Citrus Essential Oils as an Alternative Method of Control of the Fungus Alternaria alternata (Fr.: Fr.) Keissler

1. Introduction

Brazil is the largest world producer of oranges (17.1 million tons), however, it ranks seventh as a world producer of tangerines, having produced around 850 thousand tons in 2021, a much lower production than China, the first producer, with approximately 20 million tons. Ahead of Brazil are Spain, Turkey, Morocco, Egypt, and the United States, which stand out as the largest producers [1].

In the state of São Paulo, the main commercially grown mandarin varieties are the Ponkan mandarin (Citrus reticulata Blanco) and the Murcott tangor [C. reticulata x C. sinensis (L.) Osbeck], which represent about 80% of the orchards. The Rio mandarin (C. deliciosa Tenore) and Cravo mandarin (C. reticulata) are also grown, as well as smaller plantings of Fremont IAC 543 mandarin (C. clementina Hort. ex Tan. x C. reticulata), IAC 2019Maria mandarin [C. reticulata x C. sinensis (L.) Osbeck] x Pera IAC orange [C. sinensis (L.) Osbeck] and Late IAC 855 willowleaf [2].

Mandarin production in Brazil is limited to a few varieties with low genetic variability, making it vulnerable to phytosanitary problems. Among the diseases that cause significant damage to mandarins are Alternaria brown spot (ABS) [Alternaria alternata (Fr.:Fr.) Keissler] and huanglongbing (HLB) (Candidatus Liberibacter spp.), which have compromised the orchards and hindered the management and production of the crop [3,4]. Consequently, the area planted and production of mandarins in Brazil have declined in recent years [5].

As most mandarin varieties are susceptible to ABS, producers need to apply numerous fungicides for its control, around 12 to 18 times a year, increasing production costs [4]. However, the growing concern about the resistance of the fungus A. alternata to registered fungicides and their toxicity necessitates alternative means to manage the disease, besides genetic improvement. Cases of resistance of the fungus A. alternata to strobilurin group fungicides (Quinone Outside Inhibitor Resistance) have been observed since 2012 in Florida, USA [6], and were recently detected in 2019 in mandarin orchards in the state of São Paulo, Brazil [7].

Given the above, studies related to the antifungal activity of plant products have become a target of research. Essential oils (EOs) extracted from several plant species are an alternative to traditional chemical treatments, which rely on synthetic fungicides and may select resistant phytopathogenic fungi [8]. Tropical plants are a reservoir of secondary metabolites and a significant source of chemical components with different biological properties. The use of EO extracted from these plants can act as a natural fungicide, inhibiting the activity of a range of fungi and leaving no toxic residues on humans or treated food [9]. They act both by direct fungitoxic action, inhibiting mycelial growth and spore germination, and by the action of phytoalexins [10,11]. Certain terpenes present in the EOs, such as limonene, can make the cell membrane of the fungus permeable, causing the leakage of its content [12].

The objective of this study, was to evaluate the inhibitory effect of essential oils from mandarin varieties on the fungus Alternaria alternata, which causes ABS.

2. Materials and Methods

Plant Material and Extraction of Essential Oils

EOs were extracted from the ripe and unripe fruit peels of varieties showing differential responses to ABS: Murcott IAC 221 tangor (susceptible), Fremont IAC 543 mandarin, IAC 2019 Maria mandarin, and Late IAC 855 willowleaf (tolerant/resistant) grafted on Rangpur lime. The plants were located in an experimental area in Cordeirópolis, São Paulo State, Brazil, at a latitude of 22°27′35" South and a longitude of 47°24′27" West, with an average altitude of 712 m above sea level. The climate at the site is classified as subtropical Cwa with dry winters (temperatures below 18 °C) and hot summers (temperatures above 22 °C), according to the Köppen-Geiger climate classification system [13].

The EO extraction process was carried out in the Laboratory of Improvement and Analysis of Fruit Quality (LMQF) of the Sylvio Moreira Citrus Center (IAC). The EOs were extracted using the hydrodistillation method, employing the modified Clevenger apparatus [14]. 400 g of chopped peel (1 cm²) of each variety was used for oil extraction. The material was placed in 6 L flasks and kept boiling at a constant temperature of 180 ºC for three hours. After this time, the oil was collected, quantified, and stored in light-protected bottles at 4 °C.

