Mycologia, 104(5), 2012, pp. 988–997. DOI: 10.3852/11-289 # 2012 by The Mycological Society of America, Lawrence, KS 66044-8897
Arbuscular mycorrhizal colonization of giant sequoia (Sequoiadendron giganteum) in response to restoration practices Catherine Fahey1
ing light availability and carbon assimilation alter feedbacks between sapling growth and activity of AM fungi in the roots. Key words: artificial canopy gap, Glomeromycota, root colonization
Department of Plant Pathology & Plant MicrobeBiology, Cornell University, Ithaca, New York 14853
Robert A. York Center for Forestry and Department of Environmental Science, Policy, and Management, University of California at Berkeley, Georgetown, California 95634
INTRODUCTION
Teresa E. Pawlowska2
The success of restoration practices depends greatly on the quality of interactions that plants re-introduced to their native habitats establish with indigenous soil microbiota (Walker and del Moral 2003). Such interactions are of particular significance for plants that depend on microorganisms such as mycorrhizal fungi for improved mineral nutrition. Mycorrhizae are mutualistic symbioses between plant roots and fungi, in which fungi provide the plant with improved mineral nutrient and water uptake in exchange for plant-assimilated carbon (Smith and Read 2008). Associations with mycorrhizal fungi are critical for seedling establishment and growth in many tree species (Janos 1980). The goal of the present study was to evaluate the effects of restoration practices on mycorrhizal associations formed by giant sequoia, Sequoiadendron giganteum. Giant sequoia is a long-lived pioneer tree species that requires for regeneration disturbances that create distinct canopy gaps. This life history characteristic has important implications for giant sequoia management and protection. Widespread regeneration failure of giant sequoia in its native range of the western slopes of California’s Sierra Nevada has arisen due to firesuppression policies, which eliminate the discrete canopy gaps and ash substrate required by sequoia seedlings (Harvey et al. 1980). Insufficient regeneration across the native range of giant sequoia threatens the viability of their populations (York et al. 2012). While prescribed fires that burn hot enough to create canopy gaps have been effective in initiating giant sequoia regeneration (Harvey et al. 1980, Mutch and Swetnam 1995), many of these fires are currently unlikely to be within the range of frequency and diversity of fire severity that occurred before the suppression policies (Stephenson 1999, York et al. 2012). To augment prescribed fire programs or to take remedial action where fires are not a viable option (i.e. near smokesensitive areas or where severe fire is socially unacceptable), the creation of artificial canopy gaps could present a viable alternative for restoration purposes.
Department of Plant Pathology & Plant MicrobeBiology, Cornell University, Ithaca, New York 14853
Abstract: Interactions with soil microbiota determine the success of restoring plants to their native habitats. The goal of our study was to understand the effects of restoration practices on interactions of giant sequoia Sequoiadendron giganteum with arbuscular mycorrhizal (AM) fungi (Glomeromycota). Natural regeneration of Sequoiadendron is threatened by the absence of severe fires that create forest canopy gaps. Generating artificial canopy gaps offers an alternative tool for giant sequoia restoration. We investigated the effect of regeneration practices, including (i) sapling location within gaps, (ii) gap size and (iii) soil substrate, on AM fungal colonization of giant sequoia sapling roots in a native giant sequoia grove of the Sierra Nevada, California. We found that the extent of AM fungal root colonization was positively correlated with sapling height and light availability, which were related to the location of the sapling within the gap and the gap size. While colonization frequency by arbuscules in saplings on ash substrate was higher relative to saplings in mineral soil, the total AM fungal root colonization was similar between the substrates. A negative correlation between root colonization by Glomeromycota and non-AM fungal species indicated antagonistic interactions between different classes of root-associated fungi. Using DNA genotyping, we identified six AM fungal taxa representing genera Glomus and Ambispora present in Sequoiadendron roots. Overall, we found that AM fungal colonization of giant sequoia roots was associated with availability of plant-assimilated carbon to the fungus rather than with the AM fungal supply of mineral nutrients to the roots. We conclude that restoration practices affectSubmitted 2 Sep 2011; accepted for publication 7 Feb 2012. 1 Current address: Department of Biology, University of Florida, Gainesville, FL 32611 2 Corresponding author. E-mail:
[email protected]
988
