life

Article
Selection and Effect of Plant Growth-Promoting Bacteria on Pine
Seedlings (Pinus montezumae and Pinus patula)
Francisco David Moreno-Valencia 1 , Miguel Ángel Plascencia-Espinosa 2, * ,
Yolanda Elizabeth Morales-García 3,4 and Jesús Muñoz-Rojas 4, *

1 Consejo Nacional de Ciencias, Humanidades y Tecnología (CONAHCYT)—Group “Ecology and Survival of

Microorganisms”, Laboratorio de Ecología Molecular Microbiana, Centro de Investigaciones en Ciencias
Microbiológicas, Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla,
Puebla C.P. 72570, Mexico; franciscod.moreno@correo.buap.mx
2 Centro de Investigación en Biotecnología Aplicada (CIBA), Instituto Politécnico Nacional, Ex-Hacienda San
Juan Molino, Carretera Estatal Tecuexcomac-Tepetitla Km 1.5, Tlaxcala C.P. 90700, Mexico
3 Grupo Inoculantes Microbianos, Facultad de Ciencias Biológicas, Benemérita Universidad Autónoma de
Puebla, Puebla C.P. 72570, Mexico; yolanda.moralesg@correo.buap.mx
4 Group “Ecology and Survival of Microorganisms”, Laboratorio de Ecología Molecular Microbiana, Centro de
Investigaciones en Ciencias Microbiológicas, Instituto de Ciencias, Benemérita Universidad Autónoma de
Puebla, Puebla C.P. 72570, Mexico
* Correspondence: mplascencia@ipn.mx (M.Á.P.-E.); jesus.munoz@correo.buap.mx (J.M.-R.)

Abstract: Forest cover is deteriorating rapidly due to anthropogenic causes, making its restoration
urgent. Plant growth-promoting bacteria (PGPB) could offer a viable solution to ensure successful
reforestation efforts. This study aimed to select bacterial strains with mechanisms that promote plant
growth and enhance seedling development. The bacterial strains used in this study were isolated from
the rhizosphere and endophyte regions of Pinus montezumae Lamb. and Pinus patula Schl. et Cham.,
two Mexican conifer species commonly used for reforestation purposes. Sixteen bacterial strains were
selected for their ability to produce auxins, chitinase, and siderophores, perform nitrogen fixation,
and solubilize inorganic phosphates; they also harbored genes encoding antimicrobial production
Citation: Moreno-Valencia, F.D.;
and ACC deaminase. The adhesion to seeds, germination rate, and seedling response of P. montezumae
Plascencia-Espinosa, M.Á.;
Morales-García, Y.E.; Muñoz-Rojas, J.
and P. patula were performed following inoculation with 10 bacterial strains exhibiting high plant
Selection and Effect of Plant growth-promoting potential. Some strains demonstrated the capacity to enhance seedling growth.
Growth-Promoting Bacteria on Pine The selected strains were taxonomically characterized and belonged to the genus Serratia, Buttiauxella,
Seedlings (Pinus montezumae and and Bacillus. These strains exhibited at least two mechanisms of action, including the production of
Pinus patula). Life 2024, 14, 1320. indole-3-acetic acid, biological nitrogen fixation, and phosphate solubilization, and could serve as
https://doi.org/10.3390/ potential alternatives for the reforestation of affected areas.
life14101320

Academic Editor: Pabulo H. Keywords: forest species; phyto-stimulation; plant growth-promoting mechanisms; reforestation;
Rampelotto speed germination

Received: 6 September 2024

Revised: 15 October 2024
Accepted: 16 October 2024
1. Introduction
Published: 17 October 2024
The degradation of soils and the decline of native vegetation due to anthropogenic
activities is an increasingly severe environmental issue [1]. Deforestation rates from 2015 to
2020 reached an estimated 10 million hectares per year, resulting in the loss of 420 million
Copyright: © 2024 by the authors. hectares of forest since 1990 due to land-use changes [2]. These trends, coupled with global
Licensee MDPI, Basel, Switzerland. concerns over climate change and biodiversity loss, emphasize the critical role of forest
This article is an open access article cover in mitigating air pollution, carbon sequestration, and preserving ecosystem services
distributed under the terms and and habitats [3–5]. Reforestation holds significant potential for soil and water conservation,
conditions of the Creative Commons
making the forestry sector a key driver for sustainable development [6]. The most impactful
Attribution (CC BY) license (https://
strategy to counteract climate change is the expansion, restoration, and management of
creativecommons.org/licenses/by/
forests [7], which also yields long-term benefits for biodiversity conservation [8]. Despite
4.0/).

Life 2024, 14, 1320. https://doi.org/10.3390/life14101320 https://www.mdpi.com/journal/life

Life 2024, 14, 1320 2 of 21

global reforestation programs aimed at mitigating these effects [2], the expected results
have not been fully achieved [9]. The survival of forest seedlings is largely influenced
by the introduction of non-native species, insufficient post-planting care, and the lack of
stress tolerance studies on selected plant species [10]. In Mexico, forestry activities are
primarily focused on the Pinus genus, which accounts for 60% of commercially valuable
species [11]. This coniferous genus is distributed across 24 states, with two states in the
North Central region, one in the West, one in the South, and two in the Central Gulf region
standing out [12]. The economic importance of pine in Mexico is linked to its contribution
to the country’s economy and its role in the Gross Domestic Product (GDP) of the forestry
sector. Socially, its relevance stems from the communities living in forested areas who rely
on the goods and services provided by pine forests [13]. Given the importance of pine
species in Mexico, Pinus montezumae Lamb. and Pinus patula Schl. et Cham. are included
in the technological packages currently applied for soil restoration and conservation in
areas degraded by human activities. Pinus patula is widely used for timber and cellulose
production due to its high productivity and adaptability to various abiotic conditions and
non-forested soils [14,15]. Pinus montezumae has been successfully employed in several
reforestation programs aimed at watershed protection and soil restoration [16].
An effective strategy to enhance plant survival during the adaptation phase is the
application of microbial technology that supports plant growth and ensures successful trans-
plantation [17]. Plant Growth-Promoting Bacteria (PGPB) inoculants have been developed
due to their growth-promoting properties, offering both direct and indirect mechanisms
of action, primarily in agricultural crops. PGPB are classified into two groups, symbiotic
and free-living, based on their relationship with plants [18]. They are further categorized
as extracellular or intracellular PGPB depending on their location within the plant [19,20].
Extracellular PGPB are found in the rhizosphere, rhizoplane, or spaces between root cortex
cells, while intracellular PGPBs exist within the root cells [21–23]. Due to their plant growth-
promoting effects, these beneficial microorganisms are often referred to as yield-increasing
bacteria, plant health-promoting rhizobacteria, or nodule-promoting rhizobacteria, de-
pending on their mode of action on plant metabolism [24]. PGPB also enhances soil
water retention, helping to mitigate drought conditions to some extent [25]. PGPB are
further classified based on their activities as follows: as biofertilizers, they increase the
solubilization of minerals and fix nitrogen, making nutrients more accessible to plants; as
phytostimulators, they produce phytohormones such as indole-3-acetic acid (IAA), abscisic
acid, gibberellins, cytokinins, and ethylene [26,27]; and as biocontrol agents, they release
a wide variety of antibiotics and antifungal compounds that protect plants from biotic
stress, including siderophores, β-1,3-glucanase, chitinases, antibiotics, fluorescent pigments,
and cyanide [28]. Furthermore, PGPB that produce 1-aminocyclopropane-1-carboxylate
(ACC) deaminase, a critical enzyme, help reduce ethylene levels in plant roots, promoting
increased root length and growth [29,30]. Lastly, as rhizoremediators, PGPB enhance plant
growth by removing organic contaminants from the rhizosphere, and improving plant
tolerance to salinity [31], metal toxicity [32], and drought through mechanisms such as
exopolysaccharide (EPS) production [33,34], biofilm formation, and osmolyte reduction
to prevent cellular moisture loss [35,36]. These mechanisms may act simultaneously and
synergistically during different stages of plant growth. This biotechnological approach is
environmentally friendly, with no adverse effects [37,38]. Furthermore, inoculating forest
seedlings with PGPB in nurseries increases beneficial microbial populations in the plant’s
rhizosphere. As the plant serves as a vehicle for reintroducing these microbes into the soil,
it promotes early growth, reduces transplant stress, and enhances adaptation to the new
environment [39].
PGPB have been isolated from various plant-associated environments, including the
rhizosphere, endophytic, and epiphytic zones [40–42]. However, the isolation of bacteria
with the potential to promote tree growth has been less extensively studied [43–46], despite
its significant implications for the productivity of certain fruit crops and the reforestation
of forested areas. Evidence suggests that there is significant host specificity in tree species
Life 2024, 14, 1320 3 of 21

