of host roots (Theodorou & Bowen, 1987; Ishida et al., 2008). Such stimulation might be caused by chemicals secreted from host roots (e.g. flavonoids, as reported by Kikuchi et al., 2007). Given that EMF live in symbiosis with host roots, it is a reasonable strategy to germinate only in their presence. In contrast to these pioneer EMF, spores of Russula, Lactarius, Amanita and Cortinarius germinate poorly, even in the presence of host roots (Fig. 1). These poorly germinating species usually do not appear in pioneer habitats but are often dominant in many mature forests. Thus, nonpioneer EMF species may require additional stimuli that are specific to mature forests, given their ecological traits. The contrasting germination patterns between pioneer and nonpioneer EMF are inevitably reflected in infection efficiency. Indeed, spore inoculation is generally successful for pioneer EMF but not successful for nonpioneer fungi, as documented by many experiments using seedlings as well as practical applications in nurseries (Fig. 1).

Figure 1.

Spore germination, infectivity and longevity of major ectomycorrhizal fungal (EMF) genera with reference to their ecological traits.
Note that the data in this figure do not indicate that all species in each genus have the same properties. Data were derived from several representative studies using a small number of species in each genus. Direct comparison of data may be difficult owing to different experimental conditions/measures. A complete list of references for this figure is available as Supporting information online (Table S1).
*The maximum germination rate (%) recorded in each EMF genus is shown by bar graphs. If detailed data are available for a specific species/study where a maximum value was detected, upper and lower quartiles are shown by a gradation box with the mean value in the darkest color. R and M beside the maximum bars indicate stimulation with host roots and microbes, respectively. Data are mostly from Theodorou & Bowen (1987) and Ishida et al. (2008).
†Significant germination/infectivity decrease and increase after 1 yr is shown by descending and ascending arrows, respectively, P < 0.05. Data are from Ashkannejhad & Horton (2006), Ishida et al. (2008) and Bruns et al. (2009).
‡Ecological traits shown here are derived from some representative species used in spore germination and inoculation studies, and are not applicable to all species in a genus.

Dormancy and longevity of EMF spores

The term dormancy is used often in seed science, but its definition is unclear and under debate (see Finch-Savage & Leubner-Metzger, 2006). In a strict sense, dormancy is defined as the incapacity of a viable seed to germinate during a certain period of time, even under favorable external conditions. Thus, seeds can germinate under the same conditions after the dormant period ends. If the same definition is applied to EMF spores, the existence of host roots can be regarded as a type of external condition that stimulates germination. By this definition, spores of most pioneer EMF may not undergo innate dormant states because fresh spores can readily germinate in the presence of host roots immediately following spore collection (Fig. 1). However, using seedling bioassays and serially diluted spores in soil, Bruns et al. found that the infectivity of Rhizopogon spores increased with time over 4 yr. They concluded that only a small proportion of spores (1–8% at maximum estimates) were initially receptive to host roots, and receptive spores increased year by year as a result of the release of spore dormancy. This conclusion apparently contradicts some previous studies that reported high germination rates (up to 69%) and high infectivity of fresh Rhizopogon spores (e.g. Theodorou & Bowen, 1987). Although the reason for this inconsistency is unknown, further research on Rhizopogon spores may be necessary to confirm the existence of dormancy sensu stricto, as observed in some wood-decomposing Crepidotus species inhabiting temperate areas (Aime & Miller, 2002).

The longevity of EMF spores may be less controversial in definition, but this has been tested by only a few studies. Ishida et al. (2008) demonstrated a significant decrease in spore germination, after approx. 1 yr of preservation at 4°C, for many EMF species belonging to Laccaria, Inocybe, Scleroderma, Hebeloma and Russula, all of which are major EMF species in an early successional volcanic desert on Mt Fuji, Japan. Their seedling bioassays showed that the infectivity of these fungal spores also decreased after approx. 1 yr of preservation in soil under ambient conditions (Ishida et al., 2008). Furthermore, field-transplanted nonmycorrhizal seedlings remained nonmycorrhizal during the first growing season when mycorrhizal networks were unavailable, indicating that EMF spore banks are rare or absent in this desert (Nara & Hogetsu, 2004). Similarly, in mature pine forests in California, members of Russula, Lactarius, Amanita and Thelephorales tend to dominate EMF communities, but their viable spores were rarely detected in soil using seedling bioassays (e.g. Taylor & Bruns, 1999). Therefore, the spore longevity of most EMF basidiomycetes is likely to be short and insufficient to accumulate effective numbers of viable spores as soil spore banks.

In contrast to these ephemeral EMF species, Rhizopogon spores remain viable for exceptionally long periods of time; Bruns et al. report viability periods of at least 4 yr and it is suspected that longer periods are possible. The high longevity of Rhizopogon spores would enable the accumulation of abundant numbers of viable spores in soil. Indeed, Rhizopogon species are dominant in soil spore banks in pine forests in western North America, even when Rhizopogon species are almost absent from existing tree roots and sporocarps (Taylor & Bruns, 1999). Soil spore banks enable quick colonization after disturbance in advance of other EMF species (Baar et al., 1999).

It is unclear what has enabled the exceptional longevity of some Rhizopogon species. It may be interesting to compare Rhizopogon with its close relative Suillus; these genera are phylogenetically very close and exhibit host specificity to Pinaceae. Rhizopogon spores showed no decrease in infectivity when preserved for 1 yr, while the infectivity of Suillus spores decreased drastically over the same time period (Ashkannejhad & Horton, 2006). Although the mechanism of this difference in infectivity is uncertain, we know that these two EMF genera produce different types of sporocarp: Suillus produces epigeous sporocarps from which spores are dispersed mainly by wind, while Rhizopogon produces hypogeous sporocarps and largely depends on mycophagous animals for spore dispersal. Such differences in ecological traits, including niche preference, may have produced different spore longevity through evolution.

Given the great morphological and physiological variations in seed germination/dormancy mechanisms (Finch-Savage & Leubner-Metzger, 2006), it is reasonable to propose that there are similar variations in EMF spores. The findings by Bruns et al. highlight such underlying diversity of EMF spore traits by providing solid evidence of exceptionally long spore viability of Rhizopogon. Although the 4-yr time-period of monitoring was insufficient to determine the exact longevity of Rhizopogon spores, the experimental design used by Bruns et al. potentially allows to extend the monitoring over 99 years. Thus, Rhizopogon spore longevity will be determined in follow-up studies, hopefully in my lifetime.

Acknowledgements

This commentary was supported, in part, by the Japan Society for the Promotion of Science. I apologize that space limitations prohibit me from citing many other interesting references.

Ancillary