Physiological and Sanitary Attributes Evaluation of Organic Coriander Seeds Treated With Essential Oils

For seeds organic production the control of fungi with chemical fungicides is not indicated, which requires the use of biological products. In this sense, the use of essential oils derived from plants is a possibility for microorganisms control. This study evaluated technical feasibility of applying the essential oils of clove, lemongrass, rosemary, eucalyptus, ginger and Tea tree, in concentrations of 500, 1.000, 1.500 and 2.000 μL to organic coriander seeds of the Verdon variety, besides the control. At the beginning of storage and every 60 days the seeds were evaluated for water content, germination, germination velocity index and root emission, seedling emergence and seedling emergence speed index, to shoot length, rootlength, total length and sanity. The experimental design was a completely randomized (DCR), in a 6x4 + 1 factorial scheme, with six essential oils and four concentrations + control treatment, with four replications for germination and vigor analysis, and eight replications for sanitary analysis. With increasing concentration of essential oils, there was a linear reduction in germination and a reduction in the incidence of Alternaria sp. using clove and rosemary oils at a concentration of 500 μL L, eucalyptus at a concentration of 1.500 μL L and ginger with 2.000 μL L. Therefore, it is possible to use clove and rosemary essential oils up to 500 μL L to reduce the incidence of Alternaria sp. without causing significant reduction in germination.

. According to Taiz and Zeiger (2013), essential oils are often found in vegetables, in all parts of the plant, in glandular trichomes, which protrude from the epidermis and act as a sign in relation to the toxicity of the plant. These oils can be extracted from vegetables through steam distillation. In addition, some secondary metabolites of plants, present in essential oils, can act on microorganisms and on the development of plants.
However, due to different compounds present in these oils, their interaction and concentration can interfere with cell development (Miranda et al., 2015). Flávio et al. (2014) found that clove basil essential oil reduced fungal infestation in sorghum seeds, but reduced their viability and vigor. In cabbage seeds, Amini et al. (2018) verified a reduction of Xanthomonas campestris with the use of Zataria multiflora essential oil, but there was a significant reduction in seed germination.
Storage is practically a mandatory step in a seed production program, being the main concern during the period the preservation of seed quality, as infections by microorganisms can reduce seed quality (Nascimento et al., 2006). Boukaew et al. (2017) found that fumigation of clove essential oil [Syzygium aromaticum (L.) Merr. & L. M. Perry] can be applied to protect corn seeds from the fungus Aspergillus flavus during storage. However, results like these are still scarce in the literature due to the diversity of compounds present in essential oils.
The objective of this work was to evaluate the application effect of the essential oils of clove, lemongrass, rosemary, eucalyptus, ginger and Tea tree, in different concentrations, on the physiological and sanitary parameters of coriander organic seeds during storage.

Method
The freshly harvested organic Coriander (Coriandrum sativum L.) seeds of the Verdão variety supplied by the Mokiti Okada Foundation Research Center were produced in 2017 in the city of Ipeúna-SP. Product applications and seed analysis were performed at the Seed Analysis Laboratory of Ponta Grossa State University, Ponta Grossa, PR, Brazil.

Essential oils of clove [Eugenia caryophyllus (L.) Merrill & Perry], lemongrass [Cymbopogon citrates (DC)
Stapf.], rosemary (Rosmarinus officinalis L.), eucalyptus (Eucalyptus globules Labill.), ginger (Zingiber officinale Roscoe.) and Tea tree (Tea tree alternifólia Cheel.) were applied to the seeds at concentrations of 500; 1.000; 1.500 and 2.000 μL L -1 plus 1% (v/v) Tween 80 to facilitate emulsification of oils in distilled water , besides the control (no applications). The application of essential oils to seeds was by immersion for a period of three minutes. Then the seeds were placed on two sheets of sterile blotting paper for drying in a natural environment until they reached 6% of water. Subsequently, they were placed in aluminized bags for cold room storage at 10 ºC and 55% relative humidity.
Seed quality evaluations were performed immediately after treatment and at 60, 120 and 180 days after storage.

Determination of Water Content
To determine the water content, the adapted greenhouse method of Brasil (2009a) was used. For this, 2.0 grams of seeds were placed in previously identified containers and weighed in precision scales. The containers were placed open in the oven at 105±3 °C for a period of 24 hours. After the drying period, the containers were capped and placed in a desiccator until cooled for weighing. The result was expressed as a percentage of water.

