Next Article in Journal
Research and Design of a Hybrid DV-Hop Algorithm Based on the Chaotic Crested Porcupine Optimizer for Wireless Sensor Localization in Smart Farms
Previous Article in Journal
The Application of Deep Learning in the Whole Potato Production Chain: A Comprehensive Review
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Agriculture
Volume 14
Issue 8
10.3390/agriculture14081228
agriculture-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Inhibition of Botrytis cinerea and Escherichia coli by Lactic Acid Bacteria on Leafy Vegetables

by
Beata Kowalska
1,*,
Magdalena Szczech
1 and
Anna Lisek
2
1
Department of Microbiology and Rhizosphere, The National Institute of Horticultural Research, Konstytucji 3 Maja 1/3, 96-100 Skierniewice, Poland
2
Cultivar Testing, Nursery and Gene Bank Resources Department, The National Institute of Horticultural Research, Konstytucji 3 Maja 1/3, 96-100 Skierniewice, Poland
*
Author to whom correspondence should be addressed.
Agriculture 2024, 14(8), 1228; https://doi.org/10.3390/agriculture14081228
Submission received: 2 July 2024 / Revised: 16 July 2024 / Accepted: 24 July 2024 / Published: 25 July 2024
(This article belongs to the Section Crop Protection, Diseases, Pests and Weeds)
Browse Figures
Versions Notes

Abstract

:
The evaluation of the potentiality of lactic acid bacteria (LAB) strains isolated from fermented products to inhibit Botrytis cinerea and Escherichia coli O157:H7 growth on spinach and lettuce was conducted. From a total of forty LAB strains tested, three were selected due to their high inhibitory effect on plant pathogenic fungi. The identification of these isolates based on a 16S rRNA gene fragment sequence analysis confirmed the genus of Levilactobacillus sp. and Lactiplantibacillus sp. An effective method of coating LAB isolates on the lettuce and spinach surface was developed. The leaves were immersed in bacterial suspension (5.0 × 106 cfu mL−1) for 4 s and drained on tissue paper. LAB survived on lettuce and spinach leaves for 8 days at 6 log10 cfu g−1. Additionally, these bacteria decreased the number of filamentous fungi on the leaves. These isolates were found to inhibit the growth of B. cinerea and E. coli O157:H7 in vitro conditions in growing microbiological media. Their efficacy was confirmed in vivo conditions. These isolates inhibited the development of grey mould caused by B. cinerea on lettuce leaves. Two LAB isolates reduced the abundance of the pathogenic bacterium E. coli on spinach leaves by about 0.7 log10 cfu g−1. In glasshouse conditions, LAB stimulated the growth of examined plants. The lactic acid bacteria used in this study showed the capacity to be used as possible alternatives to chemical compounds in the protection of leafy vegetables against grey mould and for a decrease in E. coli O157:H7 contamination.

1. Introduction

Changes in the environment and in people’s lifestyles are bringing many challenges to food producers. Consumers demand fresh and good-quality vegetables, with high nutritional and health-promoting qualities. However, sustainable agriculture—more environmentally friendly while ensuring a sufficient food supply for the human population—requires many adaptations [1]. One of the challenges is the withdrawal of 50% of pesticides by 2030. The intensive application of chemicals in agriculture over the years has had an impact on the degradation of the environment, but on the other hand, it has ensured the better sanitation of products by destroying the microorganisms on plants that are harmful to humans. It is especially important for leafy vegetables that are eaten fresh. These are more and more popular as ready-to-eat products for their dietary and health-promoting properties. However, the consumption of leafy vegetables is often associated with the risk of illness due to microbial contamination as a result of plant colonization by human pathogenic bacteria, transferred from water, soil, or manures, or cross-contaminated during preparations for trade. In this regard, maintaining proper sanitation during field cultivation is a very important challenge. There is also an urgent need to search for alternative methods to chemical crop protection against pathogenic and spoiling microorganisms deteriorating crops “from field to fork” [2,3,4]. In the case of leafy vegetables, the application of lactic acid bacteria may be a promising method for improving the sanitary status of the products and enhancing their health-promoting qualities as an additional value.
Lactic acid bacteria (LAB) are relatively common microorganisms that are found in plants and especially in various fermented foods. They are considered probiotics—living microorganisms with health-promoting effects that might be exerted through the improvement of the intestinal microbiota and proposed modulation of immune system function [5]. The long history of using LAB for food preservation has given them the status of “generally regarded as safe” (GRAS). They are used as ingredients in different foods, edible coatings, and dietary supplements because of their protective properties for the gastrointestinal tract [6,7]. LAB have also shown great potential as preservatives or biocontrol agents in minimally processed vegetables. Their potential to inhibit the growth of foodborne pathogens, spoilage microorganisms, and plant pathogenic microorganisms has been presented in many publications [8,9,10,11]. The preservative abilities of LAB are a result of several mechanisms of action, mainly related to the production of antimicrobial compounds, organic acids, hydrogen peroxide, bacteriocins, and diacetyl. Moreover, they compete with pathogens and spoilage microorganisms for nutrients (vitamins, minerals, trace elements, and peptides) [12,13]. Protective cultures of lactic acid bacteria, to increase the safety and shelf-life of fresh greens, have been developed over the last decades [14,15,16]. For example, Siroli et al. [8] showed that Lactobacillus strains, isolated from minimally processed fruits and vegetables, increased the shelf-life of lamb’s lettuce and indicated a potential for controlling Salmonella and E. coli. Moreover, it is very important that the application of LAB does not significantly affect the sensory quality of leafy greens, e.g., their appearance, colour, smell, taste, and texture [8,17], and vegetables coated with these bacteria can be good matrices for probiotics and are an alternative for consumers who cannot eat dairy products [6].
Grey mould caused by Botrytis cinerea is one of the most important fungal diseases of vegetables, among them leafy greens or baby leaf vegetables [18,19]. Under humid conditions, the fungus produces a noticeable grey-mould fruiting layer on the affected tissues. B. cinerea also causes the secondary soft rot of vegetables in storage, transit, and the market. The control of Botrytis diseases is aided through the removal of infected debris from the field and storage rooms, and by providing conditions for the proper aeration and quick drying of plants and plant products. However, the control of Botrytis is often only partially successful, especially in cool, damp weather. Moreover, at present, due to the limitations of available chemical protectants, both in the crops and in their processing, it is necessary to develop ways of protecting and improving the marketable quality of vegetables, particularly for leafy vegetables with a short growth period, which are consumed fresh [20,21,22].
One of the indicators of the sanitary status of the vegetables is the content of Escherichia coli in the product, and its acceptable hygienic limit is 100 cfu g−1 of the product [23]. Although in this bacteria group, most strains are harmless, we can also encounter those that pose a great threat to humans [3,24,25,26]. In recent years, a significant increase in the number of E. coli infections after eating raw vegetables has been recorded. This is not only due to increased consumption and the trading market but also to other factors such as the improper application of manures [27,28], the use of contaminated water to irrigate and spray plantations [29], and the lack of proper staff hygiene during field work and processing [30,31]. Climate warming is also of great importance [1,32], especially since plant contamination is the most frequent in the summer [24]. This trend was observed in both imported and domestic leafy vegetables and was particularly strong in organic produce. Szczech et al. [33] found that the average number of E. coli and its incidence were significantly higher in organic than in conventionally cultivated vegetables. According to the Farm-to-Fork Strategy, it is very important to take care of crop quality as early as the cultivation stage. Reducing the risk of microbial contamination is an urgent problem in ready-to-eat leafy green vegetables, which are also very popular on the market as the components of fresh-cut salads [34,35,36].
From this perspective, the aims of this research were as follows: (i) to select effective pathogen inhibitors of LAB strains, isolated from fermented vegetables prepared for consumption; (ii) to evaluate the efficacy of these bacterial strains in the suppression of B. cinerea development and the reduction in E. coli O157:H7 contamination on leafy green vegetables—lettuce and spinach; and (iii) to estimate the effect of LAB strains on the growth of these plants. The significance of this research was to choose LAB isolates that possess great potential in the biological control of lettuce and spinach.

