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Dose effect in the dual inoculation of an ectomycorrhizal fungus and a mycorrhiza helper bacterium in two forest nurseries
Soil Biology and Biochemistry 31 (1999) 1555±1562
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Dose eect in the dual inoculation of an ectomycorrhizal fungus and a mycorrhiza helper bacterium in two forest nurseries Pascale Frey-Klett a,*, Jean-Louis Churin a, Jean-Claude Pierrat b, Jean Garbaye a a
INRA, Equipe de Microbiologie ForestieÁre, F54280, Champenoux, France Laboratoire de Sciences ForestieÁres, INRA-ENGREF, 14 rue Girardet, F54000, Nancy, France
b
Accepted 6 May 1999
Abstract We inoculated disinfected soil at two Douglas-®r bareroot forest nurseries with three doses (8 105, 8 107 and 8 109 cfu m ) of the rifampin-resistant mycorrhiza helper bacterium Pseudomonas ¯uorescens strain BBc6R8 and the ectomycorrhizal fungus Laccaria bicolor strain S238N. In one of the two nurseries, two doses of fungal inoculum (50 and 100 mg mÿ2 dry weight (DW) mycelium entrapped in alginate beads at the constant dose of 1 l mÿ2) were tested. For all bacterial treatments the density of P. ¯uorescens BBc6R8 in the soil, determined by dilution plating, dropped below the detection limit (10ÿ2 cfu gÿ1 DW soil) 2 weeks after inoculation. Fifteen weeks after inoculation, the introduced bacterium could be detected by enrichment only in the treatments inoculated with the highest bacterial dose. Two years after inoculation, P. ¯uorescens BBc6R8 could not be detected in the soil of any of the bacterial treatments. Five months after inoculation and sowing, bacterial inoculation signi®cantly increased the percentage of mycorrhizal short roots on plants inoculated with either low or high amounts of L. bicolor, in one of the two nurseries. The lowest bacterial dose increased mycorrhizal colonization from 45 to 70% in plants inoculated with the low amount of fungal inoculum, and from 64 to 77% in plants inoculated with the high amount of fungal inoculum. The lowest bacterial dose increased mycorrhizal colonization more than the highest bacterial dose. The same L. bicolor mycorrhizal index (70%) was obtained with 50 mg mÿ2 DW mycelium plus the bacterium than with twice this fungal dose and no bacterium (64%). Two years after inoculation, the height of the mycorrhizal Douglas-®rs in the other nursery was signi®cantly increased by the lowest bacterial dose (from 40.7 to 42.6 cm). These results indicate that co-inoculating a helper bacterium together with an ectomycorrhizal fungus can be an ecient way of optimizing controlled mycorrhization techniques for the production of highquality Douglas-®r planting stocks. They also con®rm that BBc6R8 acts at a low population density (less than 102 cfu gÿ1 soil); this contrasts with most PGPR eects discussed in the literature, where the minimal inoculation dose of 105 cfu gÿ1 soil is required to obtain the bene®cial eect. # 1999 Elsevier Science Ltd. All rights reserved. ÿ2
Keywords: Ectomycorrhizal fungus; Mycorrhizal helper bacterium; Dual inoculation; Dose eect
1. Introduction Inoculating trees with selected ectomycorrhizal fungal strains is an ecient way to improve the growth of forest seedlings in bare-root nurseries and plantations.
* Corresponding author. Tel.: +33-3-83-39-41-49; fax: +33-3-8339-40-69. E-mail address: [email protected] (P. Frey-Klett)
In France, the controlled mycorrhization of Douglas®r (Pseudotsuga menziesii (Mirbel)) Franco with Laccaria bicolor Maire P.D. Orton strain S238N is developing commercially following 10 years of experiments which con®rmed the positive eect of this fungal strain on plantation growth (Le Tacon et al., 1997). The success of fungal inoculation, i.e. fast and massive mycorrhizal establishment of the introduced fungal strain, depends on abiotic factors such as soil pH,
0038-0717/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 0 7 9 - 6
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fertility, moisture and temperature (Slankis, 1974). But it also depends on biotic factors such as soil microbial communities. Indeed, Garbaye and Bowen (1987) described how mycorrhizal infection of Pinus seedlings by three ectomycorrhizal fungi could be enhanced or impeded depending on the soil micro¯ora. Since then, the promoting eect of bacteria called Mycorrhiza Helper Bacteria (MHBs: Garbaye, 1994) on mycorrhizal establishment has been clearly demonstrated in a range of ectomycorrhizal associations (Garbaye and Bowen, 1989; RoÂzycki et al., 1994; Gagnon, 1996; Dunstan et al., 1998). Based on these results Garbaye et al. (1990) selected a mycorrhiza helper bacterial strain Pseudomonas ¯uorescens BBc6 to improve the eciency of L. bicolor S238N inoculation in French nurseries. Three types of bene®ts can be expected from using mycorrhiza helper bacteria when practising controlled mycorrhization in forest nurseries: (i) reduced need for soil fumigation due to speci®city between the fungus and MHBs (Duponnois et al., 1993); (ii) increased mycorrhizal colonization of the planting stock, and (iii) reduced quantity of fungal inoculum. The latter two only were addressed in our work, using the MHB Pseudomonas ¯uorescens BBc6 which was isolated from a sporocarp of L. bicolor S238N in a French Douglas®r plantation. This bacterial strain consistently promotes the mycorrhizal establishment of L. bicolor S238N on Douglas-®r, under gnotobiotic conditions as well as in glasshouse and nursery experiments (Duponnois and Garbaye, 1991, 1992). Frey-Klett et al. (1997a) used a spontaneous rifampin-resistant mutant BBc6R8, which phenotypically conforms to the parental strain BBc6, to show that the bacterium is not rhizospheric since its population in the soil is not increased by the presence of Douglas-®r roots. In addition, when BBc6R8 was inoculated at a rate of 107 cfu gÿ1 soil, the density of the bacterium quickly declined in the soil and in the rhizosphere of Douglas®r even though the bacterium signi®cantly promoted mycorrhizal establishment. Our aims were to determine the bacterial inoculation rate required to obtain optimal mycorrhiza formation in commercial mycorrhization of Douglas-®r with L. bicolor S238N, to monitor the behaviour of BBc6R8 in the soil and to determine whether responses to bacterial inoculation are in¯uenced by the amount of mycorrhizal fungus inoculum.
2. Material and methods 2.1. Seeds and soil Douglas-®r
seeds
from
provenance
zone
412
(Washington state, USA) were strati®ed in moist peat at 48C for about 1 month to break dormancy. In the two bare-root forest nurseries, Champenoux (eastern France) and Peyrat-le-ChaÃteau (central France), benches were ®lled with soil from the Peyratle-ChaÃteau nursery and disinfected 1 month before sowing and inoculating, in order to suppress competing resident symbionts. Soil was steam disinfected (908C at a 30 cm depth for 2 h) in Champenoux and fumigated with methyl bromide (100 g mÿ2 at 158C under a clear plastic ®lm, for 1 week) in Peyrat-leChaÃteau. 2.2. Microbial cultures and inoculum preparation The ectomycorrhizal basidiomycete L. bicolor S238N (Di Battista et al., 1996) was maintained on Pachlewsky agar medium (Pachlewski and Pachlewska, 1974). Two types of inocula were prepared. For the Champenoux nursery, the mycelium was aseptically grown in a peat±vermiculite mix as described by Duponnois and Garbaye (1991), resulting in an inoculum containing about 1 g mycelium (dry weight) lÿ1 (Mortier et al., 1988). For the Peyrat-le-ChaÃteau nursery, alginate beads containing L. bicolor mycelium (Mortier et al., 1988) were prepared to achieve a ®nal concentration of 50 and 100 mg (DW) mycelium lÿ1 inoculum. The bacterial strain BBc6R8 (Frey-Klett et al., 1997a) belongs to the biovar l of the Pseudomonas ¯uorescens species according to the BIOLOG1 identi®cation system (P. Frey-Klett, unpub. Ph.D thesis, Paris-Sud University, 1996). The BBc6R8 inoculum was prepared by growing the bacteria on King's B (KB) agar medium (King et al., 1954) with 100 mg rifampin lÿ1 at 258C for 36 h. The bacteria were then suspended in 0.1 M MgSO4 buer, washed twice, and resuspended in tap water at three dierent bacterial densities: 1.6 102 (low), 1.6 104 (medium), 1.6 106 (high) cfu mlÿ1 inoculum. Bacterial density was measured as the absorbance of the suspension at 600 nm, with reference to a standard curve calibrated by plate enumeration. 2.3. Inoculation and sowing procedures Inoculation and sowing were performed in May in both nurseries. In the Champenoux nursery, 0.5 l mÿ2 of the peat±vermiculite fungal inoculum was mixed into the top 10 cm of soil with a hand tool. In the Peyrat-le-ChaÃteau nursery, 1 l mÿ2 of the alginate inoculum beads were mixed the same way into the soil. Then in both nurseries, 5 l mÿ2 of the bacterial inoculum was applied on the surface of the soil with a watering can and the controls (fungal inoculum without bacteria) received only water. Before sowing, the