Germination Test (G)
Four repetitions of 50 seeds distributed on paper (Germitest), moistened with water, at a ratio of 2.5 times the dry paper weight were used. The paper rolls were placed in a germination chamber, where they remained at a constant temperature of 20±2 °C, with evaluations on the 21 st day after sowing, according to Brasil (2009a). The result was expressed as a percentage of normal seedlings.

Germination Test First Count (GFC)
The number of normal seedlings was recorded on the 9 th day of germination test, according to Brasil (2009a). The result was expressed as a percentage of normal seedlings.

Germination Speed Index (GSI)
The germination speed index (GSI) was calculated from the data obtained in the germination test (item 2.2). The evaluations were conducted daily, at the same time from the day when normal seedlings first emerged, whose amount was recorded and these seedlings were removed from the paper. To calculate the GSI, the formula described by Maguire (1962) was used.

Root Emission Index (REI)
Root emission index (REI) was determined from seedlings obtained in the germination test (item 2.3). The evaluations were conducted daily, at the same time from the day when normal roots first appeared, whose amount was recorded. To calculate the REI, the formula described by Maguire (1962) was used.

Seedling Emergence (SE)
Four replicates of 50 seeds were sown in 200-cell plastic trays. The seeds were sown to a depth of 1 cm and one seed per cell was placed. The substrate used was composed of peat, soil concealers, vermiculite, charcoal and pine bark.
The substrate was moistened daily with water equivalent to 60% of its holding capacity, and the trays were randomly distributed inside the greenhouse at 25 ºC and 70% relative humidity. The evaluations were performed on the 21st day after sowing and the results were expressed as percentage of emerged seedlings (Nakagawa, 1994).

Seedling Length
Seedling length was evaluated according to Nakagawa (1994). Four repetitions of 10 seeds sown on a line drawn in the upper third of the seed germination paper (Germitest), moistened with water, at 2.5 times the weight of dry paper were evaluated. They were then maintained at 20 °C, with shoot, root and seedling length evaluations on the 21 st day after sowing and the results were expressed in centimeters.

Sanitary Parameter Evaluation
To evaluate the sanitary parameter, 200 seeds (8 replicates of 25 seeds) were placed in plastic boxes, previously disinfected with sodium hypochlorite for 24 hours and cleaned with alcohol, on two sheets of moistened filter paper 2.5 times the weight of dry paper. The seeds were kept for seven days in a BOD incubation chamber, regulated at 20 °C and daily photoperiod of 12 hours. The fungi presence evaluation in the seeds was performed at seven days after sowing, using magnifying glass and microscope, classifying the fungi by gender. The results were expressed as percentage of occurrence of the observed fungi (Brasil, 2009b).

Statistical Analysis
The experimental design was a completely randomized (DCR), in a 6 × 4 + 1 factorial scheme, with six essential oils and four concentrations + control treatment, with four replications for germination and vigor analysis, and eight replications for sanitary analysis.
Data were analyzed for variance analysis to verify the significance of the interaction of essential oil and concentration factors. Essential oil concentration data, when significant, were submitted to polynomial regression analysis, up to the third degree, and when significant for essential oil, the means were compared by Tukey test (5%). When necessary, the percentage data were transformed into arcsin (x + 0.5)/100 and analyzes were performed with the aid of R Studio program.

Results
For some variables there was interaction for the factors essential oil and concentration when performed the analysis of variance (ANOVA) for coriander seeds. The results related to the first count of coriander seed germination test (9 days after sowing) indicated that there was no statistically significant difference, due to the application of oils whose concentration was 500 μl L -1 , from post-harvest to 60 days after storage (DAS) ( Table  2).
This result was also observed for concentrations of 1.000 μl L -1 in seeds stored up to 60 days. At the concentration of 1.000 μl L -1 in the seeds without storage submitted to the application of eucalyptus, ginger, clove and Tea tree oils the germination was statistically superior to the treatments with lemongrass and rosemary oils in the same concentration. For the first germination test count, the treatment with eucalyptus essential oil was statistically superior when compared to other treatments results (Table 2). Note. *Means followed by the same letter in the column, within each storage period, do not differ statistically by Tukey test at 5% significance; ns = not significant.
With increasing concentrations of essential oils applied to coriander seeds, there was a significant and linear reduction for the first post-harvest germination test count in the applications of clove, ginger and eucalyptus essential oils. At 60 DAS only the application of lemongrass oil had statistically significant variation and at 120 DAS there was a linear reduction for the germination test first count with the application of clove, lemongrass, eucalyptus and Tea tree essential oils. At 180 DAS there was a linear reduction in coriander seed germination with the application of essential oils ( Figure 1). jas.ccsenet.

