What is the yucca extract ？
Abstract
Treatment of seeds with an aqueous extract of yucca (YE), Yucca schidigera, was evaluated for antifungal activity against seedborne pathogens as well as its effect on seed germination and seedling growth of sorghum (Sorghum bicolor). The antifungal effect of YE was observed against Leptosphaeria sacchari (syn. Phoma sorghina) when the extract was applied at 2·5 and 10% concentrations. At 10% concentration, YE significantly reduced not only the incidence of L. sacchari, but also that of Fusarium spp., Cochliobolus lunatus (syn. Curvularia lunata) and Cladosporium spp. The effect of 10% YE on seedborne fungi was broader than the fungicide fludioxonil, particularly with regard to Fusarium. Furthermore, the number of normal, healthy‐looking seedlings increased in a dose‐responsive manner with YE treatment. Seedling vigour was also stimulated by YE but no correlation was observed with the concentrations tested. Under glasshouse conditions, the treatment of seeds with 10% YE increased the emergence of seedlings and plant height and reduced the number of seedlings with crown rot compared to negative controls and saponin. The positive effect was similar to the effect obtained with fungicide‐treated seeds. Treatment of seeds with synthetic saponin inhibited seedborne fungi less effectively and also negatively affected germination and vigour of the seedlings, compared to the treatment with YE. The results demonstrate an agronomic potential for the use of YE as a biofungicide for seed treatment of sorghum. The difference between the antifungal and the vigour‐stimulating effects of YE warrants further
Treatment of seeds with an aqueous extract of yucca (YE), Yucca schidigera, was evaluated for antifungal activity against seedborne pathogens as well as its effect on seed germination and seedling growth of sorghum (Sorghum bicolor). The antifungal effect of YE was observed against Leptosphaeria sacchari (syn. Phoma sorghina) when the extract was applied at 2·5 and 10% concentrations. At 10% concentration, YE significantly reduced not only the incidence of L. sacchari, but also that of Fusarium spp., Cochliobolus lunatus (syn. Curvularia lunata) and Cladosporium spp. The effect of 10% YE on seedborne fungi was broader than the fungicide fludioxonil, particularly with regard to Fusarium. Furthermore, the number of normal, healthy‐looking seedlings increased in a dose‐responsive manner with YE treatment. Seedling vigour was also stimulated by YE but no correlation was observed with the concentrations tested. Under glasshouse conditions, the treatment of seeds with 10% YE increased the emergence of seedlings and plant height and reduced the number of seedlings with crown rot compared to negative controls and saponin. The positive effect was similar to the effect obtained with fungicide‐treated seeds. Treatment of seeds with synthetic saponin inhibited seedborne fungi less effectively and also negatively affected germination and vigour of the seedlings, compared to the treatment with YE. The results demonstrate an agronomic potential for the use of YE as a biofungicide for seed treatment of sorghum. The difference between the antifungal and the vigour‐stimulating effects of YE warrants further
investigation.
Materials and methods
Seed samples
Naturally infected farm‐saved seeds of sorghum (Sorghum bicolor) used in the bioassays originated from Burkina Faso with accession numbers 48791, 48795 and 48789.
Preparation of yucca extract (YE)
A commercial liquid extract (Norponin 200 Liquid, Nor‐Natur ApS), obtained from trunks of Yucca schidigera, was used in the seed treatments. The extract was diluted in distilled water to obtain working solutions at concentrations of 0·6%, 2·5% and 10% on a v/v basis and then autoclaved at 120°C for 20 min.
Seed treatment
Approximately 12 g of naturally infected seeds of sorghum per treatment were immersed in 20 mL of 0·6%, 2·5% and 10% YE overnight at 22 ± 2°C on a rotary shaker that was set to 100 rpm. Non‐treated seeds and seeds immersed in 20 mL sterile water were incubated in a similar manner as the YE‐treated seeds and comprised the negative controls. Seeds treated with the fungicide fludioxonil (Syngenta), applied according to the label (200 mL per 100 kg seed), served as the positive control (fungicide‐treated seeds without water immersion). To expose the fungicide‐treated seeds to similar conditions as the YE‐treated seeds, another positive control was included in the experiments, when approx. 12 g of the seeds treated with fungicide were additionally immersed in 20 mL of sterile water (fungicide‐treated seeds with water immersion) and incubated under the same conditions as YE‐treated seeds. All treated seeds were air dried in a flow cabinet at 22 ± 2°C for approximately 2–4 h before use.
Evaluation of YE antifungal effect in blotter tests
A standard blotter test method described by Mathur & Kongsdal (2003) was employed to determine the antifungal effect of the different treatments on sorghum seeds. To determine the presence of fungal spores, 200 seeds (four replicates of 50 seeds; 25 seeds per Petri dish) from each of the treatments (YE‐treated, negative and positive controls) were plated on moistened filter paper (AGF 752‐85mm, Frisenette) in Petri dishes (9 cm) and incubated at alternating cycles of 12 h of near‐ultraviolet light and 12 h of darkness at 22 ± 2°C. While the negative control consisted of no treatment and seeds treated with water, the positive control constituted fungicide‐treated seeds with and without water immersion. After 7 days, the seeds were individually examined for the presence of fungal infections using stereo (magnification up to ×50) and compound binocular (magnification up to ×400) microscopes and their incidence recorded. The identification of fungi on seeds was based on the growth habit characteristics and morphology of conidia and conidiophores.
