© INRA, EDP Sciences, 2009

1. INTRODUCTION

Increased mortality has been affecting silver fir (Abies alba Mill.) forests of the Pyrenees since the mid 1980s (Camarero et al., 2002). Increased aridity of the Pyrenean region (Macías et al., 2006) has probably contributed to the mortality of this species (Camarero et al., 2002), whilst the management of these stands in the last decades of the 20th century could have predisposed certain trees of these stands to decline (Oliva and Colinas, 2007). Armillaria (Fries: Fries) Staude species are commonly observed on dead silver fir trees (Oliva and Colinas, 2007) and have been observed to cause increased mortality in fir forests of Italy (Clauser, 1980; Intini, 1988).

Armillaria species infect roots of stressed trees (Fox, 2000b) penetrating the bark with a specialized form of mycelium called rhizomorph. The infection then spreads from the infected tree to neighbouring trees through root-to-root contact or as rhizomorphs (Fox, 2000a). Root infection results in reduced tree growth and eventual death after years of gradual decline (Cherubini et al., 2002). In our context, both butt rot and girdling have been observed on Pinus and A. alba trees. Infected trees are more susceptible to other pathogenic agents. Increased damages caused by other agents reported in the Pyrenees (Camarero et al., 2003; Martín and Cobos, 1986; Oliva and Colinas, 2007) could be a result of the undetected presence of this pathogen.

There are differences in virulence amongst the species of the genus Armillaria (Guillaumin et al., 2003). Armillaria mellea (Vahl: Fries) Kummer is very destructive in broadleaf forests, fruit trees, grapevine orchards, and on ornamental trees. Armillaria ostoyae (Romagnesi) Herink is more damaging to conifer species. Other Armillaria species, such as A. gallica Marxmuller and Romagnesi (synonym: A. bulbosa (Barla) Velenovsky), A. cepistipes Velenovsky, A. borealis Marxmuller and Korhonen, A. tabescens (Scolpoli: Fries) and A. ectypa (Fries) Lamoure are generally considered secondary pathogens or saprobes. Knowledge of the abundance and distribution of the different species of the genus Armillaria in the Pyrenees would help us estimate the role of this inconspicuous fungus in silver fir mortality.

Armillaria has been reported as an influencial factor in the dynamics of several mountain forest types in Europe (Bendel et al., 2006a; Dobbertin et al., 2001) and in North America (Worrall et al., 2005). To our knowledge, its role in the dynamics of silver fir forests has never been studied. Tree-species composition of silver fir forests in the Pyrenees is changing: silver fir and Fagus sylvatica L. are increasing whilst Pinus sylvestris L. and Pinus uncinata Ram. are decreasing in abundance (Oliva and Colinas, 2007). Since there are differences in susceptibility to Armillaria among these tree species (Morquer and Touvet, 1972), Armillaria could play a role in the shifting species composition of these forests. Management has had an impact on the health of silver fir forests in the Pyrenees (Oliva and Colinas, 2007) and potentially on the availability of substrates for Armillaria species. Yet, the relationships among forest management practices, Armillaria populations, and stand health in silver fir forests have not been evaluated.

Our research aims to study the presence and distribution of the different species of the genus Armillaria in silver fir forests of the Pyrenees, and their interaction with the ecology and management of these stands.

2. MATERIALS AND METHODS

2.1. Field sampling

We studied the silver fir population of the Spanish Pyrenees. In this region, silver fir grows on northern aspects with high productivity indices. It often appears mixed with F. sylvatica in central and western locations, with P. uncinata at higher elevations, and with P. sylvestris at drier locations. Silver fir habitats are relatively cool sites (average temperature range in January is between –3 °C and 0 °C, and in August between 15 °C and 18 °C) at elevations ranging from 700 m to 2000 m above sea level (Blanco et al., 1997). We used the sampling grid described in Oliva and Colinas (2007), based on the level 1 grid used by the International Co-operative Programme on Assessment and Monitoring of Air Pollution Effects on Forests (ICP Forests) (Montoya et al., 1998), which we further subdivided at 2 km × 2 km or 1 km × 1 km . This systematic sampling grid includes 29 circular plots of 10 m diameter with at least six A. alba trees in the canopy. Dependent and independent variables measured in every plot are explained in Table I.

