Use of Rosmarinus officinalis Essential Oil and its Fractions in the Alternative Control of Grapevine Phytopathogens

Abstract

Gray rot and grape ripe rot stand out as important vineyard diseases, being caused by the phytopathogens Botrytis cinerea and Colletotrichum acutatum, respectively. Control is usually conducted with synthetic fungicides that can cause problems for human health and the environment. Thus, searching for new alternative disease management methods is necessary, including using essential oil (EO) with fungicidal activity. The present study aimed to evaluate the chemical composition and fungicidal effect of Rosmarinus officinalis EO, as well as its fractions on the mycelial growth and conidial germination of B. cinerea and C. acutatum, in vitro and in vivo. The EO had α-pinene and 1,8-cineole as major compounds. In the vacuum fractional distillation, there was a variation in the chemical composition and the relative quantity of each compound according to the fraction analyzed (fractions of the bottom and top of the column). The in vitro results showed that the EO and its fractions had a fungicidal action on B. cinerea and C. acutatum. In post-harvest tests with 'Isabella' grapes, rosemary EO and its fractions reduced the incidence and severity of gray rot and grape ripe rot in preventive and curative treatments. In the field trial conducted in an 'Isabella' grape vineyard, the concentrations of the bottom fraction of rosemary EO reduced the incidence and severity of the grape ripe rot. They did not change the qualitative parameters of the evaluated grapes, showing that the bottom fraction of rosemary EO can be applied in the vineyard as a biofungicide to control this disease.

Keywords: Botrytis cinerea; Colletotrichum acutatum; natural products; phytopathogens; viticulture; terpenes

HIGHLIGHTS

Rosemary essential oil was rectified by vacuum fractional distillation.

The top fraction had mostly non-oxygenated monoterpenes while the bottom fraction was composed mainly of oxygenated monoterpenes.

Both fractions showed an antifungal effect on B. cinerea and C. acutatum in vitro.

The bottom fraction was effective in controlling grape ripe rot in field conditions (vineyard).

INTRODUCTION

According to the Food and Agriculture Organization of the United Nations (FAO) [¹], global grape production in 2022 was 74.94 million tons from a harvested area of 6.73 million hectares. In Brazil, 1.45 million tons were produced in a harvested area of 74.8 thousand hectares in the same period. One of the main obstacles to high-quality and yield grape production is diseases caused by fungi and the damage caused by them. The incidence and severity of diseases vary according to the grapevine cultivar (resistant or tolerant) and the geographic region, as the environment plays a vital role in this context and can contribute to increasing or limiting the development of diseases [²-⁴].

Gray rot is among the most common diseases in grapevines. It is caused by the fungus Botrytis cinerea Pers. Fr. (Botryotinia fuckeliana (de Bary) Whetzel. Another prevalent disease in vineyards is grape ripe rot, caused by Glomerella sp. (perfect or sexual phase of Colletotrichum sp.) [⁵]. Colletotrichum acutatum (Simmonds) (Glomerella acutata (Guerber & Correll) in its sexual form) has been associated with the disease in several wine-growing regions of the world [⁶-⁸].

Gray rot is prevalent in vineyards worldwide, causing losses in production and grape quality [³,⁹]. B. cinerea can cause damage to all vegetative parts of the vine (branches, tendrils, leaves, inflorescences, and berries). However, reductions in grape yield and quality are mainly associated with damage caused to inflorescences or rotting of berries pre-and/or post-harvest [¹⁰-¹¹]. In table grape production, the reduction in fruit quality is caused by berry rot in the field, either during transport or storage. Furthermore, B. cinerea thrives in low temperatures (0 - 10 ºC), causing losses in refrigerated products [³-⁴,⁹].

Grape ripe rot is associated with wine-growing regions with a hot and humid climate, mainly during the grape ripening phase [⁵]. The fungus affects the quality of the grapes due to changes in the chemical composition of the berries, which have a bitter taste, making them unviable for fresh sale and, consequently, causing high economic losses [⁶-⁸]. Sporulating the fungus on ripe fruits close to harvest produces secondary inoculum sources [¹²]. The spread of this phytopathogen occurs through wind, rain, and insects [³,¹²-¹³].

Phytosanitary treatments in the vineyard are needed due to fungal diseases. The management of grapevine diseases requires the application of synthetic fungicides several times during the phenological stages to avoid losses in productivity and quality of grapes, as well as economic losses [²,¹⁴-¹⁶].

Despite the high efficacy of synthetic fungicides, several studies demonstrated their harmful effects if inappropriately used. The impacts on the environment include the reduction of biodiversity, changes in the cycling of organic matter and nutrients, the natural biological control of pests and diseases, and changes in the populations of soil and water organisms, among other deleterious effects [¹⁷-²²]. Post-harvest of grapes intended for fresh consumption, despite having no fungicides registered for disease control, sulfur dioxide (SO₂) is used for storage in cooling chambers, which, in turn, can cause phytotoxicity in fruits and allergic reactions in sensitive people [²³-²⁴].

The secondary metabolism of plants is responsible for synthesizing several bioactive substances, which protect plants against insects and pathogens and limit the growth of other plant species. Thus, substances derived from plants (extracts, resins, gums, essential oils, among others) could be used as an alternative to synthetic fungicides. Essential oil-based products are available to control plant diseases in several countries [²⁵]. Furthermore, EOs are biodegradable, with low vertebrate toxicity and environmental impact [²⁶-²⁹].

EOs are mixtures of numerous compounds with different structures and physical characteristics. This, associated with their natural origin, makes such mixtures a pool of interesting molecules as alternatives in replacing synthetic molecules widely used nowadays [²⁸-²⁹]. Fractional distillation is one of the methods for processing EOs, which aims to separate two or more molecules due to the difference in volatility between them. This process depends on the pressure and temperature of the system, as well as the physical and chemical characteristics of the molecules to be separated [³⁰-³¹]. Furthermore, the process also aims to concentrate portions of certain chemical compounds/classes and, in some cases, concentrate some constituents of the EO to high purity levels [³⁰,³²]. The method is based on volatility differences between these molecules for the separation and concentration of molecules in EOs. The process consists of heating the mixture, where initially, the molecule with the lowest boiling point is separated until finally, the one with the highest boiling point. The vapor phase is enriched by the most volatile molecule when it reaches the top of the column. Using a vacuum during the fractional distillation process reduces the boiling temperature of the mixture, which reduces the degradation of EO components due to the high temperature. Furthermore, the lower operating pressure also increases the relative volatility of the components, allowing for more efficient separation [³⁰-³¹,³³].