Figure
For the ger germinatio  For the germination speed index (GSI) the interaction between concentration and type of essential oil was significant up to 120 DAS. For seeds treated shortly in post-harvest and stored up to 60 days at a concentration of 500 μl L -1 , the results showed no significant differences regarding GSI. With the application of 1.000 μl L -1 of essential oils post-harvest and at 120 DAS, GSI results for Tea tree and clove essential oils were statistically superior to rosemary and lemongrass essential oils. However, the application of lemongrass oil at concentrations of 1.500 and 2.000 μl L -1 caused significant reduction of GSI in relation to the other treatments, maintained until 180 days of storage (Table 4). Note. *Means followed by the same letter in the column, within each storage period, do not differ statistically by Tukey test at 5% significance; ns = not significant.
There was significant interaction for essential oil and concentration for root emission index (REI) up to 120 DAS. The results were similar to those found in the GSI. From post-harvest to 120 days of storage, REI results with the application of essential oils at a concentration of 500 μl L -1 showed no significant differences. With the application of 1.000 μl L -1 , treatment with Tea tree essential oil resulted in higher REI than rosemary and lemongrass essential oil treatments in post-harvest. No differences were found in seeds stored up to 60 days when treated with these essential oils and at this concentration (Table 5).
The REI result was statistically lower for the application of lemongrass Essential Oil at concentrations of 1.500 and 2.000 μl L -1 compared to the other results and maintained until 60 days of storage. At 120 DAS lemongrass essential oils at concentrations of 1.000, 1.500 and 2.000 μl L -1 and clove with 2.000 μl L -1 resulted in statistically lower ESI than other treatments. The interaction between concentration and essential oil was not significant for REI at 180 DAS, however, there were significant differences for each variable analyzed separately. The seeds treated with eucalyptus essential oil showed a statistically higher REI than clove, lemongrass, rosemary, ginger and Tea tree oil applications (Table 5). Note. *Means followed by the same letter in the column, within each storage period, do not differ statistically by Tukey test at 5% significance; ns = not significant.
Coriander seedling emergence was also influenced by the application of essential oils. There was a significant interaction between the concentration and essential oil factors in post-harvest seed and storage of 120 to 180 days. In post-harvest, there was no significant difference in seeds treated with essential oils at concentrations of 500, 1.000 μl L -1 and control treatment. These results were also observed at 120 and 180 DAS (Table 6).
With 1.500 and 2.000 μl L -1 of clove, rosemary, eucalyptus and Tea tree essential oils the results of seedling emergence were higher than those treated with ginger and lemongrass essential oils at the same concentrations. At 120 DAS, only at the concentration of 2.000 μl L -1 there was significant variation, and the use of lemongrass oil was detrimental to seedling emergence. This was also observed at 180 DAS for concentrations of 1.500 and 2.000μl L -1 (Table 6).
There was a linear reduction in REI with increasing concentrations of eucalyptus, clove and ginger essential oils in post-harvest. At 60 DAS only the application of lemongrass essential oil adjusted to the quadratic model At 120 DAS with the application of clove and eucalyptus essential oils the seeds showed linear reduction of REI with increasing concentrations and at 180 DAS there was adjustment to the quadratic regression model, according to the regression equation derivation, the concentration of 818.96 μl L -1 caused a REI reduction (Figure 4).   There was seedlings.
After 60 (Figure 6).   Note. *Means followed by the same letter in the column, within each storage period, do not differ statistically by Tukey test at 5% significance; ns = not significant.
For coriander seeds the increase of clove essential oil concentration at post-harvest and at 180 DAS caused a linear reduction in the incidence of Cladosporium sp. (Figure 7). jas.ccsenet.

Figure 7 concen
For the in Coriander Essential o   There was in the seed in essentia