when treated with these microorganisms, which may be influenced by local environmental
and geographic conditions [47]. Thus, the mutualistic relationship between microorganisms
and plant growth processes is critical for the successful establishment of nursery-grown
seedlings in their new habitats. Root exudates are generally plant-specific and often serve
as signals to facilitate affinity with particular microorganisms [10,48]. This occurs because
plants can “select” their microbiome for beneficial bacterial colonizers, including those
residing within plant tissues [49]. Soil degradation and the loss of native vegetation
due to unsustainable human activities are escalating issues that impact geoecosystems in
Mexico and worldwide. Effective solutions are needed, such as reforestation, which plays a
vital role in soil and water conservation by sequestering carbon, improving soil fertility,
regulating river flows, and creating favorable microclimates. This paper proposes, presents,
and discusses a growth acceleration system for forest species through the isolation and
selection of plant growth-promoting bacteria, aimed at enhancing the physiological and
morphological traits of nursery seedlings. The expected outcome is an improvement in the
establishment and survival rates of forest seedlings compared to the current cultivation
methods. Additionally, the paper discusses the selection of bacterial strains based on their
plant growth-inducing mechanisms and the growth-promoting effects observed when
inoculated into P. montezumae and P. patula seedlings.

2. Materials and Methods

2.1. Bacterial Strains
Bacterial strains evaluated in this study were collected from the P. patula and P. mon-
tezumae rhizosphere and endophytic zones in the forested regions of Tlaxcala, Mexico.
A total of 35 strains from P. patula and 67 strains from P. montezumae were isolated and
cataloged. The naming convention for these strains included a sequential number based
on their isolation order, the collection site, and the tree species from which they were
collected [10]. This work is a continuation of our previously conducted study [10]; however,
a brief description of how the isolation of the strains studied was carried out is provided
below. The site for collecting plant material was chosen due to the natural distribution of
P. montezumae and P. patula. Two collections of P. montezumae were formed in Malinche
National Park, at four-week intervals between August and September 2016. Additionally,
P. patula was sampled in the Sierra de Tlaxco–Caldera–Huamantla region in November
of the same year. A field inspection was conducted to identify healthy seedlings with no
visible damage or disease. Each collected tree was evaluated for dendrometric and soil
moisture measurements, and the entire seedlings were transported to the laboratory for the
isolation of rhizospheric and endophytic colonies under aseptic conditions.
Rhizospheric bacteria were isolated from the soil most adhered to the seedling roots.
A suspension of rhizospheric soil was prepared by immersing the roots in sterile distilled
water (1:10 w/v) and homogenizing the sample. Serial dilutions were performed at a 1:7
dilution factor, and 200 µL of the sample from each dilution were spread on two different
culture media using the plate spreading technique. Endophytic bacteria were isolated
from inside the root. The root surface was sterilized by immersing it in 96% ethanol for
5 min, followed by 6.25% NaOCl for 10 min, and then it was rinsed thoroughly with
sterile distilled water. The plant material was immersed in a 0.1 M aqueous solution of
MgSO4 and ground in a porcelain mortar [50]. The resulting macerate was then inoculated
in triplicate [51,52] on Mannitol Yeast Extract Agar (pH 7) [53] and Nutrient Agar (pH
6.8) [54]. All plates were subsequently incubated at 28 ◦ C for 5 days. Different colonies that
grew on the plates were isolated, purified, and stored in a glycerol–water solution (1:4) at
−20 ◦ C for preservation and future use.

2.2. Characterization of the Isolated Bacteria Based on Their Mechanisms of Action

2.2.1. Indole Acetic Acid Production and Biosynthetic Pathway
Bacterial cells were induced by synthetic L-tryptophan (Sigma-Aldrich Production
GmbH, Buchs, Switzerland), suspended in a solution of 10% methanol/0.05% acetic acid
Life 2024, 14, 1320 4 of 21

and then sonicated and centrifuged at 12,000 rpm for 20 min. The supernatant was filtered
through a 0.22 µm PVDF membrane before analysis by reverse phase high performance
liquid chromatography and mass spectrometry (RP-HPLC–MS/MS). The analysis was
performed using a Shimadzu Nexera HPLC system coupled with a TRAP 3200Q mass
spectrometer (SCIEX, Framingham, MA, USA), equipped with a turbo ion spray interface.
A Kinetex C18 column (150 × 4.6 mm; 2.6 µm particle size) protected by a Kinetex UHPLC
Ultra C18 guard column (0.5 µm porosity × 4.6 mm inner diameter; Kinetex, Phenomenex,
Torrance, CA, USA) was used. The gradient elution and optimized parameters were
adapted from [55]. The optimized parameters for IAA and its precursors were obtained
from the Analyst software (v 1.6.3) and aligned with the proposed pathways for IAA
synthesis in plant growth-promoting bacteria [56].

2.2.2. Quantitative Testing of Phosphate Solubilization

Flasks containing Pikovskaya medium supplemented with tricalcium phosphate
(Ca3 P4 O8 ) as a P source were inoculated and incubated at 30 ◦ C and 130 rpm with continu-
ous shaking (SEV, Model 6090, Washington, DC, USA) [57]. Then, 15 mL of the bacterial
inoculants were centrifuged in 50 mL conical tubes at 10,000 rpm for eight minutes (Eppen-
dorf, Model 5804 R, Hamburg, Germany) [58]. Next, 2 mL of the supernatant were mixed
with 2 mL of reagent (1:1 v/v) and left to react for one hour. Absorbance was measured at
882 nm using a spectrophotometer (Jenway, Model 6305, London, UK). The intensity of the
blue color was correlated with the amount of P solubilized by the bacterial strains [59]. The
kinetics of phosphate solubilization by the strains were evaluated on days 0, 5, 10, and 15
after incubation using the molybdenum blue method to analyze their performance [57,60].

2.2.3. Acetylene Reduction Assay (ARA)

First, 50 µL of pure culture were inoculated into nitrogen-free mannitol semisolid agar
in 100 mL vials, which were then sealed with rubber stoppers and incubated for 48 h at
28 ◦ C. Vials showing bacterial growth were analyzed for acetylene reduction. Up to 10%
of the vial’s atmosphere was replaced with acetylene (C2 H2 ) [61]. The flame ionization
detector and injector were set to 230 ◦ C, and an HP-INNOWax Columns (Agilent, Santa
Clara, CA, USA) was used at 100 ◦ C with a 20 min cycle. The nitrogen gas flow was
adjusted to 80 psi [39,61].

2.2.4. Siderophore Production

The ability of strains to produce siderophores was qualitatively evaluated using
chrome azurol agar (CAS) [53,62,63]. Plates containing CAS agar were inoculated and
incubated at 28 ± 2 ◦ C for 48–72 h. The presence of yellow-orange halos around the
colonies on blue agar was indicative of siderophore excretion [53].

