rmfiRevista mexicana de fitopatologíaRev. mex. fitopatol0185-33092007-8080Sociedad Mexicana de Fitopatología A.C.10.18781/R.MEX.FIT.2024-18ArticlesPotential biological control Mechanisms of Bacillus paralicheniformis TRQ65 against phytopathogenic fungiValenzuela RuizValeria¹Parra CotaFannie Isela² SantoyoGustavo³Estrada AlvaradoMaría Isabel⁴Cira ChávezLuis Alberto⁵Castro LongoriaErnestina⁶de los Santos VillalobosSergio⁷ ¹Instituto Tecnológico de Sonora, 5 de febrero 818 sur, 85000, Cd. Obregón, Sonora, México.Instituto Tecnológico de SonoraInstituto Tecnológico de Sonora85000Cd. ObregónSonoraMexico²Campo Experimental Norman E. Borlaug, 85000, Cd. Obregón, Sonora, México.Campo Experimental Norman E. BorlaugCampo Experimental Norman E. Borlaug85000Cd. ObregónSonoraMexico³Instituto de Investigaciones Químico Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacán, México.Instituto de Investigaciones Químico BiológicasInstituto de Investigaciones Químico BiológicasMoreliaMichoacánMexico⁴Instituto Tecnológico de Sonora, 5 de febrero 818 sur, 85000, Cd. Obregón, Sonora, México.Instituto Tecnológico de SonoraInstituto Tecnológico de Sonora85000Cd. ObregónSonoraMexico⁵Instituto Tecnológico de Sonora, 5 de febrero 818 sur, 85000, Cd. Obregón, Sonora, México.Instituto Tecnológico de SonoraInstituto Tecnológico de Sonora85000Cd. ObregónSonoraMexico⁶Departamento de Microbiología, Centro de Investigación Científica y de Educación Superior de Ensenada, Baja California (CICESE), Ensenada, México.Centro de Investigación Científica y de Educación Superior de EnsenadaCentro de Investigación Científica y de Educación Superior de EnsenadaEnsenadaMexico⁷Instituto Tecnológico de Sonora, 5 de febrero 818 sur, 85000, Cd. Obregón, Sonora, México.Instituto Tecnológico de SonoraInstituto Tecnológico de Sonora85000Cd. ObregónSonoraMexico181220242024424RMEXFIT2024180107202428112024This is an open-access article distributed under the terms of the Creative Commons Attribution LicenseAbstract:Background/Objetive.
Bacillus paralicheniformis TRQ65 was isolated from wheat (Triticum turgidum subsp. durum) rhizosphere in commercial fields in the Yaqui Valley, Mexico. This strain was one of the most abundant bacteria in the rhizosphere. The objective of this study is to explore the potential biological control action mechanisms of Bacillus paralicheniformis TRQ65 against phytopathogenic fungi of agricultural importance, through genome sequencing and mining.
Materials and methods.
The biocontrol activity of this strain was quantified through in vitro dual assays evaluating inhibition zones against 11 agronomically important fungi. A whole-genome analysis was conducted as genomic mining to evaluate its potential for biological control.
Results.
Strain TRQ65 showed biocontrol activity against 45% of the studied fungi, where the highest inhibition was against Botrytis cinerea, 43.8% ± 9% on day 5. Based on genome sequencing and mining (antiSMASH), this bioactivity could be associated with the biosynthesis of lichenysin, bacillibactin, and/or fengycin.
Conclusion.
This research provides the first insight into the potential biological control activity of strain TRQ65. Further studies need to be carried out to validate Bacillus paralicheniformis TRQ65 as an active ingredient in sustainable bacterial inoculants for eco-friendly agriculture.
Keywords:Biological Control AgentsAgricultureBacillusFengycinsLichenysinsBacillibactin.Introduction
Sustainable agricultural practices are crucial since the growing population is estimated to reach ~ 10 billion people by 2050 (FAO, 2018). The conventional methods for food production have highly impacted agricultural land availability, agronomical soil degradation, the loss of agrochemical efficiency, and increased incidence of plant diseases (Lowry et al., 2019; Pellegrini and Fernández, 2018). Plant diseases generate yield losses of 15-25% but in some cases, up to 85% of crop yield have also been reported (Gupta et al., 2018; Mehta 2014). The most important phytopathogenic organisms agronomically are usually fungi, representing approximately 70% of plant diseases (FAO, 2016). Thus, among the most important phytopathogenic fungi due to their incidence, severity, and economic losses in subtropical and tropical regions, especially in Mexico, are Rhizoctonia, Botryosphaeria, Bipolaris, and Fusarium (Rodríguez-Guadarrama et al., 2018; Villa-Rodriguez et al., 2021; Mondragón-Flores et al., 2021).
Plant disease management worldwide in traditional agriculture is mainly focused on the intensification of the applied pesticides. Where, the use of pesticides worldwide has increased by almost 100% from 1992 to 2016 (FAO, 2018), while in Mexico the increase has been 76% in the same period (Roser et al., 2019). However, only about 0.1% reaches the agricultural crop (Gill et al., 2014); thus, the remaining pesticide is lost, resulting in negative impacts including environmental degradation, destruction of non-target organisms, the contamination of soils and aquifers with recalcitrant residues, soil degradation, and salinization (Tudi et al., 2021). The loss of biodiversity and chemical and microbial degradation in soil result in an increase in sensitivity and/or tolerance to potential external effects generating serious economic and environmental impacts (Chappell et al., 2019; Gálvez-Gamboa et al., 2018). Therefore, sustainable yield-focused strategies must be developed under an integral ecosystem approach to ensure the efficiency, quality, and yield of crops (Dietemann et al., 2024).
|In this sense, biopesticides have been a topic of interest worldwide (Thaku et al., 2024). In Mexico, 6.2% of commercial pesticides used are of biological origin (biological control agents), among which the most frequently used biological control agents are Bacillus thuringiensis, Phaecilomyces fumoroseus and Bauveria bassiana (Pérez-Olvera et al., 2011). The genus Bacillus occupies 85% of the commercially available bacterial biopesticides (Villarreal-Delgado et al., 2017). Biological control agents are beneficial organisms that help reduce the harmful effects of plant pathogens. They achieve this through antagonistic actions using various mechanisms; such as mycoparasitism, production of lytic enzymes, induction of systemic response of the plant, and/or production of δ-endotoxins, siderophores, and lipopeptides among others (Valenzuela Ruiz et al., 2020).
