J Gen Plant Pathol (2010) 76:102–111 DOI 10.1007/s10327-010-0225-6
FUNGAL DISEASES
Antifungal activities of hyoscyamine and scopolamine against two major rice pathogens: Magnaporthe oryzae and Rhizoctonia solani Fatma F. Abdel-Motaal • Soad A. El-zayat • Yuki Kosaka • Magdi A. El-Sayed • Rumi Kashima • Yukie Maeda • Mortada S. M. Nassar • Shin-ichi Ito
Received: 15 June 2009 / Accepted: 14 December 2009 / Published online: 25 February 2010 The Phytopathological Society of Japan and Springer 2010
Abstract The antifungal activities of hyoscyamine and scopolamine, major alkaloids extracted from the desert plant Hyoscyamus muticus, against two rice pathogens, Magnaporthe oryzae and Rhizoctonia solani, were studied. The minimum inhibitory concentration of hyoscyamine that resulted in distinctive inhibition (MIC50) was 1 lg/ml for both fungi. Exposure to hyoscyamine caused the leakage of electrolytes from the mycelia of both fungi. Hyoscyamine ([1 lg/ml) irreversibly delayed or inhibited conidial germination and appressorium formation in M. oryzae grown on polystyrene plates. Hyoscyamine effectively inhibited the attachment of conidia to the surface of rice (Oryza sativa) leaves and inhibited appressorium formation on the leaves. A high concentration of scopolamine (1000 lg/ml) also delayed or inhibited conidial germination in M. oryzae, but conidial germination was restored after washing the conidia with water. Antifungal activity of hyoscyamine was reduced by scopolamine. Magnaporthe oryzae infection was significantly suppressed (by[95%) in leaves of intact rice plants treated with hyoscyamine (10 lg/ml). Moreover, 10 lg hyoscyamine/ml significantly reduced the disease severity index for sheath blight to B0.2, when compared with the disease index of control plants ([7.0). Hyoscyamine ([20 lg/ml) completely inhibited sclerotial germination and development of R. solani by delaying the initiation,
F. F. Abdel-Motaal Y. Kosaka M. A. El-Sayed R. Kashima Y. Maeda S. Ito (&) Department of Biological and Environmental Sciences, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Yamaguchi 753-8515, Japan e-mail:
[email protected] S. A. El-zayat M. S. M. Nassar Department of Botany, Faculty of Science, South Valley University, Aswan 81528, Egypt
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maturation, and melanization of the sclerotia. These results suggest that tropane alkaloids may be useful for controlling blast and sheath blight diseases of rice and for studying the mechanisms that regulate conidial germination in M. oryzae and sclerotial germination and development in R. solani. Keywords Magnaporthe oryzae Rhizoctonia solani Hyoscyamine Scopolamine Alkaloid Sclerotium
Introduction Rice (Oryza sativa) forms an important part of the diet of many people worldwide. Losses in rice yield caused by diseases are one of the major constraints in rice production. Magnaporthe oryzae (a teleomorph of Pyricularia oryzae) is a causal agent of rice blast and, globally, is one of the most destructive rice diseases (Ou 1985). The infection cycle of this fungus begins when an asexual spore (a conidium) attaches itself to the surface of a rice leaf. Hydration of the conidium results in the release of adhesive mucilage from the spore tip; this mucilage has great affinity for the highly hydrophobic leaf surface (Hamer et al. 1988). Once the conidium is attached, it germinates to form a germ tube, which differentiates at the tip to produce an infective structure called the appressorium (Howard et al. 1991). The first sign of infection is the appearance of small ellipsoid lesions that become apparent 96 h after the initial infection (Peng and Shishiyama 1988; Valent et al. 1991). Another important fungal disease affecting rice is sheath blight, caused by Rhizoctonia solani (teleomorph is Thanatephorus cucumeris). The management of this disease is very difficult because the dispersal, propagation, and longterm survival of this pathogen is mediated through the sclerotium, a pigmented structure with many hyphae,
