University of Zulia, Venezuela

Bolivarian Republic of Venezuela

Keywords:

Plant growth promoting rhizobacteria

Nitrogen xation

IAA

Phosphate solubilization

Bacterial carbon solubilization

PGPR activity of coal solubilizing bacteria

Actividad PGPR de bacterias solubilizadoras de carbón

Atividade PGPR de bactérias solubilizadoras de carvão

Microbiologist, Research Group in Agricultural and

Environmental Microbiology, Popular University of Cesar,

Colombia.

Assistant Professor, Department of Microbiology, Research

Group in Agricultural and Environmental Microbiology,

Popular University of Cesar, Colombia.

Associate Professor - Faculty of Basic Sciences - University

of La Guajira.

Professor of the Department of Natural Sciences and

Environmental Education - Popular University of Cesar.

Received: 02-11-2021

Accepted: 17-05-2022

Published: 14-06-2022

Abstract

Coal solubilizing bacteria (CSB) are microorganisms to able to bio

transformed low rank coal, releasing humied organic matter in the process.

On the other hand, these bacterial genera have reported previously as plant

growth promoting bacteria. The aim of this work was to assess the Plant

Growth Promoting Rhizobacteria (PGPR) capacity of ve CSB strains:

Bacillus pumilus (CSB05), B. mycoides (CSB25), Microbacterium sp.

(CSB3), Acinetobacter sp. (CSB13) and B. amyloliquefaciens (CSB02). For

this, the PGPR traits of CSB were evaluated under laboratory conditions: the

biological nitrogen xation capacity, the reduction of acetylene, the synthesis

of indole acetic acid (IAA) and the solubilization of phosphates. In a second

experiment under plant nursery conditions, PGPR activity of strain CSB05

was evaluated in common bean plants. Under laboratory conditions, it was

evidenced that all the evaluated strains produced IAA, solubilized phosphate

in a liquid medium, presented atmospheric nitrogen xation capacity, and

only the CSB3 and CSB13 strains reduced acetylene. In the plant nursery

experiment, PGPR activity of strain CSB05 was detected in common bean

plants, reected in increases in the height of these plants. These results show

that CSB are promising in the PGPR activity, which is interesting to the design

of biological products with agricultural and environmental applications, for

the management of crops in disturbed soils of the Colombian dry Caribbean.

Helen Liliana Brito-Campo

María Fernanda Ayala-Santamaría

Katherin Julieth Barros-Escalante

Juan Guillermo Cubillos-Hinojosa

Manuel Fabián Pantoja-Guerra

Nelson Osvaldo Valero

Liliana Gómez Gómez

Rev. Fac. Agron. (LUZ). 2022, 39(2): e223932

ISSN 2477-9407

DOI: https://doi.org/10.47280/RevFacAgron(LUZ).v39.n2.10

Environment

Associate editor: MSc. Beltrán Briceño Rodríguez

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Rev. Fac. Agron. (LUZ). 2022, 39(2): e223932. April - June. ISSN 2477-9407.

2-6 |

Resumen

Las bacterias solubilizadoras de carbón (BSC) son

microorganismos capaces de biotransformar carbones de bajo rango,

liberando en el proceso materia orgánica humicada y se ha reportado

que estas bacterias podrían promover el crecimiento de las plantas.

El objetivo de este trabajo fue evaluar la capacidad Plant Growth

Promoting Rhizobacteria (PGPR) de cinco cepas de BSC: Bacillus

pumilus (BSC05), B. mycoides (BSC25), Microbacterium sp.

(BSC3), Acinetobacter sp. (BSC13) y B. amyloliquefaciens (BSC02).