From 2002 to 2003, we surveyed each of 29 stands for belowground and aboveground signs of Armillaria. The aboveground frequency for Armillaria was estimated by the percentages of stumps and dead trees colonized by Armillaria (Tab. I). Stumps and living and dead trees were assessed for mycelial fans, rhizomorphs and fruiting bodies. Butt rot produced by Armillaria on living fir trees was assessed by culturing inner-wood cores. In each plot, we extracted 2 cores per tree from the base of the stem of 6 randomly selected trees. Drilling was performed downwards and towards the stem centre. We sterilized the borer with ethanol 70% v/v between extractions and cores were kept in polyethylene bags and stored at 5 °C until they were cultured. For plots where we found no signs of Armillaria and for those in which the number of collected Armillaria tissue samples was low, we assessed silver fir saplings and seedlings and the area surrounding the plot for additional tissue samples. We used these samples when comparing environmental parameters between Armillaria species, but they were not included in the calculations of Armillaria abundance variables.

The belowground abundance was assessed by measuring rhizomorphs from soil following the methodology of Rigling et al. (1997). Soil samples were collected from four randomly selected trees per plot. Each soil sample consisted of four pooled sub-samples, collected 2 m from each selected tree, following the four cardinal directions. Each soil sub-sample was prismatic with approximate dimensions of 0.125 m × 0.125 m × 0.300 m depth. Once in the laboratory, we sieved soil samples with a 0.009 m sieve to facilitate the separation of the rhizomorphs. Once the rhizomorphs were washed, they were weighed and measured and the mean weight and the mean length per plot were calculated. A third variable characterizing Armillaria belowground abundance was the frequency of Armillaria in soil, calculated as the percentage of soil samples with rhizomorphs present.

From each soil sample, tree, or stump where signs of Armillaria were observed we attempted to culture a single Armillaria isolate. Collected tissues and wood inner cores were sterilized 10–20 s in hydrogen-peroxide 30% v/v. Sterilization was stopped by placing tissues in sterile distilled water. Surface sterilized tissues were cultured in a modified BDS selective media (Harrington et al., 1992), which consisted of 4 ppm benomyl, 0.0001% w/v streptomycin, 1.5% w/v malt extract, 1.5% w/v agar and no dichloran. Plates were incubated in the dark at room temperature (≈ 20 °C), and once any mycelial growth was observed, we transferred the isolate to malt extract agar media consisting of 1.5% w/v malt extract and 2% w/v agar. Isolates were assigned to the genus Armillaria based on their culture morphology.

2.2. Species identification by PCR-RFLP

We extracted the DNA of the isolates following the CTAB-based protocol used by Kårén et al. (1997), and we amplified a portion of the intergenic spacer region (IGS-1) of the rDNA operon using the primers LR12R and O-1 as described by Harrington and Wingfield (1995). Amplifications were performed using puReTaq Ready-to-go PCR beads (GE-Healthcare, UK) in a Biometra (Goettingen, DE) T-Personal thermal cycler. We included a negative control with no DNA with every set of PCR reactions. Isolates were typed at species level comparing their RFLP pattern with those reported by Harrington and Wingfield (1995) and Pérez-Sierra et al. (1999). For isolates that could not be identified by their IGS-1 RFLP pattern, we amplified the Internal Transcribed Spacer (ITS) region including the 5.8S gene subunit of the same rDNA operon by using ITS1F (Gardes and Bruns, 1993) and ITS4 (White et al. 1990) primers, following the cycling conditions described by Gardes and Bruns (1993).