Rosmarinus officinalis L., commonly called rosemary, is an herb native to the Mediterranean, widely disseminated in European, American, and Asian countries. It is a species used worldwide for culinary, medicinal, and commercial purposes, including the perfumery and food industries [³⁴-³⁵]. The main constituents of rosemary EO are camphor (5.0 - 21.0 wt.%), 1,8-cineole (15.0 - 55.0 wt.%), α-pinene (9.0 - 26.0 wt.%), borneol (1.5 - 5.0 wt.%), camphene (2.5 - 12.0 wt.%), β-pinene (2.0 - 9.0 wt.%) and limonene (1.5 - 5.0 wt.%), among others, in proportions that vary according to the vegetative stage and bioclimatic conditions [³⁵-³⁷]. The biological activity of R. officinalis EO depends on the chemical composition, and at least 13 different chemotypes of rosemary EO have been previously identified based on the relative percentages of α-pinene, 1,8-cineole, camphor, borneol, verbenone, and bornyl acetate [³⁴]. Furthermore, several studies have demonstrated the fungicidal effect of R. officinalis against phytopathogenic fungi such as Aspergillus flavus and Aspergillus niger [³⁸-⁴⁰], Fusarium verticillioides [⁴¹], Alternaria alternata [⁴²], Botrytis cinerea [⁴²], and Fusarium oxysporum [⁴²].

Thus, the present study aimed to evaluate the antifungal activity of rosemary (Rosmarinus officinalis) EO and its fractions (bottom and top), obtained by vacuum fractional distillation, on mycelial growth and conidia germination of Botrytis cinerea and Colletotrichum acutatum and in the control of gray rot and grape ripe rot in the post-harvest of table grapes in vitro, in vivo, and field conditions.

MATERIAL AND METHODS

Obtainment of fungal isolates

Strains of Botrytis cinerea (A58/09) and Colletotrichum acutatum (A009/13) were isolated from grapes collected in the municipality of Bento Gonçalves, Serra Gaúcha region, South Brazil. The fungal isolates were identified according to the procedures of Pedrotti and coauthors [⁴³]. They were taxonomically classified by Internal Transcribed Sequence (ITS-5.8S rDNA) sequencing, being compared to sequences deposited in the GeneBank Database using the nBLAST algorithm (NCBI). The isolates were cultivated on PDA (potato, dextrose, and agar) medium at 23±2 °C. Fungal isolates were maintained in the fungal collection of the Laboratory of Phytopathology, University of Caxias do Sul, RS, Brazil.

Source of essential oil and obtainment of the fractions

Rosemary EO (Rosmarinus officinalis), obtained by steam distillation of the aerial part of R. officinalis plants, was purchased from Tekton Company (Viamão, RS, Brazil). The raw oil and the obtained fractions were stored in a cold chamber at 5±3 °C until the conduction of the experiments.

The fractionation process and, the composition of the EO and the fractions obtained are described in detail in a previous study by Silvestre and coauthors [³¹]. In summary, the fractionation system comprised a glass column packed with Raschig rings 8 mm in diameter and package height of 8 cm, in three phases. The column was made up of 3 stages, each 8 cm high. The bottom section was a 250 mL glass flask. Heating was provided by electrical resistances wrapped around the balloon with a maximum power of 150 W. The fractions collected at each stage are condensed and stored in containers kept in a cooled bath (2 - 5 ºC). The system's internal pressure was maintained at 10 kPa with a vacuum pump. Between each step, sensors (PT-100) measured the steam temperature. In each fractionation test, 120 mL of R. officinalis essential oil was used. Each run was conducted for 1 h, and the top and bottom fractions were collected.

The raw oil and the obtained fractions were analyzed by GC/MS (qualitative analysis) and GC-FID (quantitative analysis) according to the parameters, equipment, and methods described by Silvestre and coauthors [³¹]. The identification was confirmed by comparison of each peak MS spectrum with the equipment library and by comparison of the calculated linear retention indexes (LRI) with those in the literature.

Antifungal activity of essential oil and its fractions on the mycelial growth of phytopathogens

The antifungal properties of EO and its fractions were tested according to Pedrotti and coauthors [⁴⁴]. The EO and its fractions concentrations ranged between 0.5 - 5.5 mL∙L^-1 for B. cinerea and 2.0 - 7.5 mL∙L^-1 for C. acutatum. EO was emulsified with Tween 20^® (1:1) and added to the PDA medium. The control treatment was just PDA, and Tween 20^® was equal to the highest concentration used to emulsify the EO. These emulsions were poured into glass Petri dishes (9 cm in diameter) and inoculated with 5.0 mm agar disks colonized by the phytopathogens mycelium obtained from 7-day pre-cultures. For each concentration, ten replicates were assessed. Incubation was performed at 25 °C temperature and a 12 h photoperiod for 14 days. Fungal growth was recorded on the 3^rd, 7^th, and 14^th days by measurement of the orthogonal diameter of the fungal colonies.

Transfer experiments were performed to provide a distinction between the fungistatic or fungicidal effects of EO and its fractions on the target microorganisms. For this purpose, mycelial disks that did not grow were transferred to fresh PDA medium in glass Petri dishes to assess their viability and growth after five days at 23±2 °C, with the fungal growth measured. Five days were established because both phytopathogens, under optimal growth conditions, start to develop in about 72 h [⁴⁴].

Antifungal activity of essential oil and its fractions on conidia germination

The antifungal activity of EO and its fraction on conidia germination was tested according to Pedrotti and coauthors [⁴⁴]. Conidia were harvested from 14-day-old colonies of B. cinerea and C. acutatum grown in PDA medium at 23±2 °C under a 12 h photoperiod. Then, 5.0 mL of sterile water was added to a Petri dish culture, and conidia were obtained by dislodging from the surface of cultures using a Drigalski loop. The suspension was diluted to obtain a suspension of 1∙10⁶ conidia∙mL^-1. Aliquots of conidia suspension (50 μL) were placed in microtubes containing 500 μL of PDA medium with different concentrations of EO and its fractions (0.5 to 5 mL∙L^-1 for B. cinerea and 2.0 to 10 mL∙L^-1 for C. acutatum), emulsified with Tween 20 (1:1). The negative control was composed only of PDA medium. The tubes were incubated at 23±2 °C for 24 h. The samples were placed in a hemocytometer chamber and observed under an optical microscope (100x) to assess conidia germination. All experiments were conducted in ten replicates, and 100 conidia were evaluated in each replicate. The conidia were considered germinated when the germ tube length equaled or exceeded the length of the conidia.

