Antifungal Activity of Sporogen AO-1 and p-Hydroxybenzoic acid Isolated from Penicillium sp. AF5

Antifungal Activity of Sporogen AO-1 and p-Hydroxybenzoic acid Isolated from Penicillium sp. AF5

Jae Gon Kang, Jong Hyun Hur, Bong Sik Yun¹, Ick Dong Yoo¹ and Kyu Young Kang*

Department of Agricultural Chemistry, Division of Applied Life Science, Gyeongsang National University, Jinju 660-701, Korea

1Korea Research Institute of Bioscience and Biotechnology, Yusong, Taejon 305-600, Korea Received January 15, 2003; Accepted February 21, 2003

An antagonistic fungus (AF5) was isolated from greenhouse soil. The fungus exhibited inhibitory activity against several phytopathogens in a dual culture on a potato dextrose agar medium, and was identified as Penicillium sp. based on its mycological characteristics. The antifungal compounds, sporogen AO-1 and p-hydroxybenzoic acid, were isolated from a culture broth of the Penicillium sp. AF5 and purified through solvent partition, silica gel column chromatography, and preparative TLC.

Sporogen AO-1 showed strong activity either on mycelial growth (ED50= <40 ppm)of Phytophthora capsici, Pythium ultimum, and Rhizoctonia solani or zoospore germination of Phytophthora capsici. p- Hydroxybenzoic acid exhibited weak activity.

Key words: Penicillium sp., antifungal activity, sporogen AO-1, p-hydroxybenzoic acid.

The use of agricultural chemicals is essential for increasing food production. However, heavy dosage of pesticides, which has been applied in some cases to maintain a stable production of the crop, can bring about harmful side effects and resurgence of pests due to resistance to the pesticide.

Biological control of disease is one of the means to reduce the potentially harmful fungicide use.¹⁾ Recently, there has been increasing interest in research on pest management that exploits beneficial microorganisms to protect plants from phytopathogens.^2,3) Direct application of biocontrol agents, such as Aspergillus,⁴⁾ Trichoderma,^4,5) Chaetomium,⁶⁾ and Penicillium,⁷⁾ to plant pathogens has already been attempted.

A large number of bioactive metabolites have also been isolated from various microbial sources, such as Actinomycetes, bacteria, and fungi. In addition, secondary metabolites of microbial origin have been discovered and commercialized.^8,9) These antibiotics exhibit little toxicity on the environment and plants.

The genus Penicillium is ubiquitous in all environmental niches. The genus gained notice due to its ability to produce antibiotics, namely penicillin.¹⁰⁾ Recently, new antifungal antibiotics, such as BE-31405¹¹⁾ and PF11643¹²⁾, have been isolated from the Penicillium species.

In the present study, we isolated a Penicillium sp.

antagonistic to plant pathogenic fungi and identified two antifungal compounds from a culture broth of the isolate.

Accordingly, taxonomy of the producing strain, fermentation, isolation, physico-chemical properties, and new antifungal activity of sporogen AO-1 and p-hydroxybenzoic acid are

characterized in the current paper.

Materials and Methods

Isolation and taxonomical studies of antifungal fungus.

An antifungal compound-producing strain, AF5, was isolated from a soil sample collected from a greenhouse in Jinju, Korea. Appropriate serial dilutions of the soil suspension in sterile water were spread on a rose-bengal streptomycin agar plate,¹³⁾ which plate was incubated at 28^oC for 7 days. Single colonies inhibiting growth of microorganisms nearby were isolated and screened for antifungal activity using the petri plate assay. The fungus was identified according to the morphological key and species previously described by Pitt.¹⁴⁾ Czapek-dox agar (CDA), potato dextrose agar (PDA), and malt extract agar (MEA) were used for growth and identification of the fungus. This strain was maintained on a slant of PDA at 28^oC.