In recent decades, the introduction of genomic, phylogenomic, and bioinformatics tools has contributed significantly to a better understanding of the interactions between microorganisms and plants (Sharma et al., 2020). The study of microbial species at the genetic level has not only allowed the identification of mechanisms and genes involved in root colonization, the adaptation of the host, and the improvement of plant growth (Sheppard et al., 2018; Youseif, 2018) but also, it has allowed the realization of very important advances in the characterization and taxonomic affiliation of taxa associated with beneficial effects in crops (Jagadeesan et al., 2019). Thus, the objective of this study is to explore the potential biological control action mechanisms of Bacillus paralicheniformis TRQ65 against phytopathogenic fungi of agricultural importance, through genome sequencing and mining.
Materials and MethodsMicrobial strains and culture conditions
Bacillus paralicheniformis TRQ65 was isolated from the wheat (Triticum turgidum subsp. durum) rhizosphere in commercial fields of the Yaqui Valley, Mexico (27.3692°, 110.3886°). The strain is stored in the Colección de Microorganismos Edáficos y Endófitos Nativos (COLMENA) in Mexico (www.itson.edu.mx/ COLMENA) (de los Santos Villalobos et al., 2021). To prepare bacterial cultures, 1 x 104 colony-forming units (CFU) of TRQ65 were inoculated into 10 mL of Nutrient Broth (NB). After 24 hours of incubation at 28 °C and 180 rpm, the suspension was centrifuged at 4000 rpm for 5 minutes. The bacterial pellet was then re-suspended in sterile distilled water (autoclaved at 121 °C and 15 psi for 15 minutes) and adjusted to a concentration of 1 x 108 CFU/mL (Villa-Rodriguez et al., 2021). The antagonistic activity of TRQ65 was tested against 11 phytopathogenic fungi: Fusarium brachygibbosum, Botryosphaeria rhodina, Penicillium expansum, Penicillium crustosum, Geotrichum candidum, and Cladosporium sp. (provided by the Michoacan University of San Nicolás de Hidalgo); Botrytis cinerea, Mucor rouxii, and Rhizoctonia solani (provided by the Ensenada Center for Scientific Research and Higher Education); and Neocosmospora solani and Bipolaris sorokiniana (from COLMENA). The fungi were grown on Potato Dextrose Agar (PDA) and incubated in light for 120 hours at 28°C. Spores were collected using distilled water with 0.01% Tween 20 and adjusted to a concentration of 1 x 108 spores/mL (Villa-Rodriguez et al., 2019).
In vitro dual confrontation assay of Bacillus paralicheniformis TRQ65 against phytopathogenic fungi A qualitative dual assay was carried out on Petri dishes containing Potato Dextrose Agar (PDA), where strain TRQ65 was tested against the fungal strains listed earlier. Each fungal strain was inoculated with 1 x 108 spores at the center of the Petri dish, while 1 x 108 CFU of TRQ65 was placed at equidistant points, 2 cm away from the fungal inoculum (de los Santos Villalobos et al., 2012). The inhibition zones formed around the fungal pathogens were measured quantitatively over 7 days, with three replicates for each condition. Statistical analysis was performed using Statgraphics Plus version 5.1 software. Data were analyzed using one-way ANOVA and Tukey’s Range Test (HSD) (p ≥ 0.05), and the results are presented as the mean of the replicates or independent experiments.
Bacterial genome sequencing and mining
Genomic DNA was isolated from a fresh culture of strain TRQ65, which was grown in Nutrient Broth (NB) at 28°C with shaking at 180 rpm for 24 hours, reaching a concentration of 1 x 108 CFU/mL, as described by Valenzuela- Aragon et al. (2019). The extracted DNA was then sequenced using the Illumina MiSeq platform (2 x 300 bp) at a 30x coverage depth, and the Next Generation Sequencing library preparation was carried out by using the TruSeq DNA Nano Kit for Illumina® Platforms, following the manufacturer’s specified protocols (LANGEBIO-CINVESTAV unidad Irapauto). The quality of the sequencing reads was evaluated using FastQC version 0.11.5 (Andrews, 2010). To remove adapter sequences and low-quality bases, Trimmomatic version 0.32 (Bolger et al., 2014) was applied. For de novo assembly, SPAdes version 3.10.1 (Bankevich et al., 2012) was used with the “--careful” option to minimize errors. The assembled contigs were arranged using Mauve Contig Mover version 2.4.0 (Darling et al., 2004). To detect plasmids within the genome, PLACNETw (https://castillo.dicom.unican.es/ upload/) (Vielva et al., 2017) was utilized. Genome annotation was performed using the RAST server version 2.0 (http://rast.nmpdr.org) (Aziz et al., 2008; Overbeek et al., 2013), employing the RASTtk pipeline with default settings. Contigs were scaffolded using MeDuSa, a multi-draft-based scaffolder (Bosi et al., 2015). Lastly, antiSMASH version 5.0 (https://antismash.secondarymetabolites.org) (Blin et al., 2019) was used to identify potential biosynthetic gene clusters related to biocontrol activity in the genome of the studied bacterial strains.
