Effects of Azospirillum brasilense with genetically modified auxin ...

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222 E. Baudoin et al. / Research in Microbiology 161 (2010) 219e226 For DGGE fingerprinting, abundant 16S rDNA PCR products of the expected size (550 bp) were obtained (not shown), and they were subjected to DGGE analysis, as described by Lerner et al. (2006). Relative band intensities were calculated as in ARISA. 2.9. Statistics Cell numbers were log-transformed before computing means and standard deviations. For cell numbers and plant parameters, treatments were compared with ANOVA followed (when appropriate) with Fisher’s LSD tests. Statistics were done at P < 0.05 using S-PLUS (Insightful Corporation, Seattle, WA). Principal component analysis (PCA) of ARISA and DGGE fingerprints was performed on the covariance matrix (bacterial or fungal communities as rows and bands as columns) obtained after coding the profiles according to Ranjard et al. (2001). PCA was performed using ADE-4 software (Thioulouse et al., 1997) on the most dominant (i.e. intense) ARISA peaks and all detected DGGE bands. Treatments were compared by ANOVA and (when appropriate) Fisher’s LSD tests performed separately on PC1 and PC2 scores. 3. Results 3.1. Rhizosphere colonization by the inoculants Azospirillum inoculants, which were added at 10 6 cells seed 1 , were detected at about 1 10 6 and 5 10 5 cells plant 1 in the rhizosphere at, respectively, one week and one month after inoculation (Fig. 1). At each sampling, the population size of the inoculants did not differ significantly from one another. The MPNs of the inoculants estimated in semi-solid Nfb medium were at the same level with and without rifampicin, but confirmatory evidence of the presence of Azospirillum cells under the microscope was easier to obtain in the presence of the antibiotic due to the absence of Inoculant number (log CFU per plant) 8 7 6 5 4 3 2 1 0 0 7 30 Time (days) Fig. 1. Rhizosphere colonization of inoculated wheat by A. brasilense Sp245- Rif (white bar), GM control Sp245-Rif(pFAJ0526) (hatched bar), PnptII construct Sp245-Rif(pFAJ0529) (black bar) and PsbpA construct Sp245- Rif(pFAJ0535) (bar with horizontal lines). For each treatment, means and standard deviations are shown (n ¼ 5). There was no difference between inoculants at sowing (log CFU per seed) or at any of the two samplings (log CFU per root system) based on ANOVA (P < 0.05). other bacteria in the background. In the three GM treatments, resistance to tetracycline (and cell fluorescence) was observed in all cases, indicating that the plasmid vectors were stably maintained in inoculants cells in the rhizosphere. No Azospirillum-like cells were detected in antibiotic-free Nfb when studying samples from the non-inoculated control treatment. This means that indigenous Azospirillum were absent or at levels below the detection limit (i.e. less than 10 2 cells plant 1 ), which may be the case in certain soils of temperate regions (Swędrzyńska and Sawicka, 2001). 3.2. Effect on plant growth Inoculation of wheat seeds had no significant effect on root biomass at one month, but resulted in: (i) a lower average root diameter; and (ii) in the case of Sp245-Rif(pFAJ0535), a lower root system volume (Table 1). A. brasilense Sp245-Rif and the GM control Sp245-Rif(pFAJ0526) had no impact on shoot biomass, but the latter was significantly higher for wheat inoculated with the ipdC constructs Sp245-Rif(pFAJ0529) (i.e. þ25%) or Sp245-Rif(pFAJ0535) (i.e. þ11%). 3.3. Effect on bacterial diversity in the rhizosphere The impact of the inoculants on the genetic structure of the rhizobacterial community was assessed by ARISA (Fig. 2). In comparison with the non-inoculated control, neither the wildtype Sp245-Rif, the GM control Sp245-Rif(pFAJ0526) nor the two ipdC constructs had any effect based on PCA results. In comparison with the GM control, however, the effect of the PsbpA construct Sp245-Rif(pFAJ0535) was significant. The distinction found between treatments was also evidenced when the rhizobacterial community was assessed by DGGE analysis, using selected samples (data not shown). Each Azospirillum inoculant managed to recolonize the rhizosphere once untreated wheat seeds were sown in the same pots (after removal of inoculated plants), but at population levels about 10 times lower at three weeks (data not shown). No residual impact of inoculation was found at three weeks, as there was no difference between any of the treatments based on plant growth data or assessment of ARISA data (data not shown). 3.4. Effect on fungal diversity in the rhizosphere In comparison with the non-inoculated control, neither the wild-type Sp245-Rif nor the PnptII construct Sp245- Rif(pFAJ0529) had any impact on the genetic structure of the fungal community in the wheat rhizosphere, based on PCA of ARISA data (Fig. 3). However, the effects of the GM control Sp245-Rif(pFAJ0526) and, to a lesser extent, the PsbpA construct Sp245-Rif(pFAJ0535) were significant. In comparison with the GM control Sp245-Rif(pFAJ0526), the two ipdC constructs (as well as the wild-type Sp245-Rif) displayed a significant effect on resident fungi, but the two ipdC constructs did not differ from each other.
