Inglés (pdf)
Articulo en XML
Referencias del artículo
Traducción automática
Enviar articulo por email
Citado por SciELO 
Similares en
SciELO 
Permalink
I. INTRODUCTION
Conventional agriculture has focused on the use of chemicals to solve the deficiencies of the agricultural field where continuous and indiscriminate use has been seen, which has caused damage to the environment and human health, as is the case of pesticides such as chlorobenzilate, ethylene dibromide, organochlorines, etc. [1]. 19% of Ecuador’s territory is used for agriculture, where most crop losses are caused by serious phytopathogenic diseases such as those triggered by Botrytis, Alternaria, Fusarium, Penicillium, Cercospora, Colletotrichum[2]. In Ecuador, there is a great affection for crops of interest by fungi Alternaria sp. and Botrytis sp. For example, Botrytis cinerea can infect 235 species with the socalled “Gray Rot”, where the most affected fruit is strawberries and roses. [3] [4] [5] [6] This fungus occurs in various environments, such as cold, temperate, and tropical climates. Alternaria sp. causes damage to cauliflower, beans, and tomato crops [7]. Therefore, options are being sought to control plant pathogens, where biological agents are an alternative since they will help eliminate or mitigate the damage caused by pathogenic fungi in crops [8] [9]. Bacillus subtilis is an optimal biocontroller since it can be foliar and root applied. In addition, it can colonize the rhizosphere (root-surrounding soil); it produces digestive enzymes that degrade and eliminate fungi and bacteria, while also exhibiting resistance to high temperatures, osmotic fluctuations, and other stressful environmental conditions. For this reason, it is a bacterium studied worldwide; it also produces peptide metabolites with antibiotic action (surfractins, iturins and fengicins) [10] [11]. Therefore, Bacillus subtilis is an alternative that must be considered to control various phytopathogenic fungi. Dual cultures are an excellent technique for studying the inhibitory capacity present in one microorganism against another [3]. This analysis consists of stressing a microorganism of interest, i.e., modifying certain conditions such as temperature, nutrients, and space so that it can generate metabolites that help to cope with adverse conditions, in addition it is subjected to a confrontation with another microorganism; with the main objective that they can compete for space and nutrients necessary to ensure their survival [12] [13] [14]. The metabolites generated from the dual culture can be quantified in two ways; the first is through physicochemical methods, e.g., chromatography, nuclear magnetic resonance, infrared transmission, and mass spectrophotometry [15]. The main advantage of these methods is that they identify different families of lipoproteins generated by Bacillus spp. The second way to quantify and identify lipoproteins is by molecular methods since they allow a more specific evaluation and an understanding of at what point there is a higher expression of these metabolites [13] [16]. The main molecular techniques for quantifying and identifying lipoproteins are RT-PCR and RT-qPCR [17] [18]. PCR is a tool widely used by molecular biology and, in conjunction with electrophoresis, allows the identification of the presence of genes encoding a particular lipoprotein, either surfactins, fengicins, or iturrins [16]. The second technique allows the researcher to quantify and identify in real time. RT-qPCR is a fast and efficient analysis where no other more laborious and expensive analysis is required. This analysis can be quantified using two methodologies, either absolute or relative. [19] [20]. Relative quantification is ideal for this type of analysis since it is more suitable for determining metabolic and physiological changes at the cellular level. According to [13] [21] [19]. To determine the levels above, the model 2∆∆Ct proposed by Livak and Schmittgen [22]. Where it consists of interpolating the data at a threshold point or cut-off cycle (CT) and determining the expression changes that can be generated by the microorganisms under study [16] [23]. This study aims to quantify the expression levels of the two principal genes involved in lipopeptide biosynthesis in Bacillus sp., as a way to assess their potential contribution to the control of major phytopathogenic diseases affecting agriculture. Elevated expression of these biosynthetic genes would be expected to reflect a stronger biocontrol capacity against common crop phytopathogenic in Ecuador.
