image/svg+xmlThe project method in the teaching-learning process335PAIDEIA XXIPAIDEIA XXIVol. 13, Nº 2, Lima, julio-diciembre 2023, pp. 335-343ISSN Versión Impresa: 2221-7770; ISSN Versión Electrónica: 2519-5700RESEARCH NOTE / NOTA CIENTÍFICABACTERIAL IDENTIFICATION USING 16S RRNA GENE SEQUENCING FROM BANANA PSEUDOSTEM WET ROT SAMPLESIDENTIFICACIÓN BACTERIANA MEDIANTE SECUENCIACIÓN DEL GEN 16S RRNA A PARTIR DE MUESTRAS DE PUDRICIÓN BLANDA DEL PSEUDOTALLO DEL BANANOOscar Nolasco-Cárdenas1,2*; Candy Chacon-Aguilar1,2; Linda Salas-Alva1,2; Monica Velarde1,2; Ysabel Murrugarra1,2& Ana I. F. Gutiérrez Román1.2ABSTRACTdoi:10.31381/paideiaxxi.v13i2.5873http://revistas.urp.edu.pe/index.php/Paideia1Laboratorio de Bioquímica y Biología Molecular, Facultad de Ciencias Naturales y Matemática, Universidad Nacional Federico Villarreal, Lima, Perú.2Comunidad 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/0000-0002-5672-5516Candy 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/0000-0002-8774-8729Ysabel Murrugarra-Bringas: https://orcid.org/0000-0002-3664-6730 Ana I. F. Gutiérrez Román: https://orcid.org/0000-0002-7020-7387Este 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
image/svg+xmlNolasco-Cárdenas et al.336PAIDEIA XXIin 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 – KlebsiellaLa 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 Klebsiellade 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 – KlebsiellaRESUMEN
image/svg+xmlBacterial identifcation from banana pseudostem wet rot samples337PAIDEIA XXIINTRODUCTIONBanana 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 Samsonet al. 2005 [earlierPectobacterium chysanthemi (Burkholder et al. 1953) Brenneret 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.
image/svg+xmlNolasco-Cárdenas et al.338PAIDEIA XXIMATERIALS AND METHODSSamples and bacterial isolationThe 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 cm2of 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 geneThe 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
image/svg+xmlBacterial identifcation from banana pseudostem wet rot samples339PAIDEIA XXIwith a fnal centrifugation stepfor 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 products 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 aspectsEthical 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 DISCUSSIONA 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 Klebsiellagenus, in Querecotillo 36 of 40 isolates were classifed into the Klebsiella genus, (29 Klebsiella mich-iganensisSaha et al. 2013and 7 Klebsiella variicola Rosenbluethet al. 2004). For Mambre 47 of 48 isolates were Klebsiella oxytoca (Flugge 1886) Lautrop 1956; while for Salitral 41 of 48 isolates were classifed as K. varii-cola(41 isolates) (See Table 1).This high prevalence of the genus Klebsiella was previously reported. K. variicolawas found in banana pseudostems and rhizome in tropical regions of Haiti, India, and was reported recently at Salitral province of Sullana-Piura (Fulton et al., 2020; Loganathan et al., 2021; Rafael-Rutte et al., 2022). Likewise K. variicola was involved with bacterial soft rot of carrot (Chandrashekar et al., 2018), and with wet wood disease of ironwood trees in a complex association with Ralstoniasolanacearum(Smith 1896) Yabuuchi et al.1996 emend. Safniet al. 2014 and K. oxytoca (Ayin et al., 2019)
