Seed-surface Disinfection and Germination Effects of Grapefruit Seed Extract (GSE) on Lactuca sativa Seeds Jae-Suk Choi1 1
Division of Bioindustry, College of Medical and Life Sciences, Silla University, 140, Baegyang-daero 700beon-gil, Sasang-gu, Busan 46958, Republic of Korea Correspondence and requests for materials should be addressed to J.-S. Choi (
[email protected])
Received 28 March 2017 / Received in revised form 24 May 2017 Accepted 26 May 2017 DOI 10.1007/s13530-017-0318-0 ©The Korean Society of Environmental Risk Assessment and Health Science and Springer 2017 pISSN : 2005-9752 / eISSN : 2233-7784 Toxicol. Environ. Health. Sci. Vol. 9(2), 169-175, 2017
Abstract It investigated that the seed sterilization and germination effects on Lactuca sativa seeds of two commercially available grapefruit seed extracts (GSEs) from the USA and Brazil. The total bacterial counts in the 0.05% GSE (USA) treatment groups were 0.0 CFU/g for all tested incubation times (1-6 h). All GSE treatments groups (from both the USA and Brazil) showed no or slightly harmful effects on L. sativa seed germination and seedling growth after 24 and 48 h. Additionally, seeds treated with 0.05% GSE showed slightly stronger anti-germination effects and seedling growth inhibition than those treated in a solution of the same concentration but with the pH adjusted to 7.0. We also evaluated the anti-germination effects of naringin, quercetin, and citric acid on L. sativa seeds to identify the active compounds in GSE. We observed no anti-germination effects in the naringin-treated group. In the quercetin-treated group, there was a slight dose-dependent anti-germination effect (0.01-0.1%). In groups treated with 0.01% and 0.05% citric acid (pH 4.79 and 4.03, res pectively), no and slight anti-germination effects, respectively, were observed. However, there were strong anti-germination effects among seeds treated with 0.1% citric acid (pH 4.79). We found that 0.05% GSE (from both the USA and Brazil) had strong seed sterilization effects and no anti-germination effects. Together, these results indicate that GSE may be
useful as a treatment for seed sterilization without inhibiting seed germination or seedling growth. Keywords: Seed-surface disinfection effect, Germination effect, Grapefruit seed extract, Lactuca sativa
Introduction Seed sterilization treatments, including physical scr atching of the seed coat (scarification) and heat, ozone, plasma, chemical, or pesticide treatment, are common ly used to protect seeds against various seed- and soilborne pathogens and insect infestations1-3. There are numerous reports on the use of chemicals and pesticides for seed sterilization, including chlorine gas, commercial Clorox bleach containing 5.25% NaOCl, 3% (v/v) hydrogen peroxide4, and peroxyacetic acid5. However, more effective, less toxic, and envi ronmentally friendly techniques are desirable. Grapefruit seed extract (GSE) has natural antimicro bial properties because of its flavonoids (e.g., naringin, limonoid, kaempferol, and quercetin), citric acid, and other6,7. GSE is an effective, broad-spectrum antimi crobial agent with activity against Escherichia coli, Salmonella, Candida and other fungi, herpes, and influen za8, as well as oral and skin pathogens9,10. Therefore, its use as a sanitizer in handwashes, soap, and denti frice has been investigated9,10. GSE has also been used to preserve a variety of foods, including vegetables 11,12, fruits (strawberries 6 and grapes11), fishery products13,14, pork15, chewing gum16, and Korean rice wine (Makgeolli)17. Additionally, GSE is considered environmentally safe, without toxicity to humans or animals at effective concentrations18,19. To date, few studies have described the potential of GSE as a seed-sterilizing agent. Furthermore, to the best of our knowledge, no study has evaluated the ger mination effects of GSE on Lactuca sativa, which has been reported to be sensitive to allelopathic20, toxic chemicals21 and pollutants22. Therefore, we investigated the seed sterilization effects of two commercially available GSEs on L. sativa seeds. We also evaluated the germination effects and active compounds of the two GSEs.
