Journal of Enviromental Sciences and Natural Resources

ISSN 2444-4936

Volume 5, Issue 16 – July – December – 2019

ECORFAN-Spain

Chief Editor Journal of Enviromental Sciences and VILLASANTE, Sebastián. PhD Natural Resources, Volume 5, Issue 16, July – December 2019, is a journal Executive Director edited sixmonthly by ECORFAN. 38 RAMOS-ESCAMILLA, María. PhD Matacerquillas street, Postcode: 28411.

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Journal of Enviromental Sciences and Natural Resources

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SANDOVAL – SALAS, Fabiola. PhD Universidad de Castilla

SAHAZA - CARDONA, Jorge Humberto. PhD Universidad de Antioquia

ESCOBEDO - BONILLA, Cesar Marcial. PhD Universidad de Gante

GONZALEZ - TORRIVILLA, Cesar Castor. PhD Universidad Central de Venezuela

JOVEL, Juan. PhD University of Alberta

HERNÁNDEZ - MARTINEZ, Rufina. PhD University of California

ARAUJO - BURGOS, Tania. PhD Universita Degli Studi di Napoli Federico II

GARCÍA - DE SOTERO, Dora Enith. PhD Universidad de Sao Paulo

ROMERO - PÉREZ, Diego. PhD University of California

FLORES - PACHECO, Juan Asdrúbal. PhD Universidad de Valladolid

Arbitration Committee

SÁNCHEZ - OROZCO, Raymundo. PhD Tecnológico de Estudios Superiores de Jocotitlán

DEL ÁNGEL - CORONEL, Oscar Andrés. PhD Instituto Tecnológico de Veracruz

MEDINA - SAAVEDRA, Tarsicio. PhD Universidad Nacional Autónoma de México

AVENDAÑO - ARRAZATE, Carlos Hugo. PhD Colegio de Postgraduados

RUIZ - AGUILAR, Graciela M.L. PhD Instituto Politécnico Nacional

SAHAZA - CARDONA, Jorge Humberto. PhD Universidad Nacional Autónoma de México

ACOSTA - NAVARRETE, María Susana. PhD Instituto Tecnológico de Celaya

MORÁN - SILVA, Ángel. PhD Universidad Veracruzana

CHAVEZ - SANTOSCOY, Rocío Alejandra. PhD Universidad Autónoma de Baja California

LUCIO - DOMINGUEZ, Rodolfo. PhD Universidad Michoacana de San Nicolas de Hidalgo

ROSAS - ACEVEDO, José Luis. PhD Universidad Autónoma de Guerrero

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Presentation of Content

In the first article we present, Varietal description of sorghum phenotypes for registration, by AVENDAÑO-LÓPEZ, Adriana Natividad, SANCHEZ-MARTINEZ, Jóse, PADILLA-GARCIA, José Miguel and ARELLANO-RODRIGUEZ, Luis Javier, with ascription in the Universidad de Guadalajara, as next article we present, Rhizobacteria antagonistic to the fungus Sclerotium cepivorum (Berk.t) in onion plants (Allium cepa L.), by OCEGUEDA-REYES, Martha Delia, CASAS-SOLÍS, Josefina, GONZÁLEZ-EGUIARTE, Diego R. and VIRGEN-CALLEROS, Gil, with ascription in the Universidad de Guadalajara, as next article we present, Effectiveness of Humic Acids, Fulvic Acids and lechuguilla extract (Agave lechuguilla), as biosurfactants in the Remediation of soils contaminated with Hydrocarbon, by RESENDIZ-VEGA, Marisol & GARCÍA-MELO, José Alberto, with ascription in the Universidad Tecnológica Tula – Tepejí, as next article we present, Physicochemical and antioxidant characteristics of different sprouts, by REYES-MUNGUIA, Abigail, CARRILLO- INUNGARAY, María Luisa, GARCÍA-BARRÓN, Gabriela del Socorro and TURRUBIATES- GARAY, Karla Alicia, with ascription in the Universidad Autónoma de San Luis Potosí, as next article we present, In vitro growth of Oncidium sphacelatum (Orchidaceae) in different concentrations of sucrose and nutrients, by NAVARRETE-VALENCIA, Ana Luisa, RAMÍREZ-GUERRERO, Leobarda Guadalupe, SÁNCHEZ-MONTEÓN Ana Luisa and JIMÉNEZ-MEZA, Víctor Manuel, with ascription in the Universidad Autónoma de Nayarit.

Content

Article Page

Varietal description of sorghum phenotypes for registration 1-4 AVENDAÑO-LÓPEZ, Adriana Natividad, SANCHEZ-MARTINEZ, Jóse, PADILLA- GARCIA, José Miguel and ARELLANO-RODRIGUEZ, Luis Javier Universidad de Guadalajara

Rhizobacteria antagonistic to the fungus Sclerotium cepivorum (Berk.t) in onion 5-16 plants (Allium cepa L.) OCEGUEDA-REYES, Martha Delia, CASAS-SOLÍS, Josefina, GONZÁLEZ- EGUIARTE, Diego R. and VIRGEN-CALLEROS, Gil Universidad de Guadalajara

Effectiveness of Humic Acids, Fulvic Acids and lechuguilla extract (Agave 17-25 lechuguilla), as biosurfactants in the Remediation of soils contaminated with Hydrocarbon RESENDIZ-VEGA, Marisol & GARCÍA-MELO, José Alberto Universidad Tecnológica Tula – Tepejí

Physicochemical and antioxidant characteristics of different sprouts 26-29 REYES-MUNGUIA, Abigail, CARRILLO-INUNGARAY, María Luisa, GARCÍA- BARRÓN, Gabriela del Socorro and TURRUBIATES-GARAY, Karla Alicia Universidad Autónoma de San Luis Potosí

In vitro growth of Oncidium sphacelatum (Orchidaceae) in different concentrations 30-35 of sucrose and nutrients NAVARRETE-VALENCIA, Ana Luisa, RAMÍREZ-GUERRERO, Leobarda Guadalupe, SÁNCHEZ-MONTEÓN Ana Luisa and JIMÉNEZ-MEZA, Víctor Manuel Universidad Autónoma de Nayarit

1 Article Journal of Environmental Sciences and Natural Resources December 2019 Vol.5 No.16 1-4

Varietal description of sorghum phenotypes for registration

Caracterización varietal de fenotipos de sorgo con fines de registro

AVENDAÑO-LÓPEZ, Adriana Natividad, SANCHEZ-MARTINEZ, Jóse, PADILLA-GARCIA, José Miguel and ARELLANO-RODRIGUEZ, Luis Javier

Instituto de Ciencia y Tecnología de Semillas CUCBA Universidad de Guadalajara

ID 1st Author: Adriana Natividad, Avendaño-López / ORC ID: 0000-0003-1713-1165, CVU CONACYT ID: 236981

ID 1st Coauthor José, Sánchez-Martínez / ORC ID: 00000002-1451-1149, CVU CONACYT ID: 63408

ID 2nd Coauthor: José Miguel, Padilla-García / ORC ID: 0000-0002-5627-3986, CVU CONACYT ID: 236981

ID 3rd Coauthor: Luis Javier, Arellano-Rodríguez / ORC ID: 0000-0002-3188-0245, CVU CONACYT ID: 65995

DOI: 10.35429/JESN.2019.16.5.1.4 Received June 26, 2019; Accepted December 28, 2019

Abstract Resumen

The goal of a plant breeding is the generation of El objetivo de un programa de mejoramiento new varieties that become agricultural inputs for genético es la generación de nuevas variedades que farmers. For this, it is necessary to verify the genetic lleguen a ser insumo agrícola para los productores. identity of the materials and have the breeder's Para ello es necesario comprobar la identidad registration and title. The University of Guadalajara genética de los materiales y contar con el registro y has generated varieties and quality sorghum hybrids título de obtentor. La Universidad de Guadalajara, that must be released to the public. This work was ha generado variedades e híbridos de sorgo de developed to establish the methodology of varietal calidad que deben ser liberados al público. El characterization, an essential requirement for presente trabajo se desarrolló para establecer la obtaining a breeder's title before the National Seed metodología de caracterización varietal, requisito Inspection and Certification Service (SNICS). For indispensable para obtención de título de obtentor this, 2 andro-sterile lines (A) and a fertility ante el Servicio Nacional de Inspección y restoration line (R) were described, in two locations Certificación de Semillas (SNICS). Para ello se and two production cycles. By establishing describieron 2 líneas androestériles (A) y una línea according to ranges of variation and stability, the restauradora de la fertilidad (R), en dos localidades appropriate sample size and differential phenotypic y dos ciclos de producción. Logrando establecer de characters that facilitated identification. acuerdo a rangos de variación y estabilidad, el tamaño de muestra adecuado y los caracteres fenotípicos diferenciales que facilitaron la identificación.

Sorghum, Genetic identity, Varietal description Sorgo, Descripción Varietal

Citation: AVENDAÑO-LÓPEZ, Adriana Natividad, SANCHEZ-MARTINEZ, Jóse, PADILLA-GARCIA, José Miguel and ARELLANO-RODRIGUEZ, Luis Javier. Varietal description of sorghum phenotypes for registration. Journal of Environmental Sciences and Natural Resources. 2019, 5-16: 1-4

* Correspondence to Author (email: [email protected]) † Researcher contributing first author.

2 Article Journal of Environmental Sciences and Natural Resources December 2019 Vol.5 No.16 1-4

Introduction The objective of the present work was, in addition to the characterization of the In Mexico, sorghum is a crop of great materials, to generate information that allows importance, thanks to its adaptability and its determining the sample size, that is, the nutritional properties. It is so versatile that it convenient number of plants to be used in the adapts to very adverse environmental varietal description of qualitative and conditions representing an alternative in the quantitative phenotypic characteristics to production of grain and fodder. achieve the optimization of resources and time, without sacrificing accuracy. Under the In the practice of genetic improvement hypothesis that in the development of a varietal of seeds, either commercially or description the sample size varies according to experimentally, the existence of a varietal the character under study. And increasing the description of the phenotypes of materials to be number of plants described, the coefficient of used is indispensable. Since although important variation decreases and when it presents some technological advances have been implemented stability in its values, the optimal sample size is in the identification at the molecular level, this obtained. type of analysis is used as complementary and does not replace the phenotypic description, Methodology either because of its high specialization that in some cases makes its repetitiveness difficult. The descriptions will be measured in more than one environment, for this reason the present In a varietal description, the phenotypic work was carried out in two locations: The characteristics to which the behavior of a experimental agricultural field of the University material should be adjusted are explicitly Center of Biological and Agricultural Sciences mentioned, and it is useful to make better use of CUCBA of the University of Guadalajara it, with the possibility of exploiting its where work has been done in training and potential. release of new varieties and / or sorghum hybrids; Located on the Las Agujas property, Through this document it is possible to Zapopan Latitude: 20 ° 43′12 ″ N; Longitude: legitimize and register a variety before the 103 ° 23′30 ″ O Altitude: 1,567 masl. (Figure 1) official agency, in addition to enabling its The second town was Poncitlán. Location: dissemination and properly performing its Latitude: 20º 18 '15' 'at 20º 26' 15 '' N multiplication, guaranteeing its varietal purity Longitude: 102º 16 '45' 'Altitude: 1,524 masl. not only during the production stage, but also to We worked with two androsterile lines and a strengthen the credibility of the promotion of fertility restorer (parental of commercial new varieties in the market. hybrids (ATx 626, ATX 399 and R-573). In both cases the crop was managed with the CIAT (1983) Notes that for the approval appropriate technological package for each of a new variety, it is a requirement that it be area. The production plots were of an area of clearly identifiable and distinct, as well as 500 m and the number of plants described per stable and homogeneous. The complete character was 100. certification process involves the control of the origin of the variety and the supervision during the multiplication and the benefit of it, to avoid mixing with other materials.

According to CIAT (1983), when the varietal description is made in the phenotype observing, the plants of a variety will depend on the genetic potential of each plant and its expression (phenotype), according to the environmental effects present. Therefore, the manifestation of a phenotype should be known to try to differentiate variations due to genetic effects from those that occur due to environmental effects. Figure 1 Town Las needles, Zapopan Jal.

AVENDAÑO-LÓPEZ, Adriana Natividad, SANCHEZ-MARTINEZ, ISSN 2444-4936 Jóse, PADILLA-GARCIA, José Miguel and ARELLANO-

ECORFAN® All rights reserved RODRIGUEZ, Luis Javier. Varietal description of sorghum phenotypes for registration. Journal of Environmental Sciences and Natural Resources. 2019 3 Article Journal of Environmental Sciences and Natural Resources December 2019 Vol.5 No.16 1-4

In order to provide elements that In order to know the degree of precision facilitate the identity of the materials described, achieved with the number of plants used as the the methodology proposed by UPOV and the optimal sample size, the formula proposed by International Center for Tropical Agriculture Cochran (1984) was used. CIAT was used. Varietal purity is the genetic identity of plants, it is important to note that it Where: n = (t2) (s2) does not necessarily indicate homozygous or d2 total uniformity among the population, but that the multiplied seed will faithfully reproduce the n= sample size characteristic phenotype of the variety. (Serrato, t = degree of reliability 1995). The characters described were: s2 = variance d2 = degree of accuracy desired Qualitative in seedling state Anthocyanin staining of the beetle Results Anthocyanin coloration of the back of the first leaf Anthocyanin coloration in the first leaf sheath Quantitative adult plant With the values of the mean, range and Days to anthesis coefficient of variation, it was possible to Plant height in panicle emergency Total height at maturity estimate the magnitude of variation of the Stem diameter characters, in Figure 2 it is exemplified that Length of the third sheet from the top from 40 plants the trend of the mean and the Width of the third sheet from the top coefficient of variation is maintained Visible length of the peduncle ( Weight of one thousand seeds in g Qualitative characters D7 Leaf color green D8 Extension of the discoloration in the central rib. D10 Leaf flag yellow coloration of the central rib Gluma, color in bloom Gluma, anthocyanin coloration in bloom Gluma, anthocyanin staining in pubescence Motto, edge formation. Stigma, anthocyanin staining Stigma, yellow coloring Stigma, length Flower with pedicel flower length

Panicle density at the end of flowering Panicle, branch length Panicle, density at maturity. Figure 2 Average behavior and coefficient of variation of Panicle, mature form the number of knots and leaves Caryopside, color after threshing Dorsal grain view In figure 3, the average length of the Grain Profile View Grain, germ brand size spine is shown for the character from a sample Grain, surface covered by glume size of 20 plants, the mean is not modified. Predominant endosperm texture Grain, color of crystalline albumen

Considering that the optimal sample size is one in which the coefficient of variation presents values with a tendency to stabilize, the values of the described characters were analyzed through measures of central tendency and through comparative graphs to make it possible to determine the point closer to stability or balance, resulting in the optimal sample number. Regarding the statistical Figure 3 Sample Size Behavior Regarding Averages parameters used in the varietal description, CIAT mentions that when it comes to qualitative characteristics that can be measured based on the mean (µ) standard deviation (σ) coefficient of variation (CV) and range (R)

AVENDAÑO-LÓPEZ, Adriana Natividad, SANCHEZ-MARTINEZ, ISSN 2444-4936 Jóse, PADILLA-GARCIA, José Miguel and ARELLANO-

ECORFAN® All rights reserved RODRIGUEZ, Luis Javier. Varietal description of sorghum phenotypes for registration. Journal of Environmental Sciences and Natural Resources. 2019 4 Article Journal of Environmental Sciences and Natural Resources December 2019 Vol.5 No.16 1-4

The variation in the qualitative CIAT (Centro Internacional de Agricultura characteristics was more stable even with a Tropical), 1983. Metodologías para obtener smaller sample size, which confirms that the semillas de calidad arroz, frijol, maíz, sorgo. interaction with the environment in this type of Ed. Unidad de semillas CIAT. Cali, Colombia. characteristics is smaller (Figure 3) 198 p.

