Keywords:

Antarctic microorganisms

Benecial bacteria

Plant growth

Evaluation of Antarctic strains of Bacillus sp. as plant growth promoting bacteria

Evaluación de cepas Antárticas de Bacillus sp. como bacterias promotoras del crecimiento vegetal

Avaliação de cepas antárticas de Bacillus sp. como bactérias promotoras de crescimento de plantas

Ángela Beatriz Zambrano-Solórzano

Ángel Monserrate Guzmán-Cedeño

1,2*

María Fernanda Pincay Cantos

Jonathan Gerardo Chicaiza Intriago

Diego Efrén Zambrano Pazmiño

Rev. Fac. Agron. (LUZ). 2024, 41(3): e244121

ISSN 2477-9407

DOI: https://doi.org/10.47280/RevFacAgron(LUZ).v41.n3.01

Crop production

Associate editor: Dra. Evelyn Pérez Pérez

University of Zulia, Faculty of Agronomy

Bolivarian Republic of Venezuela

Escuela Superior Politécnica Agropecuaria de Manabí

“Manuel Félix López”, 10 de Agosto N°82 y Granada

Centeno. Calceta, Manabí, Ecuador.

Universidad Laica “Eloy Alfaro” de Manabí. Ciudadela

Universitaria vía San Mateo. Manta, Manabí, Ecuador.

Received: 06-03-2024

Accepted: 10-06-2024

Published: 01-07-2024

Abstract

In agriculture, ecient microorganisms are used, among them

plant growth-promoting bacteria. This work aimed to determine, in

vitro

, the mechanism of action in strains of Bacillus sp. isolated

from Antarctica. The analyzed characteristics of the bacterium were:

catalase and hemolysis tests, Gram stain, phosphate solubilization,

growth without a nitrogen source, siderophore production, and

survival at dierent values of pH, NaCl, and temperature, which

conrmed the ecological plasticity and adaptation of these strains

in environments other than their origin. According to the desirable

characteristics, the T5, GB-70, and B-6 strains were chosen and

added to two substrates: clay and clay-compost mixture, which were

sterilized and placed in 200 mL glass bottles, and a corn seed was

planted in each of them. After two weeks, the following parameters

were evaluated: length of root (LR), seedling height (AP), and

shoot diameter (DT). The simple eect of the strains as independent

variables and their interaction did not signicantly aect the

response variables evaluated, recording the following averages:

12.84 cm (LR), 15.28 cm (AP), and 2.26 cm (DT). Considering

the substrate, the compost + clay signicantly (p<0.05) inuenced

the LR and DT characteristics of the seedlings, with averages of

14.44 and 2.38 cm, respectively. The observed mechanisms of

action distinguish promising strains that could be validated at the

eld level in agricultural production systems when inoculated in

organic fertilizers.

This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.

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2-6 |

Resumen

En agricultura, se emplean microorganismos ecientes, entre

ellos las bacterias promotoras de crecimiento vegetal. El propósito

de este trabajo fue determinar, in vitro, el mecanismo de acción

en cepas de Bacillus sp. aisladas de la Antártida. Se analizaron los

siguientes aspectos: prueba de catalasa y de hemólisis, tinción de

Gram, solubilización de fosfato, crecimiento sin fuente de nitrógeno,

producción de sideróforo, y supervivencia a diferentes valores de pH,

NaCl y temperatura, con lo cual se constató la plasticidad ecológica

y adaptación de estas cepas en ambientes distintos al de su origen.

De acuerdo con las características deseables, se escogieron las cepas

T5, GB-70 y B-6 que se añadieron a dos sustratos: arcilla y mezcla

arcilla-compost, los cuales fueron esterilizados y depositados en

botellas de vidrio con capacidad de 200 mL, colocando una semilla de

maíz en cada una. Transcurridas dos semanas, se evaluó: la longitud

de raíz (LR), la altura de la plántula (AP) y el diámetro del talluelo

(DT). El efecto simple de las cepas como variables independientes y

su interacción, no incidieron signicativamente sobre las variables

respuestas evaluadas, registrándose los siguientes promedios: 12,84

cm (LR), 15,28 cm (AP), y 2,26 cm (DT). Considerando el sustrato,

el Compost + Arcilla mostró diferencias signicativas (p<0,05) en

las características LR y DT de las plántulas, con medias de 14,44

y 2,38 cm, respectivamente. Los mecanismos de acción observados

distinguen cepas prometedoras que podrían validarse a nivel de

campo en sistemas de producción agrícola cuando se inoculan en

fertilizantes orgánicos.

Palabras clave: microorganismos antárticos, bacterias beneciosas,

crecimiento vegetal.

