Natural Bactericidal Effects of Psoralea glandulosa Essential Oil for the Control of Bacterial Canker and Speck in Tomato

Abstract

Bacterial canker and bacterial speck are diseases affecting tomato caused by Clavibacter michiganensis subsp. michiganensis and Pseudomonas syringae pv. tomato, respectively. These diseases are considered a serious threat with a strong impact on tomato production and marketing worldwide, especially because of their reduced sensitivity to traditional controls. This work reports the chemical composition of the essential oil (EO) of Psoralea glandulosa and investigates its in vitro antimicrobial activity, along with its main compound, against three strains of Clavibacter michiganensis subsp. michiganensis (CmVC533, CmVLC78, and CmVQ59) and one strain of Pseudomonas syringae pv. tomato (Pst). The results indicate that both the EO and bakuchiol have significant antibacterial capacity, especially the EO, which reaches a minimum inhibitory concentration (MIC) between 4–16 µg/mL and 128 µg/mL, and a minimum bactericidal concentration (MBC) between 8–16 µg/mL and 128 µg/mL for the strains of C. michiganensis subsp. michiganensis and P. syringae pv. tomato, respectively. The EO and bakuchiol also had an inhibitory effect when applied directly onto plates seeded with C. michiganensis subsp. michiganensis. Overall, the results from this study should be verified in the near future by in vivo studies.

1. Introduction

The tomato (Solanum lycopersicum L.) is the most widespread vegetable in the world, with the largest worldwide market. Its production increase during the latest decade is mainly due to performance improvements and, to a lesser extent, more cultivated surfaces [1,2]. In 2023, FAO published the most recent data on world tomato production (FAOSTAT): production reached 186.1 mt, an increase of 2.1% compared to the same period in 2021 [3]. The main tomato-producing country is China, which provides 36.7% of total tomato production, followed by India (11.12%) [3]. According to the International Trade Center database, world tomato exports reached 14.9 million tons in 2020, with a value of USD 16.8 billion FOB [4].

Given the economic importance of this crop, disease control is crucial to maintain the commercialization and growth of the industry worldwide [5]. An adequate disease control strategy greatly improves the productivity and quality of the final product and allows compliance with the strict standards of international markets, which demand organoleptic and minimum sanitary standards. Bacterial diseases are the biggest problem for the tomato industry, affecting plant health and growth. The most notable of these is bacterial canker [6], followed by bacterial speck [7]. Bacterial canker disease is caused by the Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis and represents a serious problem in both commercial plantations and home orchards. Symptoms are expressed as spots on leaves, stems, and fruits; an even wilting of leaves and shoots occurs, and then, in the advanced stages, the whole plant wilts and collapses [8]. On the other hand, bacterial speck is caused by the Gram-negative bacterium Pseudomonas syringae pv. tomato, which causes brown-to-black lesions on fruits, stems, pedicels, and peduncles [7]. These lesions can cause yield losses and reduce the quality of the fruit for commercial sale. These infectious diseases can spread rapidly and result in devastating losses. They are difficult to manage because there is currently no cure, and they are difficult to eradicate once they enter a greenhouse, garden, or field [9].

Chemical control using copper-based products, such as mancozeb and streptomycin, is still the most comprehensive way to manage these diseases [10]. In general, aminoglycoside antibiotics have lost their efficacy; a single mutation is sufficient to cause resistance in C. michiganensis subsp. michiganensis [10,11], and several studies confirm the resistance to copper of P. syringae pv. tomato [12]. However, applying these chemicals has increasingly produced environmental contamination and resulted in consequent health risks for agricultural workers and the population in general [13]. Because of this, there is a need for novel pesticides as alternative solutions to control tomato pests in order to replace chemical control. In this scenario, biopesticides based on natural products, such as essential oils and metabolites with antimicrobial properties, have generated substantial interest [14]. These products have shown favorable results as antimicrobial agents for controlling plant diseases in laboratory and greenhouse assays [15]. Consequently, essential oils are a promising alternative: they are cost-effective, accessible, and applicable to various pathologies.