Evaluation of the in vivo antifungal activity of the essential oil and its fractions in the post-harvest of table grapes

Grapes of the cultivar "Isabela" (Vitis labrusca × Vitis vinifera) conventionally grown in Bento Gonçalves, RS, Brazil, were used in experiments. As described above, B. cinerea and C. acutatum conidia were harvested from a 14-day-old colony. The suspension was diluted with sterile water to obtain a suspension of 1∙10⁶ conidia∙mL^-1. The antifungal activity of rosemary EO and its fractions on grapes was evaluated as curative and preventive treatments according to the methodology described by Pedrotti and coauthors [⁴⁴]. The concentrations were between 1.0 - 10.0 mL∙L^-1 for B. cinerea and 3.0 - 12.5 mL∙L^-1 for C. acutatum. Such concentrations for both phytopathogens were chosen based on the results from in vitro tests. Approximately 2.0 mm deep wounds were made on ten berries within grape clusters; each treatment consisted of 12 grape clusters. After wounding, in the post-harvest curative treatment, a conidia suspension of B. cinerea or C. acutatum was inoculated (10 μL in each wound). After 4 h, grape clusters were sprayed with different EO or fractional concentrations. In the preventive treatment, the same concentrations were sprayed in grape clusters and, after 24 h inoculated with the conidia suspension as described above. For all treatments, the grapes were placed in plastic boxes (30 cm wide x 40 cm long x 15 cm high). The boxes were incubated at 25±1 °C and 80 - 90 % relative humidity with a 16 h photoperiod for five days for those inoculated with B. cinerea and seven days for those inoculated with C. acutatum. After incubation, the incidence was evaluated for the presence or absence of symptoms of the diseases. The severity was visually assessed according to the berry area affected by the disease using a scale from zero to 100 %, as described by Pedrotti and coauthors [⁴³]. The experiment was repeated three times, independently.

Evaluation of the antifungal activity of essential oils and their fractions in the vineyard

The experiment in field conditions was conducted in a vineyard of the Isabel cultivar belonging to the Department of Agricultural Diagnostics and Research of Rio Grande do Sul State (DDPA/SEAPI). The vineyard was composed of ungrafted 'Isabella' (Vitis sp.) vines, installed in 2007 and managed in a trellis system with 2.5 m x 2.0 m spacing. The experiment for evaluation was conducted with 15 vines for each treatment, with three plots of each treatment per row, consisting of five plants in each plot. All plants were evaluated for disease incidence and severity. The treatments consisted of control (no treatment) and treatment with the bottom fraction of EO at 0.5 mL∙L^-1 and 1.0 mL∙L^-1.

Treatments started during the inflorescence elongation phase and continued until the berries fully matured. The experiment was conducted from November 18, 2019, to January 25, 2020, totaling 68 days. In this period, the average daily temperature was 22.5 °C, the average minimum temperature was 16.8 °C, the average maximum temperature was 27.6 °C, the average humidity was 67.1 %, and the accumulated rainfall was 239.1 mm according to data from the National Institute of Meteorology (INMET) [⁴⁵].

The experiment consisted of spraying the treatments directly on the clusters each week. The treatment was reapplied in case of rain. The incidence was assessed through the percentage of the number of berries that showed disease symptoms. The severity was visually evaluated according to the area of the bunch affected by the disease using a scale according to Pedrotti and coauthors [⁴³]. The results were transformed into percentages based on the control, corresponding to a natural disease occurrence.

Qualitative parameters of the grapes were also evaluated. The average number of berries per cluster was counted, the average cluster mass was measured using a digital scale, and the soluble solids content was determined using a portable refractometer.

Statistical analysis

All statistical analyses were performed using the SPSS 22.0 program. The Kolmogorov-Smirnov test was used to determine data normality. The homogeneity of variances was assessed using Levene's test. Data were analyzed by Analysis of Variance (ANOVA), and the threshold for statistical significance was set at p < 0.05 (5 %). In the case of statistical significance, Tukey's test was applied to compare treatment means.

RESULTS AND DISCUSSION

Chemical analysis of essential oils and their fractions

The chemical analysis of EOs and their fractions (bottom and top) demonstrated variations in each chemical composition and relative quantity according to the fraction analyzed.

Twenty-two compounds were identified in the EO (Table 1), among which α-pinene (58.42 wt.%) and 1,8-cineole (16.27 wt.%) were the main compounds. The chemical composition was similar to that described for this species, however, with variations in the relative amount of each compound [³¹,³⁴,⁴⁶-⁴⁷].

Table 1
Chemical composition of rosemary essential oil (Rosmarinus officinalis) and its bottom and top fractions.

Compound	IR¹	Concentration (wt.%)
		Raw essential oil	Bottom fraction	Top Fraction
α-pinene	1021	58.42±6.38	12.61±1.65	57.79±3.11
Camphene	1062	4.88±0.42	1.17±0.22	5.41±1.04
β-pinene	1105	2.61±0.11	0.54±0.07	2.32±0.68
Sabinene	1124	0.65±0.25	-	0.77±0.07
Myrcene	1167	1.88±0.39	0.37±0.10	1.61±0.14
α-terpinene	1180	0.45±0.20	-	-
Limonene	1199	3.29±0.18	1.28±0.31	3.92±0.94
1,8-cineole	1207	16.27±3.33	7.50±1.11	25.35±5.03
ρ-cymene	1274	0.88±0.35	0.72±0.08	-
δ-terpinene	1286	0.69±0.48	0.37±0.18	-
Camphor	1526	1.04±0.30	6.51±0.99	0.39±0.04
Linalool	1550	1.09±0.44	7.02±1.44	0.24±0.09
Bornyl formate	1589	0.32±0.08	3.16±1.10	-
β-caryophyllene	1608	1.18±0.69	9.87±2.01	-
Terpinyl acetate	1704	0.52±0.08	4.67±1.06	-
Verbenone	1710	1.03±0.10	8.73±1.88	-
Borneol	1721	2.00±0.11	17.30±3.94	-
Myrtenol	1800	0.29±0.04	2.44±0.65	-
Geraniol	1852	1.27±0.25	11.08±2.75	-
caryophyllene oxide	2008	-	0.96±0.13	-
Methyl eugenol	2023	-	0.41±0.06	-
Monoterpenes		73.75	17.06	71.82
Oxygenated monoterpenes		23.83	68.82	25.98
Sesquiterpenes		1.18	9.87	-
Oxygenated sesquiterpenes		-	0.96	-
Total identified		98.75	96.71	97.80

• ¹ IR: retention index determined using a mixture of n- n-alkanes (C8 - C20). Mean of triplicates. Source: adapted from Silvestre and coauthors [³¹].

