The Use of Clove and Rosemary Plant Extracts Against Colletotrichum acutatum and Botrytis cinerea

Abstract

Horticulture and agriculture are facing the challenge of growing healthy and high-quality crops. Plant extracts are currently being widely investigated as an alternative means of plant protection. Interest in these measures has increased in order to reduce the use of chemical pesticides, environmental pollution, and adverse effects on human health. Also, due to the goals of the European Green Deal and the decreasing use of chemical pesticides, it has become essential to look for safer alternatives. The aim of this study was to investigate the inhibitory effect of plant extracts of clove (Syzygium aromaticum L.) and rosemary (Rosmarinus officinalis L.) against Colletotrichum acutatum and Botrytis cinerea plant pathogens and to evaluate fungal pathogens recovery after the exposure to the extract. The plant extracts (PEs) were obtained by subcritical CO₂ extraction. The inhibitory effect of PEs was investigated in vitro at concentrations of 1200, 1600, 2000, 2400, 2800, and 3000 μL/L. Petri dishes were incubated at 25 ± 2 °C, and the mycelial growth of fungal pathogens was evaluated at 2, 4, and 7 days after inoculation (DAI). Reinoculation was then performed. The research showed that both plant extracts had an antifungal effect. However, clove PE was more effective. This allows us to say that plant-based measures can inhibit plant pathogens, but it is essential to determine the optimal concentrations and test them with different pathogens.

1. Introduction

Horticulture and agriculture are facing increasingly significant challenges in growing a large quantity of healthy and high-quality produce. This is due to the decreasing amount of free and fertile land and the constant misuse of chemical fertilizers and pesticides [1]. Due to the improper use of chemical pesticides and fertilizers, more and more pesticide-resistant species appear; often, fertilizers are used inefficiently, and large amounts are washed into the environment. The improper use of pesticides and fertilizers has an adverse impact on human health and contributes to environmental pollution [2]. To avoid these problems, the European Union has drawn up the European Green Deal, which aims to make Europe the first climate-neutral continent. One of the goals it sets is to reduce the use of chemical pesticides by 50% by 2030 [3]. As the variety of legally available chemicals for plant protection decreases, it is important to look for alternative means, such as plant essential oils and extracts [4].

Colletotrichum is a species complex comprising C. gloeosporioides, C. acutatum, and C. fragariae [5,6]. Colletotrichum spp. is a plant pathogen that can damage various crops, such as grapes, beans, onions, and pome fruits, and 80 percent of production can be lost during storage due to the ability to go through a non-pathogenic phase [5]. Colletotrichum spp. poses a significant threat to fruit trees and propagation materials in nurseries. Its characteristic symptoms are leaf spots of anthracnose, blight, and postharvest fruit rot. Colletotrichum spp. causes necrotic dark, angular, or oval lesions in all stages of plant growth. In addition, this pathogen has a hemi-biotrophic lifestyle and can change its lifestyle behavior [6]. Elevated temperatures enhance the prevalence and severity of the pathogen throughout the growing season [7]. Optimal conditions for fungal proliferation include temperatures ranging from 25 °C to 40 °C and elevated humidity in warm climate zones [8,9]. Under in vitro conditions, the ideal temperature for the growth of Colletotrichum acutatum was established at 20–25 °C. During research conducted in Lithuania [10,11], it was found that the temperature conducive to the proliferation of anthracnose ranges from 15 °C to 22 °C, with a minimum leaf wetness duration of 12 h.

Another common necrotrophic pathogen belongs to the Botrytis genus [12,13], which is causing significant postharvest yield losses, gray mold, and rot in the aerial parts of the plants. This genus damages plants such as pome fruits, tomatoes, strawberries, cucumbers, onions, leafy vegetables, and carrots [14]. Botrytis spp. causes up to 50% of strawberry and 20–50% of pear and apple losses. In addition, in Iran and Italy, it causes up to 25–37% of strawberry preharvest and postharvest losses. The optimal conditions for gray mold are 90–95% humidity and 20–30 °C. However, Botrytis spp. can survive low temperatures, such as 4 °C, during storage. Besides damaging fruits, they infect flowers, leaves, seeds, and other plant parts. As a necrotrophy, this pathogen secretes toxins and some phytotoxic compounds. The continuous application of chemical pesticides causes not only environmental concerns and handling issues, but also residual toxicity, health hazards to humans, and fungicide resistance [12].

