Revista Mexicana Ciencias Agrícolas   volume 11   number 2   February 15 - March 31, 2020

DOI: https://doi.org/10.29312/remexca.v11i2.2191

Essay

Lipopeptides produced by biological control agents of the genus Bacillus:
a review of analytical tools used for their study

Valeria Valenzuela Ruiz1

Gema Teresa Gálvez Gamboa1

Eber Daniel Villa Rodríguez1, 2

Fannie Isela Parra Cota3

Gustavo Santoyo4

Sergio de los Santos-Villalobos1§

1Sonora Technological Institute. February 5, 818 south, Cd. Obregón, Sonora, Mexico. CP. 85000. (valeriavalenzuelaruiz@gmail.com; gemateresa3@gmail.com). 2Uvex Agro International. Highway las Bocas km 45, H. Caborca, Sonora, Mexico. (eber.villa.rodriguez@gmail.com). 3Experimental Field Norman E. Borlaug-INIFAP. Cd. Obregón, Sonora, Mexico. CP. 85000. (parra.fannie@inifap.gob.mx). 4Institute of Chemical Biological Research-Michoacana University of San Nicolás de Hidalgo, Morelia, Michoacán, Mexico. (gsantoyo@umich.mx).

§Corresponding author: sergio.delossantos@itson.edu.mx.

Abstract

The genus Bacillus has a wide metabolic diversity associated with its prevalence in various ecosystems. At present, one of the most studied characteristics of this bacterial genus is its ability to reduce the incidence of diseases in agricultural crops, due to the direct or indirect repression of the growth of the causative agents of said diseases. Among the most studied mechanisms in the biological control of strains of the genus Bacillus against phyto-pathogenic agents, lipopeptides, such as surfactins, iturins and phengicins, which are low molecular weight compounds with amphiphilic characteristics that provide protection to plants both in preconditions as post-harvest. Lipopeptides can directly antagonize phytopathogenic agents and induce systemic resistance strategies in associated plants. However, the wide use of lipopeptides in the agricultural, pharmaceutical, or food sector depends on the development of efficient and economical analytical processes for the extraction, identification, and quantification of said bioactive compounds. This test presents an analysis of various analytical procedures used for the extraction, and qualitative and quantitative identification of these metabolites.

Keywords: Bacilli, biocontrol, lipopeptides.

Reception date: January 2020

Acceptance date: February 202

By 2050 it is predicted that the agricultural sector will need to increase its productivity between 70 and 100%, in order to meet the food demand of the human population, which will amount to ~10 billion people (FAO, 2017). However, one of the main limitations for the increase in agricultural production is the incidence of crop diseases caused by phyto-pathogens, causing an average yield decrease of 15-25% (Villa-Rodríguez et al., 2016; Gupta et al., 2018; Parra-Cota et al., 2018; Díaz-Rodríguez et al., 2019).

Therefore, the use of pesticides worldwide has increased from ~1.3 kg ha-1 to 2.57 kg ha-1 from 1992 to 2016; that is, 197.69% (García-Gutiérrez et al., 2012; FAO, 2018); however, only 0.1% reaching the desired objective (agricultural cultivation), where the remaining volume is mobilized by contaminating the surrounding environment, affecting other organisms, being chemically complex substances, severely impacting the environment generating pollution of soils and aquifers, soil degradation and salinization, loss of biodiversity and toxicity effects of remaining components, chemical or microbial degradation, in addition to generating loss of biodiversity (Gálvez-Gamboa et al., 2018).

Therefore, the generation of agro-biotechnological alternatives for the control of phyto-pathogens is decisive to meet the demand for current and future food in a sustainable way, focused on preserving the fertility of agricultural soils, and reducing potential effects on the environment (de los Santos Villalobos et al., 2018; Robles-Montoya et al., 2020).

Globally, the interest of the agricultural sector has increased in the use of phyto-pathogen control strategies by Biological Control Agents (ACB), particularly bacteria, as observed by the exponential increase in the world market for biopesticides, of $ 800 million in 2014 to $ 2.8 billion today (Villa-Rodriguez et al., 2019), with an estimated increase of 15% to 20% by 2020 (Gomez et al., 2018). ACB are beneficial organisms, their genes and/or products, such as metabolites, to reduce the negative effects of plant pathogens through antagonistic actions by different mechanisms, including mycoparasitism, lytic enzyme production, induction of systemic response of the plant and production of δ-endotoxins, siderophores and lipopeptides (Robles-Montoya et al., 2019; de los Santos Villalobos et al., 2019; Valenzuela-Ruiz et al., 2019; Villa-Rodríguez et al., 2019).

