# Production of Seedlings of Corymbia citriodora Inoculated with Endophytic Bacteria

^{1}

^{2}

^{*}

^{†}

## Abstract

**:**

^{−1}of IAA; and non-inoculated control without IAA and without a carrier. In the production of seedlings in a greenhouse, the treatments were the same, except for the mix, which was replaced by P. vagans strain 7URP1-6 (Pvs7), as inoculation with the mix increased the number of abnormal seedlings. In the physiological quality test of seeds, seeds inoculated with the bacteria individually did not have the physiological quality impaired and the carrier created a microenvironment around the seeds, benefiting germination percentage, germination speed index, average germination time, and average germination speed. In the greenhouse, seedlings inoculated with Pvs7, P. megaterium and E. sibiricum were taller, with a larger stem diameter and dry mass of shoot, roots, and total. Seeds inoculated with E. sibiricum had higher averages for height, chlorophyll b content, and shoot and total dry mass, as well as a greater ability to colonize the rhizosphere and roots of C. citriodora, resulting in the production of higher-quality seedlings. Inoculation of seeds of C. citriodora with endophytic bacteria proved to be a promising alternative for plant development.

## 1. Introduction

^{−1}of IAA by Rhizobium leguminosarum resulted in loss of seed vigor and the formation of abnormal lettuce seedlings [11]. The strains 7URP1-6 and 45URP4-1 of Pantoea vagans, 11URP4-2 of Priestia megaterium, 19RP3L2-7 of Exiguobacterium sibiricum, and 58CRP4-3 of Bacillus sp., used in the study, produce between 85 and 129 μg mL⁻¹ of IAA in vitro [12].

## 2. Material and Methods

#### 2.1. Physiological Quality of Seeds

#### 2.1.1. Inoculant Production and Seed Inoculation

^{®}, inputs and technology, with a control record CITRINCO8NV and germination power of 80%. The seeds were disinfested in a laminar flow chamber (Filterflux, São Paulo, Brazil). For this, the seeds were washed six times with distilled and sterilized water and immersed in 70% alcohol solution for 30 s and 5% sodium hypochlorite for 15 min and then rinsed six times with distilled and sterilized water [13].

^{9}CFU mL

^{−1}, using the saline solution as a blank [14].

^{®}papers.

#### 2.1.2. Treatments and Experimental Design

^{−1}of IAA previously dissolved in distilled and sterilized water [15]; and non-inoculated control, without IAA and without inoculation carrier. The inoculant mix was prepared using the volume of ¼ of each strain.

^{®}type moistened with distilled and sterilized water in the proportion of 2.5 times the mass of the dry paper [17]. Then, the seeds were incubated in a BOD germination chamber (Solab Sb224) at 25 °C and a photoperiod of 12 h.

#### 2.1.3. Evaluations

_{i}/t

_{i}), where n

_{i}= the number of seeds that germinated at time “i”, t

_{i}= time after installation of the test; and i = 1 ⟶ 12 days [18]. The counts were made until the germination was stabilized.

- -
- Germination percentage (GP): Calculated by the formula GP = (NG/NP) × 100, where NG = number of seeds germinated at the end of the test and NP = number of seeds placed to germinate;
- -
- Average germination speed (AGS): Calculated by the equation AGS = 1/t where t = average germination time;
- -
- Average germination time (AGT): calculated by the equation AGT = (∑n
_{i}t_{i})/∑n_{i}, where n_{i}= number of germinated seeds per day; t_{i}= incubation time; and i = 1 ⟶ 11 days; - -
- Percentage of dead seeds (PDS): PDS = (ND/NG) × 100, where ND = number of dead seeds and NG = number of seeds placed to germinate;
- -
- Entropy: calculated according to the procedure adopted by Labouriau and Valadares [19]. $\mathrm{E}={\sum}_{\mathrm{i}=1}^{\mathrm{k}}\mathrm{f}\mathrm{i}.\mathrm{l}\mathrm{o}\mathrm{g}2.\mathrm{f}\mathrm{i}$ where: E = informational entropy; fi = relative frequency of germination; and log2 = logarithm in base 2.