Plant extracts are natural measures that can improve plant growth, reduce biotic and abiotic stress influences, and decrease the need for fertilizers and pesticides [1]. Pathogenic microscopic fungi have a significant impact on crop yield loss; therefore, it is important that any alternative means used could inhibit their growth and development. Essential oils have earned attention due to their extensive applicability in industries like pharmaceuticals and food. Moreover, extensive research on essential oils has demonstrated that they are an environmentally sustainable method of plant disease control due to antiseptic, antifungal, antiviral, and antibacterial properties. Besides plant extracts and essential oils, metabolites demonstrate significant defense properties, are biodegradable, non-toxic, and inhibit plant pathogens [15,16]. Plant extracts and essential oils from clove (Syzygium aromaticum L.) and rosemary (Rosmarinus officinalis L.) are often mentioned as one of these remedies. The inhibitory effect of these measures has been observed against various genera of fungi, including Mucor, Aspergillus, Fusarium, Botrytis, and Colletotrichum [16,17,18]. Using the mycelia growth rate method, Cong-Jun Yang et al. [19] observed that the ethanol extract of Syzygium aromaticum L. had a high antifungal effect against 17 tested pathogens, with inhibition from 60% to 97%. The most active constituents of clove oil and extracts include eugenol, eucalyptol, and beta-caryophyllene. These compounds have insecticidal, antimicrobial, and nematocidal properties against diverse plant diseases, as described in investigations by Hamini et al. [20], Kacániová et al. [21], and Elnabawy et al. [22]. Rosemary is used for the extraction of essential oils and also for the production of phenolic leaf extracts that include a variety of compounds responsible for several biological features [23]. The superior antibacterial and antioxidant properties of rosemary essential oils and phenolic extracts obtained throughout the fruit maturity phase, in contrast to those collected at full bloom, were attributed to differences in the concentrations of various terpenes and phenols. Variations in rosemary terpene and phenol phenotypes result from the interplay between genes and the environment [23,24,25].

Raising concerns about the environment and human health due to the use of chemical fungicides necessitates the development of sustainable and effective alternatives in plant protection, thereby ensuring high-quality yields and food security. Eco-friendly management may serve as a viable alternative to the traditional method of using chemical fungicides for postharvest control of anthracnose. It offers many techniques that are not mutually exclusive and may align with the demands and interests of contemporary customers and the environment [11].

The aim of this study was to investigate the inhibitory effect of plant extracts of clove (Syzygium aromaticum L.) and rosemary (Rosmarinus officinalis L.) against Colletotrichum acutatum and Botrytis cinerea plant pathogens and to evaluate the recovery of fungal pathogens after the extract exposure.

2. Materials and Methods

The research was conducted at the Lithuanian Research Center for Agriculture and Forestry, Institute of Horticulture, Laboratory of Plant Protection, from 2022 to 2024.

2.1. Fungal Monoculture Isolates

The single spore isolates of B. cinerea LT12K_RUB_183 (host raspberry) and C. acutatum-18-Kau_Fur4 (host strawberry) were obtained from the Laboratory of Plant Protection isolate collection, Institute of Horticulture, Lithuanian Research Center for Agriculture and Forestry. Both isolates were identified by PCR [10,13]. The isolates were maintained on potato-dextrose agar (PDA) at 4 °C and were plated 7 days prior to the experiment on PDA at 22 °C in the dark.

2.2. Plant Extracts Preparation and Composition

The plant extracts of rosemary (Rosmarinus officinalis L.) and clove (Syzygium aromaticum L.) were obtained by subcritical CO₂ extraction. During the process, 5 kg of dried clove buds and rosemary leaves were placed in an extraction vessel. The extraction was carried out for 6 h, maintaining a pressure of 42 bars and a temperature of 10 °C. The collected plant extract was stored at 4 °C until testing [26]. The extracts’ volatile components were identified using gas chromatography/mass spectrometry (GC-MS). The Rxi-5MS capillary column (30 m × 0.25 mm; film thickness, 0.25 μm) paired with the GC-2010Plus/GCMS-QP2010 Ultra system (Shimadzu, Kyoto, Japan) (Restek, Bellefonte, PA, USA) was used for the analysis. The flow rate was 1 mL min⁻¹, and the injector temperature was 250 °C. At a rate of 5 °C per minute, the column temperature was increased from 50 °C to 160 °C, and then at a rate of 10 °C per minute, until it reached 250 °C. The split mode for injecting samples was 1:20. At 220 °C and 70 eV in electronic impact mode, mass spectra were acquired [16,18].