2.2.5. Amplification of ACC Deaminase and Some Antimicrobial Compounds’ Genes

Genomic DNA was extracted using the ZR Soil Microbe DNA Kit Miniprep™ (Zymo
Research, Orange, CA, USA), following the manufacturer’s instructions. Polymerase chain
reaction (PCR) amplifications were performed in 50 µL reaction volumes containing 1×
PCR buffer (5 µL per tube), MgCl2 , (1.5 µL), 5 µL of each primer, dNTP (1 µL), Taq DNA
polymerase (0.25 µL; Invitrogen, São Paulo, Brazil), and template DNA from each strain
(1 µL). The acdS gene amplification and fungal antibiotic-producing genes oligonucleotides
were selected based on protocols from [64–68] (Table 1). Oligonucleotides were supplied
by T4 Oligo, with the manufacturer’s analysis certificate. The cycles were run on a thermal
cycler (iCycler Thermal Cycler Firmware v 4.006, Bio-Rad Laboratories, Inc., Hercules,
CA, USA) under the following conditions: (i) Initial denaturation at 95 ◦ C for 3 min;
(ii) Denaturation at 94 ◦ C for 45 s; primer annealing temperatures were set according
to [68], with 30-s annealing for all primers; (iii) Extension at 72 ◦ C for 3 min (30 cycles) and
final extension at 72 ◦ C for 10 min. Agarose gel electrophoresis (1%) was performed in a
horizontal gel box (ENDURO™ 7.10, Labnet International, Inc., Edison, NJ, USA), using a
Life 2024, 14, 1320 5 of 21

power supply (PowerPac HC High-Current Power Supply, Bio-Rad Laboratories, Inc.) at

120 volts for 40 min in 1× TBE buffer (Thermo Fisher Scientific Inc., Waltham, MA, USA),
diluted in deionized water filtered twice with a 20 µm membrane. Bands were visualized
under UV transillumination using the Gel Doc 2000 system (Bio-Rad Laboratories, Inc.).

Table 1. Targeted genes and their corresponding primers and sequences used in this research.

Melting Amplicon
Gene Primer Primer Sequence Putative Gene Function Reference
Temp (◦ C) Size (bp)
PRND1 GGGCGGGCCGTGGTGAT Pyrrolnitrin biosynthesis
prnD 65 786 [65]
PRND2 GGACGCSGCCTGYCTGGTCTG enzyme
ACCCACCGCGCATCGTTTAT-
B2BF Polyketide synthase III
GAGC
phlD 66.5 immediate precursor to 629 [64]
CCGCCGGTATGGAAGATGA-
BPR4 2,4-diacetylphloroglucinol
AAAAGTC
Ps_up 1 ATCTTCACCCCGGTCAACG Phenazine biosynthesis
phzF 57 427 [67]
Ps_low 1 CCRTAGGCCGGTGAGAAC enzyme
AACGATCGCCCCGGTACAG-
PLTC1
AACG Polyketide synthase I
pltC 58 438 [65]
AGGCCCGGACACTCAAGA- (Pyoluteorines)
PLTC2
AACTCG
GCTCCTACTCTGTCACCTATC-
F1936f
GHGAMGACTGCAAYWSYGGC Gene encoding ACC
acdS 50 792 [68]
CTGTCGCTCTGGCTGTCACAT- deaminase
F1938r
VCCVTGCATBGAYTT

2.3. Phylogenetic Analysis

DNA extraction was carried out using the Quick-DNA Miniprep Plus kit (Zymo
Research, Irvine, CA, USA). The amplification of the 16S rDNA gene was performed
using the universal primers, 27F (forward) 5′ -AGA GTT TGA TCM TGG CTC AG-3′ and
1492R (reverse) 5′ -CGG TTA CCT TGT TAC GAC TT-3′ , and following the methodology
described in previous studies [69]. Sequence editing, assembly, and comparison using the
BLAST (Basic Local Alignment Search Tool, v 2.9.0) program against the National Center
for Biotechnology Information (NCBI) database (www.ncbi.nlm.nih.gov) were performed.
The sequences were compared with those reported in the database, and the sequences with
the highest similarity were selected for phylogenetic analysis [17]. A multiple alignment
of all selected sequences was performed on the phylogeny.fr platform with the following
steps: Sequences were aligned using multiple sequence comparison by log-expectation
computer program (MUSCLE; v3.8.31; multiple sequence alignment with high accuracy
and high throughput) configured for maximum accuracy (default MUSCLE settings). After
alignment, ambiguous regions were removed using Gblocks (v0.91b). A phylogenetic tree
was reconstructed using the phylogenetic estimation maximum likelihood method in the
PhyML software package (v3.1/3.0 aLRT). The HKY85 substitution model was selected,
assuming an estimated proportion of invariable sites (0.836) and 4 gamma-distributed rate
categories to account for rate heterogeneity among sites. The gamma shape parameter was
directly estimated from the data (gamma = 0.481). The reliability of the internal branches
was assessed using the aLRT (approximate likelihood ratio test, SH-like) method. The
graphical representation and editing of the phylogenetic tree were done with TreeDyn
(v198.3) [70–74].

2.4. Selection of Bacterial Strains for Bioassay and Preliminary Screening

Strains that exhibited at least one mechanism of action and had a good yield in the
mechanism of action tests were selected for the bioassay. Additionally, a previous test
with Pinus radiata seedlings was performed, where germinated seeds, germination speed,
Life 2024, 14, 1320 6 of 21

and root elongation were evaluated. Therefore, sixteen strains that were isolated from
the rhizosphere and endophyte regions were selected for evaluation in P. montezumae and
P. patula seeds. This was a screening to identify strains with better yields.

2.5. Adherence Assay

The seeds were placed in a container with cold water for 48 h, and empty seeds were
discarded. The seeds were stratified at temperatures between 2 and 5 ◦ C for 4 to 5 weeks as
a pre-germination treatment to break physiological dormancy [75]. The seeds were then
surface sterilized with 5% NaClO for 5 min, followed by three consecutive washes with
sterile distilled water [76,77]. The sterility of the surface-sterilized seeds was confirmed
by placing a few seeds in contact with a gelified medium. A microbial suspension was
prepared in water following the protocol by [51]. For the adhesion assay, the protocol
described by [69] was followed. The adhesion of 16 strains was tested, with four replicates
for each one. The inoculated seeds were placed in a nursery under a 12/12 h light/dark
photoperiod at 25 ◦ C. The control seeds were immersed in sterile distilled water following
the same protocol as the inoculated seeds.

2.6. Speed Germination Assay

Germination rate was examined based on the time elapsed after the seeds were
inoculated and sown in sterile vermiculite, with a seed considered germinated once the
radicle had emerged. Counts of the number of germinated seeds were conducted every
six days, including only seeds with emerged radicles. The Germination Speed Index (GSI)
was calculated according to [78].

2.7. Seedlings Inoculation

Twelve treatments with four replicates for P. montezumae seedlings and fifteen treat-
ments with four replicates for P. patula seedlings were performed. The strains were selected
for their highest yields in the germination speed and seed adhesion tests. The seedlings
were transplanted into forest soil (vitric Andosol T-4), mixed with vermiculite in a 60/40 ra-
tio. The soil was sterilized in two cycles of four hours, each at 120 ◦ C, using a semi-industrial
autoclave (Prendo Mod. AH80170, SEV-Prendo, Puebla, México). The mixture was placed
in plastic containers of 1 L capacity to transplant the seedlings of both species into each
container. The seedlings were inoculated weekly for one month with 2 mL of microorgan-
ism suspension in water [17]. Following this period, inoculations were carried out every
two weeks for two months. The seedlings were subjected to water stress to evaluate their
response to drought conditions. This qualitative method involved withholding irrigation
for two weeks. Observations focused on how the seedlings responded and adapted to
these adverse conditions, providing valuable insights into their drought resistance and
survival mechanisms.
The plants were harvested 100 days after the experiment began. The tested variables
included height, diameter, root length, and number of roots. Data were analyzed using
InfoStat 2020 version [79] to perform analysis of variance (ANOVA) and Duncan’s multiple
range test, with a significance level of α = 0.05.