II. MATERIALS AND METHODS
A. Activation of microorganisms
The microorganisms involved in the molecular analysis, such as B. subtilis, Alternaria sp., and Botrytis sp., were cryopreserved at -80 °C. These microorganisms were provided by the Life Sciences Laboratories of the Universidad Politécnica Salesiana, Quito, Ecuador [24]. The reactivation of Bacillus subtilis was prepared in a TSB medium, where 30 gL −1 was used in 1 L of distilled water and then incubated at 35°C for 24 h. After this time, it was inoculated in a petri dish with nutrient agar and incubated under the abovementioned conditions. To reactivate the phytopathogenic microorganisms, the Papa Dextrose Agar (PDA) medium was used, and incubated at 25°C.
B. Morphological identification Bacillus subtilis.
Next, a morphological identification of the microorganism was performed to ensure the absence of contaminating agents that could compromise the assay. Colony characteristics of Bacillus subtilis were examined, revealing opaque colonies with a white to grayish coloration, medium-sized (approximately 3–4 mm in diameter), circular in shape, flat, and with a dry, matte appearance. Microscopic evaluation included Gram staining, in which Bacillus subtilis exhibited the typical morphology of Gram-positive bacilli. Endospore detection was conducted using endospore staining with malachite green, allowing clear visualization of spore-forming structures. For the phytopathogenic microorganisms (Alternaria sp. and Botrytis sp.), a monosporic culture was obtained prior to analysis, and their structures were confirmed via methylene blue staining to ensure the absence of foreign or contaminant elements.
C. DNA extraction
Previously, the microorganisms to be analyzed were seeded in TSB media cultivated for 24 hours at 35°C. After this time, the PureLink™ Microbiome DNA Purification Kit (Invitrogen, USA) was used, which was essential for the extraction of the genetic material of Bacillus sp., which was later quantified by Qubit™4 Fluorometer (Invitrogen, USA), and the dsDNA HS Assay Kit (Invitrogen, USA) was used. Subsequently, the 16S rRNA gene region (V4) was amplified using the universal primers 518F and 800R to verify the presence of the purified bacterial DNA template and corroborate that the primers of the housekeeping region were amplified correctly. This confirmed the sample viability for the subsequent amplification of the genes under study [25].
D. PCR
The genes involved in the analysis of lipoproteins were ITUDI and FEND, for which primers amplifying the regions of interest were used (Table I):
In addition, the 16S gene was used to calibrate the data to assess expression levels. Amplification to determine the presence of the FEND and ITDI genes was performed using the DreamTaq™ Green PCR Master Mix (2X) (Thermo Fisher Scientific, Waltham, MA, USA). Following the manufacturer’s instructions. The amplification protocol was then carried out, with an initial denaturation of 95°C for 1 minute, continued with 45 cycles that counted with denaturation of 95°C for 30 seconds, Annealing of 60°C for a time of 30 seconds; the protocol also counted with Extension of 72°C for 30 seconds. A final Extension of 72°C for 3 minutes was added [13]. A holding cycle of 4°C for an undetermined time was programmed to end the thermal phase. The thermal phase of the PCR was carried out in the Proflex TM PCR System Applied BiosystemsTM (Thermo Fisher Scientific, Waltham, MA, USA) thermal cycler ideal for conventional PCR reactions. Once the reaction was finished, electrophoresis was performed to observe the amplification of the PCR products.
E. Antagonistic test: Dual culture
Dual culture was used to microbiologically detect the regulatory effect of B. subtilis against Alternaria sp. and Botrytis sp. 22 g of PDA were prepared in one liter of distilled water to reduce the necessary nutrients, forcing the microorganisms to fight for their survival. For this purpose, a disc of 5 mm diameter was inoculated with the fungus in the middle of the Petri dish; then, a groove was made with a separation of 3 mm [13]. The microorganisms were incubated at 25°C and were evaluated on days 1, 5, and 9 according to the methodology proposed by [16] [26]. These days, it is necessary to determine the percentage of radial growth inhibitors (PIRC) and to obtain the genetic material needed for the analysis by qPCR. To obtain the PICR, the formula proposed by Acurio [27]. Eq. 1:
R1 represents the radius of the fungus without biocontroller and R2 is the radius of the fungus with the controller.
