Current Plant Biology 27 (2021) 100209

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Current Plant Biology

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Acclimatization of Pouteria gardeneriana Radlk micropropagated plantlets:

Role of in vitro rooting and plant growth–promoting bacteria
Mariluza Silva Leite a, Tainara Eler Furtado Pinto a, Agda Rabelo Centofante a,
Aurélio Rubio Neto a, Fabiano Guimarães Silva a, Priscila Jane Romano Gonçalves Selari b, Paula
Fabiane Martins a, *
a
Instituto Federal de Educação, Ciência e Tecnologia Goiano, Rodovia Sul Goiana, Km 01, 75.901-970, Rio Verde, Goiás, Brazil
b
Instituto Federal de Educação, Ciência e Tecnologia Goiano, Rodovia GO 154, Km 03, 76.300-000, Ceres, Goiás, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Pouteria gardneriana Radlk (guapeva) is a tree native to the Brazilian Cerrado with economic, ecological, and
Galleria mellonella biotechnological importance. Guapeva yield is limited mainly by the seeds recalcitrance, that can be overcome
Micropropagation by micropropagation technique and effective acclimatization. The objective of this study was to investigate the
Acclimatization
role of in vitro rooting and inoculation of plant growth–promoting bacteria (PPGPB) during acclimatization of
Rooting
PGPB
micropropagated P. gardneriana plantlets. In the first bioassay, the presence of roots in the plantlets resulted in
higher values of biometric features, such as leaf area, numbers of leaves and secondary roots, nodal segments,
fresh and dry weights of leaves; when compared to plantlets without roots. Roots well established also resulted in
higher levels of chlorophyll a, chlorophyll b, and total chlorophyll and a higher photosynthetic rate in micro­
propagated P. gardneriana plantlets. Fr the second biassay, the PGPB inoculation was done with bacteria to
solubilize phosphate and phytohormone production, in addition to low pathogenic potential in the Galeria
mellonella larva animal model. The inoculation of the Bac109 and BP323EB consortium in plantlets rooted in vitro
had a positive effect on the root length of the plantlets during the acclimatization phase, which may favor the
long-term development of the plant. This study demonstrated that both in vitro rooting and inoculation of a
bacterial consortium may contribute to the growth of P. gardneriana during acclimatization, leading to higher
productivity and seedling quality.

1. Introduction properties [4,6].

Guapeva (Pouteria gardneriana Radlk) is a tree native to the Cerrado
The Brazilian savannah, known as Cerrado, is the second-largest that belongs to the family Sapotaceae. The potential for biotechnological
domain in the country, with immense biodiversity, and considered a application is diverse. In pharmaceutical products, guapeva has been
global biodiversity hotspot [1,2]. For this reason, deforestation in the proposed due to its high levels of phenolic compounds and condensed
region has been a major concern, and bioprospecting studies that add tannins [7]. In livestock studies, acaricidal activity from guapeva etha­
value to such diversity represent potential conservation alternatives [3]. nolic extract adsorbed in chitosan nanoparticles showed reduction in the
Plants native to the Brazilian Cerrado have unique characteristics, eggs mass of Riphicephalus (Boophilus) microplus [8]. Equally important,
such as bioactive compounds of medical importance and fruits with high species from Pouteria genus showed an ecological role as a bioindicator
nutritional content [4–6]. These features have been highlighted for their for glyphosate herbicide [9].
biotechnological potential. The food and pharmaceutical industries have Micropropagation enables large-scale multiplication of plants in vitro
invested in the development of new products from these plants, based in axenic environments and at any time of the year, overcoming the
mainly on their antioxidant, anti-inflammatory, and antimicrobial difficulties encountered in natural propagation, such as seed

* Corresponding author.
E-mail addresses: [email protected] (M.S. Leite), [email protected] (T.E.F. Pinto), [email protected] (A.R. Centofante), aurelio.
[email protected] (A.R. Neto), [email protected] (F.G. Silva), [email protected] (P.J.R.G. Selari), [email protected]
(P.F. Martins).

