Polyene production of antagonistic Streptomyces species isolated from Sphagnum peat OlaviRaatikainen, Jouko Tuomisto, Risto Tahvonen and Heikki Rosenqvist Raatikainen, 0., Tuomisto, J., Tahvonen, R. & Rosenqvist, H. Polyene production of antagonistic Streptomyces species isolated from Sphagnum peat Agric. Sci. Finl. 2: 551-560. (Dept, of Pharmacology and Toxicology, and Dept, of Pharmaceutical Chem- istry, University of Kuopio, FIN-70211 Kuopio, Finland, Division of Environmental Health, National Public Health Institute, FIN-70701 Kuopio, Finland, Inst, of Plant Protection, Agric, Res. Centre of Finland, FIN-31600 Jokioinen, Finland and Dept, of Biochemistry and Microbiology, Helsinki University ofTechnology, FIN-02150 Espoo Finland.) Several isolates of Streptomyces species, suppressive against fungal growth and ob- tained from light-coloured Sphagnum peat, produced polyene antibiotics. The mech- anism of growth suppression by these isolates is probably partially explained by antibiosis, since there was a significant difference in the antibiotic production by suppressive vs. non-suppressive isolates. The antibiotic consists of several individual components, which form an aromatic heptaene complex of the candicidin type contain- ing p-aminoacetophenone and mycosamine moieties. The minimum inhibitory concen- tration (MIC) of the antibiotic against yeasts and fungi was the same as that of candicidin. Keywords: biocontrol, antibiosis, polyenes, Streptomyces Introduction Biological control of phytopathogens by various Streptomyces species has been studied for decades, and many reports have been published about the role of antibiotics in this phenomenon (Sneh and Henis 1972, Gottlieb 1976, Rothrock and Gottlieb 1981, Rothrock and Gottlieb 1984, Williams 1986, Fravel 1988, Weller 1988). Data concerning therelationship between antibiotic productivity in vitro and biocontrol both supports and opposes this view. Polyene antibiotics are often produced by the actinomycetes isolated from dif- ferent soils (Martin and McDaniel 1977). In vitro production of a polyene antibiotic in connec- tion with the suppression of phytopathogenic fungi has been previously reported but no further charac- terization of this polyene has been performed (Sneh and Henis 1972). Hence, the correlation of the antagonistic activity of Streptomyces bacteria and their ability to produce polyene antibiotics in vitro still seems to be unclear. Finnish Sphagnum peat has been a source for a number of Streptomyces isolates which were dem- onstrated to be effective suppressors of plant dis- eases caused by Fusarium spp.,Altemaria spp. and other phytopathogenic fungi (TAHVONEN 1982a,b, Tahvonen and Avikainen 1987, Tahvonen 551 Agric. Sei. Finl. 2 (1993) https://www.c-info.fi/en/info/?token=yrOrMjrxW0xL5gwE.DvoPAG8nI5zG6Oj3cTkNGw.5o4RYCZu4AUpjwarB2o-q-_blErTPGtXXEuRx7XvBJPyVXggk8LjCcmeIDK8XiLd4gaS1CpOgvntHK89hx9WvSANBZLOYC6_INh4I0KR1ET7n48yGonHN-JWYoRBgDG-mkhqhUwkprYNwMEdFa71zkQFymGK9BvXELEHJO7gkF_6ceBzj8KjuC0vkLBBYsT8Nb2GrWx2djJoiavOVFqTVMpGMofzspP1tyG701dFcO7o8oQkgEVFZ-xIHoPzeiPOcGtp4ytCgyUKrOfTXvYq249zy4CfszkcAufNugptRKF3gnpkRfYFn282lIAmzADIuEBYpdvZarcBvVW_IA 1988). One of these isolates, identified as Strepto- myces griseoviridis (LAHDENPERÄ et al. 1991),was introduced as a biological pesticide against fungal plant diseases (Lahdenperä 1991). Preliminary experiments on agar plates indicated that the sup- pressive isolates could produce some non-volatile factors, and HPLC studies on the chemical nature of the antifungal activity of S. griseoviridis revealed the production of a candicidin type hep- taene complex (Raatikainen 1991). Thus, anti- biosis could be a partial explanation for the mech- anisms of action by the suppressive isolates due to the production of this heptaene in the environment of the growing bacteria. The production, isolation and further charac- terization of this heptaene polyene are described in this study. The production of heptaene by eight suppressive and four non-suppressive Streptomyces species, cultured on liquid or agar medium, was compared. The antifungal activity of the heptaene was estimated by determining the MIC value against yeast and mold cells. Table !. Antagonistic activity of Streptomyces isolates on Allernaria -damping-off of cauliflower. Streptomyces- Effect-% Disease index isolate no on damping-off 0-2 6 97 0.47 61 99 0.22 116 98 0.34 601 95 0.25 606 91 0.30 607 87 0.47 611 94 0.30 624 91 0.36 614 40 1.28 714 32 1.43 728 -17 1.08 740 5 1.80 Efficiency rate is described as Effect-% = (A-B)/(C-B) x 100, where A = Number of healthy plants; seeds infected with A. bras- sicicola and treated