滲調添加殺菌劑對甜椒苗立枯病與猝倒病防治之影響

種苗改良繁殖場 黃玉梅、鍾文全、陳國雄

      供試甜椒種子’天王星’、’加州王’兩品種，在滲調過程中無論添加或未添加殺菌劑處理，均可促進種子於15℃、35℃生長箱內之發芽率及發芽速率，且育苗網室之幼苗出土率亦顯著高於對照組，同時能有效防治苗立枯病(Rhizoctonia solani)與苗猝倒病(Pythium aphanidermatum)。六種防治立枯病及苗猝倒病的殺菌劑中，唯撲克拉會影響種子的發芽表現，不僅發芽率下降，發芽所需時間亦延長。滲調中添加待克利與滅達樂可抑制甜椒兩品系罹患苗立枯病及苗猝倒病之比率達77.6-86.2％與100％。將殺菌劑(賓克隆＋氫氧化銅) 複合添加於滲調液處理，對甜椒育苗期間同時控制立枯病及苗猝倒病仍然有效。試驗結果顯示：滲調添加殺菌劑不但可防治苗期病害且不影響滲調克服高溫發芽的效益，在商業種子處理應用上頗具潛力。

Combined effects of priming and fungicides on control of damping-off of sweet pepper

Seed Improvement and Propagation, Hsinshe, Taichung 42642, Taiwan

Yu-Mei Huang, Wen-Chuan Chung, and Kuo-Hsiung Chen


Abstract

This study was conducted to determine effect of seed treatment with the primer PEG 6000 with or without fungicides on seed germination in the laboratory at 15 ℃ or 35℃ as well as incidence of damping-off caused by Rhizoctonia solani or Pythium aphanidermatum and seedling growth of sweet pepper in the greenhouse, using two cultivars, Uranus and California Wonder. Seeds treated with PEG 6000 alone or PEG 6000 combined with fungicide significantly increased germination rate and germination speed at 15 ℃ and 35 ℃. Among the six fungicides tested except for Prochloraz fungicide for control of damping-off incidence, addition of fungicides to PEG 6000 solution did not significantly reduce the final germination percentage and germination speed compared with PEG 6000 primed alone treatment. PEG 6000+ Difenoconazole or PEG 6000+ Metalaxy primed seed treatment was more effective than the PEG 6000 primed seed alone in significantly decreasing Rhizoctonia or Pythium damping-off incidences by 77.6-86.2% and 100% in two cultivars, respectively. Effective control of damping-off of sweet pepper by the seed priming with PEG 6000 combined with Pencycuron and cupric hydroxide fungicides was also obtained in greenhouse. This study suggests that priming in combination with fungicides has potential for use in the commercial seed treatment.

Key words: sweet pepper, PEG 6000, fungicides, Rhizoctonia solani, Pythium aphanidermatum

Introduction

Sweet peppers (Capsicum annuum L.) are sensitive to cool, humid weather but tolerant to warm temperatures. This cool, humid condition is a strong hindrance to the cultivation of sweet pepper at nursery stage in the greenhouse as it causes poor emergence and ununiform stand due to soilborne pathogens such as Rhizoctonia solani Kühn AG-4 and Pythium aphanidermatum (Edson) Fitzpatrick, a major factor limiting commercial greenhouse production of seedlings of this crop grown in plugs in Taiwan (Chiu, 1996). Therefore, alternative control measures are needed for managing damping-off of vegetable crops grown in plugs.

Seed priming has been reported to accelerate germination, improve and enhance uniformity of seedling emergence of many crops, and raise the upper temperature, especially under unfavorable growing conditions. The technique consists of imbibing seeds in an osmotic solution that allows pregerminative metabolism to proceed, but prevents radicle protrusion through the seed coat (Parera and Cantliffe, 1994; Heydecker et al., 1975). Because of the environmental conditions developed during priming, several studies (Finch-Savage et al., 1991; Parera and Cantliffe, 1994) show that the ability of applied fungicide to establish on seeds during different priming or pregermination processes. For example, iprodione or thiram were applied directly to carrot seeds by a chemo-priming method for reducing infection of Alternaria dauci and improving emergence and yield in the field (Maude et al., 1992).