This led to the study of various microbial strains such as ACB, among which the genus Bacillus has been widely studied for its ability to sporulate, which facilitates its production and storage as a bioinoculant for long periods of time, in addition to its abundance, diversity and ubiquity in various agro-systems (soil, water and plant), its population being significantly larger compared to other microbial genera (Villarreal-Delgado et al., 2018; de los Santos Villalobos et al., 2019; Santoyo et al., 2019).

Additionally, the Bacilli unleash due to their diverse metabolic capacities, producing antibiotic antimicrobial metabolites, lytic enzymes, chitinases, cellulases, glucanases, and lipopeptides (Valenzuela-Aragon et al., 2019). Lipopeptides are low molecular weight compounds with amphiphilic characteristics that provide protection to plants both in pre-harvest and post-harvest conditions, by directly suppressing the growth of pathogens or inducing systemic resistance in host plants (Hashem et al., 2019).

Recently, Coutte et al. (2017) found 263 different lipopeptides synthesized by 11 microbial genera. Within these, Bacillus represents the most abundant producer with 98 different lipopeptides (classified in families), where in the context of biocontrol of plant diseases, the three families of Bacillus lipopeptides: surfactins, iturins and fengycina are of interest for their activity antagonist for a wide range of potential phytopathogens, including bacteria, fungi and oomycetes (Ongena and Jacques, 2008). The objective of this trial is to describe critically various analytical procedures used for the study (including the extraction and qualitative and quantitative identification) of lipopeptides produced by the genus Bacillus, focused on the production of efficient and sustainable biopesticides.

Methods used for the extraction, identification and purification of lipopeptides

Extraction and purification of lipopeptides

Extraction and purification are key stages in the study and commercialization of lipopeptides (Coutte et al., 2017). Generally, the purification of these compounds starts from a liquid matrix (eg the culture broth where the Bacillus strain was grown), although its production in solid state fermentation has also been reported (Su et al., 2018).

The purification methods are based on the physicochemical properties of lipopeptides (polarity and molecular weight). Frequently, a single method is not sufficient for the purification of these compounds. For this reason, it is common for lipopeptide purification to include multiple stages, applying methods such as i) acid precipitation; ii) use of organic solvents; iii) ultrafiltration; iv) solid phase extraction; and v) chromatography (Table 1).

Table 1. Methods of extracting and purifying lipopeptides from strains of the genus Bacillus.

Acid precipitation

One of the most common methods to start the lipopeptide purification is by acid precipitation, using concentrated hydrochloric acid (HCl) (Abdel-Mawgoud et al., 2008). This method consists in lowering the pH (~2) of the culture broth in which these compounds are dissolved. In this way, the negative charges of the lipopeptides are neutralized, decreasing their solubility in the aqueous phase, resulting in their precipitation (Biniarz et al., 2016).

Once lipopeptides precipitate, they can be recovered for further analysis or even to be subjected to other purification steps. Acid precipitation is a very effective and economical method to isolate lipopeptides. Although the use of this method allows recovering ~97% of the lipopeptides present in the culture medium, its main disadvantage is that it is not a selective method, since other compounds (eg. peptides) can coprecipitate with the lipopeptides, obtaining purities below 60% (Coutte et al., 2017).

Organic solvents

The use of organic solvents is one of the most used methods for the extraction and purification of lipopeptides. Solvents with different polarity indices such as chloroform, methanol, petroleum ether, ethyl acetate, n-hexane and ether have been used for this purpose (Biniarz et al., 2016). The use of these solvents can be used as a complementary method to acid precipitation, for example, the precipitate obtained with this method can be subjected to extraction with one of the mentioned solvents (solid-liquid extraction). However, solvent extraction can also be used as an initial step in the purification process, for example, by a liquid-liquid extraction by mixing the culture medium and the organic solvent (Alajlani et al., 2016).

Solid phase chromatography and extraction (SPE)

The extraction of lipopeptides by methods such as acid precipitation and solvent use, despite presenting high recovery efficiency, are poorly selective methods. Chromatography and solid phase extraction (SPE) are methods that allow obtaining a high degree of purity. These methods are generally used in the final stages of lipopeptide purification (Coutte et al., 2017).

Both chromatography and solid phase extraction consists of passing the lipopeptide mixture through a column, in this way the compounds of interest will interact with the solid phase of the column (resin), while the contaminating compounds will not (Poole, 2003).

Subsequently, lipopeptides that interacted with the resin can be recovered by elution with solvents (eg methanol, ethyl acetate or acetonitrile) (Poole, 2003). The success in the purification of lipopeptides with these methods depends mainly on the type of resin and the solvent mixture. It has been reported that column C18 is very efficient for adsorption of lipopeptides, due to the hydrophobic motive of these molecules (Razafindralambo et al., 1993).

