Combined use of Pinus pinaster plus

Ecological Engineering 43 (2012) 95–103 Contents lists available at SciVerse ScienceDirect Ecological Engineering journal homepage: www.elsevier.com/l...

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Ecological Engineering 43 (2012) 95–103

Contents lists available at SciVerse ScienceDirect

Ecological Engineering journal homepage: www.elsevier.com/locate/ecoleng

Combined use of Pinus pinaster plus and inoculation with selected ectomycorrhizal fungi as an ecotechnology to improve plant performance Rui S. Oliveira, Albina R. Franco, Paula M.L. Castro ∗ CBQF/Escola Superior de Biotecnologia, Universidade Católica Portuguesa, R. Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal

a r t i c l e

i n f o

Article history: Received 5 August 2011 Received in revised form 26 December 2011 Accepted 31 January 2012 Available online 27 March 2012 Keywords: Ecotechnology Forest nursery inoculation Improved plant growth and nutrition Maritime pine Pinus pinaster plus tree Selected ectomycorrhizal fungi

a b s t r a c t Pinus pinaster is an important forest species for environmental and economic reasons. Due to its importance, tree improvement plans aimed at the selection and use of phenotypically superior trees, designated by plus trees, have been established. It is known that ectomycorrhizal (ECM) fungi can improve tree survival and growth. Seeds obtained from P. pinaster plus trees have been used in forest nurseries. However, the effect of inoculation with ECM fungi on the performance of these plants has not been studied. We compared the performance of P. pinaster plants obtained from seeds of plus and non-plus trees to inoculation with different selected ECM fungi under conventional forest nursery conditions. In plants obtained from seeds of non-plus trees only those inoculated with Suillus bovinus + Laccaria laccata + Lactarius deterrimus had a significantly greater biomass and needles nitrogen concentration, while in plants obtained from seeds of plus trees this effect was seen not only in those receiving that same ECM inoculation, but also in those inoculated with Rhizopogon roseolus or Pisolithus tinctorius + Scleroderma citrinum. The best performance was that of plants obtained from seeds of plus trees and inoculated with R. roseolus or S. bovinus + L. laccata + L. deterrimus, with an increase in biomass of 2.2 and 2.0 times, respectively. This significant improvement was achieved without extra input of agrochemicals. The combined use of seeds obtained from plus trees and inoculation with selected ECM fungi can be an advantageous ecotechnological approach to improve nursery production of P. pinaster. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Pinus pinaster Ait. (maritime pine) is a relevant forest species with broad distribution in the western Mediterranean Basin, in Southern Europe and Africa, and in the Atlantic coast of Portugal, Spain and France. This species was also introduced by forestation in other European countries, Australia, New Zealand, South Africa and South America (Oliveira et al., 2000). It is the main coniferous species in France in terms of planted area and harvest yield (Pot et al., 2002) and it is the main forest species in Portugal occupying ca. 27% of the total forest area (Autoridade Florestal Nacional, 2010). The P. pinaster forest is important for environmental and economic reasons. P. pinaster has amazing performance as a pioneer tree and has been used for protection plantations and for restoration of degraded and impoverished soils (Barˇcic´ et al., 2006). Also, it is used as a main source for multiple industrial applications (e.g. wood, paper, resin) (Baptista et al., 2008; Louzada and Fonseca, 2002).

∗ Corresponding author. Tel.: +351 225580067; fax: +351 225090351. E-mail addresses: [email protected] (R.S. Oliveira), [email protected] (A.R. Franco), [email protected], [email protected] (P.M.L. Castro). 0925-8574/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.ecoleng.2012.01.021