Zhao et al. (2004) reported that combining fungicide treatments with polyethylene glycol (PEG 8000) primary caused a significant decline in the percentage of seeds infected with fungi compared to PEG priming alone. However, very little information is available on the health status of sweet pepper seeds after seed priming and fungicide treatments.
The objectives of this study were to determine effects of seed treatment with PEG 6000 combined with or without fungicides on seed germination in the laboratory at 15 ℃ or 35 ℃ as well as incidence of damping-off caused by R. solani or P. aphanidermatum and seedling growth of sweet pepper in the greenhouse, using two cultivars, Uranus and California Wonder.

Materials and Methods

Source of seeds of sweet pepper, primer and fungicides
Seeds of sweet pepper, cultivars Uranus (F1, hybrid) and California Wonder, from a commercial seed company (Know-You Seed Co., Taiwan) were used in this study. Polyethylene glycol (PEG 6000, Fluka, Switzerland) was used as primer. Six fungicides, Difenoconazole (24.9% a.i., EC, Ciba-Geigy Co., Ltd, USA), Pencycuron (23.2% a.i., SC, Bayer Co., Ltd, ), Prochloraz, (25% a.i., EC, Schering Co., Ltd, Germany), cupric hydroxide (75% a.i., WP, BASF Co., Ltd, Germany), Metalaxy (58% a.i., WP, Ciba-Geigy Co., Ltd, USA), and Etridiazole (35% a.i., EC, Great Victory Co., Ltd., Japan), were purchased from Taiwan.

Infestation of culture media with Rhizoctonia solani AG-4 Rhizoctonia solani AG-4, isolate RS-001, was isolated from a sweet pepper seedling showing damping-off symptoms in a field near Taichung, Taiwan. Agar blocks (0.5 cm in diameter) containing mycelial mats of R. solani were removed from 3-day-old cultures grown on Potato Dextrose Agar (PDA, Difco, Detroit) at 28 ℃. One block was transferred into each 250 ml flask containing 50 g of chopped and autoclaved potatoes. After incubation at 28 ℃ for 7 days, the potatoes colonized by mycelia of R. solani were added to the autoclaved BAS VAN BUUREN (BVB) No.4 medium (Visser Co., Netherlands) at a rate of 1:10 (w/w). The moisture content of the medium was adjusted to 50% (v/v) using sterile distilled water. The holes of plastic flats (45 x 35 x 15 cm; L x W x H) (SunKuan Co., Taiwan) were filled with the R. solani-infested medium. The flats were kept at room temperature for seeding. Plastic flats filled with autoclaved BVB No.4 medium without R. solani were used as control.

Production of zoospores of Pythium aphanidermatum
Pythium aphanidermatum, isolate PA-002, was isolated from a pepper seedling showing damping-off symptoms in a greenhouse near Taichung, Taiwan. Agar discs (1 cm in diameter) containing mycelial mats of P. aphanidermatum PA-02 were were removed from 2-day-old cultures grown at 30 ℃ on V8 juice agar in Petri dishes. The colony in each dish was cut into strips, 10 mm in width, and transferred into an empty Petri dish. Each dish was flooded with 20 mL of sterile distilled water. After 1 hour, the water was removed and replaced with the same amount of sterile distilled water. This process could leach out some nutrients or metabolites in the colony. The dishes were incubated at 30 ℃ under light for 24 h, then at 20 ℃ for 4 h to stimulate zoospore release (Paulitz et al., 1992). Motile zoospores were collected on a 0.45 µm filter and adjusted to the concentration of 1 X 104 zoospores mL-1 using sterile distilled water

Seed germination assay
Seeds of sweet pepper, cultivars Uranus and California Wonder, were surface sterilized in 1% sodium hypochlorite for 15 min, rinsed with sterile distilled water for 5 min, and air-dried for 10 min at room temperature. For the combined treatment of priming and fungicide, 12 g of seeds were soaked for 3 days at 20 ℃ in dark in aerated solutions of PEG 6000 at -0.5 MPa containing one of the following recommended fungicides with commercial rates: 0.125% (w/v) cupric hydroxide, 0.25% (w/v) Metalaxy, 0.033% (v/v) Etridiazole, 0.033% (v/v) Difenoconazole, 0.1% (v/v) Pencycuron, and 0.033% (v/v) Prochloraz. The treated seeds were washed under running tap water for 5 min, rinsed 3 times in sterile distilled water to remove excessive PEG and fungicides, blot-dried on filter paper (Whatman No.3), and tested for germination by incubation in dark at 15 ℃ and 35 ℃. Fifty seeds were placed on double layered, moistened filter paper (Whatman No.4) in each Petri dish (9 cm in diameter). The treatments of PEG alone and untreated seeds were used as controls. There were four dishes (replicates) for each treatment. A seed was considered germinated when the radicle tip protruded clearly from the seed coat. The germinated seeds were recorded daily for 7 days and the mean of time to germination in each treatment was calculated by the equation: GT50 = ΣTiNi/ΣNi ; where Ni is the number of newly germinated seeds at time Ti (Bradford, 1986). The experiment was repeated 3 times.