Para esto, fueron evaluados los rasgos PGPR en condiciones de

laboratorio: la capacidad de jación biológica de nitrógeno, la

reducción del acetileno, la producción de ácido indol acético (AIA)

y la solubilización de fosfatos. En un segundo experimento, en

condiciones de casa de malla, se evaluó actividad PGPR de la cepa

BSC05 en plantas de frijol común. En condiciones de laboratorio se

evidenció que todas las cepas evaluadas produjeron AIA, solubilizaron

fosfato en medio líquido, presentaron capacidad de jación de

nitrógeno atmosférico, y solo las cepas de BSC3 y BSC13 redujeron

el acetileno. En el experimento en casa malla, se observó actividad

PGPR de la cepa BSC05 en plantas de fríjol común, reejado en

incrementos en la altura de estas plantas. Estos resultados muestran

que las BSC son promisorias en la actividad PGPR, lo cual resulta de

gran interés en el desarrollo de insumos biológicos con aplicaciones

agrícolas y ambientales, para el manejo y producción de cultivos en

suelos perturbados del Caribe seco colombiano inuenciados por la

actividad minera.

Palabras clave: rizobacterias promotoras de crecimiento vegetal,

jación de nitrógeno, AIA, solubilización de fosfatos, solubilización

bacteriana de carbón.

Resumo

As bactérias solubilizadoras de carvão (BSC) são microrganismos

capazes de biotransformar carvões de baixo grau, liberando matéria

orgânica humicada no processo, além disso, tem sido relatado

que essas bactérias poderiam promover o crescimento das plantas.

O objetivo deste trabalho foi avaliar a capacidade Plant Growth

Promoting Rhizobacteria (PGPR) de cinco estirpes de BSC: Bacillus

pumilus (BSC05), B. mycoides (BSC25), Microbacterium sp.

(BSC3), Acinetobacter sp. (BSC13) e B. amyloliquefaciens (BSC02).

Para isso, foram avaliadas as características de PGPR em condições

de laboratório: a capacidade de xação biológica de nitrogênio, a

redução de acetileno, a produção de ácido indol acético (IAA) e a

solubilização de fosfatos. Em um segundo experimento em casa de

malha, avaliou-se a atividade PGPR da cepa BSC05 em plantas de

feijão comum. Em condições de laboratório, foi evidenciado que

todas as cepas avaliadas produziram IAA, fosfato solubilizado em

meio líquido, apresentaram capacidade de xação de nitrogênio

atmosférico, porém, apenas as cepas BSC3 e BSC13 reduziram

o acetileno. No experimento em casa malha, a atividade PGPR da

cepa BSC05 foi observada nas plantas de feijão comum, reetido

no aumento da altura do feijão. Estes resultados mostram que as

BSC são promissoras na atividade de PGPR, o que é de grande

interesse no desenvolvimento de insumos biológicos com aplicações

agrícolas e ambientais, para o manejo e produção de cultivos em solos

perturbados do Caribe seco colombiano inuenciados pela atividade

de mineração de carvão.

Palavras-chave: Rizobactérias promotoras do crescimento vegetal,

xação de nitrogênio, IAA, solubilização de fosfato, solubilização

bacteriana do carbono.

Introduction

The application of low rank coal (LRC) lignite-type directly

to the soil is an adequate strategy for the conservation of organic

matter in dry tropical soils (Cubillos et al., 2015), apparently the

colonization of these carbonaceous residues by coal bio-transforming

microorganisms increases the gradual release of humic substances

(HS) (Valero et al., 2014). In addition, the porosity and other

characteristics of these coals improve the physical properties of the

soil and indirectly stimulate microbial activity (Pantoja-Guerra et al.,

2019; Valero et al., 2016).

In previous bioprospecting studies of bacteria in LRC, coal

solubilizing bacteria (CSB) were isolated from the rhizosphere

of grasses that grow in environments with residual coal particles:

B. mycoides (CSB25), B. pumilus (CSB05), B. amyloliquefaciens

(CSB02), Microbacterium sp. (CSB03) and Acinetobacter sp.

(CSB13). These bacterial strains are capable to solubilize lignite,

releasing HS in the process (Valero et al., 2018; Valero et al., 2018).

Additionally, some studies have shown that these bacterial genera

have PGPR capacity (Idris et al., 2007; Tejera et al., 2013; Tejera et

al., 2011). In the case of B. pumilus, in addition to its role as PGPR,

its capacity for endophytic colonization of plant tissues has been

reported (De-Bashan et al., 2010). The PGPR capacity of some coal

solubilizing bacteria strains has also been reported (Titilawo et al.,

2020).

