Essential oils in the control of Colletotrichum gloeosporioides f. Sp. cepae in onion seeds Colletotrichum gloeosporioides

- Anthracnose, one of the major diseases in the onion, is caused by the fungus Colletotrichum gloeosporioides f. sp. cepae . The main strategy for controlling this fungus is by the use of fungicide, with this technique being used on the majority of crops. Currently, methods of disease control are being studied that are less aggressive to the environment, with the use of essential oils proving to be an alternative. As such, the aim of this study was to evaluate the direct effect of the essential oils of basil ( Ocimum basilicum ), lemongrass ( Cymbopogon citratus (DC) Stapf.), sage ( Salvia officinalis ), thyme ( Thymus vulgaris ) and citronella ( Cymbopogon winterianus ) as an alternative in the control of the fungus C. gloeosporioides f. sp. cepae , in addition to determining its effect on the physiological quality of onion seeds. The essential oils of lemongrass and sage did not affect the physiological quality of the onion seeds, and all the essential oils used helped to inhibit spore germination in C. gloeosporioides f. sp. cepae . The essential oils of thyme, lemongrass and citronella at a concentration of 2000 ppm completely inhibited mycelial growth in the fungus. Treatment with the essential oil of lemongrass in seeds inoculated with the pathogen gave greater protection against fungal attack. germination. Mycelial growth.


INTRODUCTION
Anthracnose is considered a disease of great importance in the onion (Allium cepa L.), the causative agent being the fungus Colletotrichum gloeosporioides (Penz.) Penz. Et Sacc. f. sp. cepae, which survives in the soil, in crop remains and in seeds. Until now, anthracnose has been conventionally controlled with the use of different fungicides. Among these, the most important are those based on thiophanate-methyl, copper oxychloride, folpet, and futriafol (BRASIL, 2003), and which are registered in the Ministry of Agriculture, Livestock and Food Supply (MAPA). However, the indiscriminate use of these products can generate levels of resistance, with damage to the environment.
There has recently been a growing search for alternative methods of disease control, such as the use of essential oils, due to their low environmental impact (XAVIER et al., 2012). These have allopathic characteristics, i.e. they have the capacity to interfere directly or indirectly in plants or in microorganisms through the release of chemical compounds (SIMONETO; CRUZ-SILVA, 2010) that are produced via secondary metabolism and act as allelochemicals. These compounds may also interfere with the physiological quality of the seeds and with normal plant development (SOYLU; KURT;SOYLU, 2010). For example, the essential oil of citronella (Cymbopogon nardus (L.) Rendle.) contains the compounds citronellal, geraniol and limonene, while the major component of lemongrass essential oil (Cymbopogon citratus Staph.) is citral, which is considered responsible for the allopathic activity of the plant (GUIMARÃES et al., 2008). The essential oil of basil (Ocimum basilicum L.) is composed of linalool, estragole, farnesene, eugenol and cineole, which have antifungal properties (MARTINEZ-VELAZQUEZ et al., 2011).
Research shows that essential oils may have an allelopathic effect on the physiological quality of seeds. As such, Steffen, Antoniolli and Steffen (2010) demonstrated that the essential oil of Eucalyptus gandris W. Hill ex Maiden favoured greater germination in eucalyptus seeds. According to Miranda et al. (2015), the essential oils of lemongrass (Cymbopogon citratus Staph.) and African basil (O. gratissimum L.) showed allelopathic potential on the germination and vigour of lettuce achenes that can be attributed to the content of the respective major constituents, citral and eugenol, whereas the allelopathic effect of the essential oil from basil is the joint result of all the components found in that oil.
Studies have been carried out to determine the effect of essential oils so as to develop an alternative technology for controlling pathogens. In this respect, Oliveira et al. (2017) demonstrated that lemongrass essential oil had a fungitoxic effect, inhibiting conidial germination in Alternaria solani Sorauer and Corynespora cassiicola (Bert & Curt) Wei by 40%, results which indicate that essential oils can be used for inhibiting different types of fungi.