However, due to the similarity in its structure and physicochemical properties, the purification of specific lipopeptide families is usually a complicated process. However, this has been achieved with reverse phase liquid chromatography methods. For example, recently Luna-Bulbarela et al. (2018) was able to purify different isoforms of iturines -with variants in the length of fatty acid- by reverse phase chromatography, using a C18 column as the stationary phase and a gradient of acetonitrile as the mobile phase.

Ultrafiltration

Ultrafiltration is a promising approach to the recovery of lipopeptides; however, equipment costs are high, which limits their routine use (Isa et al., 2007). Lipopeptides at concentrations above their critical concentration of micelles can be associated to form supramolecular structures such as micelles, which have nominal diameters up to two or three times larger than that of a free molecule (Coutte et al., 2017). Taking this principle into account, ultrafiltration methods focused on purifying lipopeptides in the form of micelles have been established.

The separation of lipopeptides by the filtration membrane depends on their molecular aggregation behavior and their ability to form micelles (Jauregi et al., 2013). Likewise, the pH and the concentration of lipopeptides are important factors in the formation of micelles (Sen et al., 2005). Once the lipopeptide micelles have been purified by ultrafiltration, it is possible to denature it with the use of organic solvents such as methanol or ethanol.

Ultrafiltration has been used successfully for the purification of different families of lipopeptides. For example, in one study this method was used to purify a mixture of surfactins and iturines using 10-100 kDa membranes (Jauregi et al., 2013). In general, with this method it is possible to achieve a purity of lipopeptides of up to 95% (Jauregi et al., 2013).

Identification and quantification of lipopeptides

The chromatographic methods, in addition to being used in the purification of lipopeptides, are used in their identification and quantification. Methods that use different chromatography variants have been established for lipopeptide analysis, including: thin layer chromatography (TLC) and high resolution liquid chromatography (HPLC) (Kinsella et al., 2009; Jamshidi-Aidji et al., 2019).

Due to the versatility of chromatography for lipopeptide analysis - allowing its purification, identification and quantification - this analytical tool has been the most used in lipopeptide processing. However, there are other spectroscopic methods that have been used for the analysis of these compounds, such as i) nuclear magnetic resonance (NMR) (Son et al., 2016), infrared transmission with Fourier transform (FT-IR) (Kong, 2007) and mass spectrometry (Ma et al., 2016).

Thin layer chromatography (TLC)

Thin layer chromatography is a very versatile chromatographic technique that can be used for different purposes in lipopeptide analysis. This technique consists in placing the sample of interest near one end of an aluminum plate coated with a thin layer of an adsorbent (eg. silica gel, cellulose, aluminum oxide). The sheet is placed in a closed cuvette containing one or more mixed solvents (mobile phase). As the solvent mixture rises by capillarity through the adsorbent, a differential distribution of the compounds present in the sample occurs (Ciura et al., 2017).

The samples will migrate differentially across the plate, resulting in specific values of the delay factor (Rf). The success in the separation of the compounds depends on the adsorbent (stationary phase) and the solvent mixture (mobile phase) (Ciura et al., 2017). TLC has been used to identify families of lipopeptides (Jamshidi-Aidji et al., 2019). However, it also results in a quick and simple method to evaluate the purity of these compounds during purification steps.

The identification of lipopeptides using TLC is performed by comparing the Rf value between a standard of lipopeptides and the test sample. For example, Geissler et al. (2017) established a method to simultaneously identify surfactins, iturins and phengicins in extracts from different Bacillus strains. In this study, the Rf values were specific for each family of lipopeptides, using chloroform/methanol/water (65:25:4, v/v/v) as the mobile phase and silica gel as the stationary phase.

High performance liquid chromatography (HPLC)

High performance liquid chromatography (HPLC) allows a mixture of compounds to be separated and can be used to identify, quantify and purify individual compounds from a mixture (Malviya et al., 2010). This technique consists in passing a mixture of compounds -by solvents (mobile phase) at high pressures- through a column that contains a resin inside (stationary phase). Once the compounds pass through the column, they emit signals generated by detectors (eg ultraviolet detector, photodiode matrix detector, mass detector, fluorescence detector, etc.) (Malviya et al., 2010).

HPLC has gained popularity among chromatographic techniques and is currently the main option for lipopeptide studies (Biniarz et al., 2016). This method even allows different isoforms to be separated from the same family of lipopeptides (eg. variations in the length of the fatty acid or in some amino acid).

The identification of lipopeptides with HPLC is performed by a comparison of the Rt between a standard of lipopeptides and the test sample, while quantification requires a calibration curve using different concentrations of a standard (de Souza et al., 2018). In HPLC, identification and quantification are analyzes that are carried out simultaneously. HPLC methods have been developed that allow quantification of different lipopeptide families (Table 2). For example, Yuan et al. (2011) optimized chromatographic conditions to quantify iturin A homologues, using the Eclipse XDB-C18 column as a stationary phase and an acetonitrile gradient as a mobile phase.