2.3. DNA sequencing

Several isolates resulted in a non-conclusive RFLP pattern and others were typed as A. borealis, a northern-European Armillaria species. IGS-1 and ITS amplification products of these isolates were sequenced. We also sequenced the IGS-1 amplification products of isolates typed as A. cepistipes and A. ostoyae collected near the unsuccessfully typed isolates. Genbank (NCBI) accession numbers of sequences and isolate description are presented in Table II. PCR products were sequenced in both directions with the corresponding primers using an ABI 3100 sequencer (Applied Biosystems) at the Genomic Service Facility of the Autonomous University of Barcelona (Bellaterra, Barcelona, Spain) and with an ABIPRISM 310 sequencer (Applied Biosystems) at the Sequencing and Fragment Analysis Service of the University of Malaga (Malaga, Spain). Sequences were edited and aligned first with ClustalW and then manually with the software Mega version 4 (Tamura et al., 2007). We confirmed the Alu I, Nde I and Bsm I digestion patterns observed in the electrophoresis by analysing the sequences with the software Bioedit version 7.0.0 (Hall, 1999). Isolates that could not be satisfactorily identified by their PCR-RFLP pattern were identified by performing a nucleotide BLAST search (Altschul et al., 1997), comparing their sequences with those available in NCBI database and those available in the database of the Dept. of Forest Mycology and Pathology (SLU, Sweden). In both cases, we only used the sequences of isolates collected in Europe and identified at species level by two independent methods.

2.4. Statistical analysis

We analyzed the differences in environmental and management stand characteristics amongst Armillaria species by one way ANOVA, and we compared species means by the protected least square differences method (LSD). We used the GLM procedure of SAS/STAT.

Rhizomorph weight and rhizomorph length relationships with independent variables were analysed by Poisson regression with logarithm as link function. The relationships of ecological and management variables with Armillaria frequency in soil, Armillaria stump colonisation and Armillaria dead tree colonisation were analyzed by logistic regression. Both generalized regression models were adjusted with the GENMOD procedure of SAS/STAT. Overdispersion was corrected by the ratio between the deviance and the degrees of freedom (Schabenberger and Pierce, 2001). The assumptions of the hypotheses of linearity, normality and homogeneity were met by selecting the best BOX-COX transformation of the independent variable.

The means are presented with the 95% confidence interval (CI). Variables subjected to logit or logarithmic transformations were back-transformed. In these cases, the median and the confidence limits of the median are presented. Exact confidence limits for percentages were calculated using a Bayesian approach (Clopper and Pearson, 1934).

3. RESULTS

3.1. Armillaria root rot: analyses at species level

Sampling of fungal tissues resulted in 111 samples of Armillaria. Most were from rhizomorphs (108), and only a few represented basidiome (1) and mycelia (2) tissue. From these samples, the total number of cultured isolates was 80 (Tab. III), since several samples did not yield any growth in culture, or resulted in contaminations. Out of 174 living trees assayed, Armillaria was isolated from only one individual with both inner wood cores providing positive colonisation results. Butt rot produced by Armillaria affected 0.6% (CI: 0.0–3.2) of living silver fir trees. No other Armillaria signs were observed on living trees.

Three Armillaria species were identified according to their RFLP digestion pattern: A. cepistipes, A. gallica and A. ostoyae. Two isolates showed the RFLP pattern of A. borealis. However, their IGS-1 and ITS sequencing showed greater correspondence with A. ostoyae isolates and were therefore considered A. ostoyae. Five isolates yielded the same non-reported pattern in the Alu I digestion, with fragments of 583 and 200 bp. We consider these five isolates to be A. cepistipes as their IGS-1 and ITS sequences showed greatest similarity with A. cepistipes isolates.

Armillaria cepistipes was the most frequent species (72% of isolates) in our survey. Armillaria gallica (15%) and A .ostoyae (10%) were less frequent (Tab. III). Armillaria cepistipes was predominant in all surveyed substrates: soil, stumps, dead trees, seedlings and saplings. The only sample isolated from the core of a living silver fir tree was also A. cepistipes. At the regional scale, A. cepistipes also appeared to be the most widespread of the Armillaria species as it was present in 18 out of 29 stands sampled.

Armillaria ostoyae occurred at significantly higher mean elevations than A. gallica and A. cepistipes (Tab. IV). Armillaria gallica appeared more frequently under basic soil conditions than A. cepistipes and A. ostoyae. Armillaria gallica and A. cepistipes appeared on stands with higher nitrogen index than A. ostoyae. In A. ostoyae infected stands, silver fir had lower defoliation than stands in which other Armillaria species were present. Armillaria ostoyae was associated (p = 0.066) with stands where A. alba was increasing its dominance relative to other forest tree species, but the association of silver fir dynamics index with the presence of A. ostoyae was not significant.