In Portugal, a tree improvement programme of P. pinaster has started in the early 1980s (Roulund et al., 1988). Since then, superior trees, designated as plus trees, have been selected in natural stands. The selection criteria of plus trees include characteristics such as growth rate, stem and timber quality, crown shape, resistance to diseases, to insect attack and to adverse environmental abiotic conditions, and the ability to produce fertile seeds (Lee, 2002). Field transplantation of container-grown seedlings produced from seeds in nurseries is the most common method to establish P. pinaster plantations. The use of seeds from plus trees in the production of P. pinaster can be advantageous and contribute to obtain better plants for forestry industries. This approach to improve production avoids genetic modification of plants and is generally better accepted by the public and decision makers than other approaches involving gene transfers in plant material (Koski and Rousi, 2005). Although seeds from plus trees have been used to increase forestry production of species from different genera (e.g. Betula, Pinus, Pseudotsuga) (Koski and Rousi, 2005; Lee and Connolly, 2004; Prat and Caquelard, 1995), studies are needed on the effect of nursery practices on the growth performance of seedlings obtained from seeds of plus trees. The application of ectomycorrhizal (ECM) fungi inocula on forest nursery production is becoming to be

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regarded as a good-practice management due to its potential for increasing growth and vigour of seedlings under nursery conditions (Brundrett et al., 2005; Chen et al., 2006; Duponnois et al., 2007) and for improvement in quality and performance of outplanted seedlings (Duponnois et al., 2007; Quoreshi et al., 2008; Rincón et al., 2007a). ECM fungi can improve plant survival and growth by stimulating the uptake of soil nutrients and water, and by increasing plant resistance against biotic (e.g. plant pathogens) and abiotic (e.g. presence of toxic elements) stresses (Chalot et al., 2002; Garbaye, 2000; Hall, 2002; Smith and Read, 2008). Thus, the use of ECM fungi in forest nursery production can be considered an environmentally friendly approach, as it may help to reduce the input of chemical fertilisers and pesticides, preventing the contamination of soil and water resources (Khasa et al., 2001; Oliveira et al., 2010; Sousa et al., 2012). Although potentially beneficial, nursery inoculation is not always straightforward, and requires the selection of compatible and efficient ECM fungal isolates tuned for specific target plant and growth conditions (Oliveira et al., 2011; Vosátka et al., 2008). ECM fungi are known to form symbiotic associations with P. pinaster (Nieto and Carbone, 2009; Rincón and Pueyo, 2010). However, their effect in P. pinaster plus trees has not been studied. The aims of the present study were to compare the response of P. pinaster plants obtained from seeds of plus and non-plus trees to inoculation with different selected ECM fungi under conventional forest nursery conditions; and to assess the potential of the combined use of seeds obtained from plus trees and inoculation with ECM fungi as a new ecological approach to improve the production of this important tree species. 2. Materials and methods 2.1. Experimental design In a forest nursery greenhouse, in Amarante (41◦ 16 11 N Northern Portugal, trays with 210 cm3 cells were filled with non-sterile homogenised peat (1636 mg kg−1 N-NO3 − , 1833 mg kg−1 P2 O5 , 12,528 mg kg−1 K2 O, 31,000 mg kg−1 Mg, 16,400 mg kg−1 Ca, 400 mg kg−1 Na, pH 6.47, electrical conductivity 14.4 mS cm−1 ). P. pinaster seeds were collected in the area of Ponte de Lima (41◦ 52 30 N 8◦ 50 24 W), Northern Portugal, from five adult trees classified as plus and from five adjacent adult nonplus trees. The classification criteria of the plus trees was based on volume, stem morphology, spiral grain and branch habits (Perry and Hopkins, 1967). Experimental units were arranged in a fully randomized manner using a 2 × 5 factorial design where one factor was seedling type (ST) (from plus and non-plus trees) and the second factor was fungal inoculation (FI) [non-inoculated controls (C), plants inoculated with mycelium of Thelephora terrestris Ehrh. (T), mycelium of Rhizopogon roseolus (Corda) Th. Fr. (R), a spore mixture of Pisolithus tinctorius (Pers.) Coker & Couch and Scleroderma citrinum Pers. (PS), and a mixture of mycelium of Suillus bovinus (Pers.) Roussel, Laccaria laccata (Scop.) Cooke and Lactarius deterrimus Gröger (SLL)]. Each treatment combination was replicated 10 times. These ECM fungal isolates and mixtures were chosen for their compatibility with P. pinaster in previous laboratory and greenhouse studies (Oliveira, R.S.; Franco, A.R.; Castro, P.M.L., unpublished; Sousa et al., 2012). Inoculation with a variety of ECM fungi including single/multiple species and spore/mycelium inoculum was adopted in order to try to assess a range of plant responses where possible differences in growth performance of P. pinaster seedlings obtained from seeds of plus and non-plus trees could be detected. The fungal isolates were isolated from forest ecosystems of Northern Portugal and were maintained by successive transfers in modified Melin Norkans agar (MNM, Marx, 1969). 8◦ 04 40 W),

