Effects of co-inoculation treatments on shoot biomass and root development of P. pinaster seedlings

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Assimilation of 15NH4 in bacterial biomass

The strain 111b was grown in pure culture and the evolution of [NH4+] remaining in the culture medium is given in Fig. 1. After 42h of culture, most of ammonium had disappeared from the medium. Assuming that all ammonium depleted from the medium was assimilated by the bacteria, and N contained in bacteria at the time of inoculation was close to zero, we calculated that the bacteria assimilated 74.2 mg NH4+ from the medium. Bacterial cells were grown until the [NH4+] remaining in the culture medium was very close from zero. This was observed after 83h of culture (Fig. 1). After this time, bacteria accumulated 82.3 mg of total N corresponding to 63.4 mg of 15N.
Figure 1. Depletion of ammonium concentration during the culture of Pseudomonas fluorescens, strain 111b. Ammonium was supplied as (15NH4)2SO4 labelled at 77%.

Plant parameters

Shoot biomass

The effect of inoculation on dry biomass accumulation of shoot of P. pinaster seedlings is shown in Figure 2. Shoots from non mycorrhizal plants (treatment a) accumulated significantly lower amounts of biomass than NM plants grown with bacteria (treatment c) and than mycorrhizal plants alone or grown with bacteria, bacteria and nematodes (treatments d, e and f). Culture of NM plants with P . fluorescens slightly increased the shoot biomass by 17% relative to the NM plants but this increase was not significant at p 0.05. In contrast, the co-inoculation of NM plants with P. fluorescens and nematodes strongly promoted shoot biomass by 85%. When the plants are associated with the ectomycorrhizal fungus H. cylindrosporum, shoot biomass significantly increased by 76% compared to the NM plants. The presence of P. fluorescens gave rise to the maximal shoot biomass of M plants that was increased by 131% compared to control plants. However, the addition of nematodes to M plants grown with bacteria (treatment f) tended to decrease shoot biomass compared to M plants with bacteria only (treatment e), although this effect was not significant. Finally, this last treatment (e) caused a significant increase of shoot biomass by 85% compared to the control (Fig. 2).
Figure 3. Effect of different treatments as described in figure 2 on root parameters i.e A/ number of root tips, B/ number of forks, C/ root length, D/ root elongation rate and E/ root surface area measured in P. pinaster seedlings after 35 days of co-inoculation with an ectomycorrhizal fungus, soil bacteria and bacteria-feeding nematodes. Box plots were calculated from 6 to 8 values. Different letters in boxes indicate that the corresponding means are significantly different at p » 0, 05 (ANOVA, Fisher test). Circles out of the boxes represent aberrant values.

Root architecture

Five parameters were recorded to study the effect of the inoculation treatments on root architecture (Fig. 3). Regarding the number of tips recorded after 35 d of inoculation treatments (Fig. 3A), NM and M plants presented lower average values amounting respectively to 286 and 306 tips/plant than those measured in the corresponding treatment with bacteria, whether or not associated with nematodes. However, compared to NM and M treatments, only M plants grown with bacteria (e) and bacteria with nematodes (f) had significantly more root tips per plant, with average values of 477 and 445 tips per plant, respectively. The same trend was observed regarding the number of forks but only treatment e (M+Bac) was significantly higher than the control roots, with values of 618 and 432 forks/plant, respectively (Fig. 3B).
Root length measured at 35 d was found to be highly variable between the plants of a same treatment, excepted when NM plants were grown with bacteria and nematodes (Fig. 3C). Mycorrhizal plants presented lower root length than the same plants grown with bacteria, whether or not with nematodes as well as NM plants with bacteria and nematodes. The same trend was observed regarding the root elongation rate which was about 4 cm/d in all the treatments (Fig. 3D). The lowest value (2.77 cm/d) was calculated in mycorrhizal plants (Fig. 3D).
The most pronounced effect of inoculation treatments was found on the root surface area (p=0.015) (Fig. 3E). The highest averages were recorded for treatments e (M+Bac), f (M+Bac+N) and b (NM+Bac) with values of 66.15, 63.5 and 56.53 cm2 per plant respectively. The two other treatments with NM plants, treatment a (NM) and c (NM+Bac+N) remain close to each other with values of 54.5 and 58.5cm2 per plant, respectively. Finally, the mycorrhizal association alone (treatment d) gave the minimum surface area amounting to 39.7cm2 per plant.

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Number of nematodes

The number of nematodes estimated after 35 d of treatment was not significantly different between NM and M plants, with mean values of 140 and 117 nematodes/plant in treatment c and f, respectively (for details, see appendix 1). It should be noticed that the nematode population increased during the experiment, the numbers of nematodes were 3 times greater at the end of experiment as compared to initial numbers.
Figure 4. Effect of different treatments on total nitrogen accumulation in shoots of P. pinaster seedlings, wether or not associated with an ectomycorrhizal fungus, after 35 days of co-inoculation with a bacterial strain and bacteria-feeding nematodes. The treatments are: NM plants (control), NM plants + bacteria (bac), NM plants + bacteria + nematodes (bac+nmt), M plants (mycorrhiza), M plants + bacteria (m+bac), M plants + bacteria + nematodes (m+bac+nmt). Given values are the means (n=6 to 8) with standard variation and the bars with different letters are significantly different at p 0.05 (ANOVA, Fisher test).

Effects of co-inoculation treatments on shoot biomass and root development of P. pinaster seedlings

The effect of the presence of bacteria, whether or not accompanied by nematodes that are able to feed on them, induced contrasted effects on shoot biomass of P. pinaster seedlings after only 35 d of contact. As shown in figure 2, amounts of biomass accumulated in shoots of NM plants were the lowest ones, suggesting that these plants grew very slowly. The addition of bacteria did not significantly improve shoot growth of NM plants, suggesting that the additional nutrient supply contained in bacteria remained inaccessible to the NM plants. These results also suggest that this bacterial strain has a low PGPR effect per se. In contrast, the presence of the ectomycorhizal fungus H. cylindroporum strongly stimulated the shoot growth compared to the NM plants (Fig. 2). In M plants, the presence of P. fluorescens tended to reinforce this positive effect, although it was not significant (Fig. 2). This positive effect could be due to specific relationships occurring between the hyphae of H. cylindrosporum and this bacterial strain, which was isolated from ectomycorrhizal roots collected in the field. These results are in the same line as those of Vosatka (1994) who reported that the dual inoculation (bacteria and fungi) increased the shoot biomass of plants by approximately 30% as compared with control and individual inoculation.

Table of contents :

Introduction
Material and methods
Plant and fungal material
Germination of seeds
Bacteria
Nematodes
-Isolation and multiplication of nematodes
-Sterilization and testing of nematodes with P.fluorescens
Co-inoculation experiment
Measurements
Root parameters
Nematodes counting
Nitrogen cycling
Presentation of results and statistical analysis
Results
Assimilation oh NH4 in bacterial biomass
Plant biomass
Shoot dry weight
Root architecture
Root tips, forks, surface area and length parameters
Number of nematodes at the end of experiment
Nitrogen accumulation in shoots
15N accumulation in shoot biomass
Discussions
Effects of co-inoculation treatments on shoot biomass and root development of P. pinaster seedlings
Effects of bacteria alone and with fungi on nematodes population in rhizosphere of pine
plant
Effect of inoculation treatments on total N and 15N accumulation by shoot biomass

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