F. RNA extraction
The genetic material necessary for the qPCR analysis was extracted from the dual cultures from days 1,5 and 9, to which B. subtilis was subjected to the phytopathogenic microorganisms. The Purelink RNA Mini-Kit extraction kit (Ambion, Life Technologies, USA) was used to obtain the material. The RNA obtained from the extraction was then quantified in µg(µL)−1 using the Qubit™4 Fluorometer system (Invitrogen, USA), and the RNA HS Assay Kit (Invitrogen, USA) was applied. Additionally, to ensure optimal subsequent amplification, consider that high-quality samples correspond to integrity values of RIN ≥ 7–8, which are generally required for reliable amplification and cDNA-based analyses that provide the best performance.
G. Retrotranscription
Retrotranscription was carried out using the Superscrit® III First-Strand Synthesis SuperMIX for RT-qPCR kit (Invitrogen) to obtain cDNA, and an RNA concentration of at least one µg(µL)−1 [26] was required. The reaction was then incubated at 25 °C for an estimated 10 min, 50 °C for 30 min, 85 °C for 5 min only. The reaction was cooled to 4 °C to add 1 µL of E. coli RNase H at 37 °C for 20 min to eliminate any interfering RNA template. The product obtained was quantified using the Qubit ssDNA Assay Kit (Invitrogen).
H. qPCR
The qPCR was carried out in the QuantStudio ® 5 kit (Thermo Fisher Scientific, Waltham, MA, USA). The reaction was prepared from 10 µL of Fast Sybr Green Master Mix (Thermo Fisher Scientific) following the manufacturer’s recommendations, 1 µL of Forward and Reverse primers was added, and 5 µL of cDNA, whose minimum concentration is 2 µg(µL)−1 was added. [16] completed with nuclease-free water until a final volume of 20 µL was obtained. Bacillus megaterium was included as a positive control, given its previous characterization under the same experimental protocol. The negative control consisted of a reaction with PCR-grade water, excluding the microorganism’s DNA/RNA template to confirm the absence of contamination. It was then taken to the thermal cycler, where it was set up with the following temperatures (Table II):
The gene expression levels were calculated using the method 2 − ∆∆CT [22] [19] To perform expression calculations, obtaining a calibrator group CT helps to minimize the error between samples. For this, a CT is set for both the gene of interest and the reference gene, that is Eq. 2:
The ∆CTCalibrador will be used to make the comparison between the ∆CT of the samples of the genes of interest to be evaluated in Eq. 3:
Finally, we determine the expression levels of the ∆∆CT obtained by Morales [23]. Eq. 4:
I. Statistical analysis
A 3 * 3 factorial design was used, taking the independent variable raised about the development of Bacillus subtilis against phytopathogenic microorganisms on days 1, 5, and 9, the dependent variable raised will be the levels of gene expression in lipopeptides, in addition to three replicates per treatment were raised, resulting in 27 experimental units where it will be related to the time that B. subtilis is in contact with phytopathogenic organisms, also if the time implies that there is a change at the level of gene expression. For this, ANOVA analysis was used to understand statistically how the results obtained behaved. Subsequently, Tukey’s Post Hoc test was used with a significance level of 5%, using the INFOSTAT statistical program.