Resumo

Na agricultura são utilizados microrganismos ecientes, entre

eles bactérias promotoras de crescimento de plantas. O objetivo

deste trabalho foi determinar, in vitro, o mecanismo de ação em

cepas de Bacillus

sp. isolado da Antártica. Foram analisados os

seguintes aspectos: testes de catalase e hemólise, coloração de

Gram, solubilização de fosfato, crescimento sem fonte de nitrogênio,

produção de sideróforos e sobrevivência em diferentes valores de

pH, NaCl e temperatura, o que conrmou a plasticidade ecológica

e adaptação desta cepa em ambientes diferentes de sua origem. De

acordo com as características desejáveis, foram escolhidas as cepas

T5, GB-70 e B-6 e adicionadas a dois substratos: argila e mistura

argila-composto, que foram esterilizados e acondicionados em

frascos de vidro com capacidade para 200 mL mL colocando uma

semente de milho em cada um. Após duas semanas foram avaliados:

comprimento da raiz (LR), altura da muda (AP) e diâmetro do caule

(DT). O simples efeito das deformações como variáveis independentes

e sua interação não afetou signicativamente as variáveis respostas

avaliadas, registrando-se as seguintes médias: 12,84 cm (LR), 15,28

cm (AP) e 2,26 cm (DT). Considerando o substrato, Composto +

Argila inuenciou signicativamente (p<0.05) as características de

LR e DT das mudas com médias de 14,44 e 2,38 cm, respectivamente.

Os mecanismos de ação observados distinguem cepas promissoras

que poderiam ser validadas em nível de campo em sistemas de

produção agrícola quando inoculadas em fertilizantes orgânicos.

Palavras-chave: microrganismos antárticos, bactérias benécas,

crescimento de plantas.

Introduction

The potential of microorganisms as biofertilizers is emerging as

a promising solution to gradually reducing or eliminating synthetic

fertilizers. Olanrewaju et al. (2017) argue that the adverse eects

of synthetic chemical fertilizers, such as soil nutrient depletion and

water pollution, drive the need for viable alternatives.

According to Delgado-Baquerizo et al. (2018), edaphic organisms

are fundamental to the structure and functionality of natural and

managed ecosystems (one gram of soil can contain thousands of

individual microbial taxa, including viruses). Plants interact with

various microorganisms, including bacteria and fungi, which can

have positive, negative, or neutral eects on plants. The role of

bacteria is underlined since they have various survival strategies in

adverse conditions such as low temperature, ultraviolet radiation, low

humidity, and nutrient deciencies (Chattopadhyay, 2006).

Microbial communities are rich in genetic diversity and

play a crucial role in adaptation and survival in challenging

environments; they represent a reservoir of unknown and

undescribed microorganisms, with great metabolic versatility.

These microbial communities that inhabit extreme environments are

essential for the maintenance of ecosystems (Castro et al., 2021).

The most predominant phyla in extreme (cold) environments are

Pseudomonadota, Bacteroidotas, Acidobacteriota, Cloroexota,

Planctomycetota, and Actinomycetota (Kudinova et al., 2021).

Extremophilic microorganisms also have been used as biofertilizers

(Rizvi et al., 2021) since they increase nitrogen xation and convert

insoluble phosphorus into available forms for plants. Studying the

mechanisms that generate and underlie microbial biodiversity is

critical to predicting the response of ecosystems to environmental

changes (Pincay, 2022).

Bacteria of the genus Bacillus have been widely studied for their

ability to promote plant growth and are considered a sustainable

alternative to chemical fertilizers (López-Valenzuela et al., 2019).

Bacillus sp. are a type of spore-forming plant growth-promoting

rhizobacteria (PGPR) tolerant to adverse environmental conditions.

They provide biological control against biotic and abiotic stressors

that negatively aect plant growth and development (Chattopadhyay,

2006). Therefore, the concentration of Bacillus sp. can be adjusted to

obtain optimal results in promoting the growth of Zea mays L. without

the need for chemical fertilizers (Orozco-Mozqueda et al., 2021).

Plant growth-promoting bacteria benet a host by producing

siderophores, auxins, cytokinins, and gibberellins. They also play a

crucial role in converting insoluble phosphorus into soluble forms,

xing nitrogen, and promoting plant growth. These bacteria are,

therefore, a compelling alternative to chemical fertilizers (Yu et al.,

2022).

There is growing interest in inoculating PGPR as biofertilizers to

serve as a sustainable alternative for food production (Do Amaral et

al., 2020). Literature references have shown that some Bacillus strains,

particularly B. subtilis, have great potential as growth promoters for

corn. This work aimed to determine, in vitro, the mechanism of action

of bacterial strains isolated in Antarctica.

Material and methods

This work was conducted at the Campus of the Escuela Superior

Politécnica Agropecuaria de Manabí (ESPAM)-Ecuador (0°49’8.87”

S, 80°10’53.03” W, located at 15 masl). The temperature of the place

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Zambrano-Solórzano et al. Rev. Fac. Agron. (LUZ). 2024 41(3): e244121

3-6 |

varies between 20.6 and 31.11 ºC; the average annual precipitation is

624 mm, and the relative humidity is 82.42 (Valdivieso et al., 2021).

Eighty-three strains of Bacillus sp. were used, collected on the

Greenwich, Dee, Barrientos, and Torres islands during the expedition

carried out to the territorial space of Ecuador in Antarctica in

2014. Since then, these strains have been preserved in glycerol at

-20 ºC in the Molecular Biology laboratory of ESPAM. A 100 µL

sample of each strain was inoculated in Erlenmeyers with 50 mL of

nutrient broth and maintained at 37 ºC, with shaking (Thermo Fisher

Scientic, USA) at 180 rpm for 18 h to promote growth. Colony

morphology, cell morphology (Tassadaq et al., 2013), and catalase

test of bacterial isolates from fresh cultures were evaluated (Liu et

al., 2016). A hemolysis test was performed on the microbial isolates

that solubilize tricalcium phosphate to dene the pathogenic capacity

of the bacterial strains. For this, they were grown on nutrient agar

at 37 °C, for 18 h. Then, they were inoculated onto a blood agar

base containing 5 % sterile calf blood and incubated at 37 °C, for

24 h. To determine phosphate solubilization, each bacterial strain

was inoculated in triplicate into Petri dishes containing Pikovskaya

medium (PVK, Himedia, 31.3 g.L

-1

, India) and incubated at 37 °C for

24 h and 48 h. The ability to solubilize phosphorus was observed by a

transparent halo around the bacterial colony (Teng et al., 2018). The

phosphate solubilization index (PSI) was measured and calculated

according to Afzal and Bano (2008), following the equation:

The test described by Alexander and Zuberer (1991), was carried

out to determine if the strains could produce siderophores. In this

test, the strains that had gamma hemolysis were used. Strains (T-5,

GB-70, and B-6) were punctured onto chromium azurol agar (CAS,

Thermo Fisher Scientic, 0.605 mg.mL

-1

, USA) and incubated at 37

°C for 72 h. The color change from blue to yellow-orange around

the bacterial colonies of the strains was a positive indicator of the

qualitative detection of siderophores. The diameters of the halos were

measured in mm (Abo-Zaid et al., 2020).

The strains that had the best response in phosphate solubilization

and produced siderophore were evaluated in a medium without

a nitrogen source to determine the growth capacity under these

conditions, according to Acurio et al. (2020). Each pure culture was

punctured on Ashby agar (40,7 g.L

-1

, Himedia, India), and incubation

was carried out aerobically at 37 ºC for seven days. ASHBY semi-

solid agar had the following composition (g.L

-1

): mannitol (10.0);

HPO

(0.2); MgSO

(0.2); NaCl (0.2); CaSO

(0.1); CaCO

(5.0)

and agar-agar (15.0). Nitrogen-xing bacteria were dened as those

where a 3-5 mm whitish lm was produced under the surface of the

culture medium. To determine some physiological characteristics

of the strains, the growth of the bacterial isolates was analyzed at

dierent pH (3, 5, 7, and 9), dierent temperature levels (20, 37, 50,

and 60 °C), and NaCl concentration (0, 8.5, 10, 15, and 20 %). For the

three evaluations, aliquots (5 mL) of each inoculum were placed in

Erlenmeyer vials with 45 mL of nutrient broth (8 g, Himedia, India).

The cultures were incubated at 37 °C and 180 rpm; the evaluation

was carried out at 18 h for pH and NaCl. Temperature was evaluated

at four time points: 0, 4, 16, and 24 h. Subsequently, serial dilutions

ranging from 10

-1

to 10

-8

were prepared in saline solution (0.85 %

NaCl). Then, 100 μL of the 10

-8

dilution was plated on nutrient agar

plates and spread with a Drigalski spatula. Plates were incubated at

37 °C for 18 h; after this, viable cells were counted as colony-forming

units per milliliter (CFU.mL

-1

The nal test evaluated the plant growth-promoting action of

the bacterial isolates with the best response in the preliminary tests.

The strains were grown in nutrient broth for 18 h at 37 °C. The cells

were separated by centrifugation and washed with water. Two kinds

of substrates were used (a. clay alone, and b. compost + clay), and

the substrates were sanitized using an autoclave (Yamato Scientic

America, USA) to favor the mechanism of action of the chosen

strains. The sterilized substrates were placed in glass bottles (200

mL), in which a Trueno variety corn seed was sown, considering

it as an experimental unit where 3 mL of inoculum was applied.

After two weeks, the following parameters were evaluated: shoot

diameter (DT), seedling height (AP), and length of root (LR). The

factorial experiment A (substrates: A1, A2) and B (strains: C1, C2,

C3) was conducted with a Completely Randomized Design, and the

INFOSTAT program was used for data analysis (Di Rienzo et al.,

2010).

Results and discussion

Morphological Characterization of Bacterial Isolates

By Gram staining, all strains were Gram-positive bacilli. The

strains showed positive reactions for catalase and presented a central

endospore with an ellipsoidal shape.

The results obtained coincided with those of Ratón et al. (2005),

who reported that all the strains isolated in their work were Gram-

positive and catalase-positive, even though they were from dierent

environments, which indicates that this type of bacteria has a

cosmopolitan distribution. In this regard, Gyaneshwar et al. (2002)

maintains that catalase acts on the toxic hydrogen peroxide produced

by the mitochondrial electronic transport chain and in various

oxidation and hydroxylation reactions so that it prevents its toxic

eects on organisms by decomposing it to form oxygen and water.

Some genera of Gram-positive bacteria are found in dierent

habitats, including the soil. These bacteria benet the soil by

providing nutrients or helping degrade them so that the plant can

absorb them according to its needs. In addition, these bacteria can

generate a positive impact by improving soil fertility, allowing plants

to better use nutrients (Castro et al., 2021).

Solubilization of phosphate

Of the total strains used in the study, six solubilized phosphate

and produced hydrolysis halos of 1.05 to 2.07 mm and 1.05 to 1.69

mm at 24 h and 48 h, respectively (table 1).

Table 1. Hydrolysis halo (phosphate solubilization index, P.S.I.)

produced by the strains after 24 and 48 h of incubation.

