Agronomy Journal 93:60-64 (2001)
© 2001 American Society of Agronomy
ALLELOPATHY SYMPOSIUM
Allelopathy of Aspergillus japonicus on Crops
Ren Sen Zenga,b,
Shi Ming Luoa,
Mu Biao Shia,
Yue Hong Shia,
Qiang Zenga,b and
Hui Fen Tanb
a Y.H. Shi, Inst. of Tropical and Subtropical Ecology, S. China Agric. Univ., Wushan, Guangzhou, 510642, People's Republic of China
b Elemento Organic Chem. Lab. Nankai Univ., Tianjin 300071, People's Republic of China
Corresponding author (rszeng{at}scau.edu.cn)
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ABSTRACT
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The seeds of some dicotyledons fail to germinate and grow well when contaminated by Aspergillus japonicus Saito. One strain of A. japonicus isolated from the seeds of contaminated rape (Brassica campestris L.) inhibited the seedling growth of rape and radish (Raphanus sativus L.) when the fungus was directly inoculated on the seed surface. Metabolites released from the fungus inhibited the seedling germination and seedling growth of rape and radish. The culture filtrate and mycelium acetone (C3H6O) extract inhibited the seedling growth of rape. The major allelochemical of A. japonicus was identified by spectroscopic methods as secalonic acid F (SAF). Bioassays showed that SAF at concentration 0.038 mM significantly inhibited the seedling growth of several crops.
Abbreviations: DMF, dimethyl formamide • EIMS, electron impact ionization mass spectrometry • EtOAc, ethyl acetate • NMR, nuclear magnetic resonance • SAA, secalonic acid A • SAD, secalonic acid D • SAF, secalonic acid F
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INTRODUCTION
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THE TERM ALLELOPATHY WAS COINED BY MOLISCH (1937) to refer to the biochemical interactions between all types of plants, including microorganisms (Rice, 1974, 1995). Rice (1974) pointed out that only a small amount of research had been done on the chemical inhibition of higher plants by microorganisms, except for the specialized field of plant pathology. Metabolites of many fungi may have adverse or stimulatory effects on plants (Heisey et al., 1985; Rice, 1995) such as suppression of seed germination, malformation, and retardation of seedling growth (Lynch and Clark, 1984). Many crop seeds are infected by fungi before harvest or during storage (Neergaard, 1979). If conditions are not favorable, then the situation is more serious (Kozakiewicz, 1996). Some fungi on the surface of seeds may produce mycotoxins that affect food quality (Betina, 1984), and some may produce phytotoxins that affect seed germination and seedling growth (Neergaard, 1979).
Secalonic acids, which represent a series of ergochrome pigments, are a group of fungal metabolites (Kurobane et al., 1979). They are the stereoisomers and differ only in the placement of substituting groups. The producing strains often produce one or more secalonic acids when they grow on rice (Oryza sativa L.), corn (Zea mays L.), and rye (Secale cereale L.). Secalonic acid D (SAD) is the major mycotoxin of this group of ergochromes (Betina, 1984). Secalonic acid A (SAA) was the first compound that was reported to have a highly potent phytotoxicity. It was isolated from Pyrenochaeta terrestris, the pathogen of pink root disease of onion (Allium cepa L.) and other species of Allium (Steffens and Robeson, 1987). The compound inhibited the onion seedling elongation by 4, 32, 40, 68, and 94% at concentrations of 10-9, 10-8, 10-7, 10-6, and 10-5 M, respectively. Penicillium oxalicum, which caused a storage rot of cucumber (Cucumis sativus L.) and tomato [Lycopersicon lycopersicum (L.) Karsten] fruit, also produced SAD and oxalic acid (Jarvis et al., 1990).
The genus Aspergillus is a saprophyte that occurs in and on a variety of substrates, including grains, decaying vegetation in the field, and cattle dung (Raper and Fennell, 1965; Tzean et al., 1990, p. 43). It is particularly abundant in soils in the tropics and subtropics. During routine laboratory work, we found that some dicotyledons that failed to germinate or grew poorly were infected by Aspergillus sp. In this paper, we report on the allelopathic effects of the fungus on several crops. We have demonstrated that SAF is the major chemical responsible for these effects.
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Materials and methods
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Isolation and Culture of A. japonicus
The strain of the fungus was isolated from the surface of infected radish seeds that failed to germinate and then purified by successive transfers of mycelia tips. Stock cultures were maintained on PDA slants at 28°C for 7 d and then kept at 4 to 5°C thereafter. The fermentative media consisted of: D-glucose, 20; potassium monophosphate (K2HPO4), 0.5; magnesium sulfate (MgSO4), 0.25; corn flour steep liquor, 20; and potato (Solanum tuberosum L.) liquor, 200 g L-1. Each 500-mL Erlenmeyer flask contained 100 mL of media. The flasks were incubated at 26°C in the dark for 4 d on a gyratory shaker at 180 rotations min-1.
Plant Materials
Seeds of radish, rape, cucumber, corn, and sorghum (Sorghum vulgare Pers.) were obtained from a local market in Guangzhou, China. Seeds of hairy beggarticks (Bidens pilosa L.) and barnyardgrass [Echinochloa crus-galli (L.) Beauv.] were collected at the campus of South China Agricultural University.
Inoculation of A. japonicus on Seedling
Seeds of radish and rape were surface-sterilized with 1 g L-1 mercury chloride (HgCl2) for 10 min, then with 750 g L1 ethanol (C2H5OH) for 10 s. After sterilization, the seeds were thoroughly rinsed with sterile water. Fifty seeds and one piece of filter paper were each placed in 9-cm petri dishes. Seven mL 20% (V/V) PD was added. The spores of A. japonicus were inoculated on the surface of the seeds before incubation. The controls consisted of noninoculated seeds. The dishes were incubated at 25°C in a greenhouse with 10 h of artificial light (250 µmol photons m-2 s-1) daily. The root length and seedling height of the tested plant species were measured after 5 d.
Microporous Membrane Method
A 9-cm disc of preculture A. japonicus was placed on 25% (V/V) PDA (containing 2% agar) that was covered with a sterile microporous membrane with a 0.4-µm aperture. Fungal spores were inoculated on the surface of the microporous membrane. A control was made with a disc of the same PDA without fungal spores and a microporous membrane. Five days after incubation at 28°C, the microporous membrane on which the fungi were growing (no fungal growth on the control) was aseptically removed. A piece of sterile filter, 5 mL of sterile water, and 50 surface-sterilized seeds were immediately placed on the top of the medium.
Treatment
The microporous membrane on which the fungi had grown was removed, and 10 mL of ether was added to a petri dish for 5 h to extract the fungal metabolites. The extract was moved to another dish with filter paper to evaporate the ether. After the ether had completely evaporated, 5 mL of distilled water and 50 sterile seeds were added. The seed germination, root length, and seedling height were measured 5 d after incubation with 10 h d-1 artificial light at 25°C for radish and rape and at 28°C for barnyardgrass in triplicated experiments.
Extract of Fermentative Hyphae
Hyphae that were filtered from 250 mL of liquid culture were extracted with 125 mL of acetone for 48 h. The extract was evaporated to dryness under reduced pressure at 55 to 60°C, and then the residue was dissolved in 250 mL of distilled water. The water solution was used in bioassays on rape.
Isolation and Identification of Growth Inhibition Compounds
All reagents and solvents were an analytical grade, and the silica gel for chromatography was 300 to 400 mesh. The mycelium was separated from 3000 mL of liquid culture by filtration and dried for 48 h at 58 to 60°C. Thirty-eight grams of dried mycelium was continuously extracted with ethyl acetate [C4H8O2] (EtOAc) for 3 d. The EtOAc was evaporated to dryness under reduced pressure at 55 to 60°C.
An elemental analysis for C, H, and N was taken with a CHNCORDERD MT-3 elemental analyzer. The melting points were determined with a Yanaco MP-500 apparatus and were uncorrected. Infared spectra were obtained with a Shimadza-IR 435 infrared spectrophotometer. 1H nuclear magnetic resonance (NMR) and 13C NMR were recorded on a Bruker AC-P200 spectrometer (200 MHz), using cadmium chloride (CDCl3) as the solvent and tetramethyl silicane as the internal standard. Mass spectra [electron impact ionization mass spectrometry] (EIMS) were recorded on a MSHP5988A mass spectrometer.
Bioassays
Seeds were germinated before treatment with different concentrations of SAF solutions. Ten seeds were placed in each beaker (50 mL) and kept in 5-mL SAF solutions at 28°C for 12 h of day and 12 h of night. After 4 d, the root and shoot lengths were measured. Secalonic acid F was dissolved in 1.5 g L-1 dimethyl formamide (DMF) and 1 g L-1 Tween 80 solution. The ultraviolet absorption of SAF dissolved in 1.5 g L-1 DMF did not change compared with that of SAF in 950 g L1 ethanol solution. Previous work showed that DMF and Tween 80 did not affect the phytotoxicity of SAF (unpublished data, 1999). All treatments consisted of at least three replicates.
Statistical Analysis
Bioassay data were analyzed by the student's t-test at the 0.05 and 0.01 level. To compare the effects of different concentrations of SAF, the data were subjected to an analysis of variance, and significant treatment differences were tested at the 0.05 level using the LSD test.
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Results and discussion
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Effects of A. japonicus Inoculation on Seedling Growth
When seeds of rape, radish, and cucumber were inoculated with A. japonicus, the seedling growth was significantly inhibited (Table 1). The root lengths of rape, radish, and cucumber were 18.6, 27.5, and 25% of the control, respectively.
Because the experiment was conducted in sterile conditions, the only difference was that the treated seeds were inoculated with A. japonicus. This shows that A. japonicus may affect seedling growth when it is on the seed surface. Seed germination was not affected by direct inoculation with A. japonicus. In the liquid culture experiment, we found that the color of the culture liquid was white during the first 48 h but then became yellow. The bioassays showed that the white culture liquid possessed negligible phytotoxicity. There was no significant phytotoxic difference between 96- and 144-h culture liquid, so the fungus only produces phytotoxins after 2 d and does not need to sporulate before producing phytotoxins. A. japonicus cannot affect germination because seed germination of these crops only requires 1 to 2 d. In nature, if the fungus remained on the seed surface for a sufficient amount of time, then it could inhibit seed germination.
Effects of Metabolites Released from A. japonicus on Seed Germination
In order to separate the effects of allelopathy and disease of A. japonicus, microporous membranes were used to separate the fungus and its growth substrate. The seed germination of the three crops was significantly inhibited by the metabolites released from the fungus (Table 2). The germination rates of rape, radish, and cucumber were inhibited by 54.2, 91.7, and 8.5%, respectively. Thus, the strongest effects were found on radish germination.
Table 3 show that although some seeds germinated, they stopped growing immediately after germination. The inhibition of rape root growth was more than 96%. Because there was no fungal body left on the substrate, the inhibition was only caused by the fungal exudation. The inhibition was even higher than that resulting from direct inoculation of the fungus on the seed surface. This suggests that a certain period is necessary for the fungus to produce enough phytotoxins.
Effects of Fermented Broth of A. japonicus on Seedling Growth
Fermented broth that was incubated for 4 d was filtered and diluted (x4). To exclude the effects of the broth pH, the pH of the dilution was adjusted to 7 with 1 M of sodium hydroxide (NaOH). The diluted extract (pH 2.13) and dilution (pH 7) were used for bioassay. The seedling growth of rape and radish were significantly inhibited by the culture filtrate of the fungus. The rape seedling inhibition was especially high (Table 4). Although the seedling growth was significantly improved if the broth was adjusted to a pH of 7, it was still significantly inhibited compared with control. This indicated that the inhibitory effects of the fermented broth were not caused by a low pH alone. The acetone extract of the fermentative hypha also inhibited the seedling growth of rape (Table 5).
Isolation and Identification of Allelochemicals
The EtOAc extract of 38 g of dried mycelia was evaporated to dryness to yield 1.2 g of yellow precipitate. The precipitate was fractionated by chromatography on a column of silica gel H using a petroleum ether (boiling point of 60–90°C)–EtOAc gradient (10:1–1:5) for elution. Nine hundred and thirty milligrams of yellow needles were obtained, which had the following properties: Melting point of 238 to 242°C (from acetone); [
]20D + 201.5; ultraviolet maximum (ethanol) of 337 and 268 nm; infared (KBr) of 3417 (-OH), 1746 (-COOCH3), 1725 (-C=O), 1689, and 1568 cm-1; EIMS of (relative intensity) 638.45 (calculation of 638.1635 for C32H30O14: 638.1635) (20), 579 (100), 561 (10), 501 (15), 395 (3), 377 (9), 260 (21), 151 (25), and 123 (17) m/z; and an elemental composition of 59.78, 4.71, and 35.51% for C, H, and O (calculation of 60.19, 4.70, and 35.11% for C, H, and O). Data of the 13C NMR and 1H NMR are shown in Table 6. The compound was identified as SAF compared with data reported by Andersen et al. (1977). Fig. 1
shows the structure of SAF, which was also obtained from both fermented broth and rice meal culture.
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Table 6 13C nuclear magnetic resonance (NMR) and 1H NMR data of secalonic acid F (SAF). Chemical shift in CDCl3, signal of residual CHCl3 centered at  7.24 ppm, coupling constants (Hz) in parentheses
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One thousand milliliters of fermented broth was evaporated under reduced pressure at 55 to 60°C to yield 200 mL of solution. The solution was cooled at 4 to 5°C. After 48 h, 1.45 g of colorless crystal formed. The crystals were extracted with EtOAc three times, and the extract was evaporated to dryness to yield 1.13 g of oxalic acid.
Biological Activities of Secalonic Acid F
Secalonic acid F significantly inhibited the seedling growth of the test crops at 0.038 mM (Table 7). The roots of rape seedling stopped growing after germination at 0.075 mM of SAF, and the roots of all tested crop seedlings could not grow at 0.3 mM of SAF.
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Conclusion
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The allelopathy of A. japonicus plays an important role in inhibiting the germination and seedling growth of some crops. If crop seeds are infected by the fungus and the storage conditions are humid with high temperatures, those crops may fail to germinate or may grow badly because of the allelopathy of the fungus.
We report the isolation of SAF from A. japonicus for the first time. The compound is abound in the fungal mycelia. The fungus may influence the seed germination and seedling growth of some cereal crops by means of releasing SAF.
Secalonic acid F and SAA have been demonstrated to be phytotoxic while other secalonic acids (e.g., SAD) have been demonstrated to be toxic in mammalian systems (Vev and Bolon, 1990) and are recognized as mycotoxins, elaborated by food spoilage from P. oxalicum, Aspergillus ochraceus, and Aspergillus aculeatus (Kozakiewicz, 1996). The phytotoxicity of the other ergochromes has not been demonstrated; however, in view of their close structural resemblance to SAF and SAA, they would be expected to possess significant phytotoxicity.
More research is needed to determine which of crops will be affected by the allelopathy of A. japonicus. Details about the mode of action of secalonic acids against higher plants and attention to the human and mammalian toxicity of SAF will also be required.
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ACKNOWLEDGMENTS
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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 financial support.
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NOTES
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This paper was presented orally at the Second World Congress on Allelopathy (Symposium: Allelopathy in Natural and Managed Ecosystems) held during 9–13 Aug. 1999 at Lakehead Univ., Canada.
Received for publication November 29, 1999.
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[Abstract]
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ABSTRACT
INTRODUCTION