2. Materials and Methods

2.1. Testing of LAB Antagonism against Plant Pathogenic Fungi

The isolates of LAB were obtained from the juice of fermented white head cabbage, Chinese cabbage, and cucumber. These products were bought from the local market. Plant material (25 g) with peptone water (225 cm−3) was transferred into sterile stomacher filter bags (400 cm−3). The samples were homogenized in a stomacher BagMixer 400P (8 strokes/s, 10 min). The serial dilution method was used. The LAB colonies were isolated on MRS agar (according to DeMan, Rogosa and Sharpe, Merck, Warsaw, Poland) in an anaerobic atmosphere at 30 °C. Forty isolates of LAB were used in in vitro antagonism tests towards different pathogenic fungi, i.e., Alternaria sp.; three isolates of Botrytis cinerea obtained from carrots, tomatoes, and cyclamens; Fusarium oxysporum; Penicillium sp.; Phialophora sp.; Rhizoctonia solani; and Sclerotinia sclerotiorum. All fungal strains were obtained from the culture collection of the Department of Microbiology and Rhizosphere, the National Institute of Horticultural Research. These pathogens originated from diseased plants, mainly vegetables.
The evaluation of LAB antagonism was carried out using the dual culture technique in Petri plates with MRS agar. The isolates of LAB and fungus were inoculated on opposite edges of the plate (9 cm in diameter) at a distance of approx. 6 cm from each other. Control plates were inoculated at one edge with a fungus only. Each dual culture LAB/fungus was made in triplicate. The plates were incubated at 25 °C, for 5 days. Then, the percentage of the inhibition of radial growth (PIRG) was calculated following the formula
PIRG = [(R1 − R2)/R1] × 100,
where R1 is the radial growth of the control fungal colony and R2 is the radial growth of this fungus in interaction with LAB in dual culture. The experiments were repeated.
Three of the forty LAB isolates were chosen as the most active to inhibit the growth of the tested pathogenic fungi, among others, B. cinerea. These LAB isolates were labelled as LAB-A, LAB-B, and LAB-C. They had different origins—they were obtained from fermented white head cabbage, cucumber, and Chinese cabbage, respectively. The isolates were stored at −80 °C in glycerol until their use in the next presented experiments.

2.2. Identification of LAB Bacterial Isolates Based on 16S rRNA Gene Fragment Sequence Analysis

Identification was performed for three bacterial isolates: LAB-A, LAB-B, and LAB-C. The bacterial isolates were multiplied on microbiological nutrient agar media. DNA extraction was performed using the commercial GeneMatrix Bacterial and Yeast Genomic DNA Purification Kit (EURx, Gdańsk, Poland). The identification of bacterial isolates was based on the sequence analysis of the 16S rRNA ribosomal gene fragment. The amplification of the 16S rRNA gene was performed in 35 cycles (94 °C × 1 min, 55 °C × 1 min, 72 °C × 2 min) using 27F/1492R primers [37]. The 16S rRNA gene amplification products were separated in 1.6% agarose gels. Agarose gels were stained in ethidium bromide and visualized under UV light. The identification of bacterial strains based on the obtained sequences was performed via comparison with data collected in the NCBI database (National Center for Biotechnology Information, NIH, Bethesda, MD, USA, http://www.ncbi.nlm.nih.gov; accessed on 2 March 2023).

2.3. Inhibition of B. cinerea via LAB Culture Supernatant

Fungal biomass inhibition was carried out using the cell-free supernatant of selected strains of lactic acid bacteria: LAB-A, LAB-B, and LAB-C. To prepare the cell-free supernatant, 48 h old bacterial culture in MRS broth (Merck, Warsaw, Poland) was centrifuged at 6000 rpm for 15 min at 4 °C. The supernatant was then filter-sterilized using a sterile syringe filter (pore size of 22 µm). Different concentrations (2.5, 5.0, 10.0%) of the supernatant of LAB were added to potato dextrose broth (PDB, Merck). The final volume of the medium with supernatant in the flasks was 100 mL. The medium in each flask was inoculated with a 5 mm fungal disc, from 7-day-old cultures on PDA medium (Merck). The inoculated PDB medium and PDB supplemented with 0.0, 2.5, 5.0, or 10.0% of MRS medium were served as controls. For this experiment, three isolates of B. cinerea obtained from diseased tomatoes, carrots, and cyclamens were used. The flasks were incubated at 25 °C for 12 days. Then, the fungal mat was harvested, filtered through a Whatman No. 1 filter disc (Chemland, Stargard, Poland), and dried at 50 °C for two hours. The fungal biomass of each treatment was weighed and compared with the control mycelia. Each fungal culture with or without LAB supernatant was prepared in triplicate. The experiment was conducted three times.

2.4. Inhibition of E. coli Growth via LAB Culture Supernatant

E. coli growth inhibition was carried out using the cell-free supernatant of the selected strains of lactic acid bacteria: LAB-A, LAB-B, and LAB-C. The scheme of the experiment was the same as in the methodology described in Section 2.3. The medium in each flask was inoculated with 50 µL of the 48 h old E. coli culture in NB. The flasks were incubated at 37 °C for 48 h. Then, the colony-forming unit of E. coli per one mL was determined by sowing the cultures on a nutrient agar medium (NA, Merck). After 48 h of incubation at 30 °C, the number of colonies was determined. The experiment was conducted three times.

2.5. Determination of the LAB Survival on Spinach and Lettuce Leaves Contaminated with Fungi

To prepare LAB inoculum for vegetable leaf coating, the bacteria were grown in MRS broth medium for 48 h at 30 °C. Then, the cultures were centrifuged at 6000 rpm for 15 min at 4 °C. The supernatant was discarded, and the bacterial pellet was resuspended in the same volume of 0.85% NaCl. The concentration of cells in bacterial suspensions was brought to 5.0 × 106 cfu mL−1 through the addition of 0.85% saline solution.
Lettuce and spinach plants were chosen for the experiments as leafy vegetables are most frequently used in ready-to-eat products. The fresh, detached leaves of spinach and lettuce were washed in running water and drained on tissue paper for 2 h. Then, the leaves were immersed in bacterial suspensions for 4 s and drained again on tissue paper. They were packed into polypropylene bags, 100 g of the leaves per bag, and stored at 8 °C. The leaves immersed in saline solution were only prepared as a control. Each treatment was prepared in three replications. The microbiological analyses of the leaves were conducted after 24 h, 4 days, and 8 days of incubation. Ten grams of plant material was transferred into 100 mL of peptone water in 400 mL sterile stomacher filter bags. The samples were homogenized in a stomacher BagMixer 400P (Donserv, Warsaw, Poland) with a fixed speed of 8 strokes/s for 10 min. Further decimal dilutions were prepared with the same diluents and inoculated onto the agar media: MRS agar to enumerate LAB and yeast extract glucose chloramphenicol agar (YGC) for moulds and yeasts. The results were expressed as colony-forming units per gram of plant material (cfu g−1). For statistical analysis, the data were transformed into logarithms. The experiment was conducted three times.