In this context, global demand for pesticides derived from natural sources is growing at a sustained rate of more than 10% per year, compared to only 2% for conventional pesticides. With growing concerns about environmental sustainability, biopesticides are expected to become the dominant strategy for disease control, with the global biopesticide market reaching USD 3.4 billion in 2017 and continuing to grow [16]. Among the natural products proposed to be used as biopesticides is Psoralea glandulosa Linn; this is a medicinal resinous shrub, endemic to Chile, and is known locally as “culén”. P. glandulosa is commonly used by herbalists to heal wounds and hemorrhoids. The local indigenous Mapuche communities also use it as an antiseptic in the treatment of infections and skin diseases [17]. Studies on the different products of this species have demonstrated its benefits for human health, such as its antioxidant, anti-yeast, antipyretic, antitumor, and anti-inflammatory properties [18,19,20]; however, from an agricultural point of view, with regard to its antimicrobial capacity, the resinous exudate of the aerial parts of P. glandulosa has shown potent antifungal activity against Botrytis cinerea and Phytophthora cinnamomi [21], and its essential oil has been shown to be active against Aspergillus niger [22], one of the most damaging phytopathogens for plants and vegetables.

Based on the above-mentioned background, our study aimed to determine the chemical composition of the essential oil extracted from fresh leaves of P. glandulosa and to evaluate the in vitro antibacterial activity of the oil and its main compound, bakuchiol, against C. michiganensis subsp. michiganensis and P. syringae pv. tomato bacteria, which cause bacterial canker and tomato speck, respectively.

2. Materials and Methods

2.1. Standards and Solvents

Standard pure compounds for co-injection in GC/MS, bakuchiol, caryophyllene, and caryophyllene oxide were purchased from AK Scientific Inc. (30023 Ahern Ave, Union City, CA, USA), and hexane, dichloromethane, ethyl acetate, and dimethylsulfoxide (DMSO) solvents were obtained commercially from Sigma-Aldrich Co. (St. Louis, MO, USA) and were used unpurified.

2.2. Plant Material and Extraction of Essential Oil

P. glandulosa was collected in March 2017 from Lo Orozco, Casablanca, Valparaíso Region, Chile. A voucher specimen (VALPL 2156) was deposited at the VALP Herbarium, Department of Biology, Universidad de Playa Ancha, Valparaíso, Chile. Specimens in an intact state were selected for further study. Fresh leaves (500 g) were subjected to hydrodistillation for 4 h on a Clevenger-type apparatus [23]. The oil was dried in anhydrous sodium sulfate and preserved in a sealed amber bottle at 4 °C for further analysis.

2.3. Chromatographic Analysis

The EO was diluted with dichloromethane; 1 μL essential oil was analyzed via Gas Chromatography–Mass Spectrometry (GC/MS). The analysis was carried out using a Thermo Scientific GC–MS system (GC: model Trace GC Ultra; and MS: model ISQ, Thermo Fisher Scientific, Waltham, MA, USA) operating in EI mode at 70 eV, equipped with a splitless injector (250 °C). The transfer line temperature was 250 °C. Helium was used as the carrier gas at a rate of 1.3 mL/min, and the capillary column used was an Rtx-5MS (60 m × 0.25 mm i.d., film thickness 0.25 µm). The temperature program was 40 °C (5 min) to 250 °C (8 min) at a rate of 5 °C/min. Compounds in the chromatograms were identified by the comparison of their mass spectra with those in the NIST20 library database, and by the comparison of their retention indices with those reported in the literature [24] for the same type of column with the commercial standards mentioned above.

2.4. Isolation of Bakuchiol

EO, obtained as described above (3.0 g), was chromatographed on a column of silica gel and eluted with a gradient of hexane/ethyl acetate (100:0 to 86:14). Eighty-eight fractions were collected and analyzed by TLC. Fractions 25–63 were purified by flash chromatography using a mixture of hexane/ethyl acetate (92:8) as the eluent. Twenty-five fractions were collected. Fractions 5–22 contained the active compound (1.76 g) identified as bakuchiol.

