Stayin’ alive: survival of mycorrhizal fungal propagules from 6-yr-old forest soil

Spores and sclerotia are the main propagules that allow fungi to persist through unfavorable conditions and disperse into new environments. Despite their importance, very little is known about their longevity and dormancy, especially in ectomycorrhizal fungi. To assess the viability of ectomycorrhizal fungal spores in forest soil, we collected and buried non-sterile forest soil, in pots, in the field distant from an inoculum source. After 6 yr, a subset of this soil was assayed for viable spores by baiting the fungi with Bishop pine (Pinus muricata) seedlings. Our results show that the three most frequent colonizers in year 1 continued to colonize significant percentages of seedlings in year 6: Wilcoxina mikolae (77 %), Rhizopogon vulgaris (13 %) and Suillus brevipes (9 %). While three species that colonized low percentages of seedlings in year 1, Suillus pungens (1 %), Rhizopogon salebrosus (2 %), and Thelephora terrestris (5 %) were not recovered in year 6. Laccaria proxima, a species not seen in year 1, was recovered on a single seedling in year 6. This is the first report of long-term survival of S. brevipes and W. mikolae. Our results reveal a more complete picture of ectomycorrhizal fungal spore longevity in soil spore banks. a 2012 Elsevier Ltd and The British Mycological Society. All rights reserved. Introduction propagules that can form a spore bank and potentially wait for Propagules such as spores and sclerotia are a means for fungi to escape their current environment, overcome dispersal barriers, and establish in a new favorable habitat. Dispersal may involve these propagules being carried by the air or through the guts of various animals (Claridge et al. 1992; Lilleskov & Bruns 2005). Aerial dispersal may involve the risk of spore dehydration (Ashkannejhad & Horton 2006) or UV damage (Ulevicius et al. 2004) that can lead to a loss of viability. Even if the spores are able to survive these barriers, reaching new substrata does not guarantee favorable conditions for germination, particularly in ectomycorrhizal fungi (Massicotte et al. 1994; Rusca et al. 2006). To counter this problem, it was suggested that some fungi produce resistant 3; fax: þ1 510 642 4995. u (N.H. Nguyen). ier Ltd and The British M NH, et al., Stayin’ alive: s rg/10.1016/j.funeco.201 decades before germination (Miller et al. 1994; Bruns et al. 2009). Most reports of spore banks are based on anecdotal evidence (Putnam & Sindermann 1994) and few experiments have attempted to address the question of spore survival through an extended time (Bruns et al. 2009; Nara 2009). This is especially true for ectomycorrhizal fungi (EMF). Longevity and dormancy of EMF spores have been studied using various microscopy techniques (Lamb & Richards 1974; Miller et al. 1993, 1994; Torres & Honrubia 1994) and host seedling bioassays (Castellano & Molina 1989; Ashkannejhad & Horton 2006; Ishida et al. 2008; Bruns et al. 2009). Some EMF spores remained viable from 1 month and up to 4 yr in various storage conditions. Bruns et al. (2009) showed that spores of several Rhizopogon species remained viable for at ycological Society. All rights reserved. urvival ofmycorrhizal fungal propagules from 6-yr-old forest 2.05.006 2 N.H. Nguyen et al. least 4 yr, and surprisingly, the inoculum potential of the soil mixture increased during this time. This pattern of increased inoculum potential by ectomycorrhizal spores over time has so far only been observed in Rhizopogon species, which largely depend onmammals for spore dispersal through the ingestion of basidiocarps. The genus Suillus, an epigeous relative of Rhizopogon, usually relies on air for dispersal and apparently showed an opposing pattern when dispersed in deer feces, where its spores lose viability within a year after being desiccated in deer fecal pellets (Ashkannejhad &Horton 2006). As a complement to the Rhizopogon spore longevity experiments described in Bruns et al. (2009), we tested the long-term viability of a diversity of fungal spores present in the spore bank of a natural Pinus muricata stand. Here, we report the fungi that survived after 6 yr of field incubation using pine seedling bioassays to capture the viable and receptive fungal spore community. Material and methods Soil collection and incubation In 2003, 88 l of soil was collected underneath P. muricata trees from Muddy Hollow Knoll (MHK) in Point Reyes National Seashore, CA (38 2.7360N, 122 52.1360W, WGS84 datum), brought back to the lab and homogenized with 8 l of sterile horticultural sand (11:1 ratio). Sixteen terracotta pots, measuring 16.5 cm in diameter with a volume of 1.6 l, were filled to the top with 1.6 l of the homogenized soil. Before filling the pots, the bottom holes were covered with glass microscope slides to prevent soil from leaking out, and a numbered metal tag was added to each pot to allow unequivocal identification. The tops of the pots were covered with 19 cm diameter terracotta saucers and these were secured with plastic cable binders. In the field, we observed basidiocarps of Rhizopogon vulgaris, Roccidentalis occidentalis, Suillus pungens, and Laccaria proxima in the immediate area from which the soils were collected, and we assume that spores of these species would be in the soils. However, the starting quantities and age of the inocula were not known and we assumed that all spores were deposited immediately prior to collection. The pots were buried under 15 cm of grassland soil in situ (38 11.79830N, 122 57.75170W,WGS84 datum) in an area where little or no EMF inoculum is present (Bruns et al. 2009). The burial site was about 1.25 km up-slope from a small number (<5) of young pine trees at Pita Beach; these trees were unknown to the authors at the time the experiment was setup and were only discovered via Google Earth postings that were recognized by an alert reviewer. Otherwise, the nearest known pine tree is 9 km away on the Pierce Point Road. Nevertheless, prior bioassays of the soil at the burial site showed that it was devoid on ectomycorrhizal inoculum (Bruns et al. 2009). Although this field site experiences a seasonal dry summerwith no precipitation, it does get heavy fog, which prevents the ground from becoming completely dry. Complete drying out of spores may result in loss of viability (Ashkannejhad & Horton 2006). This experimental setup allowsmaterial inside the pot to easily exchange air and Please cite this article in press as: NguyenNH, et al., Stayin’ alive: soil, Fungal Ecology (2012), http://dx.doi.org/10.1016/j.funeco.201 water with the outside soil. It also allows the spores to interact with soil microinvertebrates and the microbial community therein.