All fungal isolates used in these experiments belong to the collection of Escola Superior de Biotecnologia, and are referenced in the collection as: ref. TT-00, T. terrestris; ref. RH-01, R. roseolus; ref. SB-00, S. bovinus; ref. LL-02, L. laccata and ref. LD-02, L. deterrimus. Spores of P. tinctorius and S. citrinum were collected from a P. pinaster forest site in Caminha (41◦ 46 04 N 8◦ 35 03 W), Northern Portugal. Inoculation was performed either by injecting 6 ml of three weeks old mycelial suspensions (ca. 170 mg of fresh weight) or 10 ml of spore suspension (107 and 106 spores per seedling of P. tinctorius and S. citrinum, respectively) to the substrate of each cell. Seeds were rinsed overnight in running tap water, surface sterilised with 10% bleach solution for 15 min and washed three times with deionised sterile water. Two disinfected seeds were placed in each root tray. The experiment was initiated at the time of placing seeds and inoculum (June 2004). One month after that, plants were thinned to one seedling per cell, guaranteeing that there were 10 seedlings in each treatment. Plants were watered everyday and maintained under an average photoperiod of 8 h. Greenhouse temperature varied between 5.0 and 40.0 ◦ C and relative humidity between 10 and 80%. Trays of different treatments were periodically rotated to different bench positions to minimise differences due to their location in the greenhouse. With the exception of fungal inoculation and the use of seeds from plus trees, all the above mentioned procedures are currently used in forest nursery production. 2.2. Plant and fungal parameters All plants survived and six months after the beginning of the experiment, they were gently removed from the trays and transported to the laboratory for further analyses. The root collar diameter and shoot height were measured. The root system was separated from the shoot and washed to remove adhered substrate. The percentage of ECM fungal colonisation was assessed using a stereomicroscope (SZ30, Olympus, Japan) according to Brundrett et al. (1996). Representative ECM root tips were characterised on the basis of colour, branching, shape, presence of emanating hyphae and inner and outer mantle patterns under a stereomicroscope and by differential interference contrast microscopy (BX60, Olympus, Japan) according to Agerer (1998). The dry weights of roots and shoots were determined after drying the plant material at 70 ◦ C for 48 h and the total plant dry weight obtained as the sum of shoot and root dry weights. Oven-dried needles were finely ground and 0.2 g of material were digested according to Novozamsky et al. (1983). The digested samples were used to determine the total phosphorus (P) and nitrogen (N) concentrations in needles by colorimetry (Unicam, Helios Gamma, Cambridge, UK) (Walinga et al., 1989). 2.3. Statistical analysis The data were analysed using two-way analysis of variance (ANOVA) for each dependent variable (plant and fungal parameters) versus the independent variables [seedling type (ST) and fungal inoculation (FI)]. When a significant F-value was obtained (P < 0.05), treatment means were compared using Duncan’s multiple range test. All statistical analyses were performed using the SPSS 16.0 software package (SPSS Inc., Chicago, IL, USA). 3. Results 3.1. Plant growth and nutrition ECM inoculation and seedling type improved the growth of plants. The factor fungal inoculation showed a significant effect on

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Table 1 Two-way ANOVA significances and F values of all the measured parameters according to seedling type and fungal inoculation factors.

