Fate, tree growth effect and potential impact on soil microbial communities of mycorrhizal and bacterial inoculation in a forest plantation

Soil Biology & Biochemistry 36 (2004) 211–216 www.elsevier.com/locate/soilbio

Fate, tree growth effect and potential impact on soil microbial communities of mycorrhizal and bacterial inoculation in a forest plantation J. Heinonsaloa,*, P. Frey-Kletta, J.-C. Pierratb, J.-L. Churina, D. Vairellesa, J. Garbayea a

Centre INRA de Nancy, UMR INRA/ UHP no. 1136, Interactions Arbres/Microorganismes, F-54280 Champenoux, France b UMR INRA-ENGREF, Equipe Dynamique des Syste`mes forestie`rs, 14 rue Girardet, F-54000 Nancy, France Received 10 January 2003; received in revised form 12 September 2003; accepted 16 September 2003

Abstract The knowledge of the survival of inoculated beneficial fungal and bacterial strains in the field and the effects of their release on the indigenous microbial communities has been of great interest since the practical use of selected natural or genetically modified microorganisms has been developing. The aim of this study was to monitor, 4 years after plantation into the field site, the effects of Douglas fir (Pseudotsuga menziesii) co-inoculation with the mycorrhiza helper bacterial strain Pseudomonas fluorescens BBc6R8 and/or the fungal strain Laccaria bicolor S238N on seedling growth and on the indigenous bacterial and ectomycorrhizal communities using quantitative and qualitative approaches. The field persistence of the inoculated strains was also monitored. The seedling shoot volume estimate was statistically significantly higher in the fungal inoculated plots in comparison to the non-inoculated plots but no treatment-related changes in the quantitave or qualitative microbial measurements were observed and the inoculated strains could not be detected after 4 years. q 2003 Elsevier Ltd. All rights reserved. Keywords: Douglas fir; Laccaria bicolor; Pseudomonas fluorescens; Co-inoculation; Community; Biolog

Laccaria laccata and Laccaria bicolor have long been used in inoculation studies in North America (e.g. Molina and Chamard, 1983) as well as in Europe (e.g. Mortier et al., 1988; Villeneuve et al., 1991; Le Tacon et al., 1997), using most commonly Douglas fir (Pseudotsuga menziesii) as a host tree. L. laccata strain S238N (Di Battista et al., 1996) has been so well and widely studied, that it has become almost a model organism in forest tree inoculation research in Europe. The inoculation success in European field studies has been variable (Le Tacon et al., 1992), suggesting important unknown and uncontrolled factors in the inoculation process. Nevertheless, the commercial application of the technique has been successfully developed in France. The hypothesis of a bacterial impact on mycorrhiza formation was tested in numerous inoculation studies (Duponnois and Garbaye, 1990, 1991a,b; Duponnois et al., 1993; Frey-Klett et al., 1997, 1999) and the concept of * Corresponding author. Address: Division of General Microbiology, Department of Biosciences, University of Helsinki, P.O. Box 56, Helsinki 00014, Finland. Tel.: þ 358-9191-59201; fax: þ 358-9191-59262. E-mail address: [email protected] (J. Heinonsalo). 0038-0717/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2003.09.007

bacteria helping the ectomycorrhizal formation was presented by Garbaye (1994). In the studies mentioned, Pseudomonas fluorescens BBc6R8 was shown to increase Douglas fir-L. bicolor mycorrhiza formation even in very low population density and to improve the inoculation success under laboratory, greenhouse and nursery conditions. In recent years, more and more interest has arisen for field inoculation of different kinds of micro-organisms, genetically modified as well as naturally selected strains and more knowledge is needed to understand the environmental effects of human interventions in agriculture and forestry. Here we report the effects of Douglas fir co-inoculation with the helper bacterial strain P. fluorescens BBc6R8 and/or the fungal strain L. bicolor S238N on seedling growth and on the indigenous bacterial and ectomycorrhizal communities and the strain persistence in a field experiment 4 years after outplanting. The bacterial and fungal analyses performed were divided into two classes: quantitative and qualitative analyses of indigenous microflora. In the former, total bacterial number on tryptic soy agar (TSA) and ergosterol concentration indicating fungal biomass and