3. Results
3.1. Isolation of Bacterial Strains and Assessment of Seedling and Soil Conditions
A total of 102 bacterial strains were isolated from P. montezumae and P. patula seedlings.
Notably, 30 strains were recovered from P. montezumae, with 17 classified as rhizospheric
and 13 as endophytes. In contrast, 57 strains were isolated from P. patula, 41 from the
rhizosphere and 16 as endophytes. This demonstrates a diverse bacterial community
associated with both the roots and internal tissues of these species. The differences in the
number of strains between the two pines may be linked to the specific soil characteristics
and environmental conditions at each collection site (Table 2).
Life 2024, 14, 1320 7 of 21

Table 2. Characteristics of sampled trees and bacterial strain isolation in different forest regions.

Number of
Soil Altitudinal Sampling Forested
Tree Sample Tree Species Height (cm) Isolated
Moisture % Profile (masl) Coordinates Region
Strains
19◦ 14′ 49′′ N;
1 P. montezumae 20 60 2885 4
98◦ 05′ 44′′ O Malinche
19◦ 15′ 02′′ N; National
2 P. montezumae 25 63 2962 5
98◦ 05′ 22′′ O Park
19◦ 15′ 03′′ N;
3 P. montezumae 26 68 3030 6
98◦ 05′ 2′′ O
19◦ 15′ 04′′ N;
4 P. montezumae 30 56 3077 5
98◦ 05′ 21′′ O
19◦ 15′ 01′′ N;
5 P. montezumae 46 61 2896 3
98◦ 05′ 20′′ O
19◦ 15′ 17′′ N;
6 P. montezumae 32 54 3061 4
98◦ 04′ 55′′ O
19◦ 15′ 51′′ N;
7 P. montezumae 56 53 2931 3
98◦ 05′ 23′′ O
19◦ 41′ 31′′ N; Sierra de
8 P. patula 35 53 2923 22
98◦ 04′ 43′′ O Tlaxco-
19◦ 41′ 35′′ N; Caldera-
9 P. patula 58 51 2853 16
98◦ 04′ 44′′ O Huamantla
19◦ 41′ 34′′ N;
10 P. patula 26 54 2903 19
98◦ 04′ 43′′ O

3.2. Detected Growth-Promoting Mechanisms in Bacterial Isolates

The intracellular production of IAA via the indolepyruvic acid (IPyA) pathway, which
is a tryptophan-dependent pathway, was identified. This was observed in 16 of the eval-
uated strains, with IAA production ranging from 1 to 305 µg/mL. Results indicated that
most strains predominantly synthesize IAA through the IPyA pathway. However, the strain
C63STPp was noted to preferentially utilize the tryptamine (TAM) and indoleacetamide
(IAM) pathways (Table 3), suggesting variability in the metabolic routes employed by the
strains for IAA production.

Table 3. Growth-promoting activity and origin and identification of bacterial strains isolated from
P. patula and P. montezumae. (R = Rhizospheric; E = Endophytic; missing data indicate that no
detectable growth-promoting activity was observed for the respective strain in the tests performed).

Indole Test
Strain µg/mL Intracelular Metabolic Pathway Siderophores P Solubilizing ARA % Substrate
C1MPm 137 IPyA + - - R
C13MPm 1 IPyA - 1.5 13 R
C16MPm 186 IPyA + 1.5 - R
C18MPm 285 IPyA + 1.7 - R
C25MPm 4 IPyA + 0.4 - R
C28MPm 189 IPyA - - - E
C38STPp 4 IPyA + 0.9 - E
C39STPp 82 IPyA + 2 - R
C52STPp 78 IPyA + - - R
C54STPp 305 IPyA + 2.6 - R
C59STPp 88 IPyA + - - R
C63STPp 1 TAM IAM - 0.7 - R
C65STPp 2 IPyA + - 15 R
C68STPp 2 IPyA + - - R
C74STPp 110 IPyA + - 74 R
C99STPp 95 IPyA - - - E
Life 2024, 14, 1320 8 of 21

A diazotrophic bacterial biofilm was observed to be forming in the medium’s subsur-

face after 7 days of incubation at 30 ◦ C. The biofilm was visible within the first 1–3 days
post-inoculation, and daily growth was monitored since some bacteria exhibit accelerated
growth rates. Among the evaluated strains, eleven demonstrated acetylene-reducing activ-
ity. Six were isolated from the rhizosphere and five were endophytes, with the reduction
ranges between 8% and 29% (Table 3). Samples from positive strains were collected 96 h
after injecting 10% acetylene into the vial atmosphere to assess their performance over time.
However, an 80% decrease in performance was noted during this period, indicating that
strains exhibit greater efficiency 48 h after injection. This suggests an optimal window for
peak acetylene-reducing activity in diazotrophic strains.
Twenty-six strains tested were positive for phosphorus solubilization using the ammo-
nium molybdate and ascorbic acid method, with phosphorus solubilization concentrations
ranging from 0.1 to 2.4 mg/L. Under these incubation conditions, strain C12MPm C12MPm
was the most efficient in phosphate solubilization, followed by strains C42STPp, C44STPp,
C21MPm, and C40STPp, in order of efficiency (Table 3). The maximum strain yield was ob-
served between days 5 and 10 of incubation, where exponential phosphorus solubilization
occurred. After day 10, performance declined. However, three strains initially showed a
modest performance during the first five days, with a significant increase from the sixth
day, peaking on day 10.
Twelve strains were identified as siderophore producers based on the formation of
orange halos around their colonies. Eleven were isolated from the rhizosphere and one was
classified as endophytic. Specifically, five strains were associated with Pinus montezumae
and seven with Pinus patula (Table 3).

Amplification of ACC Deaminase and Antimicrobial Compounds Genes

The strains C13MPm, C28MPm, and C38STPp showed amplification of the prnD gene,
which is responsible for the synthesis of pyrrolnitrin (Table 4). This suggests their potential
in producing this antimicrobial metabolite. Additionally, molecular analysis revealed
that the strain C25MPm, isolated from the rhizosphere of Pinus patula, had a positive
amplification of the acdS gene, which encodes the ACC deaminase enzyme (Table 4). The
presence of this gene indicates the strain may play a crucial role in reducing ethylene stress
in plants, enhancing their growth and resilience under adverse conditions.

Table 4. Amplification of genes with antagonistic effects, amplification of the ACC deaminase
gene, and origin of bacterial strains isolated from P. patula and P. montezumae (R = Rhizospheric;
E = Endophytic).

Strain Genus prnD phlD phzF pltC acdS Substrate

C1MPm Serratia sp. - - - - - R
C13MPm N/D + - - - - R
C16MPm Serratia sp. - - - - - R
C18MPm Serratia sp. - - - - - R
C25MPm Serratia sp. - - - - + R
C28MPm Buttiauxella sp. + - - - - E
C38STPp N/D + - - - - E
C39STPp N/D - - - - - R
C52STPp Serratia sp. - - - - - R
C54STPp Serratia sp. - - - - - R
C59STPp Serratia sp. - - - - - R
C63STPp Bacillus sp. - - - - - R
C65STPp N/D - - - - - R
C68STPp N/D - - - - - R
C74STPp N/D - - - - - R
C99STPp Bacillus cereus - - - - - E
 C63STPp Bacillus sp. - - - - - R
C65STPp N/D - - - - - R
C68STPp N/D - - - - - R
Life 2024, 14, 1320
C74STPp N/D - - - - - R
9 of 21
C99STPp Bacillus cereus - - - - - E