Variations in the chemical composition of the EO may have been influenced by the plant cultivar (chemotype), environmental aspects and climatic conditions of the place where the plant species developed, vegetative stage, harvest time, post-harvest drying, storage conditions, and extraction method, among others [⁴⁸-⁴⁹].

The plant species in the present study comprises different varieties and cultivars, with morphological characteristics and chemical composition (chemotypes) that differ from each other. From the results of chemical analyses of the EO, it was possible to identify that it is the 'α-pinene' chemotype, even being the oil from a commercial source.

In the vacuum fractional distillation of rosemary EO, there was a variation in the number of compounds and the relative quantity of each compound according to the fraction analyzed (bottom and top). In the bottom and top fractions of rosemary EO, nineteen and nine compounds were identified, respectively (Table 1).

It can be seen that monoterpene hydrocarbons such as α-pinene and camphene were distilled. Such behavior occurs due to the low boiling point of these compounds (156 ºC and 158 ºC, respectively) relative to the other compounds present in the oil [³¹,⁵⁰], being collected at the top (top fraction). Other compounds, such as β-pinene, sabinene, myrcene, ρ-cymene, and limonene (hydrocarbon monoterpenes), which are more volatile, were also identified in the top fraction. Some compounds, such as 1,8-cineole, linalool, and camphor (oxygenated monoterpenes), were identified in the top and bottom fractions, demonstrating that they were not wholly distilled. Such compounds have a higher boiling point, and higher temperatures would be required to separate them. However, increasing temperature can cause the oxidation of some compounds, in addition to the possibility of distilling other compounds [³¹].

The remaining compounds (oxygenated monoterpenes, sesquiterpenes, alcohols, aldehydes, and ketones) remained in the flask (bottom fraction). This is a result of the intermolecular interactions between the different chemical groups of the compounds and because of the size of the molecules (sesquiterpenes are less volatile than monoterpenes due to the higher molar mass) [³¹,⁵¹].

Furthermore, some compounds presented a higher concentration in the bottom fraction when compared to raw EO, such as camphor (1.04 wt.% to 6.51 wt.%), linalool (1.09 wt.% to 7.02 wt.%), bornyl formate (0.32 wt.% to 3.16 wt.%), β-caryophyllene (1.18 wt.% to 9.87 wt.%), terpinyl acetate (0.52 wt.% to 4.67 wt.%), verbenone (1.03 wt.% to 8.73 wt.%), borneol (2.00 wt.% to 17.30 wt.%), myrtenol (0.29 wt.% to 2.44 wt.%), and geraniol (1.27 wt.% to 11.08 wt.%). In the top fraction, there was a higher concentration of the compounds 1,8-cineole (18.27 wt.% to 25.25 wt.%) and camphene (4.88 wt.% to 5.41 wt.%) compared to raw EO.

Similar results were obtained in vacuum fractionation studies where higher concentrations of monoterpene hydrocarbon compounds and some oxygenated monoterpene compounds that have greater volatility were identified in the top fractions, such as the EOs of Citrus deliciosa [³⁰] and Citrus sinensis [⁵¹].

Other compounds previously not detected in the raw EO were identified during the fractionation, such as caryophyllene oxide and methyl eugenol (Table 1). Silvestre and coauthors [³⁰] observed that this may occur due to chemical reactions in the mixture during the fractionation process or trace compounds that, with the concentration of the EO during separation, allowed them to be detected in GC/MS.

Evaluation of the in vitro antifungal activity of essential oils and their fractions

Mycelial growth

The antifungal activity of rosemary EO and its fractions (bottom and top) on the mycelial growth of pathogens (B. cinerea and C. acutatum) showed an inhibitory effect that increased proportionally with the concentrations and was also influenced by the duration of treatment (day of evaluation). The results of the antifungal activity of rosemary EO and its fractions on the growth of B. cinerea are shown in Table 2.

Table 2
Antifungal activity of different concentrations of Rosmarinus officinalis essential oil and its fractions on the mycelial growth of Botrytis cinerea.

Concentration (mL∙L-1)	Mycelial growth (mm)
	3rd day	7th day	14th day
Raw essential oil
Zero	59.6±4.8 a	87.0±2.8 a	90.0±0.0 a
0.5	23.6±6.7 b	86.2±2.9 a	90.0±0.0 a
1.0	17.2±5.5 bc	72.2±8.4 ab	90.0±0.0 a
1.5	18.1±7.0 bc	73.4±10.3 ab	90.0±0.0 a
2.0	12.6±4.4 c	68.6±8.8 ab	90.0±0.0 a
2.5	4.3±1.0 d	36.9±5.5 b	83.2±0.0 a
3.0	4.6±1.6 d	34.0±5.1 b	90.0±0.0 a
3.5	4.4±0.9 d	38.6±6.4 b	90.0±0.0 a
4.0	0.0±0.0 d	20.6±7.4 c	78.6±12.6 ab
4.5	0.0±0.0 d	22.6±6.6 c	67.2±8.7 b
5.0	0.0±0.0 d	0.0±0.0 d	0.0±0.0 c
5.5	0.0±0.0 d	0.0±0.0 d	0.0±0.0 c
Bottom fraction
Zero	59.6±10.6 a	87.0±6.4 a	90.0±0.0 a
0.5	13.3±5.1 b	77.1±8.5 a	90.0±0.0 a
1.0	0.0±0.0 c	0.0±0.0 b	0.0±0.0 b
1.5	0.0±0.0 c	0.0±0.0 b	0.0±0.0 b
Top fraction
Zero	59.6±5.5 a	87.0±0.5 a	90.0±0.0 a
0.5	20.5±3.7 b	50.4±6.6 b	90.0±0.0 a
1.0	11.1±3.0 c	38.3±14.5 bc	90.0±0.0 a
1.5	5.4±1.2 d	38.1±11.1 bc	90.0±0.0 a
2.0	4.7±0.9 d	32.5±10.4 bc	90.0±0.0 a
2.5	4.1±1.7 d	25.4±11.2 c	90.0±0.0 a
3.0	0.0±0.0 d	0.0±0.0 d	0.0±0.0 b
3.5	0.0±0.0 d	0.0±0.0 d	0.0±0.0 b

