Control of Alternaria brassicicola with thermotherapy and propolis and effect on the physiological quality of kale seeds

In the organic production system, the use of agrochemicals for seed treatment is prohibited. This study was carried out to evaluate the effect of heat treatment and propolis on the germination and vigor of kale seeds and the effect on the control of Alternaria brassicicola . In the treatment with wet thermotherapy, three temperatures (50, 55 and 60°C) were evaluated in separated experiments. For each temperature, five immersion times [0 (control), 15, 30, 45 and 60 minutes] were evaluated. In the dry thermotherapy treatment, seeds were submitted to constant 70ºC temperature and five times [0 (control), 24, 48, 72 and 96 hours] were evaluated. In the treatment with propolis, six concentrations (0.0, 0.2, 0.4, 0.6, 0.8 and 1.0%) were studied, with subsequent washing or not washing of the seeds. Then, seeds were inoculated with A. brassicicola and submitted to the following treatments: wet thermotherapy at 50°C for 60 minutes and at 55°C for 15 minutes and propolis at 1.0% with and without further washing. Two control treatments were included in the experiment (inoculated untreated seeds; and not inoculated and untreated seeds), totalizing six treatments. After inoculation and treatments, the incidence of the fungus on the seeds was evaluated and the effect on the physiological quality of the seeds. Both thermotherapy treatments (50°C for 60 minutes and 55°C for 15 minutes) reduced the pathogen incidence significantly. Both propolis treatments at 1% (with and without subsequent washing) did not provide a satisfactory reduction on A. brassicicola incidence in kale seeds . Thermotherapy at 55°C for 15 minutes is the best option for A. brassicicola control in kale seeds, because it does not affect seed physiological quality.

It is not always possible to obtain seed lots without the presence of undesirable microorganisms. For this reason, in most cases, the treatment of seeds is recommended, especially in vegetables, whose seeds are of high value and the cost of this treatment is very low compared to their cost (Cardoso et al., 2015). The use of agrochemicals is not allowed in the organic production system, neither in the production phase in the field, nor in the seeds treatment. Thus, studies about the control of pathogens transmitted by seeds in the organic system are necessary, in order to avoid the transmission of the pathogen and preserve the seeds sanitary quality, without harming the physiological quality of the lot.
Among the methods allowed in the organic production system, the thermotherapy is a physical treatment that consists of exposing the seeds to the action of heat (wet or dry) in combination with the treatment time (Braga et al., 2010;Spadaro et al., 2017). To be successful with this method, it is necessary to know the appropriate combination of temperature and exposure time, which may vary with the species, cultivar, lot, initial vigor, among other factors (Lanna et al., 2013;Divsalar et al., 2014). The treatment principle is based on the difference in lethal thermal points, that is, the temperature must be higher than the lethal temperature for the pathogen, without harming the seeds (Grondeau & Samson, 1994).
Although the alternative control of pathogens with heat treatment has proven to be efficient, there are few studies on this technology and, mainly, its effect on the physiological potential of the seeds (Braga et al., 2010;Lanna et al., 2013), as the heat treatment can reduce the germination and the vigor of the seeds.
In addition to heat treatment, there are other alternative seed treatments, such as the application of propolis extract, which is a product with antimicrobial properties. This product has presented good results in the control of seed pathogens (Souza et al., 2017). According to Silva et al. (2006), the product consists of a series of resinous substances, which bees use to protect the hive against the proliferation of microorganisms, including fungi and bacteria.
In view of the above, and considering the need for the eventual replacement of chemicals by alternative seed treatments in the organic production system, the objective was to evaluate the effect of heat treatment and use of propolis on the control of Alternaria brassicicola, and their effect on germination and vigor of kale seeds.

MATERIAL AND METHODS
The experiments were conducted at the Vegetable Seeds Laboratory of the Horticulture Department of the School of Agriculture (FCA) of Sao Paulo State University (UNESP), Botucatu-SP.