Production and isolation of antifungal compounds. The antifungal compounds were isolated from the culture broth of the antagonistic fungus AF5 grown in a modified Czapek-dox medium (CDM), in which asparagine was substituted for KNO3. The fungal strain was cultivated for 7 days at 28^oC in a shaking incubator. The culture broth (10 l) was filtered through cheese cloth to remove the fungal mycelia. Two antifungal compounds designated AF5C1 and AF5AE1 were isolated from a culture filtrate. To isolate AF5C1, the filtrate (10 l) was extracted with 10 l of chloroform. The chloroform extract was then combined and concentrated in vacuo to dryness to give a brown residue (ca. 2 g). This extract was applied to a silica gel (230-400 mesh) column (20× 400 mm) and eluted stepwise with hexane-ethyl acetate. Through activity-guided bioassay, the fractions (20 mg) exhibiting

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antifungal activity were combined and dried in vacuo. The residues were then loaded onto a preparative TLC (60 GF254, 20× 20 cm, 0.25 mm, Merck) and developed with toluene- ethyl acetate (3 : 1, v/v). The band showing antifungal activity was scraped off the plate and eluted with ethyl acetate. The yield was ca. 1 mg from 10 l of culture broth. To isolate AF5AE1, the culture filtrate (10 l) was adjusted to pH 2 with HCl and extracted with ethyl acetate (10 l). The ethyl acetate extract was then concentrated in vacuo to dryness to give a dark brown extract (ca. 3 g). This extract was applied to a column of silica gel and eluted stepwise with hexane-ethyl acetate. The fraction (35 mg) that exhibited antifungal activity was rechromatographed on a silica gel column and eluted with chloroform-methanol step gradients. The active fractions were combined and dried in vacuo to obtain a white solid (ca.

3 mg).

Identification of isolated compounds. UV spectrum of the purified antifungal compounds was obtained in a methanol solution using a Beckman DU-600 spectrophotometer. IR spectra were obtained using a Bruker IFS 66 spectrophotometer for samples in thin films on a KBr window.

Proton and ¹³C NMR spectra were obtained with a Bruker AW500 spectrometer. The sample was prepared as 20 mg·ml⁻¹ solution in CDCl3 for AF5C1 and in CD3OD for AF5AE1 with tetramethylsilane (Me4Si) as an internal reference. The mass spectrum was obtained on a JEOL JMS- DX300 spectrometer through direct probe method, with electron impact ionization at 70 eV.

Mycelial growth inhibition test. The serially diluted antibiotic was incorporated in 1/5 strength PDA plates at 0 to 200 ppm, and 3 mm agar plugs of five phytopathogenic fungi, Pythium ultimum, Phytophthora capsici, Rhizoctonia solani, Botrytis cinerea, and Fusarium oxysporum were placed on the center of the plates. The diameters of the mycelial growth were measured from three replicates for each treatment. ED50

values of the mycelial growth inhibition were calculated by a probit analysis.¹⁵⁾

Zoospore germination test. Spore suspensions of P.

capsici in distilled water were produced from three-day-old

cultures grown on a V8-juice agar medium. Serial dilutions of the compound in MeOH were made to obtain solutions with concentrations of 200, 100, 20, 4, 0.8, and 0.16 ppm.

Antibiotic solutions (10µl) were added to the zoospore suspension containing 10⁵ zoospore · ml⁻¹. The treated spores (100µl) were loaded onto hole-slide glasses, and placed on moistened paper in a sealed box. The zoospore suspension of P. capsici was incubated for 3 h at 25^oC, after which approximately 100 zoospores from each of the three replicates were examined under a light microscope to determine the percentage of germinated zoospores. The results are presented as mean values with a standard deviation.

Results

Isolation and identification of the antagonistic fungus.

Several antagonistic fungi isolated from the greenhouse soil were screened by the petri-plate assay against the five phytopathogenic fungi. One antagonistic fungus (AF5) showing an inhibition zone was selected for identification.

The mycological characteristics of strain AF5 were as follows.

Colonies growing on CDA attained a diameter of 20~25 mm at 28^oC in 7 days, were floccose, protuberant at the center, and bright green in color. Conidia formation was good. The bottom sides of the colonies were buff to pale brown. Exudate and soluble pigment were not produced. Colonies on MEA and PDA attained diameters of 15~20 mm at 28^oC in 7 days, were also floccose, protuberant at the center, and white to bright green. Conidia formation was moderate. The bottom sides of the colonies were bright gray. Soluble pigment was not produced. When strain AF5 was grown on PDA at 28^oC for 7 days, conidiophores were borne from the substrate hyphae, and penicilli were biverticillate. Metulae were 3~5 per stipe. Phialides were amplyform, 3 per metula. Conidia formed chains at the top of phialides, and were globose to subglobose in shape. The above characteristics of strain AF5 indicate that this fungus belongs to the genus Penicillium.