Discussion
Seed germination was 76% in post-harvest for control treatment seeds, which are common results for coriander seeds, as stated by Pereira et al. (2005). The seeds have problems related to vigor, plant establishment and disease. According to regulation N o 457 of December 18, 1986, coriander seeds must have a minimum germination of 60% for seed distribution, transportation and trade in the country (Brasil, 1986).
According to Miranda et al. (2015) seed germination or seedling development of certain species has influence of chemical alleles contained in vegetable oils. The authors concluded in their study that lemongrass essential oil reduced the germination and vigor of lettuce seeds, which can be attributed to the contents of the major constituents, citral and eugenol. The essential oil of lemongrass presented 48.7% concentration of citral, isomer of geranial (Table 1).
Linalool compounds present in lemongrass and rosemary essential oils, eugenol, present in clove and rosemary essential oils and cineol present in rosemary, Tea tree and eucalyptus essential oils, may influence the reduction of the primary root length of the seedling (Table 1). Such effect was also observed by Gomes et al. (2016) at concentrations starting at 1.5 mL L -1 with clove essential oil.
According to Nishida et al. (2005) five volatile monoterpenes, eucalyptol, α and β-pinene (present in eucalyptus essential oil), camphene (present in ginger, lemongrass and rosemary essential oil (EO)) and camphor (present in rosemary EO), showed inhibit the development of Brassica campestris roots by interfering in the synthesis of organelle and nuclear DNA within meristematic cells. Others monoterpenes, 1,8-cineole (present in the eucalyptus, Tea tree and rosemary EO) timol, geraniol (present in the EO of ginger) and camphor inhibited corn root growth and induced oxidative stress (Zunino & Zygadlo, 2004).
Among the essential oils used lemongrass reduced the physiological quality of seeds. This oil has in its geranial and neral composition ( Table 1). The geranial is a monoterpene, trans-isomer of citral. According to Chaimovitsh et al. (2012) seed germination and seedling development are inhibited in the presence of citral, which causes damage to the interphase cell microtubules of both plants and animals. Granã et al. (2013) evaluating the effect of citral on the root development of Arabidopsis thaliana, showed changes in cell division, thickening of the cell wall and reduced intercellular communication, confirming the compound's phytotoxicity. Therefore, the effects of Lemongrass Essential Oil verified in this work can be attributed to the presence of geranial and neral, which presented 48.7% and 42.2% of these substances respectively (Table 1). Brito et al. (2012) evaluating corn seeds of cultivar XGN5320, found that eucalyptus essential oil, in concentrations of 5, 10 and 15%, reduced seed germination, demonstrating that it is essential to evaluate the use of these oils through toxicity tests, because these oils have some biological activity and applications with inadequate concentration cause the abnormal development of plants. The inhibition of seed germination and seedling development were also observed by Shokouhian, Habibi & Agahi (2016). The authors verified for lettuce seeds (Lactuca sativa) that the application of essential oils of rosemary (Rosmarinus officinalis), thyme (Thymus vulgaris) and anise (Pimpinellaanisum) significantly reduced seed germination. Abbaszadeh et al. (2014) observed that the most potent inhibitory activity of thymol, carvacrol, eugenol and menthol was found for Cladosporium sp. Gomes et al. (2016) found a maximum reduction in the percentage incidence of Cladosporium sp., from concentrations of 1.53 mL L -1 to 2.0 mL L -1 with clove essential oils and basil. The same authors observed a significant reduction in the incidence of Penicillium sp. In the present work, these results were not observed with clove essential oil. According to Menezes et al. (2017) the genus Cladosporium sp. comprises a large number of fungi with worldwide distribution and which are among the most common environmental fungi. They are often isolated as contaminants, however, some species are pathogenic and toxigenic for humans. Hillen et al. (2012) found 100% inhibition in the mycelial growth of Alternaria sp. using concentrations of essential clove oil with 100, 200, 500 and 1.000 μL L -1 , and at concentrations of 20, 40 and 60 μl L -1 the inhibition gradually decreased. In the present work, these results were not observed with clove essential oil. Matusinsky et al. (2015) found a reduction in mycelial growth of isolated species of the genera Penicillium sp. and Fusarium sp. using a dose of 10.0 μL L -1 of the essential oils of rosemary (Rosmarinus officinalis) and thyme (Thymus vulgaris).
Clove oil used in this study has more than 80% eugenol, which is considered its main component (Table 1). Castro et al. (2005) found that clove essential oil was promising, significantly reducing the fungal growth of Alternaria alternata.
The results found in the present study correlating the chemical constitution and the allelopathic activity of each essential oil and its major constituents corroborate the observations made by Souza Filho et al. (2010), who stated that the effects of essential oils on seedling germination and vigor cannot be generalized and can be explained in an individualized way considering their main chemical constituents.
It is concluded that the essential oils of lemongrass, eucalyptus, ginger, rosemary and tea tree at concentrations above 500 µL L -1 reduce the germination and vigor of coriander seeds. Clove and rosemary essential oils at a concentration of 500 µL L -1 , eucalyptus at a concentration of 1.500 µL L -1 and ginger 2.000 µL L -1 are efficient for reducing Alternaria sp. in coriander seeds. Coriander seeds can be treated with clove and rosemary essential oils at concentrations up to 500 µL L -1 , without significantly affecting germination, reducing post-harvest incidence of Alternaria sp. Storage of coriander seeds treated with essential oils is not feasible.