FAHEY ET AL.: ARBUSCULAR MYCORRHIZAE IN SEQUOIA Artificial gap-based management has been used to promote regeneration of desired species such as giant sequoia and to increase spatial heterogeneity in forests (Harvey et al. 1980). Several studies have assessed the optimal size of gaps to maximize seedling regeneration while maintaining a sufficiently small gap radius to minimize potential negative effects (York et al. 2004, Mitchell et al. 2006, Forsman et al. 2010). A major determinant of seedling performance in artificial gaps is competition with mature trees in the surrounding matrix forest. Limited above- and belowground resources create gradients in availability across gaps, with resource availability being largely an effect of distance from gap edge (York et al. 2003). Plant competition for belowground resources is mediated by soil microorganisms, especially mycorrhizal fungi (Umbanhowar and McCann 2005). Thus, the interaction of resource availability gradients and mycorrhizal colonization is likely to play a role in the regeneration success of giant sequoia in gap-based management. While there has been little research on the mycorrhizal symbiosis of giant sequoia, it is known that, unlike most conifers, giant sequoia associates with arbuscular mycorrhizal (AM) fungi from the phylum Glomeromycota (Kough et al. 1985, Smith and Smith 1997). Moreover, giant sequoia seedlings have been shown to respond positively to AM inoculation under greenhouse conditions (Kough et al. 1985). Arbuscular mycorrhiza is the most common type of mycorrhizal symbiosis, occurring in the majority of land plants and mostly involving herbaceous plants and deciduous trees (Smith and Read 2008). Development of AM symbiosis is often correlated with low soil nutrients, especially phosphorus. In high phosphorus conditions, AM root colonization is usually low or absent, and AM fungi can act parasitically, acquiring carbon with little or no nutrient supplementation to the plant. Because of the plant responses to biotic and abiotic heterogeneity, gap-based management may affect the balance of give and take between the host plant and the fungus (Jones and Smith 2004, Hoeksema et al. 2010). In addition, AM root colonization is affected by light availability. A high light environment can promote ample carbohydrate supply to the fungus, resulting in higher colonization (Smith and Read 2008). Finally, AM root colonization is affected by the presence of fungal propagules in the soil that initiate the interaction. Across canopy gaps there is greater AM inoculum at gap edges than in the gap centers because of increased access to common mycorrhizal networks associated with forest matrix trees (Perry et al. 1987, Alexander et al. 1992). Thus, across a forest canopy gap, light and belowground resource gradients would be expected to have countervailing effects on AM root
989
colonization. However, it is generally thought that belowground nutrients are the major driver of AM colonization (Smith and Read 2008). In addition to the effects of light and belowground resource availability, community composition and diversity of AM fungi colonizing plant roots are important for the health of the mycorrhizal interaction. While AM fungi are known to have low host specificity, there are significant functional differences between fungal species affecting host-growth response and protection from non-AM fungi (Klironomos 2000). The overall goal of this study was to understand the effects of artificial canopy gap creation on mycorrhizal associations to provide insight into the function of AM symbiosis in giant sequoia regeneration. The specific objectives were to assess the effect of (i) sapling location within gaps, (ii) gap size and (iii) ash substrate or mineral soil on AM colonization of giant sequoia roots. We also were interested in identity of AM fungi colonizing the roots of giant sequoia saplings. We expected higher AM colonization at gap edges than at gap centers because there is greater competition with matrix forest trees at gap edges and therefore less resource availability. AM fungal inoculum associated with mature trees is also more plentiful at gap edges (Alexander et al. 1992). Gap size is expected to magnify the edge effect, so we hypothesized that AM colonization would be higher in smaller gaps than in larger gaps because of greater competition with mature trees and greater availability of fungal inoculum. Because burning creates a nutrient-rich ash layer on the soil surface, we expected that ash substrate would increase soil nutrient availability and consequently reduce AM root colonization in burned sites. In addition, high temperatures during burning may reduce the viable AM inoculum in the soil. MATERIALS AND METHODS Study site.—This study was conducted at the Whitaker Forest Research Station (36u429N, 118u569W) in the southern Sierra Nevada of California bordering Kings Canyon National Park. The site covers 100 ha, with an elevation of 1615–1830 m. The aspect is west-facing with slopes of 15–30% and granite bedrock with soil ranging from shallow to greater than 2 m deep. Annual precipitation averages 107 cm per y, ranging 40–160 cm during past 30 y at nearby Grant Grove. Before the gap creation, the forest canopy structure was two-tiered, with an emergent upper tier of approximately 1.5 large giant sequoia trees per ha of 2.44 m or greater diameter at 1.3 m height, which are estimated to be 1000 + y old. The second tier forms the main canopy and includes giant sequoia, incense cedar Calocedrus decurrens, white fir Abies concolor, sugar pine Pinus lambertiana and ponderosa pine Pinus ponderosa.
990
MYCOLOGIA center of the gap (FIG. 1). One of each pair of saplings was sampled from the bare soil treatment, the other from the ash treatment. For each pair of sub-sampled saplings, light availability was measured after two growing seasons with hemispherical photography as the percent of total transmitted photosynthetically active radiation, %TTR, (TABLE I) (York et al. 2011). Sapling heights were measured in summer 2008 and were considered to be the most relevant metric of sapling potential for recruitment into the canopy. Sampling of roots for AM colonization patterns was conducted in Jul 2009, during the saplings’ fifth growing season. We collected soil with fine roots near the base of each selected sapling by excavating with a trowel 15–20 cm deep (Carter and Gregorich 2008). Fine roots were removed from the soil and immediately fixed in a 50% ethanol solution.