Results and Discussion<bold>In vitro dual assay of <italic>Bacillus paralicheniformis</italic> TRQ65 against phytopathogenic fungi</bold>
Strain TRQ65 showed biocontrol activity against 45% of the studied phytopathogenic fungi (Fusarium brachygibbosum, Neocosmospora solani, Botryosphaeria rhodina, Botrytis cinerea, and Bipolaris sorokiniana) (Table 1). Bacillus paralicheniformis TRQ65 inhibited the mycelial growth of Botrytis cinerea in 43.8% ± 9%. This biocontrol activity started on day 4 and decreased until day 7. Furthermore, Świątczak et al. (2023), reported Bacillus paralicheniformis as a biological control agent against Botrytis cinerea in canola plants with 13% on inhibition. Similarly, Asif et al. (2023), evidenced biological control of B. paralicheniformis against Elsinoe sp., Phytophthora capsica, Botrytis cinerea, Rhizoctonia solani, Coniellla sp., Thielaviopsis sp., and Phytophthora sojae attributing its phytopathogen inhibition mainly to fengycin and bacillibactin production.
Inhibition zone (percentage) of <italic>Bacillus paralicheniformis</italic> TRQ65 against agriculturally important fungal plant pathogens.
Phytopathogen
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
Fusarium brachygibbosum
ND
ND
ND
13% ± 0%
16% ± 1%
ND
ND
Neocosmospora solani
22% ± 3%
17% ± 1%
19% ± 5%
7.6% ± 3%
ND
ND
ND
Botryosphaeria rhodina
ND
ND
7% ± 1%
ND
ND
ND
ND
Botrytis cinerea
ND
ND
ND
ND
43.8% ± 9%
34% ± 5%
31% ± 5%
Bipolaris sorokiniana
ND
ND
ND
14% ± 3%
11% ± 2%
8% ± 0%
ND
Tukey HSD test (n=3, p ≥ 0.05). The variations correspond to the standard deviation. ND = Not detected.
|On the other hand, the biocontrol of B. paralicheniformis TRQ65 against Fusarium brachygibbosum, Neocosmospora solani, Botryosphaeria rhodina, and Bipolaris sorokiniana showed variable results, where this activity started being quantifiable on day four, one, three, and four, respectively, and disappear on day five, four, three, and six, respectively (Table 1). In this sense, De la Lastra et al. (2021), reported B. paralicheniformis ability to inhibit the fungal radial growth of the pathogenic Fusarium isolates, reaching from 73 to 84% inhibition. This biocontrol activity may be associated with diffusible metabolites biosynthesized by strain TRQ65 (Kulimushi et al., 2017; Cawoy et al., 2015), where its inhibition success is highly dependent on the interacting fungal species (Andrić et al., 2020; Beauvais and Latgé 2018). However, research has shown that phytopathogens exhibit remarkable adaptability to antimicrobial metabolites produced by biological control agents, despite initial growth inhibition (Bonaterra et al., 2022). Initially, these phytopathogens may be susceptible to antibiosis or competitive exclusion by biocontrol agents (Khan et al., 2018), then they exert sophisticated mechanisms to counteract the initial inhibitory effects of biological control agents, including the synthesis of degradative enzymes capable of catabolizing or modifying inhibitory compounds, the upregulation of efflux pump systems to extrude toxic metabolites, and alterations in target site morphology or biochemistry to reduce susceptibility (Köhl et al., 2019). For instance, some Fusarium species have demonstrated the ability to produce detoxifying enzymes that neutralize antifungal metabolites produced by biological control agents (Costa Almeida et al., 2022). Thus, the efficacy of biological control can be compromised over time due to various factors, including environmental fluctuations, depletion of inhibitory compounds, complex microbial interactions within the rhizosphere, and the development of resistance by phytopathogens (Lal et al., 2020). The dynamic nature of these interactions emphasizes the importance of unraveling the molecular mechanisms behind pathogen adaptation to explain the observed loss of efficacy (Barber et al., 2024).
Bacterial genome analysis
During the last decades, biological control bacteria from the genus Bacillus with protective activities toward economically important plant pathogens have been highlighted (Finkel et al., 2017; Fira et al., 2018; Köhl et al., 2019). Thus, several studies have been carried out to explore their potential action modes. In this sense, the genome of B. paralicheniformis TRQ65 was sequenced and mined to identify promising biosynthetic gene clusters associated with its biocontrol activity.
After sequencing, the genome of B. paralicheniformis TRQ65 showed 5,079,308 total reads [2×300 base pairs (bp)]. In the quality analysis and adapter remotion, only 8.42% was dropped. After de novo assembly, a draft genome of strain TRQ65 consisted of 4,475,481 bp; 45.5% G+C content; 676,421 bp N50; 3 L50; and 32 contigs (>200 bp). The ordered and scaffolding generated 25 scaffolds. In addition, no plasmids were observed, to our understanding, the presence of plasmids has not been reported for this species (Vielva et al., 2017).
The RAST server’s genome annotation of B. paralicheniformis TRQ65 showed 91 RNAs, and 4809 predicted coding DNA sequences (CDS) distributed into 340 subsystems. The most abundant subsystem was amino acids and derivatives (341 CDS), followed by carbohydrates (338 CDS), protein metabolism (198 CDS), cofactors, vitamins, prosthetic groups, and pigments (147 genes), nucleosides and nucleotides (101), and dormancy and sporulation (96) (Figure 1). Furthermore, relating to biocontrol secondary metabolism (9 CDS) were identified including the production of auxin biosynthesis (4 CDS). A previous study carried out by Rojas Padilla et al. (2020), signaled that strain TRQ65 can produce indole acetic acid by 39.29 ± 0.30 ppm, and solubilize phosphate by 1.37 ± 0.05 %. In addition, iron acquisition and metabolism (44 CDS), including the production of siderophores (33 CDS) as bacillibactin, was detected in the genome of B. paralicheniformis TRQ65.
Subsystem category distribution of coding DNA sequences (CDS) in the <italic>B. paralicheniformis</italic> TRQ65 genome, following the RAST pipeline.