212

J. Heinonsalo et al. / Soil Biology & Biochemistry 36 (2004) 211–216

in the latter, Biolog community level physiological profiling (CLPP) for bacterial communities and morphotype analysis for ectomycorrhizas were performed. The detection of introduced strains was performed by selective plating and enrichment for the bacterial strain and morphotyping verified with PCR-RFLP methodology for the fungal strain. The site is located in the Vosges region in northeastern France. The beech (Fagus sylvatica) forest was clear-cut in the spring 1995 and the harvesting residues were removed from the experimental area. The planting of the bare rooted Douglas fir (P. menziesii, seeds originated from Washington state, provenance zone 422, USA) seedlings (30/100 m2 plot) was performed in April 1996 in a latin square design (4 treatments £ 4 replicates) with fungal (F) or bacterial (B) inoculation, bacterial – fungal (BF) co-inoculation and non-inoculated control (C) treatments. The fungal strain used, L. bicolor S238N, is described in Di Battista et al. (1996) and the bacterial strain used, P. fluorescens BBc6R8, in Frey-Klett et al. (1997). Seedlings from the F treatment were inoculated in the nursery at sowing, with the fungal strain L. bicolor S238N inoculum produced with the alginated bead method as described in Mortier et al. (1988). After 2 years in nursery, mycorrhization rate of the seedlings was estimated visually using scale 0 – 3 (0 ¼ no S238N present, 3 ¼ completely mycorrhizal with S238N) and only the healthy looking seedlings with mycorrhization rate two or three were selected for experimental use. Control seedlings were often naturally colonised by the mycorrhizal fungus Rhizopogon spp. The seedlings were chosen for out-planting according to their similarity in size. The bacterial inoculation was done by dipping the root system of the seedling into a suspension of P. fluorescens BBc6R8 (3 £ 105 cells ml 21 water) for 30 min, short before planting to the site. For treatments without bacteria, the seedlings were dipped into water. Three samplings were performed successively from 1998 to 2000. In July 1998, five seedlings from each plot were sampled for ectomycorrhizal morphotyping analysis. The second sampling period extended from May to August 2000 when altogether four seedlings and soil samples from each plot were taken. The sampling procedure was defined in order to take into account extensively the variation in time (16 sampling dates) and space in the experimental area and to allow an immediate next-day analysis of the samples in the laboratory. The third and last sampling was performed in autumn 2000, when additional four seedlings and soil samples per plot were sampled for ergosterol analysis and for L. bicolor screening. The seedlings sampled were chosen with height as close as possible to the average height within each plot in order to get a representative sample for the microbial measurements in the different treatment plots. All seedlings at site were measured for height and diameter at ground level after the growth seasons of 1996, 1997 and 1999 ðN ¼ 48 – 113Þ: The shoot volume estimate

of the trees (height £ (root collar diameter)2 ¼ HD2) was counted as reported in Stenstro¨m et al. (1990). For root sampling, one root growing uphill was dug up carefully by hand following the root from the stem toward its apical part, including the fine roots and mycorrhizas. In addition, the part of the root system in 50 cm distance from the stem was dug up and added to the sample until totally 1 l of roots and adhering soil was obtained. The samples were first kept on ice, then at þ 4 8C until analysed next day. The parts of the root systems which were not used for bacterial analysis were stored for ectomycorrhizal analysis in þ 4 8C up to 4 months in plastic bags containing humid soil, pressed tight to minimize the quantity of air inside. In the laboratory, samples were then processed sterile: roots smaller than 2 mm in diameter were cut off with scissors into a big petri-dish containing approx. 30 ml of water and rinsed. Approx. 1 dl of roots (5 – 8 g fresh wt) were put into a glass bottle (volume 125 ml), 50 ml water was added and the bottle was shaken by hand for 20 s. The water was poured-off and the rinsing was repeated three times. An aliquot of the first rinse water was kept for P. fluorescens detection (see below). Then the roots were put into a beaker containing 300 ml water and stirred for approx. 30 s to remove the last mineral soil particles. Finally, 5 g fresh wt of roots were weighed and put into a Waring blenderw together with 150 ml 0.9% NaCl, 170 ml 2% Tween 80 and 1.7 ml 10% Na5P3O10 and blended for 1 min. The suspension was filtered (Whatman No 1). The filtrate was placed at þ 4 8C prior to further bacterial analyses, which were all performed/started the same day. Bulk soil core (B 4 cm, height 12 cm) samples were taken 1.5 m uphill from the stems of the chosen seedlings, stored in a plastic bag and placed on ice until analysed. The samples were analysed as follows: avoiding the roots, 5 g fw of homogenized soil was put into a glass bottle with 50 ml 0.9% NaCl, 56 ml 2% Tween 80 and 556 ml 10% Na5P3O10 and vortexed for 2 min. Thereafter, the samples were filtered and filtrates stored as for the root samples. Bacterial enumeration was done using the Spiral system (Interscience, Saint Nom la Brete`che, France). Root and bulk soil filtrates were plated on 1/10 Tryptic Soy Broth Agar þ 100 mg l21 propiconazole (Tilt 125, a fungicide from Ciba-Geigy) l21 (Brule´ et al., 2001) and incubated for 7 days at þ 25 8C until counted. Ergosterol extraction from the soil samples was performed according to Brule´ et al. (2001). For the roots, the pre-cleaning was done as in bacterial analyses whereafter 5 g fresh wt of the rinsed roots were homogenised in 50 ml of 100% ethanol using an Ultra Turrax homogenizer (9500 rev min21). After incubating for 1 h on ice, the suspension was shaken and 5 ml of the solution was centrifuged for 15 min at 2500 rev min21. Thereafter, 1.5 ml of the supernatant was taken into a microcentrifuge tube, centrifuged at 13000 rev min21 for 5 min and filtered through a 0.45 mm filter. The samples were stored in

