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The project method in the teaching-learning process
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PAIDEIA XXI
PAIDEIA XXI
Vol. 13, Nº 2, Lima, julio-diciembre 2023, pp. 335-343
ISSN Versión Impresa: 2221-7770; ISSN Versión Electrónica: 2519-5700
RESEARCH NOTE / NOTA CIENTÍFICA
BACTERIAL IDENTIFICATION USING
16S RRNA GENE SEQUENCING FROM BANANA
PSEUDOSTEM WET ROT SAMPLES
IDENTIFICACIÓN BACTERIANA MEDIANTE
SECUENCIACIÓN DEL GEN 16S RRNA A PARTIR
DE MUESTRAS DE PUDRICIÓN BLANDA DEL
PSEUDOTALLO DEL BANANO
Oscar Nolasco-Cárdenas
1,2*
; Candy Chacon-Aguilar
1,2
; Linda Salas-Alva
1,2
;
Monica Velarde
1,2
; Ysabel Murrugarra
1,2
& Ana I. F. Gutiérrez Román
1.2
ABSTRACT
doi:10.31381/paideiaxxi.v13i2.5873
http://revistas.urp.edu.pe/index.php/Paideia
1
Laboratorio de Bioquímica y Biología Molecular, Facultad de Ciencias Naturales y Matemática,
Universidad Nacional Federico Villarreal, Lima, Perú.
2
Comunidad de conocimiento GIBBS-UNFV, Facultad de Ciencias Naturales y Matemática,
Universidad Nacional Federico Villarreal, Lima, Perú.
* Corresponding author: onolasco@unfv.edu.pe
Oscar Nolasco-Cárdenas:
https://orcid.org/0000-0002-5672-5516
Candy Chacon-Aguilar:
https://orcid.org/0000-0002-0762-1980
Linda Salas-Alva:
https://orcid.org/0009-0002-5299-293X
Monica Velarde-Vilchez:
https://orcid.org/0000-0002-8774-8729
Ysabel Murrugarra-Bringas:
https://orcid.org/0000-0002-3664-6730
Ana I. F. Gutiérrez Román:
https://orcid.org/0000-0002-7020-7387
Este artículo es publicado por la revista Paideia XXI de la Escuela de posgrado (EPG), Universidad Ricardo Palma,
Lima, Perú. Este es un artículo de acceso abierto, distribuido bajo los términos de la licencia Creative Commons
Atribución 4.0 Internacional (CC BY 4.0) [https:// creativecommons.org/licenses/by/4.0/deed.es] que permite
el uso, distribución y reproducción en cualquier medio, siempre que la obra original sea debidamente citada de su fuente original.
Wet rot affects organic banana crops in the northern tropical region of
Peru. Several bacteria have been reported to cause wet rot on different crops,
leading to severe problems, particularly in humid climates. Bacteria infect the
banana pseudostem through wounds caused during plant pruning and insect
bites. Knowledge about bacteria identif cation that causes wet rot may provide
a valuable tool for controlling this issue. The present study explored the primary
bacterial component of banana pseudostem wet rot samples at three localities
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Nolasco-Cárdenas
et al.
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PAIDEIA XXI
in the Sullana province of Piura, using 16S rRNA gene sequencing. A total of
one hundred and thirty-six sequence data were obtained from the three Sullana
localities: forty sequences of bacterial isolates from the Querecotillo locality
sample, forty-eight from Mambre, and Salitral each. The genus Klebsiella of the
Enterobacteriaceae family was identifed as the main bacterial component in
organic banana pseudostem wet rot. This fnding reinforces the need to continue
with studies that provide evidence of the bacterial role of the genus
Klebsiella
.