Fira et al. (2018) state that bacteria belonging to the genus Bacillus are well- known as producers of a wide array of antagonistic compounds of different structures, among the most important are non-ribosomally synthesized peptides and lipopeptides. To explore potential biosynthetic gene clusters associated with biocontrol activity, the genome of B. paralicheniformis TRQ65 was mined by antiSMASH (https://antismash.secondarymetabolites.org) (Blin et al., 2019). In the genome of strain TRQ65, gene clusters associated with biocontrol were detected:
i) lichenysin (100% identity), an anionic lipopeptide biosurfactant with cytotoxic, antimicrobial, and hemolytic activities (Coronel et al., 2017); ii) bacitracin (88% identity), cyclic lipopeptide antibiotic that is active against Gram-positive and some Gram-negative bacteria by inhibiting bacterial cell-wall biosynthesis (Liu et al., 2018); iii) fengycin (93% identity), a lipopeptide with fungi-toxic activity against filamentous fungi, which interact with lipid layers and alter the structure of the cell membrane and permeability (Li et al., 2019); iv) the siderophore bacillibactin (100% identity) a cyclic compound that contains catecholate groups, which have a high affinity for iron (Fe3+), in which it makes a non-suitable environment for phytopathogen colonization as it competes for iron (Félix-Pablos et al., 2022); and v) amyloliquecidin (93% identity) an antimicrobial peptide against Gram-positive bacteria. (Gerst et al., 2022). Based on the action modes of the biosynthetic gene clusters mentioned before, only lichenysin, fengycin and bacillibactin (Figure 2) have shown antifungal activities. Thus, we inferred that these three compounds could be involved in the biocontrol of the studied phytopathogenic fungi, these molecules are now being strongly studied by our team.
A) Bacillibactin structure (National Center for Biotechnology Information, 2024). Bacillibactin is built around a trilactone core formed by the cyclization of three molecules of 2,3-dihydroxybenzoic acid (DHB) linked to a central scaffold of threonine residues. The trilactone ring is formed through ester bonds between the hydroxyl groups of the threonine and the carboxyl groups of DHB. Each DHB moiety in bacillibactin contains catechol (2,3-dihydroxybenzoate) functional groups. These catechol groups are responsible for the high-affinity binding of Fe³⁺. B) Lichenysin structure (National Center for Biotechnology Information, 2024). Lichenysin contains a cyclic peptide ring consisting of 13 amino acid residues. Attached to the cyclic peptide core is a lipid tail, typically a β-hydroxy fatty acid chain. The peptide chain forms a cyclic structure through an amide bond between the carboxyl group of one amino acid and the amine group of another, creating a stable ring structure. C) Fengycin structure (National Center for Biotechnology Information, 2024). Fengycin consists of a decapeptide forming a cyclic structure through a lactone linkage. Attached to the peptide ring is a β-hydroxy fatty acid, which can vary in length, usually between 14 and 18 carbon atoms. The exact sequence of amino acids can vary slightly depending on the specific isoform of fengycin.
Furthermore, bacillibactin is a high-affinity siderophore with a trilactone core composed of 2,3-dihydroxybenzoic acid (DHB) units linked to threonine residues, where catechol groups chelate Fe³⁺ ions with high specificity, forming stable complexes that support essential metabolic processes under iron-limited conditions (Khan et al., 2016; Dimopoulou et al., 2021; Timofeeva et al., 2022). Lichenysin, an amphiphilic lipopeptide surfactant with a cyclic peptide ring and hydrophobic fatty acid chain, exhibits stability under extreme conditions and has demonstrated inhibition of fungal pathogens such as Fusarium oxysporum, Alternaria alternata, Aspergillus fumigatus, Aspergillus niger, and Neocosmospora keratoplastica (Gudiña and Teixeira, 2022; Yeak et al., 2022; Olmedo et al., 2022; Armenova et al., 2024). Fengycins, particularly fengycin A and B, are lipodecapeptides with antifungal activity against filamentous fungi, disrupting fungal cell membranes, perturbing mycelial growth, and inducing systemic resistance in plants. Studies have demonstrated their effectiveness against Botrytis cinerea, Pythium ultimum, and Monilinia fructicola, showcasing their ability to suppress spore germination and cause fungal cytoplasm leakage (Vanittanakom et al., 1986; Koumoutsi et al., 2004; Deleu et al., 2005; Ongena et al., 2005; Liu et al., 2011; Geissler et al., 2019; Andrić et al., 2020). This study provides the first insight into the potential biological control mechanism used by Bacillus paralicheniformis TRQ65 as a biological control agent; however, this study’s results indicate moderate biological control against the studied fungi. Thus, further research is necessary to explore the biological control ability, identified through genomic mining to complement the confrontation analysis results of low inhibition.
Conclusions
Bacillus paralicheniformis TRQ65 is a biological control agent that inhibits the mycelial growth of Fusarium brachygibbosum, Neocosmospora solani, Botryosphaeria rhodina, and Bipolaris sorokiniana. Based on genome mining, this bioactivity could be related to the biosynthesis of lichenysin, fengycin, and/or bacillibactin. Thus, further studies are currently being conducted by our research team to improve the ability of B. paralicheniformis TRQ65 and validate its use as an active ingredient in bacterial inoculants to migrate toward sustainable agriculture.
Acknowledgments
The authors would like to thank the funds granted by the Technological Institute of Sonora (PROFAPI_2024_0001), as well as the members of the Biotechnology Laboratory of the Microbial Resource (www.itson.mx/LBRM).
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Bacillus paralicheniformis TRQ65 fue aislada de la rizósfera del trigo (Triticum turgidum subsp. durum) en campos comerciales en el Valle del Yaqui, México. El objetivo del presente estudio es explorar los posibles mecanismos de acción de control biológico de Bacillus paralicheniformis TRQ65 contra hongos fitopatógenos de importancia agrícola, por medio de la secuenciación y explotación del genoma.
Materiales y Métodos.
La actividad de biocontrol de esta cepa se cuantificó mediante ensayos in vitro de cultivos duales, evaluando zonas de inhibición contra 11 hongos de importancia agronómica. Se realizó un análisis del genoma completo como minería genómica para evaluar su potencial de control biológico.
Resultados.