3.3.
3.3.Molecular
MolecularIdentification
IdentificationofofStrains
Strainsand
andTheir
TheirPhylogenetic
PhylogeneticComparison
Comparison
Ten strains were identified as part of the group with potential
Ten strains were identified as part of the group with potential plant plantgrowth-promoting
growth-promot-
ing abilities. These strains are closely related to the genera Serratia
abilities. These strains are closely related to the genera Serratia (C1MPm, C13MPm,C13MPm,
(C1MPm, C16MPm,
C16MPm,
C18MPm,C18MPm,C25MPm, C25MPm,
C52STPp, C52STPp,
C54STPp,C54STPp,
C59STPp),C59STPp), Buttiauxella
Buttiauxella (C28MPm),
(C28MPm), and
and Bacil-
Bacillus
lus (C63STPp, C99STPp). A phylogenetic analysis of the identified strains, usingse-
(C63STPp, C99STPp). A phylogenetic analysis of the identified strains, using se-
quences
quencesfromfrombacteria
bacteriarelated
relatedto these genera
to these and and
genera considering the habitat
considering from they
the habitat fromwere
they
isolated, revealed
were isolated, clustering
revealed into two
clustering intodistinct taxonomic
two distinct groups—γ-proteobacteria
taxonomic groups—γ-proteobacteriaand
Bacilli (Figure
and Bacilli 1). The1).
(Figure nucleotide sequences
The nucleotide for the strains
sequences were
for the submitted
strains to the GenBank
were submitted to the
database
GenBankand assigned
database andthe following
assigned theaccession
followingnumbers:
accessionPQ435155
numbers:(C1MPm),
PQ435155PQ435156
(C1MPm),
(C16MPm), PQ435157 (C18MPm), PQ435158 (C25MPm), PQ435159
PQ435156 (C16MPm), PQ435157 (C18MPm), PQ435158 (C25MPm), PQ435159 (C28MPm), (C28MPm), PQ435160
(C52STPp), PQ435161 PQ435161
PQ435160 (C52STPp), (C54STPp),(C54STPp),
PQ435162PQ435162
(C59STPp), PQ435163
(C59STPp), (C63STPp),
PQ435163 and
(C63STPp),
PQ435164
and PQ435164(C99STPp).
(C99STPp).

Figure 1. Phylogenetic tree based on 16S rDNA gene sequencing, the evolutionary relationship
between the 10 growth-promoting isolates inferred using the Phylogeny platform is observed. Evolu-
tionary distances were computed using the maximum likelihood method.

3.4. Effect of Plant Growth-Promoting Bacteria on Biomass and Root Structure of Pine Seedlings
3.4.1. Adherence and Colonization Assays
Pinus montezumae seeds germinated after 10 days, while P. patula seeds germinated
after twelve days following inoculation and planting in sterile vermiculite. In contrast, the
control group seeds germinated on day 20. The variability in germination times between
the two pine species was notable. P. montezumae exhibited a germination range of two to
four seeds per treatment, achieving an 89% success rate. In contrast, P. patula seeds reached
a 98% germination rate across all treatments.
Life 2024, 14, 1320 10 of 21

The seeds were inoculated with bacterial suspensions of 1 × 109 and 6 × 109 CFU/mL
to seeds of P. patula and P. montezumae, respectively. The seedlings of both forest species,
planted in sterile vermiculite, exhibited healthy growth, showing notable vigor. The surface
sterility test confirmed that the seeds were free of microorganisms, as no growth was
observed on the gelified medium. Additionally, bacterial adhesion was detected on the
seeds of both species in each treatment, with CFU per seed values ranging from 2 × 106 for
strain C63STPp to 4 × 106 for strain C13MPm (Table 5).

Table 5. Results of inoculation, seed adhesion, and germination rate assays for P. montezumae
and P. patula. The treatment number corresponds to the identification number assigned to the
isolated strains.

UFC/mL Germination Speed Index (GSI) Germinated Seeds

Treatment UFC/Seed
Inoculate P. montezumae P. patula P. montezumae P. patula
C1MPm 6 × 109 2 × 108 0.306 0.264 3 ± 0.25 4±0
C13MPm 6 × 109 2 × 107 0.278 0.278 3 ± 0.25 4±0
C16MPm 6 × 109 2 × 108 0.472 0.278 4±0 4±0
C18MPm 6 × 109 2 × 108 0.556 0.417 4±0 4±0
C25MPm 6 × 109 2 × 107 0.194 0.389 3 ± 0.25 4±0
C28MPm 6 × 109 2 × 109 0.306 0.264 4±0 4±0
C38STPp 6 × 109 2 × 109 0.306 0.306 3 ± 0.25 4±0
C39STPp 6 × 109 2 × 108 0.417 0.389 4±0 4±0
C52STPp 6 × 109 2 × 107 0.417 0.333 4±0 4±0
C54STPp 6 × 109 2 × 107 0.250 0.278 3 ± 0.25 4±0
C59STPp 6 × 109 2 × 109 0.250 0.194 3 ± 0.25 3 ± 0.25
C63STPp 6 × 109 2 × 108 0.417 0.250 4±0 4±0
C65STPp 6 × 109 2 × 106 0.306 0.264 4±0 4±0
C68STPp 6 × 109 2 × 107 0.306 0.306 3 ± 0.25 4±0
C74STPp 6 × 109 2 × 109 0.500 0.361 4±0 4±0
C99STPp 6 × 109 2 × 107 0.417 0.417 4±0 4±0
Control - - 0.097 0.097 2 ± 0.3 2 ± 0.3

3.4.2. PGPB Effect on Pines Seedlings

Pinus montezumae seedlings showed increased growth when inoculated with the rhizo-
spheric strains C18MPm, C52STPp, C54STPp, C74STPp, and C99STPp, (Figure 2). Strain
C74STPp, isolated from P. patula, led to a seedling height increase of 11.7 cm. Regarding root
length, five treatments resulted in elongation ranging from 26.1 to 32.1 cm, specifically with
strains, C18MPm, C28MPm, C54STPp, C63STPp, and C74STPp. Additionally, treatments
with the strains C52STPp, C54STPp, C59TPp, C63STPp, C74STPp, and C99STPp showed
increased root diameter (Table 6).
In terms of root number in seedlings, treatments with strains C16MPm, C18MPm,
C28MPm, C39STPp, C52STPp, C74STPp, and C99STPp demonstrated enhanced develop-
ment, with a range of 9 to 10 roots per seedling. For P. patula seedlings, height growth
ranged from 8.7 to 13.3 cm in the aerial parts. Treatments with strains C1MPm, C38MPm,
C39STPp, C54STPp, C74STPp, and C99STPp showed significant post-inoculation growth,
with the C74STPp strain being the most effective, achieving a height of 13.3 cm. The root
length evaluation, seedlings from inoculated treatments showed the highest yield compared
to control plants, with lengths ranging from 21.8 to 30.6 cm. The bacterial strains contribut-
ing to improved performance were C1MPm, C18MPm, C28MPm, C52STPp, C65STPp, and
C74STPp, significantly enhancing root development. For root diameter, treatments inoc-
ulated with the strains C16MPm, C18MPm, C38STPp, C39STPp, C74STPp, and C99STPp
showed notable increases. Regarding root number, treatments with the strains C38STPp,
C39STPp, C54STPp, C65STPp, C74STPp, and C99STPp resulted in a higher count, ranging
from 10 to 11 roots per seedling (Table 6).
 Life2024,
Life 14,1320
2024,14, x FOR PEER REVIEW 11
11 of 22
of 21

P. montezumae P. patula

16 14.5 14.4
13.6 13.2 13.3
14 12.6
11.6 11.7
12 10.8 10.8 11.1 10.7 10.8 11.0
10.1 10.2 10.4 10.3 10.3
9.7 9.6 9.4
10 9.0 9.0

Height (cm)
8.8 8.8
7.7
8
6 5.1 4.9

4
2
0

Treatment

Figure2.2. Evaluation
Figure Evaluation of
of stem
stem elongation
elongation in P. montezumae
in P. montezumae and P.patula
and P. patulaseedlings
seedlingsafter
aftertreatment
treatmentwith
with
growth-promoting strains.
growth-promoting strains.