The PGPR are bacterial populations present in the rhizosphere,

which have the ability to colonize the root system of plants or their

closest environment, generating increases in plant growth (Kloepper

et al., 1989). These bacteria promote plant growth by direct and

indirect mechanisms, especially in soils with nutrient limitations and

stress conditions. The most studied direct mechanisms are the nitrogen

xation, phosphate solubilization, production of phytohormones

and siderophores, and ACC-deaminase enzymes (Glick, 2012). In

addition, there are some indirect mechanisms associated with plant

protection against the activity of phytopathogenic microorganisms

(Meena et al., 2020). Therefore, the aim of this work was to evaluate

the PGPR activity of some coal solubilizing bacterial strains, so

that their potential in the design of biotechnological products of

agricultural and environmental interest can be better known.

Materials and methods

Coal solubilizing bacteria (CSB)

Strains from open-cast coal mines located in the Cesar and La

Guajira states, Colombia, identied as B. mycoides (CSB25) isolated

from the rhizosphere of Typha dominguensis; Microbacterium sp.

(CSB3) isolated from LRC; Acinetobacter sp. (CSB13) isolated from

coal wash sediments; B. pumilus (CSB05) and B. amyloliquefaciens

(CSB02) isolated from grasses rhizosphere of coal sludge in coal

mine wash areas (Valero et al., 2012). These bacteria were selected

for their ability to bio-transform LRC lignite-type in solid and liquid

media, releasing HS (Valero et al., 2014).

Evaluation of the PGPR traits of the CSB

Biological nitrogen xation (BNF) in solid medium: The ve

CSB in study were massively propagated on nutrient agar, a colony

of each strain was taken and inoculated separately in Petri dishes

with solid nitrogen-free culture medium (NFB) (Döbereiner et al.,

1976), then the strains were incubated at 37 °C for ve days to

observe growth and a change of the medium, from the initial emerald

green color to a light blue. These characteristics are characteristic of

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nitrogen-xing bacteria of free-living. The strain Stenotrophomonas

sp. (BSFG03) was used as a positive control.

Acetylene reduction test: This test was determined by the

method established by Hardy et al. (1968) whit some modications.

The strains were seeded on trypticase soy agar (TSA) and incubated

at 30 °C for 48 h. Subsequently, each one was seeded in triplicate in

hermetic vials with 2 mL of Burk liquid medium and were incubated

at 30 °C for 48 h. Then, in each vial, was injected with acetylene

and it was left to react for 1 h. The production of ethylene generated

from the reduction of acetylene by the nitrogenase enzyme was

quantied by gas chromatography. For this, a Shimadzu GC 2010 gas

chromatograph was used coupled to a mass detector, manual injection

in splitless mode (1 mL) and helium carrier gas at a constant ow of

1 mL·L

-1

. The Carbowax/20 M capillary column of 30 m, 0.250 mm

diameter and 0.25 µm. The oven temperature was set at 50 °C for

4 min at a ow rate of 36.3 cm·sec

-1

, with a run time of 4 min and

retention time of 1.8 min.

Indole-3-acetic acid (IAA) production: 500 µL of each bacterial

suspension were inoculated of trypticase soy broth supplemented with

5 mM L-tryptophan. Then, in each vial, was injected with acetylene

and it was left to react for 1 h. Then, these were incubated at 28 °C for

24 h in dark conditions. Negative control vials were used as treatment

in the same way without inoculation. After, the bacterial cultures were

centrifuged of Salkowski’s reagent were added to 1.5 mL supernatant.