La cepa TRQ65 mostró actividad de biocontrol contra el 45% de los hongos estudiados, con la mayor inhibición observada contra Botrytis cinerea (43.8% ± 9% al día 5). Basado en la secuenciación y análisis del genoma (antiSMASH), esta bioactividad podría estar asociada con la biosíntesis de lichenisina, bacilibactina y/o fengicina.
Conclusión.
Esta investigación proporciona el primer acercamiento al potencial de actividad de control biológico de la cepa TRQ65. Se necesitan estudios adicionales para validar el uso de Bacillus paralicheniformis TRQ65 como ingrediente activo en inoculantes bacterianos sostenibles para una agricultura amigable con el medio ambiente.
Palabras clave:Agentes de Control BiológicoAgriculturaBacillusFengicinasLiquenisinasBacilibactinaIntroducción
Las prácticas agrícolas sostenibles son fundamentales, ya que se estima que la población mundial alcanzará ~ 10 mil millones de personas para el año 2050 (FAO, 2018). Los métodos convencionales para la producción de alimentos han tenido un gran impacto sobre la disponibilidad de tierras agrícolas, la degradación de los suelos agrícolas, la pérdida de la eficiencia agroquímica y el aumento en la incidencia de las enfermedades vegetales (Lowry et al., 2019; Pellegrini y Fernández, 2018). Las enfermedades generan pérdidas en el rendimiento de entre 15 y 25%, aunque en algunos casos se han reportado pérdidas de hasta 85% (Gupta et al., 2018; Mehta 2014). Los organismos fitopatogénicos más importantes suelen ser hongos, que representan aproximadamente 70% de las enfermedades vegetales (FAO, 2016). Así, algunos de los hongos fitopatógenos de mayor importancia debido a su incidencia, severidad y pérdidas económicas en regiones tropicales y subtropicales, especialmente en México, son Rhizoctonia, Botryosphaeria, Bipolaris y Fusarium (Rodríguez-Guadarrama et al., 2018; Villa-Rodriguez et al., 2021; Mondragón-Flores et al., 2021).
El manejo de las enfermedades vegetales a nivel mundial en la agricultura tradicional se concentra, principalmente, en la intensificación de los pesticidas aplicados. Mientras que el uso de pesticidas a nivel mundial ha aumentado casi al 100% entre 1992 y el 2016 (FAO, 2018), en México, el aumento ha sido del 76% en el mismo periodo (Roser et al., 2019). Sin embargo, solo alrededor del 0.1% llega al cultivo agrícola (Gill et al., 2014). Así, el pesticida restante se pierde, lo cual resulta en impactos negativos, que incluyen la degradación ambiental, la destrucción de organismos no objetivo, la contaminación de suelos y acuíferos con residuos recalcitrantes, la degradación del suelo y la salinización (Tudi et al., 2021). La pérdida de la biodiversidad y la degradación química y microbiana en el suelo resultan en un aumento en la susceptibilidad y/o tolerancia a potenciales efectos externos, lo cual conduce a impactos ambientales y económicos graves (Chappell et al., 2019; Gálvez-Gamboa et al., 2018). Por lo tanto, se deben desarrollar estrategias sostenibles bajo un enofque de ecosistema integral para asegurar la eficiencia, calidad y rendimiento de los cultivos (Dietemann et al., 2024).
En este sentido, los biopesticidas han sido un tema de interés mundial (Thakur et al., 2024). En México, 6.2% de los pesticidas comerciales son de origen biológico (agentes de control biológico), entre los cuales los agentes de control biológico usados con mayor frecuencia son Bacillus thuringiensis, Phaecilomyces fumoroseus y Bauveria bassiana (Pérez-Olvera et al., 2011). El género Bacillus ocupa 85% de los biopesticidas comercialmente disponibles (Villarreal-Delgado et al., 2017). Los agentes de control biológico son organismos benéficos que ayudan a reducir los efectos dañinos de los fitopatógenos. Logran esto por medio de acciones antagónicas usando varios mecanismos, tales como el micoparasitismo, la producción de enzimas líticas, la inducción de respuestas sistémicas de la planta y/o la producción de δ-endotoxinas, sideróforos y lipopéptidos, entre otros (Valenzuela Ruiz et al., 2020).
En las últimas décadas, la introducción de herramientas genómicas, filogenómicas y bioinformáticas ha contribuido de forma significativa para entender mejor las interacciones entre microorganismos y plantas (Sharma et al., 2020). El estudio de las especies microbianas a nivel genético no solo ha permitido identificar mecanismos y genes involucrados en la colonización de raíces, adaptación de hospedantes y el mejoramiento del crecimiento de la planta (Sheppard et al., 2018; Youseif, 2018), sino que también ha permitido realizar avances muy importantes en la caracterización y la afiliación taxonómica de taxa asociados con los efectos benéficos en los cultivos (Jagadeesan et al., 2019). Así, el objetivo del presente estudio es explorar los posibles mecanismos de acción de control biológico de Bacillus paralicheniformis TRQ65 contra hongos fitopatógenos de importancia agrícola, por medio de la secuenciación y explotación del genoma.