image/svg+xmlNolasco-Cárdenas et al.340PAIDEIA XXITable 1. Principal bacterial isolates by Pseudostem sample.LocationSpecieSequence with highest blastn scores (Accession number)Numbers of bacterial isolatesQuerecotilloKlebsiella michiganensisCP054159.129Klebsiella variicola MF144432.17Pseudomonas entomophilaMN493076.12Stenotrophomonas sp.KX817899.12MambreKlebsiella oxytoca LR133932.147Kosakonia radicincitansCP040392.11SalitralKlebsiella variicolaMF144432.141Kosakonia radicincitans CP040392.13Pseudomonas entomophilaMN493076.12Pseudomonas plecoglossicidaMT367715.12In addition, genomic analyzes of Klebsiella michiganensisSaha et al.2013, obtained from diseased blackberry roots have revealed the ability of these bacteria to degrade the cell wall of plants as they have the necessary genes for the biosynthesis of type I and type II bacterial secretion systems, which would allow secreting enzymes to degrade the cell wall of plants (Luo et al., 2022; Yu et al., 2018). The potential capacity of K. variicolato be involved in or produce diseases is not limited to plants, in animals was found in Bovine mastitis disease and rare human pulmonary and urinary tract infections clinical cases as well as K. oxytocaand K. Michiganensis (Barrios-Camacho et al., 2019; Rodríguez-Medina et al., 2019; Seiffert et al.,2019; Duran-Bedolla et al., 2021; Neog et al., 2021).Nevertheless,theKlebsiella genus was also categorized by other studies as benefc bacteria due to the endophytic, nitrogen-fxing, and growth-promoting properties (Duran-Bedolla et al., 2021), so the bacterial role involving the production of enzymes that degrade the cell wall in banana pseudostem wet rot is still uncertain.The study determines the principal bacterial component while the less representative bacteria could have been excluded due to population reduction due to transport and sample dilution factors.The genus Klebsiellaof the Enterobacteriaceae family were found as the main bacterial component in organic banana pseudostem wet rot.
image/svg+xmlBacterial identifcation from banana pseudostem wet rot samples341PAIDEIA XXIAcnowledgementThis study was funded by “Proyecto de Investigación con Incentivos a nivel de Facultades (Resolución R. N°243-2022-CU-UNFV)”.Author contributions: CRediT (Con-tributor Roles Taxonomy)ONC= Oscar Nolasco-Cárdenas CCA= Candy Chacón-AguilarLSA= Linda Salas-AlvaMVV= Mónica Velarde-VilchezYMB= Ysabel Murrugarra-BringasAGR= Ana Gutiérrez-RománConceptualization: ONCData curation: CCA, LSAFormal Analysis: ONCFunding acquisition: ONC, MVV, YMB, AGRInvestigation: ONCMethodology: ONCProject administration: ONC, MVVResources: LSA, CCASoftware: ONCSupervision: ONCValidation: ONCVisualization: AGRWriting – original draft: ONC, CCA, LSAWriting – review & editing: ONC, MVV, YMB, AGRBIBLIOGRAPHIC REFERENCESAyin, C.M., Alvarez, A. M., Awana, C., Schleinzer, F.M., Marx, B.D., & Schlub, R. L. (2019). Ralstonia solanacearum, Ganoderma australe, and bacterial wetwood as predictors of ironwood tree (Casuarina equisetifolia) decline in Guam. Australasian Plant Pathology, 48(6), 625–636. Barrios-Camacho, H., Aguilar-Vera, A., Beltran-Rojel, M., Aguilar-Vera, E., Duran-Bedolla, J., Rodriguez-Medina, N., Lozano-Aguirre, L., Perez-Carrascal, O. M., Rojas, J., & Garza-Ramos, U. (2019). Molecular epidemiology of Klebsiella variicolaobtained from different sources. Scientific Reports, 9, 1–10. Basim, H., Basim, E., Bakİ, D., & Turgut, A. (2019). Wet rot disease of banana (Musa sp.) caused by Pectobacterium carotovorumsubsp. carotovorumin Turkey. Canadian Journal of Plant Pathology, 41, 174–187. Blomme, G., Dita, M., Jacobsen, K. S., Vicente, L. P., Molina, A., Ocimati, W., Poussier, S., & Prior, P. (2017). Bacterial diseases of bananas and enset: Current state of knowledge and integrated approaches toward sustainable management. Frontiers in Plant Science, 8, 1290. Chandrashekar, B.S., Prasannakumar, M. K., Puneeth, M. E., Teli, K., Priyanka, K., Mahesh, H. B., & Desai, R. U. (2018). First report of bacterial soft rot of carrot caused by Klebsiella variicola in India. New Disease Reports, 37, 21-21. Charkowski, A.O. (2018). The changing face of bacterial soft-rot diseases. Annual Review of Phytopathology, 56, 269–288.
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