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which can remain quiescent for long periods under conditions that are unfavorable for vegetative growth (Vidhyasekaran et al. 1997). The sclerotium plays a central role in the life cycle and infection cycle of sclerotiumforming fungi (Chet and Henis 1975; Willetts and Wong 1980; Willetts and Bullock 1992). Sclerotial development occurs in three distinct stages: initiation, growth to full size, and maturation and melanization (Townsend 1957). Several broad-spectrum fungicides have been used to control rice blast and sheath blight. However, increasing concern over the environmental effects of these fungicides warrants the search for alternative methods of crop protection or selective control of pathogens (Lee 2005). In addition, in a number of studies, M. oryzae was resistant to certain fungicides (Katagiri et al. 1980; Miyagi et al. 1983; Takagaki et al. 2004). Sheath blight caused by R. solani is also difficult to control because completely resistant rice cultivars are not yet available (Li et al. 1995), and the sclerotia of R. solani have a higher survival rate under various conditions (Grosch et al. 2006). Although the efficacy of many chemical and biological fungicides in controlling sclerotium formation in R. solani has been studied, no effective strategy has been developed yet. Among the rich source of bioactive chemicals we may find an alternative to our current fungicides (Kim et al. 2003). The secondary metabolites released by some plants have been shown to be fungitoxic (Field et al. 2006). Most plants of the Solanaceae family produce tropane alkaloids as secondary metabolites. A solanaceous plant Hyoscyamus muticus (Egyptian henbane) is an important medicinal plant producing tropane alkaloids, primarily hyoscyamine, which is a precursor of scopolamine. Scopolamine is an epoxide of hyoscyamine (Hashimoto and Yamada 1986) and is produced in smaller amounts (Romeike 1978). In H. muticus, concentrations of tropane alkaloids range from 1.38 to 1.58% (fresh mass), of which 90% is hyoscyamine (World Conservation Union 2005). We have recently discovered that these two compounds, particularly hyoscyamine, have antifungal activity against a broad range of fungi (Abdel-Motaal et al. 2009). The objective of the present study was to examine the antifungal activities of hyoscyamine and scopolamine against M. oryzae and R. solani.
Materials and methods Preparation of hyoscyamine and scopolamine Hyoscyamine was purchased from Tokyo Chemical Industries (Tokyo, Japan) and scopolamine hydrochloride from Sigma (St. Louis, MO, USA). Stock solutions were prepared by dissolving hyoscyamine and scopolamine
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hydrochloride in dimethylsulfoxide (DMSO) for a final concentration B0.1% (v/v). Fungicidal activity of hyoscyamine and scopolamine M. oryzae and R. solani isolates, obtained from infected rice plants in Yamaguchi, Yamaguchi Prefecture, Japan, were cultivated on slants of potato dextrose agar (PDA; Daigo, Tokyo, Japan). Either M. oryzae or R. solani from these slants was then added to 100 ml potato dextrose broth (PDB; Difco, Sparks, MD, USA) and incubated at 25C with shaking at 100 rpm. After 5 days, the mycelia were harvested from the broth using three layers of gauze cloth. The filtrate was centrifuged at 3000g for 20 min, and the mycelia were suspended in distilled water. This fungal suspension (100 ll) was added to 1 ml PDB, and the culture was incubated overnight at 25C with shaking. To assess the fungicidal activity of hyoscyamine, scopolamine, and a mixture of the two compounds, we then added different concentrations of the test compounds to the broth with the vegetative cells of the fungi. After 1 h of incubation, the fungal culture (50 ll) was mixed with 5 ll of Evans blue solution (10 mg/ml). After 5 min, the fungal cells were examined under a light microscope (Olympus BH-2, Tokyo, Japan). The percentage of dead cells (blue-stained cells) was compared between the culture treated with either of the compounds and the untreated culture (control). The treated fungal cultures were transferred to PDA and incubated at 25C for 7 days to confirm cell death. The minimum inhibitory concentration (MIC) of the compounds for fungal growth was determined according to Li and Rinaldi’s method (1999). Each experimental culture with growth inhibition (100 ll) was spread on PDA and incubated at 25C. The minimum lethal concentration (MLC), defined as the lowest concentration of the compound that causes the death of more than 95% of the colonies as compared to the control sample, was also determined. The inhibitory effect of the interaction between hyoscyamine and scopolamine was calculated on the basis of the MIC of these compounds used alone and in combination [fractional inhibitory concentration (FIC)] according to Li and Rinaldi’s method (1999). The following formula was used for this purpose FIC = [MIC100 (hyoscyamine ? scopolamine)/MIC100 (hyoscyamine)] ? [MIC100 (scopolamine ? hyoscyamine)/MIC100 (scopolamine)]. The interactions between the chemicals were classified as follows: a chemical was considered to be synergistic, addictive, indifferent, or antagonistic if its FIC was B0.5,[0.5 to B1,[1 to B2, and[2, respectively. Electrolyte leakage test Electrolyte loss was studied at 25C using a conductivity meter fitted with an immersion probe (ES-12; Horiba,