Strain codes

P.S.I. (mm)

24 h 48 h

T-1 1.10 1.18

GB-70 1.19 1.18

T-5 1.12 1.13

GC-3 1.29 1.42

GB-77 2.07 1.69

B-6 1.05 1.05

The PSI values obtained were within the solubilization rates

observed by Lara et al. (2011) in bacterial strains that produced

hydrolysis halos between 1.5 and 4.2 mm, measured after three and

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Rev. Fac. Agron. (LUZ). 2024, 41(3): e244121 July-September. ISSN 2477-9407.

4-6 |

seven days of incubation, respectively. Davis et al. (2005) show a

directly proportional relationship between the incubation time, the

size of the colony, and, therefore, its conservation, thus obtaining

excellent phosphate solubilization indices (PSI).

The values reported in the present study were within the

solubilization rates observed by Teng et al. (2018) in bacterial strains

that produced P.S.I. between 1.6 and 3.1. The PSIs of isolates B-6,

T-1, T-5, GB-70, and GC-3 were considered low (PSI ≤ 2), while

GB-77 values were considered medium (2≤PSI<3), according to the

ranges shown by Batista et al. (2021). Ramírez et al. (2008) reported

slightly higher PSI values in their study on Bacillus sp., where

they determined that this bacterial genus is an important phosphate

solubilizer.

Hemolysis test

The bacterial isolates (T-1, GB-70, T-5, GC-3, GB-77, and B-6),

with the capacity to solubilize tricalcium phosphate, were evaluated

on blood-based agar, obtaining as results that the strains T5, GB-70,

B-6 presented gamma hemolysis (no hemolytic activity, and would

not aect red blood cells in humans). In contrast, strains T-1, GB-77,

and GC-3 showed beta hemolysis (they produced transparent halos

caused by the total lysis of red blood cells), so the latter should be

discarded for agronomic use.

The percentage of strains with gamma hemolysis found in the

present study (50 %) was similar to the reported by Forbes et al.

(2007) (47 %), who maintain that the determination of this parameter

is necessary to establish the commercial use of live microorganisms

because it allows the identication of potentially pathogenic bacteria.

Qualitative assay of siderophore production

In this test, the strains with gamma hemolysis were used. The

three strains produced siderophores with values from 1.21 to 1.60

mm, demonstrating that they have iron-xing capacity that enhances

plant growth (table 2).

Table 2. Siderophore production and growth under conditions

without a nitrogen source.

Strain codes

Siderophore halo

(mm)

Growth without a nitrogen source

(mm)

Day 1 Day 5

T-5

1.21 58.23 68.00

GB-70

1.27 36.61 48.38

B-6

1.60 17.95 58.40

These results were similar to those obtained by Villarreal et al.

(2018), who observed strains with biocontrol potential belonging to the

genus Bacillus, which have demonstrated the ability to synthesize the

siderophore, regulating the concentration of iron through its chelation

(Fe

-siderophore). The selection of microorganisms with the capacity

to produce siderophores enables their use in biofertilization practices,

as indicated by Aguado-Santacruz et al. (2012), who state that

bacterial siderophores have aroused great interest in recent years due

to their potential for biological control of fungi and phytopathogenic

bacteria, constituting a growth mechanism in plant growth-promoting

rhizobacteria.

Growth of Bacillus sp in conditions without a Nitrogen source

The three strains subjected to this test grew under conditions

without a nitrogen source. Colony measurements showed values on

the rst day from 17.95 to 58.23 mm, and on the fth day from 48.38 to

68 mm (table 2). Bacillus can generally use cheap nutritional sources

as substrates for fermentation (Gu et al., 2018). Furthermore, as an

essential part of the heterotrophic Gram-positive bacteria, Bacillus

has many important applications in biological manufacturing by

fermentation (Xiao et al., 2020). The growth capacity in the absence

of a nitrogen source could be related to the benets provided by the

presence of certain strains of Bacillus sp. in the plants (Ojuederie

et al., 2019). Bacillus is a genus of bacteria that includes many

species known for their benecial eects on plants, and one of these

advantages is the ability of some strains to x atmospheric nitrogen

(Orozco-Mozqueda et al., 2021). This capacity is crucial, since

nitrogen is an essential nutrient for plant growth and its availability

can limit plant productivity in soils (Fasusi, & Babalola, 2021).

Nitrogen xation carried out by certain strains of Bacillus sp.

involves the conversion of molecular nitrogen from the air (N

) into

forms that plants can use, such as ammonium (NH

) or nitrate (NO

By supplying nitrogen to plants in this way, benecial bacteria can

improve plant growth and health, even in nitrogen-poor soils. This

is especially important in environments where nitrogen is a limiting

resource. In addition to nitrogen xation, strains of Bacillus sp. can

also promote plant growth in other ways, such as increasing the

availability of other nutrients in the soil, producing plant growth-

promoting compounds, and assisting in protection against pathogens.

All of these factors can contribute to improving the ability of plants

to grow even in the absence of available nitrogen in the soil (Bashan

et al., 2013).

Eect of pH and temperature on the growth of strains

All strains of Bacillus sp. responded well to dierent temperatures,

presenting uncountable growth (UN) due to the agglomeration of

colonies. Adaptability to high temperatures and a wide pH range is of

great relevance for bacteria of the genus Bacillus for several reasons:

a) Heat resistance: Some Bacillus species, such as B. subtilis and B.

stearothermophilus, are thermophilic, meaning they can survive and

grow in relatively high temperatures. This heat resistance allows them

to thrive in conditions where other bacteria might not survive, such

as in composts, manure piles, or in industrial sterilization processes.

b) pH tolerance: Bacteria of the genus Bacillus are known for their

ability to grow over a wide pH range (Zalma & El-Sharoud, 2021).