From the blotter tests, the percentage of sorghum seeds found naturally infected with major seedborne fungi of sorghum [i.e. Leptosphaeria sacchari (syn. Phoma sorghina), Fusarium spp. (mainly Fusarium spp. within the Gibberella fujikuroi species complex), Cochliobolus lunatus (syn. Curvularia lunata) and Cladosporium spp. (identified at genus level only)] was determined.
Evaluation of YE antifungal effect in agar test
Fifty microlitres of seed washings obtained from the immersion of the seeds in the different treatments were plated on potato dextrose agar (PDA) supplemented with chloramphenicol (500 mg of chloramphenicol per litre of PDA). The plates (four replicates per treatment) were incubated as described for the blotter tests. Five days after incubation, the antifungal effect of the different treatments was assessed by counting the total number of fungal colony forming units found in each plate. Seeds treated with water and fungicide‐treated seeds with subsequent water immersion comprised negative and positive controls, respectively.
Germination and seedling vigour tests
Two hundred seeds per treatment were germinated between two layers of filter paper as described by Mathur et al. (2003). While negative controls constituted no treatment or seeds treated with water, positive controls comprised fungicide‐treated seeds with or without water immersion. Briefly, four replicates of 50 seeds were placed on a water‐soaked filter paper (24 × 47·5 cm) and loosely covered with another layer of water‐soaked filter paper. Subsequently, the filter paper was rolled, tied with a rubber band at each end, placed in a polythene bag and incubated for 10 days in a growth chamber at 24°C. Evaluation of germination was done in accordance with the International Rules for Seed Testing by counting the number of normal (show potential for continued development into satisfactory plant under favourable conditions) and abnormal (do not show potential for continued development into satisfactory plant under favourable conditions) seedlings and dead seeds (Mathur et al., 2003). After evaluation of germination, the seedling vigour of 40 plants (four replicates of 10 plants) was determined using the equation:
image
where Ls and Lr correspond to shoot and root length in millimetres, respectively, and G relates to the number of germinated seeds as a percentage (Sparg et al., 2005).
Validation assays and comparison with pure saponin
To validate the results obtained with the seed treatment with the best performing dosage of yucca on the control of seedborne fungi, seed germination and seedling growth of sorghum, naturally infected seeds were treated as described previously with 10% YE. Experimental set up and evaluation of fungal infection, seed germination, and plant vigour were carried out as described previously in the blotter, agar and between paper methods. In order to verify if the antifungal activity of YE could be comparable with the antifungal activity of synthetic saponin (as saponin is expected to be the main active antifungal compound found in yucca extract), a saponin seed treatment was included in the experiments. The saponin suspension was prepared with 0·6 g of saponin (product number 84510, Sigma) dissolved in 20 mL sterile distilled water to obtain a 3% concentration of saponin. This saponin concentration was the equivalent amount of saponins found in 10% YE. Control treatments were no treatment, seeds immersed in water, and seeds treated with fungicide with and without water immersion.
To evaluate the effect of the different treatments on sorghum seedlings growing under glasshouse conditions, 96 seeds per treatment were sown (12 × 8) in plastic pots (13 × 14 cm) containing 500 g of soil (Lotus potting soil). The pots were placed in trays covered with plastic and incubated for 1 week in a growth chamber with cycles of 12 h light and 12 h darkness. Light was supplied by fluorescent tubes (Osram L36W/11 – 860 Lumilux plus Eco Daylight, Osram GmbH, 200 μE m−2 s−1). The growth chambers were kept at 25–27°C while the relative humidity (RH) ranged from 50–60%. All pots were watered every third day with a suspension containing liquid fertilizer (20 mL of Hornum Næring, Brøste A/S, per litre of water). While the number of emerged seedlings was assessed based on the presence of hypocotyl above ground 15 days after sowing, plant height, crown rot incidence and dry weight were determined 4 weeks after sowing. The mean height of 12 × 8 seedlings per treatment was determined by measuring the length of the stem from the soil surface to the terminal node of the last developing leaf. Crown rot incidence was assessed by counting the number of seedlings showing brown lesions around the lowest stem part (sometimes covered with mycelium growth) and dividing it by the total number of emerged seedlings per treatment. To determine the dry weight, sorghum seedlings were wrapped in aluminium foil and placed in a drying oven at 80°C. Twenty‐four to 48 h later, the dried seedlings were allowed to cool to room temperature and then the weight was determined using a Sartorius TE2101 balance.
Data analysis
All experiments were repeated at least three times. Two‐ way analysis of variance (GLM) was performed on all data and least significant differences (P < 0·05) were obtained using the Student–Newman–Keuls multiple comparison test using sas
software (Statistical Analysis System, version 9·2 for Windows, SAS Institute Inc.).
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