FIG. 1. Overhead view of the experimental layout in a canopy gap at Whitaker Forest Research Station, California. Closed circles represent giant sequoia seedlings planted in 2004. Open circles represent saplings sampled in the present study. The average basal area of the main canopy is 65 m2 per ha. This main cohort was established after logging in the 1870s that removed almost all large pines and half of the large sequoias. The subcanopy is composed mainly of the shadetolerant species such as white fir and incense cedar, whereas giant sequoias are largely absent. Experimental design.—Eighteen circular gaps, 0.04–0.41 ha, were created in 2001 and 2002. For the present study, we grouped gaps into small (0.04–0.10 ha) and large (0.18– 0.41 ha) categories. Gaps in these two classes were expected to be functionally different from each other in terms of their effects on seedling growth. Seedling growth was shown to increase with gap size up to 0.2 ha and level off in larger gaps (York et al. 2011). Trees were cut with chainsaws and removed. Stumps and roots were left intact. Slash (tops and limbs) was piled in windrows along a north-south transect and burned. Two parallel transects of giant sequoia seedlings were planted across the gaps from north to south and extending 12 m into the forest matrix on either side (FIG. 1). One transect was planted on burned substrate (ash) and the other on bare mineral soil. The seedlings were grown from seeds collected from nearby giant sequoias and raised in a nursery 1 y before transplanting into the gaps. In the nursery, seedlings were grown in sterilized soil within plastic containers and were not inoculated with AM fungi before transplanting. Seedlings were planted in the gaps in spring 2004, 2–3 y after gap creation. They were distributed every 3 m in the paired burned and unburned substrates. Sampling.—To describe AM interactions, six saplings were sampled per gap, two located within 6 m of the north and south edge of the gap respectively and two closest to the
Root processing.—A procedure was developed to clear and stain the highly pigmented giant sequoia roots as standard procedures for visualizing AM fungi were unsuccessful. The procedure was adapted from Koske and Gemma (1989) and Kough et al. (1985) with higher KOH concentrations for root clearing and higher temperatures, which were achieved by autoclaving. Roots were removed from ethanol, rinsed with tap water, placed in 20% (w/v) aqueous KOH and autoclaved at 121 C for 45 min. Darkened KOH solution was replaced with a fresh batch of KOH, and roots were autoclaved another 45 min. Roots were rinsed with tap water, bleached in 3% H2O2 for 3 h, rinsed again, and acidified in 1% HCl at least 12 h. Roots were stained in acidic glycerol with 0.05% trypan blue at 90 C for 15 min and transferred to room temperature acidic glycerol to destain for a minimum 24 h before being mounted onto slides for viewing. Microscopy observations revealed that our clearing and staining procedure caused minimal damage to the cortex, provided well cleared samples and well stained fungal structures. We recommend this procedure for future studies of arbuscular mycorrhizae in highly pigmented roots of Cupressaceae. Quantification of AM fungal root colonization.—To quantify the extent of AM fungal colonization in roots, we followed the magnified intersections method of McGonigle (1990). A total of 108 slides (one for each sapling) was examined with an average of 43 intersections per slide. We quantified the percent of root length colonized by AM arbuscules, coils, vesicles and hyphae as well as non-AM hyphae by dividing the counts of intersections with each of these structures by the total number of intersections examined per sample. We also calculated the total percent AM root colonization by adding the counts of intersections with arbuscules, vesicles, coils and mycorrhizal hyphae and dividing by the total number of intersections examined for that sample. Furthermore, we measured the frequency of root samples that contained each type of AM fungal and non-AM fungal structures. Statistical analysis.—We analyzed the response of total percent AM root colonization to position within gap (north edge, center, south edge), gap size (large or small) and soil substrate (ash or bare soil) using a linear mixed effects
ab a b a a a a (0.35) (0.37) (0.41) (0.46) (0.37) (0.38) (0.34) 0.53 0.42 0.66 0.54 0.53 0.48 0.59
hyphae coils
(1.07) (0.95) (0.67) (0.87) (0.68) (0.58) (0.78) 0.98 0.98 0.93 0.94 0.98 0.95 0.98 Only one %TTR measurement was taken for each pair of saplings on ash and mineral soil. a
Substrate
Gap size
North Center South Large Small Ash Mineral soil Within-gap location
36 36 36 48 60 54 54
47.2 (2.2) 60.6 (2.2) 31.4 (2.2) 52.8 (3.0) 40.0 (2.7) N/Aa N/Aa
a b c a b
87.3 247.6 85.7 171.8 108.6 163.2 117.2
(15.0) (14.9) (14.9) (14.0) (12.4) (10.5) (10.3)
a b a a b a b
34.2 40.3 28.6 29.1 38.5 36.5 32.2
(3.6) (3.6) (3.6) (4.1) (3.7) (3.3) (3.1)
ab a b a a a a
0.10 0.18 0.05 0.10 0.10 0.16 0.06
(0.48) (0.56) (0.72) (0.49) (0.44) (0.39) (0.47)
a a a a a a b
0.51 0.46 0.69 0.43 0.67 0.53 0.58
(0.34) (0.35) (0.37) (0.19) (0.31) (0.25) (0.27)
a a a b a a a
0.81 0.84 0.69 0.84 0.71 0.74 0.82
(0.50) (0.41) (0.33) (0.37) (0.30) (0.31) (0.39)
ab a b a a a a
hyphae vesicles arbuscules %TTR n Level Effect
991
model (PROC MIXED) implemented in the SAS statistical software (9.2, SAS Institute Inc., Cary, North Carolina) treating position within gap, gap size class and soil substrate as fixed effects and the gap number (1–18) as a random effect. Sapling height was analyzed in the same way as total percent root colonization using PROC MIXED against the same factors. All P-values were subjected to Tukey-Kramer adjustment. The presence or absence of AM fungal arbuscules, coils, vesicles, hyphae and non-AM fungal hyphae for each sample was modeled against the same factors as above using a generalized linear model (PROC GENMOD). Pearson correlation coefficients were calculated with PROC CORR.
a a a a a a a
non-AM arbuscular mycorrhizal
Total AM root colonization (%) Sapling height (cm)
Colonization frequency by
TABLE I. Mean %TTR, sapling height, total percent of giant sequoia root length colonized by AM fungi and frequency of roots colonized by different AM and non-AM fungal structures. Means are least squares means from PROC MIXED (%TTR, sapling height, total percent root colonization) and PROC GENMOD (root colonization frequencies). Values in brackets are standard errors. Letters indicate a significant difference at P # 0.05.