#### 2.1.4. Statistical Analysis

#### 2.2. Production of Seedlings in Greenhouse

#### 2.2.1. Treatments, Substrate, Conduction, Design, and Evaluations

^{−1}of IAA, synthesizing enzymes such as catalase, urease, and nitrate reductase, besides being able to solubilize inorganic and organic sources of phosphate, such as calcium phosphate and sodium phytate [12]. The inoculant production and inoculation were the same as in the previous experiment.

^{3}were filled with substrate composed of 70% coconut husk fiber and 30% carbonized rice husk and fertilized with 2 kg m

^{−3}of osmocote (NPK: 10-06-10), 1 kg m

^{−3}of Superphosphate Simple (NPK: 00-17-00) and 1 kg m

^{−3}of Mono Ammonium Phosphate (NPK: 12-61-00) and then three seeds inoculated with their respective treatments were sown in the substrate of each tube. Twelve days after sowing, once the emergence was stabilized, thinning was carried out, leaving one plant per tube. Fifteen days after sowing, a reinoculation with 5 mL per plant with its respective treatment was performed. The reinoculation was performed with the aid of a 20 mL disposable syringe coupled to a breast probe. The inoculant was applied close to the roots. Until the 60th day, the plants were fertilized weekly with Clark’s nutrient solution with ½ of the original strength and from 60° to 120° the complete Clark solution was applied [21].

#### 2.2.2. Bacterial Quantification of Shoots, Roots, and Rhizosphere

^{®}80 for two minutes for shoots and three minutes for roots and, finally, in 70% ethyl alcohol for 30 s. Then, the plant tissues were immersed in distilled and sterilized water and shaken manually for one minute; finally, they were rinsed with distilled water and sterilized four more times, changing water with each new wash [22].

_{2}PO

_{4}; 0.2 g of MgSO

_{4}; 0.1 g of NaCl; and 0.02 g of CaCl

_{2}; 2 mL of the micronutrient solution (0.04 g of CuSO

_{4}; 1.20 g of ZnSO

_{4}; 1.40 g of H

_{3}BO

_{3}; 1.00 g of Na

_{2}MoO

_{4}; and 1.175 g of MnSO

_{4}per liter of distilled water); and 4 mL of ferric EDTA (1.64% solution) and 4.5 g of KOH, with the final pH adjusted at 7.0 [22]. Then, one gram of shoot and root tissue was macerated in grail containing 9 mL of the saline above the solution.

^{−1}dilution, with serial dilution up to 10

^{−4}for shoots and 10

^{−5}for roots and rhizosphere. From the tubes of the last three dilutions of each part (shoot, roots, and rhizosphere), 0.1 mL of the suspension was removed and transferred to Petri plates with TSA medium (Tryptone Soy Agar). For each dilution, four replications were used and the plates were incubated in a BOD (Solab Sb224, São Paulo, Brazil) chamber for three days at 28 °C. After this period, the number of colonies was counted in the dilutions that allowed growth between 25 and 300 colonies. The bacterial density was calculated considering the dilution and the aliquot inoculated in the plate and expressed in the number of CFU per gram of tissue, with the average of the plates of each treatment whose number of colonies was between 25 and 300.

#### 2.2.3. Statistical Analysis

## 3. Results

#### 3.1. Physiological Quality of Seeds

#### 3.2. Production of Seedlings in Greenhouse

^{−1}of IAA. Group II: P. vagans strain 45URP4-1 and Bacillus sp. Group III: E. sibiricum. Group IV: P. vagans strain 7URP1-6 and P. megaterium (Figure 1).

