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Revista mexicana de ciencias agrícolas

versão impressa ISSN 2007-0934

Rev. Mex. Cienc. Agríc vol.7 no.4 Texcoco Mai./Jun. 2016

 

Investigation notes

Pantoea agglomerans isolated from producer surfactant pasture rhizosphere Tanzania and Llanero

José Daniel Chávez González1 

Miguel Ángel Rodríguez Barrera1 

Yanet Romero Ramírez1 

Alejandro Ayala Sánchez2 

Jesús Carlos Ruvalcaba Ledezma3 

Jeiry Toribio-Jiménez1  § 

1 Laboratorio de Microbiología Molecular y Biotecnología Ambiental, Posgrado en Biociencias. Unidad Académica de Ciencias Químico Biológicas-Universidad Autónoma de Guerrero, Chilpancingo, Guerrero, México. Av. Lázaro Cárdenas s/n, Cd. Universitaria, Chilpancingo, Guerrero, 39000, México. Tel: 52 747 4719310. Ext. 4526. (danichavez1991@gmail.com; rmiguel@gmail.com; yanetromero7@gmail.com; jtoribio@uagro.mx).

2 Campo Experimental Zacatepec-INIFAP. Carretera Zacatepec a Galeana, km 0.5. C. P. 62780. Zacatepec, Morelos, México. (ayalasa@hotmail.com).

3 Instituto de Ciencias de la Salud (UAEH) Universidad Autónoma del estado de Hidalgo, México. Ex-Hacienda la Concepción S/N, carretera a Actopan Pachuca de Soto Hidalgo, México. Tel: 5548817657. dcspjcarlos@gmail.com.


Abstract:

Pantoea agglomerans is a beneficial bacteria in various plants as it promotes plant growth, coupled with this only some strains can produce biosurfactants, these biosurfactants are capable of causing hemolysis on blood agar, foaming, dispersing the oil, reduce surface tension by the collapse of gout, emulsify hydrophobic compounds among others, these features make them interesting because they have various biotechnological applications, most notably, oil recovery, in the medical area, in the food industry and agriculture, in this last may be used for controlling phytopathogenic fungi, biosurfactants have different advantages, are compatible with the environment, tolerate high concentrations of NaCl, pH, temperature and are biodegradable, are produced by fungi, yeasts and bacteria, is why it is isolated and identified bacteria capable of producing biosurfactants in the rhizosphere of Tanzania (Panicum maximum) and Llanero (Andropogon gaynus Kunth) pastures in the Tecomate ranch in the state of Guerrero, samples a bacterial load in rhizosphere and root P. maximun was obtained of 1 x 103 a 10 x 104 and A. gaynus Kunth 5 x 103 to 21 x 104 UFC/g respectively, only 28 of these strains were able to produce hemolysis, foam collapse PPGAS drop and also present ability to emulsify diesel, gasoline, oil and vegetable oil, only five strains of P. maximun and eight of A. gaynus Kunth microbiological level were identified reporting the presence of first Pantoea aglomerans in association with the rhizosphere and root of pastures, these data serve as a basis to generate studies on its ecology and biological application to increase economic production of pastures in the state of Guerrero.

Keywords: Pantoea aglomerans; biosurfactants; pastures

Resumen:

Pantoea agglomerans es una bacteria benéfica en diversas plantas ya que promueve el crecimiento vegetal, aunado a esto solo algunas cepas pueden producir biosurfactantes, estos biosurfactantes son capaces de causar hemolisis en agar sangre, formar espuma, dispersar el aceite, disminuir la tensión superficial por el colapso de la gota, emulsificar compuestos hidrófobos entre otros, estas características los hacen interesantes dado que tienen diversas aplicaciones biotecnológicas, en las que destacan, la recuperación de crudo, en el área médica, en la industria de los alimentos y en agropecuarias, en esta última pueden ser usados para el control de hongos fitopatógenos, los biosurfactantes tienen diversas ventajas, son compatibles con el ambiente, toleran altas concentraciones de NaCl, pH, temperatura y son biodegradables, son producidos por hongos, levaduras y bacterias, es por ello que se aislaron e identificaron bacterias capaces de producir biosurfactantes en la rizosfera de los pastos Tanzania (Panicum maximun) y Llanero (Andropogon gaynus Kunth) en el rancho Tecomate en el estado de Guerrero, de las muestras se obtuvo una carga bacteriana en rizosfera y raíz en P. maximun de 1 x 103 a 10 x 104 y en A. gaynus Kunth de 5 x 103 a 21 x 104 UFC/g respectivamente, de estas solo 28 cepas fueron capaces de producir hemolisis, espuma en PPGAS y colapso de la gota, además de presentar capacidad de emulsificar diésel, gasolina, aceite vegetal y petróleo, solo cinco de las cepas de P. maximun y ocho de A. gaynus Kunth se identificaron a nivel microbiológico reportando la presencia por vez primera de Pantoea aglomerans en asociación a la rizosfera y en raíz de los pastos, estos datos sirven de base para generar estudios sobre su ecología y aplicación biológica para incrementar la producción económica de pastos en el estado de Guerrero.

Palabras clave: Pantoea aglomerans; biosurfactantes; pastos

Introduction

Pantoea aglomerans is a bacterium in the form of gram- negative bacillus, is considered ubiquitous plant and has been isolated in different environments (soil, water, insects, animals and clinical samples), some isolates have demonstrated the ability of biological control of fungi and bacteria causing of plant diseases (Theo H.M. Smits et al., 2010), have been reported in rhizosphere of Hordeum vulgare, Triticum sp. and Gloxinia alba, are able to fix nitrogen (Jimenez et al., 2007), degrade hydrocarbons and only some strains are capable of producing biosurfactant (BS) (Vasileva-Tonkova y Gesheva, 2006; Gopalakrishnan et al., 2006). The BS surface active molecules are produced by bacteria, fungi, yeast, actinomycetes and others. All BS are amphiphilic, that is, are composed of two polar parts (hydrophilic) and a nonpolar (hydrophobic), the hydrophilic group consisting of mono-, oligo- or polysaccharides, peptides or proteins, and the hydrophobic portion usually contains saturated fatty acids, unsaturated or hydroxylated fatty alcohols (Pacwa-Plociniczak et al., 2011).

The chemical structure of the BS confers affinity for interfaces which are the: lowering the surface tension of water from 72 mN/m to about 27 mN/m, reduce interfacial tension and increase the solubility of some compounds (Raiguer Iustman et al., 2009). These biosurfactants have large advantages over chemical surfactants, such as high biodegradability, low toxicity, and biocompatibility, they can be synthesized from relatively inexpensive carbon sources or from industrial waste (Raiguer Iustman et al., 2009). There are reports of bacteria capable of producing them, which include the genera Pseudomonas sp., Bacillus sp., Serratia sp., Enterobacter sp., Pantoea sp., among others (Makkar, 2011). The microbial BS have wide applications in oil recovery in the pharmaceutical, food and cosmetics, and also have potential uses in the agricultural and livestock sector (Perez-Vargas et al., 2010; Jimenez et al., 2010).

In the agriculture is documented in its application for biological control for their antimicrobial and antifungal activity and recovery of soil contaminated by pesticides or chemical fertilizers (Sachdev and Cameotra, 2013). In Mexico in the tropical region 35% of the national territory is exploited for livestock production, this in order to meet the demand for food (meat, milk, cheese and others) by the settlers, this has led farmers to exploit different forage crops for food quality and quantity, and may also meet the needs of livestock, still-post efforts in this area, other needs such as decreased protein content are added together and the lack of fodder for the seasonality of rainfall has decreased its production (Enriquez et al., 1999).