Isolation of Alternaria Alternata Fungus and Preparation of Inoculum

The Alternaria alternata fungus was isolated from typical lesions on highly susceptible Murcott tangor fruits collected in the field from a plantation located in Cordeirópolis-SP at the Sylvio Moreira Citrus Center (CCSM) of the Agronomic Institute of Campinas (IAC), where the fungus is endemic. To obtain the isolate, the methodology described by Canihos et al. [15] was used with modifications [4]. The lesions were removed from the fruits and disinfected with 70% ethanol, 3% hypochlorite, and distilled water. They were then incubated on Petri plates containing PDA medium (200g potato, 20g dextrose, and 14g L^-1 agar) with the addition of the fungicide carbendazim (640 mg L^-1), which does not affect the pathogen but other opportunistic fungi. The plates were kept in the BOD with a photoperiod of 12 hours at approximately 27ºC for 48 hours.

After 48 hours, the characteristic hyphae of the pathogen were identified on the plates using a light microscope. Serial dilution was then performed with distilled water until a concentration of 10⁵ conidia mL^-1 was obtained to obtain a single spore culture. The solution of a single spore culture was transferred to other Petri plates containing PDA. The fungus was repotted in PDA every three months using an autoclaved loop to transfer spores to the new plate. All procedures were performed in the Biotechnology Laboratory of the Citrus, in Cordeirópolis, SP.

To prepare the inoculum, inverted discs of the fungus's mycelium (8mm diameter) were transferred to Petri plates containing the same condition of the isolate and kept in the BOD for seven days with a photoperiod of 12 hours at approximately 27ºC, following the methodology of Canihos et al. [15]. After mycelial growth, 10 mL of distilled water was added to the surface of the plate, and the conidia were removed from the surface of the plate using a sterile Drigalski spatula. The suspension was filtered on a layer of sterile gauze to remove mycelial fragments from the plate, and then the concentration was adjusted to 10⁵ conidia mL^-1 with the aid of the Neubauer chamber.

Analysis of Essential Oils by GC-FID and GC-MS

The chemical composition of the EO was analyzed by gas chromatography coupled to mass spectrometry (GC-MS) and conventional gas chromatography (GC-FID) following the procedure described by Frizzo et al. [16]. A Shimadzu GC-14B gas chromatograph (Tokyo, Japan) equipped with flame ionization (FID) and data processing software (EZ-Chrom, Shimadzu Corp.) and a GC-MS QP 5050A (Shimadzu Europe) were used. Chemical identification of the components was performed by comparing mass spectra with commercial libraries and calculating the linear retention rates (LRR) on two capillary columns of different polarity: weakly polar (SE-52, Mega, Legnano, Italy) and polar (CW-20M, Mega, Legnano, Italy). Quantification of each component was performed according to Frizzo et al. [16] using tetradecane (Sigma Aldrich, USA) as an internal standard. All chromatographic analyses were performed in triplicate, and the results of the components were expressed as a relative percentage.

Inhibition of Alternaria Alternata Fungus In Vitro

In 2019 and 2020, the antimicrobial activity of the EOs from different varieties was tested against Alternaria alternata fungus by the agar diffusion method by cavity plate [17]. The effects of five concentrations (1, 2, 4, 8, and 16 μL mL^-1) on the growth and mycelial inhibition of the fungal cultures were evaluated in triplicate.

Approximately 20 mL of the potato-dextrose-agar (PDA) culture medium was added to 9 cm diameter Petri plates in an aseptic laminar flow hood. The concentrations of 0 (control), 1, 2, 4, 8, and 16 μL mL^-1 of the EO, along with Tween 80 in a 1:1 ratio, were added to the media with a micropipette. After solidification of the culture media, an 8 mm diameter inverted disc containing A. alternata mycelium (taken from a twelve-day old colony in PDA) was deposited in the center of each plate. The assay was performed in a 2x4x6x7 fully randomized design, with EOs extracted from unripe and ripe fruits of four varieties: Fremont IAC 543, Murcott IAC 221, IAC 2019Maria, and Late IAC 855, five concentrations, and the control evaluated at seven different times (24, 48, 72, 96, 120, 144, and 168 h). The plates were sealed with plastic film, identified, and incubated in a germination chamber under a 12-hour photoperiod at a temperature of 25˚C. The experiment was performed twice over two years (2019 and 2020), and because there were no statistical differences between the years, the mean values between them were used.

Preventive and Curative Control on Detached Leaves

For this experiment, new leaves of the tangor Murcott IAC 221, a susceptible variety to Alternaria brown spot (ABS) with approximately 2-3 cm, were collected from the upper third of 12-month-old plants after two weeks of pruning. The leaves were grafted onto Rangpur lime and maintained in an greenhouse. In vitro mycelial growth inhibition tests were conducted to stipulate the concentrations of essential oils (EO) from four varieties of mandarins under study for the preventive and curative control of the Alternaria alternata fungus. The concentrations used were 2, 4, 8, and 16 μL mL^-1 of EO, together with Tween 80 in a 1:1 ratio, and a control with only distilled water.