The kale seeds (cultivar Manteiga without fungicide treatment) were purchased from the company Feltrin. In the first step, an old, low vigor seed lot was used, because, according to Lanna et al. (2013), it is more sensitive to heat treatment and, therefore, better at detecting differences among treatments. For propolis treatment and the second step, a new seed lot was used to study the effect of the treatments on the control of the A. brassicicola in kale seeds. The isolate of A. brassicicola was provided by Sakata Seed Sudamerica and was initially obtained from a broccoli production field in the municipality of Senador Amaral-MG, later identified and maintained by the company's phytopathology sector. Step

1) Effect of thermotherapy (wet and dry) and propolis on seed germination and vigor
Five experiments were carried out on the treatment of kale seeds by different methods: wet thermotherapy (testing three temperatures), dry thermotherapy and propolis. I n t h e t r e a t m e n t w i t h w e t thermotherapy, three temperatures (50, 55 and 60°C) were evaluated in separate experiments. For each temperature, five seed immersion times (treatments) [0 (control), 15, 30, 45 and 60 minutes] were evaluated. The seeds were immersed in water, at constant temperature, in the time provided for each treatment. After each treatment, the seeds were cooled under distilled water and dried in a forced air seed dryer at 35ºC for 24 h. After drying, the seeds were placed in a chamber (40% RH and 20°C), until the water content stabilization at 8%.
In dry thermotherapy treatment, seeds were submitted to a constant 70ºC temperature in a forced air circulation oven, and five exposure times (treatments) [0 (control), 24, 48, 72 and 96 hours] were evaluated.
In the treatment with propolis (commercial product Apis Flora), twelve treatments were studied, resulting from the factorial 6 x 2, with six propolis concentrations (0.0, 0.2, 0.4, 0.6, 0.8 and 1.0%), with and without washing the seeds after the treatment. The seeds were stirred for 30 minutes in a becker with the propolis solution and a part of these seeds was later washed in distilled water to remove the product and the other part was not washed. After this procedure, the seeds were dried in the forced air seed dryer at 35ºC for 24 h. After drying, the seeds were placed in a chamber (40% RH and 20°C), until the water content stabilization at 8%.
In each experiment, germination tests were set up, placing 50 seeds per plot, with four replicates, evenly distributed over two sheets of germinating paper moistened to 2.5 times their dry weight with distilled water, in transparent plastic boxes (gerbox). The experiment was carried out in a Biochemical Oxygen Demand (BOD) germinator, at a temperature of 25±1°C. The total number of germinated seeds at ten days after sowing (DAS) was considered as the total germination. Germinated seeds were considered when they had primary root and aerial part. The first count of the germination test was carried out at five DAS, and considered as a vigor test (Brasil, 2009). The germination speed index (GSI) was also obtained, with the values of germinated seeds day by day, according to Maguire (1962).
Data were submitted to analysis of variance in an entirely random design and, when significant, regression analysis was performed for seed treatment times in water and dry heat and for propolis concentrations. The F test was used to compare the washing, or not, of the seeds in propolis experiment. For the analysis, the Sisvar program was used. Step

2) Control of Alternaria brassicicola in kale seeds
From the first step, four treatments were select: wet thermotherapy, at a temperature of 50°C with immersion for 60 minutes; wet thermotherapy, at a temperature of 55°C with immersion for 15 minutes; propolis treatments, at a concentration of 1.0%, with and without washing after treatment. These treatments did not harm (or harmless) seeds physiological quality (germination and vigor). Two control treatments were included in the experiment (inoculated untreated seeds; and not inoculated and untreated seeds), totalizing six treatments.
The A. brassicicola isolate was grown in Petri dishes containing PDA culture medium and, after seven days of incubation at 25°C and 12 h photoperiod, disks of 0.5 cm of fungal colony were removed and transferred to Petri dishes containing PDA plus mannitol (33.10 g L -1 ). After the fungus had colonized the entire plate, kale seeds (without chemical treatment), previously disinfected in 2% sodium hypochlorite for one minute and dried on sheets of sterile filter paper, were deposited in a single layer, remaining in contact with the colonies for 24 hours. After inoculation, the seeds were again disinfested with 2% sodium hypochlorite and dried.