SNICS. 2002. Guía técnica para la descripción Coloración de antocianinas del coleoptilo varietal para sorgo (Sorgum bicolor L.). México 25 15 p. 20 UPOV (International Union for the Protection 15 µ C.V. of New Varieties of Plants). 2000. List of TGP 10 documents and latest issue dates (TC/36/8). 5 Technical committee. April 3 to 5 Thirthy- Sixthy Session. Geneva, Swiss 0 1

Both qualitative and quantitative characters that offer a greater degree of reliability for presenting less interaction with the environment were:

Quantitative: number of secondary branches. Plant height, rachis length and number of knots and leaves

Qualitative: Predominant color of hypocotyl, predominant color of anthers, glumes and central vein, predominant habit of leaves and predominant type of head or panicle

With greater interaction and less stable: Head width, leaf area, excersion and number of nodes in the spine, considered as not very reliable characters in the identification of materials.

Conclusions

In the characters described for the 3 lines used, the optimal sample number ranges between 30 and 60

The sample size for qualitative characters was 7 with a degree of accuracy of 99%

References

CIAT. 1987. Producción de semilla genética y básica. Responsabilidad de un programa de mejoramiento. Memorias del curso avanzado sobre producción de semilla básica del 27 de Abril al 29 de Mayo. Cali Colombia.

AVENDAÑO-LÓPEZ, Adriana Natividad, SANCHEZ-MARTINEZ, ISSN 2444-4936 Jóse, PADILLA-GARCIA, José Miguel and ARELLANO-

ECORFAN® All rights reserved RODRIGUEZ, Luis Javier. Varietal description of sorghum phenotypes for registration. Journal of Environmental Sciences and Natural Resources. 2019 5 Article Journal of Environmental Sciences and Natural Resources December 2019 Vol.5 No.16 5-16

Rhizobacteria antagonistic to the fungus Sclerotium cepivorum (Berk.t) in onion plants (Allium cepa L.)

Rizobacterias antagónicas al hongo Sclerotium cepivorum (Berk.t) en plantas de cebolla (Allium cepa L.)

OCEGUEDA-REYES, Martha Delia, CASAS-SOLÍS, Josefina, GONZÁLEZ-EGUIARTE, Diego R. and VIRGEN-CALLEROS, Gil

Centro Universitario de Ciencias Biológicas y Agropecuarias, Universidad de Guadalajara. Zapopan. Jalisco, Camino Ramón Padilla Sánchez #2100 Nextipac, Zapopan, Jalisco, México.C. P: 45200

ID 1st Author: Martha Delia Ocegueda Reyes

ID 1st Coauthor: Josefina, Casas-Solís / ORC ID: 0000-0001-7880-7240, Researcher ID Thomson: U-1318-2018, CVU CONACYT ID: 218087

ID 2nd Coauthor: Diego R., González-Eguiarte / ORC ID: 0000-0001-6609-0780, CVU CONACYT ID: 3475

ID 3rd Coauthor: Gil, Virgen-Calleros / ORC ID: 0000-0003-4172-8885, CVU CONACYT ID: 74671

DOI: 10.35429/JESN.2019.16.5.5.16 Received June 26, 2019; Accepted December 30, 2019

Abstract Resumen

Rhizobacteria are microorganisms attached to the roots of Las rizobacterias son microorganismos adheridos a las plants that colonize the rhizosphere, there is a great diversity raíces de plantas que colonizan la rizosfera, existe una gran with beneficial effects on crop development and they used in diversidad con efectos benéficos sobre el desarrollo de biological control. In this study, the antagonistic effect of cultivos y se utilizan en el control biológico. En este estudio rhizobacteria as biocontrollers of Sclerotium cepivorum se determinó el efecto antagónico de las rizobacterias como Berk fungus, which cause white onion rot in large economic biocontroladoras del hongo Sclerotium cepivorum Berk, que losses, was determined. Soil samples collected from onion causan la pudrición blanca en cebolla con grandes pérdidas plants on the farm located in Santa Anita, municipality of económicas. Se colectaron muestras del suelo de plantas de Tlaquepaque, Jalisco; for isolation of rhizobacteria and S. cebollas del predio ubicado en Santa Anita, municipio de cepivorum from infected white rot bulbs. Rhizobacteria that Tlaquepaque, Jalisco; para aislamiento de rizobacterias y S. showed antagonism against the fungus, were selected 14 cepivorum a partir de bulbos infectados de pudrición blanca. treatments formed with onion plants in conditions of Se seleccionaron las rizobacterias que mostraron humidity, temperature and photoperiod constant in growth antagonismo contra el hongo. Se formaron 14 tratamientos chamber for 36 weeks. Growth promotion variables were con plantas de cebolla en condiciones de humedad, evaluated and the data analyzed by ANOVA. Rhizobacteria temperatura y fotoperiodo constante en cámara de showed an antagonistic effect against the fungus and crecimiento durante 36 semanas. Se evaluaron variables de stimulated fresh weight, root length, stem and bulb in onion promoción de crecimiento y los datos se analizaron por plants. Therefore, this PGPR is an alternative as a ANDEVA. Las rizobacterias mostraron efecto antagónico biofertilizer potential, thus avoiding degradation and contra el hongo y estimularon el peso fresco, longitud de contamination of the soil and yield of product quality and raíz, tallo y bulbo en plantas de cebolla. Por lo tanto, esta profitability for the producer. PGPR es una alternativa como potencial biofertilizante, evitando así la degradación y contaminación del suelo y rendimiento de la calidad del producto y rentabilidad para el productor.

Rhizobacteria, White rot, Sclerotium cepivorum Rizobacterias, Pudrición blanca, Sclerotium cepivorum

Citation: OCEGUEDA-REYES, Martha Delia, CASAS-SOLÍS, Josefina, GONZÁLEZ-EGUIARTE, Diego R. and VIRGEN-CALLEROS, Gil. Rhizobacteria antagonistic to the fungus Sclerotium cepivorum (Berk.t) in onion plants (Allium cepa L.). Journal of Environmental Sciences and Natural Resources. 2019, 5-16: 5-16

* Correspondence to Author (email: [email protected]) † Researcher contributing first author.

6 Article Journal of Environmental Sciences and Natural Resources December 2019 Vol.5 No.16 5-16

Introduction Background

There is a great diversity of rhizobacteria The term rhizosphere was introduced by Lorenz attached to the roots of plants that has effects on Hiltner (1904), to refer to the area where the the development of plants; In addition, they are microbial populations of bacteria that develop used in biological control is an alternative to fundamental activities around the root inhabit reduce the use of chemicals, such as agents that and activate (Arshad and Frankenberger, 1998; stimulate plant growth, soil and plant health; Saharan and Nehra, 2011). In 1978 Kloepper This favors the yield in the crops, however, the called them Plant Growth Promoting presence of diseases caused by pathogens are Rhizobacteria Rhizobacteria (PGPR), for its present in different crops, such as onions. This acronym in English. vegetable is widely cultivated and of great economic importance worldwide. In Mexico; in These beneficial soil bacteria play an the report of advances of sowing and harvest of important role in increasing plant growth and the national summary of the agricultural year increasing their defense against other disease- 2018, it refers to a tenth first place in causing microorganisms. PGPRs are classified production in the last cycle. With 20,919 according to their mechanisms of action; some hectares sown, only a total of 5,560 hectares act in the development of the plant since they were harvested (SIAP-SAGARPA, 2019). increase the availability of nutrients in the rhizosphere, inducing plant development In Mexico, the onion is vulnerable to regulated by the action of phytohormones such fungal pathogens that cause the disease called as; auxins, 3-indole-acetic acid, gibberellins, white rot caused by Sclerotium cepivorum cytokinins, abscisic acid, activity of the enzyme Berk, causing large economic losses and a 1-aminocyclopropane-1-carboxylic acid decrease in product quality; affects both foliage deaminase (ACC deaminase), which regulates and bulb (Delgado Ortiz et al., 2018); It is ethylene levels, converting ACC into α- easily disseminated through sclerotia and has a ketobutyrate and ammonium (Glick, 2014; very high viability in adverse conditions and Esquivel-Cote et al., 2013), nutritional elements can remain for long periods in the soil. such as iron, phosphorus and aluminum (Carcaño et al., 2006), in addition, increase the However, to combat this infection, amount of roots and thus the water absorption agrochemicals are used, so it is necessary to capacity and nutrients. look for biocontrol alternatives, to avoid soil contamination and decrease in product quality. Other mechanisms act to reduce the This disease causes great economic losses; harmful effect of phytopathogens through the Affected farmers opt for alternatives such as production of antagonistic substances changing crops, a strategy that is not safe (tetrasulfides, thiols, thiophenes and sulfur because the fungus is activated with exudates dioxide) and as producers of siderophores that released by the onion plant; causing serious sequester the iron present in the medium damage and also to other species of this same (Corrales-García and Ciro, 2010; Glick, 2014), genus (Granados and Wang, 2005). An as has been demonstrated, in the different alternative for biological control with mechanisms of action of a bacterial biological antagonistic microorganisms such as PGPR’s, compound that limits the development of which suppress diseases caused by pathogens as indicated by the Fungicide phytopathogens through the production of Resistance Committee (FRAC, 2018). siderophores, synthesis of antibiotics, enzymes However, the selection of microorganisms is or fungal compounds or both (Serret-López et based on the capacity for increased production al., 2016). of metabolites of interest or antagonistic factor, on the use of organic substances as a source of The present work proposed isolating nutrients and on the energy that allows them to PGPR rhizobacteria biocontrollers, as an survive and efficiently prevent the proliferation alternative for the management of S. cepivorum of phytopathogens, acting through the synthesis fungus in onion plant (Allium cepa L.) and of antibiotics, or enzymatic biocontrol have a more profitable product for the producer. processes (Desai et al., 2012), important for agriculture especially in vegetable crops.

ISSN 2444-4936 OCEGUEDA-REYES, Martha Delia, CASAS-SOLÍS, Josefina, ECORFAN® All rights reserved GONZÁLEZ-EGUIARTE, Diego R. and VIRGEN-CALLEROS, Gil. Rhizobacteria antagonistic to the fungus Sclerotium cepivorum (Berk.t) in onion plants (Allium cepa L.). Journal of Environmental Sciences and

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Onion crop Due to the problem that exists in this country, of large losses in onion crops, affected The onion (Allium cepa L.) is one of the most by the disease such as white rot caused by the cultivated vegetables worldwide. This crop fungus Sclerotium cepivorum Berk., It is belongs to the Liliaceae family, there are 600 necessary to determine and evaluate the species and it belongs to the Allium genus, antagonistic effect of rhizobacteria as composed of: onion (A. cepa L.), garlic (A. biocontrollers of this mushroom. sativum L.), leek (A. porrum L.) and chives (A. schoenoprasum L.). Its root system consists of Onion disease 20 to 200 roots that can develop from 35 to 60 cm from the soil, stimulate a low absorption of Onion crops are affected by white rot disease, nutrients and water. It has a true stem, caused by the fungus Sclerotium cepivorum cylindrical and hollow leaves, which together (Berk.) Is one of the most important (Sarmiento form the false stem at the top and bottom, the and Velandía, 2013; Castillo et al., 2016) ;; bulb develops when the conditions are optimal because it affects the genus Allium (onion, for photoperiod and temperature, the color of garlic, leek); the onion is affected at any stage the onion is associated with the variety and of development, the infection increases as the presents white flowers. radical system grows, the bulb and can cause losses up to 100% of the crops. In Mexico, it The quality of the onion is of great was first presented in Zacatecas in 1990 economic importance, a successful production (Colina, 2017; Reveles-Hernández et al., 2014). depends on the management and control of The most affected states are: Aguascalientes, pests influenced by climatic factors. In addition Chihuahua, Guanajuato, Jalisco, Morelos, to the integral application and essential Michoacán, Puebla, Querétaro, Tlaxcala and elements for the onion plant is magnesium, Zacatecas (Velásquez-Valle and Reveles manganese, zinc, boron and sulfur by foliar Hernández, 2016). route and nitrogen that provides firmness of the bulb, color, thickness of the scales and neck S. cepivorum belongs to the Mycelial that are quality factors, to achieve maximum Stenrilia Order and the Sclerotiniaceae Family performance; the absence of macronutrients and (Sarmiento and Velandía 2013; Castillo et al., micronutrients limits the nutritional balance of 2016). The sclerotia are stimulated by sulfur this vegetable, because they regulate the compounds present in the radical exudates of expression of the productive potential of the the onion plant such as: aquil cysteine and crop and the quality of the product as is the case sulfoxides, easily disseminated and that once of the bulb (Gómez, 2007). established in the soil can remain for several decades (Ortega et al., 2011; Reveles - The growth is fast and develops in Hernández et al., 2014; Hussain et al., 2017; different types of soils and climates. However, Elshahawy et al., 2017), these produce volatile it tolerates temperatures below zero, it adapts to compounds of allyl and n-propyl sulfide (Coley- regions that range between 10 and 20 ° C; Smith et al., 1990), the mycelium contains sunlight favors bulbification (Gómez, 2002; material of reserve, which allows them to SIAP-SAGARPA, 2014). This plant is typical remain at rest when the environmental of transplanting, it is done when the plants are conditions are adverse (Vera et al., 2005; 40 to 60 days (approximately 15 cm high) after Ortega et al., 2011), due to the viability of up to planting (Granados and Wang, 2008). In the 90% and being able to remain inactive for up to world more than 1,100 million tons of onion are 40 years (Reveles-Hernández et al., 2014), produced, in an area of 4.2 million hectares these are activated when the environmental (FAO). In Mexico, onion is grown in almost the conditions are favorable, in the presence of entire country, it is the third most cultivated sulfur compounds produced by the onion tuber vegetable. 95% of the production is carried out (Coley-Smith et al., 1990). And the through irrigation and in two cycles: Autumn- environmental conditions that favor the winter and spring-summer, the first being the development of the fungus is the temperature in one with the highest production (SAGARPA, the soil (18 to 22 ° C) and the optimum pH is 2 2014). Due to its culinary properties and health to 7 (Crowe et al., 1980). benefits, it has a high demand, so it represents a source of employment.

