# Assessment of mycorrhizal colonisation and soil nutrients in unmanaged fire-impacted soils from two target restoration sites

Jm Dias, Rs Oliveira, Ar Franco, Karl Ritz, Naoise Nunan, Pml Castro

► To cite this version:

Jm Dias, Rs Oliveira, Ar Franco, Karl Ritz, Naoise Nunan, et al.. Assessment of mycorrhizal coloni-sation and soil nutrients in unmanaged fire-impacted soils from two target restoration sites. Spanish Journal of Agricultural Research, 2010, 8, pp.S86-S95. bioemco-00542730

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# Assessment of mycorrhizal colonisation and soil nutrients in unmanaged fire-impacted soils from two target restoration sites

J. M. Dias<fnref n="1" />,<fnref n="2" />, R. S. Oliveira<fnref n="1" />, A. R. Franco<fnref n="1" />, K. Ritz<fnref n="2" />, N. Nunan<fnref n="3" /> and P. M. L. Castro<fnref n="1" />*

## Abstract

The mycorrhizal colonisation of plants grown in unmanaged soils from two restoration sites with a fire history in Northern Portugal was evaluated from the perspective of supporting restoration programmes. To promote restoration of original tree stands, *Quercus ilex* L. and *Pinus pinaster* Ait. were used as target species on two sites, denoted Site 1 and 2 respectively. The aim of the study was to assess whether mycorrhizal propagules that survived fire episodes could serve as *in situ* inoculum sources, and to analyse the spatial distribution of soil nutrients and mycorrhizal parameters. In a laboratory bioassay, *P. pinaster* and *Q. ilex* seedlings were grown on soils from the target sites and root colonisation by ectomycorrhizal (ECM) and arbuscular mycorrhizal (AM) fungi was determined. The ECM root colonisation levels found indicated that soil from Site 2 contained sufficient ECM propagules to serve as a primary source of inoculum for *P. pinaster*. The low levels of ECM and AM colonisation obtained on the roots of plants grown in soil from Site 1 indicated that the existing mycorrhizal propagules might be insufficient for effective root colonisation of *Q. ilex*. Different ECM morphotypes were found in plants grown in soil from the two sites. At Site 2 mycorrhizal parameters were found to be spatially structured, with significant differences in ECM colonisation and soil P concentrations between regions of either side of an existing watercourse. The spatial distribution of mycorrhizal propagules was related to edaphic parameters (total C and extractable P), and correlations between soil nutrients and mycorrhizal parameters were found.

**Additional key words:** arbuscular mycorrhiza, ectomycorrhiza, *Pinus pinaster*, post fire, *Quercus ilex*, spatial variation, soil restoration.

## Resumen

### Evaluación de la colonización micorrícica y de los nutrientes del suelo en dos zonas de restauración afectadas por fuego

Con el fin de dar soporte a un programa de restauración de dos zonas afectadas por un historial de fuego en el nor- te de Portugal se evaluó la colonización micorrícica de las plantas de cada zona. Se utilizaron *Quercus ilex* L. y *Pinus pinaster* Ait. para promover la restauración de las especies originales de las dos zonas seleccionadas (zona 1 y zon- a 2). El objetivo de este estudio fue evaluar si los propágulos de los hongos formadores de micorrizas, que habían logrado sobrevivir a los episodios de fuego, podían servir como fuente de inóculo *in situ* y analizar la distribución de los nutrientes del suelo y los parámetros micorrícicos. Se determinó la colonización por hongos ectomicorrícicos (ECM) y por hongos formadores de micorrizas arbusculares (MA) en las raíces de plántulas de *P. pinaster* y *Q. ilex* cultivadas en los suelos seleccionados. Los niveles de colonización ECM observados en los suelos de la zona 2 indi- caron que la cantidad de propágulos infectivos era suficiente como para servir de fuente primaria de inóculo para *P. pinaster*. Sin embargo, en la zona 1 se encontraron niveles muy bajos de colonización ECM y MA lo que indicaba que no había suficiente cantidad de propágulos para colonizar eficientemente las plántulas de *Q. ilex*. Se encontraron diferentes morfotipos de ECM en las plantas cultivadas en las distintas zonas. En la zona 2 se encontraron diferencias

<footnote n="1">¹ CBQF/Escola Superior de Biotecnologia. Universidade Católica Portuguesa.
Rua Dr. António Bernardino de Almeida. 4200-072 Porto. Portugal</footnote>

<footnote n="2">² National Soil Resources Institute. Cranfield University. Bedfordshire MK43 0AL. United Kingdom</footnote>

<footnote n="3">³ CNRS. Laboratoire BioEMCo, UMR7618. Bâtiment EGER. Aile B. Campus AgroParisTech.
F-78850 Thiveval-Grignon. France</footnote>

<footnote>* Corresponding author: plcastro@esb.ucp.pt</footnote>
significativas en la colonización ECM y en la concentración de P en el suelo de las regiones situadas a ambos lados del curso del agua. Se encontró una distribución espacial de los propágulos de hongos formadores de micorrizas relacionada con parámetros edáficos (C total y P extraíble) y se encontró correlación entre los nutrientes del suelo y los parámetros micorrícicos.