Materiales y métodos:Cepas microbiales y condiciones de cultivo
Se aisló a Bacillus paralicheniformis TRQ65 de la rizósfera del trigo (Triticum turgidum subsp. durum) en campos comerciales del Valle del Yaqui, México (27.3692°, 110.3886°). La cepa se conserva en la Colección de Microorganismos Edáficos y Endófitos Nativos (COLMENA) en México (www.itson.edu.mx/ COLMENA) (de los Santos Villalobos et al., 2021). Para la preparación de cultivos bacterianos, se inocularon 1 x 104 unidades formadoras de colonia (UFC) de TRQ65 en 10 mL de caldo nutritivo (NB). Después de 24 horas de incubación a 28°C y 180 rpm, la suspensión se centrifugó a 4000 rpm durante 5 minutos. Luego, el pellet bacteriano se re-suspendió en agua destilada estéril (autoclaveada a 121°C y 15 psi durante 15 minutos) y se ajustó a una concentración de 1 x 108 CFU/ mL (Villa-Rodriguez et al., 2021). La actividad antagónica de TRQ65 se probó contra 11 hongos fitopatogénicos: Fusarium brachygibbosum, Botryosphaeria rhodina, Penicillium expansum, Penicillium crustosum, Geotrichum candidum y Cladosporium sp. (proporcionados por la Universidad Michoacana de San Nicolás de Hidalgo); Botrytis cinerea, Mucor rouxii y Rhizoctonia solani (proporcionados por el Centro de Investigación Científica y de Educación Superior de Ensenada); y Neocosmospora solani y Bipolaris sorokiniana (de COLMENA). Los hongos fueron cultivados en Agar Dextrosa Papa (PDA) e incubados bajo la luz por 120 horas a 28°C. Se colectaron esporas usando agua destilada con 0.01% Tween 20 y ajustada a una concentración de 1 x 108 esporas/mL (Villa-Rodriguez et al., 2019).
<bold>Ensayo de confrontación dual <italic>in vitro</italic> de <italic>Bacillus paralicheniformis</italic> TRQ65 contro hongos fitopatógenos</bold>
Se realizó una prueba dual cualitativa en placas de Petri que contenían PDA, donde la cepa TRQ65 fue evaluada frente a las cepas fúngicas mencionadas anteriormente. De cada cepa fúngica se le inocularon 1 x 108 esporas al centro de la placa de Petri, mientras que 1 x 108 CFU de TRQ65 se colocaron en puntos equidistantes, a 2 cm del inóculo fúngico (de los Santos Villalobos et al., 2012). Las zonas de inhibición formadas alrededor de los patógenos fúngicos se midieron quantitativamente a lo largo de 7 días, con tres réplicas para cada condición. Se realizó un análisis estadístico usando el software Statgraphics Plus, versión 5.1. Los datos se analizaron usando un ANOVA de una vía y la prueba de rangos de Tukey (HSD) (p ≥ 0.05) y los resultados se presentan como la media de las réplicas o experimentos independientes.
Secuenciación y explotación del genoma bacterial
Se aisló el AND genómico de un cultivo fresco de la cepa TRQ65, que se cultivó en un caldo nutritivo (NB) a 28°C agitando a 180 rpm durante 24 horas, alcanzando una concentración de 1 x 108 CFU/mL, según lo descrito por Valenzuela-Aragon et al. (2019). Después, el ADN se extrajo con la plataforma Illumina MiSeq (2 x 300 bp) a una profundidad de cobertura de 30x, y la preparación de la biblioteca de Next Generation Sequencing se realize usando el TruSeq DNA Nano Kit plara plataformas Illumina®, siguiendo los protocolos especificados ppor el fabricante (LANGEBIO-CINVESTAV, Irapauto unit). La calidad de las lecturas de secuenciación fue evaluada utilizando FastQC versión 0.11.5 (Andrews, 2010). Para eliminar secuencias de adaptadores y bases de baja calidad, se aplicó Trimmomatic, versión 0.32 (Bolger et al., 2014). Para el ensamble de novo, se utilizó SPAdes, versión 3.10.1 (Bankevich et al., 2012) con la opción “- - careful” para minimizar errores. Los contigs ensamblados se organizaron usando Mauve Contig Mover, versión 2.4.0 (Darling et al., 2004). Para detectar plásmidos dentro del genoma, se usó PLACNETw (https://castillo.dicom.unican.es/upload/) (Vielva et al., 2017).
La anotación del genoma se realizó usando el servidor RAST, versión 2.0 (http:// rast.nmpdr.org) (Aziz et al., 2008; Overbeek et al., 2013), utilizando el pipeline RASTtk con los parámetros predeterminados. Los contigs fueron ensamblados con MeDuSa, un ensamblador basado en borradores múltiples (Bosi et al., 2015). Por último, se usó antiSMASH, versión 5.0 (https://antismash.secondarymetabolites. org) (Blin et al., 2019) para identificar posibles clústeres génicos biosintéticos relacionados con la actividad de biocontrol en el genoma de las cepas bacterianas estudiadas.
Resultados y discusión:
Ensayo dual in vitro de Bacillus paralicheniformis TRQ65 contra hongos fitopatógenos
La cepa TRQ65 presentó actividad de biocontrol contra 45% de los hongos patógenos estudiados (Fusarium brachygibbosum, Neocosmospora solani, Botryosphaeria rhodina, Botrytis cinerea y Bipolaris sorokiniana) (Cuadro 1). Bacillus paralicheniformis TRQ65 inhibió el crecimiento mycelial de Botrytis cinerea en 43.8% ± 9%. Esta actividad de biocontrol comenzó el día 4 y disminuyó hasta el día 7. Asimismo, Świątczak et al. (2023), reportó Bacillus paralicheniformis como un agente de control biológico contra Botrytis cinerea en plantas de canola un una inhibición de 13%. De forma similar, Asif et al. (2023), evidenciaron
Zona de inhibición (porcentaje) de <italic>Bacillus paralicheniformis</italic> TRQ65 contra patógenos fúngicos de plantas de
Fitopatógeno
Día 1
Día 2
Día 3
Día 4
Día 5
Día 6
Día 7
Fusarium brachygibbosum
ND
ND
ND
13% ± 0%
16% ± 1%
ND
ND
Neocosmospora solani
22% ± 3%
17% ± 1%
19% ± 5%
7.6% ± 3%
ND
ND
ND
Botryosphaeria rhodina
ND
ND
7% ± 1%
ND
ND
ND
ND
Botrytis cinerea
ND
ND
ND
ND
43.8% ± 9%
34% ± 5%
31% ± 5%
Bipolaris sorokiniana
ND
ND
ND
14% ± 3%
11% ± 2%
8% ± 0%
ND
Prueba de Tukey HSD (n=3, p ≥ 0.05). Las variaciones corresponden a la desviación estándar. ND = No detectado.
control biológico de B. paralicheniformis contra Elsinoe sp., Phytophthora capsici, Botrytis cinerea, Rhizoctonia solani, Coniellla sp., Thielaviopsis sp. y Phytophthora sojae, atribuyendo su inhibición fitopatogénica principalmente a la producción de fengicina y de bacilibactina.