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were also decreased (USA product: 3.45 mm; Brazil product: 3.60 mm) after 48 h. In the 0.05% and 0.01% treatment groups (from both the USA product and Bra zil product), there were no or only slight effects on germination and seedling growth after 24 and 48 h (Table 1). The pH values in the 5%, 1%, 0.5%, 0.1%, 0.05%, and 0.01% treatment groups using the GSE from the USA were 3.71, 4.06, 4.26, 4.59, 4.79, and 5.22, res pectively, while those using the GSE from Brazil were
Results
Anti-germination Effects of GSE In the groups treated with 1% and 5% of either GSE, the germination rates of L. sativa seeds were compl etely inhibited after 24 and 48 h (Table 1). The germi nation rates in the 0.5% GSE treatment groups were significantly lower than in the control group after 24 and 48 h (USA product: 32.71% and 75.72% germina tion, respectively; Brazil product: 43.30% and 85.73% germination, respectively). In the 0.5% GSE treatment groups, the seedling lengths were 2.15 mm (USA prod uct) and 2.30 mm (Brazil product) after 48 h, demon strating significant inhibition of seedling growth. Wea ker anti-germination effects were observed in the 0.1% GSE treatment groups (from both the USA product and Brazil product) after 24 and 48 h; the seeding lengths
3.0 2.5 TBC (log CFU/g)
Seed-surface Disinfection Effects of GSE The TBC of the group (without GSE) was 1.31 × 103 colony-forming units (CFU)/g. The TBCs in groups treated with 0.05% and 0.1% GSE (USA product) were 0.0 CFU/g for all tested incubation times (1-6 h). In the group treated with 0.01% GSE (USA product), the TBCs were 1.7 × 102, 2.1 × 102, and 2.4 × 102 CFU/g after 4, 5, and 6 h, respectively. The TBCs in the group treated with 0.1% GSE (Brazil product) were 0.0 CFU/ g for all tested incubation times (1-6 h). In the group treated with 0.05% GSE (Brazil product), the TBCs were 1.6 × 102, 1.7 × 102, and 2.9 × 102 CFU/g after 4, 5, and 6 h, respectively (Figure 1).
a
a
ab
b
ab
ab
c
2.0 1.5
b
1.0
b
c c
d d
0.5 a a a a a a a 0.0 0 1 2 3 4 5 6
Incubation time (h)
Figure 1. Seed-surface disinfection effects of grapefruit seed extract (GSE) on Lactuca sativa seeds. TBC: Total bacterial count. Control (●), 0.01% GSE (USA; ○), 0.05% GSE (USA; ▼), 0.1% GSE (USA; ▽), 0.01% GSE (Brazil; ■), 0.05% GSE (Brazil; □), and 0.1% GSE (Brazil; ◆).
Table 1. Anti-germination effects of aqueous solutions of both grapefruit seed extracts (GSEs) at different concentrations. USA product GSE
pH
Brazil product
Incubation time 24 h
48 h
pH
Incubation time 24 h
48 h
5%
3.71
0.00±0.00% (0.00±0.00 mm)
0.00±0.00% (0.00±0.00 mm)
3.88
0.00±0.00% (0.00±0.00 mm)
0.00±0.00% (0.00±0.00 mm)
1%
4.06
0.00±0.00% (0.00±0.00 mm)
0.00±0.00% (0.00±0.00 mm)
4.26
0.00±0.00% (0.00±0.00 mm)
0.00±0.00% (0.00±0.00 mm)
0.5%
4.26
32.71±9.62% (1.16±0.81 mm)
75.72±12.67% (2.15±1.34 mm)
4.44
43.30±8.45% (1.46±1.12 mm)
85.73±12.52% (2.30±1.42 mm)
0.1%
4.59
85.50±5.30% (1.20±1.06 mm)
98.50±3.25% (3.45±1.07 mm)
4.73
95.00±5.20% (1.33±0.92 mm)
100.00±0.00% (3.60±1.57 mm)
0.05%
4.79
100.00±0.00% (1.15±0.34 mm)
100.00±0.00% (9.25±1.20 mm)
4.91
100.00±0.00% (1.60±0.13 mm)
100.00±0.00% (10.60±3.42 mm)
0.01%
5.22
100.00±0.00% (1.31±0.43 mm)
100.00±0.00% (10.45±2.35 mm)
5.04
100.00±0.00% (1.69±2.21 mm)
100.00±0.00% (13.30±2.71 mm)
All measurements were performed at least three times. The results are shown as the mean±standard deviation (n≥ 3). Statistical significance compared with the control was determined using Student’s t-test at p<0.01. The negative controls had a pH value of 6.13.
