The goal of the present study was to characterize anatomical and biochemical changes in rice plant roots in response to seed treatment with rhizobacteria (
Upland rice is planted in few regions worldwide. However, it presents advantages compared to floodland rice due to its lower production costs and water consumption. The average productivity of upland rice has been under 3 ton·ha−1, whereas the productivity potential of improved cultivars is greater than 5 ton·ha−1. The low productivity is attributed to water stress, which causes low initial vigor of the seedling root, deficiency in the uptake of nitrogen in the form of nitrate (
Biofertilizers are fertilizers composed of living microorganisms that promote plant growth when in contact with seeds or roots [
Inoculation with isolates of
The characterization of histological and biochemical changes resulting from interactions between plant growth-promoting microorganisms (PGPMs) or bioagents and plant growth is important to understand the mechanisms involved in the growth process. This knowledge will be useful for the optimization of the current production system, making it more sustainable and environmentally friendly.
Bioagents constitute a sustainable option for plant production systems. In the present study, the hypothesis was advanced that bioagents R-46, R-55, and
The tested
The treatments were composed of rice seeds (cultivar. Primavera Clearfield) microbiolized with T1: a mixture of four isolates of
The tested rhizobacteria (
The four isolates of
Rice seeds were sterilized with 70% ETOH and 2% NaClO, both for 1 minute, washed in sterile water for 1 minute, and placed on sterile filter paper, where they remained for 1 hour. The seeds were then steeped in the suspensions for each treatment for 24 hours at 28°C and at 115 rpm and were subsequently sown.
Two experiments were carried out: the first one in glass tube (60 mL) and the other in polyethylene plastic tube (180 cm3). The two experiments were completely randomized, with six treatments and ten replicates; the number of samples was 20 per treatment. The assay was performed in duplicate.
The plants were collected from the test tubes 21 days following sowing. The roots were carefully washed and taken to the Botany Laboratory of the Brazilian Enterprise for Agricultural Research (Embrapa) Eastern Amazon (Laboratório de Botânica da Embrapa Amazônia Oriental), fixed in FAA50 (50 mL of 37% formaldehyde, 50 mL of glacial acetic acid, and 900 mL of 50% ethanol), and subjected to vacuum in a desiccator for 24 hours. The fixative was then discarded, and the roots were stored in 70% ethanol until further processing.
Part of the samples was rehydrated in a graded ethanolic series to obtain sections [
Another part of the samples was dehydrated in a graded increasing ethanolic series, critical point-dried (CO2), coated with a thin layer of gold, and observed using a scanning electron microscope (SEM). Cross-sections were obtained from the roots of three plants per treatment. The images obtained were used to determine the area occupied by the aerenchyma using the ANTI QUANT 2 software.
Twenty-one days after growth, ten plants were collected from each treatment, and the following measurements were performed: leaf and root length, root/leaf ratio (cm), dry mass of leaves and roots, and root/leaf ratio (g). The dry mass was measured following drying of the plant material in a forced air circulation oven for 7 days at 65°C, using an analytical scale.
Ten plants of each treatment were collected, and three replicates were performed, for each evaluation. Roots were separated from the shoot, washed, and placed in a forced air circulation oven at 65°C for 7 days, until a constant mass was reached. The roots were then ground using liquid nitrogen and stored at ±4°C.
Total lignin and lignin monomers: samples were homogenized in 50 mM sodium phosphate buffer, pH 7.0, purified in 1% Triton X-100, 1 M NaCl, and acetone, according to [
Total phenols and flavonoids: one gram of root was extracted in 20 mL methanol (80 : 20, v/v) for 15 minutes, and the extract was resuspended in 20 mL of 1% Triton X-100. The extract was used for the determination of the total phenol content using a spectrophotometer (absorbance 735 nm). A standard curve was obtained using gallic acid. Flavonoid contents were determined in extracts in aluminum chloride by a colorimetric assay (735 nm) [
An ANOVA was performed, followed by a Duncan test (
The root length and dry mass of plants treated with biopromoters were higher than control treatment. Significant differences in root length and root/leaf ratio were observed among the treatments with seeds microbiolized with the four
Length (cm) and dry mass (g) of leaves, root, and root/leaf ratio of rice plants obtained from seeds microbiolized with PGPMs, 21 days following sowing in tubes (180 cm3) containing Gernino plant substrate.