**Palabras clave adicionales:** ectomicorrizas, micorrizas arbusculares, *Pinus pinaster*, *Quercus ilex*, restauración de suelos.

## Introduction

The forest area in Portugal occupies 3.4 million ha, consisting of a mixture of tree species, which in decreasing order of abundance are *P. pinaster* Ait, *Euca-lyptus globulus* Labill., *Quercus suber* L., *Q. ilex* L. and, with a smaller representation, other coniferous and broadleaf species. In Portugal conditions are favourable for forest fires to occur (IFFN, 2005) and according to the European Forest Fires Information System (EFFIS), the burned area to mid-2004 was approximately 94,000 ha, with 35,000 ha being forest-land including forest stands and shrub land. Forest fire normally causes degradation of plant and soil microbial communities, contributing to a reduction in the density of fungal propagules (Rashid *et al.*, 1997; Stendell *et al.*, 1999; Cairney and Bastias, 2007). However, the magnitude of such negative impacts will depend on many factors such as the intensity of the fire, the amount of available fuel, the soil moisture at the time of the episode and the type of ground cover (Torres and Honrubia, 1997). Due to the negative effects of fire in plant communities and soil structure, there is an urgent need to improve restoration practices. These practices should be implemented together with an environmentally-beneficial soil restoration and conservation strategy.

Mycorrhizas are the most widespread terrestrial symbiotic associations between plants and fungi (Smith and Read, 2008) and occur at the level of roots via morphological and anatomical modifications. This relationship is known to improve plant health by increasing nutrient and water uptake, alleviating environmental stresses and enhancing plant resistance against disease (Quoreshi *et al.*, 2008; Smith and Read, 2008; Rivero *et al.*, 2009). The importance of these mutualistic associations has gained significant interest due to increasing costs of irrigation and synthetic fertilisers (McKane and Kandel, 1996; Rincon *et al.*, 2007). The

natural soil biota, which includes mycorrhizal fungi, is a very important biotechnological resource and its application represents a step forward in the use of sustainable techniques for forestry production. The most common types of mycorrhizas in nature are arbuscular mycorrhizas and ectomycorrhizas. Members of the Pinaceae are known to be obligatory with respect to ectomycorrhizal (ECM) associations (Bidartondo *et al.*, 2001). *Quercus* spp. are usually considered to be ECM, nonetheless arbuscular mycorrhizal (AM) colonisation has been found in both seedling and mature *Quercus* spp. (Ian *et al.*, 2001). Different types of mycorrhizas often co-occur in the same ecosystem (Moyersoen *et al.*, 1998) forming dual mycorrhizal associations, however, ECM fungi are apparently capable of displacing AM fungi (Lodge and Wentworth, 1990). A variety of processes may lead to species-specific spatial patterns in ECM populations, including variation in rates of genet growth (if patches are made up of one individual), internal structure of genets, or patterns of intraspecific establishment or survival that lead to clusters of individuals (Lilleskov, 2004). Relatively little is known about how mycorrhizal communities are structured spatially (Taylor, 2002) and it is therefore relevant to evaluate how individuals and communities show different spatial distributions.

The aims of this study were to assess the mycorrhizal root colonisation of plants grown in unmanaged soils from two target restoration sites with a fire history, using qualitative and quantitative approaches and to characterise the spatial variation of the mycorrhizal propagules. We hypothesised that there would be spatially-related associations between soil nutrients and mycorrhizal parameters. *Quercus ilex* and *P. pinaster* were used as target plants on laboratory bioassay studies using soils derived from two sites, since these species were extant at the sites prior to fire events.

<footnote>Abbreviations used: AM (arbuscular mycorrhizal), ECM (ectomycorrhizal), EFFIS (European Forest Fires Information System), OM (Organic matter).</footnote>
# Material and methods

## Sites description and soil sampling

The study sites were located on a mountainous region within the Serra da Cabreira, Northern Portugal (40°40'12" N, 08°3'33" W), consisting mainly of low granite mountains with steep valleys and mild climate (mean annual temperature 13 to 15°C; mean annual precipitation 1820 mm). Two contrasting sites were selected on the basis of their past fire history and their target tree species for restoration. Site 1 (860 m asl) had 4 ha targeted for restoration using *Q. ilex* and had the last fire episode in 1975, while Site 2 (830 m asl) had 5 ha targeted for restoration using *P. pinaster* and had the last fire episode in 2000. Both sites had scarce vegetation with patchily distributed herbaceous plant species such as *Pteridium aquilinum*, *Rubus* spp. and *Agrostis* spp. At Site 1, a few *Quercus* spp. were present, whereas at Site 2 some *P. pinaster* plants were identified. A uniform area was sampled at both sites, according to presence of vegetation and local topography and avoiding obvious major variation between sample

points (e.g. emergent rock, erosion points). Thirty soil samples were collected from each site on a regular grid pattern for chemical analyses and for use in a laboratory bioassay (Fig. 1). The sampling design consisted of a regularly spaced grid (30 m average) of 6 × 5 samples on Site 1. At Site 2, the design included a 6 × 2 grid at north east and a 6 × 3 grid at south west of a small existing watercourse, with an average spacing of 50 m. Soils were air-dried before the determination of their chemical properties. For loss-on-ignition, soils were oven-dried at 40°C overnight.