2.5. In Vitro Antibacterial Assay

2.5.1. Strains

Clavibacter michiganensis subsp. michiganensis (CmVC 533, CmVLC78, CmVQ59) was provided by Phytopathology Lab, Escuela de Agronomía, Pontificia Universidad Católica de Valparaíso, Chile, from their collection.

The isolates, pre-cultured on YPGA, were multiplied in 50 mL of YPG (yeast extract, 5 g; bacto-peptone, 5 g; glucose monohydrate, 10 g; distilled water, 1 L; pH 7.2) for 1 to 4 days in an orbital shaker at room temperature. The isolates were stored in 5 mL aliquots of each sample at −20 °C, and 50 µL was seeded with YPGA to determine the concentration of each strain. Scales containing 1 mL of the concentrations with 10⁸ to 10¹ cells/mL were prepared from the samples preserved at −20 °C for C. michiganensis subsp. michiganensis.

Pseudomonas syringae pv. tomato (Pst) was obtained the Laboratory of Molecular Microbiology and Environmental Biotechnology of the Technical University Federico Santa María. P. syringae pv. tomato was grown on plates with King’s B medium (KBM) containing 50 µg/mL rifampicin at 28 °C, as previously described [11]. One day prior to inoculation, a single bacterial colony was cultured in KBM broth with shaking until the exponential phase at 28 °C. Cells were collected by centrifugation at 4000× g for 10 min and suspended in 100 mL 10 mM MgCl₂.

2.5.2. Antibacterial Activity Assay

The antibacterial activity was evaluated through the broth macrodilution method [25] using two-fold serial dilutions in 1 mL of yeast–peptone–glucose broth (YPGB). The samples were dissolved in DMSO (5 mg/mL) and added to YPGB at concentrations of 1, 2, 4, 8, 16, 32, 64, 128, and 256 µg/mL. Sterile water and DMSO 5% were used as negative controls. The bacterial inoculum was prepared from an overnight culture and adjusted to a concentration of 1–5·10⁶ CFU/mL at 0, 24, 48, and 72 h of incubation in a rotatory shaker at 28 ± 2 °C. Ten microliters of each dilution was inoculated onto yeast–peptone–glucose agar (YPGA) and King’s B medium (KB) plates for C. michiganensis subsp. michiganensis and P. syringae pv. tomato, respectively. Then, the minimal inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were defined as the lowest concentrations of the compound at which microbial growth was inhibited, or reduced by 99.9%, respectively, after 72 h of incubation. The antimicrobial activity test for commercial antibiotics was performed under the same conditions as the natural products tested. All the measurements were obtained from three independent experiments, each preformed in triplicate.

2.6. Drop Test

The surface of the plate was swabbed with a 0.5 McFarland’s suspension of C. michiganensis subsp. michiganensis strain CmVC 533. Preferably, the plate was allowed to stand with the lid on for approximately 15 min to dry the inoculum. Subsequently, using a pipette or a calibrated loop, we added a 10 µL drop of the bakuchiol or EO (32, 16, 8, and 4 µg/mL) onto the surface of the inoculated plate. We left the plates for 15 min at room temperature to allow complete absorption of the drop, then inverted the plate. Then, the sample was incubated for 16 to 18 h at 35 °C. Finally, the presence or absence of a zone of inhibition was observed. All the measurements were obtained from three independent experiments, each preformed in triplicate [26].

2.7. Statistical Analysis

The statistical analysis of recovery rates was performed by comparison within isolates and between the culturing media with a Student’s t-test. Differences were considered significant at p < 0.05.

3. Results and Discussion

3.1. Extraction and Characterization of the Essential Oil

The hydrodistillation of the fresh leaves of P. glandulosa gave a yellow oil with a yield of 0.6% (v/w), and the GC-MS analysis revealed a total of nine volatile constituents (

Table 1. Constituents of the essential oil extracted from Psoralea glandulosa.