J. Heinonsalo et al. / Soil Biology & Biochemistry 36 (2004) 211–216

2 20 8C, until HPLC-analysis for ergosterol (Brule´ et al., 2001). The root and bulk soil extracts (see above) were distributed into Biologw GN2 microtitre plates (150 ml/well) (Biolog Inc., USA) and the plates were incubated for 7 days at þ 25 8C. They were read daily with Bio-Rad 550 plate reader at 590 nm wavelength and the data calculated as in Heinonsalo et al. (2001) except the area values were normalised dividing by the plate average of the 95 area values obtained. In 1996, 24 h after seedling plantation to the field site, the presence of rifampin resistent fluorescent pseudomonads in the bulk soil and root systems of control and bacterial inoculated plants was tested. Dilutions of bulk soil suspension, rhizospheric soil or root extracts (Frey-Klett et al., 1997, 1999) were plated on King’s medium B (KB þ rif þ Tilt) plates (King et al., 1954) containing 100 mg rifampin l21 and propiconazole 100 mg l21. In 2000, the presence of P. fluorescens BBc6R8 was investigated using direct plating and an enrichment procedure (Frey-Klett et al., 1997). Shortly, 100 ml sample from the first root washing solution, the soil extract before filtration (see above) and the root and soil extracts used for Biolog were taken and spread on three replicates on King’s medium B (KB þ Tilt þ rif) plates (Frey-Klett et al., 1997) and incubated for 48 h at þ 25 8C. For enrichments, approx. 1 cm3 non-washed roots or 1 g dry wt of fresh soil were put into three replicate, sterile tubes containing 3 ml KB þ Tilt þ rif liquid media and were incubated in a laboratory shaker 48 h at þ 25 8C. Three 50 ml-droplets of enrichment suspension were then placed on KB þ Tilt þ rif plates and incubated for 48 h at þ 25 8C. Finally, the presence of fluorescent colonies was checked under UV light. The ectomycorrhizal fungal community was monitored in 1998 and 2000. The roots for morphotype analysis were collected as described above. Roots , 2 mm in diameter were cut into 2 cm pieces in a water-containing petri-dish and chosen randomly for the visual observation until 300 or 200 (in 1998 and 2000, respectively) short roots, if available, were analysed. All characteristic types of mycorrhizas found were grouped based on their gross morphology (color, shape and mantle structure). Nonmycorrhizal root tips and, in 2000, old mycorrhizas (meaning types which looked necrotic or senescing: no