## Laboratory bioassay

According to the restoration scheme, *Q. ilex* and *P. pinaster* seeds were sown in soil from Site 1 and 2, respectively. Soil collected from each sampling point was homogenised and divided into three cells of labelled containers (CETAP, António Matos Lda, Espinho, Portugal). Trays with 20 cells (800 mL each) were used for *Q. ilex*, while trays with 40 cells (210 mL each) were used for *P. pinaster*. Controls were also established in

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Sampling design showing location of sampling points. Box arrow represents viewpoint for posting plots. Insets show framework for posting plots with associated sample numbers.</figcaption></figure>
order to check for possible laboratory, glasshouse or
aerial mycorrhizal root contamination. For that purpose,
soil collected from the 30 sampling points of Site 1
was mixed and autoclaved twice at 121°C for 80 min
on two consecutive days. The autoclaved soil was placed
into 15 cells of a tray. The same procedure was followed
for controls from Site 2. Twenty millilitres of a soil
suspension (prepared by shaking 100 g of unsterilised
soil in 1 litre of sterilised deionised water for 2 h and
followed by filtration (Whatman No. 1) were added to
each cell to replace soil microbial populations. Seeds
of *P. pinaster* were rinsed overnight in running tap water,
surface sterilised with 0.5% (v/v) NaOCl for 15 min
and rinsed three times with autoclaved deionised water.
Seeds of *Q. ilex* were stratified at 4°C for 6 months,
rinsed with tap water, surface sterilised with 0.5% (v/v)
NaOCl for 10 min and washed three times with auto-
claved deionised water. Each cell with soil from Site
1 received two seeds of *Q. ilex*, while each cell with
soil from Site 2 received two seeds of *P. pinaster*. If
both seeds successfully germinated in the same cell,
one was eliminated so that only one plant per cell was
grown. Plants were grown in a greenhouse (tempera-
ture and relative humidity varied from 7 to 45°C and
from 12 to 90%, respectively) at a forest nursery.
*Quercus ilex* and *P. pinaster* were harvested after 5 and
4 months of growth, respectively. *Quercus ilex* plants
were analysed for AM and ECM colonisation, whereas
*P. pinaster* plants were analysed for ECM colonisation.

Mycorrhizal analysis

The intact root system was separated from the block
of soil by immersing the roots in a beaker of water and
gently agitating them to remove the attached soil. To
analyse the ECM colonisation, roots were separated
from shoots, placed in Petri dishes with water and
maintained at 4°C until analysis. Afterwards, roots
were cleaned individually, while inspecting them vi-
sually under a dissecting microscope (Olympus SZ60,
Japan). Ectomycorrhizas were identified using a dissec-
ting microscope with a black microscope base and a
direct source of light at a magnification of 22x. Uns-
tained ECM roots were distinguished from each other
by differences in morphological characteristics. The
degree of ECM colonisation was assessed by counting
mycorrhizal root tips using the gridline intersection
method (Giovanneti and Mosse, 1980). The number of
ECM tips, total number of tips and total number of

intersections was counted to assess the percentage of
ECM colonisation and the number of ECM root tips
per metre of fine root. Representative ECM morpho-
types were characterised on the basis of type of rami-
fication, shape of unramified ends, presence of rhizo-
morphs, colour, size and features of mantle surface
(Agerer, 1998). To assess the AM root colonisation,
fresh root samples were cleared and stained with trypan
blue as described in Oliveira *et al*. (2005), placed in
Petri dishes and stored in 50% glycerol for further
analysis. The degree of AM colonisation was assessed
by counting mycorrhizal roots under a dissecting mi-
croscope (Olympus SZ60, Japan) using the gridline
intersection method.

Soil chemical analysis

The Egner-Riehm method was used to determine extractable P concentration (Riehm, 1958) and the Kjeldhal method was used to determine the total N concentration (MAFF ADAS, 1986). Organic matter (OM) was measured by loss-on-ignition (3 h at 450°C) and pH was measured in water according to the British Standard Methods (1990). The organic C content was calculated by considering 58% (w/w) of OM mass present as C.