2.6. Determination of B. cinerea Inhibition on Lettuce Leaves Coated with LAB

Detached leaves of lettuce were immersed in LAB cell suspensions (LAB-A, LAB-B, and LAB-C) at a density of 5.0 × 106 cfu mL−1, according to the method described above. The leaves were transferred to one-litre jars lined with a disc of filter paper, and moistened in sterilized distilled water (one leaf per jar). Then, the leaves were inoculated with the 7-day-old mycelium of the three abovementioned B. cinerea isolates, by putting a fragment of mycelium on a leaf. Leaves not immersed in LAB but inoculated with B. cinerea were prepared as a control. The jars were lightly covered with a screw cap and placed in a growth chamber at 10 °C in the dark. Each treatment was prepared in three replications. After 5, 10, and 15 days of incubation, grey mould development on the leaves was evaluated using a 0–5 rating scale: 0—no sign of infestation; 1—<20% of leaf surface infested with grey mould; 2—21–40% of leaf surface infested with grey mould; 3—41–60% of leaf surface infested with grey mould; 4—61–80% of leaf surface infested with grey mould; 5—>81% of leaf surface infested with grey mould. The experiment was conducted three times.

2.7. Determination of E. coli Inhibition on Spinach Leaves Coated with LAB

No pathogenic, attenuated E. coli O157:H7 isolate was used in the experiment. It was purchased from an epidemiological–sanitary station (Skierniewice, Poland). Fresh, detached leaves of spinach were washed under running water, drained, and immersed in the suspensions of tested LAB isolates at a density of 5.0 × 106 cfu mL−1, according to the method described above. Then, 50 g samples of drained leaves, spread across the plastic trays (22 × 32 cm), were sprayed with 50 µL of E. coli suspension at a density of 8.3 × 106 cfu mL−1. The leaf samples were transferred into ziplock bags and incubated at 10 °C in the dark for 48 h. The following treatments were prepared: E. coli without LAB (control), LAB-A+E. coli, LAB-B+E. coli, and LAB-C+E. coli. Each sample was prepared in three replications. After two days of incubation, a microbiological analysis was conducted to check the density of E. coli on spinach leaves. Ten grams of the leaves from each sample was transferred into 100 mL of peptone water in 400 mL sterile stomacher filter bags. The samples were homogenized in a stomacher BagMixer 400P with a fixed speed of 8 strokes/s for 10 min. Further decimal dilutions were prepared with the same diluents and inoculated onto the MacConkey agar (Merck, Warsaw, Poland). The plates were incubated for 24 h at 37 °C, and the characteristic colonies of E. coli were counted. Their number was expressed as cfu g−1 of leaves. The experiment was conducted three times.

2.8. The Effect of LAB Strains on the Growth of Spinach and Lettuce Plants

The effect of LAB strains on the growth of spinach and lettuce was evaluated in glasshouse experiments. The seeds of spinach were sown to the 1.5 L pots filled with peat substrate Klasmann TS1 (5 seeds per pot and 4 pots per treatment) and grown in a glasshouse. The lettuce was grown in the same potting medium, in multiplates. For each treatment, four micropots (90 cm3 volume each), in three replications, were used.
The LAB suspensions were prepared as described in Section 2.5. The spinach and lettuce plants were treated with LAB suspensions three times: 2, 3, and 4 weeks after sowing. The dosage was 5 mL of the suspension pipetted on the surface of each plant. After 6 weeks, the plants were cut off and their fresh weight was measured. Additionally, the number of lactic acid bacteria in the potting medium was estimated on the MRS agar medium using the serial dilution flooding method. The incubation of the samples was carried out at 30 °C. The results were presented as a number of cfu g−1 of soil dry weight. The experiment was performed three times.

2.9. Statistical Analysis

Microbial counts were analyzed in log scale (log10 cfu g−1). The results were statistically analyzed using a one-way analysis of variance with Tukey’s test (α = 0.05), using the statistical program Statistica 13.1. Data not significantly different from each other were marked with the same letters.

3. Results

3.1. LAB Antagonism against Plant Pathogenic Fungi

The inhibitory effect of 40 LAB isolates in in vitro tests was studied against 9 strains of plant pathogenic fungi: Alternaria sp., 3 strains of B. cinerea, F. oxysporum, Penicillium sp., Phialophora sp., R. solani, and S. sclerotiorum. The inhibition was expressed as the percentage of the inhibition of radial growth (PIRG). Of the 40 isolates tested, 3 of them were the most active—LAB-A, LAB-B, and LAB-C (Table 1)—and these were selected for further study. The most susceptible fungi to inhibition via LAB were Phialophora sp., B. cinerea, and R. solani. The antifungal activity against F. oxysporum was not observed at the tested concentration. Among the LAB strains, LAB-A and LAB-C showed the strongest inhibitory effects. However, LAB-B inhibited Penicillium growth, which was not observed for either LAB-A or LAB-C.

3.2. Identification of LAB Bacterial Isolates Based on 16S rRNA Gene Fragment Sequence Analysis

PCR reactions with primers 27F/1492R to amplify the 16S rRNA gene yielded products of 1492 bp (Figure 1). The sequences of the 16S rRNA gene fragment of the LAB-A isolate showed the highest similarity (99.26%) to those of the Lactiplantibacillus species (Table 2). The sequences of the 16S rRNA gene fragment of the LAB-B and LAB-C isolates showed the highest similarity (93.01–98.93%) to the sequence of bacteria of the genus Levilactobacillus. Based on the results, it was concluded that bacterial isolate LAB-A belongs to the genus Lactiplantibacillus, while bacterial isolates LAB-B and LAB-C belong to the genus Levilactobacillus.

3.3. Inhibition of B. cinerea via LAB Culture Supernatant

The degree of fungal biomass inhibition via LAB culture supernatants was related to the LAB strain tested as well as to Botrytis isolate. A great reduction in fungal biomass production was observed when treated with the cell-free medium after LAB cultivation, especially in LAB-C and LAB-A isolates. This could be due to the metabolites secreted by LAB isolates to the medium. The incubation of control in PDB for 12 days yielded a biomass of 3.15, 1.22, and 2.35 g for B. cinerea from carrots, tomatoes, and cyclamens, respectively. The addition of MRS broth medium (10%, 5%, and 2.5%) to the PDB culture slightly stimulated the biomass production of all B. cinerea strains (Figure 2A–C). Similar results were obtained in the case of 10% and 5% LAB-B supernatant addition, which also slightly stimulated the biomass production. B. cinerea from carrot biomass reduction was observed through the addition of LAB-C (10%, 5%, and 2.5%) and LAB-A (10% and 5%) cell-free media compared to treatment with an MRS addition (Figure 2A). Also, LAB-A cell-free medium 10% and 5% additions were the most effective in biomass reduction in B. cinerea from carrots and B. cinerea from cyclamens compared to control treatments. The results were statistically different. The LAB-B cell-free medium occurred to be the least effective; its 10% and 5% additions stimulate the biomass production of all studied B. cinerea strains.

3.4. Inhibition of E. coli Growth via LAB Culture Supernatant

A great reduction in the number of E. coli compared to NB and also MRS treatments was observed when treated with the LAB-A cell-free medium. The inhibition of E. coli growth was related to the concentration of supernatant, and it was significant in 10% LAB-A treatment, and then 5% and 2.5% treatment. The supernatant obtained from LAB-C also had an inhibitory effect on E. coli growth; however, this was only at the 10% and 5% concentrations. The differences also were significant. This could be due to the metabolites secreted by the LAB isolates to the medium (Figure 3).