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Tokyo, Japan) according to the procedure described by Steel and Drysdale (1988). Fungal mycelia (1 g wet mass) were suspended in 1 mM 3-(N-morpholino) propanesulfonic acid (MOPS)–KOH buffer (pH 7.0) containing different concentrations (10, 100, and 400 lg/ml) of hyoscyamine. At the end of the 24-h incubation, chloroform was added to the culture suspension (final concentration of 0.01 ml/ml of culture suspension), and the conductance was measured 2 h later to determine the total electrolyte loss. Conidial germination and appressorium formation in M. oryzae grown on polystyrene Conidia of M. oryzae were allowed to attach to a polystyrene substratum by using the method of Liu and Kolattukudy (1999). The conidia were obtained from 10-day-old fungal cultures on PDA, suspended in water (2 9 106 conidia/ml), and treated with different concentrations of hyoscyamine, scopolamine, or a mixture of the two compounds. A single drop (100 ll) of the conidial suspension was applied to three replicate polystyrene plates, which were incubated at room temperature in conditions of high humidity. Conidia were then examined over time using an inverted microscope (Olympus IX70; Olympus, Tokyo, Japan). After 24 h of incubation, the nongerminated conidia were washed off from the polystyrene plates by using sterile distilled water, and they were collected by centrifugation. The conidia thus collected were grown on another polystyrene plate, and their germination was reexamined after 24 h. Fluorescence of hyoscyamine-treated M. oryzae conidial suspension at varying times A suspension of M. oryzae conidia in water (1.2 9 106 conidia/ml) was treated with two different concentrations of hyoscyamine (1 and 10 lg/ml) in a 1.5 ml Eppendorf tube. To distinguish live cells from dead cells, the spores were stained using 0.01% (w/v) Calcofluor white M2R (Sigma) for 5 min (Fischer et al. 1985), and the percentage of live and dead conidia was determined over 3 h using epifluorescence (excitation 365 nm and emission 430 nm) microscopy (BH2-RFK; Olympus). Conidial germination and appressorium formation in M. oryzae grown on leaves Rice (‘Nipponbare’) leaves (21-day-old) were attached to a glass slide as described by Stanley et al. (2002). The conidial suspension was prepared as described and treated with hyoscyamine (0, 1, 5, or 10 lg/ml). Tween 80 was added to the treated conidia for a final concentration of
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0.025% (v/v). A drop (10 ll) of the conidial suspension (106 conidia/ml) was applied to the leaf surface; four replicate leaves were used. The leaves were incubated at room temperature under conditions of high humidity for 4–6 h. The conidia that attached to the leaf surface were stained with 0.01% (w/v) Calcofluor white for 5 min, washed thrice by flooding each plate with distilled water, and subsequently examined using an epifluorescence microscope (BH2-RFK; Olympus). This experiment was repeated thrice. In another experiment, the leaves were incubated for longer time (4 days) at 25C in a growth chamber with a 16-h light/8-h dark photoperiod. The conidial behavior on the leaf was examined using a light microscope. Effect of hyoscyamine on infection behavior of M. oryaze spores on rice plants grown in a greenhouse Rice plants were grown in a greenhouse until the V5 stage (Counce et al. 2000), where the temperature ranged between 30 and 35C, and the humidity was more than 80%. Plants were sprayed with a mixture of Tween 20 (250 mg/l) and hyoscyamine (1, 5, 10, and 20 lg/ml), allowed to dry overnight. The plants were then sprayed with a suspension of M. oryzae conidia in water (4 9 106 conidia/ml) or with water containing Tween 20 (250 mg/l). Inoculated plants were kept under conditions of high humidity ([85%) using microchambers, which were prepared by removing the screw caps and bottoms of 2-l soft drink bottles (Jia et al. 2007). The percentage of leaf blast severity and suppression were calculated 7 days after treatment with hyoscyamine. The percentage of blast disease severity was calculated for the first four leaves of each plant using the following formula, Percentage disease severity = n(1) ? n(2)… ? n(9)/ number of leaves scored 9 100, where n = number of leaves showing scores 1–9, according to the Standard Evaluation System (SES) developed by the International Rice Research Institute (IRRI 1988), Philippines. Percentage disease suppression based on a formula (controltreatment/control) 9 100 according to Karthikeyan and Gnanamanickam (2008). The experiment was conducted using five plants per pot with three replications and repeated twice. Effect of hyoscyamine on sclerotial germination and development in R. solani Sclerotia were collected from 2-week-old R. solani cultures grown on PDA and incubated for 12 h in PDB supplemented with different concentrations of hyoscyamine (1, 5, 10, 15, and 20 lg/ml). After incubation, the sclerotia were