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inoculated soil was mixed a second time to a depth of 5±10 cm. In the Champenoux nursery, strati®ed Douglas-®r seeds were then sown in rows with one seed every 2 cm for a global density of about 1000 seeds mÿ2. In the Peyrat-le-ChaÃteau nursery, seeds were broadcast spread at a density of 1500 seeds mÿ2. After sowing, seeds were covered with disinfected soil. 2.4. Plant growth conditions and experimental designs After seed germination, the nursery benches at both nurseries were shaded. In Champenoux only, natural precipitation was complemented with spray irrigation. In Champenoux, the bench was divided into 10 blocks, each containing four 0.5 m2 (1 0.5 m) plots isolated from each other by water-tight, 40 cm deep partitions. In each block, four microbial treatments of one plot each were compared: fungus alone (at the rate of about 0.5 g DW mycelium mÿ2), fungus plus low (8 105 cfu mÿ2 ), medium (8 107 cfu mÿ2) or high (8 109 cfu mÿ2) bacterial inoculation dose. In Peyrat-le-ChaÃteau, the experimental design was four blocks distributed into two parallel nursery benches and containing eight 1 m2 (2 0.5 m) plots which alternated with non-inoculated, unsown 2 m2 (2 1 m) buer plots; there were no partitions as in Champenoux. In each block, eight microbial treatments of one plot each were compared: two fungal inoculation doses (50 and 100 mg DW mycelium mÿ2) combined with four bacterial inoculation doses (0, 8 105, 8 107 and 8 109 cfu mÿ2). However, summer drought ruined some of the treatments inoculated with the high fungal dose in two blocks, located at the west extremity of the two benches and exposed to direct sunlight. 2.5. Population dynamics of P. ¯uorescens BBc6R8 Soil cores (150 cm3) were sampled in all treatments with a steel tube (12 cm long, 4 cm dia) centered on a seedling. In the Champenoux nursery, one sample per plot was taken 11 times during the whole duration of the experiment (three growing seasons). All root pieces were removed from the soil cores by sieving and homogenized before bacterial quanti®cation. In the Peyratle-ChaÃteau nursery, four cores per plot were sampled at the end of the experiment, after the ®rst growing season. Soil cores were pooled by plot, root pieces removed from cores, and homogenized before bacterial quanti®cation. For the dilution-plating quanti®cation, about 1 g of soil was subsampled, weighed and suspended in 3 ml of TS buer (TRIS, 2.42 g; NaCl, 8 g; distilled water, 1 l; pH 7.5). The samples were then vigorously homogenized for 1 min. Crude suspensions and appropriate dilutions were plated for all samples in duplicate on
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the selective medium of Simon and Ridge (1974) containing 100 mg lÿ1 of propiconazole (Tilt 125, a fungicide from Ciba), to quantify total ¯uorescent pseudomonad populations, and in triplicate on KB medium containing 100 mg rifampin lÿ1 and 100 mg propiconazole lÿ1 to quantify BBc6R8 populations. Bacterial colonies were counted after incubation at 258C for 48 h. When bacterial populations were below the detection limit (100 cfu g DWÿ1 soil) of the dilution-plating method, 0.5 g soil samples were enriched by shaking in 3 ml of KB-rifampin (100 mg lÿ1) medium at 278C for 42 h. The presence of BBc6R8 was then checked by culturing 50 ml of the enriched culture on KB-rifampin plates and detecting ¯uorescent colonies under UV radiation. 2.6. Assessment of mycorrhiza formation The mycorrhizal index (percentage of short roots mycorrhizal with L. bicolor ) was determined by randomly examining 100 short roots per 150 cm3 core sample with a stereomicroscope; L. bicolor ectomycorrhizae can be distinguished, on morphological bases, from those due to resident fungi such as Thelephora terrestris or Rhizopogon sp. In Champenoux, one core sample was randomly taken in each plot at the end of the ®rst and of the third growing seasons. In Peyrat-leChaÃteau, at the end of the ®rst growing season, three cores were randomly sampled in the plots of the four treatments inoculated with 50 mg DW mÿ2 of L. bicolor, and ®ve cores were taken per plot in the treatments with 100 mg DW mÿ2 of L. bicolor. 2.7. Eect on plant growth In Champenoux, all the plants were lifted and counted at the end of the third growing season and the shoot lengths of all the seedlings were measured. Then, all shoots and roots from each plot were dried and weighed together to calculate the mean shoot and root dry weights per plant and per plot. 2.8. Data analysis All of the statistical analyses were done at the probability level of 0.05 using the StatView1 program and all mycorrhizal index data were arcsin transformed before analysis. Data from the Peyrat-le-ChaÃteau nursery were analysed in a three-factor (bacterial dose, fungal dose, block) ANOVA with interactions (unbalanced incomplete block design). Two treatments (the control treatment inoculated with 100 mg DW mycelium mÿ2 and the treatment inoculated with 8 105 cfu mÿ2 BBcR8 bacteria and 50 mg DW mycelium mÿ2) were compared by the contrast method. Two separate two-fac-
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Table 1 Douglas-®r plants in the Champenoux nursery: dose eect of P. ¯uorescens BBc6R8 on the growth (shoot length and dry weight) after three growing seasons (gr.s.) and mycorrhizal index (percentage of short roots mycorrhizal with L. bicolor ) after one and two growing seasonsa Myc. Index (%) Bacterial dose (cfu mÿ2)
Shoot length (cm)
Root dry weight (g)
Shoot dry weight (g)
Total dry weight (g)
1 gr.s.
3 gr.s.
0 (Control) 8 105 (low) 8 107 (medium) 8 109 (high)
40.7 42.5 42.6 40.7
1.36 1.64 1.48 1.36
3.73 3.97 3.94 3.89
5.09 5.61 5.42 5.25
58 69 67 65
6 9 7 8
a Shoot length values correspond to the means of all plants in the 10 plots of each treatment (about 2800 plants per treatment); these values were analysed with a two-factor ANOVA (bacterial dose, block, P = 0.05, with individual plants as replicates in order to maximize precision). All shoots or roots in each plot were dried and weighed together, to calculate the mean shoot and root dry weights per plant for each plot; these values were analysed with a one-factor ANOVA (bacterial dose, P = 0.05, with blocks as replicates). Mycorrhizal index data were arcsin transformed before a two-factor ANOVA (bacterial dose, block, P = 0.05). In each column, means with an asterisk are signi®cantly dierent from the control ones according to the Bonferoni±Dunn test (P = 0.05).
tor ANOVA (bacterial dose, block) were also performed on the data corresponding to each fungal dose (balanced complete block design) and the dierent means were compared two by two by a Schee test. For the Champenoux nursery, shoot length data were analysed with a two-factor (bacterial dose, block) ANOVA and dry weight values were analysed with a one-factor ANOVA (bacterial dose). All the means were compared to the control ones by a Bonferoni± Dunn test.
3. Results 3.1. Dose eect of P. ¯uorescens BBc6R8 in the Champenoux nursery 3.1.1. Mycorrhizal status of the seedlings At the end of the ®rst growing season, the number of short roots colonized by L. bicolor was higher on plants inoculated with bacteria, but this dierence was not statistically signi®cant at P = 0.05 (actual
Fig. 1. Population dynamics of P. ¯uorescens BBc6R8 and total ¯uorescent pseudomonads in the soil of the Champenoux nursery, inoculated with three dierent doses of bacterial inoculum: low (8 105 cfu mÿ2), medium (8 107 cfu mÿ2) and high (8 109 cfu mÿ2). The bacterial populations were quanti®ed by the dilution-plating method. Each point represents the mean value of ten soil cores treatment (one core per plot 10 blocks). The bars are the standard deviations. Some points are below the detection limit because of nil values contributing to the mean.
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3.1.3. Survival of the introduced bacterium in the soil The population density of the introduced bacterium in the soil was above the detection limit of the dilution-plating method only with the highest inoculation dose (8 109 cfu mÿ2) and it rapidly declined after inoculation, whereas the population density of the total ¯uorescent pseudomonads remained stable (Fig. 1). With the enrichment method, BBc6R8 bacterial cells were detected 15 weeks after inoculation with the highest bacterial dose, whereas they were only detected during the ®rst 3 weeks after inoculation in the case of the two lowest bacterial doses (8 105 and 8 107 cfu mÿ2) (Fig. 2). Using the enrichment method of detection, no BBc6R8 bacterial cells were detected in the soil at the end of the third growing period, for any of the bacterial treatments.
Fig. 2. Detection of BBc6R8 bacterial cells in the soil of the Champenoux nursery, inoculated with three dierent doses of bacterial inoculum: low (8 105 cfu mÿ2), medium (8 107 cfu mÿ2) and high (8 109 cfu mÿ2). Each point represents the proportion (expressed in %) of the positive enrichments among 10 soil cores per treatment (one core per plot 10 blocks).
P = 0.07). The amount of colonization was independent of the bacterial inoculation dose (Table 1). At the end of the third growing season, the L. bicolor mycorrhizal index was low for all treatments (Table 1).