Effects of seed treatment with combined primer and fungicide on seedling emergence and damping-off of sweet pepper caused by Rhizoctonia solani Seeds of sweet pepper, cltivars Uranus and California Wonder, were treated with PEG primer containing one of the fungicides, Difenoconazole, Pencycuron, and Prochloraz, by the method described above. The treated and untreated seeds were sown in BVB No.4 media artificially infested with or without 1% (v/v) R. solani in plug trays (60 x 40 x 4.8 cm, Visser Co., Netherlands) containing 128 cells (2.5 x 2.5 x 5 cm3/cell) per tray, one seed per cell. The treatments of PEG alone and untreated seeds were used as controls. There were four replicates (plug trays) for each treatment. The trays were kept in a greenhouse (28-32 ℃) and plants were watered as required. Seedling emergence and incidence of damping-off were recorded daily for a period of 30 days. The mean rate of seedling emergence for each replicate for each treatment was calculated by the equation: ET50=ΣTiNi/ΣNi; where Ni is the number of newly seedling emerged at time Ti (Kotowski, 1926). To verify infection of seeds of plants by R. solani AG-4, unemerged seeds or seedlings showing symptoms of damping-off were collected, surface sterilized by NaOCl for 1 min, and assayed on 2% water agar plates supplemented with 200 ppm streptomycin sulfate (Sigma Co., USA) and 300 ppm tannic acid (Sigma Co., USA) using the method described by Hsieh et al. (1996). The experiment was repeated 3 times.

Effects of seed treatment with combined primer and fungicide on seedling emergence and damping-off of sweet pepper caused by Pythium aphanidermatum
Seeds of sweet pepper, cltivars Uranus and California Wonder, were treated with PEG primer containing one of the fungicides, cupric hydroxide, Metalaxy, and Etridiazole, by the method described above. The treated and untreated seeds were sown in BVB No.4 media artificially infested with or without 1 x 103 zoospores/mL of P. aphanidermatum in plug trays (60 x 40 x 4.8 cm, Visser Co., Netherlands) containing 128 cells (2.5 x 2.5 x 5 cm3/cell) per tray, one seed per cell. The treatments of PEG alone and untreated seeds were used as controls. There were four replicates (plug trays) in each treatment. The trays were kept in a greenhouse (28-32 ℃) and plants were watered as required. Seedling emergence and incidence of damping-off plants were recorded daily for a period of 30 days. The mean rate of emergence for each replicate for each treatment was calculated by the equation: ET50=ΣTiNi/ΣNi ; where Ni is the number of newly seedling emerged at time Ti. To verify infection of seeds or plants by P. aphanidermatum, unemerged seeds or seedlings showing symptoms of damping-off were collected and assayed on a selective agar plates using the method described by Stanghellini and Kronland (1985). The experiment was repeated 3 times.

Effect of seed primed with two fungicides on seedling emergence and disease incidence by Pythium aphanidermatum and Rhizoctonia solani Seeds of sweet pepper, cv. Uranus were treated with PEG primer containing two fungicides, cupric hydroxide + Difenoconazole, Metalaxy + Difenoconazole, cupric hydroxide + Pencycuron, and Pencycuron + Metalaxy, by the method described above. The treated and untreated seeds were sown in BVB No.4 media artificially infested with or without 1 x 103 zoospores/mL and 1% R. solaniin plug trays (60 x 40 x 4.8 cm, Visser Co., Netherlands) containing 128 cells (2.5 x 2.5 x 5 cm3/cell) per tray, one seed per cell. The treatments of PEG alone and untreated seeds were used as controls. There were four replicates (plug trays) in each treatment. The trays were kept in a greenhouse (28-32 ℃) and plants were watered as required. Seedling emergence and incidence of damping-off plants were recorded daily for a period of 30 days. The mean rate of emergence for each replicate for each treatment was calculated by the equation: ET50=ΣTiNi/ΣNi ; where Ni is the number of newly seedling emerged at time Ti. Unemerged seeds or seedlings showing symptoms of damping-off were collected and assayed by the method described above to verify infection of seeds or plants by P. aphanidermatum or R. solani AG-4. The experiment was repeated 3 times.