The aim of this study therefore, was to evaluate the effect of the essential oils of sage (Salvia officinalis Lineu), thyme (Thymus vulgaris Lineu), lemongrass (Cymbopogon citratus (DC) Stapf.), basil (Ocimum basilicum L i n e u ) a n d c i t r o n e l l a ( Cymbopogon winterianus Lineu) on the physiological quality of onion seeds, as well as on conidial germination and mycelial growth in C. gloeosporioides f. sp. cepae.

MATERIAL AND METHODS
The work was carried out in the Seed and Phytopathology Laboratory of Embrapa Hortaliças, in Brasilia, from August 2014 to March 2016. Onion seeds of the 'Conquista' cultivar were used, produced in 2014 in the experimental area of Embrapa Hortaliças (15º56'00" S, 48º06'00" W, at an altitude of 997.62 m).
The medicinal plants, sage, thyme, lemongrass, basil and citronella, used to obtain the essential oils, were grown in the research area for organic vegetable production (APPOH) of Embrapa Hortaliças. Choice of the plants to be used in the study was based on the control potential shown by the essential oils in other pathosystems (BRITO et al., 2010;PEREIRA et al., 2008;SEIXAS et al., 2011).
Fresh leaf material from each species was used to extract the essential oils. The plants were collected in the morning, at around 10:00, to find higher levels of essential oil (BLANK et al., 2007), and then dried in an oven at 50 °C for 84 hours (RADÜNZ et al., 2006). For each extraction, 200 g of dried leaf material and two litres of water were used, which underwent a process of hydrodistillation in a graduated Clevenger apparatus for two hours. The essential oils obtained were identified, protected from light and stored in a freezer at -20 °C until required (PEREIRA et al., 2008).
The seeds were treated with a 1000 ppm and 2000 ppm dilution of each oil, as these concentrations had no effect on the physiological quality of the onion seeds in preliminary tests. Each batch of 50 seeds was placed in a plastic bag and 0.5 mL of oil was then added for 15 minutes. The seeds were then dried at room temperature for 24 hours. The control was treated with distilled water only.
To evaluate the effect of the different concentrations of essential oils on the physiological quality of the onion seeds, the following tests were carried out: Germination -four replications of 50 seeds per treatment were sown on germination paper moistened with distilled water at 2.5 times the weight of the dry paper, and placed in plastic boxes (Gerbox ® -11 x 11 x 3.5 cm). The seeds were then incubated in a BOD (Biochemical Oxygen Demand) vertical chamber germinator at 20 °C under constant light. The evaluations were carried out 6 and 12 days after setting up the test (BRASIL, 2009).
Germination speed index -during the 12 days of the germination test, daily counts were made of the germinated seeds; the totals were divided by the respective number of days elapsed from the start of the test, using the formula proposed by Maguire (1962): GSI: G1 + G2 +...Gn/N1 + N2 +...Nn, where: G1, G2 .... Gn is the number of germinated seeds; N1, N2 ....... Nn is the number of days after seeding.
Accelerated aging -four replications of 50 seeds from each treatment were uniformly distributed over aluminium screens fitted into plastic boxes (Gerbox® -11 x 11 x 3.5 cm) with 40 mL of saturated NaCl solution (40 g NaCl / 100 ml water) at the bottom. The boxes containing the seeds were closed and kept at 41 °C for 48 hours in an ageing chamber (COSTA; TRZECIAK; VILLELA, 2008). The seeds then underwent the germination test, where the number of germinated seeds was evaluated six days after the start of the test.
Greenhouse emergence -four replications of 50 seeds per treatment were distributed over expanded-polystyrene (Styrofoam) trays of 200 cells containing Rohrbacher ® commercial substrate made from composted pine bark. The trays were irrigated twice a day. The number of emerged seedlings was evaluated 20 days after seeding, and the results expressed as a percentage of seedling emergence.
The fungus used was C. gloeosporioides f. sp. cepae from the collection of the Plant Pathology Laboratory at Embrapa Hortaliças. The isolate was recovered by spreading onto Petri dishes (9 mm in diameter) containing a culture medium of 2% oatmeal; the dishes were placed in a BOD incubator at 25 °C, where they remained for 7 days under a photoperiod of 12 hours. The conidia which formed in the dishes were then detached from the surface of the plates with a soft-bristle brush and distilled water. The conidial suspension was later filtered through a double layer of gauze and the concentration adjusted to 3.7 x 10 5 conidia mL -1 in a counting chamber.