Seed Surface Disinfection and Germination Effect of GSE
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Table 2. Anti-germination effects of aqueous solutions of both grapefruit seed extracts (GSEs) adjusted to pH 7.0 at different GSE concentrations. USA product GSE
pH
Brazil product
Incubation time 24 h
48 h
pH
Incubation time 24 h
48 h
5%
7.0
0.00±0.00% (0.00±0.00 mm)
0.00±0.00% (0.00±0.00 mm)
7.0
0.00±0.00% (0.00±0.00 mm)
0.00±0.00% (0.00±0.00 mm)
1%
7.0
0.00±0.00% (0.00±0.00 mm)
0.00±0.00% (0.00±0.00 mm)
7.0
0.00±0.00% (0.49±0.22 mm)
0.00±0.00% (0.68±0.36 mm)
0.5%
7.0
56.64±8.69% (1.52 ±1.27 mm)
90.67±12.47% (2.20 ±1.42 mm)
7.0
63.50±10.67% (1.64±0.53 mm)
93.3±11.79% (3.30±1.42 mm)
0.1%
7.0
90.00±12.3% (1.58±0.73 mm)
100.00±0.00% (3.20±1.28 mm)
7.0
95.20±5.30% (1.34±0.65 mm)
100.00±0.00% (5.33±2.54 mm)
0.05%
7.0
100.00±0.00% (1.35±0.93 mm)
100.00±0.00% (11.40±2.42 mm)
7.0
100.00±11.5% (1.24±0.83 mm)
100.00±0.00% (12.90±2.12 mm)
0.01%
7.0
100.00±0.00% (1.51±0.35 mm)
100.00±0.00% (10.65±1.78 mm)
7.0
100.00±15.3% (1.36±0.51 mm)
100.00±0.00% (12.65±1.78 mm)
All measurements were performed at least three times. The results are shown as the mean±standard deviation (n≥ 3). Statistical significance compared with the control was determined using Student’s t-test at p<0.01.
3.88, 4.26, 4.44, 4.73, 4.91, and 5.04, respectively. The pH of the negative control (0.00% GSE) was 6.13 (Table 1).
Anti-germination Effects of GSE Adjusted to pH 7.0 To ascertain whether the acidic pH of the GSE affe cted L. sativa seed germination and seedling growth, the effects of GSE solutions adjusted to pH 7.0 were evaluated. No germination was observed in the pH-ad justed 5% and 1% treatment groups using the GSE from the USA after 24 and 48 h of incubation (Table 2). The same was true for the pH-adjusted 5% treat ment group using the GSE from Brazil, while there were significant inhibitory effects on germination and seedling growth in the 1% treatment group (seedling lengths: 0.49 and 0.68 mm after 24 and 48 h, respec tively). In the pH-adjusted 0.5% treatment groups, sig nificant anti-germination effects were also observed after 24 h (USA: 56.64% germination; Brazil: 63.50% germination), while the seeding lengths after 24 h were 1.52 mm (USA) and 1.64 mm (Brazil). Weaker antigermination effects were observed in the 0.1% GSE treatment groups (from both the USA and Brazil) after 24 and 48 h, while the seeding lengths were 3.20 mm (USA) and 5.33 mm (Brazil) after 48 h. There were no significant negative effects on germination and seed ling growth in the pH-adjusted 0.05% and 0.01% treat ment groups (from both the USA and Brazil) after 24 and 48 h (Table 2).
Anti-germination Effects of Commercial Naringin, Quercetin, and Citric Acid The germination rates of seeds treated with commer cially available naringin, quercetin, and citric acid are shown in Table 3. The germination rates of L. sativa seeds treated with naringin at concentrations of 1%, 0.5%, 0.1%, 0.05%, and 0.01% (v/v) were >97.10% after 48 h of incubation, indicating no or slight antigermination effects. The germination rates were 83.33 %, 86.52%, 90.00%, 91.34%, and 95.21% in groups treated with 1%, 0.5%, 0.1%, 0.05%, and 0.01% (v/v) quercetin, respectively, after 48 h of incubation, indi cating a weak dose-dependent anti-germination effect. No germination was observed in groups treated with 1%, 0.5%, and 0.1% citric acid after 48 h. However, in the group treated with 0.05% citric acid, the germina tion rates were 96.67% and 100.0% and the seedling lengths were 2.38 mm and 4.03 mm after incubation for 24 and 48 h, respectively, indicating an inhibitory effect on seedling growth. Additional experiments were performed using 0.01% citric acid, which showed no anti-germination effects or seedling growth inhibition after 48 h.