with Streptomyces ; B = Number of healthy plants; seeds infected with A. bras- sicicola and not treated with Streptomyces C = Number of healthy plants; seeds not infected and not treated with Streptomyces Isolate 61 is previously identified as S. griseoviridis. Material and methods Strains The suppressive and non-suppressive Streptomyces isolates were selected on the basis of their antago- nistic effectiveness against the growth of phytopa- thogenic fungi in greenhouse experiments deter- mined by previously described methods (Tahvo- nen 1982a,b). The seeds were sawn into plastic pots (volume about 1 1) at a density of 36 cauli- flower seeds per pot, in quadruplicate replications of each Streptomyces isolate. Steamed peat was used as the growing substrate, and the cauliflower seedlings were grown for 3.5 weeks. The degree of infection was determined by dis- ease index at the end of the experimentusing a scale of0-2, where 0= healthy, 1 = slightly damaged foot at the stem, and 2 = severe foot damage or dead seedlings. The efficiency rate against damping-off was also determined (Table 1). Infection of the seeds was ensured by immersing cauliflower seeds in an Alternaria brassicicola sus- pension containing 2-week-old fungal mycelia grown on PDA medium in a Petri dish (100 ml of seeds/dish was used). The seeds were finally dried between filter papers before use. The solution of Streptomyces needed for the seed treatment was prepared by homogenizing the my- celia scraped from the surface of the YMG agar in sterile water. The seeds were treated by soaking them for 5 min in a suspension of Streptomyces oncontaining 10-10 cfu/ml, and finally dried over- night between filter paper sheets. Plant pathogenic isolates of Fusarium culmo- rum (W.G.Sm.) Sacc., Alternaria brassicicola (Schwein.) Wiltsch. and Botrytis cinerea Pers. ex Nocca & Balbis (Agricultural Research Centre of Finland, Jokioinen, Finland), a clinical isolate of Candida albicans TU 96942 (University of Turku, Turku, Finland) and C. albicans ATCC 10231 and Saccharomyces cerevisiae ATCC 9763 were used in the bioactivity tests. The strains were stored under liquid nitrogen or at -80°C. 552 Agric. Sei. Finl. 2 (1993) Culture conditions and extraction procedures for HPLC Eight suppressive and four non-suppressive isolates were grown at 28°C for 96 h in 10 ml test tubes containing 2 ml YMG medium (yeast extract:malt extracl:glucose, 1% each, pH 7.4) for the assess- ment of the production of heptaene polyene. Yeast extract was obtained from Difco Laboratories (MI, USA), malt extract from Laihian Mallas (Laihia, Finland) and glucose from Merck (Darmstadt, Ger- many). The medium was inoculated with 100 pi of primary culture (2 ml of YMG in 10 ml test tubes inoculated with spores scraped from the agar slants shaken at 28°C for 48 h). The cultured cell mass was centrifuged (5,000 rpm for 5 min) and the wet cell mass was weighed and homogenized in 1 ml of 0.05 M ammonium acetate (pH 3.8)-acetonitrile (40:60). The cell mass was centrifuged in 2.0 ml Eppendorf tubes for 15 min at 15,000 rpm. The supernatant containing polyenes was filtered and kept at -20°C until assayed by HPLC. Acetonitrile was HPLC grade (Rathburn, Walkerbum, UK) and ammonium acetate (Merck) was analytical grade. Water in all experiments and analysis was purified using the Milli Q system (Millipore, Molsheim, France). In addition, antibiotic production of the isolates was tested on YMG agar. The plates were rinsed with 5 ml of sterile water containing Streptomyces spores and incubated at 28°C until sporulation was achieved (typically 2 days). An area of agar of 2 .about scm (4 mm in depth) was cut out, homogen- ized with 4 ml of acetonitrile:water (60:40) and centrifuged at 15,000rpm for 15 min. The extracts were stored at -20°C until assayed by HPLC. Radioactive antibiotic was prepared by growing the suppressive isolate in the presence of 8-24kßq of | l4C]p-aminobenzoic acid ([ I4C]PABA, Amer- sham, UK) in 2 ml of YMG at 28°C for 65 h. The antibiotic was extracted as describedabove and the radioactive heptaene was analyzed by HPLC using radioactive flow detection. The cell mass for the isolation, purification and characterization of the heptaene complex was pro- duced by a commercial cultivation process at Ke- mira Research Centre (Espoo, Finland). Assay of the heptaene complex by HPLC The heptaene concentrationfrom cell mass or agar extracts was determined by HPLC (Raatikainen 1991) using candicidin (Dumex Ltd, Copenhagen, Denmark, 1366 IU/mg) as a reference standard. The potency ofcandicidin standard is designated in international units (IU) which defines the relative