Mass spectrometry (MS)

Mass spectrometry (MS) is an analytical technique that is used for the identification of analytes using the mass-charge (m/z) ratio of ionized compounds (Glish et al., 2003). A mass spectrometer is an instrument that basically consists of two elements; i) the source of ionization (eg. electrospray, plasma, laser); and ii) one or more analyzers (TOF, quadrupole) (Glish et al., 2003).

The most commonly used mass spectrometry techniques for lipopeptide analysis is matrix-assisted laser desorption-ionization (MALDI) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) (Table 2).

Table 2. Methods of identification and quantification of lipopeptides of strains of the genus Bacillus.

MALDI is a mass spectrometry method that allows the identification of compounds without structurally affecting them (El-Aneed et al., 2009). In MALDI, the sample to be analyzed is co-crystallized with a matrix compound, which is typically an organic acid that has little UV light absorption. The mixture is subsequently exposed to laser radiation, which results in the vaporization of the matrix compound that adheres to the analyte.

The matrix compound plays a key role in strongly absorbing laser energy, indirectly causing the analyte to evaporate (Hillenkamp et al., 1991). The matrix compound also serves as a donor and acceptor of protons and forces the analyte to ionize positively or negatively. MALDI is especially useful for knowing the molecular weight of purified lipopeptides, because they do not undergo structural changes during ionization.

However, this technique has also been used to know the lipopeptide profile of Bacillus extracts (Sajitha et al., 2016). ESI-MS/MS is a very complete mass spectrometry method to identify lipopeptides. When ESI-MS/MS is coupled to HPLC (HPLC-ESI-MS/MS), it allows simultaneous separation, identification and quantification of lipopeptide samples, being the most complete instrument for lipopeptide analysis (Ma et al., 2016). The quantification and separation of lipopeptides with this instrument has the same principle as HPLC, since this unit is coupled to a mass spectrometer.

The identification of lipopeptides using HPLC-ESI-MS/MS can be done using lipopeptide standards by a comparison between m/z values. However, the really useful thing about using this system is that it allows the identification of lipopeptides without the use of standards (Dunlap et al., 2019). This is achieved because with the HPLC-ESI-MS/MS system it is possible to simultaneously obtain the primary mass (MS1) of a compound - usually the intact compound in the form of an ion - and its secondary mass (MS2), generated from the compound fragmentation.

Fragmentation of the compound results in a specific mass spectrum for that molecule, which can be compared to a database such as Pubchem and Metlin, to make the compound annotation (dereplication). For example, with HPLC-ESI-MS/MS, it was possible to identify more than 10 homologs of fengicins and surfactins, without the use of commercial standards (Rangarajan et al., 2014).

Nuclear magnetic resonance imaging (NMR)

Nuclear magnetic resonance imaging (NMR) is a technique that can provide structural information of molecules in solution with a high resolution (De Faria et al., 2011). The determination of structures by NMR can be divided into the following steps: i) establish suitable conditions for recording spectra; ii) measuring a series of 1D (1H and 13C) or 2D (eg. COZY, TOCSY and ROSY) NMR spectra; iii) integrate cross peaks and transformation into upper distance limits (calibration); and iv) evaluation of the quality of the molecular structure (Biniarz et al., 2016). NMR is a method used to identify lipopeptides.

This is a tool that allows even determining structural variants in lipopeptides, such as amino acid changes in the peptide motif. For example, Volpon et al. (2000) determined variations in the lipopeptide piplastatin using NMR, named accordingly as piplastatin A and piplastatin B. With this tool they identified that these lipopeptides differ by one amino acid at position 6 of the peptide motif.

Infrared transmission with Fourier transform (FT-IR)

Fourier transform infrared transmission spectroscopy (FT-IR) is a fast and economical method to characterize the chemical structure of lipopeptides and identify the functional groups present in these compounds (Walker et al., 2009). FT-IR is a physicochemical method based on the measurement of the vibrations of a molecule excited by infrared radiation (IR) at a specific wavelength. Despite some differences in FT-IR spectra between lipopeptides and experimental conditions, the FT-IR instrument is commonly used in a range between approximately 4 000 and 400 cm (Kong, 2007).

This method is used to identify lipopeptides at the family level. For example, this technique was used successfully to identify surfactins in Bacillus circulans. The latter was achieved by comparing FT-IR spectra between a surfactin standard and a mixture of purified lipopeptides from B. circulans (Son et al., 2010).