Statistical analysis

Differences between sites and between regions either
side of the water course at Site 2 were analysed using
t-tests (α = 0.05). One-way ANOVA (α = 0.05) was used
to assess differences between samples in the same field.
Spatial analysis was also performed using the Mantel
test by comparing a distance matrix in mycorrhizal
colonisation or chemical properties with a matrix of
spatial distances among samples. This produced a nor-
malised Mantel statistic (r<sub>M</sub>) that is bounded between
-1 and +1 and behaves like a correlation coefficient
(Legendre and Legendre, 1998). It should be noted,
however, that Mantel statistics are often small, even
when highly statistically significant. The significance
of the relationship was determined by permutation of
the first of the two distance matrices (999 permutations).
A significant r<sub>M</sub> meant that there was a relationship
between physical distance and the variable analysed
(i.e. spatial structure). When spatial trends were identi-
fied, the data were analysed in more detail using t-tests.
# Results

## Soil chemical properties

At Site 1 the soil pH was 4.4 ± 0.02 and at Site 2 was 4.3 ± 0.05. Total N concentration and total organic C showed a 4-fold and 5-fold variation between samples at Sites 1 and 2, respectively, being on average 0.63 ± 0.04 % (w/w) and 11.5 ± 0.60 % (w/w) at Site 1 and 0.40 ± 0.03 % (w/w) and 9.3 ± 0.63 % (w/w) at Site 2, respectively. The total organic C content was high at both sites with Site 2 showing a significantly higher C:N ratio (24 ± 0.76 compared to 19 ± 1.02, *P* < 0.001) and Site 1 showing a significantly higher total organic C (*P* < 0.05). Extractable P values were very low at Site 2 (3.1 ± 0.39 mg P kg⁻¹ soil), considering the values adopted for agricultural crops in Portugal (LQARS, 2000). At Site 1 soil P concentration was low (8.2 ± 2.06 mg P kg⁻¹ soil) but significantly higher (*P* < 0.05) than at Site 2. A higher P concentration was however found at Site 1 in sample point 15, being 65.3 mg P kg⁻¹ soil. Extensive spatial variation in chemical properties was found across each site, with no simple or distinct patterning apparent for the majority of properties. However, the P levels in Site 2 showed a slight, but significant spatial structure at NE and SW of the watercourse (r<sub>M</sub> = 0.13, *P* = 0.022), with higher values at the NE side (Fig. 2).

## Mycorrhizal colonisation

The ECM colonisation was higher in samples from Site 2. The percentage of ECM colonisation varied between 0 and 3% in samples from Site 1 and between 10 and 57% in samples from Site 2. The number of ECM

<table><thead><tr><th>2.2</th><th>3.4</th><th>*</th><th>0.3</th><th>7.5</th><th>0.7</th></tr></thead><tbody><tr><td>3.9</td><td>4.3</td><td>*</td><td>0.1</td><td>5.1</td><td>2.4</td></tr><tr><td>4.2</td><td>9.7</td><td>*</td><td>2.2</td><td>2.5</td><td>2.4</td></tr><tr><td>4.1</td><td>5.1</td><td>*</td><td>4.2</td><td>1.6</td><td>3.7</td></tr><tr><td>5.3</td><td>2.0</td><td>*</td><td>1.0</td><td>1.0</td><td>1.2</td></tr><tr><td>4.1</td><td>4.0</td><td>*</td><td>3.1</td><td>0.7</td><td>1.7</td></tr></tbody><tfoot><tr><td colspan="3">NE</td><td colspan="3">SW</td></tr></tfoot></table>

<table><caption>**Figure 2.** Spatial patterns for extractable phosphorus (mg P kg⁻¹ soil) at Site 2 (NE and SW side of small watercourse represented by cells marked with as asterisk). Mean values: 4.4 ± 0.6 mg P kg⁻¹ (NE) and 2.3 ± 0.4 mg P kg⁻¹ (SW).</caption><thead><tr><th>Limit</th><th>Pattern</th></tr></thead><tbody><tr><td>&lt; 5</td><td></td></tr><tr><td>[5,10[</td><td></td></tr><tr><td>No data</td><td></td></tr></tbody></table>

root tips per metre of fine root ranged from 0 to 17 in samples from Site 1, and from 3 to 22 in samples from Site 2. There was no ECM colonisation on control samples. The spatial analysis showed that there was no distinct patterning for ECM colonisation at Site 1. However, at Site 2, spatial structure was identified in the percentage of ECM colonisation (r<sub>M</sub> = 0.2, *P* < 0.01) and in the number of ECM root tips per metre of fine root (r<sub>M</sub> = 0.18, *P* = 0.01), with higher values found at the NE region (Fig. 3). Additionally there was no evidence of spatial structure within both regions. Different ECM morphotypes were found in plants grown in soil from the two sites. For Site 2, a higher number of morphotypes were found (18 types at Site 2 and 10 types at Site 1). More common morphotypes were those with a simpler morphology, such as Morphotypes A and B at Site 1 (Table 1) and Morphotypes K and L at Site 2 (Table 2). Morphotypes at Site 2 also showed higher diversity in morphological characteristics than at Site 1, with some presenting rhizomorphs and bigger differences