Table In terms of
6. Results root
of the numberparameters
measured in seedlings,
of P. treatments with
montezumae and P. strains C16MPm,
patula seedlings afterC18MPm,
100 days
C28MPm, C39STPp, C52STPp, C74STPp, and C99STPp demonstrated enhanced develop-
in nursery.
ment, with a range of 9 to 10 roots per seedling. For P. patula seedlings, height growth
Height (cm) Root Length (cm) Root Diameter (mm) Number of Roots
Strain P. montezumae ranged from
P. patula 8.7 to 13.3 cm
P. montezumae in the aerial
P. patula parts. Treatments
P. montezumae P. patula with strains C1MPm,
P. montezumae C38MPm,
P. patula
C1MPm C39STPp,
12.6 ± 0.9 abc C54STPp, C74STPp,
27.7 ± 0.9 and
a C99STPp showed significant
1 ± 0.02 bc post-inoculation 8 ± 0.5growth,
bcde

C13MPm 10.8with
± 0.9 bcthe C74STPp strain23.5 being
± 0.9 the
a
most effective, achieving
1 ± 0.02 abc a height of 13.3 cm. The
7 ± 0.6 cdef
root
C16MPm 9.7 ± 0.7 ab 10.8 ± 0.6 bc 23.7 ± 0.9 abc 25.1 ± 0.9 a 1 ± 0.03 ab 2 ± 0.01 ab 9 ± 0.6 abcd 9 ± 0.5 abcde
C18MPm 11.6 ± 0.8 a 11.1length
± 0.9 abc evaluation,
32.1 ± 0.9 bcseedlings from
26.2 ± 0.9 a inoculated
1 ± 0.01 ab treatments
2 ± 0.02 ab showed 10 ±the
0.3 abhighest6 ± yield
0.9 def com-
C25MPm 9.6 ± 0.6 bcd pared
9.0 ± 0.7 bcd to control
17.5 ± 0.9plants,
c
with
22.9 ± 0.9lengths
a
1 ranging
± 0.01 ab from 1 ±21.8
0.02 bcto 30.67cm. ± 0.9 The
abcd
bacterial strains
7 ± 0.3 cdef
C28MPm 10.1 ± 0.4 ab 10.2 ± 0.9 bc 29.5 ± 0.9 ab 27.0 ± 0.9 a 1 ± 0 ab 1 ± 0.02 abc 9 ± 0.9 abcd 9 ± 0.7 abcde
C38STPp contributing
13.6 ± 0.6 ab to improved performance
23.2 ± 0.9 a were C1MPm,
2 ± 0.01 ab C18MPm, C28MPm, C52STPp,
11 ± 0.7 ab
C39STPp 9.4 ± 0.4 b 13.2C65STPp,
± 0.6 abc and
24.8 ± C74STPp,
0.9 abc significantly
21.8 ± 0.9 a 1 ±enhancing
0.01 ab 2root
± 0.02development.
ab
9 ± 0.5 abcFor root 11 ±diameter,
0.6 abc
C52STPp 10.7 ± 0.9 ab 9 ± 0.8 bcd 19.9 ± 0.9 bc 26.4 ± 0.9 a 2 ± 0.03 a 2 ± 0.01 ab 9 ± 0.5 abcd 6 ± 0.3 ef
C54STPp 10.8 ± 0.5 ab 13.3treatments
± 0.9 abc inoculated
26.1 ± 0.9 abc with
22.1 ±the
0.9 astrains 2± C16MPm,
0.02 a C18MPm,
2 ± 0.02 ab C38STPp, C39STPp,
6 ± 0.5 abcd 10 ±C74STPp,
0.5 abcd
C59STPp 10.4 ± 0.4 ab and C99STPp showed 19.7 ± 0.9 bc
notable abincreases. Regarding
2 ± 0.03 a
root number, treatments
6 ± 0.8 bcd
with the
C63STPp 10.3 ± 0.05 ab 8.7 ± 0.4 cd 26.3 ± 0.9 abc 23.0 ± 0.9 2 ± 0.02 ab 2 ± 0.01 ab 6 ± 0.4 abcd 9 ± 0.3 ef
C65STPp strains
9 ± 0.9 bcd C38STPp, C39STPp, 30.6 ± C54STPp,
0.9 a C65STPp, C74STPp,
2 ± 0.01 ab and C99STPp resulted
11 ± 0.3 abc in a
C68STPp 10.3 ± 0.8 ab 9 ±higher
0.6 bcd count,25.2 ranging
± 0.9 abc from
24.3 ±100.9toa
11 roots
2 ± 0.02per
a
seedling (Table
1 ± 0.01 abc
6).8 ± 0.9 abcd 6 ± 0.4 abcde
C74STPp 11.7 ± 0.8 a 16.7 ± 0.9 a 28.7 ± 0.9 abc 27.7 ± 0.9 a 2 ± 0.04 a 2 ± 0.01 a 10 ± 0.9 a 11 ± 0.6 a
C99STPp 11.0 ± 0.4 ab 13.3 ± 0.7 The
abc treatments
22.3 ± 0.9 abc applied 0.9 P.
23.5 ± to a patula2 ± seedlings
0.02 a showed
2 ± 0.01 ab a significant
9 ± 0.8 abcdincrease 10 ±in0.6height,
abc

Control 5.1 ± 0.6 c 5 ±with

0.8 d statistical differences
5.9 ± 0.9 d
6.4 ±supporting
0.8 b 0.6 their
± 0.01 benhanced yield
0.8 ± 0.03 c
in 2this
± 0.8aspect.
e
On thefg other
4 ± 0.9

hand,with
Values in terms
a common of root letterlength and column
within each number, are the treatmentsdifferent
not significantly applied to P. montezumae
according seed-
to Tukey's multiple
lings were the most effective, showing significant differences compared to other treat-
comparison test at p ≤ 0.05.

ments.
The treatments applied to P. patula seedlings showed a significant increase in height,
Tablestatistical
with 6. Results of the measured
differences parameters
supporting of P.enhanced
their montezumaeyield and P. inpatula seedlingsOn
this aspect. after
the100 days
other
in nursery.
hand, in terms of root length and number, the treatments applied to P. montezumae seedlings
were the most effective, showing significant differences compared to other treatments.
Height (cm) Root Length (cm) Root Diameter (mm) Number of Roots
Strain P. monte- 4. Discussion P. monte- P. monte-
P. patula P. montezumae P. patula P. patula P. patula
zumae In microorganisms, at least three Trp-dependent zumae metabolic zumae pathways for IAA biosyn-
C1MPm 12.6 ± 0.9have
thesis abc
been identified,27.7 namely± 0.9 the
a
IPyA pathway, 1 ± 0.02thebcAIM pathway, and8 the ± 0.5TAM bcde

C13MPm 10.8 ± 0.9 [80,81]. Despite the

pathway bc 23.5 ± 0.9 in prokaryotic
diversity a 1 ±metabolism,
0.02 abc
IAA biosynthesis 7 ± 0.6 cdefpre-
C16MPm 9.7 ± 0.7 ab 10.8 ± 0.6
dominantly bc 23.7 ± 0.9 two pathways,
follows abc 25.1 ± 0.9 the a 1 ± IAM
0.03 route2 ±and
ab 0.01the IPyA
ab 9 ± 0.6 abcd
route, excluding9 ± 0.5 abcdethe
C18MPm 11.6 ± 0.8 a 11.1
TAM ± 0.9 abc 32.1 and
pathway ± 0.9the 26.2 ± 0.9 a 1 ± 0.01
bc Trp-independent pathwayab 2[82].
± 0.02 10 ± 0.3 ab pattern
A abcharacteristic 6 ± 0.9
of IAAdef

C25MPm 9.6 ± 0.6 bcd 9.0biosynthesis

± 0.7 bcd 17.5 has± been
0.9 c observed 22.9 ±in 0.9plant–microbe
a 1 ± 0.01 ab 1 ± 0.02 bc 7specifically
interactions, ± 0.9 abcd related 7 ± 0.3 tocdef
the
C28MPm 10.1 ± 0.4 ab 10.2 ± 0.9
ecophysiological
bc 29.5 ± 0.9 role ab of the27.0 ± 0.9 1 ± 0
bacterial a species ab 1 ± 0.02
involved, whether
abc 9 ± 0.9
pathogenic
abcd 9 ± 0.7
or growth- abcde