FAHEY ET AL.: ARBUSCULAR MYCORRHIZAE IN SEQUOIA
Molecular identification of AM fungal species.—Soil samples with fine roots were collected in Jun 2010 from 12 haphazardly selected saplings and kept on ice until roots were removed from soil, rinsed with distilled water, patted dry and frozen in liquid nitrogen for storage at 280 C. Each root sample consisted of ten 1 cm root fragments. Three samples were analyzed per sapling. DNA was extracted with the DNeasy Plant Mini Kit (QIAGEN, Valencia, California) according to the manufacturer’s instructions with two modifications: (i) the amount of lysis buffer was increased to 600 mL and (ii) 15 mg polyvinalpolypyrrolidone (PVPP) was added to the lysis buffer (Parikka and Lemmetty 2004). The DNA isolation yield was measured with a NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, Delaware). Samples with greater than 20 ng mL21 DNA were used for PCR amplification. PCR was performed with the Phusion High-Fidelity DNA polymerase (Finnzymes Espoo, Finland) master mix with primers SSUmAf and LSUmAr (Kru¨ger et al. 2009). PCR reactions contained 1 3 Phusion HF Buffer, 800 mM dNTP mix, 0.5 mM forward and reverse primers, 0.02 U mL21 Phusion polymerase and 1 mL template DNA. Cycling conditions were 5 min initial denaturation at 99 C followed by 40 cycles of 10 s denaturation at 99 C, 30 s annealing at 60 C, 1.5 min elongation at 72 C and a final elongation at 72 C for 10 min. PCR products were examined by agarose gel electrophoresis. Samples that yielded amplicons were targeted for further processing. To minimize PCR artifacts of chimeric amplicons and nucleotide misincorporations, genomic DNA from the initial extraction was used as a template in a low-cycle PCR in which 15 cycles were performed with the same reaction mix and cycling conditions as the initial PCR, except for the denaturation step that was set at 98 C and the initial denaturation was 3 min. Low-cycle PCR products were cloned with the TOPO TA CloningH Kit for Sequencing (Invitrogen, Carlsbad, California) following the manufacturer’s instructions and were transformed into One ShotH Mach1TM2T1 R competent E. coli cells. Sixteen E. coli colonies were selected per amplicon for further analysis. Plasmid DNA was amplified with TempliPhi (GE Healthcare, Piscataway, New Jersey). TempliPhi products were purified with the QIAquick PCR Purification Kit (QIAGEN) and sequenced with BigDye Terminator 3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, California), sequencing primers T3 and T7 and the Applied Biosystems Automated 3730 DNA Analyzer.
992
MYCOLOGIA
Sequences were analyzed with the Geneious software package (Biomatters Ltd, Auckland, New Zealand). Contigs were assembled for each sample from the forward and reverse sequencing runs and subjected to nucleotide BLASTN (Altschul et al. 1990) queries of the National Center for Biotechnology Information databases to confirm their AM fungal origin. To assess taxonomic affiliations of giant sequoia AM fungi, we used MUSCLE (Edgar 2004) to align their rRNA gene sequences with published reference sequences from morphologically defined taxa representing the diversity of Glomeromycota. The alignment was adjusted manually. The ITS1 and ITS2 rRNA gene regions were excluded from the analysis due to alignment ambiguities, yielding a 1292 nucleotide alignment of 73 sequences representing the 39– fragment of the 18S, the entire 5.8S, and the 59– fragment of the 28S rRNA gene. Phylogenetic trees were constructed with MrBayes (Huelsenbeck and Ronquist 2001, Ronquist and Huelsenbeck 2003) and PhyML (Guindon and Gascuel 2003) with the general time reversible nucleotide substitution model (Tavare´ 1986), invariable sites and gamma rate variation. Bayesian analyses were carried out for 4 000 000 generations. Statistical support for the maximum likelihood tree topology was based on 1000 bootstrap replicates. Sequences generated were deposited at GenBank with accession numbers HQ895789–HQ895818.
RESULTS Patterns of root colonization.—Roots of giant sequoia saplings at the Whitaker Forest Research Station harbored AM fungal coils, vesicles and hyphae. Arbuscules were uncommon, and, where they were observed, they took the form of arbusculate coils. Total AM fungal colonization of sapling roots was 0–85.2% and averaged 34.4 6 0.2% SEM. AM fungal coils occupied 0–48.6% of root length averaging 10.9 6 0.1%, AM fungal vesicles occupied 0–29.6% of root length averaging 3.8 6 0.1% and AM fungal hyphae colonized 0–81.5% of root length averaging 30.5 6 0.2%. At least one species of non-AM fungi was common in the roots, identifiable by its highly septate hyphae. The average percent of root length colonized by nonAM fungi was 10.4 6 0.2%. A significant inverse correlation was observed between AM fungal colonization and colonization by non-AM fungi (r 5 20.3799, P # 0.0001). Sapling location within gap.—Sapling height varied significantly with the location within gaps, where saplings at the center of the gap were significantly taller than those at the north and south positions within the gap (P # 0.0001, TABLE I). Total AM root colonization also was affected by the location within the gap (P 5 0.0178), which was driven primarily by the significantly greater AM colonization in the center of gaps than on the south edge of gaps (P 5 0.0129), while
FIG. 2. The effect of giant sequoia sapling position within gap on: (a) total percent root colonization by AM fungi subdivided into fungal structures and (b) non-AM fungal colonization. Bars indicate standard error for total colonization. Bars labeled with different letters indicate a significant difference of P # 0.05.
the north edge remained intermediate (FIG. 2a). Total AM colonization was positively correlated with sapling height (r 5 0.2583, P 5 0.0072). Total AM colonization also was positively correlated with the percent of total transmitted photosynthetically active radiation, %TTR (r 5 0.2231, P 5 0.0215). The presence of coils followed the same pattern as the total AM fungal colonization, with coils significantly more common in the center of the gaps than at the south edge (P 5 0.0308, TABLE I), while the north edge remained intermediate. Presence of non-AM fungi followed the opposite pattern as the total AM colonization with significantly higher colonization at the south edge than in the center of gaps (P 5 0.0318) and intermediate colonization at the north edge (FIG. 2b, TABLE I). Gap size.—Sapling height varied significantly with gap size where saplings were significantly taller in large gaps than in small gaps (P 5 0.0038, TABLE I).