Discussion Almost 90% of global food crops are grown from seeds, which are widely distributed by national and international trade. However, many plant pathogens can
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Table 3. Anti-germination effects of commercially available naringin, quercetin, and citric acid at different concentrations. Concentration (%)
Compounds
1%
0.5%
0.1%
0.05%
0.01%
5.01
5.34
5.56
5.75
5.79
Incubation time 97.31±2.65% 24 h (3.93±0.97 mm)
96.00±4.10% (3.77±1.28 mm)
97.01±2.55% (3.00±1.10 mm)
98.24±1.35% (3.20±0.85 mm)
98.33±2.05% (3.86±1.3 mm)
48 h
98.24±1.63% (10.29±1.38 mm)
97.10±2.50% (11.83±1.91 mm)
97.33±1.78% (10.21±1.29 mm)
98.67±1.74% (10.14±1.21 mm)
98.52±1.55% (10.08±1.03 mm)
pH
4.97
5.08
5.62
Incubation time 83.33±10.28% 24 h (2.98±0.79 mm)
86.52±5.28% (3.04±0.86 mm)
90.00±8.30% (3.20±1.13 mm)
91.34±5.72% (3.50±0.94 mm)
95.21±4.35% (3.52±0.82 mm)
48 h
85.46±7.08% (10.17±1.38 mm)
89.05±8.23% (10.24±1.37 mm)
92.41±6.67% (11.15±1.08 mm)
93.33±5.46% (10.68±1.48 mm)
98.33±2.55% (11.88±1.36 mm)
pH
3.01
3.21
3.40
Incubation time 0.00±0.00% 24 h (0.00±0.00 mm)
0.00±0.00% (0.00±0.00 mm)
0.00±0.00% (0.00±0.00 mm)
96.67±5.31% (2.38±1.59 mm)
98.58±4.62% (4.86±2.41 mm)
0.00±0.00% (0.00±0.00 mm)
0.00±0.00% (0.00±0.00 mm)
0.00±0.00% (0.00±0.00 mm)
98.50±0.00% (4.03±0.59 mm)
99.67±1.03% (9.71±0.95 mm)
pH Naringin
Quercetin
Citric acid
48 h
5.97
4.03
5.82
4.79
All measurements were performed at least three times. The results are shown as the mean±standard deviation (n≥ 3). Statistical significance compared with the control was determined using Student’s t-test at p<0.01. The mean germination rates (seedling lengths) of the negative control after 24 and 48 h were 98.67±1.60% (3.09±0.54 mm) and 98.89±1.54% (10.34±1.16 mm), respectively.
be transmitted by seeds, which is a very efficient means of introducing plant pathogens into new areas and can result in considerable yield losses23,24. To protect against various seed-borne pathogens, seed sterilization treat ments such as scarification and heat, ozone, plasma, chemical, or pesticide treatment are commonly used1-3,5. However, more effective, less toxic, and environmen tally friendly techniques are desirable25. GSE has effective, broad-spectrum antimicrobial acti vity8-10 and is environmentally safe18,19. Therefore, GSE has also been used as a food preservative6,11,17 and sani tizer in external preparations9,10. Previous reports indi cated that GSE could be an effective, less toxic, and en vironmentally friendly seed-surface disinfection agent that causes no significant reduction in seed germina tion rates. Therefore, we evaluated the seed steriliza tion effects on L. sativa seeds of two commercially available GSEs from the USA and Brazil. Additional ly, we determined the anti-germination effects of GSE on L. sativa seeds and identified its active compounds. Lactuca sativa seeds treated with 0.05% GSE (from both the USA and Brazil) showed no or slightly nega tive effects in terms of germination and seedling growth after 24 and 48 h. To determine whether the acidity of
GSE affected germination, we also conducted a GSE experiment in which the pH was adjusted to 7.0. The pH-adjusted group treated with 0.05% GSE had slight ly improved germination and seedling growth than the non-adjusted group. Studies of putative allelochemicals and antimicrobi als are complex because GSE contains flavonoids, cit ric acid, and other compounds6,7, which may behave synergistically. In this study, we evaluated the antigermination effects of naringin, quercetin, and citric acid on L. sativa seeds. Only seeds treated with quer cetin showed anti-germination