Some species can tolerate an acidic pH, while others thrive in alkaline

conditions. This tolerance allows them to colonize dierent types of

soil, from acidic to alkaline, and adapt to changes in the environment,

such as those caused by human activity or industrial processes

(Guzmán et al., 2015).

This level of adaptation of the strains to extreme conditions is

similar to the physiological condition of Bacillus

sp. strains studied

by Calvo and Zúñiga (2010), who reported that 98 % of the strains

grew at 10 ºC and temperatures between 15 and 20 ºC.

Table 3. Behavior of the strains at dierent temperature levels.

Strain

codes

Temperature (ºC)

20 37 50 60

T-5

UN UN UN UN

GB-70

UN UN 1.9 10

B-6 UN 3.5 10

UN UN

UN, uncountable number of colonies.

Regarding pH, they observed that 100 % of the strains grew

well at both pH (4 and 5.5), indicating a good growth adaptation

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Zambrano-Solórzano et al. Rev. Fac. Agron. (LUZ). 2024 41(3): e244121

5-6 |

of the strains. Similar results were found by Guzmán et al. (2015)

when performing the isolation and selection of native bacteria from

Manabí-Ecuador with cellulolytic activity (table 4).

Table 4. Growth of the strains at dierent pH levels (after 18 h

of evaluation).

Strain

codes

3 5 7 9

T-5 UN 2.0 10

1.6 10

GB-70 1.0 10

2.0 10

4.1 10

2.0 10

B-6 UN UN 4.1 10

UN, uncountable number of colonies.

Eect on NaCl concentrations for the growth of Bacillus sp.

Table 5 shows the behavior of the strains at dierent levels of

NaCl. The results show that the most signicant colonies occurred

in the 20 % concentration with 7.4x10

compared to 0 % NaCl,

which obtained 1.12x10

CFU. Salt content (NaCl) and temperature

variations inuence microbial development (Ikenebomeh, 1989).

Alteration of NaCl concentration results in a change in the expression

pattern of outer membrane proteins (Hu et al., 2022). Previous ndings

have reported that several bacterial genera, including Pseudomonas,

Bacillus, Burkholderia, Enterobacter, Microbacterium, Planococcus,

and Halomonas, could produce exopolysaccharides that guarantee

their survival in salinity conditions (Qurashi & Sabri, 2012).

Table 5. Behavior of the strains at dierent concentrations of

sodium chloride.

Strain

codes

NaCl (%)

0 8.5 10 15 20

T-5 1.12 10

6.4 10

1.4 10

7.4 10

GB-70 5.9 10

6.6 10

7.9 10

5.9 10

6.2 10

B-6 2.0 10

7.0 10

7.5 10

1.57 10

Evaluation of selected bacterial strains as plant growth

promoters

When evaluating the height of corn seedlings, no statistical

dierences were found among all the sources of variation studied

(table 6). Root length and shoot diameter showed signicant

dierences (p<0.05) with the type of substrate used, placing the clay

+ compost mixture in the best statistical category.

The compost likely played a role in providing mineral elements

for the initial growth of the seedling. The ability of plants to grow

without a nitrogen source may be due to the advantages provided by

the presence of some Bacillus strains. Bacillus is a genus of plant

growth-promoting bacteria (PGPB) that form benecial associations

with plants (Joshi et al., 2023). Some Bacillus species have the ability

to x atmospheric nitrogen and convert it into a usable form for plants

and increase the eciency of nitrogen consumption in plants even in

nitrogen-decient soil conditions (Santi et al., 2013).

In addition to nitrogen xation, bacteria can promote plant growth

in other ways, such as by producing plant hormones, dissolving

nutrients in the soil, and inducing disease resistance. These benets

can help plants grow better even when the amount of nitrogen in the

soil is limited (Glick, 2012).

Table 6. Vegetative development at 15 days of age of the corn

seedlings.

Treatments

Description

Seedling

height

(cm)

Root

length

(cm)

Shoot

diameter

(cm)

Factor A NS

* *

A1 Clay 16.00 11.22

2.14

A2 Clay + Compost 14.56 14.44

2.38

Average 15.28 12.83 2.26

Factor B NS NS NS

C1 T-5 14.33 13.67 2.38

C2 GB-70 14.67 11.83 2.48

C3 B-6 16.83 13.00 1.92

Average 15.28 12.83 2.26

Factor B*Factor A NS NS NS

A1B1 Clay + AE-1 15.67 13.67 2.00

A1B2 Clay + AE-2 17.00 10.33 1.90

A1B3 Clay + AE-3 15.33 9.67 2.53

A2B1 Clay + Compost + AE-1 13.00 13.67 2.77

A2B2 Clay + Compost + AE-2 16.67 15.67 1.93

A2B3 Clay + Compost + AE-3 14.00 14.00 2.43

Average 15.28 12.84 2.26

CV % 31.9 27.3 26.17

*= Signicant statistical dierences p<0.05; NS= Non-signicant dierences;

CV= Coecient of Variation; a and b in column = dier according to Tukey at 5

% error probabilities

Hence, the presence of specic Bacillus strains in soil may be

related to the ability of plants to grow better under conditions of low

nitrogen availability, as these bacteria provide several mechanisms that

promote growth. Bacon and Hinton (2011) express that it should not

be ruled out that the genus Bacillus contains potential microorganisms

that protect the plant and could functionally be considered a biological

controller because it produces antibiotic, phosphate-solubilizing

substances that facilitate its availability to the plant, and because

it performs functions as a siderophore. Furthermore, manipulating

plant-microorganism interactions can improve plant performance in

applications ranging from climate change mitigation to agricultural

production (Ulrich et al., 2019).