3.1.2. Seedling growth Bacterial inoculation at the two lowest doses (8 105 and 8 107 cfu mÿ2) signi®cantly increased the shoot length of the Douglas-®r plants at the end of the third growing season (Table 1). The lowest dose of bacterial inoculum also signi®cantly increased the root dry weight. However, bacterial inoculation had no signi®cant eect on the shoot and the total dry weights of the plants.
3.2. Dose eect of P. ¯uorescens BBc6R8 in the Peyratle-ChaÃteau nursery 3.2.1. Mycorrhizal status of the seedlings at the end of the ®rst growing season The three-factor (bacterial dose, fungal dose, block) revealed a signi®cant eect of the bacterial dose (P = 0.0001), of the fungal dose (P = 0.03), of the block factor (P = 0.04) and no interaction, on the L. bicolor mycorrhizal index of Douglas-®r. The lower the bacterial dose, the higher the mycorrhizal helper eect. Moreover, in the treatment inoculated with 50 mg DW mycelium mÿ2 of L. bicolor and the lowest bacterial dose (8 105 cfu mÿ2), the mycorrhizal index (70%) was the same as the mycorrhizal index in the control inoculated with 100 mg DW mycelium mÿ2 (64%, P = 0.06). When plants were not inoculated with bacteria but the fungal dose increased from 50 to 100 mg DW mycelium mÿ2, the mycorrhizal index of L. bicolor on the plants increased from 45 to 64% (Table 2). When plants were inoculated with 50 mg DW mycelium mÿ2, the mycorrhizal index of L. bicolor on the plants increased with bacterial inoculation; but the bacterial
Table 2 Dose eect of P. ¯uorescens BBc6R8 on the percentage of Douglas-®r short roots mycorrhizal with L. bicolor, in the Peyrat-le-ChaÃteau nursery, 23 weeks after inoculationa Bacterial inoculation dose (cfu mÿ2) Fungal inoculation dose (mg DW mycelium mÿ2)
0 (control)
8 105 (low)
8 107 (medium)
8 109 (high)
50 100
45a 64a
70c 77b
62bc 70ab
51ab 64ab
a The values correspond to the means of 10 (treatments inoculated with 100 mg DW mycelium mÿ2) or 12 (treatments inoculated with 50 mg DW mycelium mÿ2) replicates. The percentages transformed by arcsin (square root) were analysed with a two-factor ANOVA (bacterial dose, block; P = 0.05) with interaction. A signi®cant eect of the bacterial dose, no eect of the block factor and no interaction were found by the F test. In each line, values with the same letters are not signi®cantly dierent according to a Schee test.
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eect was only signi®cant with the low and medium bacterial inoculation treatments (Table 2). For plants inoculated with 100 mg DW mycelium mÿ2, only the low bacterial inoculation treatment signi®cantly increased the mycorrhizal index of L. bicolor (Table 2). Fig. 3 shows that a negative relationship exists between the bacterial inoculation dose and the mycorrhizal helper eect (expressed as the dierence between the average mycorrhizal index with or without bacterial inoculation, and excepting the bacterial inoculation rate of 0). Lower bacterial inoculation doses were more eective at increasing mycorrhizal colonization than higher doses. This negative relationship was only signi®cant for plants inoculated at the high rate of L. bicolor inoculation (100 mg DW mycelium mÿ2). 3.2.2. Survival of the introduced bacterium in the soil The population of the introduced bacterium in the soil was analysed at the end of the ®rst growing season. Bacterial densities were below the detection limit of the dilution-plating method. With the enrichment method, the bacterium was detected in 75% of the soil samples collected in the treatment inoculated with the highest dose of bacterial inoculum, but not in the other treatments.
Fig. 3. Relationship between the absolute percent mycorrhizal increase (dierence between the average mycorrhizal indexes with and without BBc6R8) and the logarithm of the bacterial inoculation dose, in the Peyrat-le-ChaÃteau nursery, 23 weeks after inoculation. Regression analysis reveals a signi®cant relationship (shown by a dotted line) in the case of the highest fungal inoculation dose (Y = 30.781ÿ3.023X (r 2=0.998), P = 0.0276).
4. Discussion These results con®rm the ecacy of a mycorrhiza helper Pseudomonas ¯uorescens in enhancing the eect of ectomycorrhizal inoculation in bare-root forest nurseries, in terms of increased mycorrhiza formation and enhanced seedling growth. The latter eect, which can occur without the former (i.e. increased mycorrhiza formation) as shown in the Champenoux experiment, is consistent with other observations of our group in the same nursery (unpublished data) and of R. Duponnois (unpub. Ph.D thesis, Nancy University 1992). In all these cases, BBc6R8, or the wild strain BBc6 from which it was derived signi®cantly promoted Douglas-®r seedling growth when associated with L. bicolor, even without enhancing mycorrhizal establishment. This suggests that the bacteria might increase the symbiotic eciency of mycorrhizas. But the central result of our work is that, within the range of bacterial inoculation doses from 106 to 109 cfu mÿ2 (i.e. about 10 to 104 cfu cmÿ3 of soil only), the lowest doses were the most ecient ones. No bacterial inoculation must obviously have a nil eect on mycorrhiza formation; hence the negative dose±response we observed means that the response curve to bacterial dose will have a maximum. Therefore, under our experimental conditions, the doses we used were clearly super-optimal even though they were low. This result is consistent with the ®ndings of Duponnois and Garbaye (1992) who used the same organisms under similar nursery conditions. The eects we observed of bacterial inoculation on seedling growth are in agreement with C. Karabaghli (unpub. Ph.D thesis, Nancy University (ENGREF) 1997) who found that high concentrations of BBc6 were detrimental to the roots of spruce (Picea abies L.) seedlings. However, our results contrast markedly with most results with PGPRs used as rhizosphere biocontrol agents, where a minimal inoculation dose of 105 cfu gÿ1 soil is required for the bene®cial eect, and where increasing the inoculation dose generally increases plant protection (Bull et al., 1991; Raaijmakers et al., 1995). With other PGPR strains, some detrimental eects on root growth were also observed with high inoculation doses such as 108 cfu mlÿ1 (Kapulnik et al., 1985; Bashan, 1986). In our system, the eciency of the bacterial inoculation disappears when the dose of the bacterial inoculum increases. We can hypothesize that the bacterium exerts two opposite eects on plant growth: some detrimental ones toward the plant or the fungus when it is present at high densities (e.g. antibiosis or competition for nutrients), and some bene®cial ones which are independent of the cell density (such eects have not yet been determined). We found no interaction between the dose of bacterial inoculum and the amount of fungus inoculation.
