Plates were stabbed using sterile toothpicks. After 12 days, flasks containing the seedlings were subjected to ET measurements five replicates per treatment.
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Five empty flasks with filter paper and sterile water served as controls. Two independent experiments were carried out. Approximately mg ground seedling powder was extracted according to the method by Onkokesung et al. Dried rosette material of Binoculated or control WT day-old plants was used for total P analysis. Analysis was conducted using a microwave-assisted digestion. Length of the primary root and number of lateral roots of vertically Figure 1A , upper panels grown Binoculated or non-inoculated WT and 35S-etr1 seedlings were determined after 10 days of growth.
After 12 days of horizontal growth Figure 1A , lower panels , secondary leaves were counted and leaf surface area was analyzed according to the video tutorial by Zach Jarou 2 using Adobe Photoshop C5.
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Two independent experiments with four replicate Petri dishes containing at least seven vertical placement or 20 seeds horizontal placement were carried out. In vitro B55 colonization was determined for seedlings grown for 8 days on Whatman No. Effects of B55 inoculation on seedlings growth in vitro. Experimental design A. CFU, colony forming unit; FM, fresh mass; n. Endophytic bacteria were isolated following the procedure of Long et al. After 2—3 days, colony forming units CFUs of B55 were counted based on colony morphology.
The isolates were identified using the EzTaxon-e server 3 Kim et al. For glasshouse experiments Figure 3B , day-old Binoculated or non-inoculated seedlings were planted into separate TEKU pots containing sand and lecaton. At 20 dpi, plants were transferred to 10 cm diameter round pots containing lecaton and sand. Plants were fertilized every other day with 50 mL distilled water amended with 0. Survival of plants was monitored 24 dpi and length of the longest leaf or stalk height was measured every other day.
Total seed capsule number was determined at the end of the experiment 63 dpi.
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Colonization by B55 was measured 30 dpi; roots were collected and bacterial isolation was performed as described above. Bacterial identity was determined by 16S rDNA sequencing. Two independent experiments were carried out for the analysis of B55 effects on glasshouse-grown plants. The release of transgenic plants was carried under APHIS notification r-3a and the seeds were imported under permit number m.
Petri dishes containing 3-day-old Binoculated or non-inoculated WT and 35S-etr1 seedlings were shipped to the field station. Fourteen days after germination, the seedlings were transferred to pre-hydrated 50 mm peat pellets Jiffy 4 and seedlings were gradually adapted to the high light and low relative humidity of the habitat over a 2-week period.
Pre-adapted rosette-stage plants were transplanted into an irrigated field plot in size-matched quadruplets consisting of one Binoculated and one non-inoculated WT and 35S-etr1 plant in a randomized design: Figures 5A — D , 31 dpi. Survival of the plants was assessed at 46 dpi, rosette diameters were measured 46, 62, and 73 dpi; stalk height measured at 62 and 73 dpi and the number of flowers was counted at 73 dpi. B55 colonization was quantified at 47 and 73 dpi for five randomly selected quadruplets of plants.
Plants were carefully excavated and the loosely attached soil was removed before plants were wrapped in moistened paper towels and sent to the laboratory facility in Jena, Germany. Re-isolation of culturable bacteria was carried out as described, immediately after arrival 2 days after removal from the field.
Other culturable, dominant resident bacterial isolates were counted based on colony morphology and the identity of representatives was determined by 16S rDNA sequencing. The experiment was conducted once for 35S-etr1 plants field season and twice for WT and field seasons. Data analysis was carried out with the StatView software package SAS Institute with a completely randomized analysis of variance. Correlation analysis was performed with simple regression tests.
B55 was isolated from the endosphere of an ET-insensitive 35S-etr1 N. Re-isolation experiments have shown that this Gram-positive bacterium is able to colonize the endosphere and rhizoplane of N. The ET-insensitive transgenic line produces few root hairs and lateral roots Long et al. On average, Binoculated WT and 35S-etr1 roots had eight and seven times more lateral roots, respectively Figure 1D , left panels.
Interestingly, B55 colonization of the endosphere of 35S-etr1 seedlings was more than 10 times higher than in WT, while rhizosphere colonization was similar Figure 1D , right panels. In vitro cultures of B55 produced 0. Furthermore, qualitative enzyme tests revealed that B55 is able to solubilize phosphate. Effects of B55 inoculation on N. White bars represent mock-inoculated seedlings; black bars represent seedlings inoculated with B Total P content of day-old WT rosette plants C. FM, fresh mass; DM, dry mass. B55 inoculation also influenced the production of reproductive structures: Binoculated WT and 35S-etr1 plants yielded on average 2 and 1.
The seed production number of seeds per capsule was not affected by B55 Figure 3F. Effects of a B55 inoculation on glasshouse-grown plants. Timeline of experiment A. Correlation between B55 colonization and WT rosette growth C. For these in vitro and glasshouse experiments, B55 treatment prior to germination resulted in strong PGP effects.
To determine if B55 could similarly influence plant growth when plants were inoculated at a later stage of development, day-old N. Plants were colonized by ca. Timeline and experimental design A. The consistency of PGP effects observed in vitro and in the glasshouse has rarely been tested under field conditions and the PGP effects of a native root-associated bacterium had not been tested in its native host in the field.