In 83% (CI: 52–98) of the forest plots, we recovered Armillaria isolates of the same species from below- and aboveground samples of the same plot. In 17% (CI: 0–48) of the plots the aboveground isolates were identified as A. cepistipes while belowground A. cepistipes was found together with A. gallica.

The distribution of Armillaria species varied depending on the tree species and on the substrate of observation (stump or dead tree). The majority of cultures isolated from A. alba dead trees (100% CI: 65–100) and stumps (81% CI: 60–95) were identified as A. cepistipes. Armillaria gallica was only observed on A. alba stumps (18% CI: 5–40). Armillaria cepistipes was detected on a dead P. uncinata tree, on a single Populus tremula L. stump and on a single P. sylvestris stump. Armillaria ostoyae was isolated once from a dead P. uncinata tree.

3.2. Armillaria root rot: analyses at genus level

We observed significant differences (p = 0.021) among the tree species (stumps and dead trees) infected by Armillaria. Dead A. alba trees showed the highest incidence (53% CI: 32–73). It was significantly higher than the incidence observed on P. uncinata stumps and on P. sylvestris stumps, respectively of 12% (CI: 3–40) and 6% (CI: 1–45). Dead P. uncinata trees and A. alba stumps showed intermediate incidences with 41% (CI: 16–72) and 35% (CI: 19–55) of infection. Only one dead F. sylvatica tree and three stumps of P. sylvestris were found to be infected by Armillaria. Armillaria was detected neither on F. sylvatica stumps nor on dead P. sylvestris trees. The three belowground Armillaria abundance variables were positively correlated among themselves (all pairwise combinations significant at p < 0.0001).The aboveground abundance variables, dead tree and stump colonisation, did not relate significantly. Two significant relationships between below- and aboveground abundance variables were observed: a higher stump colonisation was associated with a greater frequency in soil (p = 0.040), and a greater weight in soil correlated with a higher dead tree colonisation (p = 0.018).

Stands with greater Armillaria weight in soil were those with higher slenderness values, with higher harvested BA, those that had been subjected to higher thinning intensity and those with higher LAI values (Tab. V). Higher mean lengths of Armillaria rhizomorphs in soil were observed in stands with lower density, lower BA and with lower percentage of canopy closure. Armillaria frequency was correlated with higher values of harvested BA. Dead tree colonisation was correlated with soil acidity. Stump colonisation by Armillaria occurred more frequently at lower elevations and in those stands with greater silver fir dynamic index values. Stump colonisation occurred more frequently on those stands subjected to a higher number of thinning interventions.

Pure silver fir stands showed higher median weights of Armillaria in soil (69.9 g m⁻³ CI: 44.9–108.7) than A. alba-F. sylvatica (16.3 g m⁻³ CI: 5–53.2) and A. alba-P. sylvestris mixed stands (13.3 g m⁻³ CI: 3–58.6). Median weight in pure stands was not significantly different than mean weight in A. alba-P. uncinata stands (3.4 g m⁻³ CI: 0–296.9).

4. DISCUSSION

Within Pyrenean silver fir stands, A. cepistipes was the most frequently observed species of Armillaria. Many authors have found A. ostoyae associated with conifer forests (Blodgett and Worrall, 1992; Legrand and Guillaumin, 1993; Legrand et al., 1996; McLaughlin, 2001), while we found A. ostoyae in only 20% of these silver fir stands. Legrand and Guillaumin (1993) found A. ostoyae widespread in three silver fir forests in France, with A. cepistipes only in stands with a past presence of hardwood species. This same association between A. cepistipes and the previous presence of hardwood species was noted by Rigling et al. (1997) and Tsopelas (1999) in Picea abies stands. As far as we know, there are no records of a past abundance of broadleaved species within the studied fir forests. At present, broadleaved species such as F. sylvatica, Betula pendula Roth. or P. tremula represent on average 7% of the BA of these stands, and no relation between the broadleaf component and any Armillaria species has been observed (results not shown). It seems that A. cepistipes can find suitable ecological conditions for its survival within a conifer forest, such as fir forests of Pyrenees.