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For the two fungal doses we tested, the success of the bacterial inoculation was independent of the amount of fungus inoculum used. This is dierent from some biocontrol-PGPR strains for which the bacterial eciency depends on the disease intensity and thus on the pathogen density (Raaijmakers et al., 1995). A positive dose±response relationship is generally attributed to a better colonization of the rhizosphere by the introduced microorganism (Raaijmakers et al., 1995), leading to a larger population which produces more of the eective substances (e.g. antibiotics or siderophores) either directly, because the cells are more numerous, or indirectly through quorum-sensing mechanisms within high-density micro-colonies (Chin-AWoeng et al., 1997). In our study, the dose±response relationship was negative and, with the two detection methods used, the BBc6R8 populations decreased in the soil and even in the rhizosphere after inoculation (data not shown). Since, during the same period, the number of total ¯uorescent pseudomonads remained stable, we believe that the equilibrium level of BBc6R8 is normally very low in this type of ecosystem, independent of occasional, adverse environmental factors. Two hypotheses could account for the ecacy of BBc6R8 at very low apparent population densities: (i) A large population of such bacteria exist and is metabolically active in the soil but not detected by plating because the cells are viable but non-culturable (Bloom®eld et al., 1998; Kell et al., 1998; McDougald et al., 1998) or because they occupy special niches which are not accessible to the standard extraction technique using homogenization, and (ii) The population is low but active micro-colonies (Chin-A-Woeng et al., 1997) are localized in target sites, such as walls of the fungal hyphae, where the bacteria deliver active substances. Li and Hung (1987) have shown that small populations of nitrogen-®xing bacteria were associated with the inner mantle of some ectomycorrhizas of Douglas-®r in the native area of this tree species. In our case, Frey-Klett et al. (1997b) reported that BBc6R8 bene®ts from the presence of L. bicolor mycelium in the soil and Sen et al. (1996) showed that it attaches to L. bicolor in vitro. However, BBc6R8 does not produce known N-acylhomoserine-lactones (Y. Dessaux, pers. comm.) which are needed in the synthesis of many density-dependent secondary metabolites (Swift et al., 1996). As BBc6 is Laccaria-speci®c (Garbaye and Duponnois, 1992), it can also be hypothesized that some bacterial cells are inside the hyphae, as demonstrated in the case of arbuscular endomycorrhizal fungi (Bianciotto et al., 1996; Perotto and Bonfante, 1997). As a consequence, we believe that the L. bicolor±P. ¯uorescens system presents some unusual traits which are worth studying to unveil new rhizosphere mechanisms. From the practical point of view of nursery manage-
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ment and controlled mycorrhization of forest planting stocks, bacterial inoculation at sowing is easily applied to nursery treatments as stressed by Chanway (1997). Furthermore, our results clearly show that adding a mycorrhiza helper bacterial strain to the fungal inoculum allows for the reduction of mycorrhizal inoculum while obtaining the same mycorrhizal index. Therefore, the inoculation cost could be minimized since bacteria are much easier and faster to grow than ectomycorrhizal fungi, and because our results show that a very small amount of bacteria is enough to increase plant response. Acknowledgements We are grateful to G. Catroux for reviewing the manuscript. We greatly appreciated the technical assistance of M.L. Clausse, S. Kotowski, J.L. Rousselet and E. Sidot. We also thank R. Molina and J.M. Trappe (Corvallis, Oregon) for providing strain S238, from which S238N was derived. References Bashan, Y., 1986. Signi®cance of timing and level of inoculation with rhizosphere bacteria on wheat plants. Soil Biology and Biochemistry 18, 297±301. Bianciotto, V., Bandi, C., Minerdi, D., Sironi, M., Tichy, H.V., Bonfante, P., 1996. An obligate endosymbiotic mycorrhizal fungus itself harbors obligately intracellular bacteria. Applied and Environmental Microbiology 62, 3005±3010. Bloom®eld, S.F., Stewart, G.S.A.B., Dodd, C.E.R., Booth, I.R., Power, E.G.M., 1998 The viable but non-culturable phenomenon explained?, Microbiology 1±3. Bull, C.T., Weller, D.M., Thomashow, L.S., 1991. Relationship between root colonization and suppression of Gaeumannomyces graminis var tritici by Pseudomonas ¯uorescens and P. putida. Phytopathology 81, 954±959. Chanway, C.P., 1997. Inoculation of tree roots with plant growth promoting soil bacteria: An emerging technology for reforestation. Forest Science 43, 99±112. Chin-A-Woeng, T.F.C., de Priester, W., van der Bij, A.J., Lugtenberg, B.J.J., 1997. Description of the colonization of a gnotobiotic tomato rhizosphere by Pseudomonas ¯uorescens biocontrol strain WC365, using scanning electron microscopy. Molecular Plant±Microbe Interactions 10, 79±86. Di Battista, C., Selosse, M.A., Bouchard, D., StenstroÈm, E., Le Tacon, F., 1996. Variations in symbiotic eciency, phenotypic characters and ploidy level among dierent isolates of the ectomycorrhizal basidiomycete Laccaria bicolor strain S238. Mycological Research 100, 1315±1324. Dunstan, W.A., Malajczuk, N., Dell, B., 1998. Eects of bacteria on mycorrhizal development and growth of container grown Eucalyptus diversicolor F. Muell seedlings. Plant and Soil 201, 243±251. Duponnois, R., Garbaye, J., 1991. Mycorrhization helper bacteria associated with the Douglas-®r-Laccaria laccata symbiosis: Eects in aseptic and in glasshouse conditions. Annales des Sciences ForestieÁres 48, 239±251. Duponnois, R., Garbaye, J., 1992. Application des BAM (bacteÂries
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auxiliaires de la mycorhization) aÁ l'inoculation du Douglas par Laccaria laccata S238 en peÂpinieÁre forestieÁre. Revue ForestieÁre Franc° aise 44, 491±500. Duponnois, R., Garbaye, J., Bouchard, D., Churin, J.L., 1993. The fungus-speci®city of mycorrhization helper bacteria (MHBs) used as an alternative to soil fumigation for ectomycorrhizal inoculation of bare-root Douglas-®r planting stocks with Laccaria laccata. Plant and Soil 157, 257±262. Frey-Klett, P., Pierrat, J.C., Garbaye, J., 1997a. Location and survival of mycorrhizal helper Pseudomonas ¯uorescens during establishment of ectomycorrhizal symbiosis between Laccaria bicolor and Douglas-®r. Applied and Environmental Microbiology 63, 139±144. Frey-Klett, P., BruleÂ, C., Garbaye, J., 1997b. A proposed model for the mycorrhiza helper eect of Pseudomonas ¯uorescens BBc6 on the ectomycorrhizal symbiotic system Laccaria bicolor S238NDouglas-®r. In: Ogoshi, A., Kobayashi, K., Homma, Y., Kodama, F., Kondo, N., Akino, S. (Eds.), Proceedings of the Fourth International Workshop on Plant-Growth-Promoting Rhizobacteria. Sapporo, p. 399. Gagnon, J., 1996. Stimulation of mycorhization and growth for containerized Jack Pine seedlings inoculated with Lacaria bicolor and Pseudomonas ¯uorescens. In: AzcoÂn, C., Barea, J.M. (Eds.), Mycorhizas in integrated systems from genes to plant development. European Commission EUR 16728 EN, pp. 634±637. Garbaye, J., 1994. Helper bacteria: A new dimension to the mycorrhizal symbiosis. New Phytologist 128, 197±210. Garbaye, J., Bowen, G.D., 1987. Eect of dierent micro¯ora on the success of ectomycorrhizal inoculation of Pinus radiata. Canadian Journal of Forest Research 17, 941±943. Garbaye, J., Bowen, G.D., 1989. Stimulation of ectomycorrhizal infection of Pinus radiata by some microorganisms associated with the mantle of ectomycorrhizas. New Phytologist 112, 383± 388. Garbaye, J., Duponnois, R., 1992. Speci®city and function of mycorrhization helper bacteria (MHB) associated with the Pseudotsuga menziesii±Laccaria laccata symbiosis. Symbiosis 14, 335±344. Garbaye, J., Duponnois, R., Wahl, J.L., 1990. The bacteria associated with Laccaria laccata ectomycorrhizas or sporocarps: Eect on symbiosis establishment on Douglas-®r. Symbiosis 9, 267±273. Kapulnik, Y., Okon, Y., Henis, Y., 1985. Changes in root morphology of wheat caused by Azospirillum inoculation. Canadian Journal of Microbiology 31, 881±887. Kell, D.B., Kaprelyants, A.S., Weichart, D.H., Harwood, C.R., Barer, M.R., 1998. Viability and activity in readily culturable bacteria: A review and discussion of the practical issues. Antonie van Leeuwenhoek 73, 169±187.
King, E.O., Ward, M.K., Raney, D.E., 1954. Two simple media for the demonstration of pyocyanine and ¯uorescein. Journal of Laboratory and Clinical Medicine 44, 301±307. Le Tacon, F., Mousain, D., Garbaye, J., Bouchard, B., Churin, J.L., Argillier, C., Amirault, J.M., GeÂneÂreÂ, B., 1997. Mycorhizes, peÂpinieÁres et plantations forestieÁres en France. Revue ForestieÁre Franc° aise 49, 131±154. Li, C.Y., Hung, L.L., 1987. Nitrogen-®xing (aceteylene-reducing) bacteria associated with ectomycorrhizas of Douglas-®r. Plant and Soil 98, 425±428. McDougald, D., Rice, S.A., Weichart, D., Kjelleberg, S., 1998. Nonculturability: Adaptation or debilitation? FEMS Microbiology Ecology 25, 1±9. Mortier, F., Le Tacon, F., Garbaye, J., 1988. Eect of inoculum type and inoculation dose on ectomycorrhizal development, root necrosis and growth of Douglas ®r seedlings inoculated with Laccaria laccata in a nursery. Annales des Sciences ForestieÁres 45, 301±310. Pachlewski, R., Pachlewska, J., 1974. Studies on symbiotic properties of mycorrhizal fungi of pine (Pinus sylvestris ) with the aid of the method of mycorrhizal synthesis in pure culture on agar. Forest Research Institute, Warsaw. Perotto, S., Bonfante, P., 1997. Bacterial associations with mycorrhizal fungi: Close and distant friends in the rhizosphere. Trends in Microbiology 5, 496±501. Raaijmakers, J.M., Leeman, M., van Oorschot, M.M.P., van der Sluis, I., Schippers, B., Bakker, A.H.M., 1995. Dose±response relationships in biological control of fusarium wilt of radish by Pseudomonas spp. Phytopathology 85, 1075±1081. RoÂzycki, H., Kampert, M., Strzelczyk, E., Li, C.Y., Perry, D.A., 1994. Eect of dierent soil bacteria on mycorrhizae formation in Scots pine (Pinus sylvestris L.) in vitro studies. Folia Forestalia Polonica 36, 92±102. Sen, R., Nurmiaho-Lassila, E.L., Haahtela, K., Korhonen, T., 1996. Attachment of Pseudomonas ¯uorescens strains to the cell walls of ectomycorrhizal fungi. In: AzcoÂn-Aguilar, C., Barea, J.M. (Eds.), Mycorhizas in integrated systems from genes to plant development. European Commission EUR 16728 EN, pp. 634±637. Simon, A., Ridge, E.H., 1974. The use of ampicillin in a simpli®ed selective medium for the isolation of ¯uorescent Pseudomonads. Journal of Applied Bacteriology 37, 459±460. Slankis, V., 1974. Soil factors in¯uencing formation of mycorrhizae. Annual Review of Phytopathology 12, 437±457. Swift, S., Throup, J.P., Williams, P., Salmond, G.P.C., Stewart, S.A.B., 1996 Quorum sensing: A population-density component in the determination of bacterial phenotype. Trends in Biochemical Sciences 214±219.