Subsequently, it was incubated for 20 minutes at room temperature in

dark conditions, the absorbance was read in a spectrophotometer at

520 nm, and the results obtained were compared with an AIA standard

curve at concentrations of 5 µg·mL

-1

to 100 µg·mL

-1

Solubilization of phosphates: In a preliminary test (data not

shown) the ability of the ve CSB strains to solubilize phosphate

(Ca

(PO

)2) in solid medium was evaluated. All the strains

presented a clear halo around the colonies, which was interpreted

as a presumptive result in phosphate solubilization. The phosphate

solubilization of the CSBs in liquid medium was then evaluated. One

colony of each CSB studied was inoculated in Sundara-Rao and Sinha

(SRS) broth, supplemented with tricalcium phosphate (Ca

(PO

)2) in

test tubes. After 24 h, 50 µL of inoculum were seeded in 5 mL of SRS

broth, incubated at 30 °C with shaking at 120 rpm for 24 h. Then

250 µL of the inoculum were taken and resuspended in 750 µL of

sterile deionized water and 240 µL of paramolybdate blue reagent. As

a control, tubes with 750 µL of sterile deionized water and 240 µL of

the colorimetric reagent without inoculum and the strain BSFG03 as

positive control were taken. A spectrophotometer with a wavelength

of 520 nm was used for reading. The results were compared with a

phosphorus standard curve at concentrations from 0.1 to 5 µg·mL

-1

(Kumari et al., 2018).

Evaluation of the effect of Bacillus pumillus on the growth of

Phaseolus vulgaris plants

Previous screening works (data not shown) allowed to establish the

PGPR potential of CSB strain B. pumilus (CSB05). These antecedents

allowed selecting the CSB05 strain for in planta trials, under controlled

conditions in a phytotron with common bean plants. The CSB05 strain

was seeded with nutrient agar for 48 h. Subsequently, a typical and

pure colony of B. pumilus was inoculated in an erlenmeyer ask with

200 mL of nutrient broth, incubated with shaking at 120 rpm for 72

h. Subsequently, the inoculum concentration was optimized to 1x10

UFC·mL

-1

. For this trial, an experimental design was made with two

treatments and ve repetitions, the rst treatment with the bacterial

inoculum and the second without inoculum (control treatment). The

common bean seeds were disinfected and sown in pots with 200 g of

sterile soil. Then, with a sterile syringe, each pot was inoculated with

10 mL of the biopreparation. The control treatment was inoculated

with 10 mL of sterile nutrient broth. The seedlings were kept under

plant nursery conditions. After 15 days, the height of the plant and the

total biomass were determined.

Re-isolation and identication of Bacillus pumilus as an

endophytic strain

For the recovery of B. pumilus (CSB05) as an endophytic organism,

the tissues were cut and disinfected and macerated according to

the protocol proposed by Araújo et al. (2002). Immediately serial

dilutions were made, 100 µL of sample was taken from each of the

dilutions and inoculated into Petri dishes with nutrient agar, the

boxes were incubated at 28 °C for 5 days. After 5 days, the formation

of bacterial colonies was observed, from which colonies whose

morphology presumptively agreed with B. pumilus were isolated and

puried. From these isolates, the DNA was obtained and puried

for the identication of the strain by direct amplication by PCR of

the 16S ribosomal DNA, its partial sequence (with reading in two

directions) and analysis of the sequence.

Statistical analysis

Analysis of variance (ANOVA) was performed with a 95 %

condence level in each of the experiments using the R statistical

package. Additionally, multiple comparison tests were performed:

Dunnett’s test (p<0.05) for the experiment of the acetylene reduction,

Tukey (p<0.05) for AIA production tests, solubilization of phosphates

in solid and liquid medium.

Results and discussion

Biological nitrogen xation (BNF) by CSB in solid medium

The growth of all CSB studied was observed on NFB Agar. In

addition, a shift in the bromothymol blue indicator towards a faint

blue color was observed, due to the alkalinization of the medium.

The positive control for biological nitrogen xation with the strain of

Stenotrophomonas sp. (BSFG03), showed growth in the medium and

a yellow color turn of the medium, which suggests an acidication of

the medium. NFB Agar is a selective culture medium lacking nitrogen

sources, so it is presumed that the bacteria that grew in it were able of

xing atmospheric nitrogen through the enzyme nitrogenase, capable

of incorporating atmospheric nitrogen into bacterial metabolism

(Mirza and Rodriguez, 2012)

Acetylene reduction test - nitrogenase enzyme activity

Figure 1 shows that the strains CSB3 (Microbacterium sp.)

and CSB13 (Acinetobacter baumanii) presented a higher acetylene

reduction with statistically signicant differences (p < 0.1 for both)

compared to the other evaluated CSB, which behaved similarly to the

control. With this test, the capacity to x nitrogen of the evaluated

strains was indirectly determined, measuring the activity of the

nitrogenase enzyme taking advantage of its ability to break the triple

bond of acetylene and convert it into ethylene. Therefore, it is possible

to infer that the nitrogenase activity was positive for the CSB3 and

CSB13 strains, and for the other treatments that correspond to the

Bacillus genera strains it was negative.