Nº	RT (min)	Components	% Area a	RI b	RI c	Identification
1	17.49	Caryophyllene	3.70	1476	1476	RL, MS, Co
2	18.20	α-humulene	0.57	1480	1481	RL, MS
3	19.61	δ-cadinene	0.50	1524	1524	RL, MS
4	20.55	Palustrol	0.50	1550	1550	RL, MS
5	20.85	Caryophyllene oxide	3.41	1586	1586	RL, MS, Co
6	21.24	Ledol	0.28	1590	1593	RL, MS
7	21.36	Humulene epoxide II	0.30	1613	1614	RL, MS
8	21.92	γ-muurolene	0.87	1681	1682	RL, MS
9	29.99	Bakuchiol	85.42	2138	2138	RL, MS, Co

^a Surface area of GC peak; ^b RI: retention indices relative to C₈–C₃₆ n-alkanes on the Rtx-5 MS capillary column; ^c RI: retention index from the literature; RL: comparison of the RIs with those in the literature [24]; MS: comparison of the mass spectra with those from NIST 20; Co: co-elution with standard compounds available in our laboratory.

). Monoterpenes and terpenoids account for 95.05% of the EO of P. glandulosa, and the major constituents, in descending order, were bakuchiol (85.42%), caryophyllene (3.70%), and caryophyllene oxide (3.41%).

Our results on the yield of the EO from the leaves of P. glandulosa differ from the percentages obtained for this species in the Bio-Bio region by Becerra et al. (2010); however, they agree in identifying bakuchiol (30.6%) and caryophyllene (21.8%) as the most abundant constituents, but with totally different percentages of abundance. Also in this study, the presence of ledol, naphthalene, phytol, octadecane, and eicosane is reported [22]. These compounds were not present in our oil, except for ledol. This variation in essential oil content and chemical composition is influenced by many factors, including location, plant age, climate, cultivar, distillation method, and the properties of the GC method used to determine the volatile compounds [27].

3.2. In Vitro Antibacterial Assay

Based on the above background, we tested the inhibitory and bactericidal effects of the EO of P. glandulosa and its main compound bakuchiol on four bacterial strains, three Gram-positive strains of Clavibacter michiganensis subsp. michiganensis (CmVC533, CmVLC78, and CmVQ59), and one Gram-negative strain of Pseudomonas syringae pv. tomato (Pst). The effects of the EO, bakuchiol, and the positive controls (streptomycin, mancozeb and copper sulfate) were analyzed using two parameters: MIC and MBC (

Table 2. MIC and MBC (µg/mL) values of EO of P. glandulosa and bakuchiol against phytopathogenic bacteria after 72 h of incubation.

Sample	CmVC533		CmVLC78		CmVQ59		Pst
	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC
Bakuchiol	16	32	32	32	2	4	256	256
EO	4	8	8	8	16	16	128	128
Streptomycin	2	4	2	4	4	4	128	256
Mancozeb	32	64	64	64	64	128	128	128
Copper sulphate	16	32	16	32	16	32	16	32

).

The obtained data, summarized in Table 2, clearly show that the antimicrobial activities of EO were significantly superior to the isolated compound bakuchiol against all tested bacteria, except for the CmVQ59 strain.

Comparing the antibacterial potential of the EO and bakuchiol with that of the antibiotics against the C. michiganensis subsp. michiganensis strains, only streptomycin was superior to the effect of the natural samples; however, CmVQ59 presented a higher sensitivity to bakuchiol, with an MIC value two times lower than that of the antibiotic. The EO and bakuchiol showed potent antimicrobial activity against Gram-positive strains when compared to mancozeb. Copper sulfate and bakuchiol showed comparable activity, except for CmVQ59, where bakuchiol proved to be more active. On the other hand, the EO showed lower MIC and MBC values than copper sulfate against the C. michiganensis subsp. michiganensis strains, except again for CmVQ59, where both showed similar activity. Finally, in the case of Pst, only the EO showed comparable antibacterial activity to streptomycin and mancozeb, but its value was four times lower than copper sulfate.