<figure><img src="image_3.png" /><figcaption>**Figure 3.** Spatial patterns for ectomycorrhizal (ECM) parameters at Site 2 (NE and SW side of small watercourse represented by cells marked with as asterisk). a) Mean percentage of ECM colonisation: 43 ± 2.1% (NE) and 30 ± 2.6% (SW). b) Mean number of ECM root tips per metre of fine root: 15 ± 1.2 (NE) and 10 ± 1.0 (SW).</figcaption></figure>
<table><caption>**Table 1.** Morphological characteristics and location of ectomycorrhizal morphotypes identified in the roots of *Quercus ilex* grown in soil samples from Site 1</caption><thead><tr><th>Code</th><th>Location<sup>a</sup></th><th>Type of ramification<sup>b</sup></th><th>Shape of unramified ends</th><th>Colour</th><th>Size</th><th>Mantle-surface</th></tr></thead><tbody><tr><td>A</td><td></td><td>S</td><td>Straight</td><td>White</td><td>Medium</td><td>Grainy</td></tr><tr><td>B</td><td></td><td>S</td><td>Bent</td><td>White</td><td>Variable</td><td>Grainy</td></tr><tr><td>C</td><td></td><td>S</td><td>Straight</td><td>Orange</td><td>Small</td><td>Smooth</td></tr><tr><td>D</td><td></td><td>MPI</td><td>Straight</td><td>Brown</td><td>Medium</td><td>Smooth</td></tr><tr><td>E</td><td></td><td>S/MPy</td><td>Straight</td><td>Yellow</td><td>Variable</td><td>Smooth</td></tr><tr><td>F</td><td></td><td>IPD</td><td>Bent</td><td>White</td><td>Large</td><td>Grainy</td></tr><tr><td>G</td><td></td><td>S</td><td>Straight</td><td>Brown</td><td>Variable</td><td>Smooth</td></tr><tr><td>H</td><td></td><td>D</td><td>Bent/straight</td><td>White</td><td>Small</td><td>Smooth</td></tr><tr><td>I</td><td></td><td>S</td><td>Straight</td><td>Yellow</td><td>Medium</td><td>Smooth</td></tr><tr><td>J</td><td></td><td>S</td><td>Bent/straight</td><td>White</td><td>Medium</td><td>Cottony</td></tr></tbody></table>

<sup>a</sup> Black cells show location, white cells show sampling points where morphotype was not present, grey cells show sampling points where data is not available. <sup>b</sup> MPy: monopodial-pyramidal. IPD: irregularly-pinnate dichotomous-like. D: dichotomous. MPI: monopodial pinnate. S: simple (unramified).

rences in features of the mantle-surface. Spatial distribution was assessed for each morphotype in terms of number of morphotypes at each sample point. The number of morphotypes per sample point varied from 0 to 4 at Site 1 and from 1 to 7 at Site 2. However, no simple or distinct pattern was found for either each morphotype or number of morphotypes per sample point at both sites. Arbuscular mycorrhizal colonisation in *Q. ilex* (Site 1), estimated as a percentage of root colonised, ranged from 0 to 20%. There was no AM colonisation on control samples. A spatial analysis showed that there was a significant difference (*P* < 0.05) in the percentage of AM colonisation at the Eastern, middle and Western side of the field and a higher degree of colonisation was found at the Eastern side (Fig. 4).

## Correlation between properties

Strong positive correlation was found between total N and total C at Site 1 (*r*² = 0.55, *P* < 0.001) and Site 2

(*r*² = 0.89, *P* < 0.001). No correlation was found between total N and extractable P or total C and extractable P values for both sites. Significant and positive correlations between percentage of ECM colonisation and

<table><thead><tr><th></th><th>19</th><th>4</th><th></th><th></th></tr></thead><tbody><tr><td></td><td></td><td></td><td>5</td><td></td></tr><tr><td></td><td>20</td><td></td><td>4</td><td></td></tr><tr><td></td><td>10</td><td>9</td><td>5</td><td></td></tr><tr><td></td><td></td><td></td><td>7</td><td></td></tr><tr><td></td><td>3</td><td>18</td><td>12</td><td>4</td></tr><tr><td></td><td>E</td><td>M</td><td>W</td><td></td></tr></tbody></table>

<table><thead><tr><th>Limit</th><th>Pattern</th></tr></thead><tbody><tr><td>≤ 10</td><td></td></tr><tr><td>[10,20[</td><td></td></tr><tr><td>≤ 20</td><td></td></tr><tr><td>No data</td><td></td></tr></tbody></table>