III. RESULTS AND DISCUSSIONS
A. Radial growth inhibition
The ANOVA analysis showed a p-value < 0.0001 for the variables day, growth, and their interaction, indicating a significant difference among treatments and confirming that the inhibition percentage produced by Bacillus subtilis depends on the incubation time. Tukey’s test identified four significance groups (A, B, C, and D), with the highest inhibition values occurring in group D (B. subtilis vs. Alternaria sp. on day 9, PICR = 86.57%) and group C (B. subtilis vs. Alternaria sp. on day 5, PICR = 47.16%). These values represent the strongest inhibitory effects observed in this study, demonstrating that B. subtilis can suppress the growth of Alternaria sp. by nearly 90%. It is important to emphasize that inhibition reference ranges reported in the literature are relative, as inhibition levels vary according to antagonism time and the target phytopathogen; studies have described inhibition values as low as 4.75%–25% under certain conditions, while higher ranges (33.26%–98.48%) are considered the most effective. Comparable research has shown that Bacillus sp. often exhibit stronger inhibition from day 5 onward, reaching 40%–60% against Alternaria, consistent with the patterns observed here. Additionally, references [26] and [27] indicate that several Bacillus species, including B. subtilis, are effective antagonists against multiple phytopathogens (Table III).
B. Gene expression of the FEND gene
In this study, Bacillus subtilis was shown to inhibit the growth of the phytopathogenic fungi to which it was subjected. The qPCR analysis helped to determine the gene involved in synthesizing phengicins, one of the main families of lipoproteins [28]. The study monitored the expression levels on the proposed evaluation days, which were days 1, 5, and 9. Low expression levels on the first evaluation day, as shown in Figure 1. On day 5 of the assessment, it can be found that the expression levels present higher levels of abundance of FEND, indicating a greater inhibition against phytopathogenic organisms on this day. This coincides with the findings of Chiluisa-Utreras, Medrano, et al. [16], which concluded that the day of greatest performance of the FEND gene is the fifth day of evaluation. On day 9 of the review, the expression levels are low, mainly due to [13], the highest FEN expression observed on day 5 suggests that fengycin biosynthesis occurs early in the growth kinetics. This behavior indicates that Bacillus sp. activates the fengycin production pathway before entering the stationary phase, likely in response to competition signals or early stress associated with the late exponential phase. This identifies day 5 as the optimal point for detecting or harnessing maximum fengycin production, which is consistent with the fact that Bacillus subtilis later enters a dormancy phase, making the production of this gene impossible at that stage.
A represents the average levels on the first day of the evaluation, whereas Treatment 2 (T2) is Bacillus subtilis vs Alternaria sp., which shows low expression levels. On the fifth day, the expression levels for T2 represent an increase compared to day 1. On the ninth day, the expression levels were low. The ANOVA analysis indicates a p-value of 0.0205; significant differences are evident in the day variable, which shows that the highest production of this gene is found on the fifth day (Figure 2). This study found no differences in the treatments, indicating that although phengicins are present, their yield will depend on time. Chowdhury et al. [29] argue that other Bacillus genera will have greater control against certain microorganisms. Chiluisa-Utreras, Medrano, et al. [16] determined that the yield of FEND in B. megaterium was 20 times more than that of B. subtilis, which manages to inhibit both the variable days and treatments, demonstrating that it is capable of exerting biocontrol of Alternaria sp. and Botrytis sp. In addition, B. subtilis has good efficacy in inhibiting Alternaria sp. In addition, Pedraza et al. [30] and Ley-lópez et al. [28] found that B. amyloliquefaciens, when stimulated by Collectotrichum spp. generates an increased production of fengycins capable of neutralizing its competitors (Table IV).
Fig. 2. The post hoc analysis and the Tukey test indicated that the fifth day of the trial showed higher expressions levels.