Conclusions

Currently, lipopeptides produced by strains of the genus Bacillus represent a promising and sustainable strategy for use as biopesticides in agro-systems. This will allow both the protection of crops against pathogens, with the consequent decrease in the use of synthetic fungicides and the negative effects (economic, environmental and health) associated with them. However, the wide use of lipopeptides in the agricultural, pharmaceutical, food sector, among others, depends on the development of chemical and analytical processes for the extraction, identification, quantification and production of said bioactive compounds, efficiently and economically.

Acknowledgments

The authors are grateful for the funding granted by CONACYT to project 257246 ‘Interaction wheat x plant growth promoting microorganisms: identifying genes with agro-biotechnological potential’. In addition, the master's scholarship awarded to Valeria Valenzuela Ruiz.

Cited literature

Abdel-Mawgoud, A. M.; Aboulwafa, M. M. and Hassouna, N. A-H. 2008. Characterization of Surfactin Produced by Bacillus subtilis Isolate BS5. Appl. Biochem. Biotechnol. 150(3):289-303. https://doi.org/10.1007/s12010-008-8153-z.

Alajlani, M.; Shiekh, A.; Hasnain, S. and Brantner, A. 2016. Purification of Bioactive Lipopeptides Produced by Bacillus subtilis Strain BIA. Chromatographia. 79(21-22):1527-1532. https://doi.org/10.1007/s10337-016-3164-3.

Biniarz, P.; Łukaszewicz, M. and Janek, T. 2016. Screening concepts, characterization and structural analysis of microbial-derived bioactive lipopeptides: a review. Critical Rev. Biotechnol. 37(3):393-410. https://doi.org/10.3109/07388551.2016.1163324.

Ciura, K.; Dziomba, S.; Nowakowska, J. and Markuszewski, M. J. 2017. Thin layer chromatography in drug discovery process. J. Chromatogr. A. 1520:9-22. http://dx.doi.org/10.1016/j.chroma.2017.09.015.

Coutte, F.; Lecouturier, D.; Dimitrov, K.; Guez, J. S.; Delvigne, F.; Dhulster, P. and Jacques, P. 2017. Microbial lipopeptide production and purification bioprocesses, current progress and future challenges. Biotechnol. J. 12(7):1-10. http://dx.doi.org/10.1002/ biot.201600566.

De Faria, A. F.; Teodoro-Martinez, D. S.; de Oliveira-Barbosa, G. N.; Gontijo-Vaz, B.; Serrano-Silva, I.; Simone-GarcÍa, J.; Rogério-Tótola, M.; Eberlin, M. N.; Grossman, M.; Luiz Alves, O. and Durrant, L. R. 2011. Production and structural characterization of surfactin (C14/Leu7) produced by Bacillus subtilis isolate LSFM-05 grown on raw glycerol from the biodiesel industry. Process Biochem. 46(10):1951-1957. https://doi.org/10.1016/ j.procbio.2011.07.001.

de los Santos-Villalobos, S.; Parra-Cota, F. I.; Herrera-Sepúlveda, A.; Valenzuela-Aragón, B. and Estrada-Mora, J.C. 2018. Colección de microorganismos edáficos y endófitos nativos para contribuir a la seguridad alimentaria nacional introducción. Rev. Mex. Cienc. Agríc. 9(1):191-202. http://dx.doi.org/10.29312/remexca.v9i1.858.

de los Santos Villalobos, S.; Robles, R. I.; Parra Cota, F. I.; Larsen, J.; Lozano, P. and Tiedje, J. M. 2019. Bacillus cabrialesii sp. nov., an endophytic plant growth promoting bacterium isolated from wheat (Triticum turgidum subsp. durum) in the Yaqui Valley, Mexico. Inter. J. Syst. Evol. Microbiol. 9(1):191-202. https://doi.org/10.1099/ijsem.0.003711.

de Souza, C. G.; Martins, F. I. C. C.; Zocolo, G. J.; Figueiredo, J. E. F.; Canuto, K. M. and de Brito, E. S. 2018. Simultaneous quantification of lipopeptide isoforms by UPLC-MS in the fermentation broth from Bacillus subtilis CNPMS22. Anal. Bioanal. Chem. 410(26):682-736. https://doi.org/10.1007/s00216-018-1281-6.

Díaz-Rodríguez, A.; Parra-Cota, F. I.; Santoyo, G. and de los Santos-Villalobos, S. 2019 Chlorothalonil tolerance of indole producing bacteria associated to wheat (Triticum turgidum L.) rhizosphere in the Yaqui Valley, Mexico. Ecotoxicology. 28(5):569-577. https://doi.org/10.1007/s10646-019-02053-x.

Dunlap, C. A.; Bowman, M. J. and Rooney, A. P. 2019. Iturinic lipopeptide diversity in the bacillus subtilis species group-important antifungals for plant disease biocontrol applications. Front Microbiol. 10:1794. https://doi.org/10.3389/fmicb.2019.01794.