Treatment | Length (cm) | Dry mass (g) | ||||
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Leaves | Root | Root/leaf ratio | Leaves | Root | Root/leaf ratio | |
|
33.5 ± 1.5(2) a(3) | 24.3 ± 1.9 ab | 0.73 ± 0.23 a | 2.527 × 10−2 ± 2.0 × 10−3 a | 1.079 × 10−2 ± 0.2 × 10−2 a | 0.429 ± 0.32 a |
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30.3 ± 1.5 a | 22.6 ± 1.8 ab | 0.75 ± 0.17 ab | 1.645 × 10−2 ± 7.0 × 10−3 c | 0.733 × 10−2 ± 0.4 × 10−2 c | 0.464 ± 0.86 a |
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31.1 ± 1.6 a | 17.5 ± 2.1 bc | 0.63 ± 0.26 bc | 2.120 × 10−2 ± 1.0 × 10−3 b | 0.912 × 10−2 ± 0.7 × 10−2 b | 0.412 ± 0.24 a |
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33.1 ± 1.5 a | 16.3 ± 1.6 c | 0.49 ± 0.16 c | 1.500 × 10−2 ± 3.8 × 10−3 c | 0.680 × 10−2 ± 0.4 × 10−2 d | 0.450 ± 5.33 a |
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29.7 ± 1.3 a | 14.3 ± 1.8 c | 0.48 ± 0.25 c | 1.530 × 10−2 ± 2.0 × 10−3 c | 0.615 × 10−2 ± 0.2 × 10−2 d | 0.402 ± 0.15 b |
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Control (water) | 28.2 ± 1.8 a | 14.9 ± 2.5 c | 0.53 ± 0.28 c | 1.182 × 10−2 ± 2.0 × 10−3 d | 0.464 × 10−2 ± 0.3 × 10−2 e | 0.379 ± 0.19 b |
Plants treated with the mix of the four
The root diameter increased in all the treatments with PGPMs. This change was especially pronounced for the treatments with
Root diameter, cortex and exodermis thickness, lacunae aerenchyma area (
Treatment | Root diameter ( |
Cortex thickness ( |
Exodermis thickness ( |
Aerenchyma lacunae ( |
Vascular cylinder diameter ( |
Number of protoxylem poles | Number of metaxylem vessel elements |
---|---|---|---|---|---|---|---|
|
375.05 ± 59.60(2) c(3) | 159.16 ± 17.03 b | 12.1 ± 1.07 a | 9.77 × 106 ± 18.99 × 103 a | 229.6 ± 26.97 a | 18.66 ± 6.11 a | 3.66 ± 1.10 a |
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373.94 ± 70.98 c | 161.13 ± 14.09 b | 13.28 ± 0.48 a | 9.76 × 106 ± 0.55 × 103 a | 221.4 ± 80.92 ab | 20.00 ± 1.90 a | 4.00 ± 1.90 a |
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456.63 ± 13.37 a | 197.28 ± 11.10 a | 12.3 ± 0.80 a | 9.70 × 106 ± 18.9.93 × 103 ab | 229.6 ± 26.97 a | 19.00 ± 1.90 a | 4.33 ± 1.10 a |
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369.37 ± 49.53 c | 155.48 ± 3.43 b | 11.8 ± 1.07 a | 9.72 × 106 ± 3.80 × 103 ab | 213.0 ± 53.95 ab | 18.00 ± 3.80 a | 4.66 ± 1.10 a |
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411.11 ± 63.51 b | 174.32 ± 15.58 b | 12.79 ± 0.80 a | 9.68 × 106 ± 0.38 × 103 bc | 205.0 ± 26.97 ab | 17.33 ± 2.90 a | 2.33 ± 1.10 b |
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Control (water) | 303.81 ± 46.22 d | 130.13 ± 12.32 c | 7.38 ± 0.96 b | 9.61 × 106 ± 0.76 × 103 c | 180.4 ± 26.97 b | 13.00 ± 7.60 b | 2.00 ± 1.90 b |
Plants originating from seeds microbiolized with PGPMs presented more adventitious roots, based on visual assessments, compared to control plants (Figure
Adventitious roots of rice plants originating from microbiolized seeds. (a) Control plant (nonmicrobiolized seeds). (b–f) Roots of plants from seeds microbiolized with (b)
Electromicrographs of adventitious roots of rice obtained from seeds treated with PGPMs, 21 days following sowing. (a1–a3)
The cortex and exodermis thickness and number of protoxylem poles were significantly higher in the roots of all the PGPM treatments. The treatments with
The lacunae of aerenchyma area and the number of metaxylem vessel elements increased in all the treatments with PGPMs, except for the treatment with the rhizobacteria (R-55 and R-46) and
The total phenol and flavonoid contents increased in all the plants treated with bioagents (Table
Phenols, flavonoids, lignin, and lignin monomer contents of roots of rice plants obtained from seeds microbiolized with bioagents, 21 days following sowing.