Of the 34 000 species of plants in Mexico most of them are used to feed livestock in the state of Guerrero the species Panicum maximum or grass "Tanzania" this is resistant to grazing is grown, it grows well in dry soils than too poor in nutrients of any texture even in sandy soils (Roll-Juarez et al, 2009;. Sows and Vallejos, 2011). Furthermore, the species grass Andropogon gaynus Kunth or "Llanero"; is prostrate and invasive growth that allows you to make good ground cover easily adapted to clay and sandy soils (Pérez, 2008). Have been documented few studies on the interaction of bacteria among them are lactic acid (Pasebani et al., 2010), no studies on producing species biosurfactants in rhizosphere and root of Tanzania and Llanero grasses were found as well as on their biological role and biotechnology application in livestock activities.

The ranch the Tecomate in Tierra Colorada, Guerrero, dedicated to cattle to obtain meat and are interested in increasing pasture production to meet the needs of cattle, two types of grasses to feed livestock are Tanzania and Llanero, based on the above the aim was to focus; quantify the colony forming units per gram (UFC/g) of rhizosphere or root associated with Tanzania and Llanero pastures, evaluate their ability to produce biosurfactants and identify microbiological level bacteria for the purpose of applying these in a future pasture production to strengthen Livestock in the state of Guerrero.

El Rancho the "Tecomate" in Tierra Colorada, Guerrero, Is located at the geographic coordinates 17° 08' 50.53 north latitude and 99° 37' 36.9 west longitude, with an elevation of 241 meters, with regosols soil, sandy texture silty, Aw1 climate (warm humid), and rainfall of 1300 mm annually. On that site 15 samples of rhizosphere and root were collected at random from each grass species (Tanzania and Llanero), these were deposited in polythene bags and transported to the laboratory of molecular microbiology and environmental biotechnology of the academic unit of chemical and biological sciences the UAGro. The roots were separated from the soil, and excess first with running water was removed and placed in 2% NaOCl for 15 min, finally rinsed in sterile distilled water. Subsequently 1 g of root weighed and macerated in 5 mL of sterile isotonic saline and incubated at 30 C with stirring for 12 h in Luria Bertani (LB; tryptone 10g, yeast extract 5g, NaCl 10g to pH 7.0 ).

At the same time weighed 1 g of rhizosphere and dilutions were performed serial samples macerated root and rhizosphere in the range 10-1 TO 10-4, 100 μl of each dilution was taken and were dispersed in duplicate in agar LB plates, these were incubated at 30 °C for 48 h. The microbial load was reported as UFC/g of root or rhizosphere. Bacteria isolated with the same morpho type replated blood agar to observe their hemolytic capacity at 24 h, those who had a β-hemolysis were considered as potential producers of BS (Rakeshkumar et al., 2012). Hemolytic bacteria were cultured in 50 mL of broth PPGAS (PPGAS; NH4Cl2 1.07 g, KCl 1.5 g, Tris-HCl 18.91g, MgSO4 0.19g, 5% glucose, 1% peptone pH 7.2), 30 °C for 72 h under constant stirring, to promote the production of BS (Wild et al., 1997).

The ability to produce BS was confirmed by the formation and stability of the foam, the index emulsification using diesel oil, gasoline and vegetable oil at 24 h (IE24) (Cooper and Goldenberg, 1987) and finally the size halo dispersion in oil (Figure 1), in all analyzes was included as a positive control to P. aeruginosa PAO1. The bacteria were able to produce biosurfactants were identified to genus and species using gram stain, catalase production, oxidase, with conventional biochemical for metabolic evaluation.

Figure 1 Tests for the detection of biosurfactants. A) hemolysis on blood agar; B) emulsification index; C) foaming PPGAS broth; and D) oil dispersion. 

Of isolated samples, a bacterial load root was obtained and rhizosphere in Tanzania pasture 1 x 103 a 10 x 104 and Llanero of 5 x 103 a 21 x 104 UFC/g respectively, which agrees on the average Pasebani et al. (2010), which reported 8.3 x 103 UFC/g soil of lactic acid bacteria grass Tanzania in Malaysia., Also shows that the number of UFC/g is equal in the two grass species this may be due to the same type of soil and climatic conditions.