El-Aneed, A.; Cohen, A. and Banoub, J. 2009. Mass spectrometry, review of the basics: Electrospray, MALDI, and commonly used mass analyzers. Appl Spectrosc Rev. 44(3):210-230. https://doi.org/10.1080/05704920902717872.

FAO. 2017. Food and Agriculture Organization. Introduction and overview. In: Bruinsma, J. (Ed.). World agriculture: towards 2015/2030: A FAO perspective. 1: 120 Pentonville Road London, N1 9JN, UK: Earthscan Publications Ltd. 1-28 p. http://www.fao.org/3/a- y4252e.pdf.

FAO. 2018. Food and Agriculture Organization (FAOSTAT). Pesticides. Food and Agriculture Organization of the United Nations (FAO) http://www.fao.org/faostat/en/#data/ EP/visualize.

Gálvez-Gamboa, G. T.; Sánchez-Servín, M. R.; Parra-Cota, F.; García-Pereyra, J.; Aviña-Martínez. G. N. and Santos-Villalobos, S. 2018. Pesticides in Mexican agriculture and promissory alternatives for their replacement. Biológico Agropecuaria Tuxpan. 7(11):1977-1991.

García-Gutiérrez, C. and Rodríguez-Meza, G. D. 2012. Problemática y riesgo ambiental por el uso de plaguicidas en Sinaloa Ra Ximhai. 8(3):1-10. http://www.redalyc.org/articulo.oa? id=46125177005.

Geissler, M.; Oellig, C.; Moss, K.; Schwack, W.; Henkel, M. and Hausmann, R. 2017. High-performance thin-layer chromatography (HPTLC) for the simultaneous quantification of the cyclic lipopeptides Surfactin, Iturin A and Fengycin in culture samples of Bacillus species. J. Chrom. B: 1044: 214-224. https://doi.org/10.1016/j.jchromb.2016.11.013.

Glish, G. L. and Vachet, R. W. 2003. The basics of mass spectrometry in the twenty-first century. Nat. Rev. Drug Discov. 2(2):140-150. http://dx.doi.org/10.1038/nrd1011.

Gómez, M.; Alarcón, A.; León, M.; Oehlschlager, C. and Solórzano, L. 2018. Comercialización de agentes de control biológico. In: Corporación Colombiana de Investigación Agropecuaria, ed., Control biológico de fitopatógenos, insectos y ácaros, 2nd ed. Mosquera, Colombia: Alba Marina Cotes. 762-793 pp.

Grady, E. N.; MacDonald, J.; Ho, M. T.; Weselowski, B.; McDowell, T.; Solomon, O.; Renaud, J. and Yuan, Z. 2019. Characterization and complete genome analysis of the surfactin-producing, plant-protecting bacterium Bacillus velezensis 9D-6. BMC Microbiology. 19(1):5-6. https://doi.org/10.1186/s12866-018-1380-8.

Gupta, P. K.; Chand, R.; Vasistha, N. K.; Pandey, S. P.; Kumar, U.; Mishra, V. K. and Joshi, A. K. 2018. Spot blotch disease of wheat: the current status of research on genetics and breeding. Plant Pathol. 67(3):508-531. https://doi.org/10.1111/ppa.12781.

Hashem, A.; Tabassum, B. and Fathi Abd_Allah, E. 2019. Bacillus subtilis: A plant-growth promoting rhizobacterium that also impacts biotic stress. Saudi J. Biol. Sci. https://doi.org/10.1016/j.sjbs.2019.05.004.

Hillenkamp, F.; Karas, M.; Beavis, R. C. and Chait, B. T. 1991. Matrix assisted laser desorption/ionization mass spectrometry of biopolymers. Anal Chem. 6324:1193A-1203A. https://doi.org/10.1021/ac00024a002.

Hmidet, N.; Ben Ayed, H.; Jacques, P. and Nasri, M. 2017. Enhancement of surfactin and fengycin production by Bacillus mojavensis A21: application for diesel biodegradation. BioMed Research International. 1-8 pp. https://doi.org/10.1155/2017/5893123.

Hsieh, F. C.; Li, M. C.; Lin, T. C. and Kao, S. S. 2004. Rapid detection and characterization of surfactin-producing Bacillus subtilis and closely related species based on PCR. Curr. Microbiol. 49(3):186-191. https://doi.org/10.1007/s00284-0044314-7.

Isa, M.; Coraglia, D.; Frazier, R. and Jauregi, P. 2007. Recovery and purification of surfactin from fermentation broth by a two-step ultrafiltration process. J. Membr. Sci. 296(1-2):51-57. https://doi.org/10.1016/j.memsci.2007.03.023.