• Values are the average of ten replicates per treatment. The letters compare the different concentrations of the EO and the fractions evaluated (column). Means in column followed by the same letter do not differ according to the Tukey test at a 5 % probability of error (p < 0.05). Source: authors (2024).

EO showed a fungicidal effect from a concentration of 5.0 mL∙L^-1 on the mycelial growth of B. cinerea (Table 2). Concentrations of 0.5 mL∙L^-1 to 4.5 mL∙L^-1 reduced the mycelial growth of the pathogen by more than 50 % compared to the negative control (zero) on the third day of evaluation. Similar behavior was observed on the seventh day of assessment at concentrations of 2.5 mL∙L^-1 to 4.5 mL∙L^-1. In the fourteenth-day evaluation, only the concentrations of 4.5 mL∙L^-1 or higher differed statistically from the control.

The bottom fraction of rosemary EO showed a fungicidal effect on the mycelial growth of B. cinerea from a concentration of 1.0 mL∙L^-1. The lowest concentration (0.5 mL∙L^-1) differed statistically from the control until the third evaluation day. The top fraction of rosemary EO showed a fungicidal effect from a concentration of 3.0 mL∙L^-1 and concentrations of 0.5 mL∙L^-1 to 2.5 mL∙L^-1 reduced it by more than 50 % mycelial growth of the pathogen when compared to the control on the third and seventh day of evaluation.

The results of the antifungal activity of rosemary EO and its fractions on the mycelial growth of C. acutatum are shown in Table 3.

Table 3
Antifungal activity of different concentrations of Rosmarinus officinalis essential oil and its fractions on the mycelial growth of Colletotrichum acutatum.

Concentration (mL∙L-1)	Mycelial growth (mm)
	3rd day	7th day	14th day
Zero	31.7±3.5 a	77.6±3.7 a	90.0±0.0 a
2.0	8.1±2.0 b	34.4±2.3 b	90.0±0.0 a
2.5	7.4±1.7 bc	31.8±4.0 bc	90.0±0.0 a
3.0	7.3±1.1 bc	26.7±2.7 c	90.0±0.0 a
3.5	6.1±0.8 bc	26.0±2.3 c	90.0±0.0 a
4.0	6.8±0.9 bc	19.2±1.8 d	90.0±0.0 a
4.5	6.4±0.8 bc	19.5±1.1 d	90.0±0.0 a
5.0	6.5±0.7 bc	23.2±3.9 cd	74.0±8.5 ab
5.5	6.7±0.8 bc	16.4±0.9 e	68.6±10.5 ab
6.0	6.5±0.8 bc	20.9±1.0 d	60.8±8.7 b
6.5	4.6±0.5 c	18.4±1.5 de	60.9±7.9 b
7.0	5.3±0.8 bc	15.8±1.2 e	56.1±11.0 b
7.5	4.2±0.4 c	4.8±0.8 f	33.2±8.1 c
Bottom fraction
Zero	31.7±6.6 a	77.6±2.3 a	90.0±0.0 a
2.0	5.9±4.5 b	23.7±2.8 b	66.9±3.7 c
2.5	5.4±5.0 b	22.9±3.5 b	79.4±0.3 b
3.0	0.0±0.0 b	0.0±0.0 c	0.0±0.0 d
Top fraction
0.0	31.7±6.2 a	77.6±5.0 a	90.0±0.0 a
2.0	17.9±3.5 b	40.3±8.6 b	90.0±0.0 a
2.5	14.3±3.8 b	37.3±7.4 b	90.0±0.0 a
3.0	16.5±2.9 b	39.8±8.0 b	90.0±0.0 a
3.5	15.3±3.3 b	35.4±5.9 b	72.9±5.5 b
4.0	19.2±4.0 b	36.2±6.7 b	64.9±4.2 b
4.5	18.3±6.1 b	36.2±9.2 b	62.6±6.8 b
5.0	18.6±4.4 b	41.6±4.2 b	69.6±4.4 b
5.5	16.5±5.3 b	42.2±5.5 b	71.7±6.0 b
6.0	17.7±2.9 b	39.2±6.9 b	69.1±5.9 b
6.5	16.7±3.4 b	37.9±8.3 b	68.1±3.7 b
7.0	12.7±5.0 b	32.4±7.7 b	66.5±4.8 b
7.5	0.0±0.0 c	0.0±0.0 c	0.0±0.0 c

• Values are the average of ten replicates per treatment. The letters compare the different concentrations of the EO and the fractions evaluated (column). Means in column followed by the same letter do not differ according to the Tukey test at a 5 % probability of error (p < 0.05). Source: authors (2024).

Rosemary EO showed a fungistatic effect on C. acutatum mycelial growth, where all concentrations evaluated reduced the mycelial growth of the pathogen by more than 50 % compared to the control on the third and seventh day of evaluation. On the fourteenth day of assessment, the concentrations of 6.0 mL∙L^-1 to 7.5 mL∙L^-1 differed statistically from the control (Table 3). The bottom fraction of rosemary EO showed a fungicidal effect on the mycelial growth of C. acutatum from a concentration of 3.0 mL∙L^-1. The lowest concentrations (2.0 mL∙L^-1 and 2.5 mL∙L^-1) differed statistically from the control on all days evaluated. The top fraction of rosemary EO showed a fungicidal effect from a concentration of 7.5 mL∙L^-1, and the other concentrations evaluated significantly reduced the mycelial growth of C. acutatum compared to the control on the third and seventh day of evaluation. The same behavior was observed on the fourteenth day of the assessment at concentrations of 3.5 mL∙L^-1 and 7.0 mL∙L^-1.