FAHEY ET AL.: ARBUSCULAR MYCORRHIZAE IN SEQUOIA No significant difference was observed in the total AM root colonization between the large and small gaps. However, root colonization by vesicles was significantly more common in small gaps than in large gaps (P 5 0.0223, TABLE I). All other fungal structures were not affected by the gap size. Soil substrate.—Sapling height varied with soil substrate, with saplings being significantly taller on ash than on mineral soil (P # 0.0001, TABLE I). Roots harboring arbuscules were significantly more frequent in saplings growing on ash relative to saplings on mineral soil. However, the total AM fungal root colonization was similar between the substrates as were the frequencies of roots with mycorrhizal vesicles, coils, hyphae and non-AM fungal hyphae. Molecular identification of fungi.—A total of 29 unique cloned rRNA gene sequences of AM fungi were obtained from roots of two saplings: one at the north edge of a small gap and one at the center of a large gap. Phylogenetic analyses of these sequences with reference sequences from AM fungal taxa identified morphologically revealed that giant sequoia roots harbored fungi from the genera Glomus and Ambispora (FIG. 3). Some of the Glomus-related sequences clustered with sequences from the morphologically defined taxa such as G. candidum (clade S2 in FIG. 3) and G. aggregatum (clade S6). Others were members of the lineages of G. etunicatum/ claroideum/luteum (clade S3) and G. intraradices/ irregulare (clade S4). Despite our limited sampling, we also found sequences forming a distinct clade (S5) that did not include any named reference taxa. This clade, in addition to AM fungi associated with giant sequoia, contained AM fungi found in Rubus parvifolius in the coastal sand dune system in Japan (Ahulu et al. 2006). DISCUSSION Patterns of giant sequoia root colonization.—Arbuscular mycorrhizal fungal colonization patterns in giant sequoia, which we describe here, have received little attention despite this species’ high cultural and ecological value. Our study confirmed the observation by Galluad (1905) that giant sequoia forms Paris-type arbuscular mycorrhizae with high abundance of hyphal coils and low abundance of arbuscules (FIG. 2). Nutrient transfer and growth enhancement has been observed in both Paris- and Arum-type mycorrhizae, but the functional difference between these two types is still poorly understood (Dickson et al. 2007). The total AM root colonization average of 34% was consistent with the only other study to our knowledge
993
that assessed AM colonization of giant sequoia, which observed 20% colonization in inoculated sequoia seedlings (Kough et al. 1985). In addition, similar levels of colonization have been reported in other studies of forest canopy gaps. For example, in tropical tree seedlings Whitbeck (2001) observed 20% root colonization in low light and 47% colonization in high light conditions. Lovelock and Miller (2002) found 20% AM root colonization of temperate red maple seedlings in forest understory versus 32% in canopy gaps. The strong negative correlation between the presence of AM fungi and non-AM fungi could be a sign of antagonistic interactions between AM fungi and root pathogenic fungi or fungal endophytes. Several studies have shown that AM root colonization can provide protection against plant pathogens, although mechanisms of this phenomenon are poorly understood (Whipps 2004, Herre et al. 2007, Smith and Read 2008). Two possible mechanisms could explain the negative correlation: (i) competition for colonization sites between species and (ii) activation of the plant’s defense mechanisms by the presence of AM fungi. Either of these mechanisms could help explain the observed negative correlation; however, they do suggest colonization by AM fungi before infection by the fungal pathogen. It also is possible that non-AM fungi are able to more easily infect smaller and more resource-stressed plants and the correlation pattern is circumstantial. Sapling location within gaps.—Our data showed the highest AM root colonization in the center of gaps, where light and the belowground resource availability are highest (York et al. 2003). The lowest colonization occurred at south edges of gaps, where the total resource availability is low, and intermediate at north edges, where light availability is intermediate but belowground resources are low due to competition with forest matrix trees (York et al. 2003). Such colonization patterns, along with the positive correlation with the percent of total transmitted photosynthetically active radiation, %TTR, suggest that the light availability is an important factor influencing AM fungal activity in giant sequoia roots. Low light environments can reduce AM colonization because of low amount of carbon that can be supplied by the plant to the fungal symbiont (Smith and Read 2008). AM fungi can pose a significant drain on plant hexoses, in some cases using up to 20% of the plantassimilated carbon (Pearson and Jakobsen 1993). Root colonization also was significantly correlated with sapling height, which supports the hypothesis that saplings with an ample supply of photosynthetic carbon also can support abundant AM fungi. A
994
MYCOLOGIA
FIG. 3. Taxonomic affiliation of giant sequoia AM fungi based on 18S-5.8S-28S rRNA gene sequences. A Bayesian tree is shown with posterior probabilities of 0.8 or greater displayed above branches; branches with maximum likelihood bootstrap support of 70% or greater are thickened. Sequences G11 and G19 represent cloned rRNA gene fragments of giant sequoia AM fungi, where G11 refers to a root sample taken from the center of a large gap and G19 refers to a sample taken from the edge of a small gap.