Jamshidi-Aidji, M.; Dimkić, I.; Ristivojević, P.; Stanković, S. and Morlock, G. E. 2019. Effect-directed screening of Bacillus lipopeptide extracts via hyphenated high-performance thin-layer chromatography. J. Chromatogr. A. 1605:460366. https://doi.org/10.1016/j.chroma. 2019.460366.

Jauregi, P.; Coutte, F.; Catiau, L.; Lecouturier, D. and Jacques, P. 2013. Micelle size characterization of lipopeptides produced by B. subtilis and their recovery by the two-step ultrafiltration process. Sep. Purif. Technol. 104:175-182. https://doi.org/10.1016/j.seppur.2012.11.017.

Kinsella, K.; Schulthess, C. P.; Morris, T. F. and Stuart, J. D. 2009. Rapid quantification of Bacillus subtilis antibiotics in the rhizosphere. Soil Biol. Biochem. 41(2):374-379. http://dx.doi.org/10.1016/j.soilbio.2008.11.019.

Kong, J. and Yu, S. 2007. Fourier transform infrared spectroscopic analysis of protein secondary structures. Acta Biochim. Biophys. Sin. 39(8):549-559. https://doi.org/10.11648/ j.ajaic.20150101.11.

Luna-bulbarela, A.; Tinoco-valencia, R.; Corzo, G.; Kazuma, K; Katsuhiro, K; Galindo, E.; et al. Effects of bacillomycin D homologues produced by Bacillus amyloliquefaciens 83 on growth and viability of Colletotrichum gloeosporioides at different physiological stages. Biol Control. 2018. 127:145-54. https://doi.org/10.1016/j.biocontrol.2018.08.004.

Ma, Y.; Kong, Q.; Qin, C.; Chen, Y.; Yujie, C.; Lv, R. and Zhou, G. 2016. Identification of lipopeptides in Bacillus megaterium by two-step ultrafiltration and LC-ESI-MS/MS. AMB Express. 79(6):1-15. https://doi.org/10.1186/s13568-016-0252-6.

Malviya, R.; Bansal, V.; Pal, O. P. and Sharma, P. K. 2010. High performance liquid chromatography: a short review. J. Glob. Pharma. Technol. 2(5):22-26.

Ongena, M. and Jacques, P. 2008. Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trends in Microbiology. 16(3):115-125. https://doi.org/10.1016/ j.tim.2007.12.009.

Parra-Cota, F. I.; García-Pereyra, J.; Aviña-Martínez, G. N. and de los Santos-Villalobos, S. 2018. First report of Fusarium wilt on Citrus sinensis var. Valencia in the Yaqui Valley, Mexico. Rev. Mex. Fitopatol. 37(1):193-201. https://doi.org/10.18781/r.mex.fit.1810-3.

Poole, C. F. 2003. New trends in solid-phase extraction. TrAC-Trends Anal Chem. 22(6):362-73. https://doi.org/10.1016/S0165-9936(03)00605-8.

Rangarajan, V.; Dhanarajan, G. and Sen, R. 2014. Improved performance of cross-flow ultrafiltration for the recovery and purification of Ca+2 conditioned lipopeptides in diafiltration mode of operation. J. Memb. Sci. 454(1):436-443.

Razafindralambo, H.; Paquot, M.; Hbid, C.; Jacques, P.; Destain, J. and Thonart, P. 1993. Purification of antifungal lipopeptides by reversed-phase high-performance liquid chromatography. J. Chromatogr. A 639(1):81-85. https://doi.org/10.1016/0021-9673(93)83091-6.

Robles-Montoya, R. I.; Parra-Cota, F. I. and de los Santos-Villalobos, S. 2019. Draft genome sequence of Bacillus megaterium TRQ8, a plant growth-promoting bacterium isolated from wheat (Triticum turgidum subsp. durum) rhizosphere in the Yaqui Valley, Mexico. 3 Biotech. 9(1):200-205. https://doi.org/10.1007/s13205-019-1726-4.

Robles-Montoya, R. I.; Chaparro-Encinas, L. A.; Parra-Cota, F. I. and de los Santos-Villalobos, S. 2020. Mejorando rasgos biométricos de plántulas de trigo con la inoculación de un consorcio nativo de Bacillus. Rev. Mex. Cienc. Agríc. 11(1):229- 235.

Sajitha, K. L.; Dev, S. A. and Maria Florence E. J. 2016. Identification and characterization of lipopeptides from Bacillus subtilis b1 against sapstain Fungus of rubberwood through MALDI-TOF-MS and RT-PCR. Curr Microbiol. 73(1):46-53. https://doi.org/10.1007/ s00284-016-1025-9.

