Essential oils in the management of Alternaria alternata f. sp. citri in ‘Dancy’ tangerine fruits

ABSTRACT Tangerines and their hybrids are affected by economically essential diseases. The Alternaria brown spot (Alternaria alternata f. sp. citri) deserves to be highlighted, as it is present in all tangerine production areas. This study aimed to determine the effect of essential oils on A. alternata f. sp. citri. The experiments were carried out in the Laboratories of Phytopathology and Biology and Technology of Post-Harvest of the Federal University of Paraíba, Areia, PB. Three pathogen isolates were used, and ten essential oils from grape, sunflower, eucalyptus, ginger, copaiba, mint, fennel, citronella, clove, and linseed at a concentration of 1%, diluted in potato dextrose agar for in vitro tests. The oils were diluted in distilled water for the fruit test, and the fruits were immersed in the treatments for 5 min. In addition, the fungicide Thiabendazole (400 mL/100L) and sterile distilled water were used as the control treatments. The fruits were inoculated with a conidia suspension of the pathogen (105 conidia/mL). Mycelial Growth Rate Index, colony diameter, spore production and dimensions, fruit disease severity, and enzymatic activity were evaluated. A completely randomized design was used, with 12 treatments and four replications in vivo. The results indicate that the essential oils of mint, eucalyptus, fennel, and citronella were efficient in the in vitro control of the pathogen, ultimately inhibiting the growth of fungal colonies. Eucalyptus oil efficiently managed A. alternata f. sp. citri on ‘Dancy’ tangerine fruits. Essential oils did not influence the enzymatic activity of the fruits.


INTRODUCTION
Brazil is considered the largest producer of citrus in the world, with a planted area of 7.9 million hectares.In the world production of tangerines, Brazil occupies the seventh position, with approximately 984.9 thousand tons cultivated in 52.8 thousand hectares (LANDAU et al., 2020).Tangerines are specially intended for the fresh fruit market and are used to produce concentrated juice.The Southeast region concentrates the largest national production of tangerines with 61%, producing approximately 603 thousand tons in more than 25 thousand hectares, with emphasis on the state of São Paulo, considered the main producer pole of tangerine (LANDAU et al., 2020).
The Northeast region produced 31,545 tons with an average yield of 8.45 t/ha.In Paraíba state, tangerine production was 13,219 thousand tons and a yield of 7.26 t/ha in 2019 (SILVA et al., 2019).However, the national citrus industry faces serious health problems due to the action of pathogens in the production and post-harvest phases.The most important disease of tangerines and their hybrids is Alternaria brown spot (ABS), which causes limitations in crop production and is found in all producing states in the country (CHEN et al., 2014).
The causal agent of ABS is the fungus Alternaria alternata f. sp.citri, which, when colonizing plant tissue, releases a specific toxin for the tangerine pathotype, causing cell death at the site of the attack, which can occur in all plant organs (PORCINO et al., 2017).The control of ABS is carried out mainly through registered fungicides; however, this practice has been a matter of concern due to the various risks offered to humans and the environment (XU et al., 2014).
As a result of the search for alternative sources in managing plant diseases, essential oils show promising results in controlling phytopathogens and avoiding their infection (SHARIFI-RAD et al., 2017).Several studies related to managing diseases through essential oils proved to be efficient in inhibiting mycelial growth, 87 sporulation, and spore germination in in vitro tests.Scheuermann et al. (2020) reported that when using Cymbopogon martinii and C. citratus oils at a concentration of 0.075%, they inhibited the mycelial growth of Rhizoctonia solani.
Within this context, this study aimed to determine the effect of essential oils on mycelial growth and sporulation of A. alternata f. sp.citri in vitro, and 'Dancy' tangerine fruits, in addition to verifying the induction of resistance of the treated fruits to the pathogen.
The oils were incorporated at a concentration of 1% in a semi-solid potato-dextrose-agar (PDA) medium with pH 6.0, plus two drops of Tween 80 (dispersant), then poured into Petri dishes (9 cm).Plates containing only PDA were used for the control, and the fungicide Thiabendazole (400 mL/100L) was incorporated into the culture medium.After seven days of growth of the pathogen, a disk (5 mm in diameter) of the colony was transferred to the center of each plate and placed in a Biochemical Oxygen Demand (B.O.D) incubation chamber under a 12-hour photoperiod at 25±2 °C for seven days.
The diameter of the mycelial colony (DC) was measured daily, with the aid of a millimeter ruler, in two orthogonal directions until the plate containing the control treatment was filled.As well as the measurement of the mycelial growth rate index (MGRI), according to Oliveira (1991), IVCM= (D-Da)/N, where: MGRI =mycelial growth rate index; D=current average colony diameter; Da=average diameter of the colony from the previous day; N=number of days after inoculation in the Petri dish.
To quantify conidia (spore) production, 10 mL of sterile distilled water (SDW) was added to the plates after ten days of incubation, and spores were removed using a softbristled brush.The 10 5 suspensions were filtered through a double layer of sterile gauze, and conidia were quantified in a Neubauer chamber (ALFENAS; MAFIA, 2016).The size of the spores was determined by measuring the length and width of 25 spores of each treatment on microscope slides stained with methylene blue visualized under an optical microscope.