Statistical analysis
Data of final germination percentage (FGP), time to 50% of final germination (GT50), seedling emergence percentage, time to 50% of final emergence percentage (ET50) and inhibition of damping-off were analyzed using analysis of variance (ANOVA) for a complete randomization design. Data from repeats of the same experiment were pooled when the variances were homogenous. Means of the treatments in each experiment were compared using Duncan’s multiple range test (DMRT) and Student’s t-test at P = 0.05 level. All statistical analyses were conducted using SAS/STAT® software (SAS Institute Inc., Cary, N. C., 1997).

Results
 

Seed germination assay
Seeds of sweet pepper, cultivars Uranus and California Wonder, treated with PEG6000 solution alone, PEG 6000 + Difenoconazole or PEG 6000 + Pencycuron and incubated at 15 and 35 ℃ significantly increased final percentage of germination (FGP), compared to untreated seeds (Table 1). For example, FGP of cv. Uranus at 15 C was 100, 99.5, 98,0% for the treatments of PEG 6000 alone, PEG 6000 + Difenoconazole and PEG 6000 + Pencycuron, respectively, compared to 70.5% for the untreated control. However, seeds of cvs. Uranus and California Wonder, treated with PEG 6000 + Prochloraz and incubated at 15 and 35 ℃ significantly reduced FGP, compared to untreated seeds (Table 1). For example, addition of Difenoconazole and Pencycuron, respectively, into the priming solution resulted in no significant difference compared with PEG 6000 primed seeds, whereas significant reduction of FGP of PEG 6000 + Prochloraz primed seeds were observed when two cultivars seeds were assayed at 15 and 35 ℃, ranging between 38.5-67.5% and 31.5-9.5% at any of two cultivars. The GT50 of Uranus seeds only primed in PEG 6000+Pencycuron solution were no significant difference compared with PEG 6000-treated seeds at any of two temperatures. Significant reduction of the GT50 of PEG 6000+Pencycuron seeds of California Wonder was obtained at 15 and 35 ℃, ranging from 2.54 d and 2.24 d, respectively (Table 1).
There were no significant differences in FGP between PEG 6000 primed seeds and PEG 6000+fungicide primed seeds of Uranus at any of two temperatures (Table 2). The FGP of California Wonder primed in PEG 6000+cupric hydroxide were increased ranging between 92.5 and 81.0%, respectively, at 15 and 35 ℃. The GT50 of Uranus seeds primed in PEG 6000+Cupric hydroxide or PEG 6000+Apron solution were no difference compared with PEG 6000 primed seeds at any of two temperatures, whereas GT50 of seeds primed in PEG 6000+Etridiazole solution varied with different temperatures. The reduction of GT50 of California Wonder seeds primed in PEG 6000+cupric hydroxide were faster than those of PEG 6000+other fungicide, especially incubation at 35 ℃ (Table 2).

Effect of seed primed with single fungicide on sweet pepper seedling emergence and disease incidence caused by Pythium aphanidermatum or Rhizoctonia solani
Seedling of primed with PEG 6000 or primed with PEG 6000 and fungicides seeds in both cultivars emerged more uniformly in greenhouse. There were no differences in the final emergence percentage between PEG 6000 primed seeds alone and PEG 6000+fungicide primed seeds at two cultivars. The ET50 of Uranus seeds primed in PEG 6000+Pencycuron was no significant difference compared with PEG 6000 primed seed treatment alone, whereas significant reduction of the ET50 of PEG 6000+Pencycuron seeds of California Wonder was obtained (Table 3). Damping-off by R. solani AG-4 was significantly inhibited by 77.6-86.2% with PEG 6000+Difenoconazole primed seed treatments at Califonia Wonder and Uranus, respectively, compared with PEG 6000 primed seed alone when planted in artificially infested medium (Table 3).

The final emergence percentage of seeds of Uranus primed in PEG 6000+cupric hydroxide was increased compared with PEG 6000 primed seed treatment alone, whereas no significant difference of the final emergence percentage of PEG 6000+cupric hydroxide seeds of California Wonder was obtained (Table 4). There were no differences in the ET50 between PEG 6000 primed seeds and PEG 6000+fungicide primed seeds at two cultivars. Addition of Metalaxy to PEG 6000 solution primed seeds resulted in 100% inhibition of damping-off by P. aphanidermatum at Califonia Wonder and Uranus, respectively, compared with PEG 6000 primed seeds when planted in artificially infested medium (Table 4).