Seedling type (ST) Fungal inoculation (FI) ST × FI

Root collar diameter

Shoot height

Root dry weight

Shoot dry weight

Total plant dry weight

Needles N

Needles P

% ECM root colonisation

F1,90 = 16.8*** F4,90 = 1.9 ns F4,90 = 4.4**

F1,90 = 7.5** F4,90 = 18.2*** F4,90 = 18.5***

F1,90 = 64.8*** F4,90 = 3.7** F4,90 = 3.9**

F1,90 = 2.8 ns F4,90 = 29.6*** F4,90 = 18.1***

F1,90 = 47.0*** F4,90 = 14.1*** F4,90 = 9.0***

F1,90 = 77.5*** F4,90 = 21.2*** F4,90 = 9.1***

F1,90 = 0.3 ns F4,90 = 0.9 ns F4,90 = 0.5 ns

F1,90 = 0.2 ns F4,90 = 0.6 ns F4,90 = 2.3 ns

ns, non-significant effect; ECM, ectomycorrhizal. ** Significant effect at the level of P < 0.01. *** Significant effect at the level of P < 0.001.

all measured plant growth parameters, except on root collar diameter, and the factor seedling type showed a significant effect on all measured plant growth parameters (Table 1). The interaction of both factors (seedling type and fungal inoculation) had also a significant effect on all measured plant growth parameters. There were significant differences in the root collar diameter, shoot and total dry weights of P. pinaster plants among the different inoculation treatments within each seedling type (Figs. 1a and 2b, c), whereas significant differences in shoot height and root dry weight were only observed in P. pinaster plants obtained from seeds of plus trees (Figs. 1b and 2a). Plants obtained from seeds of plus trees inoculated with R. roseolus or P. tinctorius + S. citrinum or S. bovinus + L. laccata + L. deterrimus had a significantly greater root collar diameter when compared with non-inoculated controls. However, in plants obtained from seeds of non-plus trees a significantly greater root collar diameter was only observed in the T. terrestris treatment (Fig. 1a). There were no significant differences in shoot height of plants obtained from seeds of non-plus trees among all treatments, while plants obtained from seeds of plus trees inoculated with R. roseolus or P. tinctorius + S. citrinum or S. bovinus + L. laccata + L. deterrimus showed a significantly greater shoot height (Fig. 1b). Plants obtained from seeds of plus trees inoculated with R. roseolus or S. bovinus + L. laccata + L. deterrimus showed a significant greater root dry weight (Fig. 2a). However, in plants obtained from seeds of non-plus trees there were no significant differences in root dry weight among all treatments. The shoot dry weight of P. pinaster plants obtained from seeds of non-plus trees was significantly greater when inoculated with T. terrestris or S. bovinus + L. laccata + L. deterrimus, while in plants obtained from seeds of plus trees significantly greater shoot dry weight was found when inoculated with R. roseolus or P. tinctorius + S. citrinum or S. bovinus + L. laccata + L. deterrimus (Fig. 2b). In plants obtained from seeds of non-plus trees only those inoculated with S. bovinus + L. laccata + L. deterrimus had a significantly greater total plant dry weight than non-inoculated controls, while in plants obtained from seeds of plus trees, those inoculated with R. roseolus or P. tinctorius + S. citrinum or S. bovinus + L. laccata + L. deterrimus showed a significantly greater total plant dry weight when compared with non-inoculated controls (Fig. 2c). The factors fungal inoculation, seedling type and their interaction had a significant effect on needles N concentration (Table 1). In plants obtained from seeds of non-plus trees only those inoculated with S. bovinus + L. laccata + L. deterrimus had a significantly greater needles N concentration than non-inoculated controls, while in plants obtained from seeds of plus trees, those inoculated with R. roseolus or P. tinctorius + S. citrinum or S. bovinus + L. laccata + L. deterrimus showed a significantly greater needles N concentration when compared with non-inoculated controls (Fig. 3a). The factors fungal inoculation, seedling type and their interaction had no significant effect on needles P concentration (Table 1). There were no significant differences in needles P concentration of P. pinaster