In sterile test tubes, a solution of water and Tween 80 was added, and the aliquot of EO was added to obtain the desired concentration. The mixture was then stirred until completely homogenized. A suspension of spores from a single spore culture at a concentration of 10⁵ was prepared by adding 50 mL of sterile distilled water in Petri dishes, according to Canihos et al. [15].

The trial was conducted in 2021 in an entirely randomized design with a 2x3 factorial scheme, with two control methods (preventive and curative) and three replicates per concentration. The experiment was set up according to the in vitro severity assessment model of Azevedo et al. [4] using the diagrammatic assessment scale of Martelli et al. [18].

The preventive control test was installed by applying approximately 1 mL of the solution (EO + Tween 80) per leaf, in different concentrations, on the abaxial part and spreading it with a brush. About two hours after application, when the solution had dried on the leaf surface, the pathogen was sprayed with approximately 1 mL of the conidia suspension on the leaves and then maintained in a BOD at approximately 27ºC± 2ºC with a photoperiod of 12 hours. For the curative control test, the leaves were inoculated with the conidia solution and kept for 24 hours in the BOD, along with the other control test. After 24 hours, the solution with oil was applied in the same way as in the preventive test.

Assessments and Data Analysis

The evaluations of mycelial growth (assay 1) were performed seven days after the experiment was set up by taking diametrically opposite measurements (average of two measurements) of the pathogen's mycelial growth. The percentage of mycelial growth inhibition was calculated for each concentration (treatment) compared to the control using equation 1 as follows:

Pi = (dc - dt )x100

(1)

dc

Where, Pi = Percent growth inhibition

dc = The mean diameter of the colony of the fungus in the control

dt = The mean diameter of the fungus colony in the treatment

The assessment of lesions on detached leaves, caused by the fungus, was done seven days after inoculation by observing typical symptoms of the disease and subsequently determining the injured area (% of leaf taken by the disease), as described by Martelli et al. [18]. This represented the levels of symptoms in ten illustrated grades, where "0" represented a leaf without symptoms and the severity grades ranged from 0.3 to 97% of leaf area affected by A. alternata symptoms.

All data underwent variance analysis, and when significant for concentration, regression analysis was performed. The models were selected based on the determination coefficient (R2>0.9) using the software SISVAR 4.5 [19].

3. Results and Discussion

Chemical Composition of Essential Oils

The chemical composition of the EOs was determined by GC-FID and GC-MS chromatographic analysis, which identified 48 volatile compounds (Table 1), grouped as follows: 2 acids, 12 alcohols, 6 aldehydes, 1 ketone, 20 terpenes, and 7 sesquiterpenes.

The number of compounds identified in the EOs is consistent with the literature, which reports 29 to 116 compounds in the EOs of different mandarin varieties, with limonene being the predominant compound [20,21]. The amount of limonene found in this study was similar to that reported in the literature for mandarins, which ranges from 65 to 75% [22], except for the oil of Late IAC 855 mandarin (58.9%). Myrcene or linalool are generally the second most abundant compound in citrus essential oils, with terpinene also being prominent [23]. A high percentage of linalool was observed in the IAC 2019Maria mandarin, distinguishing it from other varieties in this component.

This characterization also highlights the presence of myrcene and linalool (0.08 to 2.47%) in mandarins, and terpinene (>18%) in the oil of Late IAC 855 mandarin. Although the commercialized volumes of mandarin and mandarin oils are low, they have a high added value, mainly in the cosmetics and perfumery industry. Furthermore, their antimicrobial potential is also known [24].

It is worth noting that the inhibitory activity (antimicrobial) of an EO is explained by a complex interaction between its constituents, leading to additive, synergistic, or antagonistic effects, especially considering those present in low concentrations [25]. Therefore, it is important to study different varieties of mandarins, which present significant differences in the chemical constitution of their essential oils.

In Vitro Inhibition of Alternaria Alternata Fungus

No interactions were observed between mandarin fruit oils in fruits at different stages of maturity (unripe and ripe) and between the years of evaluation (2010 and 2020). On the other hand, significant differences were observed when comparing concentrations within the same variety and among the varieties studied.