https://doi.org/10.1016/j.cpb.2021.100209
Received 12 June 2020; Received in revised form 11 February 2021; Accepted 9 May 2021
Available online 12 May 2021
2214-6628/© 2021 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
M.S. Leite et al. Current Plant Biology 27 (2021) 100209

recalcitrance [10]. The success of micropropagation also depends on OD600 = 1.0 were inoculated onto the GELP medium, containing insol­
effective acclimatization strategies that ensure the survival of the uble phosphate for evaluation, and the plates were incubated at 28 ◦ C.
seedlings during the transfer from in vitro to ex vitro conditions, where The diameter of the translucent halo formed by phosphate solubilization
there is high light intensity, excessive water loss, and change from by the bacteria was measured after 120 h [25]. The experiment was
heterotrophic metabolism to autotrophic metabolism [11]. Alternatives conducted in triplicate. The solubilization index (SI) was calculated
to aid micropropagated seedlings during the acclimatization process according to the formula: SI = halo diameter (cm)/colony diameter (cm)
have been extensively studied to reduce losses due to mortality or to [26].
lowered productivity. One of these alternatives is the use of microor­
ganisms, such as plant growth–promoting bacteria (PGPB). 2.2.2. Synthesis of indole-3-acetic acid (IAA)
PGPB can contribute directly or indirectly to the promotion of plant The synthesis of IAA was evaluated by the colorimetric method
growth through nitrogen fixation, inorganic phosphate solubilization, described by Gordon and Weber [27]. For this, 1 mL aliquots of bacterial
production of phytohormones, siderophores and secondary metabolites culture with OD600 = 1.0 were inoculated in 5 mL of nutrient broth
with antimicrobial activity, which protect the plant against phytopath­ supplemented with 100 μg mL− 1 of tryptophan and incubated at 30 ◦ C
ogens [12–14]. PGPB may also increase plant tolerance to abiotic and 150 rpm, for 72 h in the absence of light. The control was culture
[15–17] and biotic stresses [18]. medium without bacteria. The cultures were then centrifuged (5 min,
For the successful large-scale propagation of plants, it is essential to 12,000 rpm and 10 ◦ C), and Salkowski reagent was added to the su­
establish efficient micropropagation and acclimatization protocols [19], pernatant (1.875 g FeCl3⋅6H2O, 100 mL H2O, and 150 mL H2SO4) at a
which have not yet been studied for P. gardneriana. This study aimed to ratio of 1:1.
evaluate the contribution of in vitro rooting and inoculation of PGPB The samples were incubated at room temperature, for 30 min, in the
from the Cerrado biome in the acclimatization of micropropagated absence of light, and then the absorbances were read at 530 nm in a
P. gardneriana plantlets, a species of economic and ecological impor­ spectrophotometer. The IAA concentrations synthesized were calculated
tance, native to the Brazilian Cerrado biome and a target of conservation by interpolating the absorbance results from a standard curve with
efforts. commercial IAA (Sigma-Aldrich, Germany).