Experiment II -Inoculation of the pathogen in fruits
For the in vivo test, tangerine fruits, cultivar 'Dancy' were harvested in a commercial orchard in the city of Remígio -PB (6° 54′ 10″ S and 35° 50′ 2″ W), at C2 maturation stage (SILVA et al. al., 2019).Initially, the fruits were washed with water and neutral soap, then disinfested in a 1% sodium hypochlorite solution for 5 minutes, followed by triple washing in distilled water and placed on paper towels to dry.The fruits were immersed for 5 minutes in the same treatments with essential oils previously described diluted at a concentration of 1% in SDW, plus Tween 80.
After 24 hours of treatment application, minor wounds (1 mm deep) were made in the equatorial region on opposite sides of the fruit peel.The fruits were inoculated with a conidia suspension at a concentration of 1 x 10 5 spores mL -1 with a manual sprayer and placed in polyethylene trays covered with moistened transparent plastic bags to form a humid chamber, which remained for 24 hours.Renaud et al. (2008) measured disease severity based on the diagrammatic scale Figure 1.The area under the disease progress curve (AUDPC) (SHANER; FINNEY, 1977) and the protection of the fruits by the oils were calculated by the formula: Protection = (AUDPC Treatments x 100/AUDPC Control).
For the extraction of peroxidase (POX), polyphenol oxidase (PPO), and phenylalanine ammonia-lyase (PAL) enzymes, 3.0 g of fruit peel homogenized in 10 mL of 0.1M phosphate extraction buffer, pH 6.0 were used.The suspension was centrifuged for 15 minutes at 12000 g, collecting the supernatant.The enzymatic analyzes were carried out in three evaluation periods, 5, 15, and 25 days after the application of the treatments.
The reactions to determine the enzymatic activity of POX were prepared by adding 0.25 mL of the supernatant to the reaction medium containing 0.25 mL of 1.7% guaiacol, 0.75 mL of 0.1 M phosphate buffer, pH 6.0 and 0.25 mL of 1.8% H 2 O 2 .The reactions were monitored with the aid of a spectrophotometer, observing the variation of absorbance at a wavelength of 470 nm, at 25 °C, immediately after mixing and the activity expressed in absorbance units (AU) min -1 mg -1 of protein.
To determine the PPO activity, 0.5 mL of the supernatant was added to the reaction medium containing 0.25 mL of 0.6 M S-methyl-catechol and 0.75 mL of 0.1 M phosphate buffer pH 6.8.The solution was incubated for 15 minutes at 40 °C, and the reaction stopped with the addition of 800 µL of 2N perchloric acid.The reactions were monitored with the aid of a spectrophotometer, observing the variation in absorbance, at a wavelength of 395 nm, at 25 °C, immediately after removal from the incubator and the PPO activity expressed in absorbance units (AU).min -1 .mg - of protein.
To determine PAL activity, 0.5 mL of the supernatant was transferred to test tubes, and 1.5 mL aliquots of 0.01M TRIS-EDTA buffer solution, pH 8.8, 0.5 mL of phenylalanine solution (30 μM) and 0.5 mL of distilled water were added.After incubation in a water bath at 40 °C for one hour, the reaction was stopped with 2 mL of 5M hydrochloric acid, and spectrophotometric readings were performed at 290 nm at 25 °C.Results were expressed in Absorbance Units (AU) min - 1 .mg - of protein.
The blank II was prepared using 1.5 mL of 0.01M TRIS buffer solution pH 8.8, 0.5 mL of enzyme extract, and 1.0 mL of distilled water for each treatment tested.Analyzes were performed in triplicate.The determinations of the amount of protein present in the referred extracts were carried out by the method of Bradford (1976).
The design was completely randomized.Analysis of variance was performed for the in vitro test, consisting of four replicates of three plates (12 plates per treatment).The means were compared by the Tukey test up to 5% probability.The R ® program version 2017 was used (R CORE TEAM, 2017).
The design was entirely randomized for the in vivo test, and the test on the fruits was represented by four replications of three fruits (12 fruits per treatment).The enzymatic analysis was carried out in a 7x3 factorial scheme (seven treatments and three evaluation periods), and these were performed in triplicate.The variables data were submitted for variance analysis, and the Scott-Knott test compared the means up to 5% of probability using the ASSISTAT ® program.