3.5. Survival of LAB Isolates and Fungal Contamination of Coated Spinach and Lettuce Leaves

The enumeration of lactic acid bacteria on vegetable leaves inoculated with selected LAB strains was performed after 24 h, 4 days, and 8 days. After 24 h and 4 days, the number of LAB on leaves treated with LAB was stable. The increased number of these bacteria on inoculated leaves remained significantly higher for 8 days compared to control—5.23–5.94 log10 cfu g−1 for spinach and 5.57–6.72 log10 cfu g−1 for lettuce—while the control, which was the untreated leaves of spinach and lettuce, contained 3.12 and 2.28 log10 cfu g−1 of LAB, respectively (Table 3). There were no significant differences in leaf colonization between the treatments, where different LAB strains were used. However, the strains LAB-B and LAB-C indicated a tendency for the greater colonization of spinach as well as lettuce leaves than LAB-A.
It was found that the application of LAB-A and LAB-B significantly reduced fungal contamination on both kinds of leaves (Table 3). The number of moulds in these treatments was reduced by 8–27% compared to the control. LAB-C did not exhibit such an effect, despite high leaf colonization. Yeasts were not detected in any studied sample.

3.6. B. cinerea Inhibition on Lettuce Leaves Coated with LAB

The effect of selected LAB strains on grey mould development on lettuce leaves, caused by three different strains of B. cinerea, was evaluated after 5, 10, and 15 days post-inoculation (Figure 4A–C). The development of grey mould was dependent on both B. cinerea and LAB strains. All LAB strains used in the experiment reduced grey mould expansion compared to B. cinerea-inoculated, untreated-with-LAB control treatments. The inhibitory effect of LAB strains was most pronounced in the case of B. cinerea from cyclamens. The effect was observed after 10 and 15 days of inoculation. The infection index after 10 days for treatment with LAB-A and LAB-C was 1.0, whereas for LAB-B, the symptoms of disease were not observed. The inhibition tendency was maintained for up to 15 days. The infection caused by B. cinerea isolate from tomatoes after 5 and 15 days was inhibited in treatments with LAB-A and LAB-C isolates; however, the differences were significant only after 5 days. B. cinerea from carrots was less sensitive to LAB strains, whose reductive effect was also not significant compared to the control after 10 and 15 days.

3.7. E. coli Inhibition on Spinach Leaves Coated with LAB

The coating of spinach leaves with LAB-B and LAB-C significantly decreased their contamination with E coli (Figure 5). Both of these LAB strains reduced the number of E coli by about 0.7 log10 cfu g−1, compared to the control without LAB. The strain LAB-A was not effective in E. coli inhibition.

3.8. The Effects of LAB on the Growth of Spinach and Lettuce Plants

The vegetative experiments were conducted to examine the influence of LAB strains on spinach and lettuce plants. Table 4 shows the biomass of spinach and lettuce plants treated with LAB isolates during cultivation. All studied LAB isolates had a positive influence on spinach plants. In the case of all LAB isolates, the weight of plants was higher than in the control treatment. The differences were significant. The plant weight was higher from 21.7 to 36.0%. This depended on the bacteria isolate tested and the most effective was the LAB-A strain. In the case of lettuce, it also was observed that the treatment with LAB strains stimulated its growth; however, the differences were not statistically different (Table 4). The analysis of soil obtained from spinach and lettuce cultivation showed that the LAB survived in soil. The amount of these bacteria in treated combinations ranged from 2.85 to 3.84 log10 cfu g−1 for spinach and from 2.60 to 4.92 log10 cfu g−1 for lettuce. The differences were significant (Table 4).

4. Discussion

Lactic acid bacteria strains belonging to Levilactobacillus sp. and Lactiplantibacillus sp. isolated from fermented cabbage, Chinese cabbage, and cucumber have been widely investigated for their antifungal activity and successfully proposed for increasing the microbiological quality of lettuce and spinach.
Leafy fresh vegetables have the healthy, functional, and sensory characteristics that the consumers are looking for. However, the agricultural crop conditions (e.g., manures and irrigation water) and processing operations of vegetables could favour the contamination with plant and also human pathogenic microorganisms [24,25,26]. The use of biological methods to eliminate and reduce plant pathogens and also other microbial contamination on leafy fresh vegetables is highly regarded [38,39]. Antagonistic microorganisms possess great potential to control microbial contamination. For example, some yeast strains [40,41], strains belonging to the genera Bacillus and Pantoea [22], and lactic acid bacteria [42,43] have biocontrol ability against B. cinerea. In other studies, the antifungal activity of lactic acid bacteria has been documented [44,45,46]. It is crucial that the bacteria known as biological control agents (BCAs) are able to survive and multiply at host surfaces to interfere with the pathogen [42,47]. In the present study, the ability of three lactic acid bacteria strains belonging to Levilactobacillus sp. and Lactiplantibacillus sp. to survive and multiply on the leaves of spinach and lettuce was documented. Many studies have shown that these bacteria mainly produce bacteriocins and organic acids that inhibit the growth of spoilage and also pathogenic microorganisms [12,48]. In the present study, it was also documented that the studied strains produce antifungal and antibacterial substances, thereafter releasing them into the growth medium. The inhibitory effect of them was shown towards B. cinerea (Figure 2) and E. coli (Figure 3). Moreover, it was documented that LAB-A and LAB-C reduced the growth of naturally occurring fungi on lettuce and spinach leaves (Table 3). These studies are in agreement with those conducted by Budryn et al. [16] and Świeca et al. [49], who found reduced numbers of microorganisms, including fungi, on sprouts treated with LAB.
The efficacy of added probiotic bacteria depends on inoculum level and their viability must be maintained throughout the storage of the product’s shelf-life. There is no clear agreement on the minimum concentration of the probiotic intake to achieve the beneficial effect on the host. While some researchers suggest that concentrations of higher than 106 cfu mL−1 are required, others suggest a concentration of at least 107 and 108 cfu mL−1 [50]. In the present study, the coated method allowed a high inoculum of LAB to be maintained for up to 8 days on spinach and lettuce leaves. Compared to other studies, these results are sufficient. Rößle et al. [51] used LAB solution at about 1010 cfu mL−1 to coat fresh-cut apple slices. They enumerated LAB every two days for ten days. The slices contained about 108 cfu g−1 over the test period, which is sufficient for a probiotic effect, and is comparable to counts of probiotic bacteria in commercially available dairy products. Since LAB occur naturally in many food systems and have been a part of human microbiota, in this study, the isolation of antagonistic isolates was performed from food products, which predisposes these isolates to the protection of food products.
For leaves coated with antagonistic bacteria, the present study has shown that the leaf microbiota contained less relatively abundant moulds, which are undesirable for humans. In this way, the risk of multiplication and spread on leafy green vegetables can be reduced, and the consequences of their related outbreaks in the future can be mitigated.
Because many species and strains of LAB are used in the fermentation of various foods such as dairy and bakery products, meat, seafood, and vegetables, there have been many studies of LAB for improving the storage characteristics and safety of food by inhibiting the growth of spoilage-causing microbes or pathogenic bacteria. In contrast, information on the use of LAB as biological control agents in agricultural production is relatively limited. Therefore, the present study regarding the use of bacteria to inhibit the development of grey mould can be very useful in horticultural practice.
A study conducted by Strafella et al. [52] showed that besides the well-recognized plant growth-promoting bacteria, LAB isolates from wheat rhizosphere may have potential plant growth promotion activities, too. LAB showed inhibitory activity against plant pathogenic fungi and bacteria and also may synergistically enhance the activity of compounds produced by other members of the rhizosphere. A similar phenomenon was observed in the present study, in which LAB isolates applied topically and in soil stimulated the growth of spinach and lettuce.
The number of E. coli on the spinach surface treated with LAB-A and LAB-B decreased in the presented study. Our observations were in agreement with the study conducted by Budryn et al. [16], who observed the following correlation in the case of legume sprouts—when the number of lactic acid bacteria increased by 2 log10 cfu mL−1, mould decreased by 1 log10 cfu mL−1 and pathogenic bacteria like E. coli and Klebsiella sp. decreased by 2 log10 cfu mL−1; Salmonella sp., Shigella sp., and Staphylococcus epidermidis did not appear. Yin et al. [17] proved that LAB treatment on lettuce and spinach exhibited antimicrobial efficacy against E. coli O157:H7 and Listeria monocytogenes by 2 and 1 log reduction, respectively. In the present study, LAB-B and LAB-C reduced the number of E. coli on spinach by about 0.7 log10 cfu g−1 (Figure 5). Similarly, in the study conducted by Świeca et al. [49], the sprouts enriched with probiotics were characterized by lower mesophilic bacteria counts. It has previously been shown that lactic acid bacteria and their metabolites may increase the safety and shelf-life of lamb’s lettuce [8] and spinach [38]. In the study conducted by Martins et al. [53], L. acidophilus and L. plantarum reduced coliform counts in treated fruit salad, improving biopreservation. Also, psychotrophic bacteria count fewer in the treated salad than in the control. In a study conducted by Konappa et al. [54], in field experiments, LAB isolates increased the yield of tomatoes. Moreover, LAB exhibited above 60% disease reduction in bacterial wilt in tomatoes. Based on these presented studies, the examined isolates belonging to Levilactobacillus sp. and Lactiplantibacillus sp. and their cell-free supernatant seem to be promising biocontrol agents for inhibiting B. cinerea and decreasing contamination with E. coli bacteria. On the basis of our study and the literature, LAB have great potential in biological control and in stimulating the growth of some plants [2,55,56,57].