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Chemical agents mainly mancozeb, Were incubated at 20 ° C and captan and hydrogen peroxide have been used mycelium development was observed until with positive effect for the control of white rot sclerotia formation (Ortega-Aguilar et al., (Velásquez-Valle and Reveles Hernández, 2011). 2016); and tebuconazole alone or in combination with calcium nitrate, reduced the Rhizobacterial antagonism on S. cepivorum severity of damage from 56 to 92% (Siyoum et al., 2013; Hussain et al., 2017); However, the Previously, the S. cepivorum fungus was grown inappropriate use of pesticides has had a great in ADS and a 5 mm diameter mycelium disc impact on the environment, has phytotoxicity was removed and placed in plates of the same and accumulation of residues and immersion in medium and incubated at 20 ° C; on the third the roots of edible plants (Ulacio et al., 2010; day, the plates with different cultures of Van Dyk and Pletschke, 2011; Reveles - rhizobacteria were inoculated by a single Hernández et al., 2014). Currently, native fungi groove 25 mm away around the mycelial disk and bacteria that inhabit the soil have been used and incubated at 20 ° C. Inoculation of the for their control, this depends on the capacity fungus was only used as a control and it was and environmental conditions and soil type concluded when the development covered the (Mahdizadehnaraghi et al., 2015; Vega-Celedón total of the box (Sarti and Miyazaki, 2013). The et al., 2016; Kumbhar et al., 2018). test was performed in duplicate. Halo diameters Rhizobacteria have a potential for biocontrol, of inhibition were measured for each which reduce the density of the inoculum rhizobacterium and those with the greatest avoiding the formation of sclerotia (Esquivel et antifungal effect were selected to assess al., 2013) and endomiccorrhizal fungi (Glomus antagonism and growth promotion in vivo. intraradices), capable of providing a protective effect similar to the fungicide Folicur (Jaime et Antagonism and growth promotion by al. , 2008). rhizobacteria in onion plants in growth chamber conditions Methodology Selected rhizobacteria were inoculated in Rhizobacteria and fungus isolation nutrient broth, adjusted to the McFarland scale Sclerotium cepivorum at 0.5 (10.8 CFU mL-1). Onion plants with a growth of three weeks were used with a bulb 3 Soil samples were collected from onion plants cm wide and long. By means of a cut, the size on the farm located in Santa Anita, municipality of leaves at 14 cm high and 4 cm long at the of Tlaquepaque, Jalisco, Mexico, with root was homogenized from the bulb. The bulb geographical coordinates: the 20 ° 55 'N and was made an incision of approximately 2 cm to 103 ° .44' W. For the isolation of rhizobacteria favor the treatments, 14 treatments were formed plants were selected of onion with greater stem with the rhizobacteria and fungus (Table 1). and bulb development: 10 g of soil adhered to the roots were taken, diluted in 90 mL of The rhizobacterial treatments were distilled water, homogenized and subsequently immersed in a bacterial solution (15 x 107 CFU serial dilutions (10-7) were made, one mL of mL-1) according to the treatment, for 15 The last three dilutions, which were inoculated minutes. To the treatments that were added on plates with nutrient agar (AN), were fungus, 5 multidisc myceliums containing about incubated at 37 ° C for 24 h. The colonies were 25 to 30 sclerotia were incorporated and on the selected and purified; they were kept in test other hand, a control without rhizobacteria or tubes to evaluate the antagonistic effect. fungus, subsequently transplanted into unicel containers containing 400 g of previously The isolation of Sclerotium sterilized soil ( 121 ° C / 15 min). cepivorum, the methodology described by Vimard et al. (1986), which consisted of separating sclerotia from affected onions with white rot, were disinfected with sodium hypochlorite (0.5%) for 3 min and subsequently washed in distilled water, fragmented for inoculation in Sabouraud Dextrose Agar medium (ADS). ISSN 2444-4936 OCEGUEDA-REYES, Martha Delia, CASAS-SOLÍS, Josefina, ECORFAN® All rights reserved GONZÁLEZ-EGUIARTE, Diego R. and VIRGEN-CALLEROS, Gil. Rhizobacteria antagonistic to the fungus Sclerotium cepivorum (Berk.t) in onion plants (Allium cepa L.). Journal of Environmental Sciences and

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1. Witness 6. RB1+H 11. RB2+RB3 2. Fungus 7. RB2+ H 12. RB1+ RB2+H 3. RB 1 8. RB3+ H 13. RB1+ RB3+H 4. RB 2 9. RB1+RB2 14. RB2+ RB3+H 5. RB 3 10.RB1+RB3 RB = Rhizobacteria H = Fungus Figure 1 Development of mycelium (A) and sclerotia (B) Table 1 Treatments used in onion plants Treatments of Sclerotium cepivorum and antagonism of the genus Bacillus (C and D) The plants were kept in a growth chamber of the Biotechnology Laboratory of Rhizobacterial antagonism on S. cepivorum the Department of Botany of the CUCBA for a period of 36 days; under the following Of the 656 bacterial isolates, only 20 isolates conditions: relative humidity 30%, constant showed antagonism with inhibition halos less temperature of 20 ° C and light / dark than 18 mm and three isolates with 20 to 23 photoperiods (12/12). mm on the fungus under study (Figure 1C and 1D); these last three isolates were selected and At the end of the time, the following designated as RB1, RB2 and RB3; were variables in the plants were evaluated: fresh and identified by the VITEX 2 system with the BCL dry weight, root length, stem and bulb; the reactive card (bioMérieuxMR) for positive number of leaves was measured to evaluate the Gram spore-forming bacilli, with a 95% growth promotion and the effect of S. reliability; they showed morphological cepivorum on onion plants. characteristics, such as large cream-colored colonies, smooth surface, viscous consistency, Experimental design and statistical analysis opaque density, wavy margin and dyeing as gram-positive bacilli with endospore formation. The results obtained were performed with a The three selected isolates belong to the genus completely randomized experimental design; Bacillus; the rhizobacteria RB1 and RB3 as The Sigma Plott 11.0 program was used for Bacillus subtilis and RB2 as Bacillus statistical analysis. A one-way analysis of amynololiquefaciens, these characteristics are variance (ANDEVA) was applied, for the characteristic of this genus (Tejera-Hernández comparison between the groups and the et al., 2011; Oslizlo et al., 2015). Kruskal-Wallis test in pairs, to determine the significant difference between the groups at P This antagonistic effect on S. cepivorum <0.05. may be due to the fact that the rhizobacteria isolated in this work can be producers of Results and Discussion antimicrobial molecules such as lipopeptides (Sarti and Miyazaki, 2013; Villarreal-Delgado Isolation of rhizobacteria and fungus S. et al., 2017). The Resistant Fungicide Action cepivorum Committee (FRAC) listed them to the rhizobacteria Bacillus subtilis and B. 656 rhizobacteria from the rhizosphere of onion amyloliquefaciens, as producers of fungal plants were isolated; This is related to the great substances classified in group F (F6); that act in bacterial diversity of free life that adapt and the transport or synthesis of lipids that affect colonize the soil (Saleem et al., 2007; Luna et the function and integrity of the cell membrane al., 2013). of pathogens (FRAC, 2018).

On the other hand, the S. cepivorum Other studies have shown that B. fungus was isolated and morphological subtilis acts in the control of fungi of the genus characteristics were observed showing Fusarium (Yesid et al., 2012; Mejía-Bautista et abundant whitish mycelium, formation of al., 2016; Da Silva et al., 2018). This Bacillus is black, round sclerotia, which had a diameter of an effective soil and rhizosphere colonizer with 54 nm on average per sclerotia (Figure 1A and great genetic diversity (Montor-Antonio et al., 1B ); the results coincide with what is described 2014); which favors the antagonistic activity in the taxonomic keys of Imperfect Fungi against pathogens and consequently induces the (Sarmiento and Velandía, A2013) . B developmentC of the plant.

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In addition, Sánchez et al., (2016), The root length results (Figure 3) show record the production of Iturin A by these a better development with the use of bacteria, such as fengicins that cause an rhizobacteria individually (treatments 3 to 5), osmotic imbalance in the spores and antifungal which induce root development, compared to activity against the filaments of the fungus; the control; similarly the same behavior was Surfactin inhibits spore germination (Arjona- observed in fresh weight where rhizobacteria López, 2019) and the excretion of antimicrobial individually, as well as treatment 1 (plant enzymes such as β-glucosidases and proteases without fungus, or bacteria) and when an that control leaf and root diseases (Da Silva et infection occurs with S. cepivorum al., 2018). In studies with Pseudomonas rhizobacteria try to compensate with fresh fluorescens and B. subtilis they presented weight and root length and this is closely inhibition against fungi such as Rhizoctonia related to dry weight. On the other hand, the use solani, Fusarium oxysporum, and Sclerotium in consortium form inhibits these variables sosfsii (Paredes Escalente et. Al., 2009) and compared to the development of the fungus Bacillus spp. against Macrophomina phaseolia seedling. Therefore, the best treatments were (Reyes et al., 2011), among others. those that were added separately each of the rhizobacteria. The three rhizobacteria were subjected to 24 and 37 ° C to determine if the conditions 30 A of humidity and temperatures do not affect the 25 vital processes of the genus Bacillus, as well as the effects of microbial antagonism against S. 20 cepivorum. The results showed that it does not 15 affect bacterial viability when grown at these temperatures (37 and 24 ° C) and humidity of 10 50, 75 and 100%; Some authors mention that Root length (cm) length Root 5 there is a great diversity of bacteria capable of surviving in diverse environments (Estrada 0 Bonilla et al., 2009; Calvo and Zúñiga, 2010; 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Treatments Gómez et al., 2017) and this favors the adaptation process and the ability to antagonize 2.500 B microbial (Gouda et al., 2018; Soto et al., 2018). 2.000 1.500 Rhizobacteria in the promotion of growth and control of S. cepivorum fungus on onion 1.000 plants 0.500

weight root Fresh The results of the variables studied in the 14 - treatments in the growth chamber (Figure 2), of 1 2 3 4 5 6 7 8 9 10 11 12 13 14 the onion plants, only showed a significant Treatments difference (P ≤ 0.05) in the variables: length, C fresh and dry root weight. 0.25

0.2

0.15

weight (cm) weight 0.1 root 0.05 Dry 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Figure 2 Onion plants in growth chamber Treatments

Figure 3 Treatments applied in onion plants (growth chamber)

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In the case of stem length there was no significant difference, however treatments with bacteria behaved in an equivalent manner both individually and in consortium in the development of the stem, although when the bacteria is individually and the plant is infected by fungus it is more vulnerable, but manages to survive and develop the stem despite the size of the root (Figure 4 and 5). With respect to the bulb length there was no significant difference, Figure 5 Treatment with Rhizobacteria and infection in this variable a similar behavior was shown with S. cepivorum in onion plants with and without the presence of the fungus; perhaps this result is due to the incision that This effect may be due to the fact that was made to the bulb and by submerging it and the rhizobacterium acts at the root level which impregnated with the bacterial solution it agrees with Gouda et al., (2018), which protects it against the S. cepivorum fungus. mention that the genus Bacillus improve the Ortiz et al., (2016) point out that immersion in growth of plants because it produces phenazine, bacterial solution of PGPR, benefits the carboxylic acid, antifungal compounds 2- development and health of the plant. hexium, 5 resorcinol propyl, pyrrolnitrin (Arjona-López, 2019), 2,3-butanadiol, acetoin, lipopeptides and glycopeptides such as ramnolipids and surfactins, which are effective 45.00 in inhibiting the growth of pathogenic fungi, 40.00 due to their antifungal activity and ability to 35.00 solubilize the main components of microbial 30.00 cell membranes and allow greater chance of 25.00 survival in habitats with high competition for 20.00 nutritional elements (Rios-Velasco, et al., 15.00 2016). 10.00 Stem Length (mm) Length Stem 5.00 On the other hand, the development of - 1 2 3 4 5 6 7 8 9 10 11 12 13 14 microbial populations (Salazar-Henao et al., 2016), which allows the plant to improve the Treatments absorption of nutrients as mentioned by Ortiz et al., (2016). Bacillus amyloliquefaciens and 2.50 subtilis can inhibit fungal pathogens by the production of lithic enzymes such as chitinases, 2.00 degrade the cell wall of fungi and prevent the spread of hyphae (Moreno et al., 2018). 1.50 Through these phyto-stimulants, it can also promote the protection and capacity of plants to

1.00 resist long periods of drought (Rojas-Solís et al., 2013). These results agree with Hussini et al., (2019), who report that bacterial antagonists 0.50

Largo de bulbo (mm) (mm) bulbo de Largo either individually or in consortium have the potential to develop as bioinoculants and for - ecological management. 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Treatments The soil is a complex system of beneficial effects due to interactions with

Figure 4 Treatments applied in onion plants (growth rhizobacteria, which depend on both the strain chamber) and the plant; the genus Bacillus, has influence on variable of plants of the type of mini-tubers and tubers.