213

mantle, looking dry and wilted) were also counted. The presence of L. bicolor S238N in the roots was investigated in 1998 by morphotyping and in summer and autumn 2000 by morphotyping followed by PCR-RFLP verification (Di Battista et al., 1996) using Hinf I restriction enzyme. From the samples of the third harvest from autumn 2000, only a qualitative morphological screening was done to verify the percentage of seedlings having the L. bicolor morphotype. ANOVA followed by the Tukey HSD or Bonferroni tests was used to analyse the data, which was log or p (arcsin1/10 %) transformed before statistical analysis, when needed. The discriminant analysis was performed to see if it is possible, based on the Biolog CLPPs, to distinguish different sample types (bulk soil or roots) or different treatments (C, B, F or BF). The statistical analyses were performed with the SPSS for Windows 8.0.1 and SAS software’s. Four years after outplanting, the shoot volume estimate (HD2) of the seedlings in the fungal inoculated treatment (F) were significantly higher than in the control treatment but there was no statistically significant positive effect of bacterial inoculation on seedling growth even after the first growing season in 1996 (Table 1). Immediately after the transplantation in 1996, 7.7 ^ 0.12, 7.3 ^ 0.17, 6.4 ^ 4.3 and 6.0 ^ 0.23 log cfu/g dry wt P. fluorescens BBc6R8 were found in the rhizospheric soil of B and BF treatments and in the root systems of B and BF treatments, respectively. The strain could not be found in bulk soils or in the rhizospheric soils and root systems of the non-inoculated plants. In 2000, the strain was not found in any treatment either by direct plating or in enrichment cultures (data not shown). As the inoculated bacterial strain BBc6R8 could not be detected after 4 years in any of the treatments one could speculate whether the lack of bacterial effect on seedling growth was due to the non-persistence of the inoculated bacteria. In previous experiments, where P. fluorescens BBc6R8 and L. bicolor S238N were simultaneously inoculated at sowing, FreyKlett et al. (1997) showed a positive effect of bacterial inoculation on the Douglas fir-L. bicolor symbiosis in spite of a short term survival of the bacterium up to 19 weeks in the glasshouse and nursery soil. In contrast to Frey-Klett et al. (1997), in the present work, P. fluorescens BBc6R8 was inoculated at planting on Douglas fir plants already

Table 1 Plant growth measurements Growth season

Volume (HD2, cm3) N ¼ 48 – 113

1996 1997 1999

C

B

BF

F

p-values

Mean

SD

Mean

SD

Mean

SD

Mean

SD

9.5a 52.0a 1097.3a

13.8 43.1 960.9

8.0a 50.6a 1086.5a

5.0 39.1 925.4

10.6a 61.8ab 1337.4ab

8.9 53.2 1224.8

10.7a 72.0b 1586.5b

6.9 54.9 1069.9

0.10 ,0.01 0.02

Treatment codes: C ¼ control, B ¼ bacterial, BF ¼ bacterial–fungal co-inoculation and F ¼ fungal inoculation. Different letters in the same row indicate statistically significant differences (p ¼ 0:05; one-way ANOVA followed Tukey’s HSD test).

214

J. Heinonsalo et al. / Soil Biology & Biochemistry 36 (2004) 211–216

mycorrhizal by L. bicolor S238N. Recent results have suggested that the BBc6R8 helper effect could occur early, before the root-fungus encounter, the bacteria promoting the pre-symbiotic survival of the fungus in the soil, especially under unfavourable conditions for the fungus (Brule´ et al., 2001). Therefore, the absence of positive bacterial effect in our work could be due to the too late inoculation of the bacteria on already mycorrhizal seedlings. In 1998, the Laccaria-morphotype was significantly more common in the fungal inoculated treatment (F) in comparison to the three other treatments (C, B, BF) and the abundance of Thelephora-type differed between control and BF treatments. No other differences in the morphotypes were found (Fig. 1). Therefore, it seems that co-inoculating P. fluorescens BBc6R8 and L. bicolor S238N significantly reduced the number of L. bicolor ectomycorrhizas. Frey-Klett et al. (1999) showed that the bacterial strain P. fluorescens BBc6R8 has a mycorrhiza helper effect at low population doses (10 cfu/cm3 soil) and suggested that it could have some detrimental effects toward the plant or the fungus at high population densities. In this study, the novel inoculation method used resulted in a much higher bacterial density directly applied on the mycorrhizal root system compared to the previous studies (approx. 2 £ 102 cfu/g soil dry wt) (Frey-Klett et al., 1999). As a consequence, the reduced occurrence of L. bicolor morphotype in the BF treatment compared to fungal treatment (Fig. 1) could be due to the bacterial inoculation dose which was too high. The impacts of the factors affecting the beneficial effect of the helper bacterial inoculation in natural environments needs further study. Four years after outplanting, no treatment-related trends were found in the morphotype analysis. Rhizopogon-type dominated all the seedlings (25 – 39% of mycorrhizal root