plants obtained from seeds of both plus and non-plus trees among all inoculation treatments (Fig. 3b). 3.2. Ectomycorrhizal colonisation The factors fungal inoculation and seedling type did not have a significant effect on the percentage of ECM root colonisation, neither did the interaction of both factors (Table 1). There were no significant differences in the percentage of ECM root colonisation of P. pinaster plants obtained from seeds of both plus and non-plus trees among all treatments. A total of eight different ECM morphotypes were found on the roots of P. pinaster plants (Table 2). Roots of non-inoculated control plants were colonised by ECM fungi (Fig. 4). However, only two ECM morphotypes (EM1 and EM2) were found in non-inoculated control plants (Table 2). These two ECM morphotypes were also found on the roots of plants from all treatments irrespective of seedling type. Nevertheless, their relative abundances were negligible in all treatments except in non-inoculated control plants (Fig. 5). The ECM morphotypes EM5, EM6 and EM7 were only found in the root of plants inoculated with P. tinctorius + S. citrinum, R. roseolus, S. bovinus + L. laccata + L. deterrimus, respectively, irrespective of seedling type. The ECM morphotype EM8 was only found in the root of plants inoculated with S. bovinus + L. laccata + L. deterrimus, irrespective of seedling type. 4. Discussion Inoculation with selected ECM fungi improved the growth of P. pinaster plants under nursery conditions. Previous studies have demonstrated that the growth of P. pinaster seedlings can be enhanced by ECM fungal inoculation (González-Ochoa et al., 2003; Parladé et al., 2004; Rincón et al., 2007b). The biomass of plants obtained from non-plus trees was significantly greater when inoculated with S. bovinus + L. laccata + L. deterrimus, which showed the advantage of the application of selected ECM fungi in the nursery production of P. pinaster even when the use of seeds from phenotypically superior trees is not regarded. The effect of the application of ECM fungi on the growth of plants obtained from plus trees had not been studied. Our results show that inoculation with selected ECM fungi influenced differently the growth of P. pinaster plants obtained from seeds of plus and non-plus trees. The highest values of the measured plant growth parameters of P. pinaster were those of plants obtained from seeds of plus trees and inoculated with R. roseolus or S. bovinus + L. laccata + L. deterrimus. This is in accordance with the results of González-Ochoa et al. (2003) and Rincón et al. (2007b) who showed that inoculation with L. laccata or with species of the genera Suillus or Lactarius improved the growth of P. pinaster. The biomass of inoculated plants obtained from seeds of plus trees was in general greater than that of plants obtained from seeds of non-plus trees. Furthermore, plants obtained from seeds of plus trees inoculated with R. roseolus or S. bovinus + L. laccata + L. deterrimus had a significant increase in total biomass of 2.2 and 2.0

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Shoot height (cm)

b

20

15

a

a

a

10

a

a

y

x

x

x

R

PS

x

x

x

R

PS

SLL

y

5

0 C

Root collar diameter (mm)

a

T

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PS

SLL

C

T

SLL

4

a

3

b 2

b

b

R

PS

ab

y

xy

SLL

C

T

1

0 C

T

Pinus pinaster non-plus

Pinus pinaster plus

Fig. 1. Root collar diameter (a) and shoot height (b), of Pinus pinaster plants obtained from seeds of non-plus (left) and plus (right) trees inoculated with Thelephora terrestris (T), Rhizopogon roseolus (R), mixture of Pisolithus tinctorius and Scleroderma citrinum (PS), a mixture of Suillus bovinus, Laccaria laccata and Lactarius deterrimus (SLL) and non-inoculated control (C). Columns marked with different letters within each seedling type differed significantly according to Duncan’s multiple range test at P < 0.05. Error bars are SEM.

Table 2 Ectomycorrhizal morphotypes found on the roots of Pinus pinaster plants obtained from seeds of plus and non-plus trees inoculated with Thelephora terrestris (T), Rhizopogon roseolus (R), mixture of Pisolithus tinctorius and Scleroderma citrinum (PS), a mixture of Suillus bovinus, Laccaria laccata and Lactarius deterrimus (SLL) and non-inoculated control (C). Code