Por otra parte, el biocontrol de B. paralicheniformis TRQ65 contra Fusarium brachygibbosum, Neocosmospora solani, Botryosphaeria rhodina y Bipolaris sorokiniana presentó resultados variables, donde esta actividad empezó a ser cuantificable los días cuatro, uno, tres y cuatro, respectivamente, y desaparecieron los días cinco, cuatro, tres y seis, respectivamente (Cuadro 1). En este sentido, De la Lastra et al. (2021), reportaron la capacidad de B. paralicheniformis de inhibir el crecimiento radial fúngico de los aislamientos patógenos de Fusarium de alcanzar una inhibición de entre 73 y 84%. Esta actividad de biocontrol podría estar asociada con metabolitos difusible biosintesizados por la cepa TRQ65 (Kulimushi et al., 2017; Cawoy et al., 2015), donde el éxito de su inhibición es altamente dependiente de las especies fúngicas que interactúan (Andrić et al., 2020; Beauvais y Latgé 2018). Sin embargo, existen estudios que demuestran que los fitopatógenos presentan una destacable adaptabilidad a los metabolitos antimicrobiales producidos por agentes de control biológicos, a pesar de una inhibición inicial al crecimiento (Bonaterra et al., 2022). Inicialmente, estos fitopatógenos pueden ser susceptible a la antibiosis o a la exclusión competitiva por agentes de biocontrol (Khan et al., 2018), y entonces ejercen mecanismos para contrarrestar los efectos inhibitorios iniciales de los agentes de control biológico, incluídos la síntesis de enzimas degradativas capaces de catabolizar o modificar compuestos inhibitorios, la regulación ascendente de los sistemas de bombas de eflujo para expulsar metabolitos tóxicos y las alteraciones en la morfología o bioquímica del sitio objetivo para reducir la susceptibilidad (Köhl et al., 2019). Por ejemplo, algunas especies de Fusarium han demostrado la capacidad de producir enzimas purificantes que neutralizan a metabolitos antifúngicos producidos por agentes de control biológicos (Costa Almeida et al., 2022). Así, la eficacia del control biológico puede verse comprometida con el tiempo debido a varios factores, incluyendo fluctuaciones ambientales, la disminución de compuestos inhibitorios, interacciones microbiales complejas dentro de la rizósfera y el desarrollo de resistencia por fitopatógenos (Lal et al., 2020). La naturaleza dinámica de estas interaciones enfatiza la importancia de desentrañar los mecanismos moleculares detrás de la adaptación patógena para explicar la pérdida observada de eficacia (Barber et al., 2024).
Análisis de genoma bacteriano
Durante las últimas décadas se ha destacado bacterias de control biológico del género Bacillus con actividades protectoras hacia patógenos de plantas de importancia económica (Finkel et al., 2017; Fira et al., 2018; Köhl et al., 2019). Así, varios estudios han intentado explorar sus potenciales mecanismos de acción. En este sentido, se secuenció y analizó el genoma de B. paralicheniformis TRQ65 para identificar clústers génicos biosintéticos prometedores asociados con su actividad de biocontrol.
Después de la secuenciación, el genoma de B. paralicheniformis TRQ65 presentó un total de 5,079,308 lecturas [2×300 pares base (pb)]. En el análisis de calidad y la remoción de adaptadores, solo se descartó el 8.42%. Después del ensable de novo, un genoma preliminar de la cepa TRQ65 consistió de 4,475,481 pb; 45.5% de contenido G+C; N50 de 676,421; L50 de 3; y 32 contigs (>200 bp). El orden y la estructura generaron 25 scaffolds. Además, no se observaron plasmidios y, según nuestro entendimiento, la presencia de plasmidios no ha sido reportada para esta especie (Vielva et al., 2017).
La anotación del genoma en el servidor RAST de B. paralicheniformis TRQ65 presentó 91 ARNs y 4809 secuencias de ADN codificantes predichas (CDS) distribuidas en 340 subsistemas. El subsistema más abundante fue aminoácidos y derivados (341 CDS), seguido de carbohidratos (338 CDS), metabolismo de proteína (198 CDS), cofactores, vitaminas, grupos protésicos y pigmentos (147 genes), nucleósidos y nucleótidos (101), y dormancia y esporulación (96) (Figura 1).
Distribución por categorías de subsistemas de las secuencias de ADN codificantes (CDS) en el genoma de <italic>B. paralicheniformis</italic> TRQ65, siguiendo el pipeline RAST
Además, se identificaron genes relacionados con el metabolism secundario de biocontrol (9 CDS), incluída la biosíntesis de auxinas (4 CDS). Un estudio previo desarrollado por Rojas Padilla et al. (2020) indicó que la cepa TRQ65 puede producir ácido indolacético 39.29 ± 0.30 ppm, solubilizar el fosfato en un 1.37 ± 0.05%. Además, en el genoma de B. paralicheniformis TRQ65 se detectaron genes relacionados con la adquisición y el metabolism del hierro (44 CDS), incluída la producción de sideróforos (33 CDS) como la bacilibactina.