The growth (Figure 1) and percentage of mycelial growth inhibition (Figure 2) of the fungus Alternaria alternata in vitro with different concentrations (1, 2, 4, 8, and 16 μL mL^-1) of EO from mandarins, after regression analysis, fitted linear models with a high degree of determination (R²>0.9). As the concentration increased, mycelial growth decreased after seven days (Figure 1). The oil extracted from IAC 2019Maria mandarin provided the lowest mycelial growth to the pathogen (1.10 cm) when using the highest concentration (16 μL mL^-1), followed by Late IAC 855 willowleaf (3.20 cm), Murcott IAC 221 tangor (4.53 cm), and Fremont IAC 543 mandarin (4.87 cm).

The results of mycelial growth inhibition showed that all EOs have an inhibitory effect on the fungus and indicate that the higher the concentration used in the culture media, the greater is the direct fungitoxic effect on the pathogen, inhibiting mycelial growth. Among the tested EOs, the one from IAC 2019Maria mandarin presented the best mycelial growth inhibition result (75.67%), followed by Late IAC 855 willowleaf (51.55%), Murcott IAC 221 tangor (51.34%), and Fremont IAC 543 mandarin (43.80%) (Figure 2).

Studies corroborate these results and describe that the effect of EO on citrus varieties (lemon, orange, grapefruit, and mandarin) is associated with the decrease of fungal growth with the antimicrobial potential of the tested oils [26,27]. This occurs due to the high chemical complexity of these EOs, attributing this antimicrobial effect to the synergism or antagonism among their constituents [28].

Better results were obtained by the IAC 2019Maria mandarin, which may be related to the higher concentration of isocarveol (perillyl alcohol), along with the presence of limonene and linalool in the composition of the essential oil (EO). Studies have shown the antifungal capacity of perillyl alcohol against Candida spp. strains, demonstrating fungicidal activity at concentrations ranging from strong to moderate due to the presence of bulky lipophilic groups attached to the aromatic ring, which contribute to potentiate bioactivity [29]. The fungicidal effect of EOs may be related to the presence of limonene in the constitution of oils. In several studies, the fungitoxic effect of citrus EO (C. sinensis) in the control of Asiatic Rust (Phakopsora pachyrhizi) in soybean has been observed, with limonene being a major compound with elicitor characteristics [10]. Additionally, it has been found that orange EO has a high concentration of monoterpenes and phenolic compounds, where these components inhibited the mitochondrial respiration of the fungus membrane [30], and the same concentration was found in the EOs of this study.

The fungitoxic activity of plant EOs is attributed to small terpenoids and phenolic compounds such as thymol, carvone, menthol, carvacrol, and limonene, as is the case with the majority of compounds present in the varieties studied in this work. The effects of limonene, linalool, and myrcene in the inhibition of mycelial growth and the germination of spores of Colletotrichum acutatum species isolated from Valencia orange plants are reported [31].

The fungicidal activity caused by the application of EOs is also described against a wide range of postharvest fungi, including Alternaria alternata, Colletotrichum gloeosporioides, Rhizopus stolonifer, Aspergillus spp., and several species of Penicillium spp., among others, that can be effectively controlled with the use of EOs [32,33]. It has already been found in the literature that the fungitoxic effect of the essential oil of orange, which was observed for Phakopsora pachyrhizi, was due to the presence of limonene, a majority compound with elicitor characteristics [10,11].

The components citral, linalool, and β-pinene found in the tested mandarin oils have effects against different phytopathogenic fungal species, and the antimicrobial efficacy of the combination of the chemical compounds was observed [34]. The fungitoxic effects of citrus EOs were observed in samples with a high concentration of monoterpenes and phenolic compounds, where these components inhibited the mitochondrial membrane respiration of fungi [35]. Although the characterization of the action mechanisms of the EOs is not known for sure, the accumulation of compounds of lipophilic character in the membrane causes the loss of energy by the microbial cells [36].

Limonene has antifungal activity attributed to the inhibition of pectinmethylesterase (PME), which modifies the degree of methylesterification of pectins, the main components of the cell walls of fungi [37]. Citral (a mixture of neral and geranial isomers present in EOs) reduced the mycelial growth of Fusarium oxysporum cubense, C. gloeosporioides, Bipolaris spp., and Alternaria alternata [38]. Mandarin EO with 46.7% limonene can inhibit the growth of A. alternata, Rhizoctonia solani, and Curvularia lunata [39].