amount of antibiotic present in the sample (The United States Pharmacopeial Convention 1989). Briefly, a 20 pi aliquot of the acetonitrile extract was injected into a column in a Hewlett Packard 1090 liquid chromatograph (Hewlett Packard, Waldbronn, Germany). The heptaene components were separated on an ODS Hypersil Cig column, 125 x 4 mm, containing 5 pm particles (Bischoff Chromatography, Leonberg, Germany) using isocratic or gradient elution, and the eluate was monitored at 380 nm. [ l4C]radioactivity was counted with radioactivity flow detector as de- scribed below. Isocratic elution was performed using 0.05 M ammonium acetate buffer (pH 3.8)- acetonitrile solution (62:38) as the mobile phase. Gradient elution was performed according to the method describedpreviously (Raatikainen 1991). Briefly, 0.005 M EDTA (analytical grade, Merck) containing 20% of methanol-acetonitrile (70:30) was used as the solution A and acetonitrile as the solution B in the gradient formation. Methanol was HPLC grade (Rathburn). The heptaene components were separated on a column (125 mm x 4 mm, ID) filled with ODS Hypersil phase (particle size 5 pm). Quantitative analysis was performed with gradi- ent elution (Raatikainen 1991), and the sum of the integrated areas of the individual heptaene com- ponents was used for quantification. The amount of heptaene was calculated from the total area of hep- taene peaks in the sample and from the area of dimethylsulphoxide solution of reference standards. The [ l4C]-labelled heptaene components were monitored by a radioactivity flow detector (Radi- omatic FlowOne p/CR, Radiomatic Instruments and Chemical Co., Tampa, FL, USA) using a 2.5 ml homogeneous flow cell (Raatikainen et al. 1991a). The HPLC effluent was mixed with the scintillant (Flow Scint 111, Radiomatic Instruments 553 Agric. Sei. Finl. 2 (1993) and Chemical Co.) in the ratio 1:4, respectively. The counting efficiency was about 68% and the counts between 5 and 100 keV were accepted for integration. Classification of heptaenes The UV spectrum of polyenes was measured in acetonitrile:water (60:40) by a double beam spec- trophotometer (Jasco, Tokyo, Japan). The presence of sugar and aromatic moieties was analyzed by methods described previously (Raatikainen et al. 1991a). Briefly, mycosamine was identified as its acetylated derivative from the acid hydrolysate of heptaene by GC-MS using amphotericin B (Dumex, Copenhagen, Denmark) as a reference standard. The presence of aromatic groups was de- terminedby GC-MS and HPLC-MS in the alkaline hydrolysate of the heptaene. The hydrolysate was extracted with analytical grade trichloromethane (Merck) and the fraction containing p-aminoace- tophenone was purified by semipreparative HPLC, and the purified fraction was analyzed by HPLC and GC-MS. Commercial paminoacetophenone (98%, Aldrich-Chemie, Steinheim, Germany) was used as a reference standard. Isolation of heptaenes The harvested cell mass was shaken in water (pH 3.8)-acetonitrile (40:60) and the residual cell mass was harvested by centrifugation or filtration. NaCl (analytical grade, Merck) solution (160 g/1) was added to the filtrate and the mixture was shaken until the acetonitrile layer separated. The organic layerwas concentrated to about 10% of the original volume and water was added until precipitate formed. The mixture was kept overnight at -20°C and centrifuged. The centrifugate was washed 3 times with water and finally freeze-dried at - 70°C and 10"5 bar. Additionalprecipitation was obtained by adding more water to the supernatant of the previous precipitation and by cooling overnight. The antifungal activity of the precipitate was ana- lyzed as described below. Assay ofantifungal activity by bioautography The antifungal components of the extracts contain- ing antifungal activity (as tested with agar diffu- sion, see below) were separated by thin layer chro- matography (TLC), and bioautograms were ob- tained as follows. The TLC plates (Silika K 60, 20 x 20 mm, Merck, Darmstadt, Germany) were devel- oped by trichloromethane; methanol:borate buffer (45 %vol of 0.05 M NaTfUO? (all components were analytical grade, Merck) and 55 %vol of 0.2 M boric acid, pH 8.3) (7:5:1), dried and overlaid (0.5- 1 mm) with 1% potato-dextrose-agar (PDA, Difco Laboratories) containing conidia of either A. bras- sicicola or F. culmorum.The plate was incubated at 28°C until inhibition of fungal growth was ob- served, then the Rf value for this activity was calcu- lated. Determination of antifungal activity The antifungal activities of the isolated heptaene complexes were determined by their MIC or by agar diffusion assay. A