Keyword:
Wet rot – 16S rRNA – Banana organic –
Klebsiella
La pudrición blanda afecta los cultivos de banano orgánico en la región
tropical del norte de Perú. Se ha informado que varias bacterias causan pudrición
blanda en diferentes cultivos, lo que lleva a problemas graves, particularmente
en climas húmedos. Las bacterias infectan el pseudotallo del plátano a través
de heridas causadas durante la poda de plantas y picaduras de insectos. El
conocimiento sobre la identifcación de bacterias que causan la pudrición blanda
puede proporcionar una herramienta valiosa para controlar este problema. El
presente estudio exploró el componente bacteriano primario de muestras de
pudrición blanda del pseudotallo del banano en tres localidades de la provincia
Sullana de Piura, utilizando la secuenciación del gen 16S rRNA. Se obtuvieron
un total de ciento treinta y seis datos de secuencia de las tres localidades de
Sullana: cuarenta secuencias de aislados bacterianos de la muestra de la
localidad de Querecotillo y cuarenta y ocho de Mambre y Salitral, cada una.
El género
Klebsiella
de la familia Enterobacteriaceae fue identifcado como el
principal componente bacteriano en la pudrición blanda del pseudotallo del
plátano orgánico. Este hallazgo refuerza la necesidad de continuar con estudios
que aporten evidencia del papel bacteriano del género
Klebsiella
.
Palabra clave:
Pudrición blanda – ARNr 16S – Plátano orgánico –
Klebsiella
RESUMEN
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Bacterial identifcation from banana pseudostem wet rot samples
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PAIDEIA XXI
INTRODUCTION
Banana and plantain (
Musa
sp.) are
the fourth most important food crop
with a fastest-growing in agricultural
production and world trade (Food
and Agricultural Organization of the
United Nations (FAO), 2020). Over
the past decade, the organic banana
of Cavendish triploid variety crops
promoted economic growth in Sullana
province on the tropical coast of Peru,
categorize the country among the
main producers in Latin America and
the Caribbean (MINAGRI, 2014).
However, several phytopathogens
affect banana crops with a negative
impact on performance and produc-
tivity. These are infuenced by alte
-
rations in climatic conditions that in-
crease the level of aggressiveness in
phytosanitary problems like bacterial
rot (Blomme
et al
., 2017; Martínez-
Solórzano
et al.
, 2020).
The bacterial soft and wet rot
affect the pseudostem and rhizome of
bananas and is widespread in tropical
regions worldwide. The rhizome rot
and pseudostem wet rot is due to the
secretion of bacterial pectinases that
degrade the pectin at the primary cell
walls and middle lamella (Hugouvieux-
Cotte-Pattat
et al
., 2014), causing
tissue maceration, and a wet and
foul smell of rotting (Loganathan
et
al.
, 2019). The pseudostem wet rot
is characterized by external brown or
dark brown coloration and rotting of
the internal pseudostem tissue with
amber-colored liquid (Loganathan
et
al
., 2019).
The bacterial pathogens spread
to newly planted suckers and young
plants transported by insects and
during pruning or crop management
using tools, soil, and irrigation water
contaminated with the bacterial
agents (Charkowski, 2018).
The banana pseudostem wet rot
development is associated with several
bacterial causal agents such as
Dickeya
chrysanthemi
Samson
et al. 2005
[earlier
Pectobacterium chysanthemi
(Burkholder
et al. 1953)
Brenner
et al.
1973
(Approved Lists 1980) emend.
Hauben
et al
. 1998)],
D. paradisiaca
(Fernandez-Borrero and Lopez-
Duque 1970) Samson
et al
. 2005,
Pectobacterium carotovorum subsp.
carotovorum
(Jones 1901) Hauben
et
al
. 1999 emend. Gardan
et al. 2003,
and Klebsiella variicola
Rosenblueth
et al.
2004
(Lin
et al
., 2010; Basim
et
al
., 2019; Rajamanickam
et al
., 2018;
Loganathan
et al
., 2019).
Since these bacterial pathogens
could be found together or individually
in decaying vegetables, detecting, and
identifying the principal bacterial
component is essential to understand
its contribution to banana pseudostem
wet rot. This study assesses the
primary bacterial component of three
different banana pseudostem wet rot
samples from the organic banana crop
Cavendish triploid variety in Sullana,
Piura.
The current project aims to
differentiate the isolated bacteria from
samples with soft rot symptoms and
classify them based on their 16S rRNA
sequence.