Similarly, different authors demonstrated that rosemary EO has a fungicidal effect on the mycelial growth of Aspergillus flavus, Fusarium verticillioides, Botrytis cinerea, Aspergillus flavus, and Aspergillus niger [³⁸,⁴⁰-⁴²], in addition to causing fungistatic effects on different species of Aspergillus [³⁹], Alternaria alternata and Fusarium oxysporum [⁴²]. No data assessing antifungal activity regarding rosemary EO fractions were found in the literature.

The results obtained in the present study showed that EOs and their fractions have a fungicidal or fungistatic effect on the mycelial growth of B. cinerea and C. acutatum. It was also observed that the bottom fraction showed fungicidal activity at lower concentrations than the top fraction and the EO. This effect is probably due to the compounds present in EOs and fractions that act to prevent fungal development. As proposed mechanisms for the antifungal activity of these terpenes, the literature comments on changes in cell permeability, interruption of ergosterol biosynthesis, and interference with enzymatic cell wall synthesis reactions, causing deformation of structures, osmotic disorders, loss of integrity and rigidity of the cell wall, causing lysis and leakage of the cytoplasm and the loss of macromolecules from the interior of the fungal cell [⁴⁰-⁴¹,⁵²].

In general, the results of the mycelial growth tests demonstrated that the bottom fraction had a more significant fungicidal or fungistatic effect on the pathogens. This effect is due to the composition and concentration of these fractions, which are mainly composed of oxygenated monoterpenes in high concentrations. Several studies have shown that oxygenated monoterpenes have more intense biological activity than non-oxygenated monoterpenes and, consequently, have a more pronounced antifungal activity. Furthermore, oxygenated monoterpenes with carbonyl (ketones and aldehydes, such as camphor and verbenone), ether (such as 1,8-cineole and methyl eugenol), and/or hydroxyl (alcohols/phenols, such as linalool and borneol) moieties are recognized as having potent antifungal activity [⁵³-⁵⁴].

Effect of essential oil and its fractions on conidia germination

Rosemary EO and its fractions (bottom and top) showed a dose-dependent inhibitory effect on the germination of conidia of phytopathogens (B. cinerea and C. acutatum), increasing proportionally according to the treatment concentration, demonstrating its antifungal activity.

Table 4
Antifungal activity of different concentrations of Rosmarinus officinalis essential oil and its fractions on Botrytis cinerea and Colletotrichum acutatum conidia germination.

Concentration (mL∙L-1)	Conidia germination (%)
	Essential oil	Bottom fraction	Top fraction
Botrytis cinerea
Zero	97.9±1.8 a	97.9±1.8 a	97.9±1.8 a
0.5	-	0.0±0.0 b	8.5±2.7 b
1.0	-	0.0±0.0 b	0.0±0.0 c
2.0	17.0±3.7 b	0.0±0.0 b	0.0±0.0 c
3.0	0.0±0.0 c	0.0±0.0 b	0.0±0.0 c
4.0	0.0±0.0 c	-	-
5.0	0.0±0.0 c	-	-
Colletotrichum acutatum
Zero	99.0±0.5 a	99.0±0.5 a	99.0±0.5 a
2.0	-	73.3±5.2 b	-
2.5	-	15.4±1.7 c	-
3.0	-	0.0±0.0 d	-
4.0	-	0.0±0.0 d	-
6.5	-	-	72.3±4.6 b
7.0	-	-	14.4±1.1 c
7.5	-	-	0.0±0.0 d
8.0	-	-	0.0±0.0 d
9.0	72.7±6.1 b	-	-
9.5	11.8±3.9 c	-	-
10.0	0.0±0.0 d	-	-

• Values are the average of ten replicates per treatment, with each replicate comprising 100 conidia. The letters compare the different concentrations of the EO and the fractions evaluated (column). Means in column followed by the same letter do not differ according to the Tukey test at a 5 % probability of error (p < 0.05). Source: authors (2024).

Rosemary EO completely inhibited the germination of B. cinerea conidia from a concentration of 3.0 mL∙L^-1 and a concentration of 2.0 mL∙L^-1 reduced conidial germination by 83 % (Table 4). Regarding the bottom fraction of rosemary EO, no germinated conidia were observed at all concentrations evaluated. The top fraction of rosemary EO completely inhibited the germination of B. cinerea conidia from a concentration of 1.0 m L∙L^-1, and 0.5 mL∙L^-1 reduced the germination of conidia by more than 90 %.

From a concentration of 10.0 mL∙L^-1, rosemary EO completely inhibited the conidia germination of C. acutatum, and at concentrations of 9.0 mL∙L^-1 and 9.5 mL∙L^-1, there was a significant reduction in the number of germinated conidia (27 % and 88 %, respectively) (Table 4). The bottom fraction of rosemary EO completely inhibited the conidia germination of C. acutatum from a concentration of 3.0 mL∙L^-1 and concentrations of 2.0 mL∙L^-1 and 2.5 mL∙L^-1 significantly reduced conidia germination when compared to the control (26 % and 84 %, respectively). In the top fraction, conidia germination was completely inhibited at a concentration of 7.5 mL∙L^-1, and concentrations of 6.5 mL∙L^-1 and 7.0 mL∙L^-1 reduced conidia germination by more than 25 % and 80 %, respectively.

Similarly, Bomfim and coauthors [⁴⁰-⁴¹] reported that R. officinalis EO inhibited the conidia germination of Aspergillus flavus and Fusarium verticillioides, respectively, and Sousa and coauthors [³⁸], evaluating the effect of EO on Aspergillus flavus and Aspergillus niger, reported antifungal activity. No data were found in the literature for the fractions of rosemary EO.

The results obtained in the present study showed that EOs and their fractions have an antifungal effect, inhibiting the conidia germination of B. cinerea and C. acutatum. It is suggested that this effect is due to compounds present in EOs and fractions, which affect the enzymes and amino acids responsible for the germination of conidia, leading to the death of the fungal cell [⁵⁵-⁵⁶]. As observed in the mycelial growth tests, the results of the conidial germination tests demonstrated that the bottom fraction had more prominent antifungal activity against phytopathogens.

