A native plant growth promoting bacterium, Bacillus sp. B55, rescues growth performance of an ethylene-insensitive plant genotype in nature

Abstract

Many plants have intimate relationships with soil microbes, which improve the plant's growth and fitness through a variety of mechanisms. Bacillus sp. isolates are natural root-associated bacteria, isolated from Nicotiana attenuata plant roots growing in native soils. A particular isolate B55, was found to have dramatic plant growth promotion (PGP) effects on wild type (WT) and transgenic plants impaired in ethylene (ET) perception (35S-etr1), the genotype from which this bacterium was first isolated. B55 not only improves N. attenuata growth under in vitro, glasshouse, and field conditions, but it also "rescues" many of the deleterious phenotypes associated with ET insensitivity. Most notably, B55 dramatically increases the growth and survival of 35S-etr1 plants under field conditions. To our knowledge, this is the first demonstration of a PGP effect in a native plant-microbe association under natural conditions. Our study demonstrates that this facultative mutualistic plant-microbe interaction should be viewed as part of the plant's extended phenotype. Possible modalities of recruitment and mechanisms of PGP are discussed.

Keywords: Bacillus sp.; Nicotiana attenuata; ethylene-insensitive; extended phenotype; microbial community; nature; plant growth promotion.

FIGURE 1

Effects of B55 inoculation on seedlings growth in vitro. Experimental design (A). Effect of a B55 inoculation on WT and 35S-etr1 seedlings, grown vertically and horizontally (B). Mean (±SE) seedling surface area, number of true leaves, chlorophyll a and b content (C), primary root length, number of lateral roots and B55 colonization in the rhizo- and endospheres (D) of mock- or B55-inoculated WT and 35S-etr1 seedlings. White bars represent control treatments, black bars, B55-inoculated seeds (PLSD test of an ANOVA between mock- and B55-inoculated plants: *P < 0.05; **P < 0.01; ***P < 0.001). CFU, colony forming unit; FM, fresh mass; n.d., not detected. n = 4 replicate Petri dishes containing at least 20 or 7 seeds (vertical placement), n = 5 replicate Petri dishes containing at least 20 seeds (for colonization).

FIGURE 2

Effects of B55 inoculation on N. attenuata IAA content, ET emission and P content. Mean (±SE) IAA content (A) and ethylene emission (B) of N. attenuata WT and 35S-etr1 seedlings. White bars represent mock-inoculated seedlings; black bars represent seedlings inoculated with B55. Total P content of 38-day-old WT rosette plants (C). FM, fresh mass; DM, dry mass. n = 3 for IAA; 5 for ET; n = 6 for P.

FIGURE 3

Effects of a B55 inoculation on glasshouse-grown plants. Timeline of experiment (A). Mean (±SE) length of the longest rosette leaf and stalk height (B). Correlation between B55 colonization and WT rosette growth (C). Survivorship (D), seed capsule number (E), seed number per capsule (F) and B55 colonization (G) of B55-inoculated WT and 35S-etr1 plants. Except for (G), white bars represent control treatments, black bars, B55-inoculated plants (PLSD test of an ANOVA between mock- and B55-inoculated plants: *P < 0.05; **P < 0.01; ***P < 0.001). dpi, days post infection; CFU, colony forming unit; FM, fresh mass; n.d., not detected. n = 20 for (B,F); n = 10 for (C); n > 80 for (D); n = 5 for (E).

FIGURE 4

Effects of a “late-stage” B55 inoculation on WT plants. Timeline and experimental design (A). Mean (±SE) length of the longest leaf of mock- or B55-inoculated WT N. attenuata plants cultured in the glasshouse (B). WT plants were grown in a sand-filled Teku pots until day 20, before they were inoculated with B55 and transferred to 10 cm round pots containing lecaton and sand (PLSD test of an ANOVA between mock- and B55-inoculated plants: *P < 0.05). dpi, days post infection. n = 15.

FIGURE 5

Effects of a B55 inoculation on field-grown plants (field season 2009). Timeline of experiment (A). Field plot (B). Plants were planted in rows separated by water channels (C), mock- and B55-inoculated WT and 35S-etr1 plants were planted in a quadruplets in a randomized design (D). Mean (±SE) rosette diameter and stalk height (E), survivorship (F), flower number (G), and B55 colonization (H). White bars represent control treatments, black bars, B55-inoculated plants (PLSD test of an ANOVA between mock- and B55-inoculated plants: *P < 0.05). dpi, days post-inoculation; CFU, colony forming unit; FM, fresh mass; n.d., not detected. n = 40 for WT–, WT+, and 35S-etr1+; n = 23 for 35S-etr1 at start of measurements (46 dpi); n = 5 for (H).

FIGURE 6

Effects of a B55 inoculation on field-grown WT plants (field season 2010). Mean (±SE) rosette diameter and stalk height of B55-inoculated and mock-inoculated plants. 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. Asterisk indicates a statistically significant difference between treatments as determined by a paired t-test (*P < 0.05; **P < 0.01). dpi, days post infection. n = 11 pairs.

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. dpi, days post infection; CFU, colony forming units; FM, fresh mass. n = 3.

FIGURE 8

Growth responses of different transgenic N. attenuata lines to a B55 inoculation. Relative B55-associated growth increase of different transgenic N. attenuata lines compared to non-inoculated plants. 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). Growth increase (%) of B55-inoculated plants was calculated relative to non-inoculated plants. See Table 2 for abbreviations of genes silenced by RNAi by expression of inverted repeat (ir) constructs in the transformed lines. dpi, days post infection. n = 10.