broth dilution test for deter- mining MIC was performed by serially diluting the antibiotic in a buffered peptone-glucose solution (Sabouraud-Glucose-2%-Bouillon, Merck) accord- ing to the method described by Shadomy et al. (1985). The medium was inoculated with the organ- ism to a final concentrationof 105 fungal conidia or yeast cells per ml. The tubes were incubated at 28°C for 24 h and the antibiotic concentrationof the first clear tube was considered as the MIC value. F. culmorum, A. brassicicola, C. albicans TU96942, C. albicans ATCC 10231 and S. cerevisiae ATCC 9763 were used as test organisms. Plate assay for the antifungal activity of various polyene solutions and extracts was performed on plates (diameter 9 cm) containing 20 ml of 1% PDA. The plates were rinsed with 3 to 5 ml of the solution containing the test organism at 105 cells/ml. The solution (80 pi) to be tested was pipet- ted in a well (diameter 8 mm) made in the agar with a corkbore, and the plate was incubated at 28°C, yeast for 24 hours and fungi for 48 hours, when an inhibition zone was formed. The diameter of the 554 Agric. Sei. Finl. 2 (1993) inhibition zone was taken as a measure of anti- fungal activity, where the diameter of the well (8 mm) was considered to represent no activity. C. albicans TU96942 was used as the test organism. Results Selection ofisolates The effect of Streptomyces suspension on the sever- ity of disease caused by A. brassicicola was estim- ated (Table 1). The isolates with a disease index <0.50 were classified as suppressive and those with an index >l.OO as non-suppressive. The isolates with an index of 0.5-1.0 (n =3) were not selected for further study. The efficiency rate of the isolates was analogous to the disease index, being >BO% in suppressive strains (Table 1). Isolation of heptaenes Several precipitates containing heptaene compo- nents were isolated and freeze-dried, and theirpuri- ties were tested by comparing their MIC values with their heptaene content (measured by HPLC). One inhibitory region, typically at Rf 0.2, was found in the bioautography of the extracts. HPLC analysis of the acetonitrile extract of the inhibitory region indicated the presence of heptaene a com- plex, which was probably decomposed during the TLC assay (chromatograms not shown). Chemical characterization of heptaenes The UV spectrum of the antibiotic was typical of heptaene polyenes, showing characteristic absorp- tion maxima at approximately 360,380 and400 nm (Fig. 1). The HPLC chromatogram of the heptaene components produced in the suppressive isolates is of the candicidin type (Fig. 2), and supports the previous suggestion of similarity (Raatikainen 1991). The heptaene complex contained my- cosamine similar to that of amphotericin B as shown by GC-MS and LC-MS (data not shown). Fig. 1, UV spectrum of the heptaene complex produced by the suppressive Streptomyces griseoviridis isolate 61. Fig. 2. HPLC separation of the hep- taene components produced by Streptomyces griseoviridis (lower) and reference heptaene components of candicidin (upper). The eluate was monitored by a photo diode array detector at 380 ran. The ab- sorbance of the highest peak of candicidin is 0.02. 555 Agric. Sei. Finl. 2 (1993) The GC-MS of the alkaline hydrolysate of the hep- taene complex displayed a fragmentation pattern (the three main fragments being M/Z 92, 120 and 135) for p-aminoacetophenone (Raatikainen et. al 1991a), indicating that the heptaene is aromatic (Fig. 3). The latter peak in the total ion chroma- togram (Fig. 3a) is probably due to the chlorination of the aromatic ring of p-aminoacetophenone dur- ing the extraction process after alkaline hydrolysis. This was supported by the mass spectrum with main m/z values of 169 (molecularpeak), 154 (base peak) and 126 (spectrum not shown). Formation of p-amino-monochloroacetophenone is most appar- ent as estimated from the mass spectrum. All indi- vidual components demonstrated aromaticity, as indicated by radiochromatography (data not shown), similarly to I4C-candicidin (Raatikainen et al. 1991a). Heptaene production by isolates The suppressive Streptomyces isolates were hep- taene producers when grown in liquid and on agar culture, and the heptaene containing mycelium ex- tract obtained from liquid culture inhibited the growth of C. albicans (Fig. 4). The suppressive isolate 116 produced only minute amounts of the antibiotic in both liquid and agar culture (Fig. 5). Three of the four non-suppressive isolates produced marginal amounts of polyene, and the remaining one (isolate 728) produced only trace amounts (Figs. 4 and 5). There was a significant difference (p