effects, which occurred in a dose-dependent manner (0.01-0.1%). Takahashi et al. (1998)26 found that quercetin (≤0.5 mM) used in germinating tissues of Glycine max inhi bited respiration with a mixture of substrates, includ ing NADH, L-malate, succinate, and L-glutamate, in either the presence or absence of exogenously added ADP. At concentrations >0.5 mM, the effects of quer cetin on basal respiration shifted towards stimulation. Respiration of uncoupled mitochondria was inhibited by quercetin, irrespective of the substrate type. Swell ing driven by phosphate uptake was also inhibited by quercetin. The action of quercetin on energy metabo
Seed Surface Disinfection and Germination Effect of GSE
lism in mitochondria might be the cause of reduced ATP levels in embryonic axes26. The germination-in hibiting effects of quercetin observed in G. max may also have been present in the L. sativa seeds in this study. Following treatment with 0.01% and 0.05% citric acid (pH 4.79 and 4.03, respectively), no and slight anti-germination effects, respectively, were observed; however, treatment with 0.1% citric acid (pH 4.79) had strong anti-germination effects. This suggests that the anti-germination effects of GSE are due to synergistic interactions among quercetin, citric acid, and other chemicals; however, further research is needed to con firm this. We found that 0.05% GSE (both from the USA and Brazil) had strong seed sterilization effects and no anti-germination effects. Using GSE is relatively inex pensive and does not require large-scale facilities for seed-surface disinfection. Additionally, GSE is regis tered in the Korea Food Additive Standards Codex27 as a natural additive (No. 115), and its safety has been assessed in several areas19. In conclusion, these results suggest that GSE can be used for seed sterilization with out inhibiting seed germination or seedling growth.
Materials and Methods Materials and Reagents The GSEs (DF-100) used in this study were pur chased from Chemie Research & Manufacturing Co., Inc. (Casselberry, FL, USA; glycerin: 30%, naringin: 1.97%) and QUINABRA-Quimica Natural Brasileira Ltda. (Eldorado, Brazil; glycerin: 50%, naringin: 0.48%). The chemicals used in this study were pur chased from Sigma-Aldrich (St. Louis, MO, USA) and were either high-performance liquid chromatographygrade reagents or of the highest available grade. All test solutions used ultrapure Milli-Q water (Milli-Q advantage A10; Millipore, Billerica, MA, USA). Lactuca sativa Seeds The L. sativa L. (Korean cultivar, Hongbitjeokchi masangchu) seeds used in this study were purchased from Danong (Namyangju, Korea). Seeds of uniform size were selected; damaged seeds were discarded. Seed-surface Disinfection Effects of GSE To evaluate the seed-surface disinfection effects of GSE, we measured the total bacterial counts (TBCs) of L. sativa seeds treated with GSE. Briefly, 1 g of L. sativa seeds were placed in a sterilized 50-mL glass laboratory bottle (DURAN Group GmbH, Mainz, Ger many) with 10 mL of sterilized, 0.22-μm-filtered GSE
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aqueous solution at one of several concentrations (0%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, and 5%) and then stored in a shaking incubator (150 rpm) (HB-201SF; Han Baek Scientific Co. Ltd., Bucheon-si, Gyeonggido, Korea) at 25°C. To determine the TBCs after 0, 1, 2, 3, 4, 5, and 6 h, an aliquot of the incubation solution (0.1 mL) was removed and three serial dilutions (with sterilized distilled water [DW]) were plated in dupli cate onto plate count agar (Difco, Detroit, MI, USA) and incubated at 37℃ for 48 h.