**Figure 4.** Spatial pattern for mean percentage of arbuscular mycorrhizal colonisation at the East (E), Middle (M) and West (W) side of Site 1. Mean values: 14 ± 3.3% (E), 8 ± 2.4% (M) and 5 ± 0.5% (W).
<table><caption>**Table 2.** Morphological characteristics and location of ectomycorrhizal morphotypes identified in the roots of *Pinus pinaster* grown in soil samples from Site 2</caption><thead><tr><th>Code</th><th>Location<sup>a</sup></th><th>Type of ramification<sup>b</sup></th><th>Shape of unramified ends</th><th>Rhizomorphs</th><th>Colour</th><th>Size</th><th>Mantle-surface</th></tr></thead><tbody><tr><td>K</td><td></td><td>S</td><td>Straight</td><td>Absent</td><td>Dark orange</td><td>Small</td><td>Smooth</td></tr><tr><td>L</td><td></td><td>S</td><td>Straight</td><td>Orange</td><td>Dark orange</td><td>Small</td><td>Smooth</td></tr><tr><td>M</td><td></td><td>S</td><td>Straight</td><td>Absent</td><td>Brown</td><td>Variable</td><td>Grainy</td></tr><tr><td>N</td><td></td><td>S</td><td>Straight</td><td>Absent</td><td>Dark brown</td><td>Small</td><td>Reticulate</td></tr><tr><td>O</td><td></td><td>D</td><td>Straight</td><td>Absent</td><td>Dark orange</td><td>Small</td><td>Smooth</td></tr><tr><td>P</td><td></td><td>D</td><td>Straight</td><td>Light orange</td><td>Very dark orange</td><td>Small</td><td>Smooth</td></tr><tr><td>Q</td><td></td><td>D</td><td>Straight</td><td>Absent</td><td>Dark brown</td><td>Medium</td><td>Short-spiny</td></tr><tr><td>R</td><td></td><td>D</td><td>Straight</td><td>Absent</td><td>Light orange</td><td>Medium</td><td>Short-spiny</td></tr><tr><td>S</td><td></td><td>S</td><td>Straight</td><td>Absent</td><td>Brown</td><td>Long</td><td>Smooth</td></tr><tr><td>T</td><td></td><td>D</td><td>Bent</td><td>Absent</td><td>Dark orange</td><td>Small</td><td>Grainy</td></tr><tr><td>U</td><td></td><td>S</td><td>Straight</td><td>Absent</td><td>Dark red</td><td>Medium</td><td>Stringy</td></tr><tr><td>V</td><td></td><td>D</td><td>Straight</td><td>Absent</td><td>Light pink</td><td>Small</td><td>White cottony</td></tr><tr><td>W</td><td></td><td>D</td><td>Straight</td><td>Absent</td><td>Light pink</td><td>Medium</td><td>Smooth</td></tr><tr><td>X</td><td></td><td>D</td><td>Straight</td><td>Absent</td><td>Dark brown</td><td>Medium</td><td>Smooth</td></tr><tr><td>Y</td><td></td><td>D</td><td>Straight</td><td>Absent</td><td>Dark orange</td><td>Small</td><td>Orange cottony</td></tr><tr><td>Z</td><td></td><td>D</td><td>Straight</td><td>Absent</td><td>Orange</td><td>Small</td><td>White cottony</td></tr><tr><td>A1</td><td></td><td>D</td><td>Straight</td><td>Absent</td><td>Light orange</td><td>Medium</td><td>Reticulate</td></tr><tr><td>B1</td><td></td><td>D</td><td>Straight</td><td>Absent</td><td>Very light orange</td><td>Small</td><td>Smooth</td></tr></tbody></table>

the number of ECM root tips per metre of fine root were found at Site 1 ($r^2$ = 0.83, *P* < 0.001) and at Site 2 ($r^2$ = 0.83, *P* < 0.001). No correlation was found between the percentage of ECM and AM colonisation at Site 1. Significant and positive correlation was found

between extractable P and AM colonisation at Site 1 ($r^2$ = 0.76, *P* < 0.01); the percentage of ECM colonisation and the number of ECM root tips per metre of fine root were also found to be positively correlated with extractable P only at Site 2 ($r^2$ = 0.68, *P* < 0.001 and $r^2$ = 0.64,

<footnote><sup>a</sup> Black cells show location, white cells show sampling points where morphotype is not present and grey cells represent sampling points where data is not available. <sup>b</sup> D: dichotomous. S: simple (unramified).</footnote>
$P < 0.001$, respectively). At Site 1 a positive correlation was found between the percentage of ECM colonisation and total C ($r^2 = 0.34, P < 0.1$). For the number of morphotypes per sample point, no correlation was found with any of the measured chemical properties.

## Discussion

The acidic soils studied had a relatively high organic C content and low nutrient levels. These are chemical characteristics normally found on impoverished lands, which would be expected for such mountainous lands with a history of exposure to fires (Neary *et al.*, 1999). The C:N ratio at Site 2 (more recently exposed to fire) was significantly different and higher than at Site 1, probably due to the upward movement of organic compounds caused by the transfer of heat downwards after the fire event (DeBano, 2000). The positive correlations between total N and total organic C contents suggest common microscale variability for these chemical properties (Ritz *et al.*, 2004). High levels of spatial heterogeneity in soil chemical properties were found, confirming what was reported for other ecosystems (Jackson and Caldwell, 1993; Gross *et al.*, 1995; Ritz *et al.*, 2004). The differences observed in P levels NE and SW of the small watercourse at Site 2 (Fig. 2) were probably related with hydrological characteristics of the watershed.