similar effect was shown by Whitbeck (2001) who observed increased AM colonization of a tropical lowland tree species at higher irradiance levels. Arbuscular mycorrhizal coils act as important sites of nutrient transfer between the root and fungus (Smith and Read 2008). In our study, coils followed the same pattern as total AM colonization, with coils significantly more common in the center of the gaps than at the south edges (FIG. 2). A similar effect was observed in a study by Pearson et al. (1991), where lower light intensity was correlated with fewer and
smaller arbuscules, which are comparable in function to coils in Paris-type mycorrhizae (Dickson et al. 2007). AM fungal effects on plant vigor extend beyond nutrient uptake and pathogen protection. AM fungal colonization has been shown to reduce oxidative stress in plants, indicating that high AM colonization in gap centers may be related to higher light conditions and potentially to the plant’s need for protection from oxidative stress (Porcel and RuizLozano 2004). In addition, AM fungi have been
FAHEY ET AL.: ARBUSCULAR MYCORRHIZAE IN SEQUOIA shown to increase drought tolerance by increasing the leaf water potential (Porcel and Ruiz-Lozano 2004). The water stress of the saplings at our site was shown to be lower at gap centers than at gap edges (York et al. 2011), which also may be connected with AM colonization. Because of its ability to colonize severely disturbed sites, giant sequoia is considered a long-lived pioneer species. Although giant sequoia seedlings can survive without AM fungi, their positive growth responses to AM inoculation under greenhouse conditions suggest that availability of AM inoculum during seedling establishment in nature may be important for their ultimate success (Kough et al. 1985). In our study, we found no evidence that inoculum is limiting in the gap centers. We speculate that the extensive root systems of mature giant sequoias surrounding the gaps may have been the source of inoculum for the experimental seedlings (York et al. 2010). Sequoiadendron roots can extend over 30 m horizontally and thus reach to the centers of most gaps (Hartesveldt et al. 1975). Sequoia saplings at the centers of gaps are highly successful compared to those at the edges. Saplings at the centers of gaps have both abundant belowground resources as well as sufficient carbon to support AM fungi, giving them additional competitive advantage. Higher colonization may help explain the large differences in sapling height between gap center and gap edge (i.e. saplings at the center are more than three times as tall as saplings at the edge). This suggests that the symbiosis as a whole has improved function at the centers of gaps because both partners have improved growth. Gap size.—Gap size has received a large amount of attention in silviculture because of the interest in optimizing it for tree growth (Coates 2000). Light and the belowground resource gradients are expected to be intensified in larger gaps and reduced in smaller gaps (York et al. 2003). However, in our study gap size did not significantly affect the total AM root colonization. AM fungal vesicles, which are important storage structures for the fungal symbiont, were the only structures that varied with the gap size. The higher abundance of vesicles in small gaps than in large gaps was an unexpected result because in other studies the production of storage structures (i.e. vesicles and spores) has been shown to increase with increased soil fertility and access to belowground resources compared to the nutrient transfer structures (i.e. coils) (Johnson et al. 2003, Nijjer et al. 2010). Moreover, Pearson et al. (1991) found fewer vesicles at lower light, which also is contrary to our findings. A possible explanation for these discrepan-
995
cies is that in smaller gaps AM fungi are under greater carbon stress and may act at the parasitic end of the symbiotic spectrum, using more carbon for storage structures while providing less mineral nutrients return. However, it is also important to recognize that AM fungal allocation to structures can be variable over time (Johnson et al. 2003), while our study provides a single time point observation. Soil substrate.—The soil substrate treatment was expected to have an effect on the availability of belowground resources, because giant sequoia seedlings have greater success on ash substrate (Harvey et al. 1980, York et al. 2011). In our study, the sapling height was greater on ash substrate than on mineral soil and roots with arbuscules were more frequent in saplings on ash relative to saplings on mineral soil. Otherwise, the levels of root colonization were similar between the substrates. The overall pattern of AM root colonization in the ash and mineral soil substrates, which have contrasting levels of belowground resources and sapling growth but equivalent light availability, supports the hypothesis of carbon budget-driven AM colonization. AM fungal species identification.—Identification of AM fungi that naturally associate with tree species threatened by extinction is important for their conservation and restoration to natural habitats. It also may benefit cultivation of trees for ornamental purposes. Because soil fumigation is a common practice in tree seedling nurseries and it severely reduces soil microbe abundance, inoculation with AM fungi can better prepare seedlings for transplanting into their target habitats (Menge 1982). The taxa of AM fungi identified in our study provide only a snapshot of the AM fungal community in Sequoiadendron roots. Moreover, their individual effects on the host fitness are uncertain. Nevertheless, information about taxonomic identity of these fungi can be useful for inoculating seedlings when grown in nurseries to aid in giant sequoia restoration and cultivation efforts. While some of the rRNA gene sequences, which we recovered from the giant sequoia roots, clustered with sequences from common taxa representing the lineages of Glomus etunicatum/claroideum/luteum and G. intraradices/irregulare, others could not be grouped with any known morphospecies. This finding suggests that for nursery propagation, fungi from the clades of G. etunicatum/ claroideum/luteum and G. intraradices/irregulare might be compatible with giant sequoia seedlings. In the past, Sequoiadendron seedlings were shown to respond positively under greenhouse conditions to Acaulospora trappei, G. deserticola and G. epigeum (Kough et al. 1985). However, from a conservation
996
MYCOLOGIA
standpoint, to best replicate the community of AM fungi that associate with giant sequoia in nature, nursery seedlings should be inoculated with the fungi derived from giant sequoia natural habitat. CONCLUSIONS Restoration of giant sequoia to address the centurylong failure of natural regeneration necessitates understanding of ecological processes surrounding Sequoiadendron regeneration and growth. Our study shows that restoration practices involving artificial gap creation permit AM fungal colonization of giant sequoia saplings, which is an important consideration because improved growth, stress resistance and pathogen protection of saplings are likely promoted by AM root colonization. AM fungal taxa associated with Sequoiadendron included representatives of the genera Ambispora and Glomus. We found that AM fungal colonization of giant sequoia roots was correlated with availability of plant-assimilated carbon to the fungus rather than with the AM fungal supply of mineral nutrients to the roots. Consequently we conclude that restoration practices that affect light availability and carbon assimilation alter feedback between sapling growth and activity of AM fungi in the roots. ACKNOWLEDGMENTS We thank J. Battles for help with field collections, N. VanKuren and S. Mondo for assistance with molecular work, F. Vermeylen for statistical advice, J. Morton for permission to use his unpublished Glomus aggregatum rRNA gene sequence deposited at GenBank and D. Moebius-Clune for comments on the manuscript. This research was supported by the UC Center for Forestry and Cornell University.