Effect of seed primed with two fungicides on seedling emergence and disease incidence by Pythium aphanidermatum and Rhizoctonia solani
The final emergence percentage of seeds of Uranus primed in PEG 6000+Difenoconazole+cupric hydroxide, PEG 6000+Difenoconazole+ Metalaxy and PEG 6000+Pencycuron+ Metalaxy, respectively, was no significant differences compared with PEG 6000 primed seed treatment alone, whereas treatment of seeds primed in PEG 6000 solution with cupric hydroxide and Pencycuron did significantly affect seed emergence percentage. The addition of Pencycuron and cupric hydroxide to PEG6000 solution primed seeds resulted in a significant inhibition of damping-off compared with PEG 6000 primed seeds alone (Fig. 1).
 

Discussion

Several studies reported that the effect of priming on low temperature performance of pepper seeds has ranged from no improvement to some advancement in germination percentage and rate (Yaklich and Orzolek, 1977; O’Sullivan and Bouw, 1984; Bradford et al., 1990; Khan et al., 1992). The results of this study indicated that PEG 6000 priming treatment improved the effect of high and low temperatures inducement and increased germination speed. The similar effects were observed in single or two fungicides treatments combined with PEG 6000. The study also reveals PEG 6000 solution combined with fungicide application also resulted in a significant decreased in thermodormancy and had no negative effect on seed germination compared to untreated seeds.

Beneficial effects of seed priming have been reported for pepper by Bradford et al. (1990), O’Sullivan and Bouw (1984), and Rivas et al. (1984). However, most of pepper seed priming studies have focused on improving germination and emergence of peppers while little information is available on enhancing the performance of sweet pepper seeds under low and high temperatures stress. This study revealed that the germination rate and percentage germination of PEG 6000 osmoprimed seed treatments was significantly improved under a high or low temperature compared to nontreated seeds (Table 1 and Table 2). Similar results were reported by Lin et al. (1999).

Fungicides that provide protection against vegetable seedling disease have been developed. They protect each seed or seedlings by providing a zone of protection in which pathogenic activity is inhibited (Bell and Owen, 1963). The size of the protective zone, as influenced by chemical formulation, application technique, or placement within the soil, seems to influence efficacy. Maude et al. (1992) reported that Iprodione or thiram added in PEG solution reduced infection of Alternaria dauci in carrot seeds, improved the emergence and yield of carrots in the field. Nascimento and West (1997) indicated that captan added in PEG solution eliminated species of Alternaria, Cladosporium, Epicoccum and Stemphylium on unprimed seeds of cantaloupe (Cucumis melo L.). In the study reported herein, Pythium aphanidermatum or R. solani damping-off of sweet peppers was markedly reduced when PEG 6000 osmoprimed seed were used, and protection of nearly two varieties was greater with priming than without. When combined, most of the osmoprimed and fungicides except for Prochloraz primed seed treatment were complementary and resulted in greater reduction of disease, in addition to its effect on seed germination rate (Table 3 and Table 4).

This study indicated that a combined osmoprimed-fungicide seed treatment has potential to improve disease control over a broader range of environmental conditions than currently possible with fungicide seed treatment alone. Although our lack of the mechanisms involved, several studies (Rush, 1991; Osburn and Schroth, 1988 and 1989) revealed that sugar beet seed osmoprimed with PEG 8000 germinated faster and control of preemergence damping-off of osmoprimed seed related to reduction of the rate and incidence of seed colonization by P. ultimum. This in turn was related to reduce exudation of nutrients from the primed seeds upon imbibition of water. Ananother possible explanation for reduced damping-off in primed treatments is that, due to the increased emergence rate and, seedlings may have been able to escape lethal infection. Therefore, sweet pepper seed priming with PEG 6000 and fungicide is a simple process, economically feasible, and environmentally sound. Additionally, the relative newness of the technique and the many questions concerning its mode of action and variability warrant continued research.

Acknowledgements

This study was funded by the research grant No. 93AS-1.1.5-SS-X1 of the Council of Agriculture, Executive Yuan, Taiwan, R.O.C..

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