The results obtained for the EO of P. glandulosa showed considerably low MIC and MBC values ≤ 16 µg/mL and, therefore, it was chosen for further investigation to determine its effect on the inhibition and elimination of the C. michiganensis subsp. michiganensis strain CmVC 533 in a drop assay.

The MIC values presented slight differences after hours of incubation. As can be seen in Figure 1, at T0 (0 h), inhibition was observed even at the lowest concentration (1 µg/mL) compared to the negative control assays; on the other hand, the MBC was detected at a concentration of 4 µg/mL. The same phenomena were observed at T1 (24 h). At T2 (48 h), a slight regrowth of the cultures was observed, with an MIC of 2 µg/mL, but the MBC was maintained. After 72 h of incubation, the inhibitory effect of 2 µg/mL of P. glandulosa EO was lost, and the same MBC was observed. A bactericidal effect can be observed at concentrations of 256, 128, 64, 32, 16, and 8 µg/mL of EO, and an inhibitory effect at concentrations of 4 and 2 µg/mL.

The inhibitory effect of P. glandulosa EO against C. michiganensis subsp. michiganensis may be supported by the bioactivity of the metabolites present in this oil. Its major compound, bakuchiol, has several reported biological and pharmacological applications, and interest in this compound is growing due to its potent bioactivity [28]; with respect to its antimicrobial activity, it demonstrates effectiveness against several species of Streptococcus and Enterococcus. It has been shown that bakuchiol causes cell membrane damage and DNA damage and reduces biofilm production; also, the ability of this molecule to act synergistically with other compounds in a number of medicinal applications has been demonstrated [29]. On the other hand, caryophyllene (3.70%) has exhibited strong antibacterial effects against Gram-positive and Gram-negative strains like Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, and Enterococcus faecallis [30]. Indeed, other authors have reported the significant antibacterial effects of oxide-caryophyllene (3.41%) against Staphylococcus aureus [31]. Furthermore, α-humulene (0.57%) has been shown at a concentration of 2 μg/mL to inhibit the cell growth and biofilm formation of Bacteroides fragilis strains by more than 90% [32]. Therefore, this background inclines us to propose that the strong antibacterial effect against C. michiganensis subsp. michiganensis could be a synergistic effect between the main compound and the other terpenic compounds present in the essential oil.

Valenzuela (2019) studied the effects of P. glandulosa extracts against C. michiganensis subsp. michiganensis; an MIC of 16 µg/mL was obtained with ethyl acetate extract, and in the case of dichloromethane, the MIC was even lower (4 µg/mL) [11]. Furthermore, in the study, it was also observed that the antimicrobial potential of bakuchiol is not only effective in growth inhibition, but also has a direct bactericidal effect when the compound is applied against the tested strains of this pathogen. The same was realized in our study and is shown in Figure 2, where the direct application of the essential oil of P. glandulosa and bakuchiol produces a biocidal halo on a plate seeded with C. michiganensis subsp. michiganensis strain CmVC533.

Our results are promising because our MBC results are in the range of 16–32 µg/mL for the EO and bakuchiol. Furthermore, these results suggest that the natural compounds of P. glandulosa may have a possible curative or protective effect against pathogenic bacteria in plants, meaning they could be used to replace chemical copper agents that cause contamination [11].

4. Conclusions

The inhibitory effects of the EO of P. glandulosa and the compound bakuchiol were evaluated. The EO of P. glandulosa showed MIC values between 4–16 µg/mL and 128 µg/mL for the C. michiganensis subsp. michiganensis and P. syringae pv. tomato strains, respectively, while bakuchiol showed MIC values between 2–32 µg/mL and 256 µg/mL for the C. michiganensis subsp. michiganensis and P. syringae pv. tomato strains, respectively. These results indicate that both natural products showed a significant antimicrobial effect and were generally more potent than the traditional chemical controls.

Their efficacy against C. michiganensis subsp. michiganensis at this concentration would have to be verified by in planta studies and under controlled greenhouse conditions. However, the alternative natural products studied in this work are valuable because they are new candidates for biopesticides, which generally have lower toxicity and minor environmental impact.