Polyene production of antagonistic Streptomyces species isolated from Sphagnum peat

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 Chemistry, 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.)


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 demonstrated 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  1988).One of these isolates, identified as Streptomyces 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 suppressive 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 heptaene 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.

Strains
The suppressive and non-suppressive Streptomyces isolates were selected on the basis of their antago- nistic effectiveness against the growth of phytopathogenic 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 mycelia 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 on containing 10-10 cfu/ml, and finally dried over- night between filter paper sheets.
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 8-24 kßq of | l4 C]p-aminobenzoic acid ([ I4 C]PABA, Amer- sham, UK) in 2 ml of YMG at 28°C for 65 h.The antibiotic was extracted as described above 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 produced by a commercial cultivation process at Ke- mira Research Centre (Espoo, Finland).

Assay of the heptaene complex by HPLC
The heptaene concentration from 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. [l4 C]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 described previously (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 heptaene peaks in the sample and from the area of dimethylsulphoxide solution of reference standards.
The [ l4 C]-labelled heptaene components were monitored by a radioactivity flow detector (Radiomatic FlowOne p/CR, Radiomatic Instruments and Chemical Co., Tampa, FL, USA) using a 2.5 ml homogeneous flow cell (Raatikainen et al.   1991 a).The HPLC effluent was mixed with the scintillant (Flow Scint 111, Radiomatic Instruments 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 spectrophotometer (Jasco, Tokyo, Japan).The presence of sugar and aromatic moieties was analyzed by methods described previously (Raatikainen et al. 1991 a).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 determinedby 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-aminoacetophenone 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.Additional precipitation 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.

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 organism to a final concentration of 10 5 fungal conidia or yeast cells per ml.The tubes were incubated at 28°C for 24 h and the antibiotic concentration of 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 10 5 cells/ml.The solution (80 pi) to be tested was pipetted 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 inhibition zone was taken as a measure of antifungal activity, where the diameter of the well (8 mm) was considered to represent no activity.C. albicans TU96942 was used as the test organism.

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 components were isolated and freeze-dried, and their purities 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 absorption maxima at approximately 360,380 and 400 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).  lower)  and reference heptaene components of candicidin (upper).The eluate was monitored by a photo diode array detector at 380 ran.The absorbance of the highest peak of candicidin is 0.02.
The GC-MS of the alkaline hydrolysate of the heptaene complex displayed a fragmentation pattern (the three main fragments being M/Z 92, 120 and 135) for p-aminoacetophenone (Raatikainen et.al 1991 a), 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 (molecular peak), 154 (base peak) and 126 (spectrum not shown).Formation of p-amino-monochloroacetophenone is most apparent as estimated from the mass spectrum.All indi- vidual components demonstrated aromaticity, as indicated by radiochromatography (data not shown), similarly to I4 C-candicidin (Raatikainen

Heptaene production by isolates
The suppressive Streptomyces isolates were heptaene 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<o.ol,Mann Whitney U) in the polyene productivity in liquid culture between 8 suppressive (324.5 ± 66.2 lU/ml/gram of wet cell mass) and 4 non-suppres- sive (12.0 ± 3.8 IU/ml/g) isolates.The anticandidal Fig. 3. GC-MS identification of p- aminoacetophenone released by al- kaline hydrolysis from heptaenes produced by Streptomycesgriseoviridis. Total ion chromatogram of the HPLC-purified hydrolysate (A)  and the fragmentation spectrum of p-aminoacetophenone peak (B).activity of the extracts, as indicated by the growth inhibition zone (mm) of C. albicans grown on agar plates, also differed significantly between the suppressive (23.4 ± 1.4) and nonsuppressive (11.6 ± 2.5) isolates (p<o.ol,Mann-Whitney U).The growth (as indicated by wet weight of cell mass) of the suppressive isolates (303.4 ± 17.0 mg) was greater (p<o.ol,Mann-Whitney U) than that of the nonsuppressive isolates (198.0 ± 32.6).
The results from agar (Fig. 5) were similar to those obtained from liquid culture, and there was a significant difference (p<o.ol,Mann-Whitney U) between the antibiotic productivity of the suppressive (40.9 ± 7.2 lU/ml, N=B in duplicate cultures) and non-suppressive (2.9 ± 0.6, N = 4 in duplicate cultures) isolates grown on agar.

Antifungal activity of the heptaene complex
The MIC values of the heptaene complex of this study were the same as those of candicidin and lower than the values for amphotericin B (Table 2).The MIC for the heptaenes was ten times higher against the fungi (2-4 pg/ml) compared to yeasts (0.02-0.3 pg/ml) (Table 2).