Conclusion

The mechanisms of action observed in the bacterial strains

isolated from Antarctic soils allowed us to distinguish promising

strains as bioinoculants, which is an important bacterial interaction in

organic inoculated fertilizers for agronomic purposes.

Literature cited

Abo-Zaid, G. A., Soliman, N. A., Abdullah, A. S., El-Sharouny, E. E., Matar, S.

M., & Sabry, S. A. (2020). Maximization of siderophores production

from biocontrol agents, Pseudomonas aeruginosa F2 and Pseudomonas

fluorescens JY3 using batch and exponential Fed-Batch fermentation.

Processes, 8(4), 455. https://www.mdpi.com/2227-9717/8/4/455.

Acurio Vásconez, R.D., Mamarandi Mossot, J. E., Ojeda Shagñay, A.G.,

Tenorio, E.M., Chiluisa Utreras, V.P., & Vaca Suquillo, I. D. L. Á.

(2020). Evaluación de Bacillus spp. como rizobacterias promotoras del

crecimiento vegetal (RPCV) en brócoli (Brassica oleracea var. italica)

This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.

Rev. Fac. Agron. (LUZ). 2024, 41(3): e244121 July-September. ISSN 2477-9407.

6-6 |

y lechuga (Lactuca sativa). Revista Ciencia y Tecnología Agropecuaria,

21(3), 1-16. https://doi.org/10.21930/rcta.vol21_num3_art:1465

Afzal, A., & Bano, A. (2008). Rhizobium and phosphate solubilizing bacteria

improve the yield and phosphorus uptake in wheat (Triticum aestivum).

International Journal of Agriculture and Biology. 10(1), 85 https://www.

fspublishers.org/Issue.php?y=2008&v_no=10&categoryID=39-88.

Aguado–Santacruz, G.A., Moreno–Gómez, B., Jiménez–Francisco, B., García-

Moya, E., & Preciado-Ortiz, R.E. (2012). Impacto de los sideróforos

microbianos y tosidéforos en la asimilación de hierro por las plantas:

una síntesis. Revista Fitotecnia Mexicana, 35(1), 9-21. https://

revistatotecniamexicana.org/documentos/35-1/2a.pdf.

Alexander, D. Z., & Zuberer, D. A. (1991). Use of chrome azurol S reagents to

evaluate siderophore production by rhizosphere bacteria. Biology and

Fertility of Soils, 12(1), 39-45. https://link.springer.com/article/10.1007/

bf00369386.

Bacon, C., & Hinton, D. (2011). In planta reduction of maize seedling stalk lesions

by the bacterial endophyte Bacillus mojavensis. Canadian Journal of

Microbiology, 57(6), 485-492. https://doi.org/10.1139/w11-031.

Bashan, Y., Kamnev, A.A., & de-Bashan, L.E. (2013). Tricalcium phosphate is

inappropriate as a universal selection factor for isolating and testing

phosphate-solubilizing bacteria that enhance plant growth: a proposal

for an alternative procedure. Biology and Fertility of Soils, 49, 465–479.

https://doi.org/10.1007/s00374-012-0737-7.

Batista, B.D., Bonatelli, M.L., & Quecine, M.C. (2021). Fast Screening of Bacteria

for Plant Growth Promoting Traits. In Carvalhais, L.C., Dennis, P.G. (eds)

The Plant Microbiome. Methods in Molecular Biology, 2232. Humana,

New York. https://doi.org/10.1007/978-1-0716-1040-4_7.

Calvo, P., & Zúñiga, D. (2010). Caracterización siológica de cepas de Bacillus

spp. aisladas de la rizósfera de papa (Solanum tuberosum). Ecología

aplicada, 9(1), 31-39. https://doi.org/10.21704/rea.v9i1-2.393.

Castro-Severyn, J., Pardo-Esté, C., Mendez, K. N., Fortt. J., Marquez, S, Molina,

F., Castro-Nallar, E., Remonsellez, F., & Saavedra, C. P. (2021). Living

to the high extreme: unraveling the composition, structure, and functional

insights of bacterial communities thriving in the arsenic-rich Salar de

Huasco altiplanic ecosystem. Microbiology Spectrum, 9(1), e0044421.

https://doi.org/10.1128/Spectrum.00444-21.

Chattopadhyay, M. K. (2006). Mechanism of bacterial adaptation to low

temperature. Journal of Biosciences, 31(1), 157–165. https://doi.

org/10.1007/BF02705244.

Davis, K.E., Joseph, S.J., & Janssen, P.H. (2005). Eects of growth medium,

inoculum size, and incubation time on culturability and isolation of soil

bacteria. Applied Environmental Microbiology, 71(2), 826-34. https://doi.

org/10.1128/AEM.71.2.826-834.2005.

