Agronomy Journal 93:72-79 (2001)
© 2001 American Society of Agronomy
ALLELOPATHY SYMPOSIUM
Physiological and Biochemical Mechanism of Allelopathy of Secalonic Acid F on Higher Plants
Ren Sen Zeng,
Shi Ming Luo,
Yue Hong Shi,
Mu Biao Shi and
Cong Yong Tu
Inst. of Tropical & Subtropical Ecology, S. China Agric. Univ., Wushan, Guangzhou, 510642, People's Republic of China
Corresponding author (rszeng{at}scau.edu.cn)
 |
ABSTRACT
|
|---|
Our previous work indicated that secalonic acid F (SAF) was the major allelochemical produced by Aspergillus japonicus. Studies showed that SAF markedly inhibited the seedling growth of sorghum (Sorghum vulgare Pers.), hairy beggarticks (Bidens pilosa L.), and barnyardgrass (Echinochloa crus-galli (L.) Beauv.). It significantly reduced the activities of superoxide dismutase (SOD) and peroxidase (POD) at a concentration of 0.3 mM. Secalonic acid F enhanced malondialdehyde (MDA) contents, but it lowered the content of chlorophyll (CHL) a and b as well as the photosynthetic rates of tested plants. Respiration, membrane permeability, and abscisic acid (ABA) content increased after treatment with SAF, but the reduction activity of the root system was lowered. There is no remarkable change in the soluble proteins of plants that are treated with SAF. Transmission electron microscope (TEM) observations showed that treated plants exhibited amorphous mitochondria without integral membranes and swelling chloroplasts without membranes in a disorderly arrangement. The SAF treatment also damaged the stratiform structure of the chloroplasts and the membranes and structure of the nuclei. These results suggest that SAF may weaken the protective ability of plant tissues against membrane lipid peroxidation and damage the whole membrane system of plants, resulting in the ultrastructure destruction of chloroplasts, mitochondria, and nuclei. Cell ultrastructure destruction causes a reduction of photosynthesis and root activities and an increase in respiration. These abnormal physiological processes contribute to the inhibition of plant growth.
Abbreviations: ABA, abscisic acid • DMF, dimethyl formamide • EC, electric conductivity • ELISA, enzyme-linked immunosorbent assay • MDA, malondialdehyde • POD, peroxidase • SAF, secalonic acid F • SOD, superoxide dismutase • TEM, transmission electron microscope
|
INTRODUCTION
|
|---|
ALLELOPATHY IS MEDIATED by many types of compounds with different sites and modes of biochemical action (Rice, 1984). It was originally defined as the biochemical interactions between plants of all kinds, including the microorganisms that are typically placed in the plant kingdom (Molisch, 1937). Einhellig (1986) pointed out that a clear insight into the precise physiological perturbations caused by allelochemicals had not been obtained, and it might be emphasized that much additional information is needed. One of the most significant limitations that have curtailed attempts to investigate how allelochemicals alter growth is the lack of sufficient quantities of a compound necessary to study the effects on physiological processes and cellular mechanisms (Einhellig, 1995). Several modes of action for allelochemicals are involved in the inhibition and modification of plant growth and development (Einhellig, 1986). Allelochemicals may be selective in their action, or plants may be selective in their responses. These considerations are complicated further by the presence of more than one active compound from a single plant or fungus. For example, Sorghum species contain cyanogenic glycosides, tannins, flavonoids, and a series of phenolic acids. All of these have inhibitory activity, and most of them produce different biological lesions (Einhellig, 1995).
Secalonic acids, a series of ergochrome pigments, exist in a group of food-born fungal metabolites (Betina, 1984). These fungi produce one or more secalonic acids when they grow on rice (Oryza sativa L.), corn (Zea mays L.), and rye (Secale cereale L.) (Kurobane et al., 1979). Secalonic acid A is a highly potent phytotoxic compound that is isolated from several fungi such as Aspergillus aculeatus (Andersen et al., 1977) and Pyrenochaeta terrestris (Steffens and Robeson, 1987). Secalonic acid D, a metabolite produced by Penicillium oxaium, causes storage rot of cucumber (Cucumis sativus L.) and tomato (Lycopersicon lycopersicum L.) (Jarvis et al., 1990). It is an inhibitor of protein kinase C and cyclic AMP-dependent protein kinase (Wang et al., 1996). Secalonic acid F was originally isolated from Claviceps purpurea (Aberhart et al., 1965). Our previous work indicated that SAF was a major allelochemical produced by A. japonicus (Zeng et al., 2001). Details are needed about the mode of action of secalonic acids against higher plants to deepen the understanding of the chemical interaction between fungi and higher plants. Our objective was to determine the physiological and biochemical mechanism of allelopathy of SAF.
|
Materials and methods
|
|---|
Secalonic Acid F
Secalonic acid F was isolated as yellow needles from the fermentative hyphae of A. japonicus. Its purity was determined by 1H nuclear magnetic resonance data recorded on a Bruker AC-P200 spectrometer (200 MHz) using cadmium chloride (CDCl3) as a solvent and tetramethyl silicane as an internal standard.
Secalonic acid F was dissolved in a 1.5 g L-1 dimethyl formamide (DMF) solution. Ultraviolet absorption of SAF dissolved in 10 g L-1 DMF did not change compared with SAF dissolved in a 950 g L-1 ethanol (C2H5OH) solution. A preliminary study showed that DMF did not affect the phytotoxicity of SAF (Zeng et al., 2001).
Plant Materials
The seeds of rape (Brassica campestris L.), cucumber, corn, and sorghum were obtained from a local market in Guangzhou, China. The seeds of hairy beggarticks and barnyardgrass were collected from the campus of South China Agricultural University.
Bioassays
The seeds were germinated before being cultivated in a SAF solution. Ten seeds each were placed in 50-mL beakers and kept in 5 mL of different concentrations of SAF solutions, with a temperature of 28°C and a 12-h photoperiod. The amount of photosynthetically active radiation during the daytime was 250 µmol photons m-2 s-1. After 4 d, the root and shoot lengths were measured. All treatments consisted of at least three replications.
Reduction Activities in Roots
The method used for measuring the root reduction activities has been described by Zhang (1990). The roots (0.5 g) were washed, blotter-dried, weighted, and soaked in a 10-mL mixture of 5 mL of 4 g L-1 2,3,5-triphenyl tetrazolium chloride and 5 mL of a PO4 buffer (pH 7) in darkness at 37°C for 1 h. Thereafter, 2 mL of 1.0 M sulfuric acid (H2SO4) was added to stop the reaction. The roots were removed, washed with distilled water, blotter-dried, and ground with two portions of 3 to 4 mL of ethyl acetate (C4H8O2) in a mortar and pestle. The extract was filtrated through filter paper. The volume was made up to 10 mL and the optical density of the formazan extract was read at 485 nm on the ultraviolet spectrophotometer (756 MC). An enzyme unit was defined as the quantity (mg) of tetrazolium chloride reduction per hour on a per gram root (fresh wt.) basis.
Chlorophyll Content and Photosynthetic and Respiratory Rates
The sorghum seedlings that were incubated at 28°C under 250 µmol photons m-2 s-1 artificial light for 6 d were removed to another plate with different concentrations of SAF solution. After another 6 d of incubation, 0.05 g of leaves were harvested to be extracted with a 10-mL mixture of acetone (C3H6O)-anhydrous ethanol water (45:45:10 V/V) for 48 h in the dark at 4 to 5°C. The absorption of each CHL extract was read using a spectrophotometer at 665 and 649 nm, and the values for each CHL a, CHL b, and total CHL were determined according to the method of Arnon (1949). There were three replications for each treatment, and the experiment was duplicated. Data were subjected to an analysis of variance, and significant difference was determined by nonoverlapping confidence intervals at 
.
Six-day-old seedlings were placed in one-half strength Hoagland solution with 0.038, 0.075, 0.15, and 0.3 mM SAF solution to incubate for 5 and 10 d in a greenhouse. The rate of photosynthesis was measured at 200 µmol photons m-2 s-1 and 25°C using the Li-Cor LI-6200 Portable Photosynthesis System. The respiratory rate was measured in the dark.
Malondialdehyde, Soluble Protein, and Activities of Superoxide Dismutase and Peroxidase
The plant material preparation for MDA measurement was the same as that used for CHL extraction. The receptor plants included sorghum, rice, and rape. Leaf tissue (1 g) was ground and homogenized with a chilled mortar and pestle in 5 mL of a chilled 0.05-M PO4 buffer (pH 7.8) at 1 to 5°C. The homogenate was strained through four layers of cheesecloth. The liquid suspension was centrifuged at 1900 g for 20 min. After centrifugation, the supernatant was used for measurement of MDA using the method described by Zhao et al. (1994). The supernatant preparation for SOD and POD activity measurement was the same as that used for MDA measurement. SOD activity was measured using the method described by Giannopolitis and Ries (1977). POD activity was determined using the method described by Kraus and Fletcher (1994). Soluble protein was assayed using the method of Bradford, with bovine serum albumin as the standard (Bradford, 1976).
Abscisic Acid Content
The abscisic acid content was measured using the method of enzyme-linked immunosorbent assay (ELISA). The ELISA was produced by the Nanjing Agricultural University Plant Hormone Laboratory of China. The product was suitable to measure hormones that belonged to the sopentenyladenosine group. The experimental procedures followed the method described by Zhang (1990).
Electric Conductivity
Six-day-old seedlings of rice, rape, cucumber, and sorghum were cultured in 20-mL SAF aqueous solutions for another 5 d. The control seedlings were cultured in 10 g L-1 DMF solution. Then 0.2-g seedlings were rinsed with distilled water and suspended in 30 mL of deionized water under reduced pressure for 1 h. The electric conductivity (EC) was analyzed using a conductivity meter (DDS-11A). Thereafter, the seedlings and their solution were heated to 100°C for 20 min. After the solution cooled, the EC was analyzed again.
Electron Microscopy
Six-day-old seedlings of corn, rice, and rape were cultured in 0.3 mM SAF aqueous solution for another 5 d. The root tips (3 mm) and leaf segments were fixed for 3 h in 30 g L-1 glutaraldehyde in a 0.1-mM sodium phosphate (Na3PO4) buffer (pH 7). They were postfixed for 4 h in 10 g L-1 osmic acid in a 0.1-mM PO4 buffer (pH 7.2) and then embedded. After being fixed, the specimens were examined on a JEM1010 TEM.
Statistics
The data from each experiment were subjected to an analysis of variance, with the significant differences among means identified by Duncan's multiple range test (P < 0.05).
|
Results and discussion
|
|---|
Our previous work showed that SAF inhibited the seedling growth of rape, radish (Raphanus sativus L.), and rice (Zeng et al., 2001). It also inhibited the seedling growth of barnyardgrass and hairy beggarticks at a concentration of 0.038 mM (Table 1). Even at a concentration of 0. 47 x 10-3 mM, SAF reduced the root and shoot growth of barnyardgrass seedlings by 15.8 and 19.9%, respectively. It also stimulated the root growth of sorghum at a concentration of 0.038 mM but inhibited the seedling growth at a concentration 
0.075 mM. This indicated that low concentrations of SAF would stimulate the seedling growth of some plants.
View this table:
[in this window]
[in a new window]
|
Table 1 Effects of secalonic acid F (SAF) on seedling growth of sorghum, barnyardgrass, and hairy beggarticks (cm)
|
|
Effects on Root Activities
The root reduction activities of rice seedlings were significantly reduced when they were cultured in a SAF aqueous solution. The inhibition was 14.7 and 39.1% at concentrations of 0.038 and 0.075 mM SAF, respectively (Fig. 1)
. At a concentration of 0.6 mM SAF, the rice root reduction activities were very low—only 18.9% of the normal seedling roots.
Effects on Chlorophyll Content and Photosynthetic and Respiratory Rates
Secalonic acid F obviously increased the CHL content of sorghum at a low concentration of 0.038 mM (Fig. 2)
. However, SAF significantly reduced the CHL content at a concentration of 0.075 mM. The content of CHL a and b were only 49.6 and 36.8% of the control, respectively, at a concentration of 0.15 mM SAF. The seedling leaves became yellow at this concentration. The results were consistent with the effects of SAF on seedling growth (Table 1).
View larger version (14K):
[in this window]
[in a new window]
|
Fig. 2 Effects of different conc. of secalonic acid F (SAF) on the chlorophyll (CHL) content of sorghum seedling
|
|
Figure 3
showed that SAF treatment reduced the photosynthetic rate. For dicotyls such as rape and cucumber, the reduction was drastic. The photosynthetic rates of rape and cucumber were only 12.5 and 21.6% of the control, respectively, at a concentration of 0.038 mM SAF. When the SAF concentration was >0.075 mM, the photosynthetic rates of rape were negative. This meant that the CO2 consumption of photosynthesis was less than the CO2 production of respiration. The photosynthetic rate of cucumber was negative at a concentration of 0.15 mM SAF. The photosynthetic rate of sorghum did not significantly decrease until the SAF concentration reached 0.075 mM, and it was 50% of the control at a SAF concentration of 0.30 mM. No negative photosynthetic rate of sorghum was observed. The photosynthetic rate not changing drastically in sorghum may be the result of the C-4 metabolic pathway of the plant. The photosynthetic rate of rice decreased by 34 and 75.5% at concentrations of 0.038 and 0.3 mM SAF, respectively. No negative photosynthetic rate was observed for rice.
Figure 4
shows that the respiratory rate of rape increased at a concentration of 0.075 mM SAF. When the concentration was 0.15 mM SAF, the respiratory rate of rape was 161.5% of control. The respiratory rate of cucumber significantly increased at concentrations of 0.15 and 0.30 mM SAF while the respiratory rate of sorghum changed little. The respiration of rice was sensitive to SAF at a concentration of 0.038 mM, increasing by 250%, but did not change drastically, even at a concentration of 0.3 mM SAF.
Effects on Malondialdehyde
The MDA content of rape, rice, and sorghum that were treated with 0.3 mM SAF for 5 d increased by 60, 110, and 38%, respectively (Table 2). After a 10-d treatment, it increased by 131, 151, and 89%. The MDA content of the three crops showed the similar ascending tendency when the treatment duration was longer.
View this table:
[in this window]
[in a new window]
|
Table 2 Effects of different concentrations of secalonic acid F (SAF) on the content of malondialdehyde (MDA) (nmol g-1 FW)
|
|
Effects on Superoxide Dismutase, Peroxidase, and Soluble Protein
The SOD and POD activities of all three of the tested crops significantly declined when they were treated with 0.3 mM SAF (Table 3). The SOD activities of rape and rice decreased by 49 and 71%, respectively, while they decreased by only 22.5% for sorghum. The POD activities of sorghum decreased by 21%. The soluble protein content of rape was significantly reduced by SAF treatment, whereas that of rice and sorghum did not change significantly. A reduction of SOD and POD activities may cause a mass accumulation of active O2 in plant leaves, which leads to membrane lipid peroxidation (Keppler and Novacky, 1987). Membrane lipid peroxidation results in the destruction of membrane systems (Song et al., 1996; Keppler and Novacky, 1989).
View this table:
[in this window]
[in a new window]
|
Table 3 Effects of secalonic acid F (SAF) on the activities of superoxide dismutase (SOD), peroxidase (POD) and the content of soluble proteins
|
|
Effects on Abscisic Acid Content
After 5 d of treatment with SAF, the ABA content of rice seedlings increased by 158%. When rice seedlings were treated with SAF for 10 d, the ABA content increased by 230.5%. This indicated that the ABA content in rice seedlings would increase with longer SAF treatments.
Effects on Membrane Permeability
Table 4 shows that the EC of rice and rape increased either before boiling or after boiling. The EC of rape increased by 59.6% after boiling compared with the control. And the EC of cucumber and sorghum increased by 169.2 and 80.9%, respectively, before boiling but changed little after boiling.
Effects on Cell Ultrastructure
The normal leaf cell structure and chloroplast arrangement of rape are shown in Fig. 5A
. The chloroplasts are lined closely to the cell periphery. However, the leaf cells of the treated rape have swelling chloroplasts that were in a messy distribution (Fig. 5B). Some chloroplasts combined and separated from the cell periphery. Figure 6
shows that chloroplasts of rice that were treated with 0.3 mM SAF lost their membrane—their stratiform structure became inconspicuous. Figure 7
indicates that the mitochondria of treated rice roots swelled and lost their membranes—their stratiform structure became indistinct. Figure 8
indicates that the nuclei membranes of treated corn roots became inconspicuous and incompact—the nucleolus appears to be less apparent.
|
Conclusions
|
|---|
Results indicate that SAF produced by A. japonicus interferes with many physiological and biochemical processes of higher plants. It significantly reduced the activities of SOD and POD at a concentration of 0.3 mM. Secalonic acid F enhanced MDA contents, but it lowered the contents of CHL a and CHL b as well as the photosynthetic rates of the tested plants. Respiration and ABA content increased after treatment with SAF. When cucumber and sorghum were treated with 0.15 mM SAF, their membrane permeability increased. When rice and rape were treated with 0.3 mM SAF, their cells would lose water, which resulted in the increase of EC after boiling. Secalonic acid F lowered the reduction activity of the root system, but the plants that were treated with SAF experienced no significant change in soluble proteins.
Most of the tested physiological and biochemical processes of dicotyls such as rape and cucumber were more severely affected by treatment with SAF than monocotyls such as sorghum, which was least affected. This agreed with the bioassay results and the observation that the SAF producing fungus, A. japonicus, often contaminated the seeds of some dicotyls and caused inhibition of seed germination and seedling growth. The results indicate that A. japonicus and its allelochemical SAF are selective.
The TEM observations showed that treated plants exhibited amorphous mitochondria without integral membranes and swelling chloroplasts without membranes that were in a disorderly arrangement. The SAF treatment also damaged the stratiform structure of the chloroplasts and the membranes and structure of the nuclei.
Obtained results suggest that SAF may weaken the protective ability of plant tissues against membrane lipid peroxidation and damage to the cell membrane system (Kraus and Fletcher, 1994; Song et al., 1996), resulting in an increase of membrane permeability (Keppler and Novacky, 1989) and ultrastructure destruction of chloroplasts, mitochondria, and nuclei. Ultrastructure destruction causes a reduction of photosynthesis and root activities as well as increase in respiration. These abnormal physiological processes contribute to the inhibition of plant growth. Although the exact sequence of SAF actions remains uncertain, these data suggest that some effects are more potent than others. The action of SAF on membranes is essentially responsible for the disruption of most other processes. This is consistent with the mode of action of phenolic acids (Einhellig, 1995).
|
ACKNOWLEDGMENTS
|
|---|
We thank the National Natural Science Foundation of China (39770136), Guangdong Provincial Natural Science Foundation of China (990682, 960426), and the National Laboratory of Elemento-Orangic Chemistry, Nankai University, for their financial support.
Received for publication November 29, 1999.
|
REFERENCES
|
|---|
- Aberhart, D.J., Y.S. Chen, P.D. Mayo, and J.B. Stothers. 1965. Mould metabolites: The isolation and constitution of some ergot pigments. Tetrahedron 21:1417–1432.
- Andersen, R., B. George, B. Kobbe, and A.L. Demain. 1977. Secalonic acids D and F are toxic metabolites of Aspergillus aculeatus. J. Org. Chem. 42:352–353.[ISI][Medline]
- Arnon, D.I. 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24:1–15.[Free Full Text]
- Betina, V. (ed.) 1984. Mycotoxins: Production, isolation, separation, and purification. Elsevier Sci. Publ., New York.
- Bradford, M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle protein-dye binding. Anal. Biochem. 72:248–254.[ISI][Medline]
- Einhellig, F.A. 1986. Mechanisms and modes of action of allelochemicals. p. 171–187. In A.R. Putnam and C.S. Tang (ed.) The science of allelopathy. John Wiley & Sons, New York.
- Einhellig, F.A. 1995. Mechanism of action of allelochemicals in allelopathy. p. 96–116. In Inderjit, et al. (ed.) Allelopathy: Organisms, processes, and applications. Am. Chem. Soc. Symp. Ser. 582. Am. Chem. Soc., Washington, DC.
- Giannopolitis, C.N., and S.K. Ries. 1977. Superoxide dismutase. Occurrence in higher plants. Plant Physiol. 59:309–314.[Abstract/Free Full Text]
- Jarvis, W.R., S.D. Barrie, and J.A. Traquair. 1990. Morphological and chemical studies of Penicillium oxalicum, newly identified as a pathogen on greenhouse cucumbers. Can. J. Bot. 68:21–25.
- Keppler, L.D., and A. Novacky. 1987. The initiation of membrane lipid peroxidation during bacteria-induced hypersensitive reaction. Physiol. Mol. Plant Pathol. 30:233–245.
- Keppler, L.D., and A. Novacky. 1989. Changes in cucumber cotyledon membrane lipid fatty acids during paraquat treatment and a bacteria-induced hypersensitive reaction. Phytopathology 79:705–708.
- Kraus, T.E., and R.A. Fletcher. 1994. Paclobutrazol protects wheat seedlings from heat and paraquat injury, is detoxification of active oxygen involved? Plant Cell Physiol. 35:45–52.[Abstract/Free Full Text]
- Kurobane, I., L.C. Vining, and A.G. Mclnnes. 1979. Biosynthetic relationships among the secalonic acids: Isolation of emodin, endocrocin, and secalonic acids from Pyrenochaeta and Aspergillus aculeatus. J. Antibiot. 32:1256–1266.[Medline]
- Molisch, H. 1937. Der Einfluss einer Pflanze auf die andere-Allelopathie, Gustav. Fischer. Verlag, Jena.
- Rice, E.L. (ed.) 1984. Allelopathy. 2nd ed. Academic Press, Orlando, FL.
- Song, F.M., Z. Zheng, and G.X. Chun. 1996. Role of active oxygen and membrane lipid peroxidation in plant–pathogen interactions.(In Chinese). Plant Physiol. Commun. 32:377–385.
- Steffens, J.C., and D.J. Robeson. 1987. Secalonic acid A, a vivotoxin in pink root-infected onion. Phytochemistry 26:1599–1602.
- Wang, B.H., G.M. Polya, and B.H. Wang. 1996. The fungal teratogen secalonic acid D is an inhibitor of protein kinase C and of cyclic AMP-dependent proteinkinase. Planta Med. 62:111–114.[ISI][Medline]
- Zeng, R.S., S.M. Luo, M.B. Shi, Y.H. Shi, Q. Zeng, and H.F. Tan. 2001. Allelopathy of Aspergillus japonicus on Crops. Agron. J. 93:60–64 (this issue).[Abstract/Free Full Text]
- Zhang, Z.L. 1990. Experiments and methods in plant physiology. (In Chinese), 2nd ed. Higher Educ. Press, Beijing.
- Zhao, S.J., C.C. Xu, Q. Zou, and Q.W. Meng. 1994. Improvements of method for measurement of malondialdehyde in plant tissue.(In Chinese). Plant Physiol. Commun. 30:207–210.
TOP
ABSTRACT
INTRODUCTION