According to Lopes et al. [40], the mode of action of phytoalexins on fungi includes cytoplasmic granulation, disorganization of cell contents, rupture of the plasma membrane, and inhibition of fungal enzymes. These effects are reflected in the inhibition of germination and germ tube elongation and the reduction or inhibition of mycelial growth of fungi. Sharma et al. [41] showed the inhibition of mycelial growth of the fungus Aspergillus niger when the EO of orange peels was used. The morphology of the fungus was evaluated by scanning electron microscopy, and after the test with OE, they reported that the hyphae were damaged, and in some cases, their death occurred. The same authors also evaluated the fungitoxic effect of the EO of orange on ten pathogens, observing a broad spectrum of action on microorganisms, with a minimum inhibitory concentration of 400 to 500 μg/mL^-1. Hani et al. [42] observed that the chemical components present in the oil of C. sinensis and C. reticulata inhibit intercellular and extracellular enzymes, acting as a regulator of cellular metabolism and affecting enzyme synthesis in the nucleus and/or ribosome. They also interact with nutrient uptake from the environment, affecting mycelial growth.

Similarly, in research conducted to verify the effect of 22 EOs on eukaryotic cells, it was revealed that they act as pro-oxidants, affecting mainly the cell membranes and the interior of organelles such as mitochondria, as exposed in an important review on the subject [43]. According to these authors, the cytotoxic effects of EOs in cells may be associated with changes in the intracellular oxireduction potential resulting from the activity of exposure to EOs.

In this way, essential oils can exert antimicrobial activity on a wide diversity of microorganisms. However, to obtain an expressive effect, different concentrations are necessary according to the pathogen to be inhibited. Moreover, the antifungal effect depends directly on the chemical components present in the oil, acting directly or indirectly on the biological activity and sensitivity of microorganisms [44].

Preventive and Curative Control on Detached Leaves

There was no interaction between the EOs extracted from ripe and unripe fruits; therefore, the results of these tests were grouped. When analyzing the effect of the oils in preventive and curative treatments, all the essential oils tested at the concentration of 16 μL mL^-1 provided lower values of disease severity when compared to other concentrations and the control, which showed an average severity of 57.5% of the injured leaf area (Figures 4, 5, and 6), demonstrating the aggressiveness of ABS in susceptible varieties like Murcott tangor [45].

In the preventive control test (Figure 4), it was observed that greater control of the disease was obtained at concentrations of 2, 4, and 8 μL mL^-1 when compared to the curative control test (Figure 5). At a concentration of 8 μL mL^-1, lower leaf severity was observed when using the oils of Late IAC 855 mandarin and IAC 2019Maria mandarin, with an average of 1.2% and 0.8% of the injured leaf, respectively.

The curative effect of citronella EO on rice brusone (Pyricularia grisea) was observed to reduce disease incidence by up to 50% in replicates [46]. Better results were also observed in the preventive effect of noni EO application, when compared to the curative in the control of anthracnose (C. gloeosporioides) in mango plants [47].

The use of EOs as antimicrobial agents is considered low risk because it is believed to be difficult for a pathogen to develop resistance to the complex mixture of active components that comprise these oils [48].

The IAC 2019Maria mandarin and Late IAC 855 mandarin EO showed greater effectiveness in curative control of ABS (Figure 5), leading to 2.4% and 3.5% of the infected area on the leaf, respectively, at a concentration of 8 μL mL^-1, 168 hours after inoculation. However, the application of the oil of Murcott IAC 221 tangor at lower concentrations kept the level of severity of the disease stable, preventing it from evolving in the plants and causing more damage. In the curative test, the concentration of 8 μL mL^-1 reduced the severity of the disease by half when compared to the lowest concentration used.

Díaz Dellavalle et al. [49] performed an experiment using extracts of R. officinalis on the growth of Alternaria spp., concluding that the antimicrobial action may be due to the presence of substances such as OEs in the form of alpha and beta-pinene, limonene, camphene, myrcene, terpenoids such as carnosol, and oleanic acid [50], compounds similar to the monoterpenes found in the mandarin OEs extracted in this work.

The antifungal activity of EOs is related to their hydrophobicity, which allows them to interact with lipids of the cell wall, cell membrane, and mitochondria, changing permeability and causing disturbances in these structures [12].

This study shows that EOs are a rich source of research, and many of them have shown promise and may become another option for the control of ABS. However, for the definitive and safe insertion of EOs in the recommendation for producers, studies on concentration, time of application, residual period, mechanisms of action, phytotoxicity, real safety to mammals, other vertebrates and environment, availability of products, and costs deserve more attention [51].

5. Conclusions

After seven days, the essential oil of Mandarin IAC 219Maria at a concentration of 16 μL mL^-1 showed promising results for controlling the A. alternata fungus in vitro. Furthermore, in detached leaves, the essential oil of all the varieties tested, at the highest concentration (16 μL mL^-1), provided the lowest values of disease severity on the leaves, both curatively and preventively.