Santoyo, G.; Sánchez-Yáñez, J. M. and de los Santos-Villalobos, S. 2019. Methods for detecting biocontrol and plant growth-promoting traits in Rhizobacteria. In: Reinhardt D., Sharma A. (eds) Methods in Rhizosphere Biology Research. 133-149. https://doi.org/10.1007/978-981-13-5767-1-8.

Sen, R. and Swaminathan, T. 2005. Characterization of concentration and purification parameters and operating conditions for small-scale recovery of surfactin. Process Biochem.  40(9):2953-2958. https://doi.org/10.1016/j.procbio.2005.01.014.

Sivapathasekaran, C.; Das, P. and Mukherjee, S.; et al. 2010. Marine bacterium derived lipopeptides: characterization and cytotoxic activity against cancer cell lines. Int. J. Pept. Res. Ther. 16(4):215-222. https://doi.org/10.1007/s10989-010-9212-1.

Son, S.; Ko, S. K.; Jang, M.; Kim, J. W.; Kim, G. S. and Lee, J. K. 2016. New cyclic lipopeptides of the iturin class produced by saltern-derived Bacillus sp. KCB14S006. Mar Drugs. 14(4):72-73. https://doi.org/10.3390/md14040072.

Su, Y. T.; Liu, C.; Long, Z.; Ren, H. and Guo, X. H. 2018. Improved Production of Spores and Bioactive Metabolites from Bacillus amyloliquefaciens in Solid-state Fermentation by a Rapid Optimization Process. Probiotics Antimicrob. Proteins. 11:921-930. https://doi.org/10.1007/s12602-018-9474-z.

Valenzuela-Aragon, B.; Parra-Cota, F. I.; Santoyo, G.; Arellano-Wattenbarger, G. L. and de los Santos-Villalobos, S. 2019. Plant-assisted selection: a promising alternative for in vivo identification of wheat (Triticum turgidum L. subsp. Durum) growth promoting bacteria. Plant and Soil. 435(1-2):367-384. https://doi.org/10.1007/s11104-018-03901-1.

Valenzuela-Ruiz, V.; Robles- Montoya, R. I.; Parra-Cota, F. I.; Santoyo, G.; Orozco-Mosqueda, M. C.; Rodríguez- Ramírez, R. and de los Santos-Villalobos, S. 2019. Draft genome sequence of Bacillus paralicheniformis TRQ65, a biological control agent and plant growth promoting bacterium isolated from wheat (Triticum turgidum subsp. durum) rhizosphere in the Yaqui Valley, Mexico. 3 Biotech. 9(1):436-442. https://doi.org/10.1007/s13205-019-1972-5.

Villa-Rodríguez, E.; Lugo-Enríquez, C.; de los Santos-Villalobos, S.; Para-Cota, F. I.; Figueroa-López, P. 2016. First report of Cochliobolus sativus causing spot blotch on durum wheat (Triticum durum) in The Yaqui Valley, Mexico. Plant Dis. 100(11):2329 https://doi.org/http://dx.doi.org/10.1094/PDIS-05- 16- 0634-PDN.

Villa-Rodríguez, E.; Parra-Cota, F.; Castro-Longoria, E.; López-Cervantes, J. and de los Santos-Villalobos, S. 2019. Bacillus subtilis TE3: a promising biological control agent against Bipolaris sorokiniana, the causal agent of spot blotch in wheat (Triticum turgidum L. subsp. durum). Biol. Control. 132:135-143. https://doi.org/10.1016/j.biocontrol.2019.02.012.

Villarreal-Delgado, M. F.; Villa-Rodríguez, E. D.; Cira-Chávez, L. A.; Estrada-Alvarado, M. I.; Parra-Cota, F. I. and De los Santos-Villalobos, S. 2018. The genus Bacillus as a biological control agent and its implications in agricultural biosecurity. Mexican J. Phytopathol. 36(1):95-130. https://doi.org/10.18781/R.MEX.FIT.1706-5.

Volpon, L.; Besson, F. and Lancelin, J. M. 2000. NMR structure of antibiotics plipastatins A and B from Bacillus subtilis inhibitors of phospholipase A2. FEBS Lett. 485(1):76-80.

Walker, A. M.; Yu, P.; Christensen, C. R.; Christensen, D. and McKinnon, J. J. 2009. Fourier transform infrared microspectroscopic analysis of the effects of cereal type and variety within a type of grain on structural makeup in relation to rumen degradation kinetics. J. Agr. Food. Chem. 57(15): 6871–6878. https://doi.org/10.1021/jf901461u.

Yuan, J.; Raza, W.; Huang, Q. and Shen, Q. 2011. Quantification of the antifungal lipopeptide iturin A by high performance liquid chromatography coupled with aqueous two-phase extraction. J. Chromatogr. B Anal. Technol. Biomed Life Sci. 879(26):2746-2750. http://dx.doi.org/10.1016/j.jchromb.2011.07.041.