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Dose eect in the dual inoculation of an ectomycorrhizal fungus and a mycorrhiza helper bacterium in two forest nurseries Pascale Frey-Klett a,*, Jean-Louis Churin a, Jean-Claude Pierrat b, Jean Garbaye a a
INRA, Equipe de Microbiologie ForestieÁre, F54280, Champenoux, France Laboratoire de Sciences ForestieÁres, INRA-ENGREF, 14 rue Girardet, F54000, Nancy, France
b
Accepted 6 May 1999
Abstract We inoculated disinfected soil at two Douglas-®r bareroot forest nurseries with three doses (8 105, 8 107 and 8 109 cfu m ) of the rifampin-resistant mycorrhiza helper bacterium Pseudomonas ¯uorescens strain BBc6R8 and the ectomycorrhizal fungus Laccaria bicolor strain S238N. In one of the two nurseries, two doses of fungal inoculum (50 and 100 mg mÿ2 dry weight (DW) mycelium entrapped in alginate beads at the constant dose of 1 l mÿ2) were tested. For all bacterial treatments the density of P. ¯uorescens BBc6R8 in the soil, determined by dilution plating, dropped below the detection limit (10ÿ2 cfu gÿ1 DW soil) 2 weeks after inoculation. Fifteen weeks after inoculation, the introduced bacterium could be detected by enrichment only in the treatments inoculated with the highest bacterial dose. Two years after inoculation, P. ¯uorescens BBc6R8 could not be detected in the soil of any of the bacterial treatments. Five months after inoculation and sowing, bacterial inoculation signi®cantly increased the percentage of mycorrhizal short roots on plants inoculated with either low or high amounts of L. bicolor, in one of the two nurseries. The lowest bacterial dose increased mycorrhizal colonization from 45 to 70% in plants inoculated with the low amount of fungal inoculum, and from 64 to 77% in plants inoculated with the high amount of fungal inoculum. The lowest bacterial dose increased mycorrhizal colonization more than the highest bacterial dose. The same L. bicolor mycorrhizal index (70%) was obtained with 50 mg mÿ2 DW mycelium plus the bacterium than with twice this fungal dose and no bacterium (64%). Two years after inoculation, the height of the mycorrhizal Douglas-®rs in the other nursery was signi®cantly increased by the lowest bacterial dose (from 40.7 to 42.6 cm). These results indicate that co-inoculating a helper bacterium together with an ectomycorrhizal fungus can be an ecient way of optimizing controlled mycorrhization techniques for the production of highquality Douglas-®r planting stocks. They also con®rm that BBc6R8 acts at a low population density (less than 102 cfu gÿ1 soil); this contrasts with most PGPR eects discussed in the literature, where the minimal inoculation dose of 105 cfu gÿ1 soil is required to obtain the bene®cial eect. # 1999 Elsevier Science Ltd. All rights reserved. ÿ2
Keywords: Ectomycorrhizal fungus; Mycorrhizal helper bacterium; Dual inoculation; Dose eect
1. Introduction Inoculating trees with selected ectomycorrhizal fungal strains is an ecient way to improve the growth of forest seedlings in bare-root nurseries and plantations.
* Corresponding author. Tel.: +33-3-83-39-41-49; fax: +33-3-8339-40-69. E-mail address: [email protected] (P. Frey-Klett)
In France, the controlled mycorrhization of Douglas®r (Pseudotsuga menziesii (Mirbel)) Franco with Laccaria bicolor Maire P.D. Orton strain S238N is developing commercially following 10 years of experiments which con®rmed the positive eect of this fungal strain on plantation growth (Le Tacon et al., 1997). The success of fungal inoculation, i.e. fast and massive mycorrhizal establishment of the introduced fungal strain, depends on abiotic factors such as soil pH,
0038-0717/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 0 7 9 - 6
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fertility, moisture and temperature (Slankis, 1974). But it also depends on biotic factors such as soil microbial communities. Indeed, Garbaye and Bowen (1987) described how mycorrhizal infection of Pinus seedlings by three ectomycorrhizal fungi could be enhanced or impeded depending on the soil micro¯ora. Since then, the promoting eect of bacteria called Mycorrhiza Helper Bacteria (MHBs: Garbaye, 1994) on mycorrhizal establishment has been clearly demonstrated in a range of ectomycorrhizal associations (Garbaye and Bowen, 1989; RoÂzycki et al., 1994; Gagnon, 1996; Dunstan et al., 1998). Based on these results Garbaye et al. (1990) selected a mycorrhiza helper bacterial strain Pseudomonas ¯uorescens BBc6 to improve the eciency of L. bicolor S238N inoculation in French nurseries. Three types of bene®ts can be expected from using mycorrhiza helper bacteria when practising controlled mycorrhization in forest nurseries: (i) reduced need for soil fumigation due to speci®city between the fungus and MHBs (Duponnois et al., 1993); (ii) increased mycorrhizal colonization of the planting stock, and (iii) reduced quantity of fungal inoculum. The latter two only were addressed in our work, using the MHB Pseudomonas ¯uorescens BBc6 which was isolated from a sporocarp of L. bicolor S238N in a French Douglas®r plantation. This bacterial strain consistently promotes the mycorrhizal establishment of L. bicolor S238N on Douglas-®r, under gnotobiotic conditions as well as in glasshouse and nursery experiments (Duponnois and Garbaye, 1991, 1992). Frey-Klett et al. (1997a) used a spontaneous rifampin-resistant mutant BBc6R8, which phenotypically conforms to the parental strain BBc6, to show that the bacterium is not rhizospheric since its population in the soil is not increased by the presence of Douglas-®r roots. In addition, when BBc6R8 was inoculated at a rate of 107 cfu gÿ1 soil, the density of the bacterium quickly declined in the soil and in the rhizosphere of Douglas®r even though the bacterium signi®cantly promoted mycorrhizal establishment. Our aims were to determine the bacterial inoculation rate required to obtain optimal mycorrhiza formation in commercial mycorrhization of Douglas-®r with L. bicolor S238N, to monitor the behaviour of BBc6R8 in the soil and to determine whether responses to bacterial inoculation are in¯uenced by the amount of mycorrhizal fungus inoculum.
2. Material and methods 2.1. Seeds and soil Douglas-®r
seeds
from
provenance
zone
412
(Washington state, USA) were strati®ed in moist peat at 48C for about 1 month to break dormancy. In the two bare-root forest nurseries, Champenoux (eastern France) and Peyrat-le-ChaÃteau (central France), benches were ®lled with soil from the Peyratle-ChaÃteau nursery and disinfected 1 month before sowing and inoculating, in order to suppress competing resident symbionts. Soil was steam disinfected (908C at a 30 cm depth for 2 h) in Champenoux and fumigated with methyl bromide (100 g mÿ2 at 158C under a clear plastic ®lm, for 1 week) in Peyrat-leChaÃteau. 2.2. Microbial cultures and inoculum preparation The ectomycorrhizal basidiomycete L. bicolor S238N (Di Battista et al., 1996) was maintained on Pachlewsky agar medium (Pachlewski and Pachlewska, 1974). Two types of inocula were prepared. For the Champenoux nursery, the mycelium was aseptically grown in a peat±vermiculite mix as described by Duponnois and Garbaye (1991), resulting in an inoculum containing about 1 g mycelium (dry weight) lÿ1 (Mortier et al., 1988). For the Peyrat-le-ChaÃteau nursery, alginate beads containing L. bicolor mycelium (Mortier et al., 1988) were prepared to achieve a ®nal concentration of 50 and 100 mg (DW) mycelium lÿ1 inoculum. The bacterial strain BBc6R8 (Frey-Klett et al., 1997a) belongs to the biovar l of the Pseudomonas ¯uorescens species according to the BIOLOG1 identi®cation system (P. Frey-Klett, unpub. Ph.D thesis, Paris-Sud University, 1996). The BBc6R8 inoculum was prepared by growing the bacteria on King's B (KB) agar medium (King et al., 1954) with 100 mg rifampin lÿ1 at 258C for 36 h. The bacteria were then suspended in 0.1 M MgSO4 buer, washed twice, and resuspended in tap water at three dierent bacterial densities: 1.6 102 (low), 1.6 104 (medium), 1.6 106 (high) cfu mlÿ1 inoculum. Bacterial density was measured as the absorbance of the suspension at 600 nm, with reference to a standard curve calibrated by plate enumeration. 2.3. Inoculation and sowing procedures Inoculation and sowing were performed in May in both nurseries. In the Champenoux nursery, 0.5 l mÿ2 of the peat±vermiculite fungal inoculum was mixed into the top 10 cm of soil with a hand tool. In the Peyrat-le-ChaÃteau nursery, 1 l mÿ2 of the alginate inoculum beads were mixed the same way into the soil. Then in both nurseries, 5 l mÿ2 of the bacterial inoculum was applied on the surface of the soil with a watering can and the controls (fungal inoculum without bacteria) received only water. Before sowing, the