5. Conclusions

The obtained results are also more interesting because LAB are classified as GRAS (Generally Recognized As Safe) and are often found to have beneficial effects on consumers’ health. The results also highlighted the importance of biocontrol agent isolation from commercial products—fermented cucumbers and fermented cabbage. The studied LAB isolates not only inhibited the growth of inoculated pathogens—E. coli and B. cinerea—but also spoilage microorganisms that are naturally present in the samples. These abilities predispose the isolates to colonize spinach and lettuce and survive under refrigerated storage. Moreover, lettuce and spinach leaves coated with lactic acid bacteria can be food matrices, as potential carriers of probiotic bacteria.
By conducting experiments on cut-off leaves and whole plants of lettuce and spinach, it was found that the three tested LAB isolates showed an inhibitory effect on the development of grey mould-caused B. cinerea strains and significantly increased the weight of spinach plants.
In conclusion, LAB isolates belonging to the genus Levilactobacillus sp. and Lactiplantibacillus sp. coated on spinach and lettuce leaves have great potential to protect the vegetables from grey mould and reduce their microbial contamination, especially E. coli O157:H7.
These results encourage the usage of bacterial antagonists as part of a global solution to reduce the risk of plant and human pathogens on leafy green vegetables. More studies are necessary in order to test the effect of the inoculation of these LAB strains on lettuce and spinach plants in fields.

Author Contributions

Conceptualization, B.K. and M.S.; methodology, B.K., M.S. and A.L.; validation, B.K. and A.L.; formal analysis, B.K.; investigation, B.K., M.S. and A.L.; resources, B.K.; data curation, B.K., M.S. and A.L.; writing—original draft preparation, B.K.; writing—review and editing, B.K., M.S. and A.L.; visualization, B.K.; supervision, B.K.; project administration, B.K.; funding acquisition, B.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Polish Ministry of Education and Science, under grant number ZM/2/2018 (2018–2023).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article, and further inquiries can be directed to the authors. The raw data supporting the conclusions of this article will be made available by the authors upon request.