LITERATURE CITED Ahulu EM, Gollotte A, Gianinazzi-Pearson V, Nonaka M. 2006. Cooccurring plants forming distinct arbuscular mycorrhizal morphologies harbor similar AM fungal species. Mycorrhiza 17:37–49, doi:10.1007/s00572-0060079-0 Alexander I, Ahmad N, See LS. 1992. The role of mycorrhizas in the regeneration of some Malaysian forest trees. Philos T Roy Soc B 335:379–388, doi:10.1098/rstb.1992.0029 Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J Mol Biol 215:403– 410. Carter MR, Gregorich EG. 2008. Soil sampling and methods of analysis. Boca Raton, Florida: CRC Press. 1224 p. Coates KD. 2000. Conifer seedling response to northern temperate forest gaps. Forest Ecol Manag 127:249–269, doi:10.1016/S0378-1127(99)00135-8
Dickson S, Smith FA, Smith SE. 2007. Structural differences in arbuscular mycorrhizal symbiosies: More than 100 years after Gallaud, where next? Mycorrhiza 17: 375–393, doi:10.1007/s00572-007-0130-9 Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797, doi:10.1093/nar/gkh340 Forsman JT, Reunanen P, Jokima¨ki J, Mo¨nkko¨nen M. 2010. The effects of small-scale disturbance on forest birds: a meta analysis. Can J Forest Res 40:1833–1842, doi:10.1139/X10-126 ´ tudes sur les mycorrhizes endotrophs. Rev Gallaud I. 1905. E Gen Bot 17:66–83, 123–135, 223–239, 313–325, 425, 479–500. Guindon S, Gascuel O. 2003. A simple, fast and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52: 696– 704, doi:10.1080/ 10635150390235520 Hartesveldt RJ, Harvey HT, Shellhammer HS, Stecker RE. 1975. The giant sequoia of the Sierra Nevada. Washington DC: US Department of the Interior, National Park Service. 180 p. Harvey HT, Shellhammer HS, Stecker RE. 1980. Giant sequoia ecology: fire and reproduction. Washington DC: US Department of the Interior, National Park Service. 182 p. Herre EA, Mejı´a LC, Kyllo DA, Rojas E, Maynard Z, Butler A, van Bael SA. 2007. Ecological implications of antipathogen effects of tropical fungal endophytes and mycorrhizae. Ecology 88:550–558, doi:10.1890/ 05-1606 Hoeksema JD, Chaudhary VB, Gehring CA, Johnson NC, Karst J, Koide RT, Pringle A, Zabinski C, Bever JD, Moore JC, Wilson GWT, Klironomos JN, Umbanhowar J. 2010. A meta analysis of context dependency in plant response to inoculation with mycorrhizal fungi. Ecol Lett 13:394–407, doi:10.1111/j.1461-0248.2009.01430.x Huelsenbeck JP, Ronquist F. 2001. MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics 17: 754–755, doi:10.1093/bioinformatics/17.8.754 Janos DP. 1980. Vesicular-arbuscular mycorrhizae affect lowland tropical rain forest plant growth. Ecology 61: 151–162, doi:10.2307/1937165 Johnson NC, Rowland DL, Corkidi L, Egerton-Warburton LM, Allen EB. 2003. Nitrogen enrichment alters mycorrhizal allocation at five mesic to semiarid grasslands. Ecology 84:1895–1908, doi:10.1890/00129658(2003)084[1895:NEAMAA]2.0.CO;2 Jones MD, Smith SE. 2004. Exploring functional definitions of mycorrhizas: Are mycorrhizas always mutualisms? Can J Bot 82:1089–1109, doi:10.1139/b04-110 Klironomos J. 2000. Host-specificity and functional diversity among arbuscular mycorrhizal fungi, In: C Bell, M Byrlinsky, P Johnson-Green, eds. Microbial biosystems: new frontiers. Halifax, Nova Scotia: Proceedings of the 8th International Symposium on Microbial Ecology. Atlantic Canada Society for Microbial Ecology. Koske RE, Gemma JN. 1989. A modified procedure for staining roots to detect VA mycorrhizas. Mycol Res 92: 486–488, doi:10.1016/S0953-7562(89)80195-9
FAHEY ET AL.: ARBUSCULAR MYCORRHIZAE IN SEQUOIA Kough JL, Molina R, Linderman RG. 1985. Mycorrhizal responsiveness of Thuja, Calocedrus, Sequoia and Sequoiadendron species of western North America. Can J Forest Res 15:1049–1054, doi:10.1139/x85-170 Kru¨ger M, Stockinger H, Kru¨ger C, Schu¨ßler A. 2009. DNAbased species level detection of Glomeromycota: one PCR primer set for all arbuscular mycorrhizal fungi. New Phytol 183:212–223, doi:10.1111/j.1469-8137. 