Evaluation of the in vivo antifungal activity of essential oils and their fractions in the post-harvest period of table grapes

The results of the present study demonstrated that different concentrations of rosemary EO and their fractions (bottom and top) reduced the incidence or severity of gray rot (B. cinerea) and grape ripe rot (C. acutatum) in post-harvest 'Isabella' grapes in preventive and curative treatments (Table 5).

Table 5
Antifungal activity of different concentrations of Rosmarinus officinalis essential oil and its fractions in the control of gray rot (Botrytis cinerea) and ripe grape rot (Colletotrichum acutatum) in 'Isabella' grapes at post-harvest.

Treatment	Preventative treatment		Curative treatment
	Incidence (%) *	Severity (%) **	Incidence (%) *	Severity (%) **
Botrytis cinerea
CA	0.0±0.0 e	0.0±0.0 d	0.0±0.0 d	0.0±0.0 c
CI	75.7±6.7 a	13.8±3.5 a	75.7±6.7 a	13.8±3.5 a
EO 5.0 mL∙L-1	26.7±2.8 b	3.6±1.5 c	13.0±2.6 c	3.9±1.8 b
EO 10.0 mL∙L-1	15.3±2.7 cd	1.0±0.6 cd	0.0±0.0 d	0.0±0.0 c
BF 1.0 mL∙L-1	8.7±1.9 d	0.5±0.3 d	0.0±0.0 d	0.0±0.0 c
BF 6.0 mL∙L-1	0.0±0.0 e	0.0±0.0 d	0.0±0.0 d	0.0±0.0 c
TF 3.0 mL∙L-1	26.6±3.1 b	8.7±1.6 b	34.0±5.9 b	13.9±4.1 a
TF 8.0 mL∙L-1	23.3±2.5 bc	2.5±1.0 cd	15.0±3.7 c	5.2±2.7 b
Colletotrichum acutatum
CA	0.0±0.0 d	0.0±0.0 c	0.0±0.0 d	0.0±0.0 d
CI	73.3±6.2 a	33.3±5.4 a	73.3±6.2 a	33.3±5.4 a
EO 10.0 mL∙L-1	33.0±6.0 c	1.7±0.8 c	22.0±3.7 c	6.4±3.1 c
EO 15.0 mL∙L-1	7.0±6.4 d	0.7±0.3 c	17.7±6.1 c	6.3±2.5 c
BF 3.0 mL∙L-1	0.0±0.0 d	0.0±0.0 c	0.0±0.0 d	0.0±0.0 d
BF 8.0 mL∙L-1	0.0±0.0 d	0.0±0.0 c	0.0±0.0 d	0.0±0.0 d
TF 7.5 mL∙L-1	46.3±4.1 b	11.2±5.1 b	44.3±5.0 b	18.2±4.7 b
TF 12.5 mL∙L-1	28.7±5.2 c	5.7±3.4 bc	23.3±3.9 c	7.6±2.8 c

• ^* Percentage of berries with disease symptoms (rot appearance). ^** Percentage according to the scale that evaluates the berry area affected by the disease. Values are the average of three replications. The letters compare the different concentrations of the EO and its fractions. Means in column followed by the same letter do not differ according to the Tukey test at a 5 % probability of error (p < 0.05). CA - absolute control; CI - inoculum control; EO - essential oil; TF - top fraction; BF - bottom fraction. Source: authors (2024).

In preventive treatment, 5.0 mL∙L^-1 of rosemary EO reduced the incidence by 64 % and the severity of gray rot by 74 %. At 10.0 mL∙L^-1, it reduced the incidence rate by 80 % and the severity of the disease by 92 %. In the curative treatment, the EO concentration of 5.0 mL∙L^-1 reduced the incidence by 80 % and the severity of the disease by 74 %. At the concentration of 10.0 mL∙L^-1, no disease symptoms were observed (Table 5).

In preventive treatment, the bottom fraction of rosemary EO reduced the incidence by 88 % and the severity of the disease caused by B. cinerea by 96 % at a concentration of 1.0 mL∙L^-1. At 6.0 mL∙L^-1, no symptoms of the disease were observed. In the curative treatment, the two concentrations of the bottom fraction (1.0 mL∙L^-1 and 6.0 mL∙L^-1) completely inhibited the incidence of gray rot (Table 5).

In the preventive treatment of gray rot, the top fraction of rosemary EO in the concentration of 3.0 mL∙L^-1 reduced the incidence by 65 % and the severity by 37 %. At 8.0 mL∙L^-1, the incidence decreased by 69 % and the severity of the disease by 82 %. In the curative treatment, the top fraction at a concentration of 3.0 mL∙L^-1 reduced the incidence of the disease by 55 % but could not reduce the severity. The concentration of 8.0 mL∙L^-1 reduced 80 % the incidence and 63 % the severity of gray rot (Table 5).

In preventive treatment, a concentration of 10.0 mL∙L^-1 of rosemary EO reduced the incidence by 55 % and the severity of the disease caused by C. acutatum by 95 %. At 15.0 mL∙L^-1, the EO reduced the incidence by 90 % and the severity of the disease by 98 %. In the curative treatment, a concentration of 5.0 mL∙L^-1 of EO reduced the incidence by 70 % and the severity of grape ripe rot by 81 % and, at a concentration of 15.0 mL∙L^-1, it reduced the incidence by 76 % and the severity of the disease by 81 % (Table 5).

In treatments using the bottom fraction of rosemary EO, no symptoms of the disease caused by C. acutatum were observed at both concentrations (3.0 mL∙L^-1 and 8.0 mL∙L^-1) in preventive and curative treatment (Table 5).

For the top fraction of rosemary EO, considering the preventive treatment, the concentration of 7.5 mL∙L^-1 reduced the incidence by 37 %, the severity of grape ripe rot by 66 %, and, at the concentration of 12,5 mL∙L^-1 reduced the incidence by 61 % and the severity of the disease by 83 %. In curative treatment, the top fraction at a concentration of 7.5 mL∙L^-1 reduced the incidence of the disease by 40 % and its severity by 45 % and, at a concentration of 12.5 mL∙L^-1, reduced by 68 % the incidence and 77 % the severity of the disease caused by C. acutatum.