C38STPp promoting
13.6 ± 0.6 ab bacteria [83]. Numerous 23.2 ± 0.9bacteria
a from the 2 ±taxonomic
0.01 ab classes α-Proteobacteria,
11 ± 0.7 ab
β-Proteobacteria, δ-Proteobacteria, and Bacilli ab are known toabproduce this compound [84].
C39STPp 9.4 ± 0.4 b 13.2 ± 0.6 abc 24.8 ± 0.9 abc 21.8 ± 0.9 1 ± 0.01
a 2 ± 0.02 9 ± 0.5 abc 11 ± 0.6 abc
Previously, Trp-dependent IAA production was reported in the genus Bacillus in pine
C52STPp 10.7 ± 0.9 ab 9 ± 0.8 bcd 19.9 ± 0.9 bc 26.4 ± 0.9 a 2 ± 0.03 a 2 ± 0.01 ab 9 ± 0.5 abcd 6 ± 0.3 ef
Life 2024, 14, 1320 12 of 21

species [85,86]. The results of the IAA production test in the present work highlight the sig-
nificant role of auxin-producing bacteria in promoting plant growth. The strains analyzed
demonstrated the ability to produce indole-3-acetic acid (IAA), a crucial phytohormone
involved in various plant developmental processes. This metabolic capability was de-
tected across several bacterial strains, indicating their potential as PGPB. Furthermore, the
metabolic pathway used by these strains aligns with previous studies [87–90], showing that
both rhizospheric and endophytic bacteria can synthesize IAA, thereby enhancing seed
germination and early seedling growth, which is particularly relevant for forest species
like Pinus spp. These findings reinforce the importance of microbial auxin production in
fostering mutualistic plant–microbe interactions and suggest a broader ecological role for
these bacterial isolates in forest restoration and sustainable agriculture [91].
The ARA assay has been used to identify diazotrophic bacteria isolated from P. patula,
including species such as Bacillus macerans and γ-proteobacteria (Pseudomonas sp.). A
reduction in activity peak was observed after 3 h of evaluation, with reduction ranges
varying between 110 and 120 nmol of acetylene [39]. The ARA results of our work revealed
the presence of nitrogen-fixing capabilities in the bacterial strains associated with Pinus
patula. These strains, including Bacillus macerans and Pseudomonas sp. [92–95], demonstrated
significant asymbiotic nitrogen fixation activity, a crucial mechanism for enhancing plant
nitrogen uptake. These findings emphasize the potential of these diazotrophic bacteria to
contribute to nitrogen input in forest ecosystems, which can support the growth of Pinus
species by supplementing essential nutrients, especially in nutrient-poor soils targeted
for reforestation.
The evaluation of phosphorus-solubilizing bacteria (PSB) varies over time; for instance,
maximum phosphate solubilization is typically achieved after 3 days of incubation. How-
ever, extending incubation to 5 days does not further improve the solubilization extent [57].
In our study, some strains demonstrated notable efficiency in phosphate solubilization,
with a decline in activity observed after 10 days of incubation, while others reached the
peak activity on day 10. This variation may be related to their growth capacity or gene
expression in the culture medium [38]. The primary mechanism of mineral phosphate
solubilization involves the action of organic acids synthesized by PSB, along with the
production of phosphatase enzymes [41,95]. Notable phosphate-solubilizing acids reported
include gluconic and 2-ketogluconic acids, which are consistently identified in these bacte-
ria [96]. Additionally, other organic acids with phosphate-solubilizing capacity are oxalic,
citric, butyric, malonic, lactic, succinic, malic, acetic, fumaric, adipic, and indoleacetic
acids [97,98]. There is a close link between acidic pH and effective phosphate solubilization;
a decrease in pH clearly indicates acid production, which is considered responsible for
phosphate solubilization. It is suggested that the microorganisms reducing the medium
pH during growth are effective PSB [21]. The phosphate solubilization assays of this work
revealed that several bacterial strains demonstrated significant potential as PSB, capable of
performing such a critical function for plant growth promotion. These findings highlight
the important role of PSB in enhancing nutrient availability for plants and contributing to
improved growth, particularly in phosphorus-deficient soils.
The siderophore production assays performed in the present work demonstrated that
12 bacterial strains effectively produced these iron-chelating compounds, playing a crucial
role in promoting plant growth and inhibiting pathogens [41,99]. The CAS assay revealed
strong siderophore activity, indicated by the color change from blue to orange, confirming
the ability of the strains to sequester ferric iron [100]. This process enhances iron availability
for plants, particularly in iron-deficient environments, while simultaneously limiting access
to iron for harmful microorganisms [101]. The results underscore the potential of these
strains to improve plant resilience by enhancing nutrient uptake and protecting against
pathogens, particularly in forest ecosystems.
PGPB exhibit beneficial properties for plants, linked to the expression of specific genes
that enhance nutrient uptake and mitigate the negative effects of phytopathogens [102].
ACC deaminase and IAA production are crucial in plant–bacteria interactions due to their
Life 2024, 14, 1320 13 of 21

role in promoting root elongation [95]. The unexpected results of acdS gene amplification
in our tests were surprising, as it was expected that IAA-producing strains would express
this gene. The close relationship between enzyme production and plant growth regulators
arises because ACC synthase, which converts S-adenosylmethionine (SAM) to ACC, is
stimulated by these regulators [103]. The acdS gene, frequently identified in rhizobacterial
genera from various soil types across different geographical areas, is co-regulated by
the acdR and acdB proteins [104–106]. Microbial deamination of ACC reduces ethylene
concentration, providing a beneficial mode of action for plants [107]. Additionally, strains
C13MPm, C28MPm, and C38STPp amplified the prnD gene, associated with the synthesis
of pyoluteorin, an antimicrobial involved in the biological control of soil pathogens [99].
There is a possibility that strains isolated from P. montezumae and P. patula may produce
other antimicrobials for the biological control of phytopathogens. γ-proteobacteria are
known for producing various antimicrobial compounds, such as 2,4-diacetylphloroglucinol
(DAPG), pyoluteorin (PLT), pyrolnitrin (PRN), hydrogen cyanide (HCN), and gluconic and
2-ketogluconic acids, produced via the direct oxidation of glucose pathway [108].
Studies have demonstrated the potential of Serratia strains to promote plant growth
through various mechanisms, including the following: phosphate solubilization; the
production of indole-3-acetic acid (IAA) and 1-aminocyclopropane-1-carboxylate (ACC)
deaminase; the synthesis of antimicrobial compounds, siderophores, and quorum-sensing
molecules such as N-acyl homoserine lactones (AHLs); systemic resistance (ISR) against
pathogens [109–113]; and increased drought stress tolerance [114,115]. Additionally, one
study identified a strain of Bacillus, known for its genetic diversity and spore-forming
ability, which is valuable in the production of stable bioinoculants [116,117]. Both Serratia
and Bacillus strains have been documented in the literature for their roles in the isolation
and growth promotion of pine seedlings. In this study, we present a table that contrasts
our findings with those reported in the previous research, providing a comprehensive
comparison of the effects of these strains on pine seedling growth (Table 7). This genus,
widely distributed in many ecological niches, is frequently isolated and is remarkable for
wide applications in ecology, biotechnology, industry, and clinical microbiology, with im-
portant research conducted regarding its genetic diversity [118]. Buttiauxella is a genus with
limited reports on its role as a plant growth promoter. One of the investigation reports [119]
described an endophytic strain identified as Buttiauxella sp. SaSR13, which demonstrated
successful colonization in the root elongation zone that was attributed to increased IAA
concentrations and reduced superoxide anion levels, along with improvements in the root
exudates, particularly malic and oxalic acids in Sedum alfredii. This resulted in significant
growth enhancement and cadmium accumulation. Conversely, other studies [120,121] have
shown that the Buttiauxella strains isolated from the rhizospheres of Festuca arundinacea and
Vaccinium spp. are highly effective in solubilizing inorganic phosphorus, exhibiting catalase
activity, and producing organic acids and siderophores. In our research, a Buttiauxella strain
associated with P. montezumae was characterized, demonstrating its capability to promote
the growth of both P. patula and P. montezumae, suggesting a similar beneficial potential as
the other beneficial bacteria.
The speed of seed germination is critical for seedling growth under adverse con-
ditions, reducing the risk of phytopathogenic infections or latent infections following
transplantation [122,123]. Evidence supports that faster and more uniform germination
leads to a significant reduction in seedling mortality and an increase in the number of
viable seedlings [124–126]. While increased shoot height due to bacterial inoculation may
not be essential, the health and architecture of the root system are crucial to successful
development and transplant survival. These factors greatly influence the survival post-
transplantation [47,75]. However, the effect of bacteria on shoot and seedling growth
appears to be species-specific and independent. PGPB play a vital role in enhancing
seedling growth in nurseries, as assessed through established biometric parameters such
as stem length, collar diameter, and dry weight [106]. Our results for shoot height and
root length are consistent with previous research in forest species inoculated with bacterial
Life 2024, 14, 1320 14 of 21