A significant correlation between the percentage of ECM colonisation and the number of ECM root tips per metre of fine root was found at both sites. Such correlations were previously reported (Baxter *et al.*, 1999) and suggest a common origin of microscale variability of the named parameters. At Site 2, spatial structure was identified in the percentage of ECM colonisation and in the number of ECM root tips per metre of fine root, showing significant differences between regions of either side of the watercourse. Considering the lack of spatial distribution at each side of the stream in isolation, it appears that the identified spatial structure is related to the differences between each side of the watercourse, therefore suggesting that the hydrological features are influencing soil microbiological properties. Qualitative analysis demonstrated that different ECM morphotypes occurred at both sites.

Arbuscular mycorrhizal colonisation of *Q. ilex* was low and the patterning at Eastern, middle and Western side of Site 1 suggests an influence of soil exposure to sun. For example, a greater sun exposure on one side

of the field could have altered soil structure and temperature, therefore influencing mycorrhizal propagules. However, the AM spatial structure was not confirmed when the Mantel test was used (results were not significant). Usually, an inhibition of AM fungal colonisation occurs with increasing soil P availability (Smith and Read, 2008); however, in the present study P concentration varied positively with the percentage of AM colonisation at Site 1. Since extractable P was found to be very low, it is possible that the extractable P values were not high enough to cause inhibition of AM fungal colonisation of *Q. ilex*.

The sampling scheme adopted was designed to provide a basic framework to assess spatial variation at the study sites. Other geostatistical approaches can be used to assess spatial structure in soil properties (Webster and Oliver, 2001), but they require larger numbers of samples (i.e. of the order of several hundreds). However, the data here presented can be used to infer that an appropriate starting scale for more thorough studies of spatial structure at the sites would be one that matches the basic grid size used (i.e. around 40 m), since there was a high variance at this scale for most properties.

The ECM root colonisation levels found indicated that soil from Site 2 contains ECM propagules to serve as primary source of inoculum for *P. pinaster*. The low levels of ECM and AM colonisation obtained on the roots of plants grown in soil from Site 1 indicated that the existing mycorrhizal propagules might be insufficient for effective colonisation of *Q. ilex*. Positive correlations were found between soil nutrients and mycorrhizal colonisation: extractable P and AM colonisation at Site 1, extractable P and both, the percentage of ECM colonisation and the number of ECM root tips per metre of fine root at Site 2 and the percentage of ECM colonisation and total C at Site 1.

The results from this study show that due to their spatial variation, mycorrhizal propagules that survive fire episodes are not always effective in serving as *in situ* mycorrhizal inoculum source. Therefore, assessment of the mycorrhizal colonisation potential of unmanaged fire-impacted sites should be conducted before restoration practices are implemented.

## Acknowledgements

This work was supported by Project AGRO 752, Medida 8. R. Oliveira wishes to acknowledge Funda-
ção para a Ciência e a Tecnologia and Fundo Social Europeu (III Quadro Comunitário de apoio), research grant SFRH/BPD/23749/2005. The authors wish to thank Ana Macedo and Luís Corte Real from DRAEDM. Cristina Calheiros and Gabriela Lovelace are acknowledged for helping with the procedures for the chemical analyses.

## References

AGERER R., 1998. Colour atlas of ectomycorrhizae. Einhorn Verlag, Eduard Dietenberger, Schäwbish Gmünd, Germany.

BAXTER J.W., PICKETT S.T.A., CARREIRO M.M., DIGHTON J., 1999. Ectomycorrhizal diversity and community structure in oak forest stands exposed to contrasting anthropogenic impacts. Can J Bot 77, 771-782. doi: 10.1139/cjb-77-6-771.

BIDARTONDO M.I., BAAR J., BRUNS T.D., 2001. Low ectomycorrhizal inoculum potential and diversity from soils in and near ancient forests of bristlecone pine (*Pinus longaeva*). Can J Bot 79, 293-299. doi: 10.1139/cjb-79-3-293.

BRITISH STANDARD METHODS, 1990. Test for soil for civil engineering purposes. Part 3, Chemical and Electro-chemical Tests BS 1377.

CAIRNEY J.W.G., BASTIAS B.A., 2007. Influences of fire on forest soil fungal communities. Can J For Res 37, 207-215. doi: 10.1139/X06-190.

DEBANO L.F., 2000. The role of fire and soil heating on water repellency in wildland environments: a review. J Hydrol 231, 195-206. doi: 10.1016/S0022-1694(00)00194-3.

GIOVANNETI M., MOSSE B., 1980. An evaluation of techniques for measuring vesicular-arbuscular infection in roots. New Phytol 84, 489-500. doi: 10.1111/j.1469-8137. 1980.tb04556.x.

GROSS K.L., PREGITZER K.S., BURTON A.J., 1995. Spatial variation in nitrogen availability in three successional plant communities. J Ecol 83, 357-367.

IANA., KOIDE R.T., FAYISH A.C., 2001. Vesicular-arbuscular mycorrhizal infection of *Quercus rubra* seedlings. New Phytol 151, 257-264. doi: 10.1046/j.1469-8137. 2001.00148.x.

IFFN, 2005. Fire Situation in Portugal 2005 - An overview. International Forest Fire News 34, 22-30. ISSN 1029-0864.