## 4. Discussion

^{−1}(Table 1). The best-known effect of auxins is the stimulation of rooting, increasing the area of soil exploitation by the roots and also the absorption of water and nutrients [36,37], consequently favoring plant development, as observed in the study, namely with respect to taller plants, with larger stem diameter and dry mass of the roots, shoot, and total (Figure 2). Sousa et al. [38] inoculated E. urophylla seeds with Azospirillum amazonense and Stenotrophomonas maltophilia and also observed better development of height, stem diameter, root length, and dry matter.

## 5. Conclusions

## Author Contributions

## Funding

## Data Availability Statement

## Acknowledgments

## Conflicts of Interest

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**Figure 1.**Grouping of dendrogram treatments with Euclidean distance considering the variables plant height, stem diameter, chlorophyll a and b content, and dry mass of roots, shoot, and total plants of C. citriodora inoculated with endophytic bacteria and treated with cassava starch and activated charcoal (carrier), carrier with 1.0 μg mL

^{−1}of IAA and control without inoculation (Control).

**Figure 2.**Canonical discriminant analysis of the variables plant height (PH); stem diameter (SD); shoot dry mass (SDM), root dry mass (RDM); total dry mass (TDM); chlorophyll a (Ca) and b (Cb) content of Corymbia citriodora plants treated with cassava starch and activated charcoal; the carrier with 1.0 μg mL

^{−1}of IAA and control without inoculation (GI) and inoculated with Pantoea vagans strain 45URP4-1 and Bacillus sp. (GII); Exiguobacterium sibiricum (GIII); and P. vagans strain 7URP1-6 and Priestia megaterium (GIV).

**Figure 3.**Correlation network illustrating Pearson’s correlations between the variables chlorophyll a (Ca) and b (Cb); the number of colony-forming units of the shoot (CFUSH), roots (CFUR), and rhizosphere (CFURH); stem diameter (SD); plant height (PH); and dry mass of roots (RDM), shoot (SDM), and total (TDM) of Corymbia citriodora plants inoculated with Pantoea vagans strains 7URP1-6 and 45URP4-1, Priestia megaterium, Exiguobacterium sibiricum, Bacillus sp., inoculation carrier, and the carrier with 1.0 μg mL

^{−1}of IAA and control without inoculation. The thicker greener lines represent the highest positive correlations. The red lines represent the negative correlations.

Species—Strain (GenBank Code) | Isolated From | Fertilization of the Substrate of Occurrence of the Host Plant | Synthesis of IAA In Vitro (μg mL⁻¹) | Enzymes Synthesized In Vitro | Gram Staining |
---|---|---|---|---|---|

Priestia megaterium—11URP4-2 (OQ983563) | Rooted cutting of Eucalyptus grandis × Eucalyptus urophylla | Yes | 87 | Catalase, urease | Negative |

Exiguobacterium sibiricum—19RP3L2-7 (OQ983562) | Adult plant of E. camaldulensis | No | 90 | Catalase, nitrate reductase, and phytate solubilizing phosphatase | Positive |

Pantoe vagans—45URP4-1 (OQ983564) | Rooted cutting of E. grandis × E. urophylla | Yes | 129 | Catalase, nitrogenase, urease, nitrate reductase, and calcium and phytate solubilizing phosphatases | Negative |

Bacillus sp.— 58CRP4-3 (OQ983565) | Seedlings of E. cloeziana | Yes | 95 | Catalase, urease, nitrate reductase, and producer of siderophores | Positive |

**Table 2.**Analysis of variance for germination percentage (GP), percentage of dead seeds (PDS), germination speed index (GSI), average germination time (AGT), average germination speed (AGS), number of normal (NS) and abnormal (AS) seedlings, and entropy (ENT) of Corymbia citriodora seeds inoculated with Pantoea vagans strain 45URP4-1, Priestia megaterium, Exiguobacterium sibiricum, Bacillus sp., inoculation carrier, inoculation carrier with 1.0 μg mL

^{−1}of IAA and control without inoculation.