Structure determination of isolated compounds.

Physico-chemical properties of AF5C1 and AF5AE1 are

Table. 1. Physico-chemical properties of sporogen AO-1 and p-hydroxybenzoic acid.

Properties Sporogen AO-1 p-Hydroxybenzoic acid

Appearance Colorless oil White solid

Molecular formula C15H20O3 C7H6O3

EI-MS (m/z)

calcd: 248.14 for C15H20O3 138.03 for C7H6O3

found: 248 (M+) 138(M+)

UV λmax nm (log ε) in MeOH 241 (13072) 254 (23947)

IR νmax (KBr) cm⁻¹ 3456, 2931,1667 3449, 1643

Solubility

soluble in CHCl3, EtOAc, MeOH CHCl3, EtOAc, MeOH

insoluble in Hexane, H₂O Hexane, H₂O

TLC (Rf)^a 0.2 0.34

aSilica gel TLC (Merk Art 5715): Toluene-EtOAc=3:1 for sporogen AO-1 and Hexane-EtOAc=4:1 for p-hydroxybenzoic acid

shown in Table 1. The compounds were soluble in most organic solvents such as acetone, ethyl acetate, and methanol, but insoluble in hexane and water. The Rf value of AF5C1 was 0.2 on a silica gel thin layer plate developed with toluene- ethyl acetate (3 : 1, v/v). Another compound AF5AE1 was 0.34 on a silica gel 60 thin layer plate developed with hexane- ethyl acetate (4 : 1, v/v).

Compound AF5C1 was obtained as a colorless oil. The molecular formula for AF5C1 was established as C15H20O3

based on its EI-MS and NMR spectra. The IR spectrum showed the presence of a hydroxyl group (3456 cm⁻¹) and α,β-unsaturated carbonyl group (1667 cm⁻¹). ¹H and ¹³C NMR data of AF5C1 are shown in Table 2. ¹³C NMR data and DEPT experiment revealed the presence of three methyl, three methylene, four methine, and five quaternary carbons, including one carbonyl carbon. Two methine carbons of 3-C (δC 70.96) and 6-C (δC 68.35) were assigned as oxygen- bearing carbons from their chemical shifts. On the other hand,

1H NMR spectrum disclosed the presence of a tertiary methyl group at δH 1.22, a secondary methyl group at δH 1.26, olefinic proton at δH 5.75, secondary hydroxyl group at δH 3.62, and epoxide at δH 3.33. ¹H-¹H COSY spectrum showed that 13-H (δH 1.86) was correlated to 12-H (δH 5.10), which indicates the presence of an isopropenyl group linked to δC 63.52 in HMBC spectrum. From the above spectral data, the isolated compound, AF5C1, was identified as sporogen AO-1 (Fig. 1).

Compound AF5AE1 was obtained as a white powder. The

molecular formula for AF5AE1 was established as C7H6O3

based on its EI-MS and NMR spectra. Its EI-MS spectrum showed intense fragment ions at m/z 138 (M⁺), 121 (M⁺-OH), and 93 (M⁺-COOH). IR spectrum showed the presence of a hydroxyl group (3449 cm^-1) and carbonyl group (1643 cm⁻¹).

1H and ¹³C NMR data of AF5AE1 are shown in Table 2. Two symmetric methine carbons (δC 116.07 and 133.04) and one carbonyl carbon were observed in the ¹³C NMR spectrum. ¹H NMR spectrum exhibited two symmetric methine protons (δH

7.87 and 6.81). Its proton had ortho interaction as shown by the coupling constant value of 8.7 Hz. This indicates the presence of 1,4-substituted benzene ring. Thus, the isolated compound AF5AE1 was determined as p-hydroxybenzoic acid (Fig. 1).