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Alvarado-Capo et al., (2015), who Therefore, this PGPR is an alternative as worked with potatoes, mention that Bacillus has a biofertilizer potential, thus avoiding the advantage of forming endospores which degradation and contamination of the soil and gives great stability to act as biofertilizers or yielding product quality and profitability for the biofungicides (Desai et al., 2012) and allow producer, which allows reducing the demand them to resist adverse environmental for agricultural inputs such as chemical conditions, favoring the storage of products fertilizers. However, it is important to produce (Moreno et al., 2018). Rhizobacteria favor root healthy plants to achieve greater yield and if length and fresh weight and dry weight in onion these rhizobacteria inhibit the growth of plants, which coincides with the results of pathogenic fungi, they are the alternative for the Angulo-Castro et al., (2018), who worked on management of the S. cepivorum fungus that the application and inoculation of bacterial affects the onion. strains in plants, demonstrating that there was greater development in plant height, leaf area, The inoculation of beneficial bacterial stem diameter and radical volume, they strains is recommended through the incision demonstrated that rhizospheric and endophytic and submersion of the plant tuber, in order that bacteria stimulate the emergence and growth of the plant is induced to systemic resistance, seedlings of this species. which can be used taking advantage of the stage of transplanting onions plants. Because the bacteria under study B. subtilis and B. amyloliquefaciens are producers Contribution of AIA, they activate ACC synthase, they favor the production of ACC, and therefore, of In Mexico, onion production has been affected ethylene, which inhibits root growth in large by the fungal diseases that affect this vegetable, quantities and in small amounts that favor the caused by the fungus S. cepivorum Berk., root, have an antagonistic effect against Causing large economic losses and a decrease pathogens (Tariq, 2019). in product quality, however, to combat this infection they are used agrochemicals that Many rhizobacteria show some of the affect the environment and pollute the soil. metabolic attributes that favor the growth and With this work, this problem can be solved development of plants, among which the using the rhizobacteria Bacillus subtilis and B. enzymatic activity of ACC deaminase, salinity amyloliqeifaciens as a biocontrol alternative for tolerance, AIA production and presence of the management of the S. cepivorum fungus, as siderophores are highlighted, as was well as the development and increase of onion demonstrated in previous studies by these quality (Allium cepa L.) to be sustainable and bacteruae and in the same way with Cell Free profitable for the producer. Extracts. Rhizobacteria have been shown to produce AIA from the presence of L-tryptophan References (Lavenus and Cols, 2013); this in turn activates the enzyme ACC synthetase that favors the Al-Hussini, H. S., Al-Rawahi, A. Y., Al- production of ACC and therefore regulates Marhoon, A. A., Al-Abri, S. A., Al-Mahmooli, ethylene levels, which benefits the development I. H., Al-Sadi, A. M., & Velazhahan, R. 2019. of the root (Angulo et al., (2014). Therefore, the Biological control of damping-off of tomato rhizobacteria of the genus Bacillus It is a caused by Pythium aphanidermatum by using natural alternative against diseases that affect native antagonistic rhizobacteria isolated from onion crops, such as growth promoters because Omani soil. Journal of Plant Pathology. of the benefits they have to improve the health 101(2):315-322. of these plants. Arshad, M. y W.T. Frankenberger Jr. 1998. Conclusions Plant growthregulating substances in the rhizosphere: Microbial production and Rhizobacteria showed an antagonistic effect functions. Advances in Agronomy. 62:45-151. against the fungus and stimulated fresh weight, root length, stem and bulb in onion plants. These results reflected that the application of rhizobacteria individually induces better plant development compared to a consortium. ISSN 2444-4936 OCEGUEDA-REYES, Martha Delia, CASAS-SOLÍS, Josefina, ECORFAN® All rights reserved GONZÁLEZ-EGUIARTE, Diego R. and VIRGEN-CALLEROS, Gil. Rhizobacteria antagonistic to the fungus Sclerotium cepivorum (Berk.t) in onion plants (Allium cepa L.). Journal of Environmental Sciences and

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Arjona-López, J. M., Tienda, S., Arjona- Colina, C. C. 2017. Evaluación de las Girona, I., Cazorla, F. M., & López-Herrera, C. propiedades iniinflamatorias, antiinflamatorias, J. 2019. Combination of low concentrations of antioxidantes e hipolipidémicas de cebolla fluazinam and antagonistic rhizobacteria to procesada como ingrediente funcional in vitro y control avocado white root rot. Biological en un modelo animal.Tesis doctoral. Control. 103996. Universidad Complutense. Madrid. España. 235p. Angulo-Castro, A., Ferrera-Cerrato, R., https://eprints.ucm.es/46542/1/T39618.pdf. Alarcón, A., Almaraz-Suárez, J.J., Delgadillo- Martínez, J., Jiménez Fernández, M., Garcia- Corrales-García, L. L., y Ciro, G. L. 2010. Barradas, O. 2018. Crecimiento y eficiencia Péptidos con actividad antimicrobiana fotoquímica del fotosistema II en plántulas de 2 producidos por microorganismos nativos. varidades de Capsicum annuum L. Revista Revista de la Facultad de Química Argentina Microbiologica. 50(2):178-188. Farmacéutica. Universidad de Antioquia, Medellín, Colombia. 17(2):181-190. Angulo, C. V., Sanfuentes, A. E., Rodríguez, F., Sossa, E.K., 2014. Caracterización de Crowe, F.J., Hall, D.H. 1980. Soil temperature rizobacterias promotoras de crecimiento de and moisture effects on sclerotium germination plántulas de Eucalyptus nitens. Asociación and infection of onion seedlings by Sclerotium Argentina de microbiología. 46(4):338-347. cepivorum. Phytopathology.70:74-78.

Alvarado-Capo, Y., Leiva-Mora, M., Da Silva, S. R., Moutinho, L. Brenda., Dos CruzMartín M., Mena, E., Acosta-Suárez, M., Santos, R. D., Vasconcelos-Rodrigues, I. S., Roque, B., Pichardo, T., García-Aguila, L., Talamini, V., Fernandes, F. M., Fernades, M. Jiménez-Terry, F., Hurtado, O., Veitia, N., R.P. 2018. Using antagonistic soil bacteria anda Padrón, L. 2015. Efecto de Bacillus spp. Sobre their cell-free filtrates to control the black rot el crecimiento y rendimiento agrícola de plantas pathogen Xanthomonas campestri pv. in vitro de papa cv. “Romano” en casa de campetris. Journa of Phytopathology. 166(7-8): cultivo. Biotecnología Vegetal. 15(2):115-122. 495-501.

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Natural Resources. 2019 17 Article Journal of Environmental Sciences and Natural Resources December 2019 Vol.5 No.16 17-25

Effectiveness of Humic Acids, Fulvic Acids and lechuguilla extract (Agave lechuguilla), as biosurfactants in the Remediation of soils contaminated with Hydrocarbons

Efectividad de los Ácidos Húmicos, Fúlvicos y extracto de lechuguilla (Agave lechuguilla), como agentes biosurfactantes en la Remediación de suelos contaminados con Hidrocarburos

RESENDIZ-VEGA, Marisol†* & GARCÍA-MELO, José Alberto

Universidad Tecnológica Tula – Tepejí, Chemistry Academic Corps

ID 1st Author: Marisol, Resendiz-Vega / ORC ID: 0000-0001-8199-6548, Researcher ID Thomson: T-7946-2018, CVU CONACYT ID: 819939

ID 1st Coauthor: José Alberto, García-Melo / ORC ID: 0000-003-0340-0272, CVU CONACYUT ID: 285374

DOI: 10.35429/JESN.2019.16.5.17.25 Received June 26, 2019; Accepted December 30, 2019

Abstract Resumen

The environmental pollution caused by oil and its derivatives is La contaminación ambiental ocasionada por el petróleo y sus recognized as one of the most serious problems. Once spilled derivados se reconoce como uno de los más graves problemas. on the ground, most aliphatic compounds volatilize, while other Una vez derramado en el suelo, la mayoría de los compuestos hydrocarbons such as polycyclics persist on the surface causing alifáticos se volatilizan, mientras que otros hidrocarburos como an impact on the environment and living beings. There are a los policíclicos persisten en la superficie causando un impacto variety of techniques for treatment, but they have the al ambiente y los seres vivos. Existe una diversidad de técnicas disadvantage of being expensive, so viable alternatives have para el tratamiento, pero tienen la desventaja de ser costosas, been sought such as bioremediation, which consist of making por lo que se han buscado alternativas viables como la use of microorganisms and constitutes a very competitive biorremediación, que consisten en hacer uso de technology, capable of achieving the biodegradation of microorganismos y constituye una tecnología muy competitiva, hydrocarbons contained in the soils. Depending on the capaz de conseguir la biodegradación de los hidrocarburos characteristics of the soil and the content of organic matter, the contenidos en los suelos. Dependiendo de las características del hydrocarbons of higher molecular weight and lower solubility suelo y del contenido de materia orgánica, los hidrocarburos de can adsorb in the micropores of the soil particles, resulting in mayor peso molecular y menor solubilidad pueden adsorberse this being inaccessible as carbon and energy sources for the en los microporos de las partículas del suelo, resultando con microorganisms. Therefore, surfactant agents that act by esto ser inaccesibles como fuentes de carbono y energía para los increasing bioavailability through the parallel action of the microorganismos. Por lo que se requieren agentes surfactantes desorption and solubilization of the contaminant are required. que actúan logrando incrementar la biodisponibilidad mediante This article compares 3 surfactant agents of natural origin, la acción paralela de la desorción y solubilización del which analyze and discuss the effectiveness of each of them for contaminante. En este artículo se comparan 3 agentes the treatment of soils contaminated with hydrocarbons. Once surfactantes de origen natural, que analizan y discuten la the lechuguilla extract is obtained in cold and hot, the soil is efectividad de cada uno de ellos para el tratamiento de suelos washed, fats and oils are determined by soxhlet method and the contaminados con hidrocarburos. Una vez obtenido el extracto Chemical Oxygen Demand (COD) is determined, finding a de lechuguilla en frío y caliente, se realiza el lavado de suelo, se marked effectiveness of the cold lechuguilla extract (Agave determinan grasas y aceites por método soxhlet y determina la lechuguilla). Demanda Química de Oxígeno (DQO), Encontrando una marcada efectividad del extracto de lechuguilla frío (Agave lechuguilla).

Bioremediation, Hydrocarbons, Surfactant Agent Biorremediación, Hidrocarburos, Agente Surfactante

Citation: RESENDIZ-VEGA, Marisol & GARCÍA-MELO, José Alberto. Effectiveness of Humic Acids, Fulvic Acids and lechuguilla extract (Agave lechuguilla), as biosurfactants in the Remediation of soils contaminated with Hydrocarbons. Journal of Environmental Sciences and Natural Resources. 2019, 5-16: 17-25

* Correspondence to Author (email: [email protected]) † Researcher contributing first author.

18 Article Journal of Environmental Sciences and Natural Resources December 2019 Vol.5 No.16 17-25

Introduction The contamination of the soil leads to the loss of the economic, cultural and Petroleum products are currently the most environmental values associated with the use of demanded for power generation, this has the land, for example, if the soil of a property is resulted in an increasing number of contaminated, the monetary value of these sites, contaminated soils due to spills. Rojas Molina, the local competitiveness of the area where they N. A. (2019) points out that the soils have been are located decreases, as does the quality of life turned into heavy metal sinks and other and economic activity and sustainable pollutants. This, together with the growth of the development are inhibited. population worldwide, causes great pressure on the availability of resources and land (Vreysen Not protecting the soil also means the and Maes, 2005). disappearance of environmental services, for example, to constitute the filter for the recharge Environmental pollution caused by oil of aquifers (the supply of drinking water is and its derivatives is recognized as one of the jeopardized) or to allow recreational activities most serious problems, especially when (loss of social and cultural value), (Cubero- associated with large-scale accidental spills Mata, AV (2019). (Plohl K, and Leskovsek H., 2002). One of the problems presented in the Once spilled on the ground, most methods of remediation of soils contaminated aliphatic compounds volatilize, while other with hydrocarbons is related to the hydrophobic hydrocarbons such as polycyclics persist on the nature of hydrocarbons, their low water surface (Jiménez DJ, Medina SA, Gracida JN., solubility and high adhesion to soil molecules, 2010), causing an impact on the environment are two reasons why it is necessary to increase considering their toxic effects and for living the bioavailability of these so that they are more beings (Tissot B and Welte DH., 1984). The accessible to the microorganisms responsible most studied hydrocarbons are benzene, for their degradation (Petro et al., 2014). toluene, ethylbenzene and xylene also grouped Surfactants, which are amphipathic molecules under the BTEX and polyaromatic (HAP) that have the ability to interact with appeal, naphthalene diaromatic and hydrophobic and hydrophilic compounds, being phenanthrene, anthracene and fluorene located at their interface. triaromatics (Kästner M and Mahro B.1996). However, the use of chemical According to Paul EA and Clark FE. surfactants adds an extra contaminant (1998), the extraction of hydrocarbons by (Bognolo, 1999), so the use of biosurfactants vacuum, washing of soil contaminated with would be more effective, since they have low water, incineration and electrokinetic recovery toxicity, biodegradability, biocompatibility, are among others some of the most used they are environmentally friendly and equally techniques for soil treatment. These techniques effective bioproducts in the solubilization of have the disadvantage of being expensive, so pollutants and being biodegradable the changes viable, correct, simple and economical in the structure of the treated soil are minor alternatives have been sought; and (Raiger et al., 2009). bioremediation techniques have been used, which consist of making use of microorganisms Among the microorganisms that have or plants (Bollag, J.-M. 1992), and constitutes a most commonly been isolated in soils very competitive technology, capable of contaminated with hydrocarbons are the genera: achieving the biodegradation of hydrocarbons Arthrobacter, Bacillus, Pseudomonas, contained in soils. Agrobacterium, Alcaligenes, Flavobacterium, Corynebacterium, Micrococcus, It is important to have mechanisms and Taphylococcus, Xanthomonas and techniques economically and environmentally Mycobacterium, with a total population of 1% sustainable soil remediation since pollution approximately 104 to 106 cells per gram of soil. decreases arable and productive land, thus Soils contaminated with hydrocarbons contain hindering sustainable economic development. more microorganisms than unpolluted soils, but their diversity is reduced (Messarch M and Nies L. (1997).