2.3. Antifungal Activity of Plant Extracts

The PDA medium (Liofilchem, Srl Via Scozia, Zona Industriale 64026, Roseto degli Abruzzi (TE) Italy) was used to study the inhibitory effect. The plant extract was poured into the nutrient medium, and everything was mixed well, poured into Petri dishes, and cooled. The extract in its pure form and the tested concentrations were immediately poured into the PDA medium and poured in a Petri dish. The control dish was prepared identically to the tested dish, with the exception that no plant extract was included in the PDA medium, since it served as a control version. In the antifungal activity research, four replicates (three dishes per replicate) were conducted. The plant extract was added to obtain different concentrations: 1200, 1600, 2000, 2400, 2800, 3000 μL/L. The selected concentrations were intended to evaluate the effects of the tested agents throughout a broad spectrum and to compare them with doses previously used by other researchers.

Plugs of the isolate measuring 7 mm were cut out of a dish with a grown microscopic fungal monoculture. The plugs were placed in the center of the cooled nutrient medium with the mycelium facing downwards. The prepared Petri dishes with C. acutatum and B. cinerea isolates were placed in a thermostat at 22 °C. The evaluation of mycelium growth was carried out 2, 4, and 7 DAI. The average mycelium diameter (mm) was estimated, and the inhibition was calculated using the following formulas [27]:

$$A=\left(\right(x-d)+(y-d\left)\right)/2,$$

(1)

where $A$ is the average mycelium diameter (mm); $x$, $y$ are the two perpendicular measurements of the colony diameter (mm); $d$ is the plug diameter (mm).

$$Inhibition(\%)=(\mathrm{C}-T)/\mathrm{C}\times 100\%,$$

(2)

where $\mathrm{C}$ is the radial mycelium growth of the pathogen in the control (mm), and $T$ is the radial mycelium growth of the pathogen in the treatment (mm).

2.4. Reinoculation

Reinoculation studies were performed to assess the recovery of the fungus after exposure to a plant extract. Reinoculation was performed by transferring the microscopic fungal mycelium of the test fungus from each tested variant to a new PDA nutrient medium. In this case, no additional biologically active substances were added. Plugs measuring 7 mm were cut from each variant. Two cut plugs of the same treatment were placed, with the mycelium facing downward, on new Petri dishes with PDA. In the reinoculation research, four replicates (two plates per replicate) were conducted. The prepared samples were placed in a thermostat at 22 °C. Evaluations and measurements were performed after 2 and 4 DAI. The two perpendicular measurements of the colony diameter (mm) were measured during the evaluation.

2.5. Processing of Data

All data from the conducted studies were processed using ANOVA analysis with the SAS Enterprise Guide program (SAS Institute Inc., Cary, NC, USA). The results were presented as mean values ± standard error. The Duncan test was used to analyze the data. The least significant difference of 0.05 was used. The standard error of the mean was calculated using the Excel program package.

3. Results

3.1. Chemical Composition of the Plant Extracts

The rosemary PE chemical composition was determined according to [18] and is presented in

Table 1. The composition of the plant extract compounds of rosemary (Rosmarinus officinalis L.) and clove (Syzygium aromaticum L.).