To evaluate the direct in-vitro antifungal effect of the essential oils on conidial germination, five essential oils were tested: thyme, citronella, basil, sage and lemongrass, in concentrations of 500 ppm, 1000 ppm and 2000 ppm. Tween 20 was used as emulsifier at a concentration of 1000 ppm. Two controls were used: one treatment consisting of distilled water and the other consisting of Tween 20 at 1000 ppm only.
The culture medium used was water agar (1.0 L water, 20 g agar). This was prepared first, and after cooling the essential oils were added to reach the final, pre-established concentrations, except for the controls. After homogenisation, around 25 ml of the culture medium was poured into Petri dishes (90 mm in diameter). The plates were scratched with a ballpoint pen, and divided into four quadrants. Five hundred μL of the 3.7 x 10 5 conidial suspension mL -1 was continuously deposited onto each dish and spread over the surface of the dishes with a Drigalski loop. The dishes were placed in a BOD oven at 25 ºC under a photoperiod of 12 hours. After 24 hours incubation, germination was stopped using a 0.2 mL solution of lactoglycerol, and the percentage of conidial germination determined, with 25 germinated conidia per quadrant being evaluated by trinocular stereoscopic microscope (Meiji) (PEREIRA et al., 2008). The design was completely randomised, with two dishes per treatment, each divided into four quadrants, resulting in a total of eight replications.
To evaluate the effect of the essential oils on mycelial growth in the pathogen, concentrations of 1000 ppm and 2000 ppm of the oils from thyme, citronella, basil, sage and lemongrass were tested. Tween 20 was used as emulsifier at a concentration of 1000 ppm. Two controls were used, one for the treatment consisting of distilled water and the other for the treatment comprising Tween 20 at 1000 ppm.
The culture medium used was 2.0% oatmeal, prepared and autoclaved; after cooling to 45 °C to 50 °C (BRUNELLI et al., 2006), the essential oils were added, except for the controls. The culture medium was then poured into Petri dishes (90 mm in diameter), and a mycelial disc of the pathogen (approximately 1 cm in diameter) was placed towards the centre of each Petri dish; the dishes were then sealed with plastic film and kept at 25 °C + 2 °C under a photoperiod of 12 hours.
Evaluations were made daily, where the measurements corresponded to the mean value of two diametrically opposite measurements taken with the use of a graduated rule (mm). This was repeated until the control mycelium occupied the entire surface of the dish. The mycelial speed of growth index was calculated by adapting the formula proposed by Maguire (1962). The percentage inhibition of mycelial growth was then determined relative to the control. The experiment was conducted in a completely randomised design with four replications, each unit consisting of one Petri dish.
To evaluate the effect of the essential oils on onion seeds inoculated with C. gloeosporioides, three simultaneous experiments were carried out.
Onion seeds were inoculated by means of the water restriction method, using an osmotic potential of -1.0 MPa, determined in preliminary tests. First, Petri dishes were prepared with the BDA culture medium and the -1.0 MPa mannitol solution added after autoclaving. The culture M. I. O. Lozada et al. medium with the restrictor was poured into Petri dishes (90 mm in diameter) and an agar disk from the fungal colony, 1 cm in diameter, was placed at the centre of each dish. The dishes were then incubated in a BOD at 25 °C under a photoperiod of 12 hours for seven days. Fifty onion seeds were disinfested using 1% sodium hypochlorite for 1 min, rinsed in distilled water and dried at room temperature. The seeds were distributed in a single layer over each fungus colony. Each plate was then sealed and placed in a BOD oven at 25 °C under a photoperiod of 12 hours for five days, after which they were removed from the dishes (MACHADO et al., 2007). They were then treated with the basil, citronella, lemongrass, thyme and sage oil at concentrations of 1000 ppm and 2000 ppm. Each batch of 50 seeds was treated with 0.5 mL oil for 15 minutes, dried at room temperature for 24 hours, and then subjected to health tests expressed as a percentage of infestation, germination and emergence in the greenhouse, as per the methodology described above. The experiment was carried out in a completely randomised design with 4 replications, each unit consisting of 25 seeds.
The Sisvar v.5.6 statistical software was used (FERREIRA, 2008) for statistical analysis of the data. The qualitative mean values were grouped by the Scott-Knott test (p<0.05), and regression graphs were generated for the quantitative mean values using the Excel software.