ISSN 2444-4936 RESENDIZ-VEGA, Marisol & GARCÍA-MELO, José Alberto. ECORFAN ® All rights reserved Effectiveness of Humic Acids, Fulvic Acids and lechuguilla extract (Agave lechuguilla), as biosurfactants in the Remediation of soils contaminated with Hydrocarbons. Journal of Environmental Sciences

and Natural Resources. 2019 19 Article Journal of Environmental Sciences and Natural Resources December 2019 Vol.5 No.16 17-25

Contaminants present in the non- 2. Obtain Humic Acids (AHs) from aqueous liquid phase (NAPL) or absorbed in humus, obtained from vermicompost. the soil matrix are usually not bioavailable, so 3. Obtain Fulvic Acids (AF) from humus, the degradation rate is often limited (Aronstein obtained from vermicompost. BN and Alexander M., 1992). Depending on 4. Obtain extract of Agave lechuguilla the characteristics of the soil and the content of Torrey by cold method (Ambient organic matter, hydrocarbons of greater temperature 20 ° C) and hot (50 ° C). molecular weight and lower solubility can be 5. Wash contaminated soil with absorbed in the micropores of soil particles, hydrocarbons. resulting in being inaccessible as sources of 6. Monitor the effectiveness of removal by carbon and energy for microorganisms. comparing the fat and oil content, before and after washing. 7. Monitor the effectiveness of the removal Therefore, in most cases, surfactants are by measuring the Chemical Oxygen required that act by increasing the Demand (COD) of the biosurfactant bioavailability through the parallel action of solutions used in the washing of each desorption and solubilization of the soil sample. contaminant (Singh A, et. Al. 2007, Helmy Q., 8. Analyze advantages and disadvantages et. Al. 2009) . To choose a surfactant for of each surfactant. bioremediation, the type of pollutant to be remediated, the properties of the soil, as well as Theoretical framework the properties of the surfactant itself must be taken into account, and finally the existence of Soil is defined as that unconsolidated material microorganisms that degrade the pollutant to be composed of inorganic particles, organic remedied (Volkering F, 1998). matter, water, air and organisms, which comprises from the upper layer of the earth's Surfactants are essential for the surface to different levels of depth (NOM-138- bioremediation process. Some microorganisms SEMARNAT / SS-2003). Maintains ecological produce their own surfactant to solubilize systems, since they provide chemical and hydrophobic organic compounds (Lange S and mineral components (as a result of Warger F., 1987)). This article compares 3 biodegradation); and organic complexes such as surfactants of natural origin, which analyze and humic and fulvic acids, enzymes, vitamins, discuss the effectiveness of each of them for the hormones and antibiotics; They also have a rich treatment of soils contaminated with genetic reserve. The soil constitutes a natural hydrocarbons. resource that performs various functions on the Earth's surface, providing mechanical support, Hypothesis as well as nutrients for the growth of plants and micro-organisms. The working hypothesis states that the biosurfactant extracted from Agave lechuguilla A contaminated soil can be defined as Torrey, is as effective as humic and fulvic that in which hydrocarbons or foreign materials acids. are present that, due to their quantities and characteristics, affect the nature of the soil. Objectives Land degradation can be defined as any process that decreases the current and potential capacity General of the soil to produce, quantitatively and qualitatively, goods and services. Although it To evaluate the effect of the extract of Agave can be caused by natural causes, soil lechuguilla torrey, humic acids and fulvic acids degradation is primarily the direct consequence as biosurfactant agents, in the washing of soils of man-made anthropogenic activities. The soil contaminated with hydrocarbons represents an ecosystem where, currently, a wide variety of toxic compounds can be found, Specific: including hydrocarbons derived from oil activities. It is considered a soil that should be 1. Obtain soil from the surroundings of the remedied that exceeds the limits established in Miguel Hidalgo Refinery and determine NOM-038-SEMARNAT-2003 (See table 1). its fat and oil content. ISSN 2444-4936 RESENDIZ-VEGA, Marisol & GARCÍA-MELO, José Alberto. ECORFAN ® All rights reserved Effectiveness of Humic Acids, Fulvic Acids and lechuguilla extract (Agave lechuguilla), as biosurfactants in the Remediation of soils contaminated with Hydrocarbons. Journal of Environmental Sciences

and Natural Resources. 2019 20 Article Journal of Environmental Sciences and Natural Resources December 2019 Vol.5 No.16 17-25

Table 1. Maximum permissible limits The surfactants are preferably located for hydrocarbon fractions in soil. on a surface or interface, which constitutes a hydrophilic part that makes the surfactant water Hydrocarbon Land use predominant (mg / kg dry soluble and another hydrophobic part Fraction base) (Volkering et al, 1998). Agricultural Residential Industrial Light 200 200 500 Median 1200 1200 5000 Humus: Heavy 3000 3000 6000 Conceptually the organic component of the soil Table 1 Maximum limits of hydrocarbons in soil can be defined as a set of living and dead according to their use to define if they need to be organic matter. In the dead organic matter the remedied. Source NOM-138-SEMARNAT / SS-2003 unaltered material can be differentiated, in which the morphology of the original material Remediation. It is the action of still exists, while the alteration of the remedying or working on the site, including transformed products is also called humus. digging in the mud or mud and underlying Generally soil humus is defined as a mixture of soils. Soil remediation can be divided into two dark, colloidal organic polydispersed large groups: compounds with high molecular weight and relatively resistant to decomposition (Ayuso et a) Ex situ: When the soil is excavated and al., 1996). washed outside the site where it was contaminated. In general, humus distinguishes between b) In situ: The term in situ comes from non-humic and humic substances. Non-humic Latin and means “in its original substances comprise compounds that belong to location”. biochemistry classes, such as amino acids, proteins, carbohydrates, lipids, lignin, nucleic The treatment is carried out below the acids, hormones, pigments and A variety of surface. When digging is not required. It organic acids. Humic substances are subdivided consists of the injection of washing solutions into fulvic acids (See image 2), humic acids (surfactants) to the subsoil, under controlled (See image 1) and residual humines; humines conditions in order to remove contaminants are the insoluble fraction of humic substances, adsorbed to the soil matrix. The washing of and humic acids the soluble fraction in alkaline soils in situ is a treatment that can be in situ media and acidic media at pH> 2 although not which consists of flooding the contaminated in strong acidic conditions pH <2 and fulvic soils with a solution that transports the acids is the soluble part in alkaline conditions contaminants to a determined and localized area and acids (Hayes et al., 1989). where they can be eliminated. Schulten and Leinweber in 1991, Thus, pollutants are extracted from the proposed that Humic Acids (AHs) consist of soil by passing water or other solutions isolated aromatic rings covalently linked by (surfactants) through an injection or infiltration aliphatic chains. Oxygen is present in the form system. Surfactants are substances whose of carboxyls, phenolic and alcoholic hydroxyls, molecules have both a polar group and a esters and ketones, while nitrogen is produced nonpolar group. The polar group is in general a in nitriles and heterocyclic structures. The functional group that contains heterotherms resulting carbon skeleton shows high such as O, S, N or P, if in the surfactant the microporosity with holes of various polar group is ionic with negative charge dimensions, which can trap and join other (anion) and a metal cation is called an anionic organic and inorganic soil elements, as well as surfactant, if on the contrary it has positive water. According to Cruz-Narvaez Y (2019), charge then it is a cationic, there are those that the degradation of hydrocarbons improves with do not have ionic charge to the latter are known the use of humic acids in treatment with as non-ionic surfactant (Volkering et al, 1998). bacteria. Therefore, the differences between the types of surfactants are related to the ionic charge.

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Saponosides, also known as saponins, are heterosides that consist of a glycosidic part with one or more sugars being more frequent glucose, xylose, arabinose, rhamnosa or glucoronic acid that bind to saponin (also called genic aglyconeo) through bonding glycosidic Depending on its chemical nature, sapogenin can be steroidal, steroid or triphenic glycoalkaloid. Hernández ´ et al. They report the extraction of saponins from agave extracts among them (Agave lechuguilla), from 0.1 to 1.3% dry base, establishing for this species, that only the temperature factor influences the performance of saponin extraction from foliar material. Saponins constitute a diverse group of compounds widely distributed in the plant kingdom, which are characterized by their physicochemical properties (surfactants). Obtaining extracts from ´ foliar material of Figure 1 Hypothetical 2D representation of humic acids. some agaváceas (Agave lechuguilla Torrey), for López-Salazar R., (2014) which the presence of esmilagenina (steroidal sapogenin) is reported, in addition to 8 ´ Fulvic Acids saponins plus: yucagenine, phytogenin, hecogenin, ´ tigogenin, diosgenin, gentrogenin, chlorogenin and ruizgenin (Hernández ´ et al., 2005).

Figure 2 Hypothetical 2D representation of fulvic acids. López-Salazar R., (2014) Figure 3 Classification of saponins Recovered from: Monterrosa-Brisson N. (2013) Saponins Materials and methods Agave lechuguilla Torrey is a plant that belongs to the Agavaceae family, grows in arid areas, in The study approach is quantitative experimental limestone and rocky terrain. It is exploited to descriptive since it is sought to compare and obtain fiber, known in Mexico as ixtle, which is analyze advantages and disadvantages of 3 widely used in the brush manufacturing and surfactants of natural origin. construction industries due to its abrasive characteristics and high water retention rate (65 Origin and sampling of the soil simple percent). The fiber is obtained by carving the sheet consisting of 15 percent fiber and 85 The samples were taken from soils percent pulp. The pulp contains bioactive contaminated with hydrocarbons in the compounds among which saponins with several surroundings of the Miguel Hidalgo Refinery, properties stand out. which were preserved in sterile amber bottles and processed within 24 hours of their collection / stored in refrigeration.

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Theoretical Methods

The methods used were those described in the Official Mexican Standards: Determination of pH in water (NMX-AA-008-SCFI-2011), and for COD the EPA 5220 D method; APHA, 1989. NMX-AA-005-SCFI-2013-Water analysis - measurement of fats and oils

Obtaining hot and cold extract of lechuguilla (Agave lechuguilla), extraction of humic and fulvic acids and washing of soil contaminated with hydrocarbons

At this stage the following materials were used: Figure 4 Lechuguilla extract (Agave lechuguilla), obtained cold (left) and hot (right) 250ml Pyrex brand graduated cylinder, knob,

500ml beakers (Kimax), Potentiometer, Soil wash: Blender, Conductometer, heaters with stirring, handles, Refrigerants. And the following 4 triplicate washes were performed: procedures were followed:

Washing with humic acids, fulvic acids, Cold extract lechiguilla extract obtained by room temperature method (cold method) and hot A fraction of lettuce of 220 grams is cut, the obtained lechuguilla extract thorns are washed and removed, cut into small segments and ground in a blender adding 240ml Sieve contaminated soil with sieve of de-ionized water, passed through a sieve to number 18 and 10, Weigh 10 grams of the remove solid waste sample for each beaker, 50 ml of the corresponding surfactant solution was added, it Hot extraction is stirred for a period of 2 hours. The washed soil and the residual solution are filtered and In the flat bottom ball flask 250gr of lettuce and recovered separately, labeled for fat and oil 240 ml of de-ionized water are added. It is determination and Chemical Oxygen Demand placed in a closed reflux system for 2hr. The (COD). extract is passed through a sieve to remove waste. Likewise, a triplicate series was used in

each case of a wash target with uncontaminated Obtaining Humic and Fulvic Acids: soil and a wash with deionized water.

Determination of fats and oils in the soil; before and after washing

Bring the flask to constant weight. Weigh the sample and place it in the cartridge, Install the Soxhlet, add 185ml of hexane, perform the extraction for 4hr, once it is finished, recover the hexane by distillation, let it cool inside a Figure 1 Method for obtaining humic and fulvic acids desiccator and obtain the amount of fats and Source: Self Made oils by difference of weight.

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Figure 5 Fats and oils extraction, soxhlet method, Figure 6 Hexane recovery, Figure 7 Extracted hydrocarbons

Ecuación: y= a +b2x Intercept= -0.01493 Teflon cap glass tubes were used, to Stope 4.68E-4 Standard Error=0.01141 which 2.5ml of sample, 1.5ml of digester Stope= 1.86739E-5 solution (10.216g of K2Cr2O7 + 33.3 HgSO4 and 167ml of H2SO4) and 3.5ml of sulfuric acid solution (10.142g of H2SO4 Ag2SO4), once prepared the samples are digested within a heating block at 150 ° C for 2 hours.

Subsequently, allow the samples to cool to Graphic 1 COD Calibration Curve room temperature and the absorbance is read at Source: Own Elaboration 600 nm. (EPA 5220 D; APHA, 1989). E From the graph the formula was defined Results to determine the concentration where:

COD values are obtained from a calibration curve performed with potassium biftalate (0- Gráfica 2: DQO de agentes biosurfactantes (mgO2/l) 681mg / l, equivalent to 0-800mgO2 / l). (See Graph 1). The COD values of biosurfactant agents are shown in graph 2, in which we can 2988 observe that humic acid is the one with the 2325 lowest content of organic matter 842 mgO2 / l 2247 and cold lettuce extract is the one that contains the highest amount 2988mgO2 / l, this is DQO mgO2/l because the extract is still filtered; remains of lechuguilla itself pass to it; however, it is within 842 the range of hot lettuce and fulvic acids, water 0 was used as white. ÁCIDOS HÚMICOS ÁCIDOS FULVICOS LECHIGUILLA CALIENTE LECHUGUILLA FRÍA AGUA DESIONIZADA Biosurfactante Once the soil was washed with the surfactants, we see in graph 3 that, although Graphic 2 Chemical demand of the biosurfactants the 4 agents tested work properly, there is a obtained greater removal of fats and oils in the case of Source: Self Made the soil washed with cold lettuce extract, every time that a greater amount of organic matter Gráfica 3: DQO de la solución del biosurfactante, después del lavado. passed to the aqueous extract. We could prove it by observing that the COD increased considerably from 2988mgO2 / l to 10612mgO2 / l (See graph 4). It is also the simplest and most economical process since in 10353 10612 this case obtaining lechuguilla extract does not DQO mgO2/l require heating.

3406

615 400 ÁCIDOS HÚMICOS 0.02G/500ML ÁCIDOS FULVICOS LECHIGUILLA CALIENTE LECHUGUILLA FRÍA AGUA DESIONIZADA Solución de lavado

Graphic 3 Chemical demand of the biosurfactant solution: after washing the soil contaminated with hydrocarbons Source: Self Made ISSN 2444-4936 RESENDIZ-VEGA, Marisol & GARCÍA-MELO, José Alberto. ECORFAN ® All rights reserved Effectiveness of Humic Acids, Fulvic Acids and lechuguilla extract (Agave lechuguilla), as biosurfactants in the Remediation of soils contaminated with Hydrocarbons. Journal of Environmental Sciences

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Conclusions Gráfica 4: DQO antes y despues del lavado

Agave lechuguilla Torrey contains in the pulp 14000 bioactive compounds among which saponins

12000 with surfactant properties stand out, which can be used in soil bioremediation processes 10000 10612 contaminated with hydrocarbons. 8000 10353

6000 The extract of Agave lechuguilla Torrey DQO mgO2/l

4000 obtained at room temperature turned out to have 3406 615 a better surfactant effect than the extract 2988 2000 2325 2247 842 obtained with heating. According to Hernández 4000 0 et al. (2005), Agave lechuguilla Torrey contains Ácidos Húmicos Ácidos Fulvicos Lechiguilla Caliente Lechuguilla Fría Agua Desionizada Biosurfactantes 9 saponins (esmilagenina, yucagenina, gitogenina, hecogenina, tigogenina, diosgenina, gentrogenina, chlorogenina and ruizgenina), Graphic 4 that have surfactant effect.