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PAIDEIA XXI
MATERIALS AND METHODS
Samples and bacterial isolation
The banana pseudostem samples
with wet rot symptoms (Figure 1)
were collected from three localities
of Sullana: Vichayal Querocotillo
(Latitude -4,85; Longitude -80,65),
Salitral (Latitude-4,84, Longitude
-80,67) and Mambre (Latitude -4,88,
Longitude -80,69). The samples
were transported to the Universidad
Nacional Federico Villarreal in Lima
– Peru and the bacterial isolation
was carried out at “Laboratorio de
Investigación de Bioquímica y Biología
Molecular” (LIBBM-SL10LA105).
Figure 1.
Banana pseudostem samples with wet rot symptoms.
To the
left, Organic banana with pseudostem soft wet rot symptoms. To the right,
pseudostem sample collected.
The samples were washed with
plenty of water, and 2 cm
2
of inner
section was taken in sterile conditions
and homogenized with 2 mL of
Tryptic Soy Broth (TSB). Serial tenfold
dilutions were made for each of the
cultures from the different locations
and were cultured in plates with
Tryptic Soy Agar (TSA). After 24 hours
of incubation at 25 °C, the fourth
dilution for Querecotillo and Salitral,
and the sixth dilution for Mambre,
was used for individual and aleatory
colony selection. A total of 150 colonies
were selected; 48 from Querocotillo,
51 from Mambre, and 51 from Salitral.
Molecular characterization using
16S rRNA gene
The genomic DNA of each bacteria
colony was extracted by cell rupture
through a thermal shock, each colony
was put in a test tube containing
300 μl of molecular grade water and
5 cycles of 10 minutes at -20 °C and
3 minutes at 65 °C were carried out
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PAIDEIA XXI
with a fnal centrifugation step
for fve
minutes at 10 000 rpm. Five μl of the
supernatant were used for the PCR.
Amplifcations of the 16S
rRNA gene region were carried out
using universal primers: 27F, 5’-
AGAGTTTGATCMTGGCTC -3 and 1492R,
5’ TACGGYTACCTTGTTACGACTT-3’
(Lane, 1991; Liu
et al
., 2009; Turner
et al
., 1999). The PCR reactions were
performed using the SuperFiTaq DNA
polymerase kit (Thermo Fisher Scientifc,
USA), according to the manufacturer’s
instructions. The amplifcation cycles
were performed under the following
conditions: an initial pre-denaturation
step at 98°C for 5 min, followed by 35
cycles at 94°C for 30 s, 58°C for 45 s,
72°C for 90 s, and a fnal elongation step
of 72°C for 6 min.
The amplicon prod
ucts were verifed
by 1,0% agarose gel electrophoresis,
and 20 μl of each amplifed product
were sequenced at MACROGEN
laboratories (Korea), using the Sanger
sequencing method. Three sequence
reads for PCR product were made to
achieve the maximum sequence length
(approx. 1492 bp of the 16S rRNA
gene), the sequences were aligned and
assembled in MEGA X bioinformatic
software (https://www. megasoftware.
net/), and fnally the sequences were
compared with the public sequences
using Nucleotide Basic Local
Alignment Search Tool (BLAST) at
www.ncbi.nlm.nih.gov/ BLAST, and
available from the National Center for
Biotechnology Information (NCBI).
Ethic aspects
Ethical aspects were followed in
this study to contribute positively to
both scientifc knowledge and the
well-being of the ecosystems by using
Good Laboratory Practices (GLPs).
RESULTS AND DISCUSSION
A total of 136 isolates were suc-
cessfully obtained from the three ba-
nana pseudostem samples with wet
rot symptoms. The number of isolates
selected and recovered from Queroco-
tillo was forty isolates, while for Mam-
bre, and Salitral were forty-eight iso-
lates.
The principal bacteria found was
Klebsiella
genus, in Querecotillo 36
of 40 isolates were classifed into the
Klebsiella
genus, (29
Klebsiella mich-
iganensis
Saha
et al
. 2013
and 7
Klebsiella variicola
Rosenblueth
et al.
2004). For Mambre 47 of 48 isolates
were
Klebsiella oxytoca
(Flugge 1886)