C. Gene expression of the ITUDI gene
Fengicins represent one of the main families of lipoproteins, however, analyzing the expression of genes coding for iturrins is essential, since the expression levels of this lipoprotein act in synergy with the other families of lipoproteins increasing the levels of inhibition [31]. The expression levels of the ITUDI gene show that on the first day of the evaluation, average ITUDI production was from 0.98 to 3.66 for both Botrytis sp. and Alternaria sp., respectively. By the fifth day of the assessment, the averages ranged from 2.39 to 4.58, indicating an increase in metabolite production. By the last day of the evaluation, the expression levels dropped from 1.8 to 1.6, as shown in Figure 3. The production levels of this metabolite show low levels of expression; [32] state that the production range is from 0 to 3, but its presence also indicates that other lipoproteins such as surfactins may develop. Cadena & Medrano [13] mentioned that iturins stimulate the production of fengycins and surfactins. In the present study, the gene expression levels of the ITUDI gene are not high, but their presence helps stimulate better antagonistic activity.
A represents the average levels on the first day of the evaluation, whereas Treatment 2 (T2) is Bacillus subtilis vs Alternaria sp., which shows considerable expression levels (Figure 4). On the fifth day, the expression levels for T2 represent an increase compared to day 1. On the ninth day, the expression levels present low levels in contrast to the previous evaluation days. The ANOVA analysis does not indicate a significant p-value for the variable days (Table V). However, it does show differences for the variable treatments, which suggests that B. subtilis is an excellent biocontroller for treating Alternaria sp.; B. subtilis presented low levels of expression of this gene, but Dang et al. [33] found that this Bacillus genus tends to develop better inhibition when they are in the presence of Alternaria sp. Thanks to this, we were able to see evidence that there is better control microbiologically.
Fig. 4. Post hoc analysis and Tukey test indicated that the variable trial treatments showed higher expressions levels at T2.
The greater inhibitory activity observed, despite the low ITUDI levels in B. subtilis, is consistent with the findings of Dang et al. [33], who found that this genus develops better inhibition in the presence of Alternaria sp. The expression of the ITUDI genes will depend on several factors, one of which is the species of Bacillus, since each one will develop optimal expression levels for inhibition [31] [34]. Furthermore, the increase in fungicidal capacity is due to synergistic factors: the presence of other types of lipopeptides enhances the great antagonistic capacity. Corrales [31] and Yánez-Mendizábal et al. [34] state that the presence of other lipopeptides, especially surfactins, is necessary, as their action in concert potentiates antifungal activity, which is crucial for the inhibition of filamentous fungi like those analyzed in the present study.
IV. CONCLUSIONS
The results obtained in the study showed that the FEND gene is stimulated in the presence of an external agent, i.e., Alternaria sp. stimulates the presence of this gene, and on the fifth day of the trial, the expression levels tend to be higher than on other days. The study did not find data of relevance when B. subtilis is confronted with Botrytis sp. Still, inhibition can be evidenced, suggesting a synergy between other families of lipoproteins that tend to inhibit Botrytis sp. Therefore, It is concluded that the fengycins generated by B. subtilis have an inhibitory capacity against Alternaria sp. and can be used as a biocontroller to combat this phytopathogen. However, this family of proteins is a fundamental precursor for the development of other families of lipoproteins, such as surfactins, which, together with fengycins, enhance the inhibitory development essential to combat phytopathogenic microorganisms such as Alternaria sp., Botrytis sp., Collectotrichum sp., among others, these characteristics highlight the potential of B. subtilis to contribute to more sustainable phytopathogen-management strategies through the natural production of bioactive molecules with broad inhibitory capacity, as B. subtilis–based formulations could be developed as sustainable alternatives to chemical fungicides, contributing to environmentally friendly crop protection and enabling future applications such as optimized microbial consortia or enhanced fermentation strategies to maximize lipopeptide production.
FUNDING
This research was supported by Universidad Politécnica Salesiana
CONFLICT OF INTEREST
The authors declare the following conflict of interest: statements Universidad Politécnica Salesiana, Research Group BIOARN and Master’s degree in molecular biology
ARTIFICIAL INTELLIGENCE STATEMENT
The authors declare that generative artificial intelligence tools were used for the following purposes: Translation of abstract. The tool(s) used include: Google translator. The authors take full responsibility for the content of the manuscript