Source of Variation | Mean Square | |||||||
---|---|---|---|---|---|---|---|---|

GP (%) | PDS (%) | GSI | AGT (Days) | AGS (Days^{−1}) | NS | AS ^{+} | ENT (Bits) | |

Treatment | 44.57 ** | 44.57 ** | 35.62 ** | 0.49 ** | 0.005 ** | 13.43 ** | 0.24 ** | 0.05 ^{ns} |

Residue | 9.95 | 9.50 | 3.58 | 0.07 | 0.001 | 3.07 | 0.06 | 0.04 |

CV (%) | 3.59 | 25.86 | 9.37 | 9.1 | 9.42 | 4.06 | 18.85 | 9.30 |

^{ns}not significant; CV: coefficient of variation;

^{+}statistics of data transformed by √(x + 1).

**Table 3.**Germination percentage (GP), percentage of dead seeds (PDS), germination speed index (GSI), average germination time (AGT), average germination speed (AGS), number of abnormal (AS), and normal (NS) seedlings of Corymbia citriodora seeds inoculated or not with endophytic bacteria.

Treatments | GP (%) | PDS (%) | GSI | AGT (Days) | AGS (Days^{−1}) | AS * | NS |
---|---|---|---|---|---|---|---|

Control | 85.20 b | 14.80 a | 14.52 b | 3.59 a | 0.28 b | 0.8 b | 41.80 b |

Carrier | 92.40 a | 7.60 b | 21.07 a | 2.77 b | 0.36 a | 0.4 b | 45.80 a |

Carrier with 1.0 μg mL^{−1} of IAA | 86.80 b | 13.20 a | 20.78 a | 2.68 b | 0.38 a | 0.6 b | 42.80 b |

Pantoea vagans strain 45URP4-1 | 91.60 a | 8,40 b | 23.58 a | 2.57 b | 0.39 a | 0.4 b | 45.40 a |

Bacillus sp. | 86.80 b | 13.20 a | 21.61 a | 2.69 b | 0.38 a | 0.4 b | 43.00 b |

Exiguobacterium sibiricum | 86.60 b | 16.40 a | 18.97 a | 2.84 b | 0.36 a | 0.6 b | 41.20 b |

Priestia megaterium | 88.40 b | 11.60 a | 19.61 a | 2.88 b | 0.35 a | 0.8 b | 43.40 b |

Mix | 87.40 b | 12.40 a | 21.34 a | 2.86 b | 0.35 a | 2.4 a | 41.40 b |

**Table 4.**Multivariate analysis of variance for the vectors of treatment averages of Corymbia citriodora plants inoculated with Pantoea vagans strains 7URP1-6 and 45URP4-1, Priestia megaterium, Exiguobacterium sibiricum, Bacillus sp., inoculation carrier, and inoculation carrier with 1.0 μg mL

^{−1}of IAA and control without inoculation.

Statistics | Value | ^{1}num Df | ^{2}den Df | Aprox. F | Pr > F |
---|---|---|---|---|---|

Wilks Lambda | 0.05 | 49 | 136.42 | 2.26 | 0.0001 *** |

Pillai trace | 1.72 | 49 | 224 | 1.49 | 0.03 * |

Hotelling-Lawley trace | 7.78 | 49 | 170 | 3.85 | 3.77 × 10^{−11} *** |

Roy maximum root | 6.63 | 7 | 32 | 30.33 | 2.16 × 10^{−12} *** |

^{1}num Df: degree of freedom of the numerator;

^{2}den Df: degree of freedom of the denominator. ‘***’ 0.001 ‘*’ 0.05; Pr > F: F-test significance.

**Table 5.**Means of plant height, stem diameter, chlorophyll a and b content, and dry mass of roots, shoot, and total of Corymbia citriodora plants treated with endophytic bacteria.