The Armillaria species considered most virulent, A. ostoyae, was associated with silver fir stands on more acidic soils (mean pH = 4.82) and those located at higher elevations. A. ostoyae tends to occur where A. alba is increasing its canopy dominance (lower silver fir dynamics index), so it is not surprising that, at these sites, A. alba trees had lower defoliation. At present in the Pyrenees, A. alba is increasing its dominance due to BA losses of P. uncinata and P. sylvestris (Oliva and Colinas, 2007). The former pine species is considered very susceptible to A. ostoyae (Dobbertin et al., 2001; Morquer and Touvet, 1972) and root disease centres have been historically observed in the Pyrenees (Kile et al., 1991). This Armillaria species may have indirectly favoured A. alba progression within pine forests by reducing the health of pines or even by preventing regeneration. Further research may elucidate the role of A. ostoyae driving Pyrenean pine forests’ dynamics as observed by Durrieu et al. (1985).

Armillaria gallica tended to occur in stands with relatively high soil pH (mean pH = 6.15) and at lower elevations as observed by Legrand and Guillaumin (1993), Rigling et al. (1997) and McLaughlin (2001). In the Pyrenees these two conditions are correlated (r = − 0.61, p = 0.0004) (results not shown). Therefore we cannot exclusively associate A. gallica presence with either elevation or pH.

Management variables such as harvested BA, number of interventions and thinning intensity of the average intervention correlated with Armillaria below- and aboveground abundance. However, none of the variables associated with the presence of a given Armillaria species seems susceptible to being managed, such as elevation and pH. Therefore, the Armillaria species colonising a certain stand could be in itself considered as another site characteristic. The same Armillaria genet can be present in one site for centuries (Baumgartner and Rizzo, 2001; Bendel et al., 2006b; Ferguson et al., 2003; Rizzo et al., 1998; Smith et al., 1992), and shifts in the Armillaria species composition in one site are slow due to the low survival rate of new genets (Dettman and Van der Kamp, 2001). Of our silver fir stands, 83% showed the same Armillaria species above- and belowground. This, and the fact that we found silver fir infected by three different Armillaria species, suggests that the Armillaria species infecting a certain tree species might be more dependent on the Armillaria species present in the site that on the tree species.

The incidence of Armillaria on living trees was very low (0.6%). We did not observe connection between silver fir defoliation and the abundance of any Armillaria species. We observed Armillaria colonisations concentrated on A. alba dead trees and stumps, and on P. uncinata dead trees. Colonisation of dead silver fir was mainly due to A. cepistipes. It is not clear whether Armillaria killed those trees or simply colonised them when they were weakened by other causes. Overall, A. cepistipes is generally considered a secondary pathogen and Armillaria-infected silver fir dead trees are typically of lower diameter than those living within the same stand (Oliva and Colinas, 2007). Armillaria cepistipes is probably only contributing to the self-thinning process of these forests, as suggested by Oliva and Colinas (2007) and not acting as a primary pathogen. The substantial incidence of Armillaria on P. uncinata supports the hypothesis that this pathogen is contributing to the reduction of this pine species within silver fir forests. Forest planners intending to maintain P. uncinata within silver fir forests in Pyrenees should consider the presence of Armillaria as a relevant site condition.

The abundance of A. cepistipes within silver fir forests does not seem to pose a problem at present since its virulence is low, but the frequency of Armillaria outbreaks could increase in the future. Pyrenean forests are far from being an undisturbed ecosystem since 90% of silver fir stands have been regularly managed (Oliva and Colinas, 2007). We have observed a high thinning intensity correlated with a higher biomass of Armillaria in soil, which in turn correlated with a higher incidence of Armillaria in dead trees. Increased mortality due to Armillaria associated to the onset of management activities has been reported for A. ostoyae and A. mellea (Baumgartner and Rizzo, 2001; Morrison et al., 2001), and the same process may be operating in the Pyrenean silver fir forests.

Acknowledgments

This research was partially funded by the INIA (Insituto Nacional de Investigación y Tecnología Agraria y Alimentaria) grant No. RTA01-071-C3-1 and by the sub-project S3 of the INTERREG IIIA SYLVAPYR project (I3A-1-57-E) co-funded by the European Union. Christine Fischer’s and Nicholas Rosenstock’s revision of the English writing of this manuscript is acknowledged. We thank J. Worrall and one anonymous referee for their constructive reviews.

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