Fira et al. (2018) afirman que las bacterias del género Bacillus son productoras de una amplia gama de compuestos antagonistas de diferentes estructuras; entre las más importantes se encuentran los péptidos y lipopéptidos sintetizados de forma no ribosomal. Para explorar los potenciales clústers genéticos biosintéticos asociados con la actividad de biocontrol, el genoma de B. paralicheniformis TRQ65 fue explotado por antiSMASH (https://antismash.secondarymetabolites.org) (Blin et al., 2019). En el genoma de la cepa TRQ65 se detectaron los clusters de genes asociados con el biocontrol: i) lichenisina (100% de identidad), un biosurfactante aniónico lipopeptídico con actividades citotóxicas, antimicrobianas y hemolíticas (Coronel et al., 2017); ii) bacitracina (88% de identidad), un antibiótico lipopeptídico cíclico activo contra bacterias Gram-positivas y algunas Gram-negativas, ya que inhiben la biosíntesis de la pared celular bacteriana (Liu et al., 2018); iii) fengicina (93% de identidad), un lipopéptido con actividad fungitóxica contra hongos filamentosos que interactúa con las capas lipídicas y altera la estructura de la membrana celular y su permeabilidad (Li et al., 2019); iv) el sideróforo bacilibactina (100% de identidad), un compuesto cíclico que contiene grupos catecolato con alta afinidad por el hierro (Fe3+), creando un ambiente inadecuado para la colonización por fitopatógenos al competir por el hierro (Félix-Pablos et al., 2022); y v) amiloliquecidina (93% de identidad), un péptido antimicrobiano activo contra las bacterias Gram-positivas. (Gerst et al., 2022). Con base en los modos de acción de los grupos de genes biosintéticos mencionados, solo la lichenisina, fengicina y la bacilibactina (Figura 2) han presentado actividades antifúngicas. Por lo tanto, se infirió que esos tres compuestos podrían estar involucrados en el biocontrol de los hongos fitopatógenos estudiados y nuestro equipo los está investigando en la actualidad.
Además, la bacilibactina es un sideróforo de alta afinidad con un núcleo de trilactona compuesto de unidades de ácido 2,3-dihidroxibenzóico (DHB) enlazadas a residuos de treonina, donde los grupos de catecol quelan iones de Fe³⁺ con alta especificidad, con lo que se forman complejos estables que respaldan procesos metabólicos esenciales en condiciones de hierro limitado (Khan et al., 2016; Dimopoulou et al., 2021; Timofeeva et al., 2022). Lichenisina, un surfactante lipopeptídico anfifílico con un anillo peptídico cíclico y una cadena de ácido graso hidrofóbica, presenta estabilidad bajo condiciones extremas y ha demostrado que inhibie patógenos fúngicos tales como Fusarium oxysporum, Alternaria alternata,
A) Estructura de la bacilibactina (National Center for Biotechnology Information, 2024). La bacilibactina se construye alrededor de un núcleo de trilactona formado por la ciclización de tres moléculas de ácido 2,3-dihidroxi-benzoico (DHB) enlazadas a un andamiaje central de residuos de treonina. El anillo trilactona se forma mediante enlaces de éster entre los grupos hidróxilo de la treonina y los grupos carboxilo del DHB. Cada parte del DHB en la bacilibactina contiene grupos funcionales de catecol (2,3-dihidroxi-benzoato). Estos grupos de catecol son responsables de la unión de alta afinidad dal Fe³⁺. B) Estructura de la Lichenisina (National Center for Biotechnology Information, 2024). La lichenisina contiene un anillo peptídico cíclico formado por 13 residuos de aminoácidos. A este núcleo peptídico cíclico se le une una cola lipídica, típicamente una cadena de ácido graso β-hidroxilado. La cadena peptídica forma una estructura cíclica mediante un enlace amídico entre el grupo carboxilo de un aminoácido y el grupo amina de otro, lo cual lleva a formar una estructural anular estable.
C) Estructura de la Fengicina (National Center for Biotechnology Information, 2024). La fengicina consiste en un decapéptidoque forma una estructura cíclica mediante un enlace lactona. A este anillo peptídico se le une un ácido graso β-hidroxilado, cuya longitud puede variar, generalmente entre 14 y 18 átomos de carbono. La secuencia exacta de aminoácidos puede variar ligeramente dependiendo de la isoforma específica de la fengicina.
Aspergillus fumigatus, Aspergillus niger y Neocosmospora keratoplastica (Gudiña y Teixeira, 2022; Yeak et al., 2022; Olmedo et al., 2022; Armenova et al., 2024). Por otro lado, las fengicinas, en especial las fengicinas A y B, son lipodecapeptidas con actividad antifúngica contra hongos filamentosos, que alteran las membranas celulares fúngicas, perturban el crecimiento micelial e inducen la resistencia sistémica en plantas. Existen estudios que han demostrado su efectividad contra Botrytis cinerea, Pythium ultimum y Monilinia fructicola, con lo que exhiben su capacidad para suprimir la germinación de esporas y causar la fuga del citoplasma fúngico (Vanittanakom et al., 1986; Koumoutsi et al., 2004; Deleu et al., 2005; Ongena et al., 2005; Liu et al., 2011; Geissler et al., 2019; Andrić et al., 2020). Este estudio proporciona el primer acercamiento hacia el mecanismo potencial de control biológico de Bacillus paralicheniformis TRQ65 como un agente de control biológico. Sin embargo, los resultados de este studio indican un control biológico moderado contra los hongos estudiados. Por lo tanto, se require de más estudios para explorer la capacidad de control biológico, identificado a través de la explotación genómica para complementar los resultados del análisis de confrontación de baja inhibición.
Conclusiones
Bacillus paralicheniformis TRQ65 es un agente de control biológico que inhibe el crecimiento mycelial de Fusarium brachygibbosum, Neocosmospora solani, Botryosphaeria rhodina y Bipolaris sorokiniana. Con base al aprovechamiento genómico, esta bioactividad podría estar relacionada con la biosíntesis de lichenisina, fengicina y/o bacilibactina. Así, actualmente se están llevando a cabo más estudios por parte de nuestro equipo de investigación para mejorar la capacidad de B. paralicheniformis TRQ65 y validar su uso como ingrediente activo en inoculantes bacterianos para avanzar hacia una agricultura sostenible.
Agradecimientos
Los autores agradecen los fondos brindados por el Instituto Tecnológico de Sonora (PROFAPI_2024_0001), así como los miembros del Laboratorio de Biotecnología del Recurso Microbial (www.itson.mx/LBRM).