RESULTS AND DISCUSSION
The untreated seeds had a first count of 87%, with a significant statistical difference in this count (Table 1). Seeds treated with the essential oils of thyme at the two concentrations (1000 ppm and 2000 ppm), and citronella and basil at 2000 ppm, showed 82%, 71%, 78% and 78% of germinated seeds respectively at the first reading, with no statistical difference, but did show a difference in relation to the control. These results differed from those found by Araújo Neto and Araújo (2012) in fennel (Foeniculum vulgare Mill), where treatment with a 2.5% concentration of citronella essential oil increased the percentage of germinated seeds at the first count.
The untreated seeds presented a germination of 96%. The essential oil of thyme at concentrations of 1000 ppm and 2000 ppm, and citronella at 2000 ppm, resulted in germination of 93%, 90% and 90% respectively (Table 1). It was found that citronella oil showed a decrease in germination as its concentration increased. This may be due to the presence of compounds such as citronellal and geraniol, high concentrations of which may inhibit germination (XAVIER et al., 2012).
For the germination speed index, no significant differences were found between the treatments and the control. With regard to the accelerated ageing test, the treatments did not differ from one another or in relation to the control. This test allows the vigour and storage potential of the seeds to be evaluated, and shows that germination in seeds treated with the essential oils was not affected by extreme conditions and that the seeds could be considered vigorous.
For the greenhouse emergence test, the untreated seeds had an emergence of 93% (Table 1). In this test, the basil oil at a concentration of 2000 ppm resulted in a statistical difference with the control, showing a lower number of emerged seeds (65%). The other treatments do not differ statistically from one other or from the control. Studies conducted by Blank et al. (2004) demonstrated that the essential oil of basil has great agronomic potential for linalool extraction, both due to its oil content and its being a short-cycle plant. This chemical compound can affect the germination potential of seeds treated with this oil (BRITO et al., 2010), agreeing with the results obtained in this work. Other studies were carried out by Magalhães et al. (2014), who found that basil oil affected the seedling emergence pattern of maize (Zea mays L.) as the concentration increased (0 μL L -1 , 5 μL L -1 , 10 μL L -1 , 15 μL L -1 and 20 μL L -1 ).
The treatments with the essential oils of sage and lemongrass did not differ statistically from the control in the various tests, showing no effect on the physiological quality of the onion seeds. These results agree with those of Simonetto and Cruz-Silva (2010) who verified that different concentrations (0%, 7.5%, 15%, 22.5% and 30%) of the aqueous extract of sage oil showed no statistical interaction with the germination of maize (Zea mays L.) or tomato (Solanum lycopersicum L.) seeds compared to the control.
In the in-vitro evaluation of essential oils on conidial germination and mycelial growth in C. gloesporioides f. sp.  cepae, it was found that all the essential oils caused a quadratic reduction in conidial germination as the concentration increased (Figure 1). From the concentration of 1000 ppm, the essential oils of lemongrass and citronella reduced conidial germination completely. On the other hand, the essential oils of thyme and basil caused the 100% inhibition of conidial germination from the concentration of 2000 ppm, whereas sage oil reduced conidial germination by 56% at the highest concentration (2000 ppm). The treatment consisting of Tween at 1000 ppm showed no statistical differences between the doses used and the control.
The results obtained in this experiment agree with those obtained by other authors, where the use of essential oils reduces conidial germination in different pathogens. As such, Cruz et al. (1997) found that lemongrass oil was efficient in controlling Fusarium spp., inhibiting mycelial growth and spore germination. Silva et al. (2009) found that lemongrass oil controlled C. gloeosporioides, causing the 100% inhibition of conidial germination in the pathogen. Furthermore, Guimarães et al. (2008) found pathogen inhibition with lemongrass due to the citral component which exerts an antifungal effect on the conidia.