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washed with distilled water, re-cultured on PDA, and incubated at 25C. Sclerotial germination was examined after 3 days of incubation. Sclerotial development was studied by growing R. solani on sterilized cellophane sheets placed on PDA and incubated at 25C for 2 days. Colonies that grew well on the cellophane sheet were cultured on fresh PDA plates supplemented with hyoscyamine (10, 20, or 50 lg/ml). The control PDA plates were incubated with an equal volume of DMSO. Effect of hyoscyamine on rice sheath blight Detached leaf method The second youngest leaf on V5 rice plants growing in a greenhouse was cut into a 16-cm-long segment (Jia et al. 2002). The leaf segments were immediately placed in 24 9 24 9 1.8 cm plastic dishes containing moistened filter papers. For each hyoscyamine concentration (1, 5, 10, and 20 lg/ml), in addition to the control, four leaf segments were used as a replicate. Three replicates were examined for each treatment. All leaf segments were sprayed with a mixture of Tween 20 (250 mg/l) and hyoscyamine dissolved in DMSO in the desired concentration, while a positive control was sprayed with only Tween 20 and DMSO solution. Subsequently, these solutions were allowed to dry overnight. Colonized PDA blocks, 5 mm in diameter, were excised from a 2-day-old R. solani culture with a 0.2-ml inverted disposable pipette tip, then placed near the middle of the abaxial surface of each leaf segment. The negative control was inoculated with only a PDA plug. All plates were incubated at room temperature (between 23 and 28C) under continuous fluorescent light for 72 h. The length of the lesion on each leaf was measured using a 0–9 scale, where a score of 0 indicated a leaf with no lesion and 9 indicated that 90–100% of the leaf surface was covered with lesions. Visual scores of 1–8 represented 10–80% diseased leaf area, as described previously by Jia et al. (2007). Microchamber method Three-day-old rice seedlings grown on sterilized wet filter paper were transplanted into pots containing steam-sterilized soil. For each hyoscyamine concentration (0, 1, 5, 10, and 20 lg/ml), five rice plants per pot were used as a replicate. Three replicates were examined for each treatment. Three-week-old rice plants were sprayed with a mixture of Tween 20 (250 mg/l) and hyoscyamine dissolved in DMSO in the desired concentration, while a positive control was sprayed with only Tween 20 and DMSO solution. These solutions were allowed to dry overnight. Each plant was inoculated with a round mycelial
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disk (diameter, 0.7 cm) placed at the collar region of the oldest leaf. After inoculation, each pot was covered with a 2-l soft drink bottle that served as a microhumidity chamber. The inoculated pots were kept in trays containing water in the greenhouse. Humidity in the chamber was higher than 85%, and temperature ranged between 30 and 35C. The plants were evaluated for disease reactions when the control exhibited a susceptible reaction (approximately 7–8 days after inoculation). The length of the lesion and height of the plants were measured. A disease index (0–9) was then calculated as lesion length divided by plant height and multiplied by 9, as previously described (Jia et al. 2007). A visual rating was assigned for each plant treated with hyoscyamine. On a 0–9 scale, 0 indicated absence of lesions, and 9 indicated the presence of lesions on 90– 100% of the entire plant, including the leaf. Visual scores of 1–8 indicated that 10–80% of the entire plant was diseased (Jia et al. 2007). Statistical analysis To evaluate the effect of hyoscyamine in controlling rice blast and sheath blight, data of the disease response of the detached leaf and microchamber tests were analyzed using a one-way analysis of variance (ANOVA) followed by a Dunnett comparison test using the program GraphPad Prism 3.0 (P \ 0.05 and P \ 0.01) (GraphPad Software, San Diego, CA).
Results Susceptibility of M. oryzae and R. solani to hyoscyamine and scopolamine The fungicidal activity of hyoscyamine was greater than that of scopolamine against both fungi (Fig. 1a, b). The MIC50 of hyoscyamine against both the fungi was 1 lg/ml, but M. oryzae was more susceptible than R. solani. Scopolamine inhibited the growth of the two fungi to a lesser extent than did hyoscyamine. The mixture of hyoscyamine and scopolamine resulted in an antagonistic interaction (FIC [ 2), which was determined by calculating the MIC100. No synergistic effect was observed between these two compounds. Exposure to hyoscyamine caused leakage of electrolytes from the mycelia of M. oryzae (Fig. 2a) and R. solani (Fig. 2b). This leakage increased with increasing concentrations of hyoscyamine (data not shown); it also increased steadily with an increase in the incubation time, up to 24 h. The two fungi differed in their loss of electrolytes after exposure to low concentrations of hyoscyamine. Evans blue staining of the fungal cells revealed that as the
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Fig. 1 Fungicidal activity of hyoscyamine and scopolamine. Inhibition (%) of vegetative growth of Magnaporthe oryzae (a) and Rhizoctonia solani (b) 6 h after treatment with hyoscyamine, scopolamine, or a mixture of both compounds at various concentrations, as determined by Evans blue staining. For the mixtures, axis values indicate the concentration of each compound. Bars represent the standard error (±SE)
electrolyte leakage increased with higher concentrations of the hyoscyamine, the number of dead cells also increased for both M. oryzae and R. solani (data not shown). Effect of hyoscyamine on the viability of M. oryzae conidia The number of fluorescent (viable) cells decreased with increasing concentrations of hyoscyamine (Fig. 3). Fifteen minutes after exposure to hyoscyamine, the percentage of fluorescent (viable) cells was 66% (1 lg/ml hyoscyamine) and 46% (10 lg/ml hyoscyamine), while at 3 h, this percentage was 32 and 15%, respectively. Conidial germination and appressorium formation by M. oryzae on polystyrene M. oryzae conidia exposed to 1 lg/ml hyoscyamine were able to germinate on polystyrene, although the formation of appressoria was reduced in the first 6 h (Fig. 4). In the presence of 10 lg/ml hyoscyamine, conidial germination
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Fig. 2 Effect of hyoscyamine on electrolyte leakage from Magnaporthe oryzae (a) and Rhizoctonia solani (b). Fungal mycelia (1 g wet mass) were suspended in 1 mM MOPS–KOH buffer (pH 7.0) containing hyoscyamine. After the 24-h incubation, chloroform was added to the culture suspension, and conductance was measured 2 h later to determine the total electrolyte loss. Bars represent the standard error (±SE)
was delayed, and at 8 h, 50% fewer appressoria formed in comparison to untreated conidia. Hyoscyamine at C100 lg/ml completely inhibited conidial germination; germination was not restored even after conidia were washed with water. Hyoscyamine also reduced the number of conidia adhering to the substrata to 20% (1 lg/ml hyoscyamine) and 50% (10 lg/ml hyoscyamine) compared to adherence of untreated conidia at 4 h after incubation. Germination of M. oryzae conidia was unaffected by treatment with 400 lg scopolamine/ml; however, appressorium formation was inhibited to 60% as compared to that in the control sample. A high concentration of scopolamine (1000 lg/ml) reduced conidial germination to 5% of that observed in the control sample.
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Fig. 3 Effect of hyoscyamine over time (0–180 min) on the viability of Magnaporthe oryzae conidia as determined by fluorescence of Calcofluor. Fluorescence with epifluorescence microscopy. Bars represent the standard error (±SE)
However, when washed with water, most (80%) conidia germinated, and 50% of these formed appressoria on the polystyrene surface. The mixture of hyoscyamine and scopolamine (1 lg/ml each) reduced the formation of appressoria to 50% of the levels in the control. Mixtures of hyoscyamine and scopolamine (10 lg/ml each or 100 lg/ml each) had no effect on fungal viability and conidial germination, while appressorium formation was strongly inhibited, i.e., by 93% at 6 h after the treatment. In the case of the mixture containing 400 lg/ml each of hyoscyamine and scopolamine, conidial germination was completely inhibited and was not restored after washing with water. Conidial germination and appressorium formation by M. oryzae grown on a leaf surface Conidia treated with hyoscyamine were placed on the surface of a rice leaf on a glass slide. After a 4-day incubation, hyoscyamine (1 lg/ml) effectively inhibited the attachment of conidia to the leaf surface; only 5% of the conidia formed appressoria. Hyoscyamine at 5 lg/ml completely inhibited appressorium formation, while 10 lg/ ml inhibited conidial germination, as determined using a light microscope (Fig. 5a, b). We distinguished live from dead conidia on the leaf surface after 4 h of incubation using fluorescence microscopy. Conidia attached to the leaf surface were stained using Calcofluor white, then the leaf surface was washed with water. Germination and viability were both much lower for conidia treated with hyoscyamine (1 lg/ml) than in untreated control conidia (Fig. 5c, d). The fluorescence decreased with increasing exposure to hyoscyamine at different concentrations (1 and 10 lg/ml).
Fig. 4 Effects of hyoscyamine, scopolamine, and mixtures of both compounds on conidial germination (a) and appressorium formation (b) in Magnaporthe oryzae grown on a polystyrene substrate. For the mixtures, axis values indicate the concentration of each compound. Bars represent the standard error (±SE)
At 6 h after incubation, the percentage of fluorescing conidia (i.e., viable) was [90% in the control, but was 30, 20, and 3% for conidia treated with 1, 5, and 10 lg/ml hyoscyamine, respectively (Fig. 6). Suppression of rice blast by hyoscyamine Rice plants that had been treated with hyoscyamine (10 and 20 lg/ml) were protected from M. oryzae infection; however, low concentrations of hyoscyamine (1 and 5 lg/ml) did not protect the rice plants from pathogen attack. Plants treated with 1 lg/ml hyoscyamine developed many small blast lesions and some long lesions on the leaf surface, while the one treated with 5 lg/ml had limited blast lesions. Untreated, inoculated control plants formed numerous blast lesions of different sizes on leaves ranging from pale green to yellow. Disease severity was also
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Fig. 5 Light micrographs of infection structures from hyoscyamine-treated conidia on Magnaporthe oryzae on the surface of rice leaves on a glass slide. After a 4-day incubation, melanized appressoria (MA) of M. oryzae had formed in the control (a), and germination of conidia treated with hyoscyamine (10 lg/ml) was inhibited (b). Epifluorescence microscopy after Calcofluor treatment at 4 h after the inoculation of detached leaves, conidia germinated and formed appressoria at high rates (fluorescent appressorium: FA) in the untreated control (c). Conidia treated with hyoscyamine (1 lg/ml) had a lower rate of adhesion, and conidia germinated (CG) but did not form appressoria (d)
Table 1 Effect of hyoscyamine concentrations on rice leaf blast severity and suppression Hyoscyamine (lg/ml)
Severity (%)a
Suppression (%)b
0 (control)
69.1
–
1
31.3
54.7
5
24.3
54.7
10
3.0**
95.7**
20
0**
100**
a Percentage disease severity = n(1) ? n(2)… ? n(9)/number of leaves scored 9 100, where n = number of leaves with each score from 1 to 9 according to the SES system b
Fig. 6 Percentages of viable conidia, germinated conidia, and germinated conidia forming appressoria in Magnaporthe oryzae grown on the surface of rice leaves in the presence of hyoscyamine for 6 h. Conidia that attached to the leaf surface were stained using Calcofluor and observed with epifluorescence microscopy. Bars represent the standard error (±SE)
significantly reduced in plants treated with hyoscyamine (10 and 20 lg/ml) (Table 1). Sclerotial germination after hyoscyamine treatment Mature sclerotia were treated with hyoscyamine at different concentrations; subsequently, these sclerotia were washed with sterilized water and re-cultured on PDA media. At 24 h, low concentrations of hyoscyamine (1 and 5 lg/ml) inhibited sclerotial germination in R. solani to some extent.
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Percentage of disease suppression based on formula (controltreatment/control) 9 100 according to Karthikeyan and Gnanamanickam (2008) ** P \ 0.01: significant difference from the control using Dunnett’s multiple comparison test
On the first day of incubation, the MIC50 and MIC100 of hyoscyamine were 15 and 20 lg/ml, respectively. The sensitivity of sclerotial germination to hyoscyamine decreased when the incubation time was increased to 4 days; this sensitivity was unremarkable at low concentrations of hyoscyamine but high (80 and 90%) at higher concentrations of hyoscyamine (15 and 20 lg/ml), respectively (Fig. 7). Effect of hyoscyamine on sclerotial development R. solani was grown on the surface of a sterilized cellophane sheet placed on PDA and then transferred to new PDA plates with and without hyoscyamine. The control
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Fig. 7 Effect of hyoscyamine on the germination of the sclerotia of Rhizoctonia solani. Bars represent the standard error (±SE)
sclerotia were melanized and matured on the plates without hyoscyamine after 6–8 days of culture. Cultures exposed to 10 lg/ml hyoscyamine developed normal sclerotia, but the development was delayed until 12 days after treatment, that is, 4–6 days later than the control. Higher concentrations of hyoscyamine (20 and 50 lg/ml) completely inhibited melanization and maturation of sclerotia (sclerotial development) even after 60 days of incubation. Microscopic examination revealed a clear difference in mycelial morphology between mycelia from mature (melanized) sclerotia on PDA plates not supplemented with hyoscyamine and those from immature sclerotia formed on plates containing hyoscyamine (20 lg/ml). Normal, thick hyphae were observed in the former, while thin hyphae with small structural bodies were observed in the latter (Fig. 8). Effect of hyoscyamine on rice sheath blight In detached leaf experiments, hyoscyamine significantly inhibited sheath blight (Table 2). In microchamber experiments with whole plants, hyoscyamine sprays at 5, 10, and 20 lg/ml significantly lowered the disease index (3.2, 2.0, and 1.9, respectively) (Table 2) (Fig. 9).
Discussion This study is the first to reveal the potential antifungal activity of hyoscyamine and scopolamine against the rice pathogens M. oryzae and R. solani. Hyoscyamine had greater antifungal activity than did scopolamine against mycelia and conidia of both fungi. The antifungal activity of scopolamine, the epoxide of hyoscyamine, may be lower than that of hyoscyamine because of the presence of the epoxy bridge in scopolamine. Although the mechanism
Fig. 8 Effect of hyoscyamine on sclerotial development in Rhizoctonia solani. a Control mycelia formed mature, melanized sclerotia (MS) with thick hyphae. b On PDA with 20 lg/ml hyoscyamine, immature, nonmelanized sclerotia (IMS) and thin hyphae with swollen regions formed. Mycelial morphology differed between mature (melanized) sclerotia formed on PDA plates not containing hyoscyamine (a, lower panel), and immature sclerotia formed on plates containing hyoscyamine (b, lower panel) are shown Table 2 Effects of different concentrations of hyoscyamine on sheath blight disease using the detached leaf method (lesion length) or the microchamber method (disease index) Hyoscyamine concentration (lg/ml)
Lesion length (cm)a
Disease indexb
0 (control)
10.8
7.1
1
9.6*
5.2
5
6.2**
3.2*
10
4.1**
2.0**
20
3.0**
1.9**
a
Lesion length (cm) was measured as the total lesion length on the infected leaf b Disease index was calculated based on the formula (lesion length/ culm length) 9 9, where lesion length was measured as the length of lesion along the length of the culm according to Jia et al. (2007) * P \ 0.05 and ** P \ 0.01: significant difference from the control using Dunnett’s multiple comparison test
underlying the antifungal action of hyoscyamine and scopolamine was not studied here, the fungicidal nature of hyoscyamine against M. oryzae and R. solani appears to be related to the leakage of electrolytes from the fungal cells. Electrolyte leakage from fungal cells treated with tropane alkaloids, including hyoscyamine, has not been reported as yet, even though this phenomenon has been observed in fungal cells treated with the steroidal alkaloid a-tomatine (Steel and Drysdale 1988).
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Fig. 9 Effects of various spray concentrations of hyoscyamine on sheath blight symptoms on rice 8 days after inoculation with Rhizocotonia solani in microchamber experiments. Three-week-old rice plants were sprayed with a mixture of Tween 20 and various concentrations of hyoscyamine dissolved in DMSO. Each plant was inoculated with a round mycelial disk placed at the collar region of the oldest leaf. The inoculated pots were kept in trays containing water in the greenhouse. Arrows mark lesions. Positive control plants were sprayed with no hyoscyamine and then inoculated with the fungus. Negative control plants were neither sprayed with hyoscyamine nor inoculated with the fungus
Interestingly, the antifungal activity of hyoscyamine was reduced by scopolamine. Both the compounds are known to bind and inhibit muscarinic acetylcholine receptors in animals (Reynolds 1982). Although such target site(s) of hyoscyamine in fungal cells are not known, these site(s) may be the same for scopolamine, which may explain the reduction in the activity of hyoscyamine. Adhesion of conidia to the leaf surface and subsequent germination and formation of appressoria are critical steps for the penetration of M. oryzae into the leaf epidermis (Howard and Ferrari 1989). Besides reducing appressorium formation at a high concentration (10 lg/ml), hyoscyamine inhibited conidial adhesion and delayed conidial germination at a low concentration (1 lg/ml). Germinability was not restored even when the conidia were washed with water, indicating that the antifungal activity of a high concentration of hyoscyamine (100 lg/ml) against the conidia of M. oryzae is irreversible. In contrast, washing the scopolamine-treated conidia with water restored conidial germination. A similar restoration of the germination of M. oryzae conidia on treatment with zosteric acid,
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a naturally occurring phenolic acid in eelgrass plants, has been reported (Stanley et al. 2002). The decreasing viability of conidia treated with higher concentrations of hyoscyamine (10 lg/ml and higher) may explain the reduction in conidial adhesion and germination on treatment with 10 lg/ml hyoscyamine. We also showed that hyoscyamine significantly reduced the symptoms on leaves of intact rice plants after M. oryzae infection (data not shown), suggesting that hyoscyamine can exhibit its fungicidal activity even on the leaf surface. This study is the first to report the fungicidal activity of hyoscyamine against M. oryzae in vivo. Further studies are required before using hyoscyamine as a fungicide in agriculture because this compound is toxic to animals at high doses (Reynolds 1982). For example, in rat the LD50 for hyoscyamine is 375 mg/kg. Acknowledgments We acknowledge the Department of Missions (Ministry of Higher Education and Scientific Research, Egypt) for financial support through a channel system scholarship to Ms. F. F. Abdel-Motaal.
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