Morphotype description

EM1

Dark brown, unbranched, long tortuous tips, smooth mantle surface, pseudoparenchymatous outer and inner mantle with angular cells and mounds of flattened cells, few emanating hyphae Dark brown, dichotomous branching, tortuous tips, smooth mantle surface, pseudoparenchymatous outer and inner mantle with angular cells, few emanating hyphae Dark grey, unbranched, smooth mantle surface Brown and whitish, dichotomous branching, tortuous tips, smooth mantle surface, pseudoparenchymatous outer and inner mantle with angular cells and irregularly arranged hyphae, few emanating hyphae White, dichotomous branching, long tortuous tips, smooth mantle surface, pseudoparenchymatous outer and inner mantle with angular cells, few emanating hyphae Dark orange, dichotomous branching, straight tips, grainy mantle surface, pseudoparenchymatous outer and inner mantle with angular cells and mounds of flattened cells Golden yellow, dichotomous branching, straight hairy tips, pseudoparenchymatous outer mantle, plectenchymatous inner mantle with epidermoid cells bearing a delicate hyphal net, abundant emanating hyphae Light grey, monopodial pinnate branching, tortuous tips, smooth mantle surface

EM2

EM3 EM4

EM5

EM6

EM7

EM8

Total number of morphotypes +, presence; −, absence.

Pinus pinaster non-plus

Pinus pinaster plus

C

T

R

PS

SLL

C

T

R

PS

SLL

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

− −

− +

+ −

− −

+ −

− −

− +

+ +

+ −

+ −







+









+







+









+













+









+









+









+

2

3

4

3

5

2

3

5

4

5

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a

99

x

2.0

x a

1.5 ab ab

1.0

y yz

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0 C

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PS

SLL

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SLL

1.2 1.0

x

a

0.8

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a ab

0.6

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0.2 0 C

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SLL

R

PS

SLL

1.2 x

1.0

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0.8

xyz

0.6 0.4

a a a

a

z

SLL

C

yz

a

0.2 0 C

T

R

PS

Pinus pinaster non-plus

T

R

PS

SLL

Pinus pinaster plus

Fig. 2. Root (a), shoot (b) and total plant (c) dry weights of Pinus pinaster plants obtained from seeds of non-plus (left) and plus (right) trees inoculated with Thelephora terrestris (T), Rhizopogon roseolus (R), mixture of Pisolithus tinctorius and Scleroderma citrinum (PS), a mixture of Suillus bovinus, Laccaria laccata and Lactarius deterrimus (SLL) and non-inoculated control (C). Columns marked with different letters within each seedling type differed significantly according to Duncan’s multiple range test at P < 0.05. Error bars are SEM.

times, respectively, showing the added benefit of nursery inoculation with selected ECM fungi. This significant improvement in the nursery growth of P. pinaster was achieved through the combined use of seeds from phenotypically superior trees and the inoculation with selected ECM fungi and without extra input of agrochemicals. The nutrition of P. pinaster was also improved by inoculation with ECM fungi. ECM fungi are know to improve plant uptake of N from different sources (mineral and organic) and it has been shown that the extraradical mycelium contributes to the translocation of N from soil to roots (Chalot et al., 2002; Plassard et al.,

2000). Improved N nutrition of P. pinaster plants inoculated with R. roseolus had been reported (Gobert and Plassard, 2002). In our study the greatest needles N concentration was that of plants obtained from seeds of plus trees and inoculated with R. roseolus or P. tinctorius + S. citrinum or S. bovinus + L. laccata + L. deterrimus. In plants obtained from seeds of non-plus trees only those inoculated with S. bovinus + L. laccata + L. deterrimus had improved needles N concentration, showing that inoculation with selected ECM fungi influenced differently the nutrition of P. pinaster plants obtained from seeds of plus and non-plus trees.

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5 4

a a

a

a

a

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x x

x

C

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-1

Needles P (mg g )

b

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-1

Needles N (mg g )

a

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15

R

x

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SLL

x

x

PS

SLL

12 9

b

b

b

T

R

b

a

y

y

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6 3 0 C

PS

SLL

Pinus pinaster non-plus

R

Pinus pinaster plus

Fig. 3. Needles nitrogen (a) and phosphorus (b) concentration of Pinus pinaster plants obtained from seeds of non-plus (left) and plus (right) trees inoculated with Thelephora terrestris (T), Rhizopogon roseolus (R), mixture of Pisolithus tinctorius and Scleroderma citrinum (PS), a mixture of Suillus bovinus, Laccaria laccata and Lactarius deterrimus (SLL) and non-inoculated control (C). Values are expressed in mg of element per gram of biomass of oven-dried needles. Columns marked with different letters within each seedling type differed significantly according to Duncan’s multiple range test at P < 0.05. Error bars are SEM.