2. Material and methods 2.2.3. Virulence test in G. mellonella larvae

The virulence of the strains Bac109 and BP323EB, alone and in
2.1. Biological material combination, was observed in the larvae of the G. mellonella moth. For
the experiment, larvae weighing between 150 and 200 mg were used.
2.1.1. Bacterial isolates The bacterial strains were grown in nutrient broth overnight. The
The bacterial isolates were obtained from the microorganism inoculum consisted of 10 μL of Bac109 solution at 1.3 × 106 CFU mL− 1,
collection of the Agricultural Microbiology Laboratory, Goiano Federal BP323EB at 1.3 × 108 CFU mL− 1, or the consortium of both strains at a
Institute of Education, Science and Technology (IF Gioano), Rio Verde, 1:1 ratio. The CFU concentrations were measured by plate method and
Goiás (GO), Brazil. The collection was obtained from the isolation of the ratio were chosen from preliminary tests.
endophytic bacteria from the roots of native plants from the Brazilian The bacterial solutions were injected into the proleg of the larvae
Cerrado. using a 22-gauge Gastight syringe (Hamilton, USA). For control, larvae
The bacterial species used in the present study were the Acinetobacter were injected with PBS alone, making five treatments with six larvae
lwoffii Bac109 strain, derived from Anacardium othonianum Rizzini, and each. The tests were conducted with larvae incubated at 28 ◦ C and 37 ◦ C.
the Enterobacter ludwigii BP323EB strain, isolated from Butia purpur­ Larval mortality was monitored up to 120 h. Dark-colored larvae un­
ascens, both characterized as having growth promotion potential [20, responsive to touch were considered dead [28].
21].
Although the bacterial co-cultivation has not been tested in labora­ 2.3. Establishment of P. gardneriana Radlk
tory in the present work, there are compatibility studies between the
species used. Lata et al. [22] cited both species, Acinetobacter lwoffii and 2.3.1. Seedling growth
Enterobacter ludwigii, as endophytic growth promoters of wheat (Triticum Firstly, P. gardneriana Radlk seeds were immersed in 5% sodium
aestivum), which may indicate co-growth in vivo. In laboratory cultiva­ hydroxide solution for 5 min to remove adhered mucilage [29]. Then,
tion studies, Xue et al. [23] evaluated two strains of Acinetobacter and the seeds were sown in plastic trays (53 × 37 × 8 cm), containing
Enterobacter as potential biocontrol agents against a tomato Ralstonia washed and sifted sand, and kept in a growth chamber at 25 ± 3 ◦ C,
wilt. under a photoperiod of 16 h, for 90 days. The seedlings were watered
every 15 days with nutrient solution of 50 % MS medium [30].
2.1.2. Plant material
Ripe fruits of P. gardneriana Radlk were collected between November 2.3.2. Inoculation and cultivation conditions
2015 and January 2016 at the Goiano Federal Institute of Education, The phytosanitary control was performed by spraying a systemic
Science and Technology, Rio Verde, GO, Brazil (latitude 17◦ 48′ 202′′ S, fungicidal solution (Derosal® 0.2 %) 24 h before inoculation. The nodal
longitude 50◦ 54′ 397′′ W, altitude of 749 m). segments (2 cm in length and with an axillary bud) were carefully
collected after 90 days of seedlings growing, covered with gauze and
2.2. Bacteria assays: plant growth promoting ability washed in running water for 15 min.
For disinfection, the explants were dipped in 70 % alcohol for 1 min
2.2.1. Calcium phosphate solubilization and 20 % sodium hypochlorite (brand with 2.0–2.5% of active chlorine)
The ability of Bac109 and BP323EB strains to solubilize insoluble for 20 min, and rinsed three times with sterile distilled water. All these
inorganic phosphate was tested in solid GELP medium (10.0 g L− 1 steps were carried out in a laminar flow hood.
glucose, 5.0 g L− 1 peptone, 0.05 g L− 1 yeast extract, and 15 g L− 1 water) The inoculation were made in tubes (25 × 150 mm) containing
[24]. This medium was supplemented with CaHPO4, which was gener­ 20 mL of 50 % MS medium [30], supplemented with 2.0 g L− 1 of acti­
ated by adding 50 mL of 10 % K2HPO4 and 100 mL of 10 % CaCl2 so­ vated charcoal and solidified with 3.5 g L− 1 agar. The pH was adjusted to
lution. For the inoculum, the bacterial strains were incubated in nutrient 5.7 ± 0.03, and PVC (polyvinylchloride) film was used to seal the flasks.
broth (3 g L− 1 meat extract and 5 g L− 1 peptone), at 28 ◦ C, with shaking The explants were maintained at 25 ± 3 ◦ C, with a light / dark cycle of
at 150 rpm overnight. Aliquots of 20 μL of bacterial solution at 16/8 h, 45 % relative humidity, and active photosynthetic radiation of

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M.S. Leite et al. Current Plant Biology 27 (2021) 100209