After being inoculated and treated as described above, seeds were evaluated by the "blotter test", placing in plastic Petri dishes three filter paper sheets, which had been previously moistened with distilled water and distributing 25 equidistant seeds on this substrate. Four repetitions of 25 seeds per treatment were performed. The plates containing the seeds were incubated in BOD at 20±2°C with a 12 h photoperiod for seven days. After this period, the seeds were examined individually, under a stereomicroscope, in order to evaluate the fungus incidence. The results were expressed as percentage of seeds infected with A. brassicicola.
In addition to the blotter test, seeds germination and vigor (first count of germination and GSI) were also evaluated according to Brasil (2009), as previously described.
Analysis of variance was performed for each variable (fungus incidence, germination, first count and GSI) in an entirely random design and the means were compared using the Tukey test (5%). For the statistical analysis, the Sisvar program was used.

RESULTS AND DISCUSSION
In the dry thermotherapy at 70ºC, regardless of the seed treatment time, no germinated seeds were observed, showing that this temperature was lethal to kale seeds from the tested lot. Only the control showed values of germination (52.5%), first count of germination (12.5%) and GSI (4.17) different from zero.
These results differ from those reported by Lanna (2018) who did not observe reduction in the germination of zucchini seeds treated with dry thermotherapy at 70ºC for up to 96 h. Gama et al. (2014) reported that fennel seeds did not lose quality when treated with dry thermotherapy at 70ºC for up to 15 days. However, the germination of cucumber seeds gradually decreased the longer the treatment time with dry thermotherapy at 70ºC (Shi et al., 2016). Kubota et al. (2012) also reported reduction in the germination of seeds of different cucurbits with dry thermotherapy, the greater the reduction the greater the temperature and the treatment time. However, none of these mentioned studies presented total reduction in germination as occurred with kale seeds. Another fact that can contribute to our results is that an old seed lot with low physiological quality was used and these seeds showed greater sensitivity to heat treatment. Therefore, this method should not be recommended for the treatment of kale seeds, at least at the temperature tested, which is the same tested in most researches with other species of seeds.
The germination and vigor tests showed that the wet heat treatment at 60°C is lethal for kale seeds with more than 15 minutes treatment (Table 1). However, even with 15 minutes, germination has already been drastically reduced, showing that treatment with this temperature is not feasible. Grondeau & Samson (1994) described that wet thermotherapy should be applied at a temperature from 45 to 60ºC for a maximum exposure period of 60 minutes, regardless of the species. Although the temperature is within the range recommended by these authors, the use of 60°C is not recommended for the treatment of kale seeds. Spadaro et al. (2017) recommend that the treatment of organic seeds by this method should be carried out with temperatures ranging from 50 to 55 o C, not recommending 60 o C. Nega et al. (2003), Braga et al. (2010), Soriano et al. (2011) andLanna (2018) also related this same conclusion for different species. However, Cunha et al. (2017) reported that the immersion of zucchini seeds at 60 o C for up to 18 minutes did not affect the physiological quality of the seeds.
As the time of exposure of the seeds to the wet heat treatment at 55°C increased, a reduction in the rate of germination and vigor was observed (Figure 1), the reduction being more pronounced after 15 minutes treatment. Regarding the observed values (Table  1), it can be seen that after 15 minutes germination was even numerically superior to the control without treatment for the first count of germination and GSI.
On the other hand, for all evaluated characteristics (germination, first count and GSI) no significant difference was observed by the F test (5%) and regression analysis for the immersion times tested (15 to 60 minutes) when the seeds were treated in water at 50 o C (Table 1). For the first count of germination, an average of 14.1% was observed at five days after sowing, total germination showed an average of 51.5% and for the GSI, an average of 4.2. Thus, wet thermotherapy at a temperature of 50ºC did not affect the physiological quality of the seeds, even for this low quality seed lot, and this treatment can be recommended for kale seeds.
The results observed in the literature vary according to temperature, treatment time and the species studied. Braga et al. (2010) demonstrated that tomato seeds treated at 52, 53, 54 and 55°C for 30 minutes presented lower germination than those not treated with thermotherapy. Corn seeds showed a loss of vigor when treated at 70 o C for 3 minutes, but not when the treatment was at 50 o C and 60 o C (Vieira et al., 2019).