strains [17,39,47,127,128]. Although the duration of trials may vary, the evaluated strains
show plant growth improvements, even in short-term trials. Strains isolated from the
rhizosphere of Pinus patula in Colombia have also shown enhanced plant performance
following inoculation [39]. The results of this study highlight the importance of seed germi-
nation speed in Pinus species to improve seedling growth under adverse conditions. The
bacterial strains evaluated demonstrated improvements in seedling growth, confirming
their potential as plant growth-promoting agents in forest species.

Table 7. Comparison of treatment yields with similar works.

Height Range Root Length Root Diameter Duration of

Reference Strains Total Treatments Forestry Species
(cm) Range (cm) Range (mm) Trials (Months)
Serratia marcescens,
Bacillus subtilis, Pinus taeda L. and
[47] Paenibacillus macerans, 12 Pinus elliottii 13–14.8 15.6–17.9 3
Bacillus pumilus, (Engelm.)
Bacillus sphaericus
Enterobacter intermedius,
Pseudomonas fluorescens, Quercus ilex ssp.
[128] Chryseobacterium 4 Ballota and Pinus 17.52–19.72 4
balustinum, pinea
Phosphorobacillus latus
Bacillus sp.,
Curtobacterium sp.,
[127] Arthrobacter sp., 10 Pinus pinea 20–26 5
Staphylococcus sp.,
Burkholderia sp.
Pseudomonas sp., Bacillus
macerans., Enterobacter
[39] 11 Pinus patula 7.3–18.7 12
agglomerans., Suillus
luteus., A. chroococcum.
Cupriavidus basilensis.,
Rhodococcus qingshengii.,
Pseudomonas spp., Pinus
[17] Pseudomonas gessardii., 10 pseudostrobus 4.6–6.6 5
Stenotrophomonas (Lindl.).
rhizophila., Rhodococcus
erythropolis., Cohnella sp.
This Serratia sp., Buttiauxella Pinus montezumae
16 7.7–14.5 17.5–29.5 1–2 4
study sp., Bacillus sp. and Pinus patula

The ANOVA test revealed significant differences in the growth responses of the
Pinus patula and Pinus montezumae seedlings to the treatments applied, highlighting specific
patterns for each species. The most notable outcome for P. patula was the significant increase
in seedling height, indicating that the applied treatments had a clear and statistically
significant effect on this growth parameter. This suggests that the factor being tested,
likely the inoculation conditions, played a crucial role in promoting height in P. patula
seedlings. In contrast, P. montezumae seedlings showed better performance in terms of root
length and the number of roots, although these differences were not statistically significant
in the other growth parameters. This indicates that while the treatments had a positive
effect on the root system of P. montezumae, this effect was not strong enough to reflect
significant changes in additional traits, such as stem height or overall biomass. Thus, the
treatments appear to be more effective in influencing root morphology in P. montezumae,
but further refinement may be required to observe broader impacts. Interestingly, no
significant differences were observed in root diameter for either species. This suggests that
root diameter might be less sensitive to the treatments applied, or that other factors, such
as genetic traits play a more dominant role in determining this particular parameter. The
lack of statistical significance for root diameter may also imply that the methods used need
further optimization to influence this trait. From these observations, several perspectives
emerge. First, the treatments could be optimized to achieve more consistent results across
all growth parameters. While height and root length responded positively in each species,
focusing on improving other traits, like root diameter, could lead to more comprehensive
growth enhancements. Additionally, further exploration of species-specific responses
Life 2024, 14, 1320 15 of 21

could provide insights into why P. patula responded better in terms of height, while P.
montezumae showed more robust root growth. Moreover, incorporating additional variables
beyond height and root traits, such as photosynthetic rate, nutrient uptake, or stress
tolerance, could offer a more complete picture of how the treatments affect plant health
and development. This would also provide valuable information for refining treatment
protocols in both nursery and field conditions. These findings could have significant
implications for reforestation and forest management programs. By tailoring treatments to
enhance specific traits, such as height in P. patula for timber production or root growth in P.
montezumae for soil stabilization, can improve the success rates of these programs.
This study highlights the significant role of PGPB in promoting growth and improving
resilience in forest species such as P. montezumae and P. patula. Key mechanisms include
phosphate solubilization, siderophore production, nitrogen fixation, and the synthesis of
indole-3-acetic acid (IAA). These bacterial activities enhance nutrient availability, stimulate
root development, and improve seed germination, which are crucial for successful refor-
estation, especially in nutrient-deficient soils. The potential application of these bacteria in
forest restoration demonstrates their ecological importance and offers a promising approach
to sustainable forest management. Further work will be needed to demonstrate that these
mechanisms are indeed associated with the growth promotion we have observed in situ.
However, it is likely that several of the growth-promoting mechanisms observed in vitro
for the bacteria explored in this study are also occurring in association with pine plants, as
reported in other works [129,130].

5. Conclusions
This study identified and characterized the key mechanisms by which isolated strains
from Pinus patula and Pinus montezumae promote plant growth. The evaluated strains
exhibited plant growth-promoting mechanisms, including auxin production, phosphate
solubilization, and siderophore production. Ten strains with the potential to enhance
pine growth were selected for further molecular characterization, with seven belonging
to the genus Serratia, one to Bacillus, and notably, one to the less commonly associated
genus Buttiauxella. These findings suggest a promising approach to enriching soil microbial
populations in reforestation efforts by utilizing nursery-grown plants inoculated with
beneficial microorganisms. By introducing plants with a robust rhizospheric microbiota,
not only is the reintroduction of beneficial microbes into the soil facilitated, but early plant
growth is also enhanced, mitigating stress and leading to better soil adaptation compared
to the non-inoculated plants.

Author Contributions: Conceptualization: F.D.M.-V. and Y.E.M.-G.; methodology, F.D.M.-V. and

Y.E.M.-G.; software, F.D.M.-V.; formal analysis, F.D.M.-V. and M.Á.P.-E.; investigation, F.D.M.-V.,
J.M.-R. and M.Á.P.-E.; data curation, F.D.M.-V. and J.M.-R.; writing—original draft preparation,
F.D.M.-V. and M.Á.P.-E.; writing—review and editing, F.D.M.-V., Y.E.M.-G. and J.M.-R.; visualization,
Y.E.M.-G. and M.Á.P.-E.; supervision, J.M.-R. and M.Á.P.-E. All authors have read and agreed to the
published version of the manuscript.
Funding: This project was funded by the Scientific Research and Technological Development Projects
of the Secretariat of Research and Postgraduate Studies (SIP) of the National Polytechnic Institute,
Mexico, SIP Projects 20180951 and 20170573. This work was supported by the Internationalization of
Research program at VIEP-BUAP (VIEP-Muñoz-Rojas 100425788).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The raw data supporting the conclusions of this article will be made
available by the authors on request.
Life 2024, 14, 1320 16 of 21

Acknowledgments: Francisco David Moreno-Valencia acknowledges CONAHCYT, which provided

a postdoctoral scholarship (CVU: 309635), and the Meritorious Autonomous University of Puebla
and Microbiological Sciences Research Centre, for providing the laboratories and resources to carry
out the postdoctoral work.
Conflicts of Interest: The authors declare no conflicts of interest.

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