Plant Extract	Rosmarinus officinalis L.		Syzygium aromaticum L. [18]
Component	PA 1 (%)	RT 2	PA 1 (%)	RT 2
α-pinene	8.92	6.730	0.26	6695
Camphene	2.98	7.083
β-pinene	3.11	7.813
1-octen-3-ol	0.20	7.959
3-octanone	0.15	8.043
Myrcene	1.52	8.159
p-cymene	2.70	9.213
α-terpinene	0.38	8.894
Eucalyptol	41.28	9.426	0.36	9285
Linalool	1.33	11.284
Camphor	16.62	12.640
Borneol	3.94	13.239
Terpinen-4-ol	1.06	13.509
α-terpineol	4.16	13.944
Bornyl acetate	0.28	16.428
Ε-caryophyllene	3.39	20.038
α-Humulene	0.42	20.858
β-Bisbaolene	0.21	22.121
Caryophyllene oxide	0.60	24.071	0.52	24.134
Caryophylla-4(12),8(13)-dien-5α-ol	0.17	25.595
Ʒ-Methyl jasmonate	0.18	25.759
Caryophyllene-14-hydroxy-Ʒ	0.40	26.130
Caryophyllene-14-hydroxy-9-epi-E	0.25	26.472
Oleic acid	0.45	32.984
trans-Ferruginol	0.46	35.012
Squalene			0.53	33.304
trans-caryophyllene			17.80	20.168
Germacrene D			0.27	21.568
α-cubebene			0.82	18.163
Eugenol			52.88	18.787
Eugenol acetate			21.95	22.822
α-copaene			0.93	18.935
α-humulene			2.00	20.922
Other 3	4.84		1.49
Total Identified	100.00		99.81

¹ PA—peak area. ² RT—retention time. ³ The compounds that were less than 0.14% of the quantity of the plant extract. The results are presented as mean (n = 3).

. In total, 100.00% of rosemary PE components were identified. Eucalyptol 41.28%, camphor 16.62%, and α-pinene 8.92%, three dominant compounds, were determined in rosemary PE. The chemical composition of the volatile compounds of clove PE was eugenol 52.88%, eugenol acetate 21.95%, and trans-caryophyllene 17.80% [18].

3.2. Inhibitory Effect of Clove Plant Extract

The inhibitory effect (Figure 1) and reinoculation (Figure 2) of clove PE against C. acutatum were evaluated. The C. acutatum was completely inhibited at the various concentrations tested. In the tested concentration range between 1200 μL/L and 3000 μL/L, the calculated inhibition reached 100%. This suppression persists during the 2, 4, and 7 DAI and is statistically significant. The C. acutatum growth in the control continuously increased from 2 DAI to 7 DAI.

Following the pathogen inhibition, a reinoculation study was conducted, and the data obtained are presented in Figure 2. Reinoculation is needed to monitor the recovery after PE inhibition and evaluate whether it is fungistatic or fungicidal. The results show that C. acutatum cannot recover and grow at the tested concentrations. This trend can be observed at all six concentrations tested, both at 2 DAI and 4 DAI.

The inhibitory effect of clove PE against B. cinerea is presented in Figure 3. Our data shows that PEs have a strong inhibitory effect against the tested isolate. No B. cinerea growth was observed at 2 DAI at all concentrations. At 4 DAI, slight mycelial growth was detected at a concentration of 1200 μL/L; in this case, the inhibition reached 93%. At concentrations of 1200 μL/L and 1600 μL/L, slight growth of microscopic fungal mycelia was observed 7 DAI. The inhibition reaches 90% and 96% at these concentrations, respectively. At higher concentrations, B. cinerea is completely inhibited (inhibition reaches 100%).

The evaluation of reinoculation (Figure 4) shows that mycelial growth of B. cinerea was visible 2 DAI at the concentration of 1200 μL/L. However, the growth was 36% less compared to the control. The viability of the B. cinerea was also visible at 4 DAI at concentrations of 1200 μL/L and 1600 μL/L. Stronger mycelial growth of B. cinerea was observed at 4 DAI at a concentration of 1200 μL/L. Mycelium growth increased by 13% in this case. The growth of the B. cinerea was significantly reduced to a concentration of 1600 μL/L and reached 56%. At concentrations of more than 1600 μL/L, it can be observed that the microscopic fungus can no longer grow normally.

3.3. Inhibitory Effect of Rosemary Plant Extract

The effect of the rosemary PE against C. acutatum is presented in Figure 5. Our data indicated that the rosemary PE has an inhibitory effect against C. acutatum. However, there are no significant differences between the concentrations, except for at 2 DAI at a concentration of 2000 μL/L, in which case, the inhibition reaches 78%. At 4 DAI, at the three lowest concentrations of 1200–2000 μL/L, the inhibition reached 54%, 56%, and 57%, respectively. At 7 DAI, the inhibition was 47%, 50%, and 50%, respectively. Meanwhile, at the three higher concentrations, the inhibition was 54%, 60%, and 56% (7 DAI). The 4 DAI and 2 DAI results showed that the inhibition at different concentrations was 77%, 82%, 79%, 84%, 89%, and 86%, respectively.