Mycelial growth inhibition test. The antifungal activities of compounds sporogen AO-1 and p-hydroxybenzoic acid are Table 2. ¹H and ¹³C NMR data for sporogen AO-1and p-hydroxybenzoic acid.^a

Position Sporogen AO-1 (CDCl3) Position p-Hydroxybenzoic acid (CD₃OD)

13C (δ) ¹H (δ) ¹³C (δ) ¹H (δ)

1 30.96 2.51 (1H, dddd, J=2.0, 5.0, 14.4, 14.4Hz) 1 122.97

2+6 133.04 7.87 (2H, d, J=8.7 Hz) 2.33 (1H, ddd, J=2.9, 4.0, 14.4Hz)

3+5 116.07 6.81 (2H, d, J=8.7 Hz)

2 35.27 2.15 (1H, m)

4 163.35

1.43 (1H, m)

7 170.33

3 70.96 3.62 (1H, ddd, J=4.4, 10.8, 10.8 Hz)

4 44.38 1.81 (1H, m)

5 41.02

6 68.35 3.33 (1H, s)

7 63.52

8 192.78

9 121.22 5.75 (1H, d, J=1.9 Hz)

10 163.00

11 139.11

12 114.47 5.10 (2H, m)

13 19.78 1.86 (3H, t, J=1.1 Hz) 14 18.80 1.22 (3H, s)

15 11.28 1.26 (3H, d, J=6.7 Hz)

a 1H and ¹³C NMR spectra were measured at 500 and 125 MHz, respectively.

Fig. 1. Structures of sporogen AO-1 and p-hydroxybenzoic acid.

shown in Table 3. ED50 values of sporogen AO-1 against P.

capsici, P. ultimum, and R. solani were 6, 39, and 24 ppm, whereas those of p-hydroxybenzoic acid were 136, 106, and over 200 ppm, respectively.

Zoospore germination test. The zoospore germination inhibition activities of sporogen AO-1 and p-hydroxybenzoic acid against P. capsici were evaluated (Fig. 2). The Inhibition of the zoospore germination by sporogen AO-1 was over 80%

at a dosage of 20µg · ml⁻¹, whereas that of p-hydroxybenzoic acid was rather weak.

Discussion

In recent years, biological control of soil-borne pathogens has received increasing attention as a promising alternative to chemical control. Disease suppression by microorganisms in agriculturally important crops has been studied in detail.^16-18)

A study on screening and investigation of the antagonistic microorganisms against plant pathogenic fungi in the greenhouse was performed. This paper presents the isolation and characterization of a Penicillium strain AF5 that secrets antifungal compounds against plant pathogenic fungi. The strain AF5 exhibited significant inhibitory activities against P.

capsici, R. solani, P. ultimum, and B. cinerea based on dual culture on potato dextrose agar.

The isolated compound, sporogen AO-1, was active against all fungi tested, except for B. cinerea and F. oxysporum. In

particular, sporogen AO-1 was as potent as metalaxyl in the mycelial growth inhibition of P. capsici (Table 3). The zoospore germination inhibition activity of sporogen AO-1 was excellent over 80% at a dosage of 20µg · ml⁻¹, slightly lower than ED50 of mycelial growth, whereas that of p- hydroxybenzoic acid was weak (Fig. 2).

p-Hydroxybenzoic acid has previously been isolated from P.

patulum¹⁹⁾ and used as an intermediate for organic syntheses and fungicides. Sporogen AO-1 has also been isolated from the culture broth of Aspergillus oryzae (NOY-2) and used as a phialospore formation-stimulator.^20,21) Sporogen AO-1 exhibited significant sporogenic activity at a dose of 4.4µg per disc against A. oryzae, whereas no activity at 50µg per disc against Penicillium sp. (data not shown). These data suggest that sporogen AO-1 have sporogenic activity against specific strains such as Aspergillus. Accordingly, this is the first report on the isolation of sporogen AO-1 from a culture broth of a Penicillium sp. along with its fungicidal activity against phytopathogenic fungi.

Acknowledgments. This work was supported by a grant from the Province of Gyeongsangnam-do, Republic of Korea.

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