Delgado-Baquerizo, M., Oliverio, A., Brewer, T., Benavent-González, A.,

Eldridge, D., Bardgett, R., Maestre, F., Singh, B., & Fierer, N. (2018). A

global atlas of the dominant bacteria found in soil. Science, 359(6373),

320-325. https://www.science.org/doi/10.1126/science.aap9516.

Di Rienzo, J., Balzarini, M., Gonzales, L., Casanoves, F., Tablada, M. y Robledo,

C. (2010). InfoStat versión 2018 [Programa]. Grupo InfoStat. https://

www.infostat.com.ar/index.php?mod=page&id=34.

Do Amaral, F. P., Tuleski, T. R., Pankievicz, V. C., Melnyk, R. A., Arkin, A. P.,

Gritts, J., Tadra-Sfeir, M. Z., Maltempi de Souza, E., Deutschbauer, A.,

Monteiro, R. A., & Stacey, G. (2020). Diverse bacterial genes modulate

plant root association by benecial bacteria. mBio, 11(6). https://doi.

org/10.1128/mbio.03078-20.

Fasusi, O. A., & Babalola, O. O. (2021). The multifaceted plant-benecial

rhizobacteria toward agricultural sustainability. Plant Protection Science,

57(2), 95-111. https://doi.org/10.17221/130/2020-PPS.

Forbes, B., Sahm, D., & Weissfeld, A. (2007). Bailey & Scott’s. Diagnostic

microbiology. Mosby, Elsevier.

Gu, Y., Xu, X., Wu, Y., Niu, T., Liu, Y., Li, J., Du, G., & Liu, L. (2018). Advances

and prospects of Bacillus subtilis cellular factories: From rational design

to industrial applications. Metabolic Engineering, 50, 109-121, https://

doi.org/10.1016/j.ymben.2018.05.006.

Guzmán, A., Zambrano, D., Rivera, R., Rondón, A., Laurencio, M., Pérez, M., &

León, R. (2015). Aislamiento y selección de bacterias autóctonas de Manabí-

Ecuador con actividad celulolítica. Cultivos Tropicales, 36(1), 7-16. https://

ediciones.inca.edu.cu/index.php/ediciones/article/view/934/pdf.

Gyaneshwar, P., Naresh, G., Kumar, L., Parekh, J., & Poole, P. (2002). Role of soil

microorganisms in improving P nutrition of plants. Plant and Soil, 245(1),

83-93. https://doi.org/10.1023/A:1020663916259.

Glick, B. R. (2012). Plant growth-promoting bacteria: mechanisms and

applications. Scienti.ca, 1, 963401. https://doi.org/10.6064/2012/963401.

Hu, S., Li, Y., Wang, B., Yin, L., & Jia, X. (2022). Eects of NaCl Concentration

on the Behavior of

Vibrio brasiliensis and Transcriptome Analysis. Foods,

11(6), 840. https://doi.org/10.3390/foods11060840.

Ikenebomeh, M.J. (1989). The inuence of salt and temperature on the natural

fermentation of African locust bean. International Journal of Food

Microbiology, 8(2), 133-9. https://doi.org/10.1016/0168-1605(89)90067-6.

Joshi, S., Gangola, S., Bhandari, G., Bhandari, N. S., Nainwal, D., Rani, A., Malik,

S., & Slama, P. (2023). Rhizospheric bacteria: the key to sustainable

heavy metal detoxication strategies. Frontiers in Microbiology, 14,

1229828. https://doi.org/10.3389/fmicb.2023.1229828 .

Kudinova, A. G., Dolgih, A. V., Mergelov, N. S., Shorkunov, I. G., Maslova, O.

A., & Petrova, M. A. (2021). The abundance and taxonomic diversity of

lterable forms of bacteria during succession in the soils of Antarctica

(Bunger hills). Microorganisms, 9(8), 1728. https://doi.org/10.3390/

microorganisms9081728.

Lara, C., Esquivel, L., & Negrete, J. (2011). Bacterias nativas solubilizadores de

fosfatos para incrementar los cultivos en el departamento de Córdoba-

Colombia. Biotecnología en el sector agropecuario y agroindustrial,

9(2), 114-120. https://revistas.unicauca.edu.co/index.php/biotecnologia/

article/view/789/413.

Liu, F. P., Liu, H. Q., Zhou, H. L., Dong, Z. G., Bai, X. H., Bai, P., & Qiao, J. J.

(2016). Isolation and characterization of phosphate-solubilizing bacteria

from betel nut (Areca catechu) and their eects on plant growth and

phosphorus mobilization in tropical soils. Biology and Fertility of Soils,

50(6), 927–937. https://doi.org/10.1007/s00374-014-0913-z.

López-Valenzuela, B. E., Armenta-Bojórquez, A. D., Hernández-Verdugo, S.,

Apodaca-Sánchez M. A., Samaniego-Gaxiola, J. A., & Valdez-Ortiz, A.

(2019). Trichoderma spp. y Bacillus spp. como promotores del crecimiento

en maíz (Zea mays L.). Phyton, 88(1), 37-46. https://doi.org/10.32604/

phyton.2019.04621.

Olanrewaju, O. S., Glick, B. R., & Babalola, O. O. (2017). Mechanisms of Action

of Plant Growth Promoting Bacteria. World Journal of Microbiology and

Biotechnology, 33, 197. https://doi.org/10.1007/s11274-017-2364-9.