JACKSON R.B., CALDWELL M.M., 1993. The scale of nutrient heterogeneity around individual plants and its quantification with geostatistics. Ecology 74, 612-614. doi: 10.2307/1939320.

LEGENDRE P., LEGENDRE L., 1998. Numerical ecology, 2nd English ed. Elsevier Science Publishers. 551 pp. doi: 10.2307/1939320.

LILLESKOV E.A., BRUNS T.D., HORTON T.R., TAYLOR D.L., GROGAN P., 2004. Detection of forest stand-level

spatial structure in ectomycorrhizal fungal communities. FEMS Microbiol Ecol 49, 319-332. doi: 10.1016/j.femsec.2004.04.004.

LODGE D.J., WENTWORTH T.R., 1990. Negative associations among VA-mycorrhizal fungi and some ectomycorrhizal fungi inhabiting the same root system. Oikos 57, 347-356.

LQARS, 2000. Manual de fertilização das culturas. Laboratório Químico Agrícola Rebelo da Silva, Lisboa.

MAFF ADAS, 1986. The analysis of agricultural materials, 3rd ed. Ministry of Agriculture, Fisheries and Food, Agricultural Development and Advising Service Reference Book, London, 427, 183-185.

MCKANE L., KANDEL J., 1996. Microbiology essentials and applications, 2nd ed. McGraw-Hill, Inc, USA.

MOYERSOEN B., FITTER A.H., ALEXANDER I.J., 1998. Spatial distribution of ectomycorrhizas and arbuscular mycorrhizas in Korup National Park Rain Forest, Cameroon, in relation to edaphic parameters. New Phytol 139, 331-320. doi: 10.1111/j.1469-8137.1998.00190.x.

NEARY D.G., KLOPATEK C.C., DEBANO L.F., FOLLIOTT P.F., 1999. Fire effects on belowground sustainability: a review and synthesis. For Ecol Manage 122, 51-71. doi: 10.1016/S0378-1127(99)00032-8.

OLIVEIRA R.S., CASTRO P.M.L., DODD J.C., VOSÁTKA M., 2005. Synergistic effect of *Glomus intraradices* and *Frankia* spp. on the growth and stress recovery of *Alnus glutinosa* in an alkaline anthropogenic sediment. Chemosphere 60, 1462-1470. doi: 10.1016/j.chemosphere.2005.01.038.

QUORESHI A.M., PICHE Y., KHASA D.P., 2008. Field performance of conifer and hardwood species 5 years after nursery inoculation in the Canadian prairie provinces. New For 35, 235-253. doi: 10.1007/s11056-007-9074-3.

RASHID A., AHMED T., AYUB N., KHAN A.G., 1997. Effect of forest fire on number, viability and post-fire re-establishment of arbuscular mycorrhizae. Mycorrhiza 7, 217-220. doi: 10.1007/s005720050183.

RIEHM H., 1958. Die ammoniumlaktatessigsäure - methode zur bestimmung der leichtlöslichen phosphorsäure in karbonathaltigem boden. Agrochimica 3, 49-65.

RINCON A., PARLADE J., PERA J., 2007. Influence of the fertilisation method in controlled ectomycorrhizal inoculation of two Mediterranean pines. Ann For Sci 64, 577-583. doi: 10.1051/forest:2007035.

RITZ K., MCNICOL J.W., NUNAN N., GRAYSTON P., MILLARD P., ATKINSON D., GOLLOTE A., HABESHAW D., BOAG B., CLEGG C.D., GRIFFITHS B.S., WEATLEY R.E., GLOVER L.A., MCCAIG A.E., PROSSER J.I., 2004. Spatial structure in soil chemical and microbiological properties in an upland grassland. FEMS Microbiol Ecol 49, 191-205. doi: 10.1016/j.femsec.2004.03.005.

RIVERO S.H.T., MOORILLÓN V.G.N., BORUNDA E.O., 2009. Growth, yield, and nutrient status of pecans fertilized with biosolids and inoculated with rizosphere
fungi. Bioresour Technol 100, 1992-1998. doi: 10.1016/
j.biortech.2007.12.078.

SMITH S.E., READ D.J., 2008. Mycorrhizal symbiosis,
3rd ed. Academic Press, London.

STENDELL E.R., HORTON T.R., BRUNS T.D., 1999. Early
effects of prescribed fire on the structure of the ectomy-
corrhizal fungus community in a Sierra Nevada pondero-
sa pine forest. Mycol Res 103, 1353-1359. doi: 10.1017/
S0953756299008618.

TAYLOR A.F.S., 2002. Fungal diversity in ectomycorrhizal
communities: sampling effort and species detection. Plant
Soil 244, 19-28. doi: 10.1023/A:1020279815472.

TORRES P., HONRUBIA M., 1997. Changes and effects of
a natural fire on ectomycorrhizal inoculum potential of
soil in a *Pinus halepensis* forest. For Ecol Manage 96,189-
196. doi: 10.1016/S0378-1127(97)00058-3.

WEBSTER R., OLIVER M.A., 2001. Geostatistics for en-
vironmental scientists. John Wiley, Chichester, UK.