E. Baudoin et al. / Research in Microbiology 161 (2010) 219e226 223 Table 1 Effect a of inoculation with A. brasilense Sp245-Rif, GM control Sp245-Rif(pFAJ0526), PnptII construct Sp245-Rif(pFAJ0529) or PsbpA construct Sp245- Rif(pFAJ0535) on wheat plants at one month after sowing (mean standard deviation; n ¼ 8). Plant biomass Root fresh weight Shoot fresh weight Units g plant g plant 1 1 Non-inoculated control Sp245-Rif GM control Sp245-Rif(pFAJ0526) PnptII construct Sp245-Rif(pFAJ0529) PsbpA construct Sp245-Rif(pFAJ0535) 1.43 0.29 1.30 0.35 1.37 0.25 1.39 0.25 1.30 0.41 1.57 0.30 a 1.56 0.22 a 1.48 0.27 a 1.96 0.46 b 1.74 0.43 b Root architecture Total root length cm plant 1 899 158 1019 317 1017 158 996 157 961 241 Root surface area cm 2 plant 1 148 45 133 43 125 24 129 29 118 35 Average root diameter mm 0.519 0.092 a 0.419 0.033 b 0.392 0.051 b 0.403 0.052 b 0.399 0.035 b Total root volume cm 3 plant 1 2.00 0.90 a 1.40 0.48 ab 1.24 0.36 ab 1.34 0.43 ab 1.15 0.40 b Number of roots 2325 328 2920 837 2835 587 2412 728 2439 726 a When statistical differences were found between treatments (ANOVA and Fisher’s LSD tests), they are indicated by letters a and b (P < 0.05). 4. Discussion Understanding the ecological impact of Azospirillum phytostimulators modified for IAA overproduction is an important issue when attempting to better define relevant usage conditions for these inoculants (Morrissey et al., 2002). Here, inoculant densities in the rhizosphere were rather high one month after sowing (i.e. about 3.7 10 5 cells g 1 fresh root), which means ecological conditions were favorable for expression of their plant-beneficial traits (Jacoud et al., 1999). Importantly, bacterial count data indicated that (i) the GM plasmids were maintained in all inoculant cells and (ii) the presence of a GM plasmid had no negative effect on the rhizosphere fitness of Sp245-Rif derivatives. A similar behavior was also described for Pseudomonas and GM derivatives in the cucumber rhizosphere (Beyeler et al., 1999). Strain Sp245-Rif as well as the GM control Sp245- Rif(pFAJ0526) had no effect on the biomass of wheat shoots, but the higher IAA production potential of the two GM strains did translate into significantly enhanced shoot biomass of winter wheat. This indicates that the GM inoculants were ecologically functional as phytostimulators in the experiment (which is also indicated by their effects on microbial rhizosphere community). Comparable effects on plant were obtained on spring wheat with similar ipdC constructs in A. brasilense Sp245 (Spaepen et al., 2008) and on cucumber with Pseudomonas modified for IAA overproduction (Beyeler et al., 1999). Two related versions of the PnptII and PsbpA constructs (i.e. without the eyfp gene) were previously shown to produce more IAA than the wild-type strain under laboratory conditions (Spaepen et al., 2008). In the current work, the A. brasilense inoculants influenced root system architecture, as they resulted in thinner roots, but 0.05 PC2 12% AB 0.025 AB 0 -0.1 -0.08 -0.06 -0.04 -0.02 0 0.02 0.04 0.06 0.08 AB A B -0.025 PC1 50% Fig. 2. Effects of Azospirillum brasilense inoculants on the rhizosphere bacterial community as indicated by PCA ordination of ARISA fingerprints (79 dominant peaks analyzed). Symbols refer to non-inoculated wheat (empty circle), wheat inoculated with A. brasilense Sp245-Rif (empty square), GM control Sp245- Rif(pFAJ0526) (full square), PnptII construct Sp245-Rif(pFAJ0529) (full triangle) and PsbpA construct Sp245-Rif(pFAJ0535) (full diamond). For each treatment, means and standard errors are shown (n ¼ 8). Treatments were compared by ANOVA and Fisher’s LSD tests performed separately on PC1 and PC2 coordinates. Letters A and B indicate the statistical relations between treatments along axis PC1 (P < 0.05). -0.05
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