Reinoculation (Figure 6) shows that C. acutatum is not suppressed and can continue to develop mycelium after the fungal mycelium has been transferred to a PDA medium without the tested active substances. Mycelial growth was significantly inhibited at a concentration of 2400 μL/L, reaching 28%, at 4 DAI. At this concentration, the inhibition of 2 DAI reached 44%. With an amount of 2400 μL/L, the C. acutatum may have shown heightened sensitivity to the clove PE. The defensive systems were more effective at both lower and higher concentrations than at this concentration. At this level of concentration, it is believed that one defensive mechanism transfers to another, since the former is unable to sustain development.

The inhibitory effect of rosemary PE against B. cinerea is presented in Figure 7. This extract also shows an inhibitory effect against the tested genus. No significant differences are observed at a concentration of 1200–2000 μL/L, and the inhibition at 4 DAI and 7 DAI was 63%, 63%, 76%, 47%, 52%, and 57%, respectively. At these concentrations, no microscopic fungal growth was visible 2 DAI. At the three higher concentrations (2400–3000 μL/L), the inhibition of 2 DAI also had no significant differences between concentrations and reached 91%, 88% and 92%, respectively. At 4 DAI and 7 DAI, inhibitions were 79%, 78%, 86%, and 41%, 43%, 50%, respectively.

Reinoculation (Figure 8) shows that B. cinerea is not suppressed and can continue to develop mycelium after transferring the fungal mycelium. At 2 DAI, at concentrations of 1200 μL/L, 1600 μL/L, 2000 μL/L, no significant differences in mycelium growth are visible, but a more intensive mycelium growth of 17% is observed at a concentration of 1600 μL/L, at 4 DAI. A more intensive mycelium growth of 108% is also observed at a concentration of 2800 μL/L, at 4 DAI. In this case, we observed that fungi grow faster after reinoculation at concentrations of 1600 μL/L and 2800 μL/L than in other treatments. The B. cinerea grown at these concentrations may have more robustly activated stress response systems that enhance mycelial development. At alternative doses, these response mechanisms are not entirely efficacious. The reinoculation shows the effect of PEs on B. cinerea growth conditions after returning to primary conditions (without PEs).

4. Discussion

As fewer and fewer chemical pesticides are available for plant protection, there is growing interest in alternative measures to control pathogens. Remedies such as plant essential oils and extracts are often mentioned. For this reason, these studies focus on plant extracts. Two plant extracts were selected for this study: rosemary (Rosmarinus officinalis L.) and clove (Syzygium aromaticum L.). Their inhibitory effect against two common pathogens, C. acutatum and B. cinerea, was investigated. The results of the studies showed an intensive inhibitory effect of these measures against the tested isolates. The effect of plant extract on two major pathogens C. acutatum and B. cinerea was evaluated. In our opinion, PEs exhibit lower phytotoxicity compared to essential oils, which is attributable to their reduced concentration of active compounds.

Various plant extracts and essential oils are effective against fungal pathogens. The essential oils of Thymus vulgaris L., Hyssopus officinalis L., and Juniperus communis L. were even effective against seedborne pathogens in different vegetable seeds such as carrots and tomatoes. Moreover, Thymus vulgaris L. was completely effective against Alternaria spp. from 200 μL/L [15]. The different concentrations of clove extract showed that complete inhibition of the Botrytis cinerea mycelium is achieved with a concentration as low as 600 μL/L [18]. In our studies, the efficacy of clove PE was tested against C. acutatum and B. cinerea, and it was confirmed that this extract has inhibitory properties against the tested genera. It was also found that C. acutatum from a concentration of 1200 μL/L and B. cinerea from a concentration of 2000 μL/L completely inhibited mycelial growth (no mycelial growth observed).