Experiment I
In the in vitro test, the Alternaria alternata f. sp.citri differed statistically between treatments in mycelial growth.Isolate I8 (PB) showed a higher mycelial growth rate index (MGRI) compared to the others; their averages ranged from 0 to 2.63 cm (Figure 2A).This result can be justified due to the similarity between the temperature conditions of the in vitro culture and the conditions in the field where the isolate was collected.The essential oils (EOs) of citronella, fennel, and mint prevented the in vitro growth of the pathogen, presenting fungitoxic action and equating to the fungicide effect, confirming its direct action in the suppression of the pathogen in the tested experimental conditions.
All isolates showed statistical differences in the diameter of the pathogen colonies.The EOs of ginger, citronella, copaiba, fennel, and mint reduced the mycelial growth of the three isolates, similar to the fungicide treatment (Figure 3).
The EOs present chemical compounds responsible for the efficiency in the management of phytopathogens and may present two or more main components.These compounds are considered the majority and may be responsible for the fungitoxic effect exerted by essential oils.However, some oils present these compounds in small amounts, called trace components, which may have the same effect as the major compounds (BAPTISTA et al., 2015;YU et al., 2017).
Inhibition of mold growth is a standard method of reducing mold growth.Studies pointed out that the antifungal activity against A. alternata could be attributed to eugenol and eugenol acetate, which showed excellent results (SHARMA et al., 2017;JINGA et al., 2018).Perina et al. (2014), observed that the EO of Thymus vulgaris inhibited the growth of A. alternata, in addition to delaying the beginning of the infection process, preventing its penetration.Essential oils act on the cell wall disrupting the plasma membrane that can attack specific organelles in the cytoplasm of pathogens (JINGA et al., 2018).
For spore production, a significant difference was observed between the isolates.All treatments were efficient in inhibiting I8 sporulation (Figure 4A).EOs of eucalyptus, grape seed, copaiba, and linseed induced sporulation of isolates 28 and 30 (Figures 4B and C).There was no statistical difference between treatments and isolates analyzed for the variable conidia diameter of A. alternata f. sp.citri.It is important to emphasize that greater growth of the fungal colony does not reflect in greater spore production due to factors intrinsic to the pathogen.Studies describing the antimicrobial action of EOs from Cinnamomum zeylanicum and Eugenia caryophyllus were effective in vitro and in vivo in inhibiting the development of A. alternata in the epidermis of Hylocereus undatus fruits (CASTRO et al., 2017).

Experiment II
Regarding the area under the disease progress curve (AUDPC), there was a statistical difference between the treatments evaluated for I-8 (Figure 5).The other treatments did not differ statistically and did not promote fruit protection compared to the control.An ecological alternative of great potential is the use of EOs in the control of phytopathogens, which is associated with other disease management practices, results in greater efficiency in the control of these microorganisms, in addition to minimizing the emergence of new races of phytopathogens, collaborating in the production of organic products (KACEM et al., 2016;SCHEUERMANN et al., 2020).
According to Xu et al. (2014), Laurus nobilis oil (500 μg mL -1 ) was effective in protecting cherry tomato (Solanum lycopersicum var.cerasiforme) fruits from A. alternata infection, with an inhibition rate of 33.9%.Chen et al. (2014), found that citronella oil showed vigorous inhibition activity on A. alternata at a concentration of 1.5 μL mL -1 in tomato fruits (S.Lycopersicum L.) with a 52% reduction in disease severity, corroborating with the present research.
The antimicrobial action exerted by essential oils is due to the substances present in their composition, which, once in contact with microorganisms, the lipophilic capacity can allow their penetration into fungal membrane structures, causing damage to it, such as the leakage of ions and other cellular components, affecting the integrity of cell membranes (JINGA et al., 2018).
The results obtained in the in vitro and in vivo tests showed the potential of treatments with essential oils to reduce mycelial growth, sporulation, and the severity of Alternaria brown spot in tangerine fruits and confirm the importance of OEs in the management of A. alternata.f. sp.citri in organic production fields since it is an economically viable alternative, easy to apply, compatible with other disease control strategies, and environmentally safe (YU et al., 2017).
Regarding enzymatic activity, no significant differences were observed between the treatments used (Table 1).However, there was a significant difference (p<0.01) between the evaluation periods, with an increase in enzymatic activity in the second period (Table 1).
The increase in enzymatic activity in the second evaluation period (15 days after harvest) can be explained by the increase in fruit respiration due to senescence and starch degradation, causing physiological changes, increasing the respiratory capacity of the fruits, and providing greater enzymatic action (MENEGASSI et al., 2017).
Table 1.Activity of peroxidase (POX), polyphenol oxidase (PPO), and phenylalanine ammonia-lyase (PAL) enzymes in 'Dancy' tangerine fruits treated with essential oils (1%) and thiabendazole fungicide (400 mL/100L), in different periods. 1 *Evaluations performed at 5 (1st period), 15 (2nd period), and 25 (3rd period) days after the application of treatments.Means followed by the same lowercase letter in the column and uppercase letter in the lines belong to the same cluster by the Scott-Knott test up to 5% significance.