All the treatments with the essential oils inhibited mycelial growth in C. gloeosporioides in relation to the control, which showed no inhibition of the pathogen (Table  2). At a concentration of 2000 ppm, the essential oils of thyme, lemongrass and citronella completely inhibited mycelial growth in C. gloeosporioides. Sage oil at 2000 ppm inhibited growth of the fungus by 64.39% relative to the control, followed by the essential oil of thyme at 1000 ppm, with 49.01% inhibition. The essential oil of basil at the concentration of 1000 ppm inhibited fungus growth by 32.41%. The essential oils of sage, lemongrass and citronella at the concentration of 1000 ppm, and of basil at 2000 ppm, inhibited growth of the pathogen by 18.99%, 18.62%, 18.01% and 17.21% respectively, not differing from one other, but differing from the control. The standard treatment with Tween at 1000 ppm did not significantly inhibit mycelial growth in the pathogen.
Agreeing with the results of this study, Silva et al. (2009) evaluated the effect of lemongrass essential oil on mycelial growth in C. gloeosporioides at concentrations of 1 μL mL -1 , 3 μL mL -1 , 5 μL mL -1 and 10 μL mL -1 in the passion fruit (Passiflora edulis Sims), demonstrating that from the concentration of 1 μL mL -1 , the essential oil inhibited growth of the pathogen by 100%. According to Itako (2009), lemongrass oil resulted in a fungitoxic effect on germination and mycelial growth in Cladosporium fulvum Cooke, with a reduction of 96.30% in spore germination at a concentration of 26.46% in relation the control. On the other hand, the oil at a concentration of 40.67% showed 20.03% inhibition of mycelial growth in the pathogen. Pereira et al. (2011), evaluated the effect of citronella, thyme and lemongrass oil at concentrations of 1000 μL L -1 , 1500 μL L -1 and 2000 μL L -1 on the control of Cercospora coffeicola, and found that the oils totally inhibit conidial germination in Cercospora coffeicola, besides reducing mycelial growth in the pathogen. On the other hand, studies Mean values followed by the same letter do not differ by Scott-Knott test, p<0.05 Table 2 Nascimento, Nery and Rodrigues (2008) on the control of Colletotrichum gloeosporioides from papaya fruit with basil-leaf extract showed no statistical difference in mycelial growth for eight days compared to the control.
For the seeds inoculated with C. gloeosporioides f. sp. cepae, each treatment differed from the control, which showed 65% germination (Table 3), except for the treatment with basil oil at 2000 ppm which resulted in 72% of germinated seeds. Seeds treated with the essential oils of lemongrass and basil at 1000 ppm, and the essential oil of citronella at both concentrations (1000 ppm and 2000 ppm), showed 88%, 84%, 85% and 88% germination respectively. These oils differed from the control and the other treatments, having the highest values for germination. The essential oils of sage and thyme at the two concentrations, and of lemongrass at 2000 ppm, had 80%, 79%, 79%, 78% and 81% germination respectively, without differing statistically from one another, showing a greater number of germinated seeds compared to the control.
In the health test, each treatment differed from the control, which showed more infected seeds with a rate of 73%, proving that essential oils have an effect in controlling the pathogen, as they had a lower percentage infection (Table 3). The treatments with the essential oils of lemongrass at the two concentrations (1000 ppm and 2000 ppm), and of basil and citronella at 2000 ppm, did not differ statistically from one other, but differed from the control, having the lowest number of seeds with symptoms of the pathogen, respectively 5%, 2%, 11%, and 10%; the treatment with sage oil at both concentrations presented 54% and 45% respectively in relation to the control. These two treatments had the highest number of infected seeds in comparison with the other essential-oil treatments, and were considered the treatments that exerted the least control over the pathogen, followed by the essential oils of citronella and basil at the concentration of 1000 ppm, and thyme at both concentrations (1000 ppm and 2000 ppm), with a percentage infection of 22%, 19%, 31% and 20% respectively.
From the results of the test for emergence (Table 3), all the treatments with essential oils showed a statistical difference in relation to the control, which had a percentage emergence of 12%. The treatment with lemongrass oil at 1000 ppm, showed the highest percentage emergence of 71% compared to the control, with the treatment with sage oil at 1000 ppm being the least effective of the oil treatments, reflected in its percentage seedling emergence of 26%. The remaining treatments showed no statistical differences between one another.

Treatment
Concentration ( Table 3 -Mean values for percentage germination, seed infection and emergence in onion seeds (Allium cepa) inoculated with Colletotrichum gloeosporioides f. sp. cepae and treated with essential oils