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4-6 |

behaved similar to each other, showing the ability to solubilize

tricalcium phosphate. Solubilization showed lower values at 24 h

except for the positive control (BSFG03), which behaved similarly

over time. In the gure 3 is showed that the highest solubilization

occurred at 48 h, but then at 72 h there was a decrease in soluble

inorganic phosphorus values, which is conrmed by Castagno et

al. (2011), the strains studied by them had a maximum peak at 48

h and a decrease at 72 h. This phenomenon can be explained by

alluding to the fact that in an initial stage (up to 48 h of incubation)

the phosphate was solubilized to be consumed later (Tejera et al.,

2013).

Figure 2. Production of indole-3-acetic acid (IAA) by CSB by

colorimetric reaction with Salkowsky’s reagent.

The letters above the bars represent statistically

signicant differences (Tukey p<0.05).

Figure 3. Quantitative determination of phosphate solubilization

by CSB in liquid medium over time (24, 48, 72 h).

The letters show the signicant differences between

treatments (Tukey p<0.05).

The genus Bacillus is one of the most studied regarding the ability

to solubilize phosphates (Tejera et al., 2013). The solubilization

of phosphates by PGPR allows not only a better assimilation

Figure 1. Quantitative biological nitrogen xation (ethylene

generated from the reduction of acetylene) of

CSB. The asterisks (***, **, *) indicate signicant

differences of the treatments with respect to the

control. (Dunnett <0.01 *** - <0.05 ** - <0.1 *).

In other studies, with B. pumilus has been reported that is able

of reducing acetylene to ethylene (Acuña et al., 2010) and other

species of the genera Bacillus isolated from wheat, corn and rice,

have been characterized with high nitrogenase activity (Corrales-

Ramirez et al., 2016). Although in this test it could be inferred that

the CSB25, CSB05 and CSB02 strains do not have the ability to

reduce acetylene, it is important to clarify that the Burk`s medium

used in this test was devoid of nitrogen, which is a factor that

activates sporulation in the genera Bacillus, this limits its metabolic

activity (Lalloo et al., 2009). This result could be the effect of an

underestimation.

Production of indole-3-acetic acid (IAA)

All the CSB evaluated were IAA producers, the results showed

in gure 2 have the net IAA production, and that is, the estimate in

the control treatment (without inoculation) was subtracted to avoid

the artifact effect of a possible coloration in the medium. According

to gure 2, the production of IAA behaved in a similar way in all

the strains, except for CSB3, which was statistically superior to

the other treatments, while in the other strains they did not present

signicant differences between them. The genera Bacillus, it

is not generally a great producer of IAA, however, some strains

capable of producing up to 16 µg·mL

-1

(Tejera et al., 2011). The

production of IAA from rhizosphere microorganisms can improve

root architecture, increasing the total surface of the root, which

consequently can improve the absorption of water and nutrients

(Naveed et al., 2015)

Quantitative determination of phosphate solubilization of

CSB in liquid medium

All strains presented phosphate solubilization values higher than

the negative control. The CSB3 strain had a lower solubilization

value than the rest of the strains over time, inferring that this strain

does not present a signicant capacity to solubilize phosphates. The

other treatments (CSB25, CSB13, CSB05, CSB02 and BSFG03)

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Brito-Campo et al. Rev. Fac. Agron. (LUZ). 2022, 39(2): e2239325-6 |

of insoluble phosphorus from the soil but also the possibility of

progressively minimizing the amount of chemical fertilizers used

daily in conventional agriculture (Kumari et al., 2018).