Fig. 1. Morphotypes of the Douglas fir mycorrhizae from the summer 1998, divided into five classes: 1 ¼ Laccaria-, 2 ¼ Thelephora-, 3 ¼ Rhizopogon-type, 4 ¼ Non-identified and 5 ¼ Non-mycorrhizal. N ¼ 20: Different letters above the standard deviation bars within the morphotypeclass indicate statistically significant differences (p ¼ 0:05; one-way ANOVA followed Bonferroni test). C ¼ control, B ¼ bacterial, BF ¼ bacterial–fungal co-inoculation and F ¼ fungal inoculation.

Fig. 2. Morphotypes of the Douglas fir mycorrhizae from the summer 2000, divided into nine classes: 1 ¼ Laccaria-, 2 ¼ Old Laccaria-, 3 ¼ Cenococcum-, 4 ¼ Rhizopogon-, 5 ¼ Scleroderma-type, 6 ¼ Non-identified beige, 7 ¼ Other non-identified, 8 ¼ Old mycorrhizas and 9 ¼ Nonmycorrhizal. N ¼ 16: Different letters above the standard deviation bars within the morphotype-class indicate statistically significant differences (p ¼ 0:05; one-way ANOVA followed Tukey’s HSD test). C ¼ control, B ¼ bacterial, BF ¼ bacterial – fungal co-inoculation and F ¼ fungal inoculation.

tips), Cenococcum-type being the second most common (around 10% of mycorrhizas) irrespective of the treatment (Fig. 2) and the L. bicolor morphotype was detected only in low abundancy without any treatment-related differences. Morphotyping is considered an appropriate identification method for going through a large number of samples, especially in case of L. bicolor mycorrhizas which have distinct morphological characteristics. The disadvantage of morphotyping is the uncertainty in distinguishing closely related or similar looking types and therefore the DNAbased additional analyses are needed. Our morphotype results contrasts with previous report of long-lasting persistence of strain L. bicolor S238N in the field based on a sporophore survey (Selosse et al., 1998). Two assumptions could explain the temporary low number of L. bicolor ectomycorrhizas in our work in 2000. First, fluctuating and seasonal environmental factors in the site might have caused a temporary decline of the introduced strain during the sampling periods. High occurrence of the dead looking mycorrhizas in the summer months argues for further studies of seasonal dynamics of ectomycorrhizal roots. Secondly, since the non-destructive and representative root sampling was a challenge, some young, active L. bicolor S238N mycorrhiza could have been left in the soil, especially from the outer parts of the root system. The two assumptions are supported by the observations in the autumn sampling, when Laccaria-ectomycorrhizas were again frequent in the fungal inoculated plots (Table 2). However, the strain S238N of L. bicolor was not detected in any sample from the summer or autumn 2000 by PCRRFLP, suggesting the disappearance of the strain under the detection level, while local Laccaria species took over on the Douglas fir root system.

J. Heinonsalo et al. / Soil Biology & Biochemistry 36 (2004) 211–216

215

Table 2 Quantitative microbial measurements, Biolog community-level parameters and monitoring of the introduced fungal strain Microbial parameters

Total bacterial cfus

Fungal ergosterol

Biolog (bacterial community)

Introduced strains L. bicolor morphotype

N ¼ 16

Roots (cfu log 10 g21 dry wt) Bulk soil (cfu log 10 g21 dry wt) Roots (mg g21 dry wt) Bulk soil (mg g21 dry wt) Roots (no. of C-sources utilised of 95) Bulk soil (no. of C-sources utilised of 95) Roots (lag-period in wells, days) Bulk soil (lag-period in wells, days) Present in plants (%)