45− 55 μmol m-2 s− 1. The approach used for in vitro rooting consisted in 2.4.2.1. Physiological analyses. Gas exchange and chlorophyll a fluo­
kepping the culture medium without plant growth regulators and rescence were measured on the second fully expanded pair of leaves in
replacement, along 90 days. the period between 8 and 10 h. Chlorophyll a and b and total chlorophyll
Contamination during the in vitro propagation was not observed due were measured in the same leaf areas where leaf gas exchange and
to maintenance of aseptic conditions from a protocol adapted for chlorophyll a fluorescence were determined.
micropropagation of native plants of the Cerrado [29,31]. In addition, Gas exchange was assayed to determine the photosynthetic rate (A,
contamination by endophytic microorganisms was also not present, and μmol m-2 s-1), transpiration rate (E, mmol m-2 s-1), stomatal conduc­
all explants were feasible for bioassays. tance (gs, mol H2O m-2 s-1), and the ratio of internal CO2 concentrations
to external concentrations (Ci/Ca), using a portable open-system
2.4. Effect of in vitro rooting on acclimatization infrared gas analyzer (LI-6400, LI-COR Inc., Lincoln, NE, USA), under
an ambient CO2 concentration and room temperatures of 24–26 ◦ C. All
2.4.1. Preacclimatization phase of the measurements were conducted under artificial saturating photon
A preacclimatization phase (Fig. 1A) were carried out to improve the irradiance (1000 μmol m-2 s-1) at the leaf level.
plantlets adaptation from in vitro to ex vitro conditions. Three small holes The chlorophyll a fluorescence parameters were determined using a
were made in the PVC film to allow gas exchanges between the atmo­ portable fluorometer with a modulated pulse MINI-PAM (Walz®,
sphere inside and outside the tube. The growth conditions were the same Effeltrich, Germany) equipped with a leaf-clip 2030-B [32,33]. The leaf
as described before. At third day, the holes in PVC film have been was dark-adapted for 30 min and exposed to a weak far-red light pulse
enlarged. The plantlets remained under incubation for five days. (0.5 μmol m-2 s-1) for the determination of the initial fluorescence (F0).
A saturating light pulse (0.8 s; 2400 μmol (photons) m-2 s-1) was applied
2.4.2. Acclimatization assay to estimate the maximum emitted fluorescence (Fm). Using these pa­
The acclimatization steps are summarized in the Fig. 1B. The PVC rameters, the maximum quantum yield of photosystem II (PSII) was
film was removed and the plantlets were carefully taken out from tubes. calculated (Fv/Fm) [34].
Then, the roots were thoroughly washed with tap water and transferred In light-adapted leaves, the steady-state fluorescence yield (Fs) was
to 500 mL plastic pots. Three treatments were tested to see the effect of measured after a saturating white light pulse (2400 μmol m – 2 s–1,
rooting for seedling acclimatization: i) plantlets with well established 0.8 s) was applied to achieve the light-adapted maximum fluorescence
roots (Fig. 2A); ii) plantlets with poorly established roots (standardized (Fm′ ). The actinic light was turned off, and far-red illumination was
to 2 cm) (Fig. 2B); and iii) plantlets without roots (Fig. 2C). The roots applied (2 μmol m – 2 s–1) to measure the light-adapted initial fluo­
were standardized by pruning when it was required. rescence (F0′ ). Using these parameters, the effective quantum yield of
The plastic pots containing Bioplant® substrate (Fig. 2D–F) were PSII (ΔF/Fm’) was calculated [34]. ΔF/Fm’ was used to estimate the
covered with a transparent polythene bag (20 × 30 cm) to build a moist apparent electron transport rate (ETR)[36], and non-photochemical
chamber. However, after 7 days, the corners of the polythene bags were quenching (NPQ) was calculated according to Bilger and Bjorkman [37].
cut. Subsequently, the openings in the polythene bags were enlarged Chlorophyll a and b and total chlorophyll were determined using a
weekly, and after 30 days, the polythene bag were completely removed. portable meter (ClorofiLOG1030®, Falker®, Porto Alegre, Brazil) and
The plantlets were cultivated in a greenhouse, under irradiance that expressed as the Clorofilog index.
varied throughout the day, from a minimum of 29 μmol m− 2 s-1 and a
maximum of 322 μmol m− 2 s-, at 26 ◦ C (average), 66 % relative hu­ 2.5. Effect of PGPB on acclimatization
midity, and 50 % shading. Manual irrigation was performed by applying
10 mL / pot, once a week, and became daily, after removing plastic bag. Plantlets with well established roots were used to evaluate the effect
In addition, fertirrigation was performed with 10 mL / pot of nutrient of strains Bac109 and BP323EB on acclimatization. The bacterial strains
solution composed of 50 % MS medium salts, each 15 days. were cultured in nutrient broth overnight, at 30 ◦ C and 150 rpm for
At the end of the experiment, day 53, the following parameters were preinoculum production, and four treatments were tested: control
evaluated: survival rate (%); number of main and secondary roots; (absence of bacterial inoculant), Bac109 strain inoculation, BP323EB
length of the longest root (cm); number of leaves; number of segments; strain inoculation, and inoculation of both strains.
seedling length (cm); fresh and dry weights of leaves, roots, and stems The bacterial solutions were centrifuged for 10 min at 4000 rpm. The
(g); leaf and root area (cm2). The leaf and root areas were calculated supernatant was discarded and washed four times with 0.85 % saline
from the integration of the images in ImageJ software [32]. The dry solution. The bacterial pellet was then resuspended in saline until
weight was determined after drying the material in an oven at 65 ◦ C reaching an OD600 of 1.0. For standardization of the inoculum, 108 cells
until it reached a constant weight. mL− 1 were used.
After preacclimatization stage described before, and each plantlets