There was no effect of ECM fungal inoculation on the P concentration of needles and there were no differences in plants obtained from seeds of both plus and non-plus trees. It is generally accepted that mycorrhizal plants are more efficient in P uptake than non-mycorrhizal plants (Chalot et al., 2002). In a study with P.

pinaster seedlings, Casarin et al. (2004) reported improved P nutrition after inoculation with R. roseolus. However, no improvement in P nutrition of mycorrhizal plants has also been reported (Walker, 2001). These seemingly contradictory results may be due to several reasons such as P availability in the nursery substrate and the

% ECM root colonisation

80

60

a

a

a

a

a

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x

x

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40

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Pinus pinaster non-plus

SLL

C

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SLL

Pinus pinaster plus

Fig. 4. Percentage of ectomycorrhizal root colonisation of Pinus pinaster plants obtained from seeds of non-plus (left) and plus (right) trees inoculated with Thelephora terrestris (T), Rhizopogon roseolus (R), mixture of Pisolithus tinctorius and Scleroderma citrinum (PS), a mixture of Suillus bovinus, Laccaria laccata and Lactarius deterrimus (SLL) and non-inoculated control (C). Columns marked with the same letters within each seedling type did not differ significantly according to Duncan’s multiple range test at P < 0.05. Error bars are SEM. ECM, ectomycorrhizal.

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Fig. 5. Relative abundance of ectomycorrhizal morphotypes found on the roots of Pinus pinaster plants obtained from seeds of non-plus (left) and plus (right) trees inoculated with Thelephora terrestris (T), Rhizopogon roseolus (R), mixture of Pisolithus tinctorius and Scleroderma citrinum (PS), a mixture of Suillus bovinus, Laccaria laccata and Lactarius EM3, EM4, EM5, EM6, EM7, EM8. deterrimus (SLL) and non-inoculated control (C). ECM, ectomycorrhizal.  EM1,  EM2,

heterogeneity of P uptake capacities among different ECM fungi (Cairney and Smith, 1993; Courty et al., 2010). In order to potentiate the applicability of this study, with the exception of fungal inoculation and the use of seeds from plus trees, we employed practices currently used in forest nurseries. These include the use of substrates which often have high P concentration as it was recorded in our study (Vaario et al., 2009). Under high soil P levels the contribution of ECM fungi for the plant P nutrition can be reduced, irrespective of the ECM fungal species (Jones et al., 1990), which may explain why there was no effect of inoculation on P nutrition in our experiments. All plants including non-inoculated controls became colonised by ECM fungi. This was expected as the experiments were conducted under conventional forest nursery conditions using nonsterile substrate. The two ECM morphotypes found in the roots of non-inoculated control plants were also found with low relative abundance in plants from all treatments irrespective of seedling type, suggesting that ECM fungal propagules which were probably present in the substrate or airborne entered our systems and colonised the roots of P. pinaster plants irrespective of inoculation treatments or seedling type, such as previously reported (Stottlemyer et al., 2008). Quantitatively there was no effect of ECM fungal inoculation on ECM colonisation and there were no differences between plants obtained from seeds of plus and non-plus trees. However, the observed relative abundances of ECM morphotypes in the roots of P. pinaster plants suggest that different ECM fungi were present in the different inoculation treatments. The characteristics of the observed morphotypes together with the descriptions found in the literature (Agerer, 1998; Nieto and Carbone, 2009; Pera and Alvarez, 1995) did not allow the unequivocal identification of the individual inoculated ECM fungi in the roots of P. pinaster. However, the observed morphotypes resemble those described in the roots of P. pinaster and in other pine species in association with the same fungal species or with fungal species from the same genera (Agerer, 1998; Nieto and Carbone, 2009; Pera and Alvarez, 1995), suggesting that the inoculated ECM fungi formed ectomycorrhizas in the roots of P. pinaster in our systems. Molecular analysis using DNA extraction and PCR with ITS primers could help to ascertain the fungal species associated with each morphotypes. Using these techniques, Kohout et al. (2011) found that though the majority of the ECM morphotypes found in