Once the soil was washed, fats and oils A better option for the washing of soils were determined by finding a lower amount in contaminated with hydrocarbons was the the soil washed with lechuguilla extract extract of Agave lechuguilla Torrey, compared obtained at room temperature (See graph 4). to the hot extract and the solution of humic and The amount of fat decreased in the case of the fulvic acids. The extract obtained at room soil washed with a humic acid solution from temperature was able to remove the 694.7 g / kg to 178g / kg, in the case of the soil hydrocarbons since the COD of the extract washed with a solution of fulvic acids it passed from 2988mgO2 / l to 10612mgO2 / l decreased from 694.7 g / kg to 155.5 g / kg , in when washing the soil and the soil was finally the case of hot lettuce extract the decrease was with 65.6 g / Kg of fats and oils. from 694.7 g / kg to 139.2g / kg and in the case of washing with the extract of lettuce obtained Finally, the treatment of the extract will at room temperature (cold lettuce) the decrease be necessary to degrade the hydrocarbons that was from 694.7g / kg to 65.6g / kg A soil were removed from the soil. sample was also taken that was considered uncontaminated within the University campus References and yet we found that it contained 145.8 g / kg, this may be due to herbicides that are used in Aronstein BN and Alexander M. (1992). garden areas or contaminated irrigation waters "Surfactants at Low Concentrations Stimulate (See Graphic 5) Biodegradation of Sorbed Hydrocarbons in Samples of Aquifer Sands and Soil Gráfica 5: Grasas y aceites remanentes en el suelo lavado Slurries,"Environ. Toxicol. Chem. 11, 1227- 800 1233. 694.7 700

600 Bollag, J.M. (1992). Decontaminating soil with enzymes: An in situ method using phenolic and 500 anilinic compounds. Environ. Sci. Technol. 400 26:1876–1881.

300 GrasasAceites g/Kgy 200 178 145.8 155.5 139.2 Cubero-Mata, A. V. (2019). Identificación del

100 65.6 peligro por contaminación de metales pesados y

0 bacterias patógenas en los sedimentos del Suelo no contaminado Suelo contaminado Suelo lavado con Ácidos Suelo lavado con Ácidos Suelo lavado con extracto de Suelo lavado con extracto de Húmicos Fúlvicos lechuguilla caliente lechuguilla fría embalse de Central Hidroeléctrica Platanar de Muestras de suelo COOPELESCA RL, Costa Rica.

Graphic 5 Fat remaining in the soil washed with the Cruz Narváez, Y. (2019). Construcción de un different surfactants: Humic acids, Fulvic acids, Hot and biorreactor con bacterias inmovilizadas para la cold Lechuguilla extract biodegradación de hidrocarburos.

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Helmy Q, Kardena E, Wisjnuprapto. (2009). Rojas Molina, N. A. (2019). Validación de una Performance of petrofilic consortia and effect metodología para la determinación de benceno of Surfactant tween 80 addition in the oil sludge en suelos mediante HS-GC-FID y su aplicación removal Process. J. of Applied Sciences in en biorremediación en suelos co-contaminados Environ. Sanitation, 4 (3): 207-218. con Hg (ii).

Jiménez DJ, Medina SA, Gracida JN. (2010). Secretaría de Medio Ambiente y Recursos Propiedades, aplicaciones y producción de Naturales (SEMARNAT), 2012, Norma Oficial biotensoactivos. Int. Contam. Ambient. 26 (1) Mexicana que establece los límites máximos 65-84. permisibles de hidrocarburos en suelos y las especificaciones para su caracterización y Kästner M and Mahro B. (1996). Microbial remediación (NOM-138-SEMARNAT/SS- degradation of polycyclic aromatic 2003). México, D.F. Diario Oficial de la hydrocarbons in soils affected by the organic Federación, 29 de marzo de 2003, 21 matrix of compost. Appl Microbiol Biotechnol. Recuperado 44:668–675. de http://www.dof.gob.mx/nota_detalle.php?co digo=5313544&fecha=10/09/2013 Lange S and Warger F. (1987). Structure and Properties of Biosurfactants. In Biosurfactants Singh A, Van Hamme JD, Ward O. (2007). and Biotechnology, Marcel Dekker Inc., N.Y Surfactants in microbiology and biotechnology: Part 2. Biotechnology Advances, 25. 99 – 121. López-Salazar R., (2014). Efecto de un humato Tissot B, Welte DH. (1984). Petroleum de calcio y fulvato de hierro en la calidad y formation and occurrence. New York USA: producción de chile jalapeño (capsicum annum Sppringer-Verlang. l.) y piquin (capsicum annum l. aviculare) y en la porosidad en un suelo calcisol. (Tesis Volkering F, Breure AM, Rulkens WH. (1998). Doctoral). Universidad Autónoma de Nuevo Microbiological aspects of surfactant use for León, Nuevo León Monterrey. biological soil remediation. Biodegradation, 8. 401–417. Messarch M and Nies L. (1997). Modification of heterothropic plate counts for assessing the bioremediation potencial of petroleum contaminated soils. Environ. Technol. 18, 639- 646.

Monterrosa-Brisson N. (2013), Evaluación de especies de Agavesobre los procesos fisiopatológicos asociados al Alzheimer, (Tesis Doctoral), Instituto politécnico Nacional, Ciudad de México.

Paul, E.A. and Clark, F.E. (1989) Soil Microbiology and Biochemistry, Academic Press, San Diego.

Paul EA, Clark FE. (1998), Soil microbiology and biochemistry, Academic Press, San Diego. Plohl K, and Leskovsek H. (2002). Biological degradation of motor oil in water. Acta Chim. Slov., 49, 279-289

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Physicochemical and antioxidant characteristics of different sprouts

Características fisicoquímicas y antioxidantes de diferentes germinados

REYES-MUNGUIA, Abigail†*, CARRILLO-INUNGARAY, María Luisa, GARCÍA-BARRÓN, Gabriela del Socorro and TURRUBIATES-GARAY, Karla Alicia

Universidad Autónoma de San Luis Potosí, Unidad Académica Multidisciplinaria Zona Huasteca. Romualdo del Campo 501, 79060 Cd. Valles S.L.P.

ID 1er Autor: Abigail, Reyes-Munguia / ORC ID: 0000-0002-2151-7979, CVU CONACYT ID: 46743

ID 1er Coautor: María Luisa, Carrillo-Inungaray / ORC ID: 0000-0002-0636-0141, CVU CONACYT ID: 53406

ID 2nd Coauthor: Gabriela Del Socorro, García-Barrón

ID 3rd Coauthor: Karla Alicia, Turrubiates-Garay

DOI: 10.35429/JESN.2019.16.5.26.29 Received June 26, 2019; Accepted December 30, 2019

Abstract Resumen

In the germination of seeds an increase in their En la germinación de semillas se ha observado un nutritional value has been observed, such as the incremento en su valor nutritivo, como el aumento increase in protein and micronutrients. It has been de proteína y micronutrientes. Se ha encontrado que found that continuous intake of some types of la ingesta continua de algunos tipos de germinados sprouts helps prevent certain chronic diseases, such ayuda a prevenir ciertas enfermedades crónicas, as colon cancer, Alzheimer's, among others. The como el cáncer de colon, el alzheimer, entre otras. objective is to evaluate the physicochemical and El objetivo es evaluar las características antioxidant characteristics of alfalfa, chia, lentil and fisicoquímicas y antioxidante de los germinados de broccoli sprouts. For the seed germination process a alfalfa, chía, lenteja y brócoli. Para el proceso de container was placed with water and covered with a germinación de semillas se colocó un recipiente con cloth so that a firm surface remained and on this the agua y se cubrió con una tela de tal manera que seeds were placed. The seeds were sprayed with quedara una superficie firme y sobre esta se water twice a day until favorable growth was noted. colocaron las semillas. Las semillas se rociaban con Broccoli sprouts obtained the highest values of agua dos veces por día hasta notar crecimiento DPPH radical inhibition and polyphenol content. favorable. El germinado de brócoli obtuvo los The physicochemical and antioxidant characteristics valores más altos de inhibición al radical DPPH y el vary according to germination days. contenido de polifenoles. Las características fisicoquímicas y antioxidantes varían de acuerdo a los días germinación.

Sprouts, Health, Antioxidants Germinados, Salud, Antioxidants

Citation: REYES-MUNGUIA, Abigail. CARRILLO-INUNGARAY, María Luisa, GARCÍA-BARRÓN, Gabriela del Socorro and TURRUBIATES-GARAY, Karla Alicia. Physicochemical and antioxidant characteristics of different sprouts. Journal of Environmental Sciences and Natural Resources. 2019, 5-16: 26-29

* Correspondence to Author (email: [email protected]) † Researcher contributing first author.

27 Article Journal of Environmental Sciences and Natural Resources December 2019 Vol.5 No.16 26-29

Introduction It has been found that the continuous intake of some types of sprouts such as The diet consists of a set of practices, customs, sprouted brown rice which helps prevent certain ideologies and availability of food for chronic diseases, such as colon cancer, individuals, during any stage of life nutrients Alzheimer's, due to its higher content of y- will be required to ensure the nutritional needs aminobutyric acid (Park, 2015; Yodpitak et al., are met (Bergero, 2017), the nutritional needs 2019). Therefore, the objective of this work was of each individual found related to energy to evaluate the physicochemical and antioxidant expenditure, need for essential nutrients and characteristics in different seed sprouts such as limited by fat, cholesterol, salt and alcohol lentils, chia, broccoli and alfalfa. (Carcamo Vargas & Mena Bastías, 2006). It is necessary to choose habits of eating food that in Methodology addition to meeting these needs provide a sufficient content of antioxidants and fiber that Preparation of sprouts prevent cardiovascular and chronic degenerative diseases, these foods should Lentils (Lens culinaris), chia (Salvia hispanica), preferably be of plant origin (WHO / FAO, broccoli (Brassica oleracea var. Italica) and 2003). alfalfa (Medicago sativa) seeds were placed to germinate, for which a container with water was placed and covered with cloth in such a Seed sprouts and legumes are widely way that a firm surface will remain and on this used in oriental cuisine in hot dishes and salads. the seeds were placed. The seeds were sprayed Domestic germination is mainly carried out in with water twice a day until favorable growth underdeveloped countries due to its low cost was noted. and benefits such as activation of nutritional factors, increase dietary fiber, B vitamins and Preparation of extracts promotes greater mineral bioavailability (Valera, Torres, & Cova, 2018). In the Once the sprouts had considerable growth, they germination of cereal or leguminous seeds an were cut, weighed and crushed with the help of increase in their nutritional value has been a mortar and water added to a 1: 3 ratio, with observed, such as the increase in protein and the exception of alfalfa sprouts which was micronutrients (Barrón-Yáñez et al., 2009). prepared at a 1:10 solution of water, then the Some authors have compared the nutritional extracts were placed in a shaker at 200 rpm for value between sprouts and plants, such is the 30 minutes, and then placed in a centrifuge at case of broccoli where their nutrients in sprouts 3000 rpm for 30 minutes to obtain a less cloudy are 10 to 100 times more than adult plants extract. (Hinojosa-Dávalos et al., 2019). In addition, germination can significantly improve the total Physicochemical Determinations Humidity phenolic content, antioxidant activity, as well as the amount of phenolic acids and flavonoids Its determination was made using the method detected in the outbreaks which are higher in indicated by the AOAC. the sprouts than in non-germinated counterparts (Pajak, et al., 2019 ). Ash

The increase in flavonoids and Its determination was made using the method polyphenols give the plant a greater bioactive indicated by the AOAC. effect and are currently considered as desirable compounds in human food for their antioxidant Reducing and total sugars activity and more and more studies suggest that the exogenous consumption of antioxidants Its determination was made using the method from vegetables and fruits helps to prevent indicated by the AOAC. degenerative diseases since they exert various physiological effects in humans, such as in the Acidity prevention of oxidation due to the damage of low density lipids and lipoproteins (Coronado, Its determination was made with the AOAC et al., 2015; Yodpitak et al., 2019). method.

ISSN 2444-4936 REYES-MUNGUIA, Abigail. CARRILLO-INUNGARAY, María ECORFAN® All rights reserved Luisa, GARCÍA-BARRÓN, Gabriela del Socorro and TURRUBIATES- GARAY, Karla Alicia. Physicochemical and antioxidant characteristics of different sprouts. Journal of Environmental Sciences and Natural Resources. 2019 28 Article Journal of Environmental Sciences and Natural Resources December 2019 Vol.5 No.16 26-29

Antioxidant activity (DPPH radical) The readings were made at 510 nm absorbance on a UV / visible spectrophotometer The activity of the extracts was measured (Re et al., 1999). according to the methodology described by Brand-Williams et al. (1995) through the Results inhibition of the stable radical 2,2 diphenyl-1- pricrylhydracil (DPPH •) that has a violet The sprouts of the four different broccoli, chia, coloration with a maximum absorbance of 515 alfalfa and lentil seeds grew over a period of 3 nm and that once reacts with an antioxidant, it to 6 days, with the lentil being the fastest is reduced and its absorption to it disappears. growing. 2.9 ml of a methanolic solution of DPPH • was placed in a quartz cell and reacted with 0.1 ml of the aqueous extract of the sprouts. The mixture was allowed to react and the change in absorbance was monitored on a UV / visible spectrophotometer for a period of half an hour every 5 minutes. This test was performed on each germinated extract in triplicate.

The percentage of DPPH inhibition was calculated using the following equation:

% INH = [(Ac ‐ As) / Ac] x 100

The physicochemical characteristics of Where: each of the sprouts evaluated in this Ac = DPPH absorbance • before the reaction investigation are reported in Table 1. Of which As = absorbance of the DPPH mixture • with similar characteristics are observed among the sample. them.