As for the production of BS only 28 (13 Tanzania and 15 Llanero) bacterial strains were able to produce hemolysis on blood agar, these all foamed broth PPGAS and dispersed oil which demonstrates the capacity of BS (Figure 1), only 13 strains were able to emulsify hydrophobic compounds to 24h. All strains isolated from the rhizosphere of grasses Tanzania were able to emulsify gasoline, vegetable oil and oil unlike isolated in Llanero pasture, this is one of the first reports of bacteria isolated from grasses with ability to emulsify hydrophobic compounds and that they can be used for bioremediation processes (Table 1). The data obtained in this study are consistent with Vasileva-Tonkova and Victoria Gesheva (2006) which describe a strain of Pantoea sp., A-13 isolated from soil of the islands, Dewart, Antarctica, Gopalakrishnan et al. (2006) report two strains of Pantoea sp., 1 and 2 producers of BS isolated from the coast of India able to emulsify hydrophobic compounds.

Table 1 Index emulsification at 24 h (IE24) strains isolated from the rhizosphere of Tanzania and Llanero pastures of Rancho el Tecomate. 

Toribio-Jimenez et al. (2014) report a strain of Pantoea vagans producing BS resistant isolated heavy metals from the tailings El Fraile in Mexico, all these strains have been proposed as alternatives to accelerate bioremediation processes, but have not evaluated their ability to promote plant growth. We report thirteen strains of P. agglomerans with 99% identity, such as species Pantoea sp already said., They have been reported as endophytes of various crops, which stands Pantoea ananatis isolated from leaves and seeds of Panicum virgatum known as grass "rod" in Table 1. Index emulsification at 24 h (IE24) strains isolated from the rhizosphere of Tanzania and Llanero pastures of Rancho el Tecomate.

Canada by Gagne-Bourgue et al. (2013), confirming the vertical transmission to the next generation of hosts, the above is consistent with our findings because only one species as best producer of BS in the rhizosphere of grasses Tanzania and Llanero identified.

As for the IE24 all strains were able to primarily emulsify vegetable oil, diesel, gasoline and oil respectively, highlighting including M1C6 strains and M3C1 isolated from Tanzania pastures, these data are consistent with those described in Pseudomonas aeruginosa isolated from hospital surfaces (Toribio-Jimenez et al., 2015). Jacobucci et al. (2009) describe a strain of P. agglomeras isolated from soil contaminated with oil able to grow and produce BS using kerosene as sole carbon source, what bioremediate proposed as candidate for hydrocarbon contaminated sites, it is possible that the strains isolated in this study are able to grow in hydrocarbons as sole carbon source for their ability also shown in emulsification. It described that the BS produced by Pantoea sp., Is of glycolipid type (Dewart et al., 2006, Gomes de Almeida et al., 2015), so presumably the BS produced by native strains are the glycolipids type. In addition to the production of BS by strains of Pantoea sp., It is important to delve into his study as potential in promoting plant growth as many are capable of solubilizing phosphates, fix nitrogen and produce plant hormones in order to encourage production and biological control of fodder for the livestock industry in Mexico.

Conclusion

It reported first thirteen strains Pantoea agglomerans capable of producing biosurfactant associated with the rhizosphere of Tanzania and Llanero pastures in the "Tecomate" ranch dedicated primarily to production systems grazing animals in the state of Guerrero, between strains highlighted the M1C6 and M3C1, these data reflect poor study about finding producing bacteria BS in pasture, and on the microbial ecology of the rhizosphere on pasture, so it is important to isolate and identify bacteria the biosurfactant and its biological role on pasture or forage production as an alternative in the livestock sector in the state of Guerrero.

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Received: March 2016; Accepted: June 2016

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