Pinus spp. corresponded to a single fungal taxon, one (a suilloid morphotype) was separated into eight species of Suillus and Rhizopogon based on 200 internal transcribed spacer (ITS) sequences. This study also emphasises the importance of using selected ECM fungal isolates previously screened for compatibility with the target plant. Most of the inoculant fungi tested in our experiments had already been used in laboratory, greenhouse and field trials with P. pinaster (Sousa et al., 2012; Vosátka et al., 2008). A careful selection of mycobionts prior to inoculation is important to ensure mycorrhization and to help develop combinations of ECM fungal inocula adequate for the target conditions (Vosátka et al., 2008). The results from the present study show that the response of P. pinaster plants obtained from seeds of plus and non-plus trees to inoculation with ECM fungi differed. They also suggest that plants obtained from seeds of plus trees take more advantage from the ECM symbiosis than those obtained from seeds of non-plus trees. However, further field studies to assess the performance of mycorrhizal plus trees and the follow-up of field transplanted inoculated plants of P. pinaster obtained from seeds of plus trees would be necessary to investigate the reasons why a seedling grows into a phenotypically superior tree. In case of P. pinaster obtained from seeds of non-plus trees the inoculation with ECM can also be advantageous, however, it is advisable to use S. bovinus + L. laccata + L. deterrimus, which was the inoculation treatment that yielded the greater promoting effect to plant vigour. Among all treatments tested in this study, inoculation with R. roseolus or S. bovinus + L. laccata + L. deterrimus resulted in the greater improvement in the growth and nutrition of P. pinaster plants obtained from seeds of plus trees. Since there were no significant differences in improvement of growth and nutrition of P. pinaster plants between the two treatments (R. roseolus and S. bovinus + L. laccata + L. deterrimus), inoculation with R. roseolus is preferred for being more practical to perform. Further field studies to assess the performance of outplanted P. pinaster inoculated with R. roseolus and S. bovinus + L. laccata + L. deterrimus could help to decided on the most adequate inoculation treatment. The more diverse ECM morphotypes found in plants inoculated with S. bovinus + L. laccata + L. deterrimus in comparison with those found in R. roseolus inoculated plants could play an important role in plant fitness and survival after transplanting to forest conditions. More diverse ECM morphotypes that include different exploration types

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may contribute to a more efficient foraging strategy and, therefore, improve plant performance under field conditions (Agerer, 2001). Nevertheless, a cost/benefit analysis to assess the economic feasibility of the inoculation with R. roseolus and S. bovinus + L. laccata + L. deterrimus in P. pinaster nursery production would be recommended. The ultimate objective of forest nurseries is the production of higher quality stock. If P. pinaster plants obtained from seeds of plus trees and inoculated with the appropriate ECM fungi are used, it can result in the production of more vigorous plants and in a decreased time to planting without extra input of agrochemicals. This improvement in plant vigour is particularly important when the production of P. pinaster plants is targeted for restoration or rehabilitation of forests in disturbed ecosystems. The use of plants obtained from plus trees (considered phenotypically superior) is an advantageous strategy to obtain better plants for forestry industries. ECM fungi are an integrant part of forest ecosystems, vital for tree survival and development and as such should be considered in forestry. Therefore, we propose the use of P. pinaster plants obtained from seeds of plus trees and inoculated with R. roseolus as an ecotechnology to improve nursery production of P. pinaster.

Acknowledgements The authors wish to acknowledge Fundac¸ão para a Ciência e a Tecnologia and FSE (III QCA), research grant of R.S. Oliveira (SFRH/BPD/23749/2005). This work was supported by the FCT Project PTDC-AGR-CFL-111583-2009.

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