Rosemary essential oil and extract are mentioned as one of the alternative means of combating pathogenic microscopic fungi. The effectiveness of these measures is mentioned in studies by scientists [28,29,30]. It was shown that rosemary plant extract at a concentration of 1000 μL/L achieved a 50 percent inhibition of mycelial growth against Alternaria spp. after seven days [30]. In our studies, the effect of the same extract against C. acutatum and B. cinerea was investigated using higher concentrations of the extract. It was found that the inhibition of C. acutatum reached 56%, 7 DAI at 3000 μL/L. The inhibition of B. cinerea reached 50%, 7 DAI at 3000 μL/L. In other studies, it is showed that ethanol extracts of Syzygium aromaticum L. inhibit Valsa mali 96.3% and Fusarium graminearum 97.4% [19]. The S. aromaticum L. essential oils show antimicrobial activity against Penicillium expansum at 250 and 500 L/mL concentrations [20].

Rosemary bioactive compounds (terpenes and rosmarinic acid) inhibit Alternaria alternata and Pseudomonas viridiflava [23]. In addition, rosemary essential oil and its polyphenolic extract have an antimicrobial effect on Listeria monocytogenes serovar 4b (CECT 935), Staphylococcus aureus (CECT 240), Salmonella serotype Typhimurium (CECT 443), and Escherichia coli O157:H7 (CECT 4267) [24].

In our research, rosemary PE was less effective than clove PE. At 7 DAI, at a concentration of 3000 μL/L, the clove extract completely inhibited mycelial growth (100%) against both tested isolates, while rosemary inhibited Colletotrichum spp. by 56%, and Botrytis spp. by 50% under the same conditions. Other researchers found a stronger effect of clove extract compared to rosemary extract against B. cinerea. They investigated the inhibitory effect of clove and rosemary essential oils and presented the research results, which showed that the use of 2% clove emulsion inhibited Botrytis spp. by 46.22% and 2% rosemary emulsion inhibited micelle activity by 19.44% [31]. To control secondary metabolism and adjust to environmental changes that impact growth, survival, and host adaptability, fungi employ epigenetic processes such as DNA methylation and histone alterations. Epigenetic control can influence pathogenicity and prevent fungal infections, whereas specific settings stimulate secondary metabolism to produce distinct chemicals [32].

In our experiments, clove PE 1200–3000 μL/L inhibited the growth of B. cinerea and C. acutatum mycelium. Moreover, the reinoculation of clove PE also shows promising inhibition results. Additionally, rosemary PE at the same concentration was less effective than clove PE. Our results suggest that PE may have the potential to suppress plant pathogens and could serve as an alternative plant protection measure. Our results confirmed that it is important to test various concentrations, as it acts differently on various fungal species. The reason for using such different concentrations was that the isolates used in this research originated from several areas or distinct plant hosts. Distinct isolates, even within the same species, may exhibit divergent reactions to stress, and their susceptibility to the used agents will differ. Ensuring the consistency of the PEs’ composition is challenging, since the developing raw material may experience varying stressors and acquire disparate levels of metabolites, which are crucial for effective inhibition. Therefore, it is important to work with the same material and PEs. Moreover, different extraction methods may yield extracts with distinct compositions. The use of various solvents, such as CO₂, will result in a higher concentration of the composition in the extract. There are a lot of challenges with plant material and extraction methods; therefore, it is essential to be accurate. Also, by-products could be a more sustainable option for plant material used for PE extraction.

5. Conclusions

The results showed that the clove and rosemary plant extracts have an inhibitory effect against C. acutatum and B. cinerea. The plant extract of clove completely inhibited C. acutatum at a concentration of 1200 μL/L. The fungus no longer grew at these concentrations, even in the reinoculation. The effect of this extract against B. cinerea showed that it can completely inhibit mycelial growth from a concentration of 2000 μL/L. From the concentration of 2000 μL/L, the fungal mycelium also stopped growing in the reinoculation. The rosemary extract has a lower inhibitory effect on the tested isolates than the plant extract from cloves. The results show that the intensive inhibition of mycelial growth of C. acutatum is achieved by the extract from a concentration of 1200 μL/L. However, no significant differences were observed when the concentrations were increased. Reinoculation results show that the fungus recovers and can grow normally. Rosemary extract has a similar effect against B. cinerea, but in this case, a more potent mycelial inhibition is observed at a concentration of 3000 μL/L extract. Based on this data, different PEs and their concentrations have different effects against the tested isolates. Therefore, it is important to investigate the impact of different PEs against different genera of microscopic fungi in the future. Future studies should focus on examining the effects of various products in experimental settings, assessing their impact on disease control in inoculated and naturally infected plants, and exploring the phytotoxic potential impact associated with different concentrations.