Fig. 1. Preacclimatization (A) and acclimatization (B) workflows of Pouteria gardeneriana Radlk micropropagated plantlets.

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Fig. 2. P. gardneriana Radlk seedlings cultured in vitro. (A and D) Seedlings with well established roots, (B and E) seedlings with poorly established roots (2 cm), and
(C and F) seedlings without roots transferred to plastic pots containing Bioplant® substrate. Bar: 2 cm.

received 2 mL of inoculation solution directly in the substrate near the 3. Results

roots [38]. The control group received only saline solution. The condi­
tions for cultivation were the same as previously described, including 3.1. Characterization of bacterial isolates
the moisture chamber steps. At the end of 60 days, the number of leaves,
shoot and root lengths, and fresh and dry weights of stems, roots, and Solubilization of inorganic phosphate was detected by radial diffu­
leaves were evaluated. sion in the culture medium and formation of a solubilization halo.
A. lwoffii Bac109 did not form a phosphate solubilization halo, so its SI
2.6. Statistical analyses value was equal to 0 for all replicates. The mean SI value of E. ludwigii
BP323EB was 1.12. A. lwoffii Bac109 produced 4.34 μg mL− 1 IAA, while
The larval survival curve in the virulence experiment was drawn in BP323EB strain produced 17.33 μg mL− 1.
GraphPad Prism 6 using the Kaplan-Meier method. The curves were
compared using the log-rank test (Mantel-Cox) and Bonferroni correc­ 3.2. Virulence test from bacteria against G. mellonella larvae
tion. To evaluate the effect of in vitro rooting on acclimatization, the
experimental design used was completely randomized, with three To test the pathogenicity in the animal model, the isolates were
treatments (and five replicates with six seedlings each, totaling 90 injected into G. mellonella larvae. After seven days of incubation at 28 ◦ C,
seedlings). For the evaluation of the effect of the PGPB, the experimental the survival rate of larvae inoculated with A. lwoffii Bac109 was 100 %
design consisted of blocks with four treatments in five blocks of four and of those inoculated with E. ludwigii BP323EB was 50 % (Fig. 3).
plantlets each, totaling 20 plantlets per treatment, for a total of 80 When the two strains were inoculated together, larval survival dropped
plantlets. The material was selected to ensure homogenization and to 33.3 %. At 37 ◦ C, 83.3 % of the larvae survived treatment with the
respecting the principle of randomness. The mean values of IAA syn­ Bac109 strain, and 33.3 % survived treatment with the BP323EB strain.
thesis, phosphate solubilization, and biometric data were statistically The larval survival rate was 16.7 % when the larvae were inoculated
evaluated by analysis of variance, applying the F test, with comparison with the bacterial consortium. All control larvae (untreated or inocu­
of means by Tukey’s test (P < 0.05), within SISVAR software [39]. lated with PBS) survived throughout the experiment at both tempera­
tures. Survival with PGPB differed from survival in the control group at
the P < 0.001 level and showed that bacterial virulence was
temperature-dependent, with the highest mortality rate at 37 ◦ C.