Germination Bioassay Germination bioassays were performed from Novem ber 2015 to September 2016. To avoid microbial con tamination before the germination tests, seeds were sur face-sterilized with a solution of 10% sodium hypo chlorite (425044; Sigma-Aldrich) for 5 min and then washed five times with DW28,29. On a clean bench, ten sterilized L. sativa seeds were evenly placed on filter paper (55 mm in diameter; Toyo Roshi Kaisha, Tokyo, Japan) in sterile Petri dishes (6 cm in diameter; SPL Life Science, Pocheon, Korea) and moistened with 1.2 mL of each test solution. The Petri dishes were then kept in an incubator (VS-1203PFC-L; Vision Scientific Co. Ltd., Daejeon, Korea) in the dark at 25±1℃. Germi nation was considered to have occurred only after the radicle had protruded by at least 1 mm28,30. Germina tion rates were determined by counting the number of germinated seeds at 24-h intervals over a 48-h period. The percentage germination following each treatment was calculated and compared to that of the control, which was treated with DW. The percentage germina tion was calculated using the following equation: Germination rate (%) = n/N × 100,
where n is the number of seeds germinated and N is the number of seeds sown23.
Determining the Anti-germination Effects of GSE To evaluate the anti-germination effects of GSE on L. sativa seeds, ten sterilized seeds were placed uni formly on filter paper in sterile Petri dishes and moist ened with 1.2 mL of a 0%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, or 5% (v/v) aqueous solution of one of the two commercial GSEs. The germination rates were deter mined as described above. Because the pH values of the GSE aqueous solutions were acidic (Table 1), they were adjusted to pH 7.0 by adding an appropriate amount of 1 M NaOH to avoid effects of acidity on seed germination. Then the above-mentioned germina tion assay was repeated using the pH-adjusted GSE solutions.
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Determining the Anti-germination Effects of Naringin, Quercetin, and Citric Acid To identify the compounds in GSE responsible for anti-germination effects on L. sativa seeds, we investi gated the anti-germination effects of commercially available naringin, quercetin, and citric acid on L. sativa seeds. Germination assays were conducted follow ing the methods mentioned above, excluding the fol lowing concentrations: 1%, 0.5%, 0.1%, 0.05%, and 0.01% (v/v).
Acknowledgements Publication of this article was co-sponsored by the ToxEHS.
Statistical Analyses All experiments were performed independently at least three times. The significance of the results was calculated using Student’s t-test. Results were consid ered statistically significant vs. the control at p<0.01.
Conflict of Interest I have no conflict of interest in the work.
References 1. Dhayal, M., Lee, S.-Y. & Park, S.-U. Using low-pres sure plasma for Chartamus tinctorium L. seed surface modification. Vacuum 80, 499-506 (2006). 2. Kim, D.-H. & Lee, J.-M. Effects of seed sterilization treatment on germination and seedling growth of bottle gourd (Lagenaria siceraria). J. Kor. Soc. Hort. Sci. 42, 131-136 (2001). 3. Kang, M. H. et al. Differential inactivation of fungal spores in water and on seeds by ozone and arc dis charge plasma. PLoS ONE 10, 1-16 (2015). 4. Barampuram, S., Allen, G. & Krasnyanski, S. Effect of various sterilization procedures on the in vitro germi nation of cotton seeds. Plant Cell Tiss. Organ Cult. 118, 179-185 (2014). 5. Hopkins, D. L., Thompson, C. M., Hilgren, J. & Lovic, B. Wet seed treatment with peroxyacetic acid for the control of bacterial fruit blotch and other seedborne dis eases of watermelon. Plant Dis. 87, 1495-1499 (2003). 6. Jang, S. A., Shin, Y. J. & Song, K. B. Effect of rape seed protein-gelatin film containing grapefruit seed extract on ‘Maehyang’ strawberry quality. Int. J. Food Sci. Technol. 46, 620-625 (2011).