Determination of polyphenols (Folin- Parameters Alfalfa Lentil Chia Broccoli ciocalteau) pH 6.23 6.6 6.01 6.17 Total solids (° Brix) 4.18 5.22 5.4 5.61 It was performed according to the Folin- Humidity (%) 9.2 8.5 7.4 7.9 Ciocalteau methodology (Singleton et al., Ashes (%) 4.34 5.71 5.16 5.21

1999). 1 ml of each sample of triplicate extracts Table 1 Physicochemical characteristics of alfalfa, lentil, was placed in a test tube, and 5 ml of diluted chia and broccoli sprouts reagent (1:10) of Folin-Ciocalteau was added, allowed to stand for 7 min and 4 ml of the Antioxidant activity solution was added of 7.5% sodium carbonate until a homogeneous mixture is achieved. The Table 2 shows the results of the antioxidant tubes were covered in order to protect them properties for sprouts, with broccoli sprouting from light and incubated for 2 hours at room which has higher values for the percentage of temperature. Absorbance readings were read at inhibition (63.49 ± 1.43%) and the total 740 nm on a UV / visible spectrophotometer. polyphenol content (76.41 ± 5.03 mq GAE / L) comparison to the lentils, chia and alfalfa Flavonoid Determination sprouts, shown in table 2.

5% Sodium nitrite (NaNO2) was prepared with Sprouted Radical Polyphenols distilled water, 10% Aluminum Chloride Inibition mq GAE / L % DPPH (AlCl3) with distilled water and 1M sodium Lentil 5.07 ± 0.64 22.17 ±0.11 hydroxide (NaOH). 250 µl of the extracts of the Chia 47.25 ± 4.8 23.79 ± 0.46 sprouts were used, then 75 µl of 5% NaNO2 Alfalfa 8.013 ± 0.75 28.73 ± 0.45 was added and allowed to stand for 5 minutes, Broccoli 63.49 ± 1.43 76.41 ± 5.03 GAE: Gallic acid equivalent 150 µl of 10% AlCl3 was added and kept at rest for 6 minutes and finally added 500 μl of 1M Table 2 Antioxidant properties of alfalfa, lentil, chia and NaOH plus 275 μl of distilled water. broccoli sprouts

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When germinating, the seeds increase OMS/FAO. (2003). DIETA, NUTRICIÓN Y the content of some nutrients with respect to the PREVENCIÓN DE ENFERMEDADES seeds from which they originate, such as CRÓNICAS. Ginebra . vitamins and assimilable minerals, as well as a considerable increase in enzyme levels. Pajak, P., Socha, R., Broniek, J., Królikowska, K., & Fortuna, T. (2019). Antioxidant Conclusions properties, phenolic and mineral composition of germinated chia, golden flax, evening primrose, The physicochemical and antioxidant phacelia and fenugreek. Food chemistry, 279, characteristics of the sprouts evaluated depend 69-76. on the germination conditions and days. Singleton, V. L., Orthofer, R., & Lamuela References Raventos, R. M. (1999). Analysis of total phenols and other oxidation substrates and Barrón-Yáñez, M., Villanueva-Verduzco, C., antioxidants by means of folinciocalteau García-Mateos, M., & Colinas-León, M. reagent. Methods in Enzymology, 199, 152-178. (2009). Valor nutricio y contenido de saponinas en germinados de huauzontle (Chenopodium Valera, D., Torres, A., & Cova, A. (2018). nuttalliae Saff.), Calabicita (Cucurbita pepo L.), Efecto del proceso de germinación de granos de Canola (Brassica napus L.), Amaranto Cajanuscajan en la composición nutricional, (Amaranthus leucocarpus S. Watson syn. ácidos grasos, antioxidantes y bioaccesibilidad hypochondriacus L.). Chapingo Serie mineral. Revista Chilena de Nutrición, 323-330. Horticultura, 15(3), 237-243. Yodpitak, S., Mahatheeranont, S., Boonyawan, Bergero, M. &. (2017). Los significados sobre D., Sookwong, P., Roytrakul, S., & Norkaew, la alimentación saludable de mujeres de un O. (2019). Cold plasma treatment to improve barrio periférico de la ciudad de Córdoba germination and enhance the bioactive (Argentina). Perspectivas En Nutrición phytochemical content of germinated brown Humana(2), 151. Obtenido de rice. Food chemistry, 289, 328-339. https://doi.org.creativaplus.uaslp.mx/10.17533/ udea.penh.v19n2a03

Brand Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of free radical method to evaluate antioxidant activity. Lebensmittel Wissenschaft and Technologie, 25-30.

Carcamo Vargas, G. I., & Mena Bastías, C. P. (2006). Alimentacion saludable. Horizontes Educacionales.

Coronado, M., Vega, S., Gutiérrez, R., Vázquez, M., & Radilla, C. (2015). Antioxidantes: perspectiva actual para la salud humana. Revista chilena de nutrición, 42(2), 206-212.

Hinojosa-Dávalos, J., Cardona-López, M., Gutiérrez-Lomelí, M., Barrera-Rodríguez, A., & Robles-García, M. (2019). Identificación del perfil fitoquímico y efecto del estrés lumínico sobre la capacidad antioxidante del germinado de brócoli en un dispositivo germinador rotatorio tipo tambor. Biotecnia, 31(3), 67-75.

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In vitro growth of Oncidium sphacelatum (Orchidaceae) in different concentrations of sucrose and nutrients

Crecimiento in vitro de Oncidium sphacelatum (Orchidaceae) en diferentes concentraciones de sacarosa y nutrientes

NAVARRETE-VALENCIA, Ana Luisa†*, RAMÍREZ-GUERRERO, Leobarda Guadalupe, SÁNCHEZ-MONTEÓN Ana Luisa and JIMÉNEZ-MEZA, Víctor Manuel

Universidad Autónoma de Nayarit, Unidad Académica de Agricultura

ID 1st Author: Ana Luisa, Navarrete-Valencia / ORC ID: 0000-0003-2976-7275

ID 1st Coauthor: Leobarda Guadalupe, Ramírez-Guerrero / ORC ID: 0000-0003-1764-477X, Research ID Thomson: S- 7947-2018, CVU CONACYT ID: 211448

ID 2nd Coauthor: Ana Luisa, Sánchez-Monteón / ORC ID: 0000-0003-3781-2400, CVU CONACYT ID: 44857

ID 3rd Coauthor: Víctor Manuel, Jiménez-Meza / ORC ID: 0000-0002-6925-9809, CVU CONACYT ID: 160315

DOI: 10.35429/JESN.2019.16.5.30.35 Received June 26, 2019; Accepted December 30, 2019

Abstract Resumen

The objective of the present work was to offer an in vitro El objetivo del presente trabajo tuvo por finalidad ofrecer propagation alternative for the Oncidium sphacelatum una alternativa de propagación in vitro para la orquídea orchid through the evaluation of the response to the crop in Oncidium sphacelatum por medio de la evaluación de la different concentrations of sucrose and the nutrient medium respuesta al cultivo en diferentes concentraciones de of Murashige and Skoog. Initially the seeds were sown in sacarosa y del medio nutritivo de Murashige y Skoog. this culture medium at 100% of their concentration with the Inicialmente las semillas se sembraron en este medio de addition of 3.0% sucrose, once formed the protocorms the cultivo al 100% de su concentración con la adición de 3.0% response to the application of different sucrose de sacarosa, una vez formados los protocormos se evaluó la concentrations was evaluated (3.0, 2.25, 1.5, 0.75 and 0%) respuesta a la aplicación de diferentes concentraciones de and of the nutrients of the culture medium (100, 75, 50, 25 sacarosa (3.0, 2.25, 1.5, 0.75 y 0%) y de los nutrientes del and 0%) under a completely randomized experimental medio de cultivo (100, 75, 50, 25 y 0%) bajo un diseño design of eight treatments with four repetitions. The analysis experimental completamente al azar de ocho tratamientos of variance showed statistical differences between con cuatro repeticiones. El análisis de varianza mostró treatments for the formation of buds in the protocorms, diferencias estadísticas entre tratamientos para la formación number of leaves per bud, plant height, root length and de brotes en los protocormos, número de hojas por brote, formation of pseudobulbs in the shoots. The highest altura de planta, longitud de raíz y formación de outbreak formation in the protoorms was observed in pseudobulbos en los brotes. La mayor formación de brotes treatments with 3.0% sucrose and nutrient concentrations at en los protocormos se observó en los tratamientos con 3.0% 50 or 25%. For the growth of the leaves and the root the de sacarosa y concentraciones de nutrientes al 50 ó 25%. concentration of 3.0% of sucrose was required combined Para el crecimiento de las hojas y la raíz se requirió la with the concentrations of nutrients at 25, 50 and 100%. The concentración de 3.0% de sacarosa combinada con las formation of pseudobulbs in the outbreaks only occurred in concentraciones de nutrientes al 25, 50 y 100%. La the treatment with 3.0% sucrose combined with the 25% formación de pseudobulbos en los brotes sólo se presentó en nutrient concentration. el tratamiento con 3.0% de sacarosa combinada con la concentración de nutrientes al 25%.

Orchid, in vitro nutrition, Sucrose Orquídea, nutrición in vitro, Sacarosa

Citation: NAVARRETE-VALENCIA, Ana Luisa, RAMÍREZ-GUERRERO, Leobarda Guadalupe, SÁNCHEZ- MONTEÓN Ana Luisa and JIMÉNEZ-MEZA, Víctor Manuel. In vitro growth of Oncidium sphacelatum (Orchidaceae) in different concentrations of sucrose and nutrients. Journal of Environmental Sciences and Natural Resources. 2019, 5-16: 30- 35

† Researcher contributing first author.

31 Article Journal of Environmental Sciences and Natural Resources December 2019 Vol.5 No.16 30-35

Introduction In Oncidium baueri, treatment with 40 gL-1 of sucrose at half the concentration of the The Orchidaceae family is one of the largest MS medium was the most efficient for families in the plant kingdom, worldwide vegetative growth and rooting (Sorace et al., between 25,000 and 30,000 species are 2008). According to Debegh (1991), sucrose is reported, represented with more than 725 an important component as a source of carbon genera (Dressler, 1981). In Mexico it is and energy. The experiments with the Diuris estimated that there are about 1,200 species, of longifolia orchid showed increases in rooting which 40% are endemic. Likewise, it is one of by increasing the concentration of sucrose from the families with the highest number of species 20 to 40 gL-1. in extinction and one of the most monitored within CITES (Miceli et al., 2009). In this research work, the Oncidium sphacelatum Lindley orchid was selected, it is Justification an epiphytic or lithophyte species, which usually forms colonies, its foliage is lush and Orchid seeds are among the smallest in the colorful, blooms from January to April, the plant kingdom, basically they consist of an inflorescence reaches up to 1.5 m long With embryo and the testa, lacking a nutrient reserve numerous small and slightly fragrant flowers, (Arditti and Abdul-Ghani, 2000). Therefore, for yellow with reddish brown spots in the middle their germination they require an obligatory basal part, it is distributed in Mexico, Belize, symbiotic relationship with mycorrhizal fungi Guatemala, El Salvador, Honduras and of the Rhizoctonia genus (Hadley, 1997; Suárez Venezuela (Miceli et al., 2009; Alayón, 2011). et al., 2006; Valadares et al., 2013). In orchids, the germination process begins when the seed is According to Mc. Vaugh (1985) hydrated and turns green, resulting in an develops in the oak and pine-oak forests from undifferentiated tissue called a protoorm, from 550 to 1700 m in the states of Jalisco, Nayarit which the seedling originates after going and Sinaloa; this author reported abundance of through five stages of development (Seaton and this species in the surroundings of Compostela Ramsay, 2005): (1) formation of protoorm, (2) in the state of Nayarit. appearance of rhizoids, (3) appearance of apical meristem, (4) development of the first leaf and Objective (5) appearance of the first root. From stage 5, the seedling is considered to be formed. The objective of the present work was to offer Knowing the in vitro germination process of an in vitro propagation alternative for the orchid seeds is essential to improve the Oncidium sphacelatum Lindley orchid by propagation protocols for these species evaluating the response to the crop in different (Bertolini et al., 2016). concentrations of sucrose and the nutrient medium of Murashige and Skoog. Problem Methodology to be developed Under conditions of in vitro culture, the composition of the culture medium for the The experimental work was carried out in the germination of the seeds and the growth of the Tissue Cultivation Laboratory of the Academic plants is very important, being generally Unit of Agriculture ’of the‘ Autonomous constituted by macro and micronutrients, University of Nayarit’. vitamins, amino acids, sucrose and a gelling agent (Sorace et al. , 2008). The nutritious 82-day-old plants (in a protoormal state) medium of Murashige and Skoog (1962) has of an average size of two to three millimeters in been used successfully in the in vitro culture of diameter were used, which were obtained from orchids (Billard et al., 2014). In works carried mature seeds germinated in vitro out with the Laelia cinnabarina orchid, asymptotically, in the MS culture medium satisfactory results have been obtained when (Murashige and Skoog, 1962). using this nutrient medium at half of its concentration (Stancato and Feria, 1996) or a third of its concentration with orchids of the genus Phalaenopsis (Bressan, et al., 1999).