To conduct such a test, we examined the effect of B55 inoculation of seeds on the growth of WT plants, during two field seasons, and 35S-etr1 plants during one field season in their native habitat in SW Utah, USA.
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B55 inoculation strongly enhanced the survival and growth of the ET-insensitive 35S-etr1 plants, whereas during the first field season , effects on WT plants were barely detectable Figure 5. During the second field season , Binoculated WT plants exhibited significant increases in both rosette diameter and stalk height compared to controls Figure 6. Effects of a B55 inoculation on field-grown plants field season Field plot B.
Plants were planted in rows separated by water channels C , mock- and Binoculated WT and 35S-etr1 plants were planted in a quadruplets in a randomized design D. Effects of a B55 inoculation on field-grown WT plants field season Plants were grown in a paired design. Only pairs in which both plants survived until the end of the experiment were included in the analysis. The number of flowers was evaluated 62 dpi; and no significant difference was found between Binoculated and non-inoculated WT plants.
Interestingly, the non-inoculated 35S-etr1 plants did not produce any flowers until the end of the experiment, while the Binoculated 35S-etr1 plants produced almost as many as the WT non-inoculated plants Figure 5G.
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Re-isolation experiments revealed that with 4. Surprisingly, Blike colonies were also identified from some roots of non-inoculated WT and 35S-etr1 plants Figure 5H , suggesting that Bacillus sp. We examined the influence of a B55 inoculation on the culturable, endophytic, naturally associated plant microbial community of plants after 73 days of growth in the field. B55 inoculation strongly affected the most abundant culturable bacterial taxa associated with WT and 35S-etr1 roots. The analysis focused on bacterial strains found to colonize roots to the same degree as the introduced B Binoculated plants harbored twice as many bacteria of a greater diversity of bacterial taxa, compared to controls Figure 7.
In addition to B55, inoculated WT roots harbored Pantoea sp. Utah and Pantoea sp. Utah and 35S-etr1 roots harbored Pantoea sp. Utah and Pseudomonas sp. Utah for accession numbers see Table 1. These bacterial genera were not detected in roots of control plants. No difference in colonization by B55 of the inoculated plants was observed, and B55 was not found amongst the dominant bacterial taxa of non-inoculated plants.
The culturable bacterial communities of WT and 35S-etr1 control plants were dominated by one isolate: Enterobacter sp. Utah There was no difference in the extent of colonization by Enterobacter sp.
Utah of the non-inoculated WT and the 35S-etr1 plants Figure 7. Effects of a B55 inoculation on the resident culturable bacterial community of field-grown plants. Colony forming units of B55 and resident isolates at 73 dpi; see Table 1 for accession numbers of isolates. All lines inoculated with B55 late-stage inoculation showed positive growth responses. Figure 8 shows the increase in growth of each line compared to non-inoculated plants. While the JA-deficient N. Interestingly, and in contrast to the results from the seed inoculation procedure, 35S-etr1 plants benefited less from the interaction compared to WTev, when inoculated at a later stage of development 20 days.
Growth responses of different transgenic N. Relative Bassociated growth increase of different transgenic N. Twenty day old plants were inoculated with B55 or mock-inoculated late-stage inoculation. Size of the longest leaf was measured on the day of inoculation and at the end of the experiment 12 or 14 dpi. See Table 2 for abbreviations of genes silenced by RNAi by expression of inverted repeat ir constructs in the transformed lines. Numerous studies have reported on bacterial-mediated PGP effects in vitro and in the glasshouse, however, PGPB frequently fail under field conditions, probably due to their inability to colonize roots properly in a competitive environment Compant et al.
Furthermore, bacterial formulations, previously reported to promote plant growth, often exhibited negative effects when applied in a natural environment. Our in vitro experiments demonstrated that many parameters associated with PGP e. Furthermore, chlorophyll a and b contents, previously reported to be lower in ET-insensitive plants Grbic and Bleecker, were restored to WT control levels by B55 inoculation.
Two types of explanations could account for the greater endosphere colonization despite similar rhizosphere colonization Figure 1D : B55 was originally isolated from a 35S-etr1 plant and hence this genotype may have phenotypes that reflect its natural host. Native N. Baldwin, unpublished results and it would not be surprising if natural ET-deficient ecotypes, similar to those of the 35S-etr1 also occurred in these populations.
Similar host-specific associations have recently been reported by Weyens et al. Second, N. Similar results were found in an ET-insensitive Medicago truncatula line, which was hypercolonized by rhizobia or Klebsiella pneumoniae , respectively Penmetsa, ; Iniguez et al. Interestingly, the rhizospheres of WT and 35S-etr1 plants were similarly colonized by B Hence the greater apparent benefit obtained by 35S-etr1 plants compared to WT may simply have resulted from the greater endosphere B55 colonization.
Glasshouse experiments with Binoculated and non-inoculated WT and 35S-etr1 plants were consistent with our in vitro findings. Inoculated 35S-etr1 plants grew similarly to non-inoculated WT plants and Binoculated plants yielded more seed capsules than their respective controls. Again, 35S-etr1 plants gained a greater growth benefit from B55 inoculation than WT plants did. Further reviews describe technologies to produce inoculants, the biocontrol of post harvest pathogens as a suitable alternative to agrochemicals, and the restoration of degraded soils.
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