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Fig. 3. Survival of G. mellonella larvae inoculated with A. lwoffii Bac109 and E. ludwigii BP323EB at 28 ◦ C and 37 ◦ C. Untreated: larvae were not inoculated; PBS:
larvae were inoculated with PBS only; BP323EB: larvae were inoculated with E. ludwigii BP323EB; Bac109: larvae were inoculated with A. lwoffii Bac109. Significance
was assessed by the log-rank test (Mantel-Cox) with Bonferroni correction (P < 0.001).

3.3. Effect of in vitro rooting on acclimatization The highest levels of chlorophyll a, chlorophyll b, total chlorophyll
and photosynthetic rate (A) were obtained in the treatment of seedlings
Even without the addition of growth regulators, all explants had with well established roots (means of 25.28, 7.02, 32.30 μg cm− 2, and
rooted by 90 days of incubation. The differences in root growth ranged 1.72 μmol m− 2 s-1, respectively) and/or partial roots (mean 21.45; 5.41;
from 0.5 to 20 cm. Shoot growth did not show a relationship with root 26,86 μg cm− 2, and 1.22 micromol m− 2 s-1), indicating the importance
size. In acclimatization bioassay, after 53 days in the greenhouse, the of roots to acclimatization. The lowest means, 18.10, 4.31,
plants exhibited visible root growth (Fig. 4). 22.41 μg cm− 2, and 0.03 μmol m− 2 s-1, respectively, were obtained when
The number of leaves and segments, leaf area, secondary root the micropropagated seedlings were transplanted without roots (Fig. 6A
number and fresh and dry weights of leaves did not differ between and B).
plantlets with well established roots and with poorly established roots, There were no differences in transpiration rate, stomatal conduc­
and also between plants with poorly established roots from the group tance, or the Ci/Ca ratio between the evaluated treatments. The observed
without roots (Fig. 5A, C and B). In addition, for these same features, means for these characteristics were 0.41, 0.34 mol m− 2 s-1, and
plants with well established roots differed from plants without roots. 0.79 mol, respectively (Supplementary Table 2).
Root area (Fig. 5C) and main-root length (Supplementary Table 1)
differed among the three treatments tested, being higher in plantlets 3.4. Effect of microbial inoculants on acclimatization
with well established roots, followed by seedlings with poorly estab­
lished roots and finally by seedlings, keeping the differences observed The root length was favored by bacterial consortium (Bac109 +
when started the experiment. The highest values for root area and main- BP323EB) inoculation, but not differed among the treatments Control,
root length were observed in seedlings acclimatized with well estab­ Bac109 and Bac323 (Fig. 7B). The number of leaves were not affect
lished roots, with means of 3.33 cm2 and 11.12 cm, respectively. It is positively by bacterial inoculation (Fig. 7A), but the plant length was
important to note that the plantlets from the treatment without roots, at higher in the treatments with bacterial consortium (Bac109 + BP323EB)
the end of 53 days, showed growth in this organ, reaching an average of (Fig. 7C).
1.00 cm2 for the main-root length and 2.82 cm2 for root area. The bacterial strains did not increase the fresh weight of
Shoot length, number of main roots, survival rate, and stem dry and P. gardneriana leaves, stems, or roots (Supplemental Table 3), nor did
fresh weights did not vary as a function of treatment (Supplementary they increase the dry weight of the plantlets (Supplementary Table 4).
Table 1). The species showed a high percentage of survival (mean of
62.2 %) during acclimatization, regardless of the root conditions of the
plantlets obtained from the in vitro culture.

Fig. 4. P. gardneriana Radlk seedlings acclimatized in a greenhouse at 53 days. Plants originated from in vitro culture with well established roots. (A), with poorly
established roots (B), and without roots (C). Bar: 4 cm.

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M.S. Leite et al. Current Plant Biology 27 (2021) 100209

Fig. 5. Biometric data at 53 days of acclimatization of P. gardneriana seedlings that originated from micropropagated seedlings with well established roots, poorly
established roots or without roots. (A) Number of leaves and segments. (B) Root fresh and dry weight. (C) Leaf and root area. (D) Leaf fresh and dry weight. zMeans
followed by the same letter do not differ by Tukey’s test at 0.05 probability.