Acknowledgments

The authors express many thanks to Jolanta Winciorek and Anna Michalska for their technical support.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Cavicchioli, R.; Ripple, W.J.; Timmis, K.N.; Azam, F.; Bakken, L.R.; Baylis, M.; Behrenfeld, M.J.; Boetius, A.; Boyd, P.W.; Classen, A.T.; et al. Scientists’ warnings to humanity: Microorganisms and climate change. Nat. Rev. Microbiol. 2019, 17, 569–589. [Google Scholar] [CrossRef]
  2. Julien-Javaux, F.; Gerard, C.; Campagnoli, M.; Zuber, S. Strategies for the safety management of fresh produce from farm to fork. Curr. Opin. Food Sci. 2019, 27, 145–152. [Google Scholar] [CrossRef]
  3. Alegbeleye, O.O.; Odeyemi, O.A.; Strateva, M.; Stratev, D. Microbial spoilage of vegetables, fruits and cereals. Appl. Food Res. 2022, 2, 100122. [Google Scholar] [CrossRef]
  4. Sobiczewski, P.; Iakimova, E.T. Plant and human pathogenic bacteria exchanging their primary host environments. J. Hortic. Res. 2022, 30, 11–30. [Google Scholar] [CrossRef]
  5. Arshad, F.; Mehmood, R.; Hussain, S.; Khan, M.A.; Khan, M.S. Lactobacilli as probiotics and their isolation from different sources. Br. J. Res. 2018, 5, 43. [Google Scholar] [CrossRef]
  6. Bernal-Castro, C.; Espinosa-Poveda, E.; Gutiérrez-Cortés, C.; Díaz-Moreno, C. Vegetable substrates as an alternative for the inclusion of lactic acid bacteria with probiotic potential in food matrices. J. Food Sci. Technol. 2024, 61, 833–846. [Google Scholar] [CrossRef]
  7. Pavli, F.; Tassou, C.; Nychas, J.E.; Chorianopoulos, N. Probiotic incorporation in edible films and coatings: Bioactive solution for functional foods. Int. J. Mol. Sci. 2018, 19, 150. [Google Scholar] [CrossRef] [PubMed]
  8. Siroli, L.; Patrignani, F.; Serrazanetti, D.I.; Tabanelli, G.; Montanari, C.; Gardini, F.; Lanciotti, R. Lactic acid bacteria and natural antimicrobials to improve the safety and shelf-life of minimally processed sliced apple and lamb’s lettuce. Food Microbiol. 2015, 47, 74–84. [Google Scholar] [CrossRef]
  9. Murray, K.; Wu, F.; Shi, J.; Xue, S.J.; Warriner, K. Challenges in the microbiological food safety of fresh produce: Limitations of post-harvest washing and the need for alternative interventions. Food Qual. Saf. 2017, 1, 289–301. [Google Scholar] [CrossRef]
  10. Marin, V.R.; Ferrarezi, J.H.; Vieira, G.; Sass, D.C. Recent advances in the biocontrol of Xanthomonas spp. World J. Microbiol. Biotechnol. 2019, 35, 72. [Google Scholar] [CrossRef]
  11. Lopez-Seijas, J.; Garcia-Fraga, B.; Da Silva, A.F.; Sieiro, C. Wine lactic acid bacteria with antibacterial activity as potential biocontrol agents against Fusarium oxysporum f. sp. lycopersici. Agronomy 2020, 10, 31. [Google Scholar] [CrossRef]
  12. Issaoui, K.I.; Senhaji, N.S.; Zinebi, S.; Haoujar, I.; Amajoud, N.; Abrini, J.; Khay, E.O. Potencial application of bacteriocin produced from lactic acid bacteria. Microbiol. Biotechnol. Lett. 2020, 48, 237–251. [Google Scholar] [CrossRef]
  13. Daranas, N.; Rosello, G.; Cabrefiga, J.; Donati, I.; Frances, J.; Badosa, E.; Spinelli, F.; Montesinos, E.; Bonaterra, A. Biological control of bacterial plant diseases with Lactobacillus plantarum strains selected for their broad-spectrum activity. Ann. Appl. Biol. 2018, 174, 92–105. [Google Scholar] [CrossRef] [PubMed]
  14. Tsuda, K.; Tsuji, G.; Higashiyama, M.; Ogiyama, H.; Umemura, K.; Mitomi, M.; Kubo, Y.; Kosaka, Y. Biological control of bacterial soft rot in Chinese cabbage by Lactobacillus plantarum strain BY under field conditions. Biol. Control 2016, 100, 63–69. [Google Scholar] [CrossRef]
  15. Asare, P.T.; Greppi, A.; Stettler, M.; Schwab, C.; Stevens, M.J.A.; Lacroix, C. Decontamination of minimally-processed fresh lettuce using reuterin produced by Lactobacillus reuteri. Front. Microbiol. 2018, 9, 1421. [Google Scholar] [CrossRef]
  16. Budryn, G.; Klewicka, E.; Grzelczyk, J.; Gałązka-Czarnecka, I.; Mostowski, R. Lactic acid fermentation of legume seed sprouts as a method of increasing the content of isoflavones and reducing microbial contamination. Food Chem. 2019, 285, 478–484. [Google Scholar] [CrossRef]
  17. Yin, H.B.; Chen, C.H.; Gu, G.; Nou, X.; Patel, J. Pre-harvest biocontrol of Listeria and Escherichia coli O157 on lettuce and spinach by lactic acid bacteria. Int. J. Food Microbiol. 2023, 387, 110051. [Google Scholar] [CrossRef] [PubMed]
  18. Cheung, N.; Tian, L.; Liu, X.; Li, X. The destructive fungal pathogen Botrytis cinerea—Insights from genes studies with mutant analysis. Pathogens 2020, 9, 923. [Google Scholar] [CrossRef] [PubMed]
  19. Xie, X.W.; Zhang, Z.X.; Chai, A.L.; Shi, Y.X.; Li, B.J. Grey mould of leaf mustard caused by Botrytis cinerea, a new disease in China. Austral. Plant Dis. Notes 2016, 11, 23. [Google Scholar] [CrossRef]
  20. Hua, L.; Yong, C.; Zhanquan, Z.; Boqiang, L.; Guozheng, Q.; Shipping, T. Pathogenic mechanisms and control strategies of Botrytis cinerea post-harvest decay in fruits and vegetables. Food Qual. Saf. 2018, 3, 111–119. [Google Scholar] [CrossRef]
  21. Garfinkel, A.R. The history of Botrytis taxonomy, the rise of phylogenetics, and implications for species recognition. Phytopathology 2021, 111, 437–454. [Google Scholar] [CrossRef] [PubMed]
  22. Moura, G.G.D.; Barros, A.V.; Machado, F.; Martins, A.D.; Silva, C.M.; Durango, L.G.C.; Forim, M.; Alves, E.; Pasqua, M.; Doria, J. Endophytic bacteria from strawberry plants control gray mold in fruits via production of antifungal compounds against Botrytis cinerea L. Microbial. Res. 2021, 251, 126793. [Google Scholar] [CrossRef] [PubMed]
  23. Commission Regulation No2073/2005 of 15 November 2005 on Microbiological Criteria for Foodstuffs. Available online: https://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CONSLEG:2005R2073:20130701:EN:PDF (accessed on 14 July 2024).
  24. Denis, N.; Zhang, H.; Leroux, A.; Trudel, R.; Bietlot, H. Prevalence and trends of bacterial contamination in fresh fruits and vegetables sold at retail in Canada. Food Control 2016, 67, 225–234. [Google Scholar] [CrossRef]
  25. Elias, S.O.; Noronha, T.B.; Tondo, E.C. Salmonella spp. and Escherichia coli O157:H7 prevalence and levels on lettuce: A systemic review and meta-analysis. Food Microbiol. 2019, 84, 103217. [Google Scholar] [CrossRef]
  26. Azimirad, M.; Nadalian, B.; Alavifard, H.; Panirani, S.N.; Bonab, S.M.V.; Azimirad, F.; Gholami, F.; Jabbari, P.; Yadegar, A.; Busani, L.; et al. Microbiological survey and occurrence of bacterial foodborne pathogens in raw and ready-to-eat green leafy vegetables marked in Tehran, Iran. Int. J. Hyg. Environ. Health 2021, 237, 113824. [Google Scholar] [CrossRef] [PubMed]
  27. Mootian, G.; Wu, W.H.; Matthews, K.R. Transfer of Escherichia coli O157:H7 from soil, water, and manure contaminated with low numbers of the pathogen to lettuce plants. J. Food Protect. 2009, 72, 2308–2312. [Google Scholar] [CrossRef] [PubMed]
  28. Hruby, C.E.; Soupir, M.L.; Moorman, T.B.; Pederson, C.; Kanwar, R. Salmonella and fecal indicator bacteria survival in soils amended with poultry manure. Water Air Soil Pollut. 2018, 229, 32. [Google Scholar] [CrossRef]