2009.02835.x Lovelock CE, Miller R. 2002. Heterogeneity in inoculum potential and effectiveness of arbuscular mycorrhizal fungi. Ecology 83:823–832, doi:10.1890/0012-9658 (2002)083[0823:HIIPAE]2.0.CO;2 McGonigle TP, Miller MH, Evans DG, Fairchild GL, Swan JA. 1990. A new method which gives an objective measure of colonization of roots by vesicular-arbuscular mycorrhizal fungi. New Phytol 115: 495– 501, doi:10.1111/j.1469-8137.1990.tb00476.x Menge JA. 1982. Effect of soil fumigants and fungicides on vesicular-arbuscular fungi. Phytopathology 72:1125– 1132. Mitchell RJ, Hiers JK, O’Brien JJ, Jack SB, Engstrom RT. 2006. Silviculture that sustains: the nexus between silviculture, frequent prescribed fire and conservation of biodiversity in longleaf pine forests of the southeastern United States. Can J Forest Res 36:2724–2736, doi:10.1139/x06-100 Mutch LS, Swetnam TW. 1995. Effects of fire severity and climate on ring-width growth of giant sequoia after burning. Missoula, Montana: Symposium on Fire in Wilderness and Park Management. USDA Forest Serv Gen Tech Rep INT-GTR-320. p 241–246. Nijjer S, Rogers WE, Siemann E. 2010. The impacts of fertilization on mycorrhizal production and investment in western Gulf Coast grasslands. Am Midl Nat 163:124– 133, doi:10.1674/0003-0031-163.1.124 Parikka P, Lemmetty A. 2004. Tracing latent infection of Colletotrichum acutatum on strawberry by PCR. Eur J Plant Pathol 110:393–398, doi:10.1023/B:EJPP. 0000021073.67137.d2 Pearson JN, Jakobsen I. 1993. Symbiotic exchange of carbon and phosphorus between cucumber and three arbuscular mycorrhizal fungi. New Phytol 124:481–488, doi:10.1111/j.1469-8137.1993.tb03839.x ———, Smith SE, Smith FA. 1991. Effect of photon irradiance on the development and activity of VA mycorrhizal infection in Allium porrum. Mycol Res 95: 741–746, doi:10.1016/S0953-7562(09)80824-1 Perry DA, Molina R, Amaranthus MP. 1987. Mycorrhizae, mycorrhizospheres and reforestation—current knowledge and research needs. Can J Forest Res 17:929–940, doi:10.1139/x87-145 Porcel R, Ruiz-Lozano JM. 2004. Arbuscular mycorrhizal influence on leaf water potential, solute accumulation and oxidative stress in soybean plants subjected to
997
drought stress. J Exp Bot 55:1743–1750, doi:10.1093/ jxb/erh188 Ronquist F, Huelsenbeck JP. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574, doi:10.1093/bioinformatics/ btg180 Smith FA, Smith SE. 1997. Structural diversity in (vesicular)arbuscular mycorrhizal symbioses. New Phytol 137:373– 388, doi:10.1046/j.1469-8137.1997.00848.x Smith SE, Read DJ. 2008. Mycorrhizal symbiosis. New York: Academic Press. 800 p. Stephenson NL. 1999. Reference conditions for giant sequoia forest restoration: structure, process and precision. Ecol Appl 9:1253–1265, doi:10.1890/10510761(1999)009[1253:RCFGSF]2.0.CO;2 Tavare´ S. 1986. Some probabilistic and statistical problems in the analysis of DNA sequences. In: Miura RM, ed. New York: American Mathematical Society: lectures on mathematics in the life sciences. p 57–86. Umbanhowar J, McCann K. 2005. Simple rules for the coexistence and competitive dominance of plants mediated by mycorrhizal fungi. Ecol Lett 8:247–252, doi:10.1111/j.1461-0248.2004.00714.x Walker LR, del Moral R. 2003. Primary succession and ecosystem rehabilitation. Cambridge, UK: Cambridge Univ. Press. 442 p. Whipps JM. 2004. Prospects and limitations for mycorrhizas in biocontrol of root pathogens. Can J Bot 82:1198– 1227, doi:10.1139/b04-082 Whitbeck JL. 2001. Effects of light environment on vesicular-arbuscular mycorrhiza development in Inga leiocalycina, a tropical wet forest tree. Biotropica 33: 303–311. York RA, Battles JJ, Eschtruth AK, Schurr FG. 2011. Giant sequoia (Sequoiadendron giganteum) regeneration in experimental canopy gaps. Restor Ecol 19:14–23, doi:10.1111/j.1526-100X.2009.00537.x ———, ———, Heald RC. 2003. Edge effects in mixed conifer group selection openings: tree height response to resource gradients. Forest Ecol Manag 179:107–121, doi:10.1016/S0378-1127(02)00487-5 ———, Fuchs D, Battles JJ, Stephens SL. 2010. Radial growth responses to gap creation in large, old Sequoiadendron giganteum. Appl Veg Sci 13:498–509, doi:10.1111/j.1654-109X.2010.01089.x ———, Heald RC, Battles JJ, York JD. 2004. Group selection management in conifer forests: relationships between opening size and tree growth. Can J For Res 34:630– 641, doi:10.1139/x03-222 ———, Stephenson NL, Meyer M, Hanna S, Moody T, Caprio T, Battles JJ. 2012. Giant sequoia. In: Panek JA, Battles JJ, Austin JT, Sydoriak CA, Nydick NR, Esperanza Am, Saah DD, eds. National Park Service. Fort Collins, Colorado: A natural resource condition assessment for Sequoia and Kings Canyon national parks. (In press).