Fig. 6. Physiological data at 53 days of acclimatization of P. gardneriana seedlings that originated from micropropagated seedlings with well established roots, with
poorly established roots, or without roots. (A) Chlorophyll a, b, and total. (B) Photosynthetic rate. zMeans followed by the same letter do not differ by Tukey’s test at
0.05 probability.

Fig. 7. Biometric data at 60 days of acclimatization of P. gardneriana seedlings that originated from seedlings micropropagated inoculated with bacteria. (A) Leaves.
(B) Root length. (C) Plant length. zMeans followed by the same letter do not differ by Tukey’s test at 0.05 probability.

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4. Discussion 37 ◦ C, and more than 80 % of the larvae survived, which demonstrates

that the strain is not pathogenic to the model tested. After seven days of
P. gardneriana is a plant species with economic, ecological, and the experiment, E. ludwigii BP323EB had killed 50 % of the larvae at
biotechnological importance, but its propagation is limited due to seed 28 ◦ C and approximately 70 % at 37 ◦ C, revealing greater pathogenicity
recalcitrance. One way to overcome this difficulty is through in vitro than A. lwoffii. When inoculated together, larval mortality was approx­
micropropagation techniques and efficient acclimatization protocols. imately 70 % at 28 ◦ C and 85 % at 37 ◦ C. The endophytic isolate
Acclimatization is a critical phase for the success of micro­ B. seminalis TC3.4.2R3 has multiple functions that promote plant
propagation because it can result in the death of most transplanted growth, but when Gonçalves et al. [48] tested it in G. mellonella, it killed
seedlings. In this process, in vitro rooting of seedlings may be critical, 100 % of the larvae in 24 h at 37 ◦ C and at 65 h at 28 ◦ C when inoculated
increasing the survival capacity and development of the plants under the at 107 CFU mL− 1. The authors then tested the bacteria in mice at the
new environmental conditions [11,40]. During transplanting, roots are same concentration, and after 30 days of experiment, the bacterium had
usually pruned, which can decrease the vegetative growth of some tree not killed any mouse [48]. These findings demonstrate that an endo­
species but can also lead to an increase in the number of new roots and phytic bacterium highly virulent in larvae will not necessarily be path­
root biomass [41]. Therefore, understanding the influence of rooting on ogenic in mammals. Compared to B. seminalis TC3.4.2R3, the isolates
acclimatization is important for determining the key factors for the tested in this study were much less virulent, since after seven days, 100
success of P. gardneriana establishment in the field. % mortality was not observed in any of the treatments. A
The presence of roots had a positive effect on the biometric charac­ temperature-dependent behavior of virulence was also observed, as the
teristics of acclimatized P. gardneriana plantlets. When the plantlets did mortality was higher at 37 ◦ C than at 28 ◦ C, as reported by Gonçalves
not present roots, several characteristics, such as leaf area, number of et al. [48].
leaves, number of nodal segments, and leaf fresh and dry weights were Based on these results, Bac109 and BP323EB strains were used as
negatively affected. In addition, plantlets acclimatized with well or inoculants in P. gardneriana seedlings since the production of IAA pro­
poorly established roots showed greater investment in the formation of motes the development of roots during the acclimatization phase. Plants
new roots, increase the leaves and the segments number, while the roots inoculated with the consortium A. lwoffii Bac109 and E. ludwigii
absence hindered several growth parameters. BP323EB showed significant increases in root and plant length,
Seedlings with roots also resulted in higher levels of chlorophyll a, demonstrating that in the long term, inoculated plants would have a
chlorophyll b, and total chlorophyll and higher photosynthetic rates. De more developed and efficient root system, which could lead to better
Klerk [42] describes that apple and rose microcuttings rooted in ex vitro adaptation to the environmental conditions experienced during accli­
condition have less survival and growth during acclimatization than matization. Micropropagated apple seedlings inoculated with rhizobia
when they have been rooted in vitro, what can be related to role of the have shown greater in vitro rooting, with increased length, biomass, and
newly formed roots for replenishing water that is lost from leaves with number of roots, which is essential for the next phase, acclimatization