From the results obtained, we can observe that the effect of rosemary EO and its fractions (bottom and top) are dose-dependent, where, with an increase in its concentrations, there was a more pronounced reduction in the incidence and severity of post-harvest diseases caused by B. cinerea and C. acutatum on grapes. Furthermore, it can be observed that curative treatments with rosemary EO and its fractions were more efficient, resulting in a greater reduction in the incidence and severity of diseases compared to preventive treatments. Additionally, as observed in vitro tests (mycelial growth and conidial germination), the results of the post-harvest test demonstrated that the bottom fraction of the EO showed a more pronounced antifungal activity, reducing or eliminating the incidence and severity of gray rot and grape ripe rot when compared to the results obtained with rosemary EO (raw) and the top fraction.

Similar results were obtained by Sousa and coauthors [³⁸], who evaluated the effect of R. officinalis EO in the control of Aspergillus flavus and Aspergillus niger in table grapes (Vitis sp. 'Isabella'). The authors reported a reduction in the infection rate by pathogens, which aligns with the results obtained in this study.

From our results, it can be observed that the EO and its fractions have a fungicidal effect in vivo, reducing the incidence and severity of gray rot (B. cinerea) and grape ripe rot (C. acutatum) in preventive and curative treatments in the post-harvest of table grapes ('Isabella'). According to Regnier and coauthors [⁵⁷], the compounds present in EOs inhibit post-harvest pathogens due to their effects on cellular metabolism, directly affecting mycelial growth and conidial germination. Thus, R. officinalis EO and its fractions can be a tool potential in the post-harvest chain of table grapes, in the storage or packaging process, such as biofungicides to control rot and other diseases caused by phytopathogenic fungi.

Notably, the fractionation process generated a bottom fraction with more pronounced antifungal/ fungistatic activity, which implies lower dosages to control these phytopathogens. Thus, the fractionation of EO allows for obtaining a product with control potential (bottom fraction), allowing the use of the remaining material (top fraction) for other applications, such as perfumery, cosmetics, medicine, and the chemical and pharmaceutical industries.

Evaluation of the antifungal activity and quality of grapes treated with the bottom fraction of R. officinalis essential oil in the vineyard

Based on the observed antifungal activity in the tests of mycelial growth and conidial germination and in the post-harvest of 'Isabella' grapes test, the bottom fraction of rosemary EO was used for the treatments in the test carried out at an 'Isabella' vineyard. Data relating to the test in the vineyard are compiled in Table 6.

Table 6
In vivo antifungal activity of the bottom fraction of Rosmarinus officinalis essential oil in controlling grape ripe rot (Colletotrichum acutatum) in an 'Isabella' vineyard and quality parameters of the treated berries.

Treatment	Incidence * (%)	Severity ** (%)	Berries by bunch	Bunch mass (g)	Soluble solids (°Brix)
Control	34.9±5.2 a	92.1±10.3 a	32.4±10.5 a	59.85±7.29 a	13.73±1.22 a
Bottom 0.5 mL∙L-1	30.0±5.9 b	91.9±9.8 a	29.5±8.6 a	52.70±5.65 a	14.44±1.68 a
Bottom 1.0 mL∙L-1	8.5±1.4 c	53.3±6.7 b	29.7±7.9 a	55.08±4.81 a	13.95±2.30 a

• ^*
  Percentage of berries with disease symptoms (rot appearance). ^** Percentage according to the scale that evaluates the bunch area affected by the disease. Values are the average of three replications. The letters indicate the comparison between the different treatments. Means in column followed by the same letter do not differ according to the Tukey test at a 5 % probability of error (p < 0.05). Source: authors (2024).

Regarding the different treatments, no symptoms of gray rot caused by B. cinerea were observed. Therefore, evaluations were carried out only on grape ripe rot caused by C. acutatum, where shriveled and/or mummified berries characterized the symptoms of the disease.

When evaluating the quality of the grapes, no significant differences were observed between the different treatments in all parameters assessed, demonstrating that the bottom fraction of rosemary EO did not interfere with the quality of the grapes (Table 6).

The present study also demonstrated that different concentrations of the bottom fraction of rosemary EO (0.5 mL∙L^-1 and 1.0 mL∙L^-1) efficiently controlled grape ripe rot disease (Table 6). The concentration of 0.5 mL∙L^-1 reduced the incidence of the disease by 15 %. However, this treatment was unable to reduce the severity. At a concentration of 1.0 mL∙L^-1, there was a 75 % reduction in the incidence and a 42 % reduction in the severity of the disease, demonstrating that the bottom fraction of the EO efficiently controlled grape ripe rot when applied in the field. Similar results were obtained by Pedrotti and coauthors [⁵⁸], who showed that E. staigeriana EO was able to reduce the incidence and severity of gray rot caused by B. cinerea and the severity of grape ripe rot caused by C. acutatum when applied in the field to 'Tannat' (Vitis vinifera) grapes, intended for fine wines production.

The reduction in the incidence and severity of C. acutatum in the field test demonstrates the potential for using the bottom fraction of rosemary EO, most likely due to removing the non-oxygenated monoterpenes concentrated in the top fraction. Considering that the bottom fraction showed greater control efficiency over phytopathogens in vitro, in post-harvest and field tests, the reduction in application doses, as well as obtaining two products, makes the fractionation of rosemary EO and the use of the bottom fraction as a promising control agent for use in greener agriculture and less dependent on synthetic molecules.

CONCLUSION

The EO's vacuum fractional distillation (fractionation) resulted in a variation in the chemical composition and the relative quantity of each compound according to the fraction analyzed (bottom and top). Rosemary EO and its fractions showed fungicidal or fungistatic action on B. cinerea and C. acutatum, which varied according to concentration. The EO and its fractions can be applied in the post-harvest chain of table grapes, in the storage or packaging process, as biofungicides to control rot caused by B. cinerea and C. acutatum. The bottom fraction reduced the incidence and severity of grape ripe rot in the vineyard without effects on the qualitative parameters of the grapes, demonstrating that this fraction can be applied as a biofungicide to control C. acutatum.

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• Funding:
  This research received no external funding.

Edited by

• Editor-in-Chief:
  Bill Jorge Costa

• Associate Editor:
  Jane Manfron Budel

Publication Dates

• Publication in this collection
  08 Nov 2024
• Date of issue
  2024

History

• Received
  09 Jan 2024
• Accepted
  26 Aug 2024