Treatment | Phenols | |||
---|---|---|---|---|
Total phenols | Flavonoids | Lignin | Lignin monomers | |
|
64 × 10−2 ± 0.15 × |
10.93 × 10−2 ± 0.67 × 10−2 a | 2.93 × 10−2 ± 1.05 × 10−2 a | 1.44 × 10−2 ± 1.37 × 10−2 c |
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8.15 × 10−2 ± 1.21 × 10−2 ab | 8.05 × 10−2 ± 0.62 × 10−2 b | 0.40 × 10−2 ± 0.10 × 10−2 b | 0.49 × 10−2 ± 0.30 × 10−2 c |
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9.11 × 10−2 ± 1.47 × 10−2 a | 8.16 × 10−2 ± 0.34 × 10−2 b | 0.42 × 10−2 ± 1.82 × 10−2 b | 2.47 × 10−2 ± 1.83 × 10−2 b |
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8.92 × 10−2 ± 2.27 × 10−2 a | 8.06 × 10−2 ± 0.74 × 10−2 b | 2.18 × 10−2 ± 1.82 × 10−2 a | 4.05 × 10−2 ± 0.56 × 10−2 a |
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9.48 × 10−2 ± 1.96 × 10−2 a | 7.54 × 10−2 ± 0.37 × 10−2 c | 0.90 × 10−2 ± 1.45 × 10−2 b | 0.38 × 10−2 ± 0.73 × 10−2 c |
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Control | 6.80 × 10−2 ± 0.77 × 10−2 b | 6.79 × 10−2 ± 0.19 × 10−2 d | 0.74 × 10−2 ± 0.30 × 10−2 b | 0.47 × 10−2 ± 8.00 × 10−2 c |
All the results are presented in mg per g dry mass.
Treatment with
These root modifications promoted by PGPMs result in positive physiological responses of plants. This response was observed in
All the tested PGPMs resulted in an expansion of the root cortex up to 30% and of the exodermis up to 68% compared to the control, 21 days following sowing. This change was more pronounced for
Ethylene was identified as the hormonal signal mediating formation of aerenchyma in corn and rice [
The vascular cylinder diameter increased 27% in the treatments with
The plants treated with
All the tested PGPMs increased the levels of at least two phenols in roots. The maximum total phenol content was 42% in the plants treated with
The increase in lignin content was approximately 300% in the plants treated with
The rhizobacteria
All the root architecture modifications resulting from the interaction between seedlings and bioagents (rhizobacteria and
Brazilian Enterprise for Agricultural Research
Hypersensitivity reaction
Indol-3-acetic acid
Induced systemic resistance
Plant growth-promoting microorganisms
Plant growth-promoting rhizobacteria
Reactive oxygen species
Scanning electron microscopy.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors thank the National Council for Scientific and Technological Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)), Amazon Research Foundation (Fundação Amazônia Paraense de Amparo à Pesquisa (Fapespa)), and the Rural Federal University of Amazon (Universidade Federal Rural da Amazônia (UFRA)) for the research funding and the Brazilian Federal Agency for the Support and Evaluation of Graduate Education (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)) for the granting of a Master’s scholarship.