Ojuederie, O. B., Olanrewaju, O. S., & Babalola, O. O. (2019). Plant growth

promoting rhizobacterial mitigation of drought stress in crop plants: im-

plications for sustainable agriculture. Agronomy, 9(11), 712. https://doi.

org/10.3389/fpls.2022.875774.

Orozco-Mozqueda, M., Flores, A., Rojas-Sánchez, B., Urtis-Flores, C., Morales-

Cedeño, L., Valencia-Marín, M., Chávez-Ávila, S., Rojas-Solís, D., &

Santoyo, G. (2021). Plant Growth-Promoting Bacteria as Bioinoculants:

Attributes and Challenges for Sustainable Crop Improvement. Agronomy.

11(6), 1167. https://doi.org/10.3390/agronomy11061167.

Pincay, M. (2022). Diversidad microbiana presente en agua residual proveniente

de industria atunera. Revista Espamciencia, 13(1), 70-76. https://doi.

org/10.51260/revista_espamciencia.v13i1.312.

Qurashi, A.W., & Sabri, A.N. (2012). Bacterial exopolysaccharide and biolm

formation stimulate chickpea growth and soil aggregation under salt

stress. Brazilian Journal of Microbiology, 43(3), 1183-91. https://doi.

org/10.1590/S1517-838220120003000046.

Ramírez, M., Roveda, G., Bonilla, R., Cabra, L., Peñaranda, A., López, M., &

Díaz, C. A. (2008). Uso y manejo de biofertilizantes en el cultivo de la

uchuva. Corporación Colombiana de Investigación Agropecuaria. http://

hdl.handle.net/20.500.12324/12852.

Ratón, T., Portuondo, I., Salas, D., Ramos, N., & Giro, Z. (2005). Aislamiento de

cepas del género Bacillus sp. con potencialidades para la bioprotección

y la estimulación del crecimiento vegetal. Revista Cubana de Química,

17(1), 189-195. https://cubanaquimica.uo.edu.cu/index.php/cq.

Rizvi, A., Ahmed, B., Khan, M. S., Umar, S., & Lee, J. (2021). Psychrophilic

Bacterial Phosphate-Biofertilizers: A Novel Extremophile for Sustainable

Crop Production under Cold Environment. Microorganisms, 9(12), 2451.

https://doi.org/10.3390/microorganisms9122451.

Santi, C., Bogusz, D., & Franche, C. (2013). Biological nitrogen xation in non-

legume plants. Annals of Botany, 111(5), 743-767. https://doi.org/10.1093/

aob/mct048.

Tassadaq, H., Aneela, R., Shehzad, M., Iftikhar, A., Jafar, K., Veronique, E. H.,

Kim, K. Y., & Muhammad, A. (2013). Biochemical characterization and

identication of bacterial strains isolated from drinking water sources of

Kohat, Pakistan. African Journal of Microbiology Research, 7(16), 1579–

1590. https://doi.org/10.5897/AJMR12.2204.

Teng, Z., Chen, Z., Zhang, Q., Yao, Y., Song, M., & Li, M. (2018). Isolation and

characterization of phosphate solubilizing bacteria from rhizosphere

soils of the Yeyahu Wetland in Beijing, China. Environmental Science

and Pollution Research, 26(33), 33976-33987. https://doi.org/10.1007/

s11356-018-2955-5.

Ulrich, D.E., Sevanto, S., & Ryan, M. (2019). Las interacciones planta-microbio

antes de la sequía inuyen en las respuestas siológicas de la planta a la

sequía severa posterior. Informe cientíco, 9(1), 249. https://www.nature.

com/articles/s41598-018-36971-3.

Valdivieso, S., Cedeño, G., & Guanoluisa, A. (2021). Análisis Estadístico de los

datos climáticos históricos de la ESPAM MFL. https://www.manabi.gob.

ec/wp-content/uploads/2021/11/1VF_Analisis-Estadistico-de-los-datos-

climaticos-historicos-de-la-ESPAM-MFL.pdf.

Villarreal, M., Villa, E., Cira, L., Estrada, M., Parra, F., & Santos, S. (2018). El

género Bacillus como agente de control biológico y sus implicaciones en

la bioseguridad agrícola. Revista Mexicana de Fitopatología, 36(1), 95-

130. https://www.rmf.smf.org.mx/ojs/index.php/RMF/article/view/100.

Xiao, F., Li, Y., Zhang, Y., Wang, H., Zhang, L., Ding, Z., Gu, Z., Xu, S., & Shi, G.

(2020). Construction of a novel sugar alcohol-inducible expression system

in Bacillus licheniformis. Applied Microbiology and Biotechnology,

104(12), 5409–5425. https://doi.org/10.1007/s00253-020-10618-8.

Yu, H., Wu, X., Zhang, G., Zhou, F., Harvey, P., Wang, L., Fan, S., Xie, X., Li, F.,

Zhou, H., Zhao, X., & Zhang, X. (2022). Identication of the phosphorus-

solubilizing bacteria strain JP233 and its eects on soil phosphorus

leaching loss and crop growth. Frontiers in Microbiology, 13, 892533.

https://doi.org/10.3389/fmicb.2022.892533.

Zalma, S. A., & El-Sharoud, W. M. (2021). Diverse thermophilic Bacillus

species with multiple biotechnological activities are associated within

the Egyptian soil and compost samples. Science Progress, 104(4),

368504211055277. https://doi.org/10.1177/00368504211055277.