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inoculated soil was mixed a second time to a depth of 5±10 cm. In the Champenoux nursery, strati®ed Douglas-®r seeds were then sown in rows with one seed every 2 cm for a global density of about 1000 seeds mÿ2. In the Peyrat-le-ChaÃteau nursery, seeds were broadcast spread at a density of 1500 seeds mÿ2. After sowing, seeds were covered with disinfected soil. 2.4. Plant growth conditions and experimental designs After seed germination, the nursery benches at both nurseries were shaded. In Champenoux only, natural precipitation was complemented with spray irrigation. In Champenoux, the bench was divided into 10 blocks, each containing four 0.5 m2 (1 0.5 m) plots isolated from each other by water-tight, 40 cm deep partitions. In each block, four microbial treatments of one plot each were compared: fungus alone (at the rate of about 0.5 g DW mycelium mÿ2), fungus plus low (8 105 cfu mÿ2 ), medium (8 107 cfu mÿ2) or high (8 109 cfu mÿ2) bacterial inoculation dose. In Peyrat-le-ChaÃteau, the experimental design was four blocks distributed into two parallel nursery benches and containing eight 1 m2 (2 0.5 m) plots which alternated with non-inoculated, unsown 2 m2 (2 1 m) buer plots; there were no partitions as in Champenoux. In each block, eight microbial treatments of one plot each were compared: two fungal inoculation doses (50 and 100 mg DW mycelium mÿ2) combined with four bacterial inoculation doses (0, 8 105, 8 107 and 8 109 cfu mÿ2). However, summer drought ruined some of the treatments inoculated with the high fungal dose in two blocks, located at the west extremity of the two benches and exposed to direct sunlight. 2.5. Population dynamics of P. ¯uorescens BBc6R8 Soil cores (150 cm3) were sampled in all treatments with a steel tube (12 cm long, 4 cm dia) centered on a seedling. In the Champenoux nursery, one sample per plot was taken 11 times during the whole duration of the experiment (three growing seasons). All root pieces were removed from the soil cores by sieving and homogenized before bacterial quanti®cation. In the Peyratle-ChaÃteau nursery, four cores per plot were sampled at the end of the experiment, after the ®rst growing season. Soil cores were pooled by plot, root pieces removed from cores, and homogenized before bacterial quanti®cation. For the dilution-plating quanti®cation, about 1 g of soil was subsampled, weighed and suspended in 3 ml of TS buer (TRIS, 2.42 g; NaCl, 8 g; distilled water, 1 l; pH 7.5). The samples were then vigorously homogenized for 1 min. Crude suspensions and appropriate dilutions were plated for all samples in duplicate on
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the selective medium of Simon and Ridge (1974) containing 100 mg lÿ1 of propiconazole (Tilt 125, a fungicide from Ciba), to quantify total ¯uorescent pseudomonad populations, and in triplicate on KB medium containing 100 mg rifampin lÿ1 and 100 mg propiconazole lÿ1 to quantify BBc6R8 populations. Bacterial colonies were counted after incubation at 258C for 48 h. When bacterial populations were below the detection limit (100 cfu g DWÿ1 soil) of the dilution-plating method, 0.5 g soil samples were enriched by shaking in 3 ml of KB-rifampin (100 mg lÿ1) medium at 278C for 42 h. The presence of BBc6R8 was then checked by culturing 50 ml of the enriched culture on KB-rifampin plates and detecting ¯uorescent colonies under UV radiation. 2.6. Assessment of mycorrhiza formation The mycorrhizal index (percentage of short roots mycorrhizal with L. bicolor ) was determined by randomly examining 100 short roots per 150 cm3 core sample with a stereomicroscope; L. bicolor ectomycorrhizae can be distinguished, on morphological bases, from those due to resident fungi such as Thelephora terrestris or Rhizopogon sp. In Champenoux, one core sample was randomly taken in each plot at the end of the ®rst and of the third growing seasons. In Peyrat-leChaÃteau, at the end of the ®rst growing season, three cores were randomly sampled in the plots of the four treatments inoculated with 50 mg DW mÿ2 of L. bicolor, and ®ve cores were taken per plot in the treatments with 100 mg DW mÿ2 of L. bicolor. 2.7. Eect on plant growth In Champenoux, all the plants were lifted and counted at the end of the third growing season and the shoot lengths of all the seedlings were measured. Then, all shoots and roots from each plot were dried and weighed together to calculate the mean shoot and root dry weights per plant and per plot. 2.8. Data analysis All of the statistical analyses were done at the probability level of 0.05 using the StatView1 program and all mycorrhizal index data were arcsin transformed before analysis. Data from the Peyrat-le-ChaÃteau nursery were analysed in a three-factor (bacterial dose, fungal dose, block) ANOVA with interactions (unbalanced incomplete block design). Two treatments (the control treatment inoculated with 100 mg DW mycelium mÿ2 and the treatment inoculated with 8 105 cfu mÿ2 BBcR8 bacteria and 50 mg DW mycelium mÿ2) were compared by the contrast method. Two separate two-fac-
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Table 1 Douglas-®r plants in the Champenoux nursery: dose eect of P. ¯uorescens BBc6R8 on the growth (shoot length and dry weight) after three growing seasons (gr.s.) and mycorrhizal index (percentage of short roots mycorrhizal with L. bicolor ) after one and two growing seasonsa Myc. Index (%) Bacterial dose (cfu mÿ2)
Shoot length (cm)
Root dry weight (g)
Shoot dry weight (g)
Total dry weight (g)
1 gr.s.
3 gr.s.
0 (Control) 8 105 (low) 8 107 (medium) 8 109 (high)
40.7 42.5 42.6 40.7
1.36 1.64 1.48 1.36
3.73 3.97 3.94 3.89
5.09 5.61 5.42 5.25
58 69 67 65
6 9 7 8
a Shoot length values correspond to the means of all plants in the 10 plots of each treatment (about 2800 plants per treatment); these values were analysed with a two-factor ANOVA (bacterial dose, block, P = 0.05, with individual plants as replicates in order to maximize precision). All shoots or roots in each plot were dried and weighed together, to calculate the mean shoot and root dry weights per plant for each plot; these values were analysed with a one-factor ANOVA (bacterial dose, P = 0.05, with blocks as replicates). Mycorrhizal index data were arcsin transformed before a two-factor ANOVA (bacterial dose, block, P = 0.05). In each column, means with an asterisk are signi®cantly dierent from the control ones according to the Bonferoni±Dunn test (P = 0.05).
tor ANOVA (bacterial dose, block) were also performed on the data corresponding to each fungal dose (balanced complete block design) and the dierent means were compared two by two by a Schee test. For the Champenoux nursery, shoot length data were analysed with a two-factor (bacterial dose, block) ANOVA and dry weight values were analysed with a one-factor ANOVA (bacterial dose). All the means were compared to the control ones by a Bonferoni± Dunn test.
3. Results 3.1. Dose eect of P. ¯uorescens BBc6R8 in the Champenoux nursery 3.1.1. Mycorrhizal status of the seedlings At the end of the ®rst growing season, the number of short roots colonized by L. bicolor was higher on plants inoculated with bacteria, but this dierence was not statistically signi®cant at P = 0.05 (actual
Fig. 1. Population dynamics of P. ¯uorescens BBc6R8 and total ¯uorescent pseudomonads in the soil of the Champenoux nursery, inoculated with three dierent doses of bacterial inoculum: low (8 105 cfu mÿ2), medium (8 107 cfu mÿ2) and high (8 109 cfu mÿ2). The bacterial populations were quanti®ed by the dilution-plating method. Each point represents the mean value of ten soil cores treatment (one core per plot 10 blocks). The bars are the standard deviations. Some points are below the detection limit because of nil values contributing to the mean.
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3.1.3. Survival of the introduced bacterium in the soil The population density of the introduced bacterium in the soil was above the detection limit of the dilution-plating method only with the highest inoculation dose (8 109 cfu mÿ2) and it rapidly declined after inoculation, whereas the population density of the total ¯uorescent pseudomonads remained stable (Fig. 1). With the enrichment method, BBc6R8 bacterial cells were detected 15 weeks after inoculation with the highest bacterial dose, whereas they were only detected during the ®rst 3 weeks after inoculation in the case of the two lowest bacterial doses (8 105 and 8 107 cfu mÿ2) (Fig. 2). Using the enrichment method of detection, no BBc6R8 bacterial cells were detected in the soil at the end of the third growing period, for any of the bacterial treatments.
Fig. 2. Detection of BBc6R8 bacterial cells in the soil of the Champenoux nursery, inoculated with three dierent doses of bacterial inoculum: low (8 105 cfu mÿ2), medium (8 107 cfu mÿ2) and high (8 109 cfu mÿ2). Each point represents the proportion (expressed in %) of the positive enrichments among 10 soil cores per treatment (one core per plot 10 blocks).
P = 0.07). The amount of colonization was independent of the bacterial inoculation dose (Table 1). At the end of the third growing season, the L. bicolor mycorrhizal index was low for all treatments (Table 1).
3.1.2. Seedling growth Bacterial inoculation at the two lowest doses (8 105 and 8 107 cfu mÿ2) signi®cantly increased the shoot length of the Douglas-®r plants at the end of the third growing season (Table 1). The lowest dose of bacterial inoculum also signi®cantly increased the root dry weight. However, bacterial inoculation had no signi®cant eect on the shoot and the total dry weights of the plants.