C

B

BF

F

p-values

Mean

SD

Mean

SD

Mean

SD

Mean

SD

5.8a

0.5

5.8a

0.4

5.6a

0.4

5.9a

0.4

0.30

3.5a

0.6

3.5a

0.7

3.6a

0.7

3.6a

0.5

0.96

0.2a

0.1

0.3a

0.3a

0.1

0.2a

4.1 £ 1024 4.8

1.2 £ 1023 a 87.1a

5.1 £ 1024 a 85.0a

3.3 £ 1024 10.3

1.0 £ 1023 a 86.7a

6.9 £ 10-2 9.4 £ 1024 7.4

0.56

5.4 £ 1024 a 86.6a

9.9 £ 10-2 9.0 £ 1024 6.7

0.87

71.6a

24.8

73.9a

15.4

73.2a

15.7

74.2a

15.4

0.98

3.1a

0.8

2.6a

0.7

2.9a

0.9

3.0a

0.9

0.26

3.4a

1.0

3.3a

0.8

3.1a

0.8

3.3a

0.7

0.58

31a

28

19a

13

38a

48

75a

29

0.16

0.05

Treatment codes: C ¼ control, B ¼ bacterial, BF ¼ bacterial–fungal co-inoculation and F ¼ fungal inoculation. Different letters in the same row indicate statistically significant difference (p ¼ 0:05; one-way ANOVA followed Tukey’s HSD test).

The total number of TSA-culturable bacteria, the ergosterol concentrations, the total number of different carbon sources utilised and the average lag period of the bacterial growth on different carbon sources did not differ

significantly between the different treatments either in the bulk soil or in the root samples (Table 2). In the discriminant analysis of the Biolog data, only the differences detected by the first axis were significant ðp ¼ 0:0054; Fig. 3). On that

Fig. 3. Projection of the samples in the first two axes (DA) obtained by the discriminant analysis of the Biolog multivariate data. Capitalized letters indicate the samples from the roots, small italic letters the samples from the bulk soil. C ¼ control, B ¼ bacterial, BF ¼ bacterial–fungal co-inoculation and F ¼ fungal inoculation.

216

J. Heinonsalo et al. / Soil Biology & Biochemistry 36 (2004) 211–216

axis, all the root samples have negative values whereas the bulk soil samples have positive values showing that the microbial communitites in root and bulk soil samples are significantly different. In contrast, the analysis does not separate the different treatments significantly. In conclusion, the fungal strain L. bicolor S238N and the bacterial strain P. fluorescens BBc6R8 have disappeared from the soil and the Douglas fir rhizosphere 4 years after plantation. Nevertheless, the fungal inoculation has significantly improved the Douglas fir growth from the second growing season. In contrast, the bacterial inoculation had no significant effect. Both microbial inoculations did not affect the soil and rhizosphere microbial communities 4 years after the establishment of the plantation suggesting that the indigenous microbial communities have either high resistance to change or ability to go rapidly back to normal conditions. This result has practical importance since it shows that in the studied case of controlled mycorrhization in a forest plantation, human interventions do not risk induction of long-term perturbation of the soil microbial communities.

Acknowledgements This study was financed by Academy of Finland, Marjatta ja Eino Kolli foundation, Association FrancoFinlandaise pour la Recherche Scientifique et Technique, Region Lorraine (Soutien aux jeunes e´quipes) and INRA Nancy. Yves Le Tacon is acknowledged for the morphotype analysis performed in 1998, Chantal Brule´ and Marie-Lise Clausse for help in starting the experiment and measurements and Jesus Diez for useful discussions on the manuscript. We also thank R. Molina and J.M. Trappe (Corvallis, Oregon) for providing the strain S238, from which S238N was derived.

References Brule´, C., Frey-Klett, P., Pierrat, J.C., Courrier, S., Ge´rard, F., Lemoine, M.C., Rousselet, J.L., Sommer, G., Garbaye, J., 2001. Survival in the soil of the ectomycorrhizal fungus Laccaria bicolor and the effects of a mycorrhiza helper Pseudomonas fluorescens. Soil Biology and Biochemistry 33, 1683–1694. Di Battista, C., Selosse, M.-A., Bouchard, D., Stenstro¨m, E., Le Tacon, F., 1996. Variations in symbiotic efficiency, phenotypic characters and ploidy level among different isolates of the ectomycorrhizal basidiomycete Laccaria bicolor strain S 238. Mycological Research 100, 1315–1324. Duponnois, R., Garbaye, J., 1990. Some mechanisms involved in growth stimulation of ectomycorrhizal fungi by bacteria. Canadian Journal of Botany 68, 2148–2152. Duponnois, R., Garbaye, J., 1991a. Mycorrhization helper bacteria associated with the Douglas fir-Laccaria laccata symbiosis: effects in