7. Cvetnić, Z. & Vladimir-Knežević, S. Antimicrobial activity of grapefruit seed and pulp ethanolic extract. Acta. Pharm. 54, 243-250 (2004). 8. Krajewska Kulak, E., Lukaszuk, C. & Niczyporuk, W. Antifungal activity of 33% grapefruit-water glycerol solution. J. Eur. Acad. Dermatol. Venereol. 17, 486487 (2003). 9. Ha, Y. M. et al. Antimicrobial activity of grapefruit seed extract and processed sulfur solution against human skin pathogens. J. Life Sci. 19, 94-100 (2009). 10. Lee, B. B. et al. Antimicrobial activity of test dentifrice product containing grapefruit seed extract and processed sulfur solution against oral pathogens. J. Life Sci. 19, 956-962 (2009). 11. Xu, W. T. et al. Postharvest grapefruit seed extract and chitosan treatments of table grapes to control Botrytis cinerea. Postharvest Biol. Technol. 46, 86-94 (2007). 12. Lee, D. S., Hwang, Y. I. & Cho, S. H. Developing anti microbial packaging film for curled lettuce and soy bean sprouts. Food Sci. Biotechnol. 7, 117-121 (1998). 13. Cho, S. H., Seo, I. W., Choi, J. D. & Joo, I. S. Antimi crobial and antioxidant activity of grapefruit and seed extract on fishery products. Bull. Korean Fish. Soc. 23, 289-296 (1990). 14. Corbo, M. R. et al. Study on the synergic effect of nat ural compounds on the microbial quality decay of packed fish hamburger. Int. J. Food Microbiol. 127, 261-267 (2008). 15. Hong, Y. H., Lim, G. O. & Song, K. B. Physical prop erties of Gelidium corneum-gelatin blend films contain ing grapefruit seed extract or green tea extract and its application in the packaging of pork loins. J. Food Sci. 74, C6-10 (2009). 16. Jin, M. S. et al. Antimicrobial and anti-gingivitis effect of chewing gum containing grapefruit seed extract and xylitol. J. Korean Acad. Periodontol. 33, 485-497 (2003). 17. Choi, J.-S. et al. Antibacterial effect of grapefruit seed extract (GSE) on Makgeolli-brewing microorganisms and its application in the preservation of fresh Mak geolli. J. Food Sci. 79, M1159-M1167 (2014). 18. Ionescu, G., Kiehl, R., Wichmann-Kunz, F., Williams, C. & Baum, L. Oral citrus seed extract. J. Orthomol. Med. 5, 230-238 (1991). 19. Heggers, J. P. et al. The effectiveness of processed grapefruit-seed extract as an antibacterial agent, II. Mechanism of action and in vitro toxicity. J. Altern. Complement. Med. 8, 333-340 (2002). 20. Dandelot, S., Robles, C., Pech, N., Cazaubon, A. & Verlaque, R. Allelopathic potential of two invasive alien Ludwigia spp. Aquat. Bot. 88, 311-316 (2008). 21. Valerio, M. E., García, J. F. & Peinado, F. M. Determi nation of phytotoxicity of soluble elements in soils, based on a bioassay with lettuce (Lactuca sativa L.). Sci. Total Environ. 378, 63-66 (2007). 22. ISO. Soil qualit - Determination of the effects of pol lutants on soil flora - Screening test for emergence of lettuce seedlings (Lactuca sativa L.). ISO 17126 (2005).
Seed Surface Disinfection and Germination Effect of GSE
23. Rennie, W. J. & Cokerell, V. in The epidemiology of plant diseases (eds Cooke, B. M., Jones, D. G. & Kaye, B.) 357-372 (Springer, Netherlands, 2006). 24. Maude, R. B. in Seedborne diseases and their control: principles and practice (CAB international, Walling ford, 1996). 25. Jo, Y.-K. et al. Use of silver nanoparticles for manag ing Gibberella fujikuroi on rice seedlings. Crop Prot. 74, 65-69 (2015). 26. Takahashi, L., Sert, M. A., Kelmer-Bracht, A. M., Bracht, A. & Ishii-Iwamoto, E. L. Effects of rutin and quercetin on mitochondrial metabolism and on ATP levels in germinating tissues of Glycine max. Plant
175
Physiol. Biochem. 36, 495-501 (1998). 27. KFDA. Korea Food and Drug Administration. in Food additive standards codex (Seoul, Korea, 2012). 28. Choi, J.-S. & Choi, I. S. Inhibitory effect of marine green algal extracts on the germination of Lactuca sativa seeds. J. Environ. Biol. 37, 207-213 (2016). 29. Möller, M. & Smith, M. L. The significance of the min eral component of seaweed suspensions on lettuce (Lactuca sativa L.) Seedling Growth. J. Plant Physiol. 153, 658-663 (1998). 30. Gallardo, K. Proteomics of Arabidopsis seed germina tion. A comparative study of wild-type and gibberellindeficient seeds. Plant Physiol. 129, 823-837 (2002).