3.2. Dose eect of P. ¯uorescens BBc6R8 in the Peyratle-ChaÃteau nursery 3.2.1. Mycorrhizal status of the seedlings at the end of the ®rst growing season The three-factor (bacterial dose, fungal dose, block) revealed a signi®cant eect of the bacterial dose (P = 0.0001), of the fungal dose (P = 0.03), of the block factor (P = 0.04) and no interaction, on the L. bicolor mycorrhizal index of Douglas-®r. The lower the bacterial dose, the higher the mycorrhizal helper eect. Moreover, in the treatment inoculated with 50 mg DW mycelium mÿ2 of L. bicolor and the lowest bacterial dose (8 105 cfu mÿ2), the mycorrhizal index (70%) was the same as the mycorrhizal index in the control inoculated with 100 mg DW mycelium mÿ2 (64%, P = 0.06). When plants were not inoculated with bacteria but the fungal dose increased from 50 to 100 mg DW mycelium mÿ2, the mycorrhizal index of L. bicolor on the plants increased from 45 to 64% (Table 2). When plants were inoculated with 50 mg DW mycelium mÿ2, the mycorrhizal index of L. bicolor on the plants increased with bacterial inoculation; but the bacterial
Table 2 Dose eect of P. ¯uorescens BBc6R8 on the percentage of Douglas-®r short roots mycorrhizal with L. bicolor, in the Peyrat-le-ChaÃteau nursery, 23 weeks after inoculationa Bacterial inoculation dose (cfu mÿ2) Fungal inoculation dose (mg DW mycelium mÿ2)
0 (control)
8 105 (low)
8 107 (medium)
8 109 (high)
50 100
45a 64a
70c 77b
62bc 70ab
51ab 64ab
a The values correspond to the means of 10 (treatments inoculated with 100 mg DW mycelium mÿ2) or 12 (treatments inoculated with 50 mg DW mycelium mÿ2) replicates. The percentages transformed by arcsin (square root) were analysed with a two-factor ANOVA (bacterial dose, block; P = 0.05) with interaction. A signi®cant eect of the bacterial dose, no eect of the block factor and no interaction were found by the F test. In each line, values with the same letters are not signi®cantly dierent according to a Schee test.
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eect was only signi®cant with the low and medium bacterial inoculation treatments (Table 2). For plants inoculated with 100 mg DW mycelium mÿ2, only the low bacterial inoculation treatment signi®cantly increased the mycorrhizal index of L. bicolor (Table 2). Fig. 3 shows that a negative relationship exists between the bacterial inoculation dose and the mycorrhizal helper eect (expressed as the dierence between the average mycorrhizal index with or without bacterial inoculation, and excepting the bacterial inoculation rate of 0). Lower bacterial inoculation doses were more eective at increasing mycorrhizal colonization than higher doses. This negative relationship was only signi®cant for plants inoculated at the high rate of L. bicolor inoculation (100 mg DW mycelium mÿ2). 3.2.2. Survival of the introduced bacterium in the soil The population of the introduced bacterium in the soil was analysed at the end of the ®rst growing season. Bacterial densities were below the detection limit of the dilution-plating method. With the enrichment method, the bacterium was detected in 75% of the soil samples collected in the treatment inoculated with the highest dose of bacterial inoculum, but not in the other treatments.
Fig. 3. Relationship between the absolute percent mycorrhizal increase (dierence between the average mycorrhizal indexes with and without BBc6R8) and the logarithm of the bacterial inoculation dose, in the Peyrat-le-ChaÃteau nursery, 23 weeks after inoculation. Regression analysis reveals a signi®cant relationship (shown by a dotted line) in the case of the highest fungal inoculation dose (Y = 30.781ÿ3.023X (r 2=0.998), P = 0.0276).
4. Discussion These results con®rm the ecacy of a mycorrhiza helper Pseudomonas ¯uorescens in enhancing the eect of ectomycorrhizal inoculation in bare-root forest nurseries, in terms of increased mycorrhiza formation and enhanced seedling growth. The latter eect, which can occur without the former (i.e. increased mycorrhiza formation) as shown in the Champenoux experiment, is consistent with other observations of our group in the same nursery (unpublished data) and of R. Duponnois (unpub. Ph.D thesis, Nancy University 1992). In all these cases, BBc6R8, or the wild strain BBc6 from which it was derived signi®cantly promoted Douglas-®r seedling growth when associated with L. bicolor, even without enhancing mycorrhizal establishment. This suggests that the bacteria might increase the symbiotic eciency of mycorrhizas. But the central result of our work is that, within the range of bacterial inoculation doses from 106 to 109 cfu mÿ2 (i.e. about 10 to 104 cfu cmÿ3 of soil only), the lowest doses were the most ecient ones. No bacterial inoculation must obviously have a nil eect on mycorrhiza formation; hence the negative dose±response we observed means that the response curve to bacterial dose will have a maximum. Therefore, under our experimental conditions, the doses we used were clearly super-optimal even though they were low. This result is consistent with the ®ndings of Duponnois and Garbaye (1992) who used the same organisms under similar nursery conditions. The eects we observed of bacterial inoculation on seedling growth are in agreement with C. Karabaghli (unpub. Ph.D thesis, Nancy University (ENGREF) 1997) who found that high concentrations of BBc6 were detrimental to the roots of spruce (Picea abies L.) seedlings. However, our results contrast markedly with most results with PGPRs used as rhizosphere biocontrol agents, where a minimal inoculation dose of 105 cfu gÿ1 soil is required for the bene®cial eect, and where increasing the inoculation dose generally increases plant protection (Bull et al., 1991; Raaijmakers et al., 1995). With other PGPR strains, some detrimental eects on root growth were also observed with high inoculation doses such as 108 cfu mlÿ1 (Kapulnik et al., 1985; Bashan, 1986). In our system, the eciency of the bacterial inoculation disappears when the dose of the bacterial inoculum increases. We can hypothesize that the bacterium exerts two opposite eects on plant growth: some detrimental ones toward the plant or the fungus when it is present at high densities (e.g. antibiosis or competition for nutrients), and some bene®cial ones which are independent of the cell density (such eects have not yet been determined). We found no interaction between the dose of bacterial inoculum and the amount of fungus inoculation.
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For the two fungal doses we tested, the success of the bacterial inoculation was independent of the amount of fungus inoculum used. This is dierent from some biocontrol-PGPR strains for which the bacterial eciency depends on the disease intensity and thus on the pathogen density (Raaijmakers et al., 1995). A positive dose±response relationship is generally attributed to a better colonization of the rhizosphere by the introduced microorganism (Raaijmakers et al., 1995), leading to a larger population which produces more of the eective substances (e.g. antibiotics or siderophores) either directly, because the cells are more numerous, or indirectly through quorum-sensing mechanisms within high-density micro-colonies (Chin-AWoeng et al., 1997). In our study, the dose±response relationship was negative and, with the two detection methods used, the BBc6R8 populations decreased in the soil and even in the rhizosphere after inoculation (data not shown). Since, during the same period, the number of total ¯uorescent pseudomonads remained stable, we believe that the equilibrium level of BBc6R8 is normally very low in this type of ecosystem, independent of occasional, adverse environmental factors. Two hypotheses could account for the ecacy of BBc6R8 at very low apparent population densities: (i) A large population of such bacteria exist and is metabolically active in the soil but not detected by plating because the cells are viable but non-culturable (Bloom®eld et al., 1998; Kell et al., 1998; McDougald et al., 1998) or because they occupy special niches which are not accessible to the standard extraction technique using homogenization, and (ii) The population is low but active micro-colonies (Chin-A-Woeng et al., 1997) are localized in target sites, such as walls of the fungal hyphae, where the bacteria deliver active substances. Li and Hung (1987) have shown that small populations of nitrogen-®xing bacteria were associated with the inner mantle of some ectomycorrhizas of Douglas-®r in the native area of this tree species. In our case, Frey-Klett et al. (1997b) reported that BBc6R8 bene®ts from the presence of L. bicolor mycelium in the soil and Sen et al. (1996) showed that it attaches to L. bicolor in vitro. However, BBc6R8 does not produce known N-acylhomoserine-lactones (Y. Dessaux, pers. comm.) which are needed in the synthesis of many density-dependent secondary metabolites (Swift et al., 1996). As BBc6 is Laccaria-speci®c (Garbaye and Duponnois, 1992), it can also be hypothesized that some bacterial cells are inside the hyphae, as demonstrated in the case of arbuscular endomycorrhizal fungi (Bianciotto et al., 1996; Perotto and Bonfante, 1997). As a consequence, we believe that the L. bicolor±P. ¯uorescens system presents some unusual traits which are worth studying to unveil new rhizosphere mechanisms. From the practical point of view of nursery manage-