ISSN 2444-4936 NAVARRETE-VALENCIA, Ana Luisa, RAMÍREZ-GUERRERO, ECORFAN® All rights reserved Leobarda Guadalupe, SÁNCHEZ-MONTEÓN Ana Luisa and JIMÉNEZ- MEZA, Víctor Manuel. In vitro growth of Oncidium sphacelatum (Orchidaceae) in different concentrations of sucrose and nutrients. Journal of Environmental Sciences and Natural Resources. 2019 32 Article Journal of Environmental Sciences and Natural Resources December 2019 Vol.5 No.16 30-35

This medium was supplemented with 30 Treatment Composition gL-1 sucrose, 8gL-1 agar, 100 mgL-1 T-1 distilled water gelled with agar myoinositol, 0.5 mgL-1 nicotinic acid, 0.5 mgL- T-2 25% MS with 7.5 gL-1 sucrose 1 -1 T-3 50% MS with 15 gL-1 sucrose, pyridoxine and 0.5 mgL thiamine, the pH T-4 75% MS with 22.5 gL-1 sucrose was adjusted to 5.7 with 0.1 N KOH. The T-5 100% MS with 30 gL-1 sucrose, culture medium was sterilized in an autoclave at T-6 75% MS with 30 gL-1 sucrose, 121 ° C and 1 kg cm2 of pressure for 15 T-7 50% MS with 30 gL-1 sucrose minutes and was distributed in 25 mL quantities T-8 25% MS with 30 gL-1 sucrose in glass bottles of 120 mL capacity. Table 1 Treatments under evaluation The sowing and disinfection of the capsule containing the seeds was carried out in Variables under study the horizontal laminar flow hood, placed in a 1.2% sodium hypochlorite solution for ten The variables studied at 115 days of culture in minutes, then three rinses were applied with these treatments were the following: sterile water, then the capsule was placed on a sterile paper napkin to absorb the surrounding Survival of protoorms or shoots water and make a cross-section to distribute the seeds on the surface of the jars with culture The count of protoorms or protoorms with medium, which were covered with two layers vegetative bud formation that showed green of transparent plastic film and fastened with a color in its different shades was performed and league of rubber to be taken to the incubation calculated in percentage with respect to the area. initial number of 20 protoorms per experimental unit. During all phases of the in vitro culture from seed to the formation of whole plants in Number of shoots the incubation area the temperature was maintained from 23 to 30 ° C, under a The number of protoorms with bud formation photoperiod of 16 hours, and were illuminated per experimental unit was counted. with fluorescent light lamps that provided a luminous intensity of 66 Wm-2. Number of leaves per bud

The seeds turned green from 20 days The number of leaves in the shoots per after sowing. To maintain the supply of water experimental unit was counted and and nutrients, they were transferred to fresh subsequently the average number of leaves per medium with the same composition after 40 shoot was obtained for statistical analysis. days of cultivation. The green seeds developed to the protoorm phase from 60 days after Plant height sowing, so the next change to fresh medium under different concentrations of sucrose and The plant height was determined by using a nutritive medium was planned. ruler and the unit of mediation established was in millimeters (mm). This was determined from Experimental design and experimental unit the base to the apex of the youngest leaf in the shoots per experimental unit and subsequently A completely randomized experimental the average height was obtained for statistical design with four repetitions was used, the analysis. treatments are shown in Table 1. Root length The experimental unit was formed by a flask containing a group of 20 plants (in a state The root length was determined by the use of a of proto-form of a size of 2 to 3 mm in ruler and the unit of mediation established was diameter). in millimeters (mm). This was determined from the base to the apex of the root of greater length in the shoots per experimental unit and subsequently the average length was obtained for statistical analysis.

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Pseudobulb formation Treatment Number of shoots Number of leaves per bud * * The number of protoorms with bud formation T-1 3.11 c 1.58 b T-2 3.38 c 1.96 a and pseudobulb per experimental unit was T-3 3.35 c 1.99 a counted. T-4 3.35 c 2.12 a T-5 3.99 b 1.87 a b Statistic analysis T-6 3.91 b 1.58 b T-7 4.52 a 1.72 b The data obtained from the variables for each of T-8 4.52 a 1.72 b the treatments were evaluated using an analysis of variance. For the variables number of shoots Table 2. Test of multiple comparisons of and number of leaves per bud, data means by the Tukey method (α = 0.05) for the transformation was performed using the square number of shoots and the number of leaves per root of the value of x + 0.05. shoot. * Means with the same letter are statistically equal. A means comparison test was also carried out according to the Tukey test (α = Treatment Plant height Root Length (mm) (mm) 0.05) with the support of EXCEL® statistical T-1 7.25 c 0.00 d tools. T-2 10.00 b 2.00 c d T-3 10.75 b 2.75 c d Results T-4 13.00 b 4.25 c T-5 17.75 a 12.00 a After 197 days of in vitro culture, 100% T-6 9.00 c 13.75 c T-7 15.00 a 8.00 b survival of both protoorms and outbreaks was T-8 15.00 a 10.00 a b observed in all treatments under evaluation. Table 3. Test of multiple comparisons of The analysis of variance for the means by the Tukey method (α = 0.05) for plant variables number of shoots, number of leaves height and root length. * Means with the same per bud, plant height and root length showed letter are statistically equal. highly significant statistical differences (P <0.01) between treatments (Tables 2 and 3). For the variable plant height, the treatments with the lowest concentration of For the variable number of outbreaks, nutritive medium and 30 gL-1 of sucrose, the Tukey test identified three groups, Fraguas et al., 2003, obtained satisfactory treatments with the concentration of 50 and -1 growth of plants resulting from the cross 25% of nutrient medium and 30 gL of sucrose between the Cattleya labiata and Laelia developed the largest number of outbreaks and itambana orchids, in between MS culture with formed the first group, the second was formed 20 gL-1 sucrose. by treatments with 75 and 100% nutrient -1 medium with 30 gL of sucrose and the third For the variable number of leaves per group is composed of treatments with the bud, treatments T4, T2 and T5 stand out with lowest concentrations of sucrose. the highest response to this variable.

Similarly, Sorace et al., 2008 evaluated For root length, treatment T5 and T8 the number of Oncidium Bauer outbreaks and showed a greater response, they coincide in the obtained the best results with the most dilute contribution of 30 gL-1 of sucrose but differ in levels of the MS medium with 40 gL-1 sucrose. the concentration of the nutrient medium.

Del Rosario and De Guzmán (1981) obtained a positive effect promoted by the high concentration of sugar, in rooting embryos of coqueiro "makapuno" using nutritious medium MS.

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The results of the growth behavior of Acknowledgments Oncidium baueri root show that there were no differences for the concentration of sucrose, but The experimental work was supported by the the treatment containing the MS medium at ½ Agronomist Engineer Aldo Gabriel Tejeda with the addition of 40 gL-1 sucrose presented Cervantes, with the contribution of the capsules the greatest number of roots , and is statistically with the seeds of the species Oncidium different from the concentration of 30 gL-1, do sphacelatum Lindley and his experience in not report statistical differences between the cultivation both under natural and in vitro concentrations of 30 and 60 gL-1 and differ conditions of these species. Likewise, the statistically with the concentration of 40 gL-1. support in the activities of preparation of solutions, means of cultivation and sterilization According to Oliveira (1994), high of the Agronomist Silvia Ayala Castillo, both, sugar concentrations can inhibit root formation members of the administrative staff of the in vitro. Academic Unit of Agriculture. The Autonomous University of Nayarit is thanked The only treatment where pseudobulb for giving the working conditions that allowed formation was observed was T8 (25% MS with the development of this research. 30 gL-1 sucrose) which has facilitated the process of acclimatization to the outside References environment of the Oncidium sphacelatum orchid. Alayón, G.J.A.(2011). Orquídeas en huertos familiares de Calakmul Campeche, México. Conclusions Ecosur. Campeche, México.135 pp.

The growth of protoorms and the development Arditti. J. & A Abdul-Ghani, A.K. (2000). of buds with leaves, roots and pseudobulbs of Transley Review No. 110. Numerical and the Oncidium sphacelatum Lindley orchid was physical properties of orchid seeds and their achieved with the supplement to the culture biological implications. New Phytol, medium of 30 gL-1 sucrose. 145(1),367 – 421. Doi:10.1046/j.1469- 8137.2000.00587.x The growth of protoorms and the development of shoots with leaves, roots and Bertolini, V. Valle, M.J.R. y Rojas V.A.N. pseudobulbs was achieved with the contribution (2016).Aplicación de visión por computadora of nutrients to the culture medium of Murashige en la evaluación del desarrollo in vitro de and Skoog (1962) at 25% of the original protocormos de Oncidium sphacelatum Lindl., concentration, this implies the decrease in cost (Orchidaceae). Acta Agron. 65(3), 261- and a reagent use savings. 267.doi:htpp://dx.doi.org/10.15446/acag.v65n3. 52212. The results obtained indicate that the sucrose supplement is essential for the orchid Billard, C.E., Dalzotto, C.A. y Lallana, V.H. under study for normal growth and (2014). Desinfección y siembra asimbiótica de development of plants under artificial semillas de dos especies y una variedad de cultivation conditions. With respect to the orquídeas del género Oncidium. Polibotánica contribution of mineral nutrients, the decrease 38:145-157. of these in the nutritive environment is not affected, while maintaining the contribution of Del Rosario, A.G., Guzman, E.V. The growth the energy source with the sucrose supplement. of Makapuno coconut embryos in vitro as affected by mineral composition and sugar level of the médium during the liquid and solid culture. Philippine Journald of Cience. Manila. 105:215-222.

Debergh, P.C. (1991). Control of in vitro plant propagation. In: Crocomo, O.J., Sharp, W.R. (Ed.). Biotecnologia para producao vegetal. Piracicaba. CebTEC/FEALQ.

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Dressler, R.L. 1981. The Orchids. Nature Suárez , J.P. Weib. M. Abele. A. Garnica. S. Hystory and Classification. Harvard University Oberwinkler. F. & Kottke, I. (2006). Diverse Press, Cambridge. tulasnelloid fungi form mycorrhizas with epiphytic orchids in an Andean cloud forest. Fraguas, C.B.,Villa, F., Souza, A.V.,Pasqual, Mycol Res. 110, 1257 – 1270. Doi: M.,Dutra, L,F. (2003). Crescimiento in vitro de 10.1016/j.mycres. 2006.08.004. plántulas de orquídeas oriundas de hídricos, entre Cattleya labiata e Laelia itambana. Ceres, Valadares, R.B.S. Perotto. S. Santos, E.C. & Lavras. V.50(292): 719-726. Lambais, M.R. (2013). Proteome changes in Oncidium sphacelathum (Orchidaceae), at Hadley, G.(1997). Orchid mycorrhiza. En different trophic stages of symbiotic Arditti, J y Pridgeon, A.M. (Eds.) Orchid germination. Mycorrhiza, 24(5),349- Biology:Reviews and Perspectives vol 2. 360.Doi:10.1007/s00572-013-0547-2. Cornell University Press. Ithaca, Nueva York. 83 – 118 p. Zepeda, Q.V.H. (2003). Micropropagación de la orquídea Galeandra batemanii Rolfe, una Mc Vaugh, R. (1985). Flora Novo-Galiciana a especie amenazada (Tesis de Licenciatura). descriptive account of the vascular plants of Universidad Autónoma de Nayarit, Facultad de Western Mexico. W.I. Anderson (Ed.) The Agricultura. Xalisco, Nayarit, México. 1-80 pp. University of Michigan Press. Vol 16. 363 pp.

Miceli, M.C.L., C. Orantes G. y R. Pérez L. 2009. Orquídeas y bromelias del Parque Nacional Cañón del Sumidero. Universidad de Ciencias y Artes de Chiapas.Tuxtla Gutiérrez, Chiapas.155p.

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Objectives Objectives Methodology Methodology Contribution Contribution

Keywords (In English) Keywords (In Spanish)

Indicate 3 keywords in Times New Roman and Bold No. Indicate 3 keywords in Times New Roman and Bold No. 10 10

Citation: Surname (IN UPPERCASE), Name 1st Author, Surname (IN UPPERCASE), Name 1st Coauthor, Surname (IN UPPERCASE), Name 2nd Coauthor and Surname (IN UPPERCASE), Name 3rd Coauthor. Paper Title. Journal of Enviromental Sciences and Natural Resources. Year 1-1: 1-11 [Times New Roman No.10]

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Introduction

Text in Times New Roman No.12, single space.

General explanation of the subject and explain why it is important.

What is your added value with respect to other techniques?

Clearly focus each of its features Figure 1 Title and Source (in italics)

Clearly explain the problem to be solved and Should not be images-everything must be editable. the central hypothesis.

Explanation of sections Article.

Development of headings and subheadings of the article with subsequent numbers Table 1 Title and Source (in italics)

[Title No.12 in Times New Roman, single Should not be images-everything must be editable. spaced and bold] Each article shall present separately in 3 Products in development No.12 Times New folders: a) Figures, b) Charts and c) Tables in Roman, single spaced. .JPG format, indicating the number and sequential Bold Title. Including graphs, figures and tables- Editable For the use of equations, noted as follows:

r In the article content any graphic, table and Yij = α + ∑h=1 βhXhij + uj + eij figure should be editable formats that can change size, type and number of letter, for the (1) purposes of edition, these must be high quality, not pixelated and should be noticeable even Must be editable and number aligned on the reducing image scale. right side.

[Indicating the title at the bottom with No.10 Methodology and Times New Roman Bold] Develop give the meaning of the variables in linear writing and important is the comparison of the used criteria.

Results

The results shall be by section of the article.

Annexes

Tables and adequate sources Graphic 1 Title and Source (in italics) Thanks Should not be images-everything must be editable. Indicate if they were financed by any institution, University or company.

ISSN 2444-4936 Surname (IN UPPERCASE), Name 1st Author, Surname (IN ECORFAN® All rights reserved UPPERCASE), Name 1st Coauthor, Surname (IN UPPERCASE), Name 2nd Coauthor and Surname (IN UPPERCASE), Name 3rd Coauthor. Paper Title. Journal of Enviromental Sciences and Natural Resources. Year [Times New Roman No. 8] Instructions for Scientific, Technological and Innovation Publication

Conclusions

Explain clearly the results and possibilities of improvement.

References

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Journal Name Article title Abstract Keywords Article sections, for example:

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Author Name (s) Email Correspondence to Author References

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Journal of Enviromental Sciences and Natural Resources

“Varietal description of sorghum phenotypes for registration” AVENDAÑO-LÓPEZ, Adriana Natividad, SANCHEZ-MARTINEZ, Jóse, PADILLA-GARCIA, José Miguel and ARELLANO-RODRIGUEZ, Luis Javier Universidad de Guadalajara

“Rhizobacteria antagonistic to the fungus Sclerotium cepivorum (Berk.t) in onion plants (Allium cepa L.)” OCEGUEDA-REYES, Martha Delia, CASAS-SOLÍS, Josefina, GONZÁLEZ- EGUIARTE, Diego R. and VIRGEN-CALLEROS, Gil Universidad de Guadalajara

“Effectiveness of Humic Acids, Fulvic Acids and lechuguilla extract (Agave lechuguilla), as biosurfactants in the Remediation of soils contaminated with Hydrocarbon” RESENDIZ-VEGA, Marisol & GARCÍA-MELO, José Alberto Universidad Tecnológica Tula – Tepejí

“Physicochemical and antioxidant characteristics of different sprouts” REYES-MUNGUIA, Abigail, CARRILLO-INUNGARAY, María Luisa, GARCÍA-BARRÓN, Gabriela del Socorro and TURRUBIATES-GARAY, Karla Alicia Universidad Autónoma de San Luis Potosí

“In vitro growth of Oncidium sphacelatum (Orchidaceae) in different concentrations of sucrose and nutrients” NAVARRETE-VALENCIA, Ana Luisa, RAMÍREZ-GUERRERO, Leobarda Guadalupe, SÁNCHEZ-MONTEÓN Ana Luisa and JIMÉNEZ-MEZA, Víctor Manuel Universidad Autónoma de Nayarit