malfunctioning stomata. [40]. Inoculation of A. lwoffii Bac109 in consortium with Pantoea
On the other hand, a study with Eucalyptus showed that the presence agglomerans Bac131 in cashew plants promoted a great increase in bio­
of vascular cambium in eucalyptus mini-cuttings rootted became an metric parameters [21].
advantage of vascular connection for adventitious roots, which has an The inoculation of PGPB into Manihot esculenta Crantz seedlings
affect on the proper functionality of the root, influencing the develop­ during acclimatization increased the height, diameter, dry weight, and
ment of plant in normal conditions and at low temperatures [43]. The nitrogen concentration, helping the seedlings overcome the stress of this
results of this study indicate that in vitro rooting is the most effective phase [49]. Diazotrophic bacteria increased the shoot and root dry
treatment to increase biometric parameters, as it contributes to better weight of micropropagated pineapple seedlings during acclimatization
root development in the acclimatization phase. [50]. Similar results have been observed during acclimatization of
The survival of the seedlings during the analyzed period was not Ananas comosus (L.) Merr. seedlings inoculated with Azotobacter chroo­
affected by the differences in the roots of seedlings, and shoot growth coccum: Bacterial inoculation affected both physiological and
did not show a relationship with root size in the micropropagated biochemical parameters, with increases in the indicators of photosyn­
seedlings. However, as the root structure is related to nutrients uptake thesis and in the levels of carbohydrates, amino acids, and minerals
and with plant performance in stress conditions [15,44], the root [51].
absence can be a negative factor in acclimatization process if a long time
is considered. 5. Conclusions
Acinetobacter and Enterobacter have multiple functional traits for
promoting plant growth, such as calcium phosphate solubilization, The presence of roots in P. gardneriana Radlk micropropagated
phytohormone synthesis, and biocontrol [20,44]. When the bacterial seedlings increased biometric characteristics, such as shoot and root
strains A. lwoffii Bac109 and E. ludwigii BP323EB were inoculated in biomas, as well as physiological characteristics, such as photosynthetic
micropropagated P. gardneriana seedlings, the seedlings showed good rate. The positive effect endorse the importance of root development in
root development. Initially, the plant growth–promoting potential of in vitro protocol for better plant development in green house conditions,
both bacteria was analyzed, and only E. ludwigii BP323EB solubilized during acclimatization. The PGPB consortium inoculation, A. lwoffii
phosphate in vitro. However, the two species did produce IAA. Acineto­ Bac109 and E. ludwigii BP323EB, also contributed to plant growth with
bacter and Enterobacter have multiple functional traits that promote increase of root and plant length. The association of micropropagation
plant growth, such as calcium phosphate solubilization, phytohormone protocol to obtain P. gardneriana Radlk seedlings rooted and tha inoc­
synthesis, and biocontrol [20,21,45]. ulation of PGPB can be used to increase survival rate and seedling
To be considered a potential inoculant in agriculture, microorgan­ quality for economic and biotechnological purposes.
isms need to be tested for pathogenicity in animals. Alternatives to
mammalian animal models, especially larvae of the moth G. mellonella, Funding
have been used for this purpose because their innate immune systems
have similarities with those of mammals, they do not come with any This work was supported by Goiás Research Foundation (Fapeg) and
ethical considerations, and they are inexpensive. These larvae can the National Council for Scientific and Technological Development
develop at both 28 ◦ C, which is considered the ambient temperature, (CNPq).
and 37 ◦ C, which is the body temperature of mammals [46,47].
A. lwoffii Bac109 showed no virulence to the larvae at 28 ◦ C, and at

7
M.S. Leite et al. Current Plant Biology 27 (2021) 100209

Data availability and yield by altering root auxin sensitivity and the xylem structure in transgenic
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Declaration of Competing Interest
endophytic and rhizospheric microbial isolates associated with Butia purpurascens
roots for promoting plant growth, Antonie van Leeuwenhoek Int. J. Gen. Mol.
The authors report no declarations of interest. Microbiol. 111 (2018) 2157–2174, https://doi.org/10.1007/s10482-018-1108-7.
[21] P.S.A. Faria, V.D.O. Marques, P.J.R.G. Selari, P.F. Martins, F. Silva, J.F. Sales,
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