In Brassicas, Soriano (2016) reported decreased germination the longer the seed exposure time to treatment at 55°C. Lanna et al. (2013) found that thermotherapy at a temperature of 58°C was lethal to cabbage seeds, and at 55°C there was loss of germination and vigor the longer the time of exposure to heat treatment. Nega et al. (2003) and Soriano et al. (2011) do not recommend seed treatments at 55°C for more than 40 minutes for Brassicas seeds.
According to Coutinho et al. (2007), the treatment with hot water can cause denaturation of the external tissues and/ or rupture of the seed coat, deteriorating the seeds quickly in comparison to the seeds not treated by thermotherapy. The higher the temperature and the treatment time, the greater the probability of causing damage to the seeds. Based on the observed results, dry thermotherapy at 70 o C is not recommended. The treatment at 50 o C did not affect the seed's germination and vigor, being chosen the longest time (60 minutes) to continue the research, because the longer the time, the greater the probability of controlling fungi in the seeds. On the other hand, at 55 o C a gradual reduction in seed quality was observed the longer the treatment time; therefore, the shorter time (15 minutes) was chosen because having less germination reduction compared to the other tested times.
In propolis treatments, no significant difference was observed for germination, first count and GSI, with an average of 81.5%, 26.9% and 7.3, respectively.
Thus, the highest concentration used, 1.0%, and the factors with and without washing did not negatively affect the physiological quality of kale seeds, as observed by Souza et al. (2017) and Fraga et al. (2016) with cucumber and carrot seeds.
A. brassicicola incidence in uninoculated, untreated seeds was 0.8%, showing that the seed lot used had a low incidence of this fungus, whereas in the inoculated, untreated control the incidence was 92.0%, showing that the seed inoculation was efficient (Table 2). Treatment with 1.0% propolis without washing reduced the incidence of the pathogen by almost half (43.2%) compared to the inoculated control (92.0%). There was no A. brassicicola control when seeds were washed after being treated with 1.0% propolis. The fungus could be observed in 88% of seeds from that treatment, not differing from the inoculated, untreated control (Table 2). Therefore, the washing of seeds after treatment with propolis, caused lost of fungus control in kale - 14.9 7.9 6.5 F 0.6 ns 0.5 ns 0.2 ns ns = not significant at 5% probability; ** = significant at 1% probability; CV = coefficient of variation.
seeds. Souza et al. (2017) observed that the higher the concentration of propolis (0 to 20%), the lower the mycelial growth in vitro of Penicillium sp., and at the concentration of 20% there was no development of the fungus. The probable reason is the concentration of flavonoids contained in propolis, which have an antimicrobial effect. On the other hand, Carvalho et al. (2019) reported that propolis extract (up to 25%) was not Averages followed by the same letter, in columns, do not differ from each another, by the Tukey test at 5% probability. CV = coefficient of variation. effective in controlling Aspergillus sp. in onion seeds. In brassica seeds, mycelium of A. brassicicola can be found growing superficially, but also internally (Kohl et al., 2010); propolis has a protective effect, acting on seed surface but, with washing after seed treatment, there was no effect on fungi control. Therefore, there are divergences among the different researches cited. However, each research has its particularities, that is, different pathogens were evaluated, different concentrations of propolis extract, treatment exposure times, in addition to the fact that, many times, the evaluation is done only in vitro, that is, with the fungus in petri dish, not in seeds. In addition, Fernandes et al. (2006) also state that the effectiveness of propolis may vary according to the place of production. The chemical composition of propolis is extremely complex, because of the great variability of the Brazilian flora and the seasonal conditions of the area. Silva et al. (2006) analyzed propolis for physico-chemical composition and antimicrobial activity according to the sample collection period; he detected variable flavonoid content (bioactive compound) in samples collected in the winter, which varied between 0.27% and 0.38%, while in the summer it varied from 0.19% to 0.52%.
Both thermotherapy treatments, 50ºC for 60 minutes and 55ºC for 15 minutes, reduced the incidence of the fungus to 0.8%, not differing significantly from the uninoculated control (Table 2), showing the efficiency of these treatments almost eradicating A. brassicicola in the seeds. Soriano (2016) also reported control of Xanthomonas campestris pv. campestris in kale seeds with thermotherapy at 50 o C for 20 and 40 minutes.