Evaluation of the effect of B. pumilus (CSB05) on the growth

of P. vulgaris plants

The endophytic CSB05 showed PGPR activity on common

bean seedlings under plant nursery conditions, showing signicant

statistical differences regarding to the control. The variable studied:

height was higher than the control treatment as shown in gure 4.

Figure 4. Stem length (left) and total dry weight (right) of

Phaseolus vulgaris, after 15 days of inoculation with

Bacillus pumilus (CSB05) compared to the control.

This growth promotion carried out by the endophytic strain CSB05,

regarding to the increase in the length of the stem in bean plants

could be due to its ability to produce auxin and AIA, which inuences

both cell division, growth and differentiation and are involved in the

growth of stems and lateral roots (Spaepen et al., 2014). Although

there are few studies on B. pumilus as a growth promoter in common

bean plants, some studies have shown the ability of B. pumilus strains

to promote different growth, types of plants, increasing stem and

root elongation, and sprout germination, using various mechanisms

such as IAA production, phosphate solubilization, production of

pathogen biocontroller metabolites (Ansari et al., 2019) and even

improve the adjacent microbiota after inoculation (De-Bashan et

al., 2010). Likewise, it is important to note that the CSB05 strain in

vitro tests presented the ability to solubilize phosphates (gure 3), a

key mechanism for plant development in degraded soils, where the

microbiota has been negatively affected by the use of fertilizers by

current agricultural techniques (Ramírez et al., 2014,).

Re-isolation and identication of CSB05 as an endophytic

strain

From the processing of plant tissues, it was possible to isolate

a bacterial morphotype, with the microscopic and macroscopic

morphology characteristic of B. pumilus (gure 5), while in plants

without bacterial inoculation, no bacterial isolation with these

characteristics was recovered. Comparison with the Ribosomal

Data Project (RDP) 16S sequence database, using the SeqMatch

tool against cultured isolates, indicates that the assembled problem

sequence has greater homology with sequences from the species B.

pumilus (gure 5).

Figure 5. Comparison of the sequence of the 16s rRNA of B.

pumilus CSB05 against SeqMatch-RD.

Endophytic bacteria establish a close relationship with plants

and can positively inuence their development, in phytoremediation

processes, acting as plant growth regulators, and also as biological

controllers, which provides an agronomic benet that can inuence

the decline of chemical fertilizers (Pérez and Chamorro, 2013). In

similar studies, B. pumilus has been reported as endophytic bacteria

and also as an effective biological controller producing metabolites

that inhibit the activity of phytopathogenic microorganisms (Ren et

al., 2013).

PGPR bacteria have been projected as a strategy for the

phytoremediation of soils with mining residues (De-Bashan et al.,

2010). Based on the results of the plant nursery evaluation of the

endophytic strain B. pumilus CSB05, in this study it is concluded that

it can increase the growth of the P. vulgaris plant. Likewise, in the

results of the in vitro tests, it was evidenced that the studied strains

present between two and three attributes of PGPR, for which it is

necessary to implement future greenhouse and eld trials to evaluate

their PGPR capacity. The results presented in this work agree with

those obtained by Titilawo et al. (2020), who reported PGPR traits in

coal biotransformer bacteria strains.

These PGPR traits allow these bacteria to be screened for

further studies and subsequent development of dual-purpose organic

amendments, which not only help to release HS in the rehabilitation of

soils disturbed by mining, but also allow the effective establishment

of species plants in soils disturbed by anthropic activity.

Conclusions

The coal solubilizing bacteria strains Microbacterium sp. (CSB3),

Acinetobacter sp. (CSB13), B. mycoides (CSB25), B. pumilus

(CSB05) and B. amyloliquefaciens (CSB02) show PGPR traits.

The endophytic strain B. pumilus (CSB05), stimulates the increase

in stem length in P. vulgaris plants after 15 days of inoculation and

is postulated as a potential biotechnological CSB and PGPR for the

design of useful products in the treatment of degraded soils.

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Acknowledgments

To Grupo de Investigación Microbiología Agrícola y Ambiental

(MAGYA) of the Universidad Popular del Cesar from Colombia

and the Servicio Nacional de Aprendizaje (SENA) La Salada Caldas

- Antioquia, Colombia for the support in the development of this

research.

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