aseptic and in glasshouse conditions. Annales des Sciences Forestie`res 48, 239–251. Duponnois, R., Garbaye, J., 1991b. Effect of dual inoculation of Douglas fir with the ectomycorrhizal fungus Laccaria laccata and mycorrhization helper bacteria (MHB) in two bare root forest nurseries. Plant and Soil 138, 169–176. Duponnois, R., Garbaye, J., Bouchard, D., Churin, J.-L., 1993. The fungusspecificity of mycorrhization helper bacteria (MHBs) used as an alternative to soil fumigation for ectomycorrhizal inoculation of bareroot Douglas-fir planting stocks with Laccaria laccata. Plant and Soil 157, 257–262. Frey-Klett, P., Pierrat, J.-C., Garbaye, J., 1997. Location and survival of mycorrhiza helper Pseudomonas fluorescens during establishment of ectomycorrhizal symbiosis between Laccaria bicolor and Douglas fir. Applied and Environmental Microbiology 63, 139 –144. Frey-Klett, P., Churin, J.-L., Pierrat, J.-C., Garbaye, J., 1999. Dose effect in the dual inoculation of an ectomycorrhizal fungus and a mycorrhiza helper bacterium in two forest nurseries. Soil Biology and Biochemistry 31, 1555–1562. Garbaye, J., 1994. Tansley Review No 76. Helper bacteria: a new dimension to the mycorrhizal symbiosis. New Phytologist 128, 197 –210. Heinonsalo, J., Jørgensen, K., Sen, R., 2001. Microcosm-based analyses of Scots pine seedling growth, ectomycorrhizal fungal community structure and bacterial carbon utilization profiles in boreal forest humus and underlying illuvial mineral horizons. FEMS Microbiology Ecology 36, 73–84. King, E.O., Ward, M.K., Raney, D.E., 1954. Two simple media for the demonstration of pyocyanine and fluorescein. Journal of Laboratory and Clinical Medicine 44, 301–307. Le Tacon, F., Alvarez, I.F., Bouchard, D., Henrion, B., Jackson, R.M., Luff, S., Parlade, J.I., Pera, J., Stenstro¨m, E., Villeneuve, N., Walker, C., 1992. Variations in field response of forest trees to nursery ectomycorrhizal inoculation in Europe. In: Read, D.J., Lewis, D.H., Fitter, A.H., Alexander, I.J. (Eds.), Mycorrhizas in Ecosystems, CAB International, Oxford, pp. 119 –134. Le Tacon, F., Mousain, D., Garbaye, J., Bouchard, D., Churin, J.-L., Argillier, C., Amirault, J.-M., Ge´ne´re´, B., 1997. Mycorhizes, pe´pinie`res et plantations forestie`res en France. Revue Forestie`re Francaise no. sp, 131 –154. Molina, R., Chamard, J., 1983. Use of ectomycorrhizal fungus Laccaria laccata in forestry. II. Effects of fertilizer forms and levels on ectomycorrhizal development and growth of container-grown Douglas-fir and ponderosa pine seedlings. Canadian Journal of Forest Research 13, 89 –95. Mortier, F., Le Tacon, F., Garbaye, J., 1988. Effect of inoculum type and inoculation dose on ectomycorrhizal development, root necrosis and growth of Douglas fir seedlings inoculated with Laccaria laccata in a nursery. Annales des Sciences Forestie`res 45, 301 –310. Selosse, M.-A., Jacquot, D., Bouchard, D., Martin, F., Le Tacon, F., 1998. Temporal persistence and spatial distribution of an American strain of the ectomycorrhizal basidiomycete Laccaria bicolor in a French forest plantation. Molecular Ecology 7, 561–573. Stenstro¨m, E., Ek, M., Unestam, T., 1990. Variation in field response of Pinus sylvestris to nursery inoculation with four different ectomycorrhizal fungi. Canadian Journal of Forest Research 20, 1796–1803. Villeneuve, N., Le Tacon, F., Bouchard, D., 1991. Survival of inoculated Laccaria bicolor in competition with native ectomycorrhizal fungi and effects on the growth of outplanted Douglas-fir seedlings. Plant and Soil 135, 95–107.