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ment and controlled mycorrhization of forest planting stocks, bacterial inoculation at sowing is easily applied to nursery treatments as stressed by Chanway (1997). Furthermore, our results clearly show that adding a mycorrhiza helper bacterial strain to the fungal inoculum allows for the reduction of mycorrhizal inoculum while obtaining the same mycorrhizal index. Therefore, the inoculation cost could be minimized since bacteria are much easier and faster to grow than ectomycorrhizal fungi, and because our results show that a very small amount of bacteria is enough to increase plant response. Acknowledgements We are grateful to G. Catroux for reviewing the manuscript. We greatly appreciated the technical assistance of M.L. Clausse, S. Kotowski, J.L. Rousselet and E. Sidot. We also thank R. Molina and J.M. Trappe (Corvallis, Oregon) for providing strain S238, from which S238N was derived. References Bashan, Y., 1986. Signi®cance of timing and level of inoculation with rhizosphere bacteria on wheat plants. Soil Biology and Biochemistry 18, 297±301. Bianciotto, V., Bandi, C., Minerdi, D., Sironi, M., Tichy, H.V., Bonfante, P., 1996. An obligate endosymbiotic mycorrhizal fungus itself harbors obligately intracellular bacteria. Applied and Environmental Microbiology 62, 3005±3010. Bloom®eld, S.F., Stewart, G.S.A.B., Dodd, C.E.R., Booth, I.R., Power, E.G.M., 1998 The viable but non-culturable phenomenon explained?, Microbiology 1±3. Bull, C.T., Weller, D.M., Thomashow, L.S., 1991. Relationship between root colonization and suppression of Gaeumannomyces graminis var tritici by Pseudomonas ¯uorescens and P. putida. Phytopathology 81, 954±959. Chanway, C.P., 1997. Inoculation of tree roots with plant growth promoting soil bacteria: An emerging technology for reforestation. Forest Science 43, 99±112. Chin-A-Woeng, T.F.C., de Priester, W., van der Bij, A.J., Lugtenberg, B.J.J., 1997. Description of the colonization of a gnotobiotic tomato rhizosphere by Pseudomonas ¯uorescens biocontrol strain WC365, using scanning electron microscopy. Molecular Plant±Microbe Interactions 10, 79±86. Di Battista, C., Selosse, M.A., Bouchard, D., StenstroÈm, E., Le Tacon, F., 1996. Variations in symbiotic eciency, phenotypic characters and ploidy level among dierent isolates of the ectomycorrhizal basidiomycete Laccaria bicolor strain S238. Mycological Research 100, 1315±1324. Dunstan, W.A., Malajczuk, N., Dell, B., 1998. Eects of bacteria on mycorrhizal development and growth of container grown Eucalyptus diversicolor F. Muell seedlings. Plant and Soil 201, 243±251. Duponnois, R., Garbaye, J., 1991. Mycorrhization helper bacteria associated with the Douglas-®r-Laccaria laccata symbiosis: Eects in aseptic and in glasshouse conditions. Annales des Sciences ForestieÁres 48, 239±251. Duponnois, R., Garbaye, J., 1992. Application des BAM (bacteÂries
1562
P. Frey-Klett et al. / Soil Biology and Biochemistry 31 (1999) 1555±1562
auxiliaires de la mycorhization) aÁ l'inoculation du Douglas par Laccaria laccata S238 en peÂpinieÁre forestieÁre. Revue ForestieÁre Franc° aise 44, 491±500. Duponnois, R., Garbaye, J., Bouchard, D., Churin, J.L., 1993. The fungus-speci®city of mycorrhization helper bacteria (MHBs) used as an alternative to soil fumigation for ectomycorrhizal inoculation of bare-root Douglas-®r planting stocks with Laccaria laccata. Plant and Soil 157, 257±262. Frey-Klett, P., Pierrat, J.C., Garbaye, J., 1997a. Location and survival of mycorrhizal helper Pseudomonas ¯uorescens during establishment of ectomycorrhizal symbiosis between Laccaria bicolor and Douglas-®r. Applied and Environmental Microbiology 63, 139±144. Frey-Klett, P., BruleÂ, C., Garbaye, J., 1997b. A proposed model for the mycorrhiza helper eect of Pseudomonas ¯uorescens BBc6 on the ectomycorrhizal symbiotic system Laccaria bicolor S238NDouglas-®r. In: Ogoshi, A., Kobayashi, K., Homma, Y., Kodama, F., Kondo, N., Akino, S. (Eds.), Proceedings of the Fourth International Workshop on Plant-Growth-Promoting Rhizobacteria. Sapporo, p. 399. Gagnon, J., 1996. Stimulation of mycorhization and growth for containerized Jack Pine seedlings inoculated with Lacaria bicolor and Pseudomonas ¯uorescens. In: AzcoÂn, C., Barea, J.M. (Eds.), Mycorhizas in integrated systems from genes to plant development. European Commission EUR 16728 EN, pp. 634±637. Garbaye, J., 1994. Helper bacteria: A new dimension to the mycorrhizal symbiosis. New Phytologist 128, 197±210. Garbaye, J., Bowen, G.D., 1987. Eect of dierent micro¯ora on the success of ectomycorrhizal inoculation of Pinus radiata. Canadian Journal of Forest Research 17, 941±943. Garbaye, J., Bowen, G.D., 1989. Stimulation of ectomycorrhizal infection of Pinus radiata by some microorganisms associated with the mantle of ectomycorrhizas. New Phytologist 112, 383± 388. Garbaye, J., Duponnois, R., 1992. Speci®city and function of mycorrhization helper bacteria (MHB) associated with the Pseudotsuga menziesii±Laccaria laccata symbiosis. Symbiosis 14, 335±344. Garbaye, J., Duponnois, R., Wahl, J.L., 1990. The bacteria associated with Laccaria laccata ectomycorrhizas or sporocarps: Eect on symbiosis establishment on Douglas-®r. Symbiosis 9, 267±273. Kapulnik, Y., Okon, Y., Henis, Y., 1985. Changes in root morphology of wheat caused by Azospirillum inoculation. Canadian Journal of Microbiology 31, 881±887. Kell, D.B., Kaprelyants, A.S., Weichart, D.H., Harwood, C.R., Barer, M.R., 1998. Viability and activity in readily culturable bacteria: A review and discussion of the practical issues. Antonie van Leeuwenhoek 73, 169±187.
King, E.O., Ward, M.K., Raney, D.E., 1954. Two simple media for the demonstration of pyocyanine and ¯uorescein. Journal of Laboratory and Clinical Medicine 44, 301±307. Le Tacon, F., Mousain, D., Garbaye, J., Bouchard, B., Churin, J.L., Argillier, C., Amirault, J.M., GeÂneÂreÂ, B., 1997. Mycorhizes, peÂpinieÁres et plantations forestieÁres en France. Revue ForestieÁre Franc° aise 49, 131±154. Li, C.Y., Hung, L.L., 1987. Nitrogen-®xing (aceteylene-reducing) bacteria associated with ectomycorrhizas of Douglas-®r. Plant and Soil 98, 425±428. McDougald, D., Rice, S.A., Weichart, D., Kjelleberg, S., 1998. Nonculturability: Adaptation or debilitation? FEMS Microbiology Ecology 25, 1±9. Mortier, F., Le Tacon, F., Garbaye, J., 1988. Eect of inoculum type and inoculation dose on ectomycorrhizal development, root necrosis and growth of Douglas ®r seedlings inoculated with Laccaria laccata in a nursery. Annales des Sciences ForestieÁres 45, 301±310. Pachlewski, R., Pachlewska, J., 1974. Studies on symbiotic properties of mycorrhizal fungi of pine (Pinus sylvestris ) with the aid of the method of mycorrhizal synthesis in pure culture on agar. Forest Research Institute, Warsaw. Perotto, S., Bonfante, P., 1997. Bacterial associations with mycorrhizal fungi: Close and distant friends in the rhizosphere. Trends in Microbiology 5, 496±501. Raaijmakers, J.M., Leeman, M., van Oorschot, M.M.P., van der Sluis, I., Schippers, B., Bakker, A.H.M., 1995. Dose±response relationships in biological control of fusarium wilt of radish by Pseudomonas spp. Phytopathology 85, 1075±1081. RoÂzycki, H., Kampert, M., Strzelczyk, E., Li, C.Y., Perry, D.A., 1994. Eect of dierent soil bacteria on mycorrhizae formation in Scots pine (Pinus sylvestris L.) in vitro studies. Folia Forestalia Polonica 36, 92±102. Sen, R., Nurmiaho-Lassila, E.L., Haahtela, K., Korhonen, T., 1996. Attachment of Pseudomonas ¯uorescens strains to the cell walls of ectomycorrhizal fungi. In: AzcoÂn-Aguilar, C., Barea, J.M. (Eds.), Mycorhizas in integrated systems from genes to plant development. European Commission EUR 16728 EN, pp. 634±637. Simon, A., Ridge, E.H., 1974. The use of ampicillin in a simpli®ed selective medium for the isolation of ¯uorescent Pseudomonads. Journal of Applied Bacteriology 37, 459±460. Slankis, V., 1974. Soil factors in¯uencing formation of mycorrhizae. Annual Review of Phytopathology 12, 437±457. Swift, S., Throup, J.P., Williams, P., Salmond, G.P.C., Stewart, S.A.B., 1996 Quorum sensing: A population-density component in the determination of bacterial phenotype. Trends in Biochemical Sciences 214±219.