  29. Decol, L.T.; Casarin, L.S.; Hessel, C.T.; Batista, A.C.F.; Allende, A.; Tondo, E.C. Microbial quality of irrigation water used in leafy green production in Southern Brazil and its relationship with produce safety. Food Microbiol. 2017, 65, 105–113. [Google Scholar] [CrossRef] [PubMed]
  30. Alegbeleye, O.O.; Singleton, I.; Sant’Ana, A.S. Sources and contamination routes of microbial pathogens to fresh produce during field cultivation: A review. Food Microbiol. 2018, 73, 177–208. [Google Scholar] [CrossRef]
  31. Machado-Moreira, B.; Richards, K.; Brennan, F.; Abram, F.; Burgess, C.M. Microbial contamination of fresh produce: What, where, and how ? Compr. Rev. Food Sci. Food Saf. 2019, 18, 1727–1750. [Google Scholar] [CrossRef]
  32. Liu, C.; Hofstra, N.; Franz, E. Impacts of climate change on the microbial safety of pre-harvest leafy green vegetables as indicated by Escherichia coli O157 and Salmonella spp. Int. J. Food Microbiol. 2013, 163, 119–128. [Google Scholar] [CrossRef] [PubMed]
  33. Szczech, M.; Kowalska, B.; Smolińska, U.; Maciorowski, R.; Oskiera, M.; Michalska, A. Microbial quality of organic and conventional vegetables from Polish farms. Int. J. Food Microbiol. 2018, 286, 155–161. [Google Scholar] [CrossRef] [PubMed]
  34. Hoagland, L.; Ximenes, E.; Ku, S.; Ladisch, M. Foodborne pathogens in horticultural production systems: Ecology and mitigation. Sci. Hortic. 2018, 236, 192–206. [Google Scholar] [CrossRef]
  35. Mogren, L.; Windstam, S.; Boqvist, S.; Vågsholm, I.; Söderqvist, K.; Rosberg, A.K.; Linden, J.; Mulaosmanovic, E.; Karlsson, M.; Uhlig, E.; et al. The hurdle approach—A holistic concept for controlling food safety risks associated with pathogenic bacterial contamination of leafy vegetables. A review. Front. Microbiol. 2018, 9, 1965. [Google Scholar] [CrossRef] [PubMed]
  36. Kowalska, B.; Szczech, M. Differences in microbiological quality of leafy green vegetables. Ann. Agric. Environ. Med. 2022, 29, 238–245. [Google Scholar] [CrossRef] [PubMed]
  37. Lane, D.J. 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics; Stackebrandt, E., Goodfellow, M., Eds.; John Wiley & Sons Ltd.: Chichester, UK, 1991; pp. 115–175. [Google Scholar]
  38. Uhlig, E.; Kjellström, A.; Nurminen, N.; Olsson, C.; Oscarsson, E.; Canaviri-Paz, P.; Mogren, L.; Alsanius, B.; Molin, G.; Håkansson, A. Use of bacterial strains antagonistic to Escherichia coli for biocontrol of spinach: A field trial. Innov. Food Sci. Emerg. Technol. 2021, 74, 102862. [Google Scholar] [CrossRef]
  39. Mendoza, I.C.; Luna, E.O.; Pozo, M.D.; Vásquez, M.V.; Montoya, D.C.; Moran, G.C.; Romero, L.G.; Yépez, X.; Salazar, R.; Romero-Peña, M.; et al. Conventional and non-conventional disinfection methods to present microbial contamination in minimally processed fruits and vegetables. LWT Food Sci. Technol. 2022, 165, 113714. [Google Scholar] [CrossRef] [PubMed]
  40. Parafati, L.; Vitale, A.; Restuccia, C.; Cirvilleri, G. Biocontrol ability and action mechanism of food-isolated yeast strains against Botrytis cinerea causing post-harvest bunch rot of table grape. Food Microbiol. 2015, 47, 85–92. [Google Scholar] [CrossRef] [PubMed]
  41. Ristić, D.; Vučurović, I.; Aleksić, G.; Nikolić, B.; Durović, S.; Starović, M. Application of different combinations of lactic acid, phototrophic bacteria and yeast mixtures in control of seed and seedlings pathogens of tomato and pepper. Pestic. Phytomed. 2021, 36, 73–82. [Google Scholar] [CrossRef]
  42. De Simone, N.; Capozzi, V.; de Chiara, M.L.V.; Amodio, M.L.; Brahimi, S.; Colelli, G.; Drider, D.; Spano, G.; Russo, P. Screening of lactic acid bacteria for the bio-control of Botrytis cinerea and the potential of Lactiplantibacillus plantarum for eco-friendly preservation of fresh-cut kiwifruit. Microorganisms 2021, 9, 773. [Google Scholar] [CrossRef]
  43. Haidar, R.; Fermaud, M.; Calvo-Garrido, C.; Roudet, J.; Deschamps, A. Modes of action for biological control of Botrytis cinerea by antagonistic bacteria. Phytopathol. Mediterr. 2016, 55, 13–34. [Google Scholar] [CrossRef]
  44. Barrios-Roblero, C.; Rosas-Quijano, R.; Salvador-Figueroa, M.; Gálvez-Lόpez, D.; Vázquez-Ovando, A. Antifungal lactic acid bacteria isolated from fermented beverages with activity against Colletotrichum gleosporioides. Food Biosci. 2019, 29, 47–54. [Google Scholar] [CrossRef]
  45. Manjarres Melo, J.J.; Alvarez, A.; Ramirez, C.; Bolivar, G. Antagonistic activity of lactic acid bacteria against phytopathogenic fungi isolated from cherry tomato (Solanum lycopersicum var. cerasiforme). Curr. Microbiol. 2021, 78, 1399–1408. [Google Scholar] [CrossRef] [PubMed]
  46. Deepthi, B.V.; Poornachandra Rao, K.; Chennapa, G.; Naik, M.K.; Chandrashekara, K.T.; Sreenivasa, M.Y. Antifungal attributes of Lactobacillus plantarum MYS6 against fumonisin producing Fusarium proliferatum associated with poultry feeds. PLoS ONE 2016, 11, e155122. [Google Scholar] [CrossRef] [PubMed]
  47. Marin, A.; Plotto, A.; Atares, L.; Chiralt, A. Lactic acid bacteria incorporated into edible coating to control fungal growth and maintain postharvest quality of grapes. HortScience 2019, 54, 337–343. [Google Scholar] [CrossRef]
  48. Dubourg, G.; Elsawi, Z.; Raoult, D. Assessment of the in vitro antimicrobial activity of Lactobacillus species for identifying new potential antibiotics. Int. J. Antimicrob. Agents 2015, 46, 590–593. [Google Scholar] [CrossRef] [PubMed]
  49. Świeca, M.; Kordowska-Wiater, M.; Pytka, M.; Gawlik-Dziki, U.; Bochnak, J.; Złotek, U.; Baraniak, B. Lactobacillus plantarum 299V improves the microbiological quality of legume sprouts and effectively survives in these carriers during cold storage and in vitro digestion. PLoS ONE 2018, 13, e0207793. [Google Scholar] [CrossRef]
  50. Alegre, I.; Vinas, I.; Usall, J.; Anguera, M.; Abadias, M. Microbiological and physicochemical quality of fresh-cut apple enriched with the probiotic strain Lactobacillus rhamnosus GG. Food Microbiol. 2011, 28, 59–66. [Google Scholar] [CrossRef]
  51. Rößle, C.; Auty, M.A.E.; Brunton, N.; Gormley, R.T.; Butler, F. Evaluation of fresh-cut apple slices enriched with probiotic bacteria. Innov. Food Sci. Emerg. Technol. 2010, 11, 203–209. [Google Scholar] [CrossRef]
  52. Strafella, S.; Simpson, D.J.; Khanghahi, M.Y.; De Angelis, M.; Gänzle, M.; Minervini, F.; Crecchio, C. Comparative genomics and in vitro plant growth promotion and biocontrol traits of lactic acid bacteria from the wheat rhizosphere. Microorganisms 2021, 9, 78. [Google Scholar] [CrossRef]
  53. Martins, E.M.F.; Ramos, A.M.; Stringheta, P.C.; Lago-Vanzela, E.S.; Oliveira, P.M.; Martins, M.L. Fresh cut fruit salads as a promising vehicle for Lactobacillus acidophilus and Lactobacillus plantarum. Int. Food Res. J. 2017, 24, 2185–2192. [Google Scholar]
  54. Konappa, N.M.; Maria, M.; Uzma, F.; Krishnamurthy, S.; Nayaka, S.C.; Niranjana, S.R.; Chowdappa, S. Lactic acid bacteria mediated induction of defense enzymes to enhance the resistance in tomato against Ralstonia solanacearum causing bacterial wilt. Sci. Hortic. 2016, 207, 183–192. [Google Scholar] [CrossRef]