Variables | Group | |||
---|---|---|---|---|

I | II | III | IV | |

Plant height (cm) | 14.08 | 14.44 | 16.97 | 16.04 |

Stem diameter (mm) | 1.20 | 1.41 | 1.42 | 1.46 |

Chlorophyll a | 29.4 | 28.89 | 28.75 | 28.09 |

Chlorophyll b | 8.43 | 8.37 | 8.71 | 8.23 |

Roots dry mass (g) | 1.04 | 1.02 | 1.04 | 1.05 |

Shoot dry mass (g) | 3.78 | 4.31 | 4.79 | 4.12 |

Total dry mass (g) | 4.82 | 5.33 | 5.83 | 5.16 |

^{−1}of IAA and control without inoculation; GII: Pantoea vagans strain 45URP4-1 and Bacillus sp.; GIII: Exiguobacterium sibiricum; GIV: P. vagans strain 7URP1-6 and Priestia megaterium.

**Table 6.**Density of bacteria (CFU g of tissue

^{−1}) in the rhizosphere, roots, and shoot of Corymbia citriodora plants inoculated or not with endophytic bacteria.

Treatments | CFU g of Tissue^{−1} | ||
---|---|---|---|

Rhizosphere | Root | Shoot | |

Control | 4.0 × 10^{7} e | 4.2 × 10^{7} e | 6.4 × 10^{4} d |

Carrier | 1.6 × 10^{8} d | 4.2 × 10^{7} e | 4.4 × 10^{5} b |

Carrier with 1.0 μg mL^{−1} of IAA | 2.2 × 10^{7} e | 1.8 × 10^{8} c | 3.0 × 10^{5} c |

Pantoea vagans strain 7URP1-6 | 7.1 × 10^{7} e | 2.9 × 10^{8} b | 6.1 × 10^{5} a |

Priestia megaterium | 1.9 × 10^{8} c | 7.8 × 10^{7} d | 4.3 × 10^{5} b |

Exiguobacterium sibiricum | 4.2 × 10^{8} a | 5.3 × 10^{8} a | 6.6 × 10^{4} d |

P. vagans strain 45URP4-1 | 2.9 × 10^{8} b | 1.5 × 10^{8} c | 2.0 × 10^{5} c |

Bacillus sp. | 2.3 × 10^{8} c | 9.4 × 10^{7} d | 1.3 × 10^{5} d |

CV (%) | 22.8 | 17.6 | 31.3 |

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## Share and Cite

**MDPI and ACS Style**

de Oliveira, A.M.; de Abreu, C.M.; Grazziotti, P.H.; de Andrade, G.F.P.; Gomes, J.V.; Avelino, N.R.; Menezes, J.F.S.; Barroso, G.M.; dos Santos, J.B.; da Costa, M.R.
Production of Seedlings of *Corymbia citriodora* Inoculated with Endophytic Bacteria. *Forests* **2024**, *15*, 905.
https://doi.org/10.3390/f15060905

**AMA Style**

de Oliveira AM, de Abreu CM, Grazziotti PH, de Andrade GFP, Gomes JV, Avelino NR, Menezes JFS, Barroso GM, dos Santos JB, da Costa MR.
Production of Seedlings of *Corymbia citriodora* Inoculated with Endophytic Bacteria. *Forests*. 2024; 15(6):905.
https://doi.org/10.3390/f15060905

**Chicago/Turabian Style**

de Oliveira, Augusto Matias, Caique Menezes de Abreu, Paulo Henrique Grazziotti, Gabriel Faria Parreiras de Andrade, Jaqueline Vieira Gomes, Natanielly Rodrigues Avelino, June Faria Scherrer Menezes, Gabriela Madureira Barroso, José Barbosa dos Santos, and Márcia Regina da Costa.
2024. "Production of Seedlings of *Corymbia citriodora* Inoculated with Endophytic Bacteria" *Forests* 15, no. 6: 905.
https://doi.org/10.3390/f15060905