Discussion
The limited usefulness and correlation of culture assays in antibiotic production and biocontrol ac- tivity of the test strains have been noted earlier (Gottlieb 1976,Fravel 1988).The antibiotic ac- suppressive and non-suppressive (dashed line) isolate groups are different (p<o.ol,Mann-Whitney U).Production in liquid culture is expressed as the concentration (lU/ ml) of antibiotic in the aceto- nitrile:water extract from I gram of wet mycelium.The antifungal activity of the extracts was tested against the clinical isolate Can- dida albicans TU 96942 and is shown as the diameter (mm) of the inhibition zone formed in agar dif- fusion assay.Fig. 5. Production of aromatic heptaenes by suppressive and nonsupprcssive (dashed line) isolates on agar plates is significantly different (p<o.ol,Mann-Whitney U).Production on agar culture is expressed as the concentration (lU/ml) of antibiotic in the acetonitrile:water extract from agar.tivity, or antagonism, on agar did not correlate with the suppression of plant diseases by test organisms in greenhouse experiments in a study conducted by Rothrock and Gottlieb (1981).In that study, one strain (Streptomyces noursei) was a polyene (nystatin) producer, but the strain was not effective in controlling plant diseases.The earlier results of Sneh and Henis (1972) indicated that polyene production was typical of the antagonists used.They could not, however, demonstrate the actual production of polyenes in non-sterile soils or substrates.
The present study suggests that the growth inhibition of the plant pathogens by suppressive Streptomyces isolated from Finnish Sphagnum peat can be partially explained by antibiosis.Cultures of 7 suppressive Streptomyces isolates were producers of a candicidin type heptaene complex.The struc- tures of candicidin D (Fig. 6), one of the main components of the antibiotic complex, and some other aromatic heptaenes have been determined previously (Omura and Tanaka 1984).Although one isolate (116) was considered to be suppressive, it produced only small amounts of heptaene both in liquid and in agar culture.In addition to isolate 116, some contradiction could be seen between the antifungal activity and polyene production of certain suppressive isolates (Fig. 4), but the reason could not be clarified in this study.This contradiction may be due to differences in the genetic control of the antibiotic biosynthesis causing variation in the antibiotic productivity.Another exception was the nonsuppressive isolate 714 which exhibited some anticandidal activity in the bioassay (Fig. 4).It did not produce heptaene, indicating the presence of some unknown antifungal factor.The reason for enhanced growth of the suppressive isolates in small scale liquid culture remains unclear.Perhaps they are simply more viable than the non-suppressive isolates in this medium, since their growth on agar was also better.
Suppressive isolates typically produced similar patterns of individual heptaene components as indicated by HPLC (data not shown).The aromatic group in all individual components was p-aminoacetophenone and the sugar moiety was mycosamine, reflecting the similarity with candicidin (Fig. 6).Thus the identification of the aromatic group by HPLC-MS provides a technique to deter- mine the total amount of aromatic heptaenes present in soil samples.
Our results did not eliminate the possibility of non-suppressive isolates producing heptaene in the presence of other carbon sources.When a rich medium, containing yeast extract, malt extract and glucose was used as the growth medium, no significant production of polyene was found.The nutritional environment, however, may affect the antibiosis and biocontrol as suggested by Fravel  (1988).Studies on the nature of the bald mutant of one of the suppressive S. griseoviridis (isolate 61) indicated that the carbon source of minimal me- Fig. 6.The structure of candicidin D, showing the aromatic (left) and sugar moieties (right) attached to the macrolide ring (Omura and   Tanaka 1984).
It is known that the heptaene polyene antibiotics bind to the ergosterol molecules in the fungal or animal cell membrane, and together they form ion conducting pores that make the plasma membrane leaky to monovalent and divalent cations (Bolard   1986, Raatikainen et al. 1991 b).This apparently stimulates fungal cell respiration, increasing the energy demand, ultimately resulting in cell death.
In addition to antibiosis by heptaenes, the other known mechanisms of antagonism (eg.hyperparasitism, production of siderophores, competition) are also likely, and it has been suggested that one of the suppressive isolates produced extracellular en- zymes with lytic characteristics (Tapio and Pohto- Lahdenperä 1991).The production of chitinase was supported by the growth of S. griseoviridis isolate 61 on chitin (unpublished results), though further research on the activities of extracellular enzymes is needed to firmly establish this mech- anism.
The production of polyene antibiotics can be used as a marker in screening of the biocontrol activity among isolates of Streptomyces.Although a significant correlation between suppressiveness and polyene productivity exists, only limited con- clusions may be drawn on the role of antibiosis in this antagonism by these results.Studies with non- producing mutants, originating from suppressive strains, could clarify the mechanism of their an- tagonistic action.On the other hand determinations of the heptaene components in greenhouse sub- strates are a promising way to assess the role of antibiosis.

Table ! .
Antagonistic activity of Streptomyces isolates on Allernaria -damping-off of cauliflower.
= Number of healthy plants; seeds not infected and not treated with Streptomyces Isolate 61 is previously identified as S. griseoviridis.

Table 2 .
MIC values (ug/ml) of isolated heptaene produced by Slreplomycesgriseoviridis on the growth of F. culmorum, C. albicans and S. cerevisiae.