  55. Lamont, J.R.; Wilkins, O.; Bywater-Ekegärd, M.; Smith, D.L. From yogurt to yield: Potential applications of lactic acid bacteria in plant production. Soil Biol. Biochem. 2017, 111, 1–9. [Google Scholar] [CrossRef]
  56. Ma, L.; Zhang, M.; Bhandari, B.; Gao, Z. Recent development in novel shelf life extension technologies of fresh-cut fruits and vegetables. Trends Food Sci. Tech. 2017, 64, 23–38. [Google Scholar] [CrossRef]
  57. Raman, J.; Kim, J.-S.; Choi, K.R.; Eun, H.; Yang, D.; Ko, Y.-J.; Kim, S.-J. Application of lactic acid bacteria (LAB) in sustainable agriculture: Advantages and limitations. Int. J. Mol. Sci. 2022, 23, 7784. [Google Scholar] [CrossRef]
Agriculture 14 01228 g001
Figure 1. The amplification of the 16S rRNA gene of bacterial isolates. PCR products of size 1492 bp obtained via reaction with primers 27F/1492R; 1 kb—size marker.
Figure 1. The amplification of the 16S rRNA gene of bacterial isolates. PCR products of size 1492 bp obtained via reaction with primers 27F/1492R; 1 kb—size marker.
Agriculture 14 01228 g001
Agriculture 14 01228 g002
Figure 2. Effect of the cell-free supernatants obtained from LAB-A, LAB-B, and LAB-C cultures on mycelia biomass of B. cinerea from carrots (A), tomatoes (B), and cyclamens (C). Different letters above the bars indicate a significant difference between means at α = 0.05 via Tukey’s test. Statistical calculations were made separately for each B. cinerea isolate.
Figure 2. Effect of the cell-free supernatants obtained from LAB-A, LAB-B, and LAB-C cultures on mycelia biomass of B. cinerea from carrots (A), tomatoes (B), and cyclamens (C). Different letters above the bars indicate a significant difference between means at α = 0.05 via Tukey’s test. Statistical calculations were made separately for each B. cinerea isolate.
Agriculture 14 01228 g002
Agriculture 14 01228 g003
Figure 3. Effect of the cell-free supernatants obtained from LAB-A, LAB-B, and LAB-C cultures on E. coli growth. Different letters above the bars indicate a significant difference between means at α = 0.05 via Tukey’s test.
Figure 3. Effect of the cell-free supernatants obtained from LAB-A, LAB-B, and LAB-C cultures on E. coli growth. Different letters above the bars indicate a significant difference between means at α = 0.05 via Tukey’s test.
Agriculture 14 01228 g003
Agriculture 14 01228 g004aAgriculture 14 01228 g004b
Figure 4. The effect of selected LAB strains on grey mould development on lettuce leaves caused by strains of B. cinerea from tomatoes, carrots, and cyclamens after 5 (A), 10 (B), and 15 (C) days after inoculation by B. cinerea. Different letters above the bars indicate a significant difference between means at α = 0.05 via Tukey’s test. Statistical calculations were made separately for each B. cinerea isolate and for each term.
Figure 4. The effect of selected LAB strains on grey mould development on lettuce leaves caused by strains of B. cinerea from tomatoes, carrots, and cyclamens after 5 (A), 10 (B), and 15 (C) days after inoculation by B. cinerea. Different letters above the bars indicate a significant difference between means at α = 0.05 via Tukey’s test. Statistical calculations were made separately for each B. cinerea isolate and for each term.
Agriculture 14 01228 g004aAgriculture 14 01228 g004b
Agriculture 14 01228 g005
Figure 5. The number of E. coli bacteria on spinach leaves coated with LAB strains. Different letters above the bars indicate a significant difference between means at α = 0.05 via Tukey’s test.
Figure 5. The number of E. coli bacteria on spinach leaves coated with LAB strains. Different letters above the bars indicate a significant difference between means at α = 0.05 via Tukey’s test.
Agriculture 14 01228 g005
Table 1. Percentage of inhibition of radial growth (PIRG) of pathogenic fungi in dual culture with selected strains of LAB.
Table 1. Percentage of inhibition of radial growth (PIRG) of pathogenic fungi in dual culture with selected strains of LAB.
Tested FungiPIRG (%)
LAB-ALAB-BLAB-C
Alternaria sp.7.0 b0.0 a10.5 c
B. cinerea from carrots12.1 c4.4 b16.5 c
B. cinerea from tomatoes15.8 c4.7 b11.5 c
B. cinerea from cyclamens5.7 b0.7 a14.1 c
F. oxysporum0.0 a3.0 b0.0 a
Penicillium sp.0.0 a28.0 c0.0 a
Phialophora sp.54.6 d18.9 c54.6 d
R. solani33.3 d0.0 a15.0 c
S. sclerotiorum7.0 b0.0 a3.0 b
Means followed by a different letter in the same column indicate a significant difference at α = 0.05 using Tukey’s test.
Table 2. Identification of bacterial isolates based on comparison with NCBI data.
Table 2. Identification of bacterial isolates based on comparison with NCBI data.
IsolateSequence Length (bp)Genus/Species with the Highest SimilaritySequence Number NCBIIdentity (%)Identification
LAB-A405Lactiplantibacillus pingfangensisNo_179289.199.26Lactiplantibacillus sp.
Lactiplantibacillus plantarumNo_113338.199.26
Lactiplantibacillus pentosusNo_029133.199.26
LAB-B501Levilactobacillus brevisNo_044704.294.64Levilactobacillus sp.
Levilactobacillus angrenensisNo_180286.193.01
Levilactobacillus yonginensisNo_109452.193.01
LAB-C467Levilactobacillus brevisNo_116238.198.93Levilactobacillus sp.
Levilactobacillus fujinensisNo_180290.197.64
Levilactobacillus tangyuanensisNo_180287.197.64
Table 3. The number of lactic acid bacteria and moulds on the leaves of spinach and lettuce after 8 days of incubation.
Table 3. The number of lactic acid bacteria and moulds on the leaves of spinach and lettuce after 8 days of incubation.
TreatmentLactic Acid BacteriaMoulds
The Density of Microorganisms (log10 g−1)
SpinachLettuceSpinachLettuce
Control (untreated by LAB)3.12 a2.28 a4.00 b4.76 b
LAB-A5.23 b5.57 b3.40 a3.99 a
LAB-B5.77 b6.72 b3.67 a3.48 a
LAB-C5.94 b6.40 b4.04 b4.76 b
Means followed by a different letter in the same column indicate a significant difference at α = 0.05 using Tukey’s test.
Table 4. The influence of LAB strain inoculation on plant weight and number of LAB in soil.
Table 4. The influence of LAB strain inoculation on plant weight and number of LAB in soil.
TreatmentWeight of One Plant (g)Number of LAB (log10 cfu g−1)
SpinachLettuceSpinachLettuce
control8.2 a6.4 a0.00 a0.00 a
LAB-A11.1 c7.3 a2.85 b4.81 c
LAB-B9.9 b7.4 a3.21 c2.60 b
LAB-C10.4 bc6.6 a3.84 c4.92 c
Means followed by (a) different letter(s) in the same column indicate a significant difference at α = 0.05 via Tukey’s test.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Kowalska, B.; Szczech, M.; Lisek, A. Inhibition of Botrytis cinerea and Escherichia coli by Lactic Acid Bacteria on Leafy Vegetables. Agriculture 2024, 14, 1228. https://doi.org/10.3390/agriculture14081228

AMA Style

Kowalska B, Szczech M, Lisek A. Inhibition of Botrytis cinerea and Escherichia coli by Lactic Acid Bacteria on Leafy Vegetables. Agriculture. 2024; 14(8):1228. https://doi.org/10.3390/agriculture14081228

Chicago/Turabian Style

Kowalska, Beata, Magdalena Szczech, and Anna Lisek. 2024. "Inhibition of Botrytis cinerea and Escherichia coli by Lactic Acid Bacteria on Leafy Vegetables" Agriculture 14, no. 8: 1228. https://doi.org/10.3390/agriculture14081228

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop