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Characterization of a fungistatic substance produced by Aspergillus flavus isolated from soil and its significance in nature
New Biotechnology Volume 28, Number 6 October 2011
RESEARCH PAPER
Research Paper
Characterization of a fungistatic substance produced by Aspergillus flavus isolated from soil and its significance in nature Yen-Ting Chen, Mei-Ju Lin, Ching-Hui Yang and Wen-Hsiung Ko Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan
A fungus capable of using vegetable tissues for multiplication in soil was isolated and identified as Aspergillus flavus based on morphological characteristics and sequence similarity of ITS and 28S. When grown in liquid medium prepared from the same vegetable tissues used in soil amendment, the isolate of A. flavus produced a substance capable of preventing disease development of black leaf spot of mustard cabbage caused by Alternaria brassicicola and inhibiting the germination of A. brassicicola conidia. The inhibitory substance was fungistatic, and was very stable under high temperature and high or low pH value. It was soluble in ethanol or methanol, moderately soluble in water, and insoluble in acetone, ethyl acetate or ether. The inhibitor is not a protein and has no charges on its molecule. This is the first discovery of the production of a fungistatic substance by this deleterious fungus. Results from this study suggest the possession of a strong competitive saprophytic ability by A. flavus, which in turn may explain the widespread occurrence of this fungus in soils. Production of a fungistatic substance when A. flavus was grown in medium prepared from vegetable tissues suggests the importance of antibiotic production in its competitive saprophytic colonization of organic matters in soils.
Introduction Aspergillus flavus is an important deleterious fungus due to its production of aflatoxins especially aflatoxin B1 which is the most toxic and most potent naturally occurring carcinogen characterized [1,2]. It is also a common cause of invasive and non-invasive aspergillosis in human and animals, and an opportunistic pathogen of many agricultural crops such as corn, cotton, peanuts as well as tree nuts [3–5]. The fungus is found throughout the world in soils including agricultural, forest, orchard, grassland and wetland soils [6,7]. It was also isolated from virgin as well as cultivated desert soils [8–10]. The widespread occurrence of A. flavus in soil has been associated with its ability to colonize plant debris in soil [11–13]. However, little is known about the competitive saprophytic ability [14] of this fungus in soils. During our recent research, microorganisms capable of utilizing vegetable tissues in soil for multiplication were selectively isolated. Broth prepared from the same vegetable tissues was used to cultiCorresponding author: Ko, W.-H. ([email protected]) 1871-6784/$ - see front matter ß 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.nbt.2011.02.005
vate these organisms. Triturated cultures were screened for ability to control black leaf spot of mustard cabbage caused by Alternaria brassicicola. The culture of a fungus, which was subsequently identified as A. flavus, was found to be very effective in controlling the disease and its extract was strongly inhibitory to the pathogen. This is the first discovery of the production of a fungistatic substance by this important fungus. In this report, we describe the isolation and identification of A. flavus from soil, the production and characterization of the fungistatic substance produced by this fungus and the significance of the discovery in the explanation of its strong competitive saprophytic ability in soil.
Materials and methods Isolation of soil microorganisms Soil samples collected from six locations in central Taiwan were taken from a depth of 0–10 cm, sifted and moistened to about 65% water-holding capacity. Media for selective isolation of fungi, bacteria and actinomycetes were prepared as previously described www.elsevier.com/locate/nbt
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[15]. Approximately 1.3 g soil was mixed with 100 ml sterile distilled water in an Omni mixer chamber at 5000 rpm for 30 s. The suspension was diluted to 10 4, 10 5 and 10 6 for fungi, 10 5, 10 6 and 10 7 for actinomycetes and 10 6, 10 7 and 10 8 for bacteria to determine the dilution needed to obtain soil suspension essentially free of each group of microorganisms. A 1-ml aliquot of diluted soil suspension was mixed with 20 ml of a molten selective medium at 458C in a Petri plate. Five plates were used for each treatment. Vegetables including fruit of tomato (Lycopersicon esculentum), tubers of sweet potato (Ipomoea batatas), and leaves and stems of spinach (Spinacia oleracea), ong choy (Ipomoea aquatica) and common purslane (Portulaca oleracea) were purchased from local market and chopped into small pieces. For soil amendment, about 500 g soil was mixed with 4% of each chopped vegetable in a 1000ml bottle and incubated at 248C for at least two weeks before use. To isolate microorganisms with ability to utilize amended nutrients for multiplication, suspension of amended soil was diluted to the concentration pre-determined for each group of microorganisms and plated on the selective medium as described above. After incubation at 248C for seven days, colonies appeared were individually transferred to 10% V-8 agar (10% V-8 juice, 0.02% CaCO3 and 2% agar) plates.
Culture of isolated microorganisms in liquid medium Liquid medium was prepared by triturating 4 g each of the five chopped vegetables in 100 ml water in an Omni mixer at 4000 rpm for 3 min and dispensing 50 ml broth in a 250-ml flask. After autoclaving, each flask was inoculated with two loopfuls of bacterium, or a piece (ca. 4 5 3 mm) of actinomycete or fungus agar culture. Inoculated flasks were incubated on a shaker for two weeks. After incubation, cultures were separately ground in an Omni mixer at 4000 rpm for 1 min before being used to test their ability to reduce disease incidence.
New Biotechnology Volume 28, Number 6 October 2011
kept in the greenhouse. The number and the size of lesions that developed at the inoculated sites were recorded three days after inoculation. Two leaves were used for each treatment, and all the experiments were repeated at least twice.
DNA extraction The DNA of A. flavus isolate V5F-13 was extracted from 0.1 g threeday-old mycelia grown on cellophane placed on PDA [18] by the plant genomic DNA extraction kit (GeneMark Technology Co., Taichung, Taiwan). The ITS region was amplified with primers ITS1 and ITS4 [19]. PCR was performed in a 50 ml volume reaction containing 2 ml DNA, 1 pmol of upstream and downstream primers and 2.5 units of SuperTaq polymerase (Protech Technology Enterprise Co., Ltd, Taipei, Taiwan) with buffer system recommended by the manufacturer. Cycling conditions of PCR were: initial denaturation at 948C for 2 min, 30 cycles at 948C for 30 s, 558C for 30 s, 728C for 1 min, and a final elongation at 728C for 6 min. The PCR product was analyzed by electrophoresis in a 1.2% agarose gel. In the same manner the large ribosomal subunit 28S was analyzed with primer pairs LROR and LR7 [20]. The annealing temperature was changed to 508C.
Bioassay A 10-ml aliquot of conidial suspension (2 104 spores ml 1) of A. brassicicola was mixed with 10 ml of culture or extract in a cavity of a sterile eight-cavity slide. Slides with spores were kept moist by placing each on an L-shaped glass rod in a 9-cm Petri plate containing 10 ml sterile distilled water. Germination was recorded after incubation at 288C for four hours, and 100 spores were counted in each of three replicates.
Extraction of inhibitory substance
A. brassicicola (Schw.) Wiltshire (isolate Aba-31) was grown on 10% V-8 agar at 248C under light for four to six days for the production of conidia. A conidial suspension was prepared by placing two pieces of culture blocks (ca. 5 5 3 mm) in 5 ml sterile distilled water in a test tube and by agitating the test tube for 30 s with a Vortex mixer. The concentration of conidia was adjusted to 3 conidia ml 1 with a Pipetman microliter pipette (West Coast Scientific, Oakland, CA) [16].
Each flask of liquid culture of A. flavus was tested for the ability to inhibit spore germination of A. brassicicola to assure the presence of the inhibitory substance before freeze drying. One gram of dry powder, obtained from approximately 50 ml of culture, was extracted with 50 ml of water, ethanol, methanol, acetone, ethyl acetate or ether in a 250-ml flask by shaking on a shaker for 24 hours. The mixture was centrifuged at 1500 g for 5 min to obtain clear extract. For bioassay and characterization of the inhibitory substance, 10 ml extract was evaporated to 2 ml followed by the addition of 2 ml water and evaporation to 2 ml again. For control, water was similarly extracted with each solvent and tested for the ability to inhibit spore germination.
Disease control assay of liquid cultures
Characterization of the inhibitor
Seeds of mustard cabbage [Brassica juncea (L.) Coss.] were grown in 8-cm pots containing a mixture of peat moss and vermiculite (9:1, v/v). Two leaves of a four-week-old plant were sprayed to run off with liquid culture of a test microorganism daily three times before inoculation on the fourth day. Each mustard cabbage leaf was inoculated with five 2-ml drops of conidial suspension of A. brassicicola along the edge of the leaf, and a 10-ml drop of molten agar consisting of 1% agar and 1% V-8 juice at 608C was added to each inoculum drop to fix the inoculum on the target site [17]. Leaves of mustard cabbage sprayed with liquid medium were inoculated with conidia of A. brassicicola and used as controls. Inoculated plants were placed in moist chambers and
To study the ability of different adsorptive materials to remove the inhibitor from the culture extract of A. flavus, 5 g of Diaion SK1B cation exchange resins (equivalent to Amberlite 1R-120), Diaion SA 12A anion exchange resins (equivalent to Amberlite 1RA-420; Tai-Young Chemical Co., Kaohsiung, Taiwan) or activated charcoal (Sigma–Aldrich) was washed with 50 ml of distilled water three times by shaking over a six-hour period to remove possible inhibitory substances [21]. Ten milliliter extract was shaken with 1 g cation exchange resins, anion exchange resins or activated charcoal in a 150-ml flask at 100 strokes min 1 for 24 hours and filtered through a Whatman no. 1 filter paper. The filtrates were then used for germination tests.
Inoculum preparation
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Results Effect of liquid cultures of isolated microorganisms on disease incidence From the six soil samples, 71 fungi, 69 actinomycetes and 101 bacteria which were able to use vegetable tissues for multiplication in soils were isolated. Most liquid cultures of isolated microorganisms were ineffective in reducing the disease incidence, when sprayed on mustard cabbage leaves. However, the cultures of one bacterium and five fungi were able to reduce disease incidence of black leaf spot caused by A. brassicicola from 90% in the control to 30% or less, and lesion diameter from 18 mm to 10 mm or less (Table 1). Although all of them were able to reduce the germination rate of A. brassicicola conidia to certain extent, only the culture of the fungus V5F-13 was capable of reducing the germination rate from more than 90% in the control to 0%. It was, therefore, selected for further studies.
Identification of the fungus On Czapek’s agar, the fungus V5F-13 grows 3.8–4.0 cm in diameter in 14 days at 248C. The mycelium was white, but the colony appeared pyrite yellow. Stipes measured 220–370 mm, pale brown and rough. Vesicles were subglobose or pyriform, 12.0–28.0 mm wide. Aspergilla were usually biseriate, occasionally uniseriate or with both forms in the same vesicle. Phialides (7.8–10.4 2.1– 5.0 mm) covered about 3/4 of the whole vesicles surface. Conidia were globose, rough and 3.5–5.6 mm in diameter. The morphological characteristics of V5F-13 fitted the description of A. flavus Link [22]. Its ITS sequence (HQ 395774) matched the ITS sequence of FJ216392 of A. flavus in GenBank with a 99.6% similarity. Its 28S sequence (HQ 395773) also matched the 28S sequence EU071389 TABLE 1
Effect of six liquid cultures of soil microorganisms from the first selection on disease incidence of black leaf spot of mustard cabbage caused by Alternaria brassicicolaa Isolate
Disease incidence (%)
Lesion size (mm, diam.)
V3B-34
20
10
TABLE 2
Effectiveness of different solvents in extracting inhibitory substances from freeze-dried powder of liquid culture of Aspergillus flavus against germination of Alternaria brassicicola conidia Solvent
Water
Germination (%) Extract
Control
57
100
Ethanol
0
98
Methanol
0
100
Acetone
89
98
Ethyl acetate
86
99
Ether
85
91
Water (control)
95
Research Paper
To determine whether the effect of the inhibitor is fungistatic or fungicidal, conidia of A. brassicicola in 1 ml of the extract (105 spores ml 1) in a 1.5 ml centrifuge tube were incubated at 288C. After 24 or 48 hours, the inhibitor was removed by centrifugation of the spore suspension at 1500 g for 5 min and replacement of the supernatant with the same amount of sterile distilled water. Conidia in 0.1 ml of the suspension were then spread on a 1.5% water agar plate. Germination was recorded after incubation at 288C for 24 hours.
of A. flavus in GenBank with a 99.6% similarity. Therefore, the fungus V5F-13 was identified as A. flavus. A culture of the isolate was deposited at Culture Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan, as BCRC34704.
Isolation of the inhibitory secondary metabolite For studying the nature of the inhibitory secondary metabolite, the inhibitory substance was isolated by freeze drying the liquid culture of A. flavus and extracting the dry powder with different organic solvents. After the evaporation of solvents, the extracts were tested for their ability to support spore germination of A. brassicicola. Extracts of ethanol or methanol showed complete inhibition of germination of A. brassicicola conidia. Water extract was moderately inhibitory, while extracts of acetone, ethyl acetate or ether showed much less inhibitory or close to not inhibitory. Controls with each solvent were not inhibitory (Table 2).
Characterization of the inhibitory secondary metabolite Exposure of A. brassicicola conidia to the ethanol culture extract of A. flavus for 24 or 48 hours inhibited germination completely in the extract. However, these condia germinated nearly completely after being transferred to water agar (Table 3), indicating that the effect of the inhibitory metabolite was fungistatic. When the pH of the extract was adjusted from the original 5 to 4 or 3 with 1 N HCl, or 6 with 1 N NaOH, germination of A. brassicicola was still inhibited completely. However, when the pH was adjusted to 7, 8 or 9, germination increased. The extract was no longer inhibitory when its pH was adjusted to 10 (Table 4). When the pH of the extract was adjusted to 2 or 12 for 24 hours and then re-adjusted back to 5, the extract still completely inhibited conidial germina-
V3F-3
0
0
V4F-13
30
10
V5F-13
0
0
Ability of Alternaria brassicicola conidia to germinate after exposure to culture extract of Aspergillus flavus for 24 or 48 hours
V5F-14
0
0
Exposure time (h)
V5F-15
0
0
90
18
Liquid medium (control) a
Two leaves were sprayed once with a liquid culture daily for three days before inoculation on the fourth day. Each leaf was inoculated at five locations, and the disease incidence and lesion size were recorded after three days.
TABLE 3
Germination (%) Extract
Water agar
24
0
81
48
0
82
98
97
Water (control)
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New Biotechnology Volume 28, Number 6 October 2011
TABLE 4
TABLE 6
Conidial germination of Alternaria brassicicola in culture extract of Aspergillus flavus adjusted to various pH values
Effects of exchange resins and activated charcoal treatments of culture extract of Aspergillus flavus on its inhibitory activity against germination of Alternaria brassicicola conidia
pH value after adjustment
Germination (%) Water (control)
Extract 3.0
0
Treatment
pH value after Treatmenta
91
Germination (%)
4.0
0
89
Extract without treatment
5.0 (original)
0
91
Cation exchange resins
4.6
94
Anion exchange resins
4.9
0
6.1
82
5.0
96
6.0
0
Research Paper
7.0
20
93
Activated charcoal
8.0
20
93
Water (control)
9.0
56
87
10.0
100
93
TABLE 5
Effects of extreme pH and high temperature treatments of culture extract of Aspergillus flavus on its inhibitory activity against germination of Alternaria brassicicola conidia Treatment
Treatment Duration
a
Germination (%)
pH 2
24 hours
0
pH 12a
24 hours
0
1008C
30 min
0
Autoclave
15 min
0
Non-treated extract Water (control) a
0 97
The pH value was adjusted back to the original 5 after treatment.
tion of A. brassicicola (Table 5). Conidia of A. brassicicola still failed to germinate after the extract was heated at 1008C for 30 min or autoclaved for 15 min (Table 5). The inhibitory effect of the extract on germination of A. brassicicola was not affected after treatment with cation exchange resins or anion exchange resins. However, the extract was essentially no longer inhibitory after treatment with activated charcoal (Table 6). No positive bands were observed following SDS–PAGE of the inhibitory extract indicating that the inhibitory metabolite is not a protein.
Discussion A. flavus V5F-13 was among the microorganisms capable of colonizing vegetable tissues mixed in soil. Because soil contains abundant and diverse microorganisms [23], the ability to colonize plant tissues under such environment is a good indication that A. flavus possesses a strong competitive saprophytic ability [14]. This may explain its widespread, occurrence in soil [6–10]. The A. flavus isolated in this study produced stable fungistatic substance when cultured in liquid medium prepared from the same kinds of vegetable tissues used in the soil amendment. This indicates the possibility of production of such fungistatic substance by A. flavus during its colonization of plant tissues in soil. It is considered possible that suppression of substrate competitors by this fungistatic substance may account at least in part for the strong com682
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a
5.0
0 0
The pH value was adjusted to the original 5.0 before spore germination test.
petitive saprophytic ability of A. flavus in soil. Such possibility deserves further investigation. The inhibitory substance produced by A. flavus was very stable under high or low pH value or high temperature, indicating that the compound may last very long in nature after its application to the plant leaves. This may explain why the inhibitory substance was very effective in controlling black leaf spot of mustard cabbage even though the inhibitor was only fungistatic against A. brassicicola. Although the compound was effective in controlling black leaf spot of mustard cabbage in the greenhouse, its possible development into a commercial product is far from certain. Its disease control efficacy under field conditions and its toxicity to the non-target organisms in nature remain to be investigated. The substance produced by A. flavus was effective against A. brassicicola at pH 3–6, but was less effective at pH 7–9 and completely ineffective at pH 10. However, when the pH of the extract was changed back to pH 5 after 24 hours at pH 12, it was still strongly inhibitory to A. brassicicola. This suggests that the effect of high pH was probably on the susceptibility of the test organism, rather than on the inhibitor per se. Several antibiotics are more active against microorganisms in acidic medium than in the more alkaline medium [24]. Results from the extraction of freeze dry powder of liquid culture of A. flavus show that the inhibitory substance produced by this fungus is soluble in ethanol or methanol, moderately soluble in water and insoluble in acetone, ethyl acetate or ether. The inhibitory effect of the extract was not affected by treatment with cation or anion exchange resins, indicating that the inhibitor has no positive or negative charge on its molecule. SDS–PAGE of the inhibitory substance suggests that the inhibitor is not a protein. On the basis of its solubility in different solvents and nonexchangeability with cation and anion exchange resins, the inhibitory substance appears to be a hydrophilic compound with hydrophobic functional groups. This is the first report of the production of a fungistatic substance by this important deleterious fungus.
Acknowledgments The authors thank Dr. S. S. Tzean, Department of Plant Pathology and Microbiology, National Taiwan University for assistance in fungus identification. This study was supported in part by a grant from the National Science Council of Taiwan (NSC 97-2321-B-005007-MY3).
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1 Squire, R.A. (1981) Ranking animal carcinogens: a proposed regulatory approach. Science 214, 877–880 2 Bennett, J.W. and Klich, M.A. (2003) Mycotoxins. Clin. Microbiol. Rev. 16, 497–516 3 Scheidegger, K.A. and Payne, G.A. (2003) Unlocking the secrets behind secondary metabolism: a review of Aspergillus flavus from pathogenicity to functional genomics. J. Toxicol-Toxin Rev. 22, 423–459 4 Yu, J. et al. (2005) Aspergillus flavus genomics: gateway to human and animal health, food safety, and crop resistance to diseases. Rev. Iberoam. Mycol. 22, 194– 202 5 Hedayati, M.T. et al. (2007) Aspergillus flavus: human pathogen, allergen and mycotoxin producer. Microbiology 153, 1677–1692 6 Klich, M.A. (2002) Biogeography of Aspergillus species in soil and litter. Mycologia 94, 21–27 7 Horn, B.W. (2003) Ecology and population biology of aflatoxigenic fungi in soil. J. Toxicol-Toxin Rev. 22, 351–379 8 Ranzoni, F.V. (1968) Fungi isolated in culture from soils of the Sonoran Desert. Mycologia 60, 356–371 9 Klich, M.A. (1998) Soil fungi of some low-altitude desert cotton fields and ability of their extracts to inhibit Aspergillus flavus. Mycopathologia 142, 97–100 10 Abdel-Azeem, A.M. and Ibrahim, M.E. (2004) Diversity of terrophilous mycobiota of Sinai. Egypt J. Biol. 6, 21–31 11 Griffin, G.J. and Garren, K.H. (1976) Colonization of rye green manure and peanut fruit debris by Aspergillus flavus and Aspergillus niger group in field soils. Appl. Environ. Microbiol. 32, 28–32 12 Abbas, H.K. et al. (2004) Spatial variability of Aspergillus flavus soil populations under different crops and corn grain colonization and aflatoxins. Can. J. Bot. 82, 1768–1775
13 Abbas, H.K. et al. (2009) Ecology of Aspergillus flavus, regulation of aflatoxin production, and management strategies to reduce aflatoxin contamination of corn. Toxin Rev. 28, 142–153 14 Garrett, S.D. (1970) Pathogenic Root-Infecting Fungi. Cambridge University Press 15 Ko, W.H. et al. (2010) Activity and characterization of secondary metabolites produced by a new microorganism for control of plant diseases. New Biotechnol. 27, 397–402 16 Ann, P.J. et al. (2010) Reevaluation of the report of the A2 mating type of Phytophthora infestans on tomato in Taiwan. Bot. Stud. 51, 203–207 17 Xu, X.L. and Ko, W.H. (1998) A quantitative confined inoculation method for studies of pathogenicity of fungi on plant. Bot. Bull. Acad. Sin. 39, 187–190 18 Wang, P.H. et al. (2010) Severe decline of wax apple trees caused by Fusarium solani in northern Taiwan. Bot. Stud. 51, 75–80 19 White, T.J. et al. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols, a Guide to Methods and Applications (Innis, M.A. et al. eds), pp. 315–322, Academic Press 20 Vilgalys, R. and Hester, M. (1990) Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Bacteriology 172, 4238–4248 21 Ho, W.C. et al. (2007) Effect of oriental medicinal plant extracts on spore germination of Alternaria brassicicola and nature of inhibitory substance from speedweed. Plant Dis. 91, 1621–1624 22 Tzean, S.S. et al. (2005) Fungal Flora of Taiwan, vol. 4. Basidiomycota and Anamorphic Fungi. Taiwan National Science Council 23 Alexander, M. (1977) Introduction to Soil Microbiology (2nd ed.), John Wiley 24 Poala, P. et al. (1970) Effect of pH of medium and size of inoculum on activity of antibiotics against group D Streptococcus (Enterococcus). Appl. Microbiol. 19, 623–637
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References
RESEARCH PAPER
Research Paper
Characterization of a fungistatic substance produced by Aspergillus flavus isolated from soil and its significance in nature Yen-Ting Chen, Mei-Ju Lin, Ching-Hui Yang and Wen-Hsiung Ko Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan
A fungus capable of using vegetable tissues for multiplication in soil was isolated and identified as Aspergillus flavus based on morphological characteristics and sequence similarity of ITS and 28S. When grown in liquid medium prepared from the same vegetable tissues used in soil amendment, the isolate of A. flavus produced a substance capable of preventing disease development of black leaf spot of mustard cabbage caused by Alternaria brassicicola and inhibiting the germination of A. brassicicola conidia. The inhibitory substance was fungistatic, and was very stable under high temperature and high or low pH value. It was soluble in ethanol or methanol, moderately soluble in water, and insoluble in acetone, ethyl acetate or ether. The inhibitor is not a protein and has no charges on its molecule. This is the first discovery of the production of a fungistatic substance by this deleterious fungus. Results from this study suggest the possession of a strong competitive saprophytic ability by A. flavus, which in turn may explain the widespread occurrence of this fungus in soils. Production of a fungistatic substance when A. flavus was grown in medium prepared from vegetable tissues suggests the importance of antibiotic production in its competitive saprophytic colonization of organic matters in soils.
Introduction Aspergillus flavus is an important deleterious fungus due to its production of aflatoxins especially aflatoxin B1 which is the most toxic and most potent naturally occurring carcinogen characterized [1,2]. It is also a common cause of invasive and non-invasive aspergillosis in human and animals, and an opportunistic pathogen of many agricultural crops such as corn, cotton, peanuts as well as tree nuts [3–5]. The fungus is found throughout the world in soils including agricultural, forest, orchard, grassland and wetland soils [6,7]. It was also isolated from virgin as well as cultivated desert soils [8–10]. The widespread occurrence of A. flavus in soil has been associated with its ability to colonize plant debris in soil [11–13]. However, little is known about the competitive saprophytic ability [14] of this fungus in soils. During our recent research, microorganisms capable of utilizing vegetable tissues in soil for multiplication were selectively isolated. Broth prepared from the same vegetable tissues was used to cultiCorresponding author: Ko, W.-H. ([email protected]) 1871-6784/$ - see front matter ß 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.nbt.2011.02.005
vate these organisms. Triturated cultures were screened for ability to control black leaf spot of mustard cabbage caused by Alternaria brassicicola. The culture of a fungus, which was subsequently identified as A. flavus, was found to be very effective in controlling the disease and its extract was strongly inhibitory to the pathogen. This is the first discovery of the production of a fungistatic substance by this important fungus. In this report, we describe the isolation and identification of A. flavus from soil, the production and characterization of the fungistatic substance produced by this fungus and the significance of the discovery in the explanation of its strong competitive saprophytic ability in soil.
Materials and methods Isolation of soil microorganisms Soil samples collected from six locations in central Taiwan were taken from a depth of 0–10 cm, sifted and moistened to about 65% water-holding capacity. Media for selective isolation of fungi, bacteria and actinomycetes were prepared as previously described www.elsevier.com/locate/nbt
679
RESEARCH PAPER
Research Paper
[15]. Approximately 1.3 g soil was mixed with 100 ml sterile distilled water in an Omni mixer chamber at 5000 rpm for 30 s. The suspension was diluted to 10 4, 10 5 and 10 6 for fungi, 10 5, 10 6 and 10 7 for actinomycetes and 10 6, 10 7 and 10 8 for bacteria to determine the dilution needed to obtain soil suspension essentially free of each group of microorganisms. A 1-ml aliquot of diluted soil suspension was mixed with 20 ml of a molten selective medium at 458C in a Petri plate. Five plates were used for each treatment. Vegetables including fruit of tomato (Lycopersicon esculentum), tubers of sweet potato (Ipomoea batatas), and leaves and stems of spinach (Spinacia oleracea), ong choy (Ipomoea aquatica) and common purslane (Portulaca oleracea) were purchased from local market and chopped into small pieces. For soil amendment, about 500 g soil was mixed with 4% of each chopped vegetable in a 1000ml bottle and incubated at 248C for at least two weeks before use. To isolate microorganisms with ability to utilize amended nutrients for multiplication, suspension of amended soil was diluted to the concentration pre-determined for each group of microorganisms and plated on the selective medium as described above. After incubation at 248C for seven days, colonies appeared were individually transferred to 10% V-8 agar (10% V-8 juice, 0.02% CaCO3 and 2% agar) plates.
Culture of isolated microorganisms in liquid medium Liquid medium was prepared by triturating 4 g each of the five chopped vegetables in 100 ml water in an Omni mixer at 4000 rpm for 3 min and dispensing 50 ml broth in a 250-ml flask. After autoclaving, each flask was inoculated with two loopfuls of bacterium, or a piece (ca. 4 5 3 mm) of actinomycete or fungus agar culture. Inoculated flasks were incubated on a shaker for two weeks. After incubation, cultures were separately ground in an Omni mixer at 4000 rpm for 1 min before being used to test their ability to reduce disease incidence.
New Biotechnology Volume 28, Number 6 October 2011
kept in the greenhouse. The number and the size of lesions that developed at the inoculated sites were recorded three days after inoculation. Two leaves were used for each treatment, and all the experiments were repeated at least twice.
DNA extraction The DNA of A. flavus isolate V5F-13 was extracted from 0.1 g threeday-old mycelia grown on cellophane placed on PDA [18] by the plant genomic DNA extraction kit (GeneMark Technology Co., Taichung, Taiwan). The ITS region was amplified with primers ITS1 and ITS4 [19]. PCR was performed in a 50 ml volume reaction containing 2 ml DNA, 1 pmol of upstream and downstream primers and 2.5 units of SuperTaq polymerase (Protech Technology Enterprise Co., Ltd, Taipei, Taiwan) with buffer system recommended by the manufacturer. Cycling conditions of PCR were: initial denaturation at 948C for 2 min, 30 cycles at 948C for 30 s, 558C for 30 s, 728C for 1 min, and a final elongation at 728C for 6 min. The PCR product was analyzed by electrophoresis in a 1.2% agarose gel. In the same manner the large ribosomal subunit 28S was analyzed with primer pairs LROR and LR7 [20]. The annealing temperature was changed to 508C.
Bioassay A 10-ml aliquot of conidial suspension (2 104 spores ml 1) of A. brassicicola was mixed with 10 ml of culture or extract in a cavity of a sterile eight-cavity slide. Slides with spores were kept moist by placing each on an L-shaped glass rod in a 9-cm Petri plate containing 10 ml sterile distilled water. Germination was recorded after incubation at 288C for four hours, and 100 spores were counted in each of three replicates.
Extraction of inhibitory substance
A. brassicicola (Schw.) Wiltshire (isolate Aba-31) was grown on 10% V-8 agar at 248C under light for four to six days for the production of conidia. A conidial suspension was prepared by placing two pieces of culture blocks (ca. 5 5 3 mm) in 5 ml sterile distilled water in a test tube and by agitating the test tube for 30 s with a Vortex mixer. The concentration of conidia was adjusted to 3 conidia ml 1 with a Pipetman microliter pipette (West Coast Scientific, Oakland, CA) [16].
Each flask of liquid culture of A. flavus was tested for the ability to inhibit spore germination of A. brassicicola to assure the presence of the inhibitory substance before freeze drying. One gram of dry powder, obtained from approximately 50 ml of culture, was extracted with 50 ml of water, ethanol, methanol, acetone, ethyl acetate or ether in a 250-ml flask by shaking on a shaker for 24 hours. The mixture was centrifuged at 1500 g for 5 min to obtain clear extract. For bioassay and characterization of the inhibitory substance, 10 ml extract was evaporated to 2 ml followed by the addition of 2 ml water and evaporation to 2 ml again. For control, water was similarly extracted with each solvent and tested for the ability to inhibit spore germination.
Disease control assay of liquid cultures
Characterization of the inhibitor
Seeds of mustard cabbage [Brassica juncea (L.) Coss.] were grown in 8-cm pots containing a mixture of peat moss and vermiculite (9:1, v/v). Two leaves of a four-week-old plant were sprayed to run off with liquid culture of a test microorganism daily three times before inoculation on the fourth day. Each mustard cabbage leaf was inoculated with five 2-ml drops of conidial suspension of A. brassicicola along the edge of the leaf, and a 10-ml drop of molten agar consisting of 1% agar and 1% V-8 juice at 608C was added to each inoculum drop to fix the inoculum on the target site [17]. Leaves of mustard cabbage sprayed with liquid medium were inoculated with conidia of A. brassicicola and used as controls. Inoculated plants were placed in moist chambers and
To study the ability of different adsorptive materials to remove the inhibitor from the culture extract of A. flavus, 5 g of Diaion SK1B cation exchange resins (equivalent to Amberlite 1R-120), Diaion SA 12A anion exchange resins (equivalent to Amberlite 1RA-420; Tai-Young Chemical Co., Kaohsiung, Taiwan) or activated charcoal (Sigma–Aldrich) was washed with 50 ml of distilled water three times by shaking over a six-hour period to remove possible inhibitory substances [21]. Ten milliliter extract was shaken with 1 g cation exchange resins, anion exchange resins or activated charcoal in a 150-ml flask at 100 strokes min 1 for 24 hours and filtered through a Whatman no. 1 filter paper. The filtrates were then used for germination tests.
Inoculum preparation
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Results Effect of liquid cultures of isolated microorganisms on disease incidence From the six soil samples, 71 fungi, 69 actinomycetes and 101 bacteria which were able to use vegetable tissues for multiplication in soils were isolated. Most liquid cultures of isolated microorganisms were ineffective in reducing the disease incidence, when sprayed on mustard cabbage leaves. However, the cultures of one bacterium and five fungi were able to reduce disease incidence of black leaf spot caused by A. brassicicola from 90% in the control to 30% or less, and lesion diameter from 18 mm to 10 mm or less (Table 1). Although all of them were able to reduce the germination rate of A. brassicicola conidia to certain extent, only the culture of the fungus V5F-13 was capable of reducing the germination rate from more than 90% in the control to 0%. It was, therefore, selected for further studies.
Identification of the fungus On Czapek’s agar, the fungus V5F-13 grows 3.8–4.0 cm in diameter in 14 days at 248C. The mycelium was white, but the colony appeared pyrite yellow. Stipes measured 220–370 mm, pale brown and rough. Vesicles were subglobose or pyriform, 12.0–28.0 mm wide. Aspergilla were usually biseriate, occasionally uniseriate or with both forms in the same vesicle. Phialides (7.8–10.4 2.1– 5.0 mm) covered about 3/4 of the whole vesicles surface. Conidia were globose, rough and 3.5–5.6 mm in diameter. The morphological characteristics of V5F-13 fitted the description of A. flavus Link [22]. Its ITS sequence (HQ 395774) matched the ITS sequence of FJ216392 of A. flavus in GenBank with a 99.6% similarity. Its 28S sequence (HQ 395773) also matched the 28S sequence EU071389 TABLE 1
Effect of six liquid cultures of soil microorganisms from the first selection on disease incidence of black leaf spot of mustard cabbage caused by Alternaria brassicicolaa Isolate
Disease incidence (%)
Lesion size (mm, diam.)
V3B-34
20
10
TABLE 2
Effectiveness of different solvents in extracting inhibitory substances from freeze-dried powder of liquid culture of Aspergillus flavus against germination of Alternaria brassicicola conidia Solvent
Water
Germination (%) Extract
Control
57
100
Ethanol
0
98
Methanol
0
100
Acetone
89
98
Ethyl acetate
86
99
Ether
85
91
Water (control)
95
Research Paper
To determine whether the effect of the inhibitor is fungistatic or fungicidal, conidia of A. brassicicola in 1 ml of the extract (105 spores ml 1) in a 1.5 ml centrifuge tube were incubated at 288C. After 24 or 48 hours, the inhibitor was removed by centrifugation of the spore suspension at 1500 g for 5 min and replacement of the supernatant with the same amount of sterile distilled water. Conidia in 0.1 ml of the suspension were then spread on a 1.5% water agar plate. Germination was recorded after incubation at 288C for 24 hours.
of A. flavus in GenBank with a 99.6% similarity. Therefore, the fungus V5F-13 was identified as A. flavus. A culture of the isolate was deposited at Culture Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan, as BCRC34704.
Isolation of the inhibitory secondary metabolite For studying the nature of the inhibitory secondary metabolite, the inhibitory substance was isolated by freeze drying the liquid culture of A. flavus and extracting the dry powder with different organic solvents. After the evaporation of solvents, the extracts were tested for their ability to support spore germination of A. brassicicola. Extracts of ethanol or methanol showed complete inhibition of germination of A. brassicicola conidia. Water extract was moderately inhibitory, while extracts of acetone, ethyl acetate or ether showed much less inhibitory or close to not inhibitory. Controls with each solvent were not inhibitory (Table 2).
Characterization of the inhibitory secondary metabolite Exposure of A. brassicicola conidia to the ethanol culture extract of A. flavus for 24 or 48 hours inhibited germination completely in the extract. However, these condia germinated nearly completely after being transferred to water agar (Table 3), indicating that the effect of the inhibitory metabolite was fungistatic. When the pH of the extract was adjusted from the original 5 to 4 or 3 with 1 N HCl, or 6 with 1 N NaOH, germination of A. brassicicola was still inhibited completely. However, when the pH was adjusted to 7, 8 or 9, germination increased. The extract was no longer inhibitory when its pH was adjusted to 10 (Table 4). When the pH of the extract was adjusted to 2 or 12 for 24 hours and then re-adjusted back to 5, the extract still completely inhibited conidial germina-
V3F-3
0
0
V4F-13
30
10
V5F-13
0
0
Ability of Alternaria brassicicola conidia to germinate after exposure to culture extract of Aspergillus flavus for 24 or 48 hours
V5F-14
0
0
Exposure time (h)
V5F-15
0
0
90
18
Liquid medium (control) a
Two leaves were sprayed once with a liquid culture daily for three days before inoculation on the fourth day. Each leaf was inoculated at five locations, and the disease incidence and lesion size were recorded after three days.
TABLE 3
Germination (%) Extract
Water agar
24
0
81
48
0
82
98
97
Water (control)
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New Biotechnology Volume 28, Number 6 October 2011
TABLE 4
TABLE 6
Conidial germination of Alternaria brassicicola in culture extract of Aspergillus flavus adjusted to various pH values
Effects of exchange resins and activated charcoal treatments of culture extract of Aspergillus flavus on its inhibitory activity against germination of Alternaria brassicicola conidia
pH value after adjustment
Germination (%) Water (control)
Extract 3.0
0
Treatment
pH value after Treatmenta
91
Germination (%)
4.0
0
89
Extract without treatment
5.0 (original)
0
91
Cation exchange resins
4.6
94
Anion exchange resins
4.9
0
6.1
82
5.0
96
6.0
0
Research Paper
7.0
20
93
Activated charcoal
8.0
20
93
Water (control)
9.0
56
87
10.0
100
93
TABLE 5
Effects of extreme pH and high temperature treatments of culture extract of Aspergillus flavus on its inhibitory activity against germination of Alternaria brassicicola conidia Treatment
Treatment Duration
a
Germination (%)
pH 2
24 hours
0
pH 12a
24 hours
0
1008C
30 min
0
Autoclave
15 min
0
Non-treated extract Water (control) a
0 97
The pH value was adjusted back to the original 5 after treatment.
tion of A. brassicicola (Table 5). Conidia of A. brassicicola still failed to germinate after the extract was heated at 1008C for 30 min or autoclaved for 15 min (Table 5). The inhibitory effect of the extract on germination of A. brassicicola was not affected after treatment with cation exchange resins or anion exchange resins. However, the extract was essentially no longer inhibitory after treatment with activated charcoal (Table 6). No positive bands were observed following SDS–PAGE of the inhibitory extract indicating that the inhibitory metabolite is not a protein.
Discussion A. flavus V5F-13 was among the microorganisms capable of colonizing vegetable tissues mixed in soil. Because soil contains abundant and diverse microorganisms [23], the ability to colonize plant tissues under such environment is a good indication that A. flavus possesses a strong competitive saprophytic ability [14]. This may explain its widespread, occurrence in soil [6–10]. The A. flavus isolated in this study produced stable fungistatic substance when cultured in liquid medium prepared from the same kinds of vegetable tissues used in the soil amendment. This indicates the possibility of production of such fungistatic substance by A. flavus during its colonization of plant tissues in soil. It is considered possible that suppression of substrate competitors by this fungistatic substance may account at least in part for the strong com682
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a
5.0
0 0
The pH value was adjusted to the original 5.0 before spore germination test.
petitive saprophytic ability of A. flavus in soil. Such possibility deserves further investigation. The inhibitory substance produced by A. flavus was very stable under high or low pH value or high temperature, indicating that the compound may last very long in nature after its application to the plant leaves. This may explain why the inhibitory substance was very effective in controlling black leaf spot of mustard cabbage even though the inhibitor was only fungistatic against A. brassicicola. Although the compound was effective in controlling black leaf spot of mustard cabbage in the greenhouse, its possible development into a commercial product is far from certain. Its disease control efficacy under field conditions and its toxicity to the non-target organisms in nature remain to be investigated. The substance produced by A. flavus was effective against A. brassicicola at pH 3–6, but was less effective at pH 7–9 and completely ineffective at pH 10. However, when the pH of the extract was changed back to pH 5 after 24 hours at pH 12, it was still strongly inhibitory to A. brassicicola. This suggests that the effect of high pH was probably on the susceptibility of the test organism, rather than on the inhibitor per se. Several antibiotics are more active against microorganisms in acidic medium than in the more alkaline medium [24]. Results from the extraction of freeze dry powder of liquid culture of A. flavus show that the inhibitory substance produced by this fungus is soluble in ethanol or methanol, moderately soluble in water and insoluble in acetone, ethyl acetate or ether. The inhibitory effect of the extract was not affected by treatment with cation or anion exchange resins, indicating that the inhibitor has no positive or negative charge on its molecule. SDS–PAGE of the inhibitory substance suggests that the inhibitor is not a protein. On the basis of its solubility in different solvents and nonexchangeability with cation and anion exchange resins, the inhibitory substance appears to be a hydrophilic compound with hydrophobic functional groups. This is the first report of the production of a fungistatic substance by this important deleterious fungus.
Acknowledgments The authors thank Dr. S. S. Tzean, Department of Plant Pathology and Microbiology, National Taiwan University for assistance in fungus identification. This study was supported in part by a grant from the National Science Council of Taiwan (NSC 97-2321-B-005007-MY3).
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