Treatment by wet thermotherapy has been an efficient method in different vegetable species. Silva et al. (2018) reported 100% control of Fusarium oxysporum f. sp. cepae on onion seeds with thermotherapy treatment at 60ºC. Nega et al. (2003) observed reduction over 85% of different species of Alternaria in carrot seeds with hot water treatments at 50°C for 30 minutes and 53°C for 10 minutes. These results show the efficiency of thermotherapy in the control of different Alternaria species in vegetable seeds, confirming the result obtained in the present research.
Schaad & Alvares (1993) state that cabbage, broccoli and Brussels sprouts seeds should be treated at 50°C for 25 minutes, and cauliflower, kohlrabi, kale, turnip and turnip greens seeds should be treated for 15 minutes. In this research, the treatment at 50°C for 60 minutes was effective in controlling A. brassicicola in kale seeds.
According to Kohl et al. (2010), A. brassicicola is a pathogen of great importance in the organic production of brassica, especially when transmitted by seeds. Heat treatment can be an alternative to this fungus control in brassica seeds in the organic system. However, it will only be efficient as part of the management strategy, which begins in the field of seed production, with preventive measures against infection by this fungus in the seeds.
An accentuated reduction in seeds germination and vigor was observed comparing inoculated untreated seeds to the not inoculated, untreated control ( Table 2), showing that infection by this pathogen harms seeds physiological quality. According to Kohl et al. (2010), in organic seed production of Brassica vegetables, infections by A. brassicicola can cause severe losses in seed quality (germination and vigor). Nega et al. (2003) observed that the germination and the first count of germination in some brassica seeds were lower when inoculated with the bacterium X. campestris pv. campestris, in relation to the control without inoculation. Infection of seeds by fungi and bacteria, in addition to promoting their dissemination, also harms their physiological quality.
T h e r m o t h e r a p y o r p r o p o l i s treatments did not affect the germination of the seeds compared to the control (Table 2). However, the treatment with thermotherapy at 50ºC for 60 minutes resulted in lower values in the first count of germination and in the GSI, that is, less vigor, similar to inoculated, untreated treatment. Kohl et al. (2010) reported that the heat treatment at 50 o C did not affect the germination of cabbage seeds naturally infected by A. brassicicola.
Considering that the minimum standard for kale seed germination for commercialization is 80% (Brasil, 2013), only seeds from inoculated, untreated treatment or submitted to thermotherapy treatment at 50ºC for 60 minutes (Table 2) could not be commercialized, besides the inoculated and not treated seeds.
The reduction in germination and vigor in the inoculated and untreated seeds occurred because the fungus colonized the seed. For the thermotherapy treatment, the longer treatment time (60 minutes) harmed germination. According to Coutinho et al. (2007), the treatment with hot water can cause denaturation of the external tissues and/or rupture of the seed coat, impairing the vigor of the seeds, as observed in the heat treatment at 50ºC for 60 minutes, due to the longer exposure time of the seeds to hot water. Furthermore, according to Nakagawa (1999), the successive treatments (disinfestation, inoculation and thermotherapy) can compromise the germination of the lots, an effect similar to the study of accelerated aging in seeds, which causes stress through the exposure of the seeds immersed in water, and reduces significantly seed germination and vigor. So, it can explain the results observed in thermotherapy at 50ºC for 60 minutes different from the first step.
Treatment with 1.0% propolis did not affect the germination and vigor of the kale seeds, similar to that observed by Souza at al. (2017) where the application of propolis extract solutions (up to 20% concentration) did not affect the physiological quality of cauliflower seeds. Fraga et al. (2016) did not observe reduction in the physiological quality of carrot seeds when submitted to solutions of propolis extract in concentrations of 2, 4, 8 and 16 mL L -1 distilled water.
Therefore, thermotherapy at 55ºC for 15 minutes